16 Human Intervention in Evolution

CHAPTER 16 HUMAN INTERVENTION IN EVOLUTION
FIGURE 16.1 In January 2009, a private company in South Korea (RNL Bio Co) announced the first ever successful cloning of dogs using adipose (fat) stem cells. These two Beagle pups, Magic and Stem, were the only successes from 84 cloned embryos that were transplanted into surrogate mother dogs. In this chapter, we will examine human intervention in evolutionary processes using procedures including selective breeding and various technologies through which the normal processes of natural selection are overridden.
KEY kNOWLEDGE
This chapter is designed to enable students to: gain an overview of human interventions in evolutionary processes enhance understanding of gene technology and recognise applications that intervene in evolutionary processes acquire knowledge of selective breeding and reproductive technologies, and recognise how they intervene in evolutionary processes distinguish between reproductive and therapeutic cloning.
Lambs in spring
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The Booroola gene appears to have been introduced by Bengal sheep imported to Australia from India in the 1792. Records show that the Booroola merino flock can be traced back to the merinos owned by Rev. Samuel Marsden (17641838) who also owned a Bengal flock.
Early spring is the lambing season in Australia. In certain sheep breeds, such as merino, a female sheep or ewe typically produces just a single lamb each season. However, these days, it is not uncommon to see merino ewes with twins, sometimes triplets and, in some cases, even quads. What has caused this change? It is human intervention through artificial selection of an allele for increased f ecundity by selective breeding with rams and ewes that have that particular allele. The gene concerned is known as the Booroola gene. Its action in producing increased fecundity was recognised by the Seears brothers (see figure 16.2) in the merino flock at their Booroola property, near Cooma, New South Wales in the 1950s. The Booroola gene is located on the number-6 sheep chromosome and its rare BB allele increases ovulation rate while its more common B+ allele has no effect on ovulation rate. In heterozygous BBB+ ewes, the ovulation rate is increased by a factor of 1.5, while in homozygous BBBB ewes, their ovulation rate is increased by a factor of 3.0. (The ovulation rate in homozygous B+B+ ewes is 1.0.)
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FIGURE 16.2 (a) A Booroola ewe with quintuplets, 1958. The merino ewe had the allele of a gene that increased her ovulation rate, making her a more prolific breeder than normal. Was this ewe more likely to be homozygous or heterozygous for the BB allele? (b) Dick Seears (left), his son Michael, and Jack Seears (right) at Cooma in 1959, with two 13-year-old ewes. One ewe had produced 27 lambs, the other 24 lambs.
The Booroola gene is formally known as BMPR-1B and its alleles are denoted as FecBB and FecB+ but, for simplicity, they are shown here as BB and B+.
Using selective breeding techniques, the BB allele has now been incorporated into other sheep breeds that normally produce a single lamb from each mating. This was done by crossing these breeds with sheep known to have the BB allele. Selective breeding is an example of artificial selection. In this procedure, only those animals that display a particular trait in their phenotype or are known carriers of the trait are chosen to reproduce. Selective breeding is just one example of how humans can intervene in natural evolutionary processes. Later in this chapter, we will examine other examples of human intervention in normal evolutionary processes in a variety of species.
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Selective breeding in action

From early times, artificial selection was carried out to improve herd quality. Farmers selected the best males for mating with their breeding females. In the case of beef cattle, bulls were chosen for their genetic superiority in terms of desirable market characteristics, such as meat yield and nonfatty carcass. For dairy cattle, desirable market characteristics included milk yield and butterfat content. Merino rams are chosen for the fineness (thinness) of their wool fibre and the yield of greasy fleece. Males were also selected based on other inherited features, including good conformation (form, outline or shape), high fertility based on sperm counts and the absence of any known genetic defects. Through artificial selection, farmers could improve the quality of their herds. The deliberate selection by a breeder of specific animals to provide the genetic material for the next generation is a process known as selective breeding or artificial selection. This is in contrast to the random breeding that occurs when any male animal in a popu lation has an equal chance of mating with any female.
Selective breeding: Australian sheep

More than 200 recognised breeds of sheep exist, derived from wild sheep or mouflons (Ovis musimon) that were domesticated at least 6000 years ago. Mouflon sheep are native to parts of Europe and central Asia and are still living today. About 75 per cent of the sheep in Australia today are merino sheep, which are prized for their wool quality. The first merinos were imported to Australia by John Macarthur in 1796 these were Spanish merinos from South Africa. In Australia, several strains (breeds) of merino are recognised, including the Peppin merino, the South Australian merino and the Saxon merino. Selective breeding (artificial selection) over generations has enhanced particular features of these breeds and their survival in particular climatic conditions. For example, the large South Australian merino, which has been bred to thrive in the arid regions of that state and in similar regions in others, produces greasy wool with strong (thick) fibres. In contrast, the much smaller Saxon merino thrives in wetter regions and has been bred for wool of increasing fineness (thin fibres). Table 16.1 shows some of the different wool characteristics of several sheep breeds, including some merino breeds.
TABLE 16.1 Wool characteristics of some sheep breeds. A staple is a collection of a large number of wool fibres. Typically, the fleece grows to a fixed length (like the coat of cats and dogs). In contrast, the fleece of Drysdale sheep continues to grow (like human hair) and so the staple length is given as growth per year. Which breed produces the finest (thinnest) wool?
Breed of sheep Trait body mass (kg) greasy fleece weight (kg) diameter of fibre (m) length of staple (mm) Drysdale 116 12.0 40.0 400 (annual) Saxon merino 60 5.0 17.4 75 Peppin merino 132 18.6 22.0 150 Strong Wool merino 147 20.0 23.5 95 Polwarth 130 12.0 24.6 150 Corriedale 104 13.0 28.0 175
Data from the National Wool Museum (Geelong, Victoria)
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The value of a wool fleece is determined principally by two traits, namely, clean fleece weight the heavier the better and fibre diameter the thinner the better! These are inherited traits but, because they are polygenic, the rate of change that can be achieved through selective breeding over many generations is slower than with monogenic traits. (Can you suggest why?). For example, selection programs at the Triangle Research Station in New South Wales using several merino strains produced gains in these two important traits. The results of selective breeding, with an equal emphasis on increasing clean fleece weight and decreasing fibre diameter, are shown in table 16.2. Although the gains may appear small, they represent significantly increased profits for woolgrowers.
TABLE 16.2 Results of a 10-year selective breeding program on two strains of merino sheep with an equal emphasis on increasing clean fleece weight and decreasing fibre diameter
Merino strain Fine wool merino Medium wool merino Group Experimental Control Experimental Control Clean fleece weight (kg) 4.0 3.5 5.0 4.7 Fibre diameter (microns) 18.3 19.7 19.3 20.8
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The first merino sheep brought to the Australian colony in 1796 had fleeces that yielded about 1.5 to 2.0 kilograms wool per year. Today, fleeces of merino sheep can commonly yield up to 10 kilograms wool per sheep per year.
(Data from NSW DPI Prime Facts 579, March 2007)
Several sheep breeds have been developed in Australia, including the Poll Dorset, a meat producer, and the White Suffolk, a dual-purpose wool and meat producer. Suffolk sheep have black faces and legs (see figure 16.3a). This breed produces superior, fast-growing, lean lambs but growers were not receiving an appropriate market price for these lambs because their wool included some dark fibres. By selective breeding over many generations involving crosses of blackfaced Suffolks and white-faced Poll Dorsets, a new breed, the White Suffolk, was produced in Australia. This new breed has all the features of the Suffolk breed except for the black face and legs (see figure 16.3b).
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FIGURE 16.3 Sheep breeds: (a) Suffolk sheep with their black face and legs (b) White Suffolk, which has the features of the Suffolk except for the black face and legs. The White Suffolk was derived from a program of selective breeding and artificial selection and was developed for a range of Australian pastoral conditions.
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Aurochs are now extinct. The last member of this species was killed by a Polish poacher in 1627.
Selective breeding: Australian cattle

The many different breeds of cattle are believed to have been derived by artifi cial selection from a wild species, known as the auroch (Bos taurus), that was domesticated at least 6500 years ago. The original cattle brought to Australia were British breeds that were suited to the temperate regions but did not thrive in tropical Australia. Selective breeding has produced a number of breeds for the tropics. These new breeds include the Australian Bradford, which was bred in Queensland in the mid-twentieth century. The Australian Bradford breed combines features of the Brahman, such as the hump, loose skin, short coat and heat and tick resistance, with features of the Hereford, including its colour markings. Another cattle breed developed in Australia is the Australian Milking Zebu, which resulted from selected crosses between the Asian dairy cattle and European dairy cattle. This breed is a reliable milk producer in tropical regions, it is heat resistant because it is able to sweat and its loose skin makes it resistant to ticks, which it can easily shed. Artificial selection occurs when breeders and farmers favour particular inherited features in their show animals and livestock because of their economic value or aesthetic appeal, and use selective breeding to enhance those features and to increase their frequency. Artificial selection is in contrast to natural selection, which favours only those inherited features that enhance survival and reproduction in the wild. Features that are economically important or aesthetically appealing but that do not contribute to survival and reproduction are not favoured by natural selection and hence are not seen in populations in the wild. Inherited characteristics that are the goal of selective breeding programs are not necessarily those features that best equip animals for survival and reproduction under natural conditions in the wild. In some cases, features deemed desirable by animal breeders would in fact be selected against in the wild. In chickens, for example, artificial selection over many generations has transformed wild fowls to farmed chickens with very different egg production capabilities. During their short annual breeding season, wild fowls produce only about 12 eggs; in contrast, domesticated chicken varieties lay several hundreds of eggs continuously throughout the year. Artificial selection in domesticated species, particularly in so-called show varieties, sometimes favours features that are clearly disadvantageous for survival and reproduction and would be positively selected against in the wild. Examples of features maintained only by artificial selection can be seen in domesticated animals, such as: Jacobin pigeons, whose distinctive arrangement of neck feathers forms a ruff that masks their faces, except from immediately in front (see figure 16.4a) hairless cats (see figure 16.4b) and dogs (see figure 13.6a, page 471) English bulldogs, whose greatly shortened muzzles result in breathing problems (see figure 16.4c). Features such as these are only maintained in the gene pool of the populations through human intervention using selective breeding.
(c) FIGURE 16.4 (a) Jacobin pigeon. Compare this image with figure 14.9b on page 512, which shows a Jacobin pigeon in 1835. Continued selective breeding over many generations has enhanced the size of the feather ruff that surrounds the face. The survival of this variety is now due to artificial selection by pigeon breeders. (b)Sphynx cat. These cats are essentially hairless but most have some very fine, soft hair on their noses and ears. Hairlessness in cats has arisen as a spontaneous mutation several times in different parts of the world. (c) English bulldog. The characteristic short muzzle of this breed has several disadvantages, including impaired breathing.
eLesson eles-1567 Ancient resurrection
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To collect sperm from a stud animal, a teaser animal is used to entice the stud to mate. When the stud tries to mount the teaser, his penis is re-directed into a warmed tube that is used to collect the semen.
Technologies in selective breeding

In commercial herds and flocks, new reproductive technologies resulting in selective breeding include: artificial insemination sex selection through sperm sorting multiple ovulation and embryo transfer oestrus synchronisation.
Artificial insemination (AI)

Natural breeding in mammals can be modified by artificial insemination (AI). This technique brought about changes in herd management by altering the how, when and where of breeding. AI involves collecting semen from a selected stud animal and then introducing this semen by artificial means into the reproductive tract of females of the same species. When first developed, the technique of AI involved the use of fresh semen only. The use of AI increases the number of offspring that one stud animal could produce. One ejaculate from a male bull contains sufficient sperm to fertilise ten eggs. This volume of semen can be divided into ten portions and used to artificially inseminate ten cows and so can produce ten offspring. In contrast, in a natural one-to-one mating, that same volume of ejaculate would normally produce just one offspring. In 1949, a successful technique was developed for freezing semen. Successful meant that, when the frozen semen was thawed, it contained motile sperm that were capable of fertilising eggs. The freezing technique involves adding semen to a special solution with a controlled pH and that comprises a mixture of various chemicals, including glycerol. Samples of this diluted semen (0.25 mL volume) are taken up in straws, frozen rapidly and stored in liquid nitrogen at 196 C. Under these circumstances, semen samples can be stored for many years and still retain their ability to fertilise an egg after thawing. Through AI technology using frozen semen, physical and temporal barriers to mating are removed. This technology means that one prize stud animal can: fertilise many more females than under natural conditions fertilise females located hundreds or thousands of kilometres distant from that stud animal because its frozen sperm can be easily transported over great distances (see figure 16.5) fertilise female animals and produce offspring long after its death.
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By means of AI, a single ram can mate with 2000 ewes as compared with a maximum of 200 ewes through natural matings.
Embryo transfer in livestock (MOET)

AI and the use of frozen semen can greatly increase the contribution of particular bulls and rams to the genetic makeup of herds and flocks. In a similar way, a technique known as multiple ovulation and embryo transfer (MOET) allows high-quality cows and ewes to make a much greater than normal contribution to the future generations. Multiple ovulation refers to a process whereby a female receives injections of the follicle-stimulating hormone (FSH) that stimulate her to super-ovulate, or produce multiple eggs. An injection of gonadotrophinreleasing hormone (GnRH) is also given to make all the eggs mature at the same time. Embryo transfer refers to the process through which embryos at days 6 to 7 of development are removed from the reproductive tract of a female and transplanted into the tracts of other females of the same species. These females act as surrogate mothers and carry the embryos to term and give birth.
FIGURE 16.5 Transport of frozen semen and artificial insemination has geographically extended the reproductive capacity of prize studs.
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In sheep, AI can be achieved through either an intra-uterine or a cervical technique. In intra-uterine AI, semen is placed directly in the uterus of a ewe through use of a laparoscope, with average pregnancy rates of 70 to 90 per cent. With cervical AI, semen is introduced via the vagina, with average pregnancy rates of 55 to 65 per cent.
MOET is another example of human intervention in the evolutionary process. In the process of MOET in sheep, for example, a high-quality donor ewe is treated so that she super-ovulates. When her eggs are released, they are fertilised, typically through AI with sperm from a selected ram. The fertilised eggs develop within the ewes uterus for about 6 days. At the end of that time, the embryos are flushed from the ewes uterus. On average, about seven embryos can be collected from a single flush. These embryos are immediately transferred directly into the uterus of young recipient ewes (see figure 16.6) that will be the surrogate mothers of these embryos. Embryos not transferred to recipient ewes are frozen in liquid nitrogen and stored for later use. (Frozen embryos can be stored indefinitely.)
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MOET has made the movement of genetic stock between countries easier because quarantine regulations are less stringent for frozen embryos than for live animals.
FIGURE 16.6 Transfer of an embryo into the uterus of a recipient or surrogate ewe
The same donor ewe can be used for MOET procedures several times during a breeding season. Over a normal reproductive lifetime, one ewe might produce 30eggs. Multiple ovulation, however, greatly increases this egg output. The advantages of embryo transfer are that genetically important female lines can be multiplied at much faster rates than can occur through normal reproduction and that valuable embryos can be stored. Under conditions of natural selection, this would not be possible.
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The sex of an embryo produced by IVF can be identified at a very early stage of its development before it is transferred to a female. A single cell is taken from the embryo, cultured and the sex chromosome make-up is identified.
Sex selection through sperm sorting

Which sex do you want? Under normal circumstances, a sex ratio of about one male to one female is expected in live-born mammals. In the beef industry, however, male calves are preferred because they have more beef (muscle) on their carcasses at a given age than females. In contrast, in the dairy industry, female calves are necessary for milk production (see figure 16.7, page 628). Sex selection is now possible. After semen has been collected from a stud bull, for example, it is possible to treat the semen and separate the sperm
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with X chromosomes from those with Y chromosomes. Sperm cells are first labelled with a harmless fluorescent dye that binds to DNA. The X chromosome in mammals is larger and contains more DNA than the Y chromosome. As a result, the sperm with X chromosomes fluoresce more brightly than those with Y chromosomes. After labelling, the sperm are then separated into two groups depending on their fluorescence. The use of this sperm separation technique has allowed sex selection to occur on a large scale (see figure 16.8).
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(b) beef cattle
FIGURE 16.7 (a) Dairy cattle, and
eLesson eles-1568 Sexual selection
FIGURE 16.8 A FlowSight cytometer with its visual display used for the rapid and accurate separation of cells. Cells are stained with a fluorescent dye and this instrument uses a laser beam to sort cells according to differences in their fluorescence. If sperm cells were being sorted using fluorescence from a DNA-binding dye, which sperm would fluoresce more sperm with an X or a Y chromosome?
Manipulating breeding cycles

The oestrous cycle was discussed in Nature of Biology Book 1, Fourth Edition, chapter 12, pages 4056.
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Release of the hormone oestrogen from ovarian follicles stimulates the release of luteinising hormone (LH) from the pituitary gland. Release of LH in turn causes ovulation and suppresses oestrogen production.
It is now possible to synchronise the time of oestrus or sexual receptivity of female farm animals, such as cattle and sheep. Oestrus synchronisation results in all sexually mature females being in oestrus within a predictable and narrow time frame, with the result that the time of fertilisation in a herd or flock, either by AI or natural mating, can be more efficiently managed. Synchronisation is also necessary for MOET procedures so that the intended embryo donor and the recipient surrogate mothers come into oestrus at the same time. Advantages of synchronisation include: less time (and hence lower labour costs) needed to test animals to see if they are in oestrus higher fertilisation rates and birth rates more uniform and manageable crops of calves or lambs, since all young are born within a short period lower mortality rates because greater oversight of all newborns is possible. Oestrus synchronisation can be achieved in a number of ways. One method depends on the fact that the hormone progesterone inhibits ovulation by stopping production of another hormone, oestrogen, which is needed to bring female animals into oestrus. By adding an external source of progesterone to female livestock, oestrus production and the associated ovulation are suppressed. When the source of progesterone is simultaneously removed from a group, mature females go into oestrus and ovulate within a short time period. How is this external progesterone delivered?
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Methods of supplying progesterone to farm livestock include: feeding using a dietary supplement implants under the skin sponges inserted into the vagina CIDRs (controlled internal drug-releasing devices) inserted into the vagina. In the case of CIDR (pronounced cee-dar) use in cattle, the insert is left in place for seven days. When the insert with its supply of progesterone is removed, the level of circulating progesterone drops and oestrus begins within three days.
Artificial pollination in plants

FIGURE 16.9 Dr Kath Cooper, triticale breeder, harvesting triticale trial plots. Its scientific name is X Triticosecale (where X denotes that it is a hybrid).
A process similar to AI is used by plant breeders with populations of cultivated plants. In plants, the process is termed artificial pollination and it is another example of human intervention in the evolutionary process. Unlike AI, artificial pollination has been used for centuries. We have seen (in chapter 10, pages 33844) that Gregor Mendel used artificial pollination in part of his breeding experiments with edible pea plants. Artificial pollination is still carried out essentially as it was done by Mendel. The process of artificial pollination involves: removal of unripe stamens from the plant to be fertilised protection of the stigma of the selected female plant from stray pollen collection of pollen to be used in the artificial pollination transfer of the donor pollen onto the stigma of the female parent. Refer back to figure 10.3 (page 338) to check on these details.
Creating new species

Artificial pollination is used in the creation of new plant species. In this case, pollen is collected from one species and it is transferred to the stigma of a second closely related species. One example of this was the creation of a wheatrye hybrid plant. A wheat species (Triticum turgidum) was artificially pollinated using rye (Secale cereale) (see figure 16.9). The result of this artificial pollination was a new plant species with one set of wheat chromosomes and one set of rye chromosomes. Such a plant would be infertile because its chromosomes could not undergo the normal pairing that occurs during meiosis (see figure 16.10a, page 630). By using a specific chemical treatment, a doubling of the chromosome number in the plant cells occurred so that the cells then contained two sets chromosomes and two sets of rye chromosomes. As a result, the of wheat mature plant would be fertile because it could undergo normal meiosis (see figure 16.10b, page 630). The new species is known as triticale (triti- from the wheat parent and -cale from the rye parent). This new species combined the desirable genetic qualities of wheat with the inherited hardiness of rye. Rye can grow in cold climates and on low nutrient soils. In contrast, wheat is grown mainly in temperate parts of the world. Triticale is the first artificially created cereal crop to be developed and is grown in a number of countries. Artificial pollination combined with the use of chemical treatment to double the chromosome number in cells accelerates evolution. This technique allows genetic material from two species that would naturally have remained reproductively isolated to be artificially combined.
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Treatment of cells with the compound colchicine results in a doubling of the chromosome number because it stops spindle formation during mitosis. Colchicine is extracted from bulbs of the Mediterranean plant Colchinium.
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Doubling of whole sets of chromosomes in plants does not affect their viability. In contrast, this process leads to inviability in animal species.
Wheat (Triticum)
Rye (Secale) RR
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FIGURE 16.10 (a) Wheat cells have two sets of chromosomes (WW) and rye has two sets (RR). Pollen and eggs from these plants have one set (W and R). Cells of the hybrid wheatrye plant contain one set of wheat chromosomes, denoted as W, and one set of rye chromosomes, denoted as R. Because normal pairing cannot occur during meiosis, this plant is sterile. (b) Chemical treatment doubles the chromosome number. Cells of the treated wheatrye hybrid plant contain two sets of wheat chromosomes, denoted as WW, and two sets of rye chromosomes, denoted as RR. Normal meiosis can occur so this plant can reproduce sexually. Durum wheat (Triticum turgidum), a tetraploid species (4x = 28), is most commonly used as the female parent, while rye (Secale cereale) is a diploid species (2n = 14).
Sterile hybrid WR
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Treatment with colchicine
Fertile hybrid (Triticosecale) WWRR
Genetic impact of selective breeding

Genetic variation refers to the genetic differences present in the members of a
population. In the gene pool of a population, the greater the number of different alleles of each gene, the greater the genetic variation in that population. Selective breeding of domesticated animals, such as cattle and sheep, occurs when the parental input to the next generation is restricted to a small number of animals, selected on the basis of their superiority for a limited number of
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inherited traits. This process of artificial selection can result in a loss of genetic variation in the gene pool of the herd or flock concerned. The loss of genetic variation may not be noticed immediately and may become apparent only when there is a change in the environmental conditions. If, for example, members of an animal population have never been exposed to a particular disease-causing virus, the loss of an allele that confers resistance to that disease would not be noticed. The value of the allele in question would be realised only after the virus comes into the environment. Genetic variation can also be lost when plant crops are cultivated from commercially produced seeds that come from just one, or a small number, of the many varieties of that plant species. Loss of genetic variation can be an unintended consequence of the use of reproductive technologies, such as artificial insemination (AI) combined with the transport of frozen semen, which increases the genetic influence of a small number of stud males in animal herds and flocks. Similarly, the use of multiple ovulation and embryo transfer (MOET) restricts the number of breeding females in animal herds and flocks. Likewise, the use of artificial pollination with a limited number of plants as the pollen source may reduce the genetic variation of a plant population under cultivation.
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Does loss of genetic variation matter?

The death of more than one million Irish peasants from starvation in the period from 1846 to 1851 is a tragic reminder that genetic variation matters. Potato was the staple diet of Irish peasants. When an outbreak of late potato blight occurred, caused by the fungus Phytophthora infestans, potato tubers turned black and rotted in the ground. Potatoes are endemic to South America, where hundreds of species grow wild. This food crop was introduced to Europe, first by the Spaniards in 1570 and later, in about 1590, by the English. All potatoes grown in Europe were plants descended from the European potato (Solanum tuberosum). When potato blight broke out in Ireland in 1846, this potato variety was genetically uniform for susceptibility to fungal infection so that all the potato tubers rotted. Natural populations of the wild relatives of plants can be sources of alleles for potentially valuable traits. For example, in the 1940s, resistance to the late potato blight disease was identified in Solanum demissum, which grows wild in the Andes and is a close relative of the European potato. Cross breeding was used to introduce this allele into the commercial varieties of potato, providing them with resistance to blight.
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Saving genetic variation

As wild populations are destroyed through land clearance and as larger areas are devoted to the cultivation of smaller numbers of commercial crop varieties, the safeguarding of genetic variation in wild populations is critical. In the case of plant varieties, some contribution to this is being achieved through the establishment of seed banks. In 1992, Western Australia became the first state to set up a genetic diversity bank when the Threatened Flora Seed Centre was established at the WA Herbarium. Seeds of rare and endangered plant groups are collected from various populations of the plant groups concerned. (Why collect from more than one population?) After collection, the seeds are carefully cleaned and counted in a laboratory (see figure 16.11a). The seeds are then dried to reduce their water content then stored in sealed aluminium foil packs at low temperatures (see figure 16.11b). An important step in the preservation of the genetic variation and biodiversity of the worlds major crop plants and their wild relatives occurred in
FIGURE 16.11 (a) Processing and testing of seed in the laboratory prior to storage (b) The freezer. Extracted and dried seed is sealed in laminated aluminium foil packages and frozen at 18 C for the long term.
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A rc
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SWEDEN Spitsbergen (Norway) NORWAY
February 2008 when the Svalbard Global Seed Vault (SGSV) was opened on remote Spitsbergen Island close to the North Pole (see figure 16.12). The SGSV facility has large underground storage areas dug into the side of a mountain and these are maintained at a temperature of 18 C (see figure 16.13). Here, duplicate samples of seeds from seed banks throughout the world can be placed in secure long-term storage at no cost. This storage acts as an insurance against the loss of the valuable genetic variation in food crop plant varieties and their wild relatives.
FIGURE 16.12 The Norwegian island of Spitsbergen, north of the Arctic Circle, is the location of the Svalbard Global Seed Vault.
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FIGURE 16.13 (a) Entrance to the Svalbard Global Seed Vault on the island of Spitsbergen. (b) Diagram showing the three storage areas at the end of a tunnel deep within a mountain. This facility will ensure that the genetic variation in plant crops is preserved for future generations.
At capacity, SGSV can store more than 4 500 000 samples, each containing up to 500 seeds. During the first year of its operation, various national and international groups deposited more than 400 000 unique seed samples. The first seeds from Australia were deposited in the SGSV in early 2011 and these were duplicate samples of seeds from the Australian Temperate Field Crops Collection at Horsham, Victoria (see figures 16.14a and b).
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FIGURE 16.14 (a) The Australian Temperate Field Crops Collection (ATFCC) building at Horsham, Victoria (b) The first Australian consignment of seeds, taken from ATFCC, packaged ready for delivery to SGSV in Norway. From left: Dr Bob Redden (Curator, ATFCC), Hon Peter Walsh (Victorian Minister for Agriculture and Food Security), Hon Hugh Delahunty (MP for Lowan) and Dr Tony Gregson who delivered this seed consignment to Norway in February 2011.
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The following box outlines the importance of conserving the genetic diversity of crop plant species essential insurance for the worlds food supply.
GENE BANkS fOR cROP GENETIc RESOURcES

The green revolution of the 1960s led to adoption of improved higher yielding varieties of wheat, rice and other crops in developing countries that, combined with improved management, enabled former food importers such as India, China and Pakistan to become self-sufficient and even to export. In this process, traditional farmervillage plant varieties (landraces) were largely displaced by just a few modern varieties across national and international cropping zones. However, many of these landraces had been grown for centuries and were adapted to the myriad climatesoil environments in each locality. These landraces were recognised as important to conserve, as collectively they contain genetic variation for coping with different climates and extreme climatic stresses and have much more diverse genetic diversity than modern varieties. This is the rationale for collecting and conserving the landraces of agricultural crops in gene banks, from field grain crops to vegetable and horticultural crops. Seed samples of landraces were collected directly from farmers fields on joint collecting missions with the host country, shared with the host if an external gene bank was involved, then deposited in the national and other gene banks. The introduction of genetic resources to another country may have quarantine requirements. In Australia, for nearly all crops, a full generation is required in a secure quarantine facility, and only seed from disease-free plants is harvested and passed to the gene bank. Most countries have gene banks. The largest gene banks are at international crop research institutes, such as those for wheat in Mexico and for rice in the Philippines, with over 100 000 separate seed samples each. Australia is currently reorganising various statebased gene banks into a national gene bank system. For field grain crops, the Australian Genebank Grains (AGG) will be located at Horsham, Victoria, and will combine the temperate field crops collection from Horsham, the tropical crops collection from Biloela, Queensland, and the cereals collection from Tamworth, New South Wales, to have over 100 000 accessions of many crops. AGG is important because almost all of Australias food crops were introduced with European settlement. Imported genetic diversity is needed to breed varieties that can cope with new climatic stresses, that can be selected for improved nutrition, and that are resistant to new strains of pathogens that can release millions of spores from susceptible crops each year. Seed samples in gene banks are bar coded and passport information on the origin and characteristics of the collection sites is filed. Samples of 1000 seeds are dried to 6% seed moisture and then stored in vacuum-sealed, triple-foil aluminium pouches at both 2C and 18 C. These procedures enable seed to remain viable for over 25 years at 2 C (see figure 16.15).
FIGURE 16.15 Part of the 2 C storage area at the Australian

Temperate Field Crop Collection at Horsham, Victoria
Small seed samples of 100 seeds are distributed free on request to plant breeders, scientists and students in Australia, and also exchanged with other gene banks and scientists internationally (see figures 16.16a and b). The seed is sent with a Standard Material Transfer Agreement, by which the recipient pays a small royalty into a special international trust fund if the genetic resource becomes a parent of a commercially released new variety. This trust fund helps collect and conserve genetic resources in developing countries, often where the initial domestication of respective crops began up to 11 000 years ago. Seeds in the gene bank are grown for seed increase (regeneration) when stocks become low as a result of meeting requests or because old seed stocks are losing viability (see figure 16.17). The collections must maintain live seed to be useful. This regeneration provides an opportunity to record important data, such as time to flowering and to maturity, height and agronomic traits, and grain yield when grown in field plots. Seed characters are also recorded. This information adds value to the collection and is published as a database on an internet site that assists scientists in choosing germplasm for study or for breeding improved varieties.
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(a)
(b)
FIGURE 16.17 Regeneration igloo where seeds from the gene bank are grown. What are the purposes of this regeneration?
Gene banks also store improved released varieties and special genetic stocks such as those for mapping the genes on chromosomes. Breeding lines may be stored; however, they are not regenerated because breeding programs produce further improved material each year. The collection size must be contained to focus on the critical diversity that needs to be conserved for future needs in this and subsequent centuries.
FIGURE 16.16 (a) Dr Bob Redden, Curator, showing some of

the seed collection from the gene bank at Horsham to scientists from the Chinese Academy of Agricultural Science (b) Seed samples of lentil varieties
KEY IDEAS
Selective breeding is one form of artificial selection used in plant and animal breeding. Artificial selection using selective breeding can maintain inherited features in a population that, under natural conditions, would be selected against. Several new reproductive technologies are available to assist selective breeding in commercial flocks and herds. Reproductive technologies, such as AI, artificially increase the contribution of selected animals to the gene pool of the next generation. Use of reproductive technologies can result in reduced genetic variation in a gene pool. Loss of genetic variation in a population may result in failure of the population to survive environmental change.
QUIck-cHEck
1 Define the following terms. a artificial insemination b artificial pollination 2 What is the likely effect on the genetic variation of a herd produced by AI using semen from one prize stud? 3 Identify two consequences for AI when technology allowed semen to be successfully frozen. 4 Give two other examples of human intervention in the evolutionary processes through selective breeding. 5 What is meant by the term MOET? 6 a Why did the potato crop in Ireland fail in the 1840s? b What does this show about genetic variation?
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Natural embryo splitting in mammals occurs when the cells of a two-cell or four-cell stage of an embryo spontaneously separate and each cell develops individually into identical twins (or triplets or quads).
No sex at all! Cloning

Some reproductive technologies, such as artificial insemination and artificial pollination, involve modifications to the sexual reproduction that occurs in animal and plant populations. These technologies use artificial means to transfer the selected sperm and pollen to the female reproductive structures. In contrast, other reproductive technologies, such as cloning, involve methods of asexual reproduction in which the genetic information of the new organism comes from one parent cell only. Mammals are normally produced through a sexual route: that is, from the fertilisation of an egg by a sperm, with the fertilised egg then developing into a new embryo. However, other techniques exist in which a new mammalian embryo does not arise from a single fertilised egg but from other artificially created cell types. These techniques are typically referred to as reproductive cloning but, as we will see below, different cloning techniques exist. These techniques include: cloning by embryo splitting cloning by somatic cell nuclear transfer (SCNT). These reproductive cloning techniques are yet another example of how humans can intervene in the evolutionary processes.
(a) Enucleating a cell
Embryo splitting: identical twins, triplets, quads or quins

Enucleated egg cell
Egg cell with nucleus
Embryo splitting occurs when the cells of an
(b) Nuclear transfer
Somatic cell Enucleated egg cell Egg cell with somatic cell nucleus
early embryo are artificially s eparated. Typically, the embryo is produced through in-vitro fertilisation (IVF) and, using a very fine glass needle, the cells of the early embryo are separated in the laboratory. Each single cell is then implanted into the uterus of a surrogate female parent where embryonic development continues. As a result, organisms produced through the splitting of one embryo are identical. Embryo splitting has been used for some years in the livestock industry. In cattle, for example, embryo splitting enables the genetic output from one mating of a top bull and a prize cow to be multiplied. Instead of just one calf from such a mating, several calves can be produced using surrogate mothers.
(c) Cell fusion
Electric pulse Enucleated egg cell Somatic cell Egg cell fused with somatic cell
Cloning using somatic cell nuclear transfer techniques
FIGURE 16.18 (a) Producing an enucleated cell (b) Nuclear transfer between two cells (c) Cell fusion. Fusion of two cells is commonly done using a short electric pulse.
Some possibilities exist to manipulate cells and their nuclei. It is possible, for example, to: remove the nucleus from a cell (when this occurs the cell is said to be enucleated) (see figure 16.18a) transfer the nucleus from one cell to an enucleated cell to form a re-designed nucleated cell (see figure 16.18b) fuse a somatic cell with an enucleated cell (see figure 16.18c).
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(a)
The birth of two sheep, Megan and Morag (see figure 16.19), in 1995 marked a significant scientific milestone. These two sheep were the first mammals ever to be cloned using nuclear transfer technology. Each of these sheep developed from an unfertilised enucleated egg cell that was fused with an embryonic cell that contained its nucleus. In each case, the embryonic cell used came from the culture of one embryonic cell line; as a result, Megan and Morag were identical twins.
(b)
Donor embryo cells are separated. Donor cell placed next to enucleated egg
Nucleus extracted from unfertilised egg
Donor cell fuses with enucleated cell by an electric pulse.
Embryo develops as in a fertilised egg.
FIGURE 16.19 (a) Megan and Morag, two

Welsh mountain ewes, born in August 1995 (b) Megan and Morag were not born as a result of a normal mating between a ram and a ewe, but were created using nuclear transfer cloning. What cells were involved in their production?
FIGURE 16.20
Dolly and her first lamb, Bonnie. Bonnie was born in April 1998 after a natural mating of Dolly with a Welsh mountain ram.
. . . and then came Dolly!

The scientific world was stunned after the announcement in February 1997 of the existence of Dolly, a FinnDorset female lamb born the previous year at the Roslin Institute in Scotland (see figure 16.20). The scientists who created Dolly were Ian Wilmut, Keith Campbell and their colleagues from Roslin Institute. Why was Dolly the lamb famous? Read what Ian Wilmut wrote about Dolly:
Dolly seems a very ordinary sheep ... yet, as all the world acknowledged, ... she might reasonably claim to be the most extraordinary creature ever to be born ... Dolly has one startling attribute that is forever unassailable: she was the first animal of any kind to be created from a cultured, differentiated cell taken from an adult. Thus she confutes once and for all the notion virtual dogma for 100 years that once cells are committed to the tasks of adulthood, they cannot again be totipotent. (extract from Wilmut I., Campbell K. and Tudge C., The Second Creation: Dolly and the Age of Biological Control, Harvard University Press, Cambridge, Mass., 2000)
Totipotent refers to a cell that is able to give rise to all different cell types.
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eLesson eles-1569 The Shine brothers
While cloning via nuclear transfer had occurred successfully in the past, those earlier cases involved embryonic or fetal cells, never adult somatic cells. The use of adult somatic cells, such as skin cells, to construct new organisms represents remarkable human intervention in the evolutionary processes. Through this means, cells from sterile animals or from animals past their reproductive period, or even dead animals, can provide all the genetic information of new organisms. In nature, the normal evolutionary processes would not allow these events to occur. How was Dolly created? The artificial cloning of mammals involves: obtaining the nucleus from a somatic (body) cell of an adult animal this is the donor nucleus removing the nucleus from an unfertilised egg cell, typically of the same species this is the enucleated egg cell transferring the donor nucleus into the enucleated egg cell culturing the egg cell with its donor nucleus until it starts embryonic development transferring the developing embryo into the uterus of a surrogate animal where it completes development. The genetic information in the cloned animal comes from the nucleus of the adult body cell and so the genotype of the cloned animal is determined by the donor nucleus, not by the egg into which the nucleus is transferred. The procedure in the case of Dolly is shown in figure 16.21. An unfertilised egg from a Scottish Blackface Scottish Blackface ewe had its nucleus removed. A cell was taken from ewe the culture of mammary cells derived from the udder (mammary gland) of a FinnDorset ewe. Using a short electric pulse, the cultured mammary cell was fused with the enucleated egg cell to form a single cell. This reconstructed cell was cultured for a short time and was then implanted into the uterus of a surrogate Blackface ewe where the embryo developed. At 5 pm on 5 July 1996, this surrogate Scottish Blackface ewe gave birth to Dolly, a FinnDorset lamb, Remove the first mammal to be produced by cloning using an adult DNA from unfertilised somatic cell.
egg
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Dolly was named in fun after Dolly Parton, because she was derived from an udder (mammary gland) cell.
FinnDorset ewe
Remove udder cell from FinnDorset ewe
After Dolly what next?

Who are Matilda, Suzi and Mayzi, cc and Snuppy? Matilda the sheep was the first lamb to be cloned in Australia and was born in April 2000 (see figure 16.22). Suzi and Mayzi (see figure 16.22b) were Australias first calves to be artificially cloned from the skin cells of a cow fetus. Suzi and Mayzi are identical twins but were born two weeks apart in April 2000. Why were they not born on the same day? cc (short for carbon copy) was the first cat to be arti ficially cloned using a cumulus cell from an adult female cat, Rainbow, as announced by a group of American scientists in February 2002 (see figure 16.22c). Snuppy, the Afghan hound, was the first dog to be arti ficially cloned from an ear cell of a 3-year-old Afghan hound, as announced by a group of South Korean scientists in August 2005 (see figure 16.22d). Snuppy is short for Seoul National University puppy.
Use electricity to fuse cells Single cell
Culture containing early embryo Implant in surrogate
Dolly, a clone of FinnDorset mother
FIGURE 16.21 Technique of animal cloning by nuclear transfer
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(a)
(b)
(c)
(d)
FIGURE 16.22 A picture gallery of mammalian clones since Dolly: (a) Matilda, the first lamb to be cloned in Australia, was born by caesarian section at the Turretfield Research Centre of the South Australian Research and Development Institute (SARDI). She is pictured here with Dr Teija Peura, one of the scientists responsible for the achievement. (b) Suzi and Mayzi, genetically identical Holstein calf clones, were derived from the skin cell of one cow fetus. (c) cc, shown on the right at one year old, was the worlds first cat produced by somatic cell cloning, using a cumulus cell from Rainbow (left). (d) Snuppy, the worlds first dog produced by somatic cell cloning, is shown with the Afghan dog that supplied the ear cell (left) and his surrogate mother, a labrador (right).
Cloning: the downside

The success rate in initiating development of the egg cell after transfer of the donor nucleus is low. For example, in the case of an artificially cloned calf, known as Second Chance, 189 implantations were made into surrogate cows before a pregnancy was achieved. This case, however, was remarkable because the adult cell that provided the donor nucleus came from a 21-year-old Brahman bull called First Chance. This was an extremely old adult cell to use as the starting point for cloning. Because of testicular disease, First Chance had been castrated so that he was sterile when one of his body cells was successfully cloned. The kitten cc, produced by somatic cell cloning, was the only one of 87 embryos implanted into surrogate mothers that survived to term. To get Snuppy, 123 dog embryos were surgically implanted into surrogate females and, of these, only three survived for a significant period, with one dying before birth, one dying soon after birth, and the sole survivor being Snuppy. Dolly was
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At the time of birth of the cloned calf Second Chance, the bull First Chance that provided the donor nucleus from one of his somatic cells was dead.
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One estimate is that the ends (telomeres) of human chromosomes progressively shorten by tens or hundreds of base pairs per year.
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Dollys stuffed remains are now on display in the Royal Museum in Edinburgh.
the only live birth from a series of 277 cloned embryos. Clearly, somatic cell cloning is presently far from routine, with fewer than one per cent of the cloned embryos surviving beyond birth. Of the clones that survive beyond birth, many have abnormalities that can cause death early in life. One institution reported in 2003 that, for every healthy lamb clone born, about five had abnormalities. Abnormalities reported include impaired immune system function and the large offspring syndrome in which clones have abnormally large organs. There is evidence that, each time a mammalian cell divides, the specialised ends of their chromosomes lose some DNA base pairs and become shorter. These ends, which are known as telomeres, do not carry structural genes. Some scientists suggest that the shortening of the chromosome ends is associated with ageing. Will ageing be more rapid in a cloned animal that originates from an adult cell that already has shortened chromosome ends than in a normal organism? The death of Dolly in February 2003 suggested that this may be the case. Six-year-old Dolly was put to sleep because of a deteriorating lung disease and arthritis, unusual conditions for a sheep of Dollys age and one that was housed indoors, since sheep can live for about 12 years. Matilda, the cloned lamb (see figure 16.22a) that was born in March 2000, died less than 3 years later.
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Under the Prohibition of Human Cloning Act 2002, the maximum penalty for offences relating to human cloning is 15 years imprisonment.
Attitudes to cloning
Public attitudes to animal cloning are mixed. Some people support the concept because they believe that it will benefit people by providing a source of tissues for transplantation or other products. Other people oppose the concept for various reasons, such as their belief that cloning is interfering with nature. When people are questioned about the cloning of human beings, there is a very high level of opposition to it. Some governments, including Australias, have banned experiments directed to producing human clones, and leaders of some religious groups have opposed human cloning. The Prohibition of Human Cloning Act 2002, passed by the Australian Parliament in December 2002, bans human cloning. This Act took effect on 16 January 2003.
Cloning plants
eLesson eles-1570 Cloning
Cloning of plants can occur both naturally and artificially. Natural cloning occurs through cuttings, runners and suckers. Artificial cloning of plants involves the culturing of a piece of adult plant. As this piece grows, it can be further sub divided so that a large number of genetically identical plants can be produced from the original piece. If large numbers of plants are produced through natural or artificial cloning, the members of the resulting population are genetically identical. As a result, these populations have very limited genetic variation compared with a popu lation that has been produced by sexual reproduction.
KEY IDEAS
Artificial cloning of mammals is a new technology of asexual reproduction. Two types of artificial cloning techniques are embryo splitting and nuclear transfer. Embryo splitting involves the artificial separation of embryo cells in vitro. Nuclear transfer involves the transfer of a nucleus from an adult somatic cell to an egg cell that has had its own nucleus removed. Legislation of the Australian parliament prohibit human cloning. Artificial cloning of plants involves subdividing cultured plant tissue. Cloning of plants produces organisms that are genetically identical to each other and to the original cultured plant tissue.
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QUIck-cHEck
7 Identify the following as true or false. a Cloning mammals involves fusing an intact egg cell with an intact somatic cell. b The genotype of a cloned mammal is determined by the egg cell. c An enucleated cell is one that has had its nucleus removed. d Dolly was produced by a process of embryo splitting. e Megan and Morag were produced by a process of cloning using nuclear transfer. f Cloning of mammals uses a different technique from that used in cloning plants.
Transferring genes between species

Orchids, people, parrots, crocodiles, ferns, toads, salmon, oak trees, sharks, starfish, oysters, shrimp, squid, dragonflies, leeches, wheat, pine trees, mosses, seaweed . . . the genetic material of all kinds of organism is the nucleic acid DNA. In chapter 12, we discussed techniques for manipulating DNA. We saw that scientists can cut genetic material of any species into pieces, can find a particular piece, can join pieces, can make multiple copies of one particular piece, and can insert a DNA segment from one organism into the DNA of an organism belonging to the same or a different species. In addition, through gene technology, scientists can also manipulate the DNA of organisms.
Gene transfer between species

The complete set of genes and the noncoding DNA that each species possesses make up its genome. Under normal conditions, genes of one species can be transferred only to another member of that species, for example, from parents to offspring. Transfer of genes between different species normally does not occur. The restrictions that normally prevent gene transfer between different species are known as the species barrier. These restrictions include the inability of different species to mate and the inability of gametes from one species to fertilise those of another species. Genetic engineering technology, however, has made this species barrier irrelevant. Genetic engineering technology allows the genetic material to be manipulated and enables genes to be transferred between any two species. Examples of these gene transfers include the transfer of a human gene into bacteria, the transfer of a human gene into cows, the transfer of a bacterial gene into cotton plants and the transfer of a jellyfish gene into mice (see figure 16.23). Any organisms that possess a foreign gene or segment of foreign DNA in their genome as a result of human experimentation are termed transgenic organisms (TGOs). The introduction and incorporation of external DNA into a cell can result in permanent genetic changes. If the cells concerned are prokaryotic cells, such as bacterial cells, they are said to be transformed. If the cells are eukaryotic cells, when external DNA is added to the cells they are said to be transfected.
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Transgenic pets for sale! In 2004, pet stores in the United States began to sell transgenic zebrafish, known as GloFish, that were transfected with a fluorescent sea anemone gene.
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FIGURE 16.23 Mouse pups with the jellyfish gene showing green fluorescence when placed in particular illumination. Transgenic mice can fluoresce; nontransgenic mice, which lack the gene, cannot fluoresce.
IS IT A GMO OR A TGO OR BOTH?

The term genetically modified organism (GMO) refers to any organism whose genetic make-up has been artificially changed. So, all transgenic organisms (TGOs) are GMOs but not all GMOs are TGOs. Why? GMOs include organisms whose genotypes have been modified but the modification does not involve insertion of gene(s) from a different species. Such modifications can include the switching off (or silencing) of a gene that is normally active in an organism. In 2004, an American biotechnology company started taking orders for genetically modified cats in which the gene controlling production of a cat allergen was silenced. However, the company did not succeed in producing hypo-allergenic GMcats. Instead, the company screened cats to find those lacking the Fel 1d protein, which is the cause of allergy in most people, and used classic selective breeding to produce hypo-allergenic, but nonGMO, kittens for sale.
Im nonGMO
Buy me, and silence those sneezes!
Techniques for gene transfer

eLesson eles-1571 What has Darwin done for us?
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The gun that fires genes! Scientists at the University of Adelaide transferred rye genes (Secale cereale) into wheat embryos (Triticum aestivum). Copies of the rye gene were coated onto microscopic gold pellets that were placed on a special membrane above the embryos. A particle gun then propelled these pellets into the wheat embryo.
Various techniques exist for transferring genes into a host cell. These include: micro-injection of the DNA of a gene into a cell, such as an egg cell (refer back to figures 12.21 and 12.22 on page 434) or a somatic cell transfer using a virus, either a retrovirus or an adenovirus, to carry the gene (retroviruses are RNA viruses, while adenoviruses are DNA viruses) use of an electric pulse (electroporation) use of ballistics (the gene gun see the Odd Fact on this page). These various techniques are hit and miss. In the past, when scientists wished to create a genetically modified transgenic mammal, the only method available was micro-injection of the DNA of the gene concerned into newly fertilised eggs. The eggs were then implanted into females and the embryos were allowed to develop to term. It was only after the baby mammals were born that they were tested to see if the gene had been taken up. Success rates using this method were not high, perhaps just one in 100. However, all this changed one day in Scotland . . . when Polly was born.
Cloned transgenic mammals

In early July 1997, yet another remarkable cloned lamb, a Poll Dorset called Polly (not Dolly!), was born at the Roslin Institute in Scotland. This lamb was cloned from a fetal cell using the nuclear transfer technique. In addition, Polly had a
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human gene and a marker gene in all her cells. So, Polly was a transgenic cloned lamb. How was this done? This was achieved by genetically engineering the fetal cells by drenching many of them with the DNA of the genes concerned. Then, only those fetal cells that had taken up the genes were selected to be cloned. Artificial cloning has been applied to transgenic organisms. Soon after development begins, the cells of a transgenic embryo can be artificially sepa rated so that the single cells then develop into a number of identical organisms. George and Charlie were the first transgenic cows to be artificially cloned. They were derived from cattle body cells that had been genetically altered to incorporate the human gene for a particular blood protein known as serum albumin. Clint, Arnold and Danny were the first goats to be artificially cloned from adult goat cells. They were also transgenic as their cells contained the spider gene for silk production. Mira, Mira and Mira were three genetically identical female goats produced by artificial cloning of an adult goat cell that incorporated a human gene that controls production of a protein that prevents blood clotting. The milk produced by these goats contains this human protein. Human genes and genes of other species have been engineered into mammalian cells, such as hamster cells and mouse cells. Human genes have also been engineered into mammalian clones, such as cattle, sheep and goats. These events are not possible under the normal evolutionary processes. Why has this been done? This was carried out with a view to the transgenic cells (or the cloned transgenic mammals) making the protein products of these genes. Lets look at two case studies.
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In the United States on 2 October 1992, twins Karine and Cedric were born. This birth was the first ever to a woman who had been treated with recFSH.
Case study 1: transgenic hamster cells as factories

Andrew and Diane were keen to start a family but, after 18 months of trying without success to conceive, the young couple sought medical advice. Andrew was found to have a normal sperm count and sperm motility but Dianes tests showed that she was not producing adequate levels of follicle-stimulating hormone (FSH) and so was not ovulating. In the past, Diane would have been treated with hormone isolated from the urine of menopausal women. However, in 1994, FSH that was produced using recombinant DNA techniques was approved for use in several countries. In Australia, it is marketed as Puregon. Recombinant FSH (recFSH) is available in larger quantities, the supply is more reliable and it is purer than the urine-derived product because it does not contain the extraneous materials that were often present in the latter. The production of human recFSH occurs in transgenic hamster cells. These cells have been genetically engineered so that they contain a specific human gene, namely, the human gene that controls production of folliclestimulating hormone. In table 12.2 (on page 432), we identified some other transgenic cells that have been engineered to contain human genes. In each case, the various transgenic cell lines were established to produce human gene products in higher quantities, at higher purity levels and at lower costs than are possible by extracting the substance from human tissue or fluids.
Case study 2: transgenic mammals as factories

Recent transgenic work has involved the production of transgenic cows and goats whose cells carry foreign genes for specific protein products. If these genes are expressed in the milk produced by these transgenic mammals, they function as mini-pharmaceutical factories to produce particular protein products. George and Charlie are cloned transgenic cows with the gene for a human protein known as serum albumin. These transgenic cows have expressed this gene in their milk. It has been estimated that one
FIGURE 16.24
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transgenic cow can produce about 80 kilograms of this human protein per year and so herds of transgenic cows could become an important source of this protein, which would otherwise have to be obtained from human blood donations (see figure 16.24). Likewise, the cloned goats Mira, Mira and Mira, have produced milk containing a human protein that prevents clotting, which is used in patients undergoing heart surgery.
KEY IDEAS
Many different kinds of transgenic organisms (TGOs) have been engineered. Transgenic cell lines have been produced for the purpose of producing specific gene products for medical purposes. Transgenic mammals have been cloned for several purposes, such as the production of various gene products, including human proteins.
QUIck-cHEck
8 Who are Mira, Mira and Mira? 9 Identify one human protein that has been produced by transgenic cows.
Gene therapy or gene transfer

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In September 1999, 18-yearold Jesse Gelsinger, who had a very mild form of an enzyme deficiency disorder, became the first person to die in tragic circumstances as a result of a gene transfer experiment.
eLesson eles-1572 Cystic fibrosis
Another human intervention that has the potential to change natural evolutionary processes is gene therapy. Gene therapy or gene transfer is a process by which the function of a faulty allele in an organism is replaced by addition of a normally functioning allele of the gene concerned. It is a technique that aims to treat inherited disorders by directly targeting the genotype. This is in contrast to conventional treatments for inherited disorders that act at the level of the phenotype by ameliorating the symptoms of the disorder. Even more, gene therapy provides the prospect for treating inherited disorders for which no treatment presently exists. Technical difficulties must be solved: How can a gene be targeted to cells of the affected tissue? How can a gene be targeted to a position where it does not interfere with the function of another essential gene? In addition, ethical issues must be resolved: Should gene therapy be restricted to somatic tissues only, so that the introduced gene is not transmitted to the next generation? At present, gene therapy affecting germline cells is banned. Before gene therapy is permitted, there must be an assessment of the safety of the patient and the general public and the expected benefit to the patient is compared with the likely risk. At present, gene therapy aims to add copies of the normal allele of a gene into the cells of a target tissue, switching them on to produce the functional protein that is missing in a person with a particular disorder, such as a clotting factor in persons with haemophilia, or a tumour suppression agent in persons suffering from certain cancers. In searching for possible new treatments for several diseases, clinical trials involving gene therapy have taken place (see table 16.3).
TABLE 16.3 Examples of some disorders for which clinical trials of gene therapy are occurring in various institutes
Symptoms abnormally thick mucus secretions lung infections and lack of pancreatic digestive function abnormally high blood cholesterol fatty deposits in arteries and elevated risk of heart attack haemophilia lack of blood clotting factor VIII excessive bleeding severe combined immune deficiency (SCID) depressed immune system inability to ward off even minor infections Disorder cystic fibrosis (see box on page 645) hypercholesterolaemia
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In vivo gene therapy
Ex vivo gene therapy Cloned gene D+
Gene transfer Cloned gene D+ Cells removed Patient cells D
Some cells now D+ Select D+ cells and allow to reproduce
Cloned gene
Return genetically modified cells to patient
D+
D+
D+
FIGURE 16.25 Gene therapy is possible either in vivo (shown at left) or ex vivo (shown at right). In vivo gene therapy is used for cells that cannot be removed from a patient.
There are two broad categories of gene therapy: in vivo gene therapy given directly to a patient ex vivo gene therapy in which a patients cells are manipulated outside the body and then returned into the individual. The general action of both in vivo and ex vivo gene therapy is outlined in figure 16.25. A patient is D and lacks the ability to make protein D. To cure the patient, a D+ allele must be inserted into appropriate cells.
RNA version of cloned gene
Retrovirus
Using viruses to transfer genes into cells
The functional piece of DNA to replace a faulty allele is specially prepared and is called a cloned gene. How does the cloned gene get into a patients cells? One of the various techniques for transferring genes into cells uses viruses that have been modified to ensure they cannot cause Cloned RNA inserted disease. Both RNA retroviruses and DNA adenoviruses can be used into RNA of retrovirus as vectors to carry cloned genes into cells.
In the patients cell: 1. Retrovirus transforms RNA into DNA 2. DNA incorporated into cell chromosome Nuclear membrane disintegrates during cell replication
Retroviruses
Once inside a cell, a retrovirus carrying an RNA copy of a cloned gene transforms this RNA into an equivalent DNA molecule and integrates that DNA into the host cell chromosome (see figure 16.26). Once integrated, this DNA can replicate with the mitotic cycle of the host cell. Retroviruses can be used only in cells that are reproducing and so they are used to carry cloned genes into cells that can be taken from a patient and cultured outside the body, such as bone marrow cells. Host cells that have incorporated the cloned genetic material can then be isolated and replicated before being returned to the patient.
Inject engineered cells into patient
Adenoviruses
Bone marrow
FIGURE 16.26 Cloned RNA version of a gene is inserted into a retrovirus that has had some of its RNA removed. After infecting a cell, the retrovirus transforms its RNA into DNA that can then be incorporated into the host cells chromosomes.
In contrast, when adenoviruses are used as vectors to carry DNA into cells, the viral DNA and its cloned gene remain separate from the host DNA. If the transfer into the nucleus is successful, the cloned DNA becomes active and produces a functional protein. Although this type of gene therapy can be successful, the result may be short lived. Why? This small piece of free DNA does not replicate and so, if the host cells replicate, the next generation of cells lack the replacement cloned gene.
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CYSTIc fIBROSIS AND GENE THERAPY

The inherited disorder cystic fibrosis (CF) is one of the human CF condition. Experiments with knockout the most common single-gene disorders in human mice showed that, when copies of the normal CF gene lations that originated in western Europe. CF is are transferred into cells lining their airways, their popu inherited as a Mendelian recessive condition and in symptoms are reduced. Australia occurs in about 1 in 2000 births. Persons Based on the results with knockout mice, clinical with CF have defective transport of chloride ions and trials began on people with CF. Copies of the normal CF secrete thick mucus that blocks their airways and gene were delivered to their airway cells as an aerosol pancreatic ducts. The major signs of CF include pro- via the nasal cavities. Most people receiving gene duction of very salty sweat, blockage of airways with therapy for CF show a reduction in their symptoms. consequent lung infection and heart complications, and However, because the normal gene does not become malabsorption from the digestive tract. The pancreatic incorporated into the DNA of their chromosomes, the defect can be treated by taking tablets that supply the gene therapy is only temporary and must be repeated. missing enzymes. Side effects of gene therapy for CF can include immune The gene responsible for cystic fibrosis is located reactions if viruses are used to carry the genes. on the number-7 chromosome and contains the coded genetic instructions for making a protein that binds to cell membranes and transports chloride ions out of cells. The normal form of the protein consists of a chain of 1480 amino ??? acids. Persons with CF produce Im just going to Id prefer cheese! an altered form of this protein inhale my CFTR gene . . . that is not able to function as a transport protein. Mouse embryos missing a particular gene can be engineered and are known as knockout mice. Knockout mice missing the normal CF gene have been produced and they provide an animal model that can be used to investigate
FIGURE 16.27
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Why stem? Stem cells are so named because all other kinds of cell stem from these undifferentiated cells.
Stem cells: a new approach

As people age, a number of degenerative disorders appear more commonly, such as Parkinsonism or Parkinson disease. This particular disorder results from the death of certain brain cells that normally produce a chemical (dopamine) that controls muscle movements. Persons with Parkinsonism show impairment of their motor movements, balance and speech. Early treatment for Parkinsonism involved administering dopamine to affected persons. This treatment gave only short-term improvement. Is there a way in which the lost dopamine-producing cells can be replaced? Experimental work is now proceeding on the potential use of stem cells to replace the lost cells in the brain. Other conditions and disorders in which stem cells might play a role in restoring normal function include Alzheimer disease, stroke, insulin-dependent diabetes and spinal cord injuries.
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What are stem cells?

Stem cells are undifferentiated or precursor cells that have the ability to differentiate into many different and specialised cell types, such as nerve cells, blood cells, bone cells, heart cells, skin cells and so on. Table 16.4 describes stem cells according to the range of different cell types that they have the power to produce. The first human stem cells were identified in the 1960s and these cells were in the bone marrow. One type of stem cell in the bone marrow can differentiate into (or give rise to) red blood cells, white blood cells and platelets (refer back to figure 8.10 on page 250). Since then, stem cells have been found in other human tissues, such as fat tissue, in skin and in the circulating bloodstream, but in very low numbers. In 1998, scientists discovered how to isolate stem cells from embryonic tissue. Stem cells also exist in other mammalian species and have been widely studied in mice. Because stem cells have the potential to differentiate into specialised cells of various kinds, their potential use to replace faulty or dead cells is great and much research is presently occurring. In September 2005, for example, scientists at the University of California reported that, following the injection of human stem cells from nerve tissue into the spinal cords of paralysed mice, the test group of mice displayed better mobility than the non-injected controls after just nine days and, after four months, the test group of mice could walk. The stem cells migrated up the spinal cord and developed into different kinds of cells including those cells that form insulating layers of myelin around nerve cells. Figure 16.28 shows the growth of myelin around nerve cells in the damaged region of a mouse spinal cord following injection of stem cells. Table 16.4 shows how stem cells are described as totipotent, pluripotent or multipotent in terms of their power or potency to produce various cell types.
Shiverer
FIGURE 16.28 Injured spinal cord of mouse following injection of human stem cells. These stem cells developed into myelin-producing cells that form a wrapping (green) around nerve cells (red) (see the areas marked by arrowheads). Other nerve cells remained without a myelin wrapping (see the areas indicated with arrows).
(a)
TABLE 16.4 Categories of stem cells according to their power to produce various cell types. The most powerful stem cells are totipotent cells. Why?
Description of stem cell totipotent (toti = all) Power or potency of stem cell can give rise to all cell types can give rise to most cell types can give rise to certain cell types Example fertilised egg and cells of a two-, four-or eight-cell embryo cells from the inner cell mass of an early embryo adult/somatic cells, such as bone marrow stem cells and adipose stem cells
pluripotent (pluri = most)

(b)
multipotent (multi = many)
FIGURE 16.29 (a) Branching

mammary gland produced from a single mouse breast stem cell (b) Section through mammary tissue produced from stem cell with different cell types shown by arrows (L = lumen)
In late 2005, scientists at the Walter and Eliza Hall Institute of Medical Research in Melbourne identified a cell line within mouse breast tissue that included multipotent stem cells. Remarkably, they found that just one of these cells introduced to fat tissue in the laboratory could produce a branching mammary gland (see figure 16.29a). These stem cells were able to give rise to the various cell types present in mammary glands (see figure 16.29b). Stem cells can also be grouped as follows: embryonic stem cells, which can be obtained from the inner cell mass of an early embryo known as a blastocyst (see figure 16.30). A single cell is isolated from the inner cell mass of a blastocyst and is grown in culture, dividing by mitosis to produce a culture of stem cells. Embryonic stem cells are pluripotent; this means that they can give rise to many different cells types found in a mammal, such as blood cells, skin cells and liver cells.
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Scientists have discovered that proteins produced by three key genes maintain pluripotent stem cells in their undifferentiated state. These proteins, known as Oct4, Sox2 and Klf4, repress or silence the genes needed for embryonic development. When production of these proteins stops, the stem cells start to differentiate and are no longer stem cells. (Information from the journal Cell, 8 September 2005)
Egg nucleus
Inner cell mass (forms embryo) Polar body
Outer cells (form placenta)
Fertilised egg
Egg cytoplasm
Two-cell stage
Morula (1030 cells) (day 4)
Blastocyst (day 5)
FIGURE 16.30 The development of a fertilised cell to a blastocyst stage. To see some real cells, use the Embryos weblink for this chapter in your eBookPlus.
adult stem cells (more accurately called somatic stem cells), which can be obtained from various sources such as bone marrow, skin and umbilical cord blood (see figure 16.31). Somatic stem cells are multipotent; this means that they can give rise to certain cell types such as various kinds of blood cells or various kinds of skin cells. Cord blood, for example, contains mainly bloodcell-producing stem cells.
EMBRYONIC STEM CELLS
Stem cells removed from inner cell mass of blastocyst
Bone cells Stem cells cultured in laboratory
Nerve cells Skin cells
ADULT (SOMATIC) STEM CELLS
FIGURE 16.31 Stem cell lines

can be created from various sources. Somatic stem cells is the preferred term rather than adult stem cells. Why?
Blood cells Stem cells removed from umbilical-cord blood and bone marrow
Therapeutic cloning for stem cell therapy

Depending on its purpose, cloning can be separated into reproductive cloning and therapeutic cloning. Reproductive cloning occurs when the purpose of the cloning is to produce a new organism (this was described on pages 6359). In contrast, the purpose of therapeutic cloning is to produce stem cells for use in treatment. Therapeutic cloning involves the creation, through the nuclear transfer technique, of an embryo for the purpose of obtaining stem cells from that embryo. These stem cells are intended for use in treating a patient who has a spinal cord injury or brain injury, has suffered a stroke or has a degenerative disease. The cell that provides the nucleus in therapeutic cloning is a healthy cell from the patient who is to receive treatment. As a result, the embryo that is created is a genetic match to the patient and these cells do not cause an immune response. Figure 16.32 (page 648) shows the process of therapeutic cloning. Therapeutic cloning raises possibilities for new treatments for diseases. Application of this technique would mean that normal evolutionary pressures of natural selection would no longer act on people with particular disorders. However, therapeutic cloning also raises major ethical issues.
ODD fAcT
In 2010, Spanish researchers reported that they used cells extracted from the human heart to reprogram adipose stem cells into forming heart muscle cells.
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FIGURE 16.32 Therapeutic cloning involves the creation of an embryo that is genetically identical to a patient. The patients cell is fused with an enucleated egg cell and develops into an early embryo. Stem cells are then taken from the inner cell mass of the early embryo (blastocyst) and grown in culture as pluripotent stem cells. Would these cultured cells be expected to cause an immune response if injected into the patient? Why?
Disease-free cells taken from patient
Enucleation of egg cell
Fusion of cell and egg
Embryo cultured and stem cells removed
Required cell types introduced into patient
Embryonic stem cells cultured and specific types obtained
Ethical issues are raised

The use of early embryos as a source of stem cells raises many ethical issues since establishing an embryonic stem cell line destroys an embryo. Likewise, ethical issues arise because this procedure involves the artificial creation of an embryo solely for the purpose of obtaining stem cells, a process that destroys the embryo. In December 2002, the Research Involving Human Embryos Act 2002 was passed in the Australian parliament. This Act established a framework that regulated the use of excess embryos. An excess embryo is one that both: 1. was originally created by artificial reproductive technology for use in IVF procedures 2. has been identified in writing by all responsible people as being in excess to the needs of the couple for whom the embryo was first created. Provisions of the Research Involving Human Embryos Act 2002 include the following: only persons holding a special licence may carry out research on embryos where that research may damage or destroy the embryo, only excess embryos created before 5 April 2002 may be used embryos cannot be created solely for research purposes. The provisions of the Research Involving Human Embryos Act 2002 are monitored by the National Health and Medical Research Council (NHMRC) Licensing Committee. Notice that, under the provisions of this Act, therapeutic cloning is not permitted in Australia.
Using a patients own stem cells

If it were possible to use a patients own stem cells in the treatment of a degenerative disease affecting that person, this would overcome two problems, by: removing any possibility of tissue rejection owing to immune responses avoiding the ethical issues that arise in the use of embryonic stem cells. However, as indicated in table 16.4 on page 646, somatic stem cells are more limited than embryonic stem cells because the range of cell types into which multipotent somatic stem cells can differentiate is more limited than that of pluripotent embryonic stem cells. So, two key questions are: Do sources of somatic stem cells exist that can be easily obtained with minimal surgical intervention? Yes. Stem cells can be obtained from bone marrow, and somatic stem cells can also be obtained from adipose (fat) tissue. Fat tissue is relatively easy to harvest from a person using liposuction and the adipose stem cells can be readily harvested from this tissue and multiplied in culture. Can multipotent somatic stem cells be made pluripotent?
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Yes. In 2009, researchers showed that somatic stem cells could be induced or genetically reprogrammed to become pluripotent by treatment with specific proteins known as transcription factors (see Odd Fact on page 647). These induced pluripotent stem cells (iPS cells) appear to be similar to embryonic stem cells. Because of their ability to differentiate into several clinically useful cell types, iPS cells have potential for use in the personalised treatment of a range of human degenerative diseases. Pointers to their potential use come from various research studies, such as the finding that, when human iPS cells were transplanted into the brains of rats suffering from Parkinson disease, these iPS cells generated functional dopamine neurons and reversed the disease symptoms. (Parkinson disease is caused by the loss of dopamine-producing neurons from the midbrain.) However, although it is still at an early stage, research on the therapeutic use of iPS cells continues. Issues under scrutiny include the lower efficiency and lower reliability of iPS cells in producing differentiated cells when compared with embryonic stem cells. While the use of iPS cells had not been approved for routine therapeutic use in Australia or the United States by 2011, these cells continue to provide an important tool for research in disease modelling and in the screening of drugs for use in the treatment of particular diseases.
KEY IDEAS
Gene therapy currently involves the introduction of copies of a normal allele of a gene into the cells of a target tissue to compensate for a defective allele. Both technical and ethical issues are raised in relation to gene therapy. Stem cells are undifferentiated cells that have the ability to form various cell types. Stem cells vary in their potential to form cell types and may be classified as totipotent, pluripotent or multipotent. Therapeutic cloning of stem cells has potential for treatment of particular disorders and other conditions. Legislation in Australia places strict controls on the use of embryos in research.
QUIck-cHEck
10 Identify two human inherited disorders for which clinical trials of gene therapy have been conducted. 11 a What is a knockout mouse? b Briefly explain how knockout mice are being used in initial testing of gene therapy techniques. 12 Identify the following as true or false. a Therapeutic cloning is currently legal in Australia. b Stem cells are undifferentiated precursor cells. c Each stem cell can give rise to all the different types of specialised cells. 13 Identify one location where you would expect to find: a embryonic stem cells b somatic stem cells. 14 Explain the difference between the members of the following pairs. a reproductive cloning and therapeutic cloning b totipotent and pluripotent stem cells
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The BRCA1 (BReast CAncer 1) gene is located on the number-17 chromosome while the BRCA2 (BReast CAncer 2) gene is located on the number-13 chromosome (see figure 16.33). Both are associated with a higher risk of breast and ovarian cancer in women and of prostate cancer in men.
Genetic screening
Ellen, a young woman in good health makes an informed decision to have a double mastectomy or breast removal. Why? Her family medical history shows that her maternal grandmother, her mother and two of her mothers sisters died of breast cancer. Only a few cancers have an inherited genetic component and one of these is breast cancer. About 5 to 10 per cent of women who are diagnosed with breast cancer have a hereditary form of that disease. About 80 per cent of women with a heritable form of breast cancer have a mutation in one of two genes, either BRCA1 or BRCA2, that makes them far more susceptible to developing breast and other cancers. Genetic screening can be done for women who, based on their family medical history, may be at an increased risk of breast cancer. In Ellens case, her family background put her in the high-risk category. At the time, Ellen did not have breast cancer but the genetic screening test on a sample of her blood showed that she had inherited the mutant form of the BRCA1 gene from her mother, which put her at a high risk of developing the breast cancer that killed so many members of her family. Genetic screening is a procedure in which a DNA sample is analysed to detect the presence of one or more alleles associated with an inherited disorder. Genetic screening may be carried out as follows: adult screening, to identify carriers of an inherited disease where a couple wish to determine if one or both of them can transmit an inherited disease to their children embryo biopsy or pre-implantation genetic screening, in which a single cell is removed from an embryo conceived by IVF to determine that the embryo will not later be affected by certain inherited diseases prenatal screening, to identify the genetic status of a fetus where a specific inherited disorder is suspected to be present, using chorionic villus sampling or amniocentesis (see the case of haemophilia in chapter 12, pages 4368) predictive screening, to identify persons at risk of developing a lateonset disease, such as Huntington disease (see chapter 12, pages 43840).
p BRCA2 p
BRCA1
Chromosome 13
Chromosome 17
FIGURE 16.33
Human chromosomes showing the BRCA2 and BRCA1 gene loci
KEY IDEAS
Genetic screening involves testing for the presence of a faulty allele that is the cause of an inherited human disorder. Genetic screening is carried out for various purposes and can use embryonic, fetal or adult cells as a source of DNA.
QUIck-cHEck
15 Which of the following statements best describes genetic screening? a Genetic screening cures genetic disorders. b Genetic screening detects genetic disorders. c Genetic screening detects the presence of alleles that produce genetic disorders. 16 How does an embryo biopsy differ from prenatal screening?
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Technology in human reproduction

Stopping conception
If a couple have normal reproductive systems, the only sure way not to reproduce is to abstain from having sexual intercourse. This may be considered as extreme action by a sexually active couple who wish to delay having a baby or to limit the size of their family once they have children. The couple will probably choose to use some other way to prevent having a baby. In the past, this was achieved by preventing gametes from meeting each other by the use of mechanical or some other means. A wider range of techniques is now available for couples. Some methods inhibit the production of gametes; others allow gamete production and fertilisation to occur but prevent implantation of any zygote formed. The production of a zygote and its implantation into the uterus wall is called conception. Any technique designed to prevent this from happening is called contraception. A range of contraceptive methods is available information about some of these is given in table 16.5. A couple may take a number of factors into account when selecting a contraceptive. For example, the success rates of different methods vary (see table 16.5). A success rate of 98 per cent for a method means a failure rate of 2 per cent. This means that two in 100 couples who use the method achieve a pregnancy; that is, the method failed to prevent conception in those cases. Advice should always be sought from a medical practitioner to reduce the likelihood of any problems related to the use of various forms of contraception. It is important to understand that many methods of contraception are no different from treatment for a variety of situations; there may be side effects from the chemicals or mechanical devices used and the detrimental effect may be greater in some people than in others. Some individuals may have particular medical conditions that rule out a particular method of birth control. In addition, a couple may also want to be reassured that use of a contraceptive method will not have a deleterious effect on any subsequent pregnancy they hope to achieve. You should also be aware that many contraceptive methods fail from time to time even when couples follow the instructions for a particular method. You can obtain more information about contraception from a Family Planning Clinic in your area.
ODD fAcT
If a woman breastfeeds her child on demand for up to six months after delivery and has not menstruated, she has a 98 per cent chance of not conceiving a success rate similar to that of many modern methods of contraception.
TABLE 16.5 Comparison of the success rates of different contraceptive methods

Method vasectomy (vas deferens cut and sealed) tubal ligation (fallopian tubes cut and sealed) contraceptive pill (all types combined) intra-uterine device (IUD) condom with spermicide diaphragm with spermicide condom alone (rubber sheath over penis) diaphragm alone (cap over cervix) spermicides alone safe period natural family planning withdrawal douching no contraception Effectiveness extremely effective highly effective Success rate (%) 99.8 99.8 98 9599 9798 9798 8590 8590 80 75 7082 6070 2050
moderately effective
unreliable
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Overcoming infertility
Infertility is the inability to conceive or carry a pregnancy to a live birth. This means that a breakdown in any of the steps from gamete formation to birth can lead to infertility. Although the cause of infertility may be unknown in many cases, about 3040 per cent of infertility problems relate to male factors and about 50 per cent to female factors. Sometimes both male and female factors are involved. About 1015 per cent of infertility in females is associated with ovulation and about 35 per cent have fallopian tube problems. Many of these fallopian tube problems have arisen from pelvic inflammatory disease (PID). Pelvic inflammatory disease is a broad term used to describe infections of the reproductive tract and is caused by bacteria that are often sexually transmitted. Other cases of infertility involve hostile mucus, in which the mucus of a woman interacts with the sperm of her partner and produces an environment in which the sperm do not survive. Although about 80 per cent of the cases of infertility in males have no known cause, many of the remainder are related to defects in sperm production. A typical fertile male produces semen with more than about 20 million sperm per millilitre, more than 60 per cent of motile sperm and more than 60 per cent of sperm with a normal structure. Because the number of sperm in semen can vary from time to time, a sperm analysis of a male would be repeated one or more times before a reliable diagnosis relating sperm production to infertility could be made. If the cause of infertility is known, such as blocked fallopian tubes, which prevents sperm and egg from coming into contact with each other, treatment of some kind may be available. In other cases, a problem may disappear of its own accord and fertility is restored. Another problem may be the lack of a uterus, as in the case of Maggie Kirkman whose story you can read on pages 6567. The wish to have a child can lead a couple to investigate avenues by which they can change their infertility. They may explore the possibility of adoption, although an adopted child will not have genetic material inherited from the new parents. Also, the number of children available for adoption has decreased significantly in recent years. A range of technologies has developed that offer some solution to at least some couples. Some of these technologies are outlined in the following section.
Donor insemination
If infertility is due to faulty sperm from the male partner, donor insemination with a success rate of about 80 per cent may be a procedure acceptable to the couple. Donor insemination involves fertilisation of the woman with donor sperm at the time when she is most likely to be ovulating. The donors physical characteristics, that is, those of the biological father, are carefully matched to the physical characteristics of the social father, the partner or husband of the woman. A child born as a result of donor insemination has 50 per cent of its genetic material inherited through the mothers egg and 50 per cent inherited from the biological fathers sperm. The child has no genetic material from the social father. Legally, a child born after donor insemination is the child of the mother and the husband at the time of the birth.
IVF in-vitro fertilisation

Some partners both produce normal healthy gametes but cannot achieve a pregnancy. Hostile mucus may prevent the meeting, or it may be a different problem such as damaged fallopian tubes or lack of a uterus. In-vitro f ertilisation (IVF) with or without associated techniques may assist such couples. Read about IVF in the box on pages 6534. If more than one egg is
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collected from a woman in the IVF process, it can be fertilised and then the resulting embryo can be frozen. The technique for freezing involves transferring an embryo, usually at the eight-cell stage of development, through several solutions of a special fluid containing a cryoprotectant. This protects the embryo during the freezing process by preventing it from being damaged by the formation of ice crystals. Storage eventually takes place in liquid nitrogen at 196 C. At this temperature, all metabolic processes cease and the embryo can be stored frozen for many years. The advantage of using this technique for storage is that fewer collections are necessary, only some of the eggs need to be used at any one time, and the lihood of multiple pregnancies can be reduced. like
ODD fAcT
The first authenticated birth of a normal baby from a thawed embryo took place at the Queen Victoria Medical Centre in Melbourne in March 1984.
Donation of eggs
A woman who is unable to produce eggs may become pregnant with an egg that has been donated anonymously by another woman on the IVF program. The egg is fertilised using sperm from the recipients husband. Whose genetic material does the baby contain? IVF is a multistep process and failure can occur at any stage; for example, failure may occur at the egg collection stage, at the in-vitro fertilisation stage, at the transfer stage or during pregnancy. The most significant factor in the success of IVF treatment is the mothers age. Figure 16.34 shows the pregnancy and birth rates for women in different age groups after successful invitro fertilisation and transfer of the embryo to the mother. Notice that the likelihood of a successful birth falls to below 30 per cent for women aged 35 and over.
The pregnancy and birth rates for IVF procedures 50% 45% 40% 35% 30% 25% 20% 15% 10% 5% 0% 2529 3034 3537 3839 4041 Female age (years)
Rates per transfer
Pregnancy rates Birth rates
4243
4445
FIGURE 16.34 Pregnancy and birth rates for 11 369 women after IVF treatment and embryo transfer. Data are the national results for transfers carried out by the Monash IVF Group for the two-year period January 2006 to December 2008.
BIOTECH
Assisting reproduction in humans
A woman entering an IVF treatment cycle has daily blood tests to determine the maturity of the eggs that are developing in her ovaries. She is given artificial hormones to stimulate the ovaries and blood tests are carried out to determine when the eggs are about to be released by the ovary. An egg or eggs are removed from the womans ovary by a technique called laparoscopy (see figure 16.35) and stored under special conditions in the laboratory. In laparoscopy, an instrument called a laparoscope is inserted through a small incision in the abdominal wall. The laparoscope looks a little like a telescope, but it has the lens of a microscope. A light attached to the laparoscope enables the doctor to see blisters on the ovary. These blisters contain fluid in which the eggs develop. The fluid and the egg it contains are sucked out of the ovary. For fertilisation to be possible, it is critical that the eggs be collected at the precise time they are ripe.
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Sperm is then produced by the husband. The sperm are treated to remove the outer protein coat because that is what happens as sperm move through a females reproductive tract. This evidently makes it easier for a sperm to penetrate an egg. In addition, the semen may be centrifuged to produce a concentrated sample that is more likely to lead to fertilisation. The process of fertilisation takes place outside the mothers body in a small glass tube or dish (see figure 16.36).
Laparoscope Forceps
The fertilised egg is incubated in the laboratory and begins to reproduce by mitosis. A four-cell embryo is obtained about 35 to 46 hours after insemination, and an eight-cell embryo after about 48 to 60 hours. Embryos are usually transferred to the mothers uterus at the two- or four-cell stage. The worlds first IVF baby was Louise Brown, born in Britain in 1978. The first in Australia and fourth in the world was born in Melbourne in June 1980.
Needle Vacuum
Ovary
FIGURE 16.35 When eggs are required for in-vitro fertilisation, they are sucked from the surface of the ovary with a needle. The ovaries are held in place with forceps and viewed through a laparoscope during the procedure.
Transfer to mother
(a)
(b)
(c)
FIGURE 16.36 (a) An egg is incubated with sperm in appropriate culture fluid. (b) After fertilisation occurs the fertilised egg has its band of protective cells removed. (c) The zygote undergoes mitosis and a two-celled embryo results. Each of the two cells reproduces by mitosis to form a four-cell embryo, and so on. Transfer of the embryo back into the mother takes place usually at the two- or four-cell stage.
Depending on what numbers are used, the success rate may be quoted from 487 out of 1369 (35.6 per cent) to 575 out of 1100 (52.3 per cent). Some would argue the latter figure is the correct one because loss during pregnancy is a natural feature of pregnancy. The older a woman is, the greater the chance that she will fail to achieve pregnancy with IVF techniques. Another concern is the possible increase in defects found in children born through IVF techniques. Although some of these children are born with defects, the proportion is not significantly different from the proportion of defects found in children conceived naturally.
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BIOTECH
Reducing the chance of implanting a defective embryo
During the early cleavage stage of an embryo generated by assisted reproduction (generally the eight-cell stage), one or two cells may be removed for chromosomal or DNA analysis. The technique is called pre-implantation genetic diagnosis (PGD) and is carried out using fluorescence in situ hybridisation (FISH). Tests can be carried out to identify specific genetic disorders such as cystic fibrosis or to check for chromo somal abnormalities, particularly those that occur in embryos of older women.
Surrogacy
Fibroids are benign tumours that develop in the wall of the uterus, usually when a woman is in her thirties or forties. If they grow large, tumours may press on nearby organs or cause profuse bleeding in the uterus.
A surrogate mother is a woman who agrees to have a baby for another woman who is generally infertile. If there is no financial reward for the woman having the baby, it is called altruistic surrogacy. Maggie Kirkman lacks a uterus. It had been removed because of advanced fibroids. Nevertheless, she longed to have a child. This became possible because one of her sisters, Linda, agreed to be a surrogate mother. Maggie believes her parenthood became possible through biology, technology and generosity. Read her story on pages 6567. Maggie and Linda have also written a book about the surrogacy and the legal steps they had to take to make it possible. Their book is entitled My Sisters Child: A Story of Full Surrogate Motherhood Between Two Sisters Using In-vitro Fertilisation (Penguin, Ringwood, Vic., 1988). Linda made no contribution to the genetic material of Alice, Maggies daughter, as Maggies own egg was used. However, some surrogate arrangements do not involve IVF technologies; donor sperm may be used with the surrogates own egg. Cases have occurred in America where a woman who has used her own egg for surrogacy has changed her mind about relinquishing the baby after its birth.
Gamete intrafallopian transfer

Gamete intrafallopian transfer (GIFT) is another technique used for some infertile couples. A couple may produce viable egg and sperm that are prevented from meeting by an inability of the egg to enter and pass down the fallopian tube. Gametes can be collected from the couple and then introduced into the fallopian tube of the female. Fertilisation and normal development may then occur. The success rate is about 28 per cent.
Intracytoplasmic sperm injection

This technique is used where males produce insufficient sperm to achieve fertilisation naturally. A trained embryologist chooses a sperm on the basis of its morphology and motility. The sperm is injected into the cytoplasm of an oocyte. This is followed by standard IVF techniques. The technique is also used for couples who have had previous failures with IVF. The success rate is about 24 per cent. Social, moral and legal considerations Because the techniques discussed on pages 6515 deal with the creation of human life, many questions are raised. Should we interfere with nature? When does life begin? What should happen to frozen embryos on the death or divorce of the parents? Recent legal issues related to reproductive technologies include questions such as: Should a woman be allowed to use the frozen sperm of her dead husband? Should sperm be collected from a dead person because a family makes the request? The law needs to change as the technology changes and continues on many of the issues that arise. debate
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PERSONAL STORY
Maggie Kirkman parenthood through biology, technology and generosity
Maggie Kirkman wrote the following story in 1999, when her daughter was 11 years old. of information from New Scientist and worked out that such a thing would be possible. Almost immediately I thought that Linda might be interested; she is an adventurous person. Linda had two young children and her husband had had a vasectomy, indicating that they wanted no more children of their own. There is a whole book between mentioning the subject to Linda and carrying out Sevs amazing plan. You can imagine the reaction of the infertility specialist: Excuse me; we have neither sperm nor a uterus. Can you help us to have a baby? One of the issues we dealt with along the way was the relative significance of genes and gestation. Here, we were reminded how important individual differences can be; Linda was prepared to gestate a baby for me but could not have used her own eggs, whereas my other sister, Cynthia, offered to donate eggs but knew she could not give up a baby she had gestated. Because we were using my eggs, I had to go on the in-vitro fertilisation program to ensure that I produced more than one egg and that their development could be monitored. A fine example of biological serendipity was that my hormone cycle and Lindas coincided in such a way that eggs could be removed ertilised with sperm from a donor, and the from me, f resulting two embryos transferred to Linda, all without chemical manipulation of Lindas hormones. One of those embryos implanted itself in Lindas uterus. Unfortunately, it grew too close to her cervix (a condition called placenta praevia, which can cause lifethreatening haemorrhages). As a result, about the last six weeks of Lindas pregnancy had to be spent in or near a hospital, and my daughter, Alice, was delivered by caesarean section a month prematurely, in May 1988. The long spell in hospital was the only problem we experienced with what was then a unique procedure (excluding: the legal difficulties, although we even tually were allowed to adopt Alice; the objections from religious conservatives, who were concerned that this was adultery by remote control and turned children into commodities; the claims by radical feminists that I was treating my sister as an object; and the shock of being sought suddenly for media interviews). We, as an extended family, have found this to be a wonderful way of creating a new human being, a much-wanted child who was welcomed by the whole family. To those who worry that Linda has been coerced into using her body this way, Linda says, I am an intelligent, well-educated, confident, assertive and articulate woman who is not easily pushed around. Linda estating a niece or nephew, knew that she was g
FIGURE 16.37 Maggie Kirkman (left) with her daughter Alice, and her sister Linda (right) in 1999
I never doubted that I would be a mother; I even had visions of six children. When I first tried to become pregnant, at the age of 24 in the early 1970s, I assumed that I would be as fertile as my mother and my sisters, each of whom had become pregnant immediately upon trying (if not before). After a year of disappointment, I went to a gynaecologist who found nothing wrong with me, but put me on clomiphene to boost my fertility. (IVF was not available in those days.) Six fruitless years later, I had a hysterectomy in response to very advanced fibroids. Although it seems to me now that the clomiphene may have caused their development and rapid growth, I dont regret having taken it. Clomiphene was a chance to achieve a goal that I valued. One of the casualties of the problems and distress arising from infertility was my first marriage, which ended. However, I met and married my second husband, Sev, in 1985, finding symmetry in my lack of a uterus and his lack of sperm. Sevs discovery of azoospermia when he volunteered to be a sperm donor was shattering, less traumatic only than the death of his father when he was eight years old. It was obvious to me that we would grow old as a childless couple. It was not obvious to Sev. A few months after we married, he suggested that we investigate the possibility of having children. We were both too old to adopt. I thought he had overdosed on science fiction. (My youngest sister, Linda, more kindly describes Sev as a fabulously creative lateral thinker.) What Sev suggested was surrogate motherhood, using IVF and sperm donation. It was a new concept to me; Sev had linked bits and pieces
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although Sev and I would not have forced her to relinquish the baby had she come to see him or her as a son or daughter instead. At the age of 11, Alice loves the X-Files, Good News Week and the Simpsons. Her hair is dyed with purple streaks. She takes the manner of her birth for granted, and, contrary to some predictions, she is not bullied at school because her aunt gave birth to her. Lindas children, Heather and Will, have always accepted that Alice is their cousin, not their sister, and are slightly disdainful of others who cannot understand what to them is perfectly obvious.
I am sure that the clear understanding of relationships within our family has developed because Linda and Alice have remained in close contact as aunt and niece; Linda has not been left to wonder what became of the child she bore, and Alice will never have to search for her birth mother. As for the five other children that I had planned, I am thrilled to have one. There is no lingering sense of an incomplete family, just joy and amazement that a fertile imagination and a generous sister (in combination with extraordinary technology and medical skill) have made me a mother to such a special human being.
KEY IDEAS
Many techniques are available to people to prevent conception. Infertility in humans has a variety of causes. Several techniques have been developed to overcome infertility in humans.
QUIck-cHEck
17 List four causes of infertility in humans. 18 Explain the term in-vitro fertilisation. 19 Explain briefly how IVF technology has overcome infertility in some couples.
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BIOCHALLENGE
1 The image shows a tobacco plant with a firefly gene that causes the plant to glow. a Is this plant an example of a TGO? Explain. b Suggest how this plant could have been produced. c Is this gene transfer a natural evolutionary process? 3 The image shows some transfected cells. a Identify two ways in which foreign DNA can be introduced to cells. b Is this a natural evolutionary process?
4 Horses do not have normally have twins, yet identical twin horses were produced at Colorado State University. a Explain how this may have occurred. b Are the twin horses an example of TGOs? c Is the process by which the twins were produced a natural evolutionary process?
2 The diagram above shows of a cloning technique. a Identify the process that is occurring here. b What will be the result of this process? c Is this a natural evolutionary process?
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CHAPTER REVIEW
Key words
adult stem cells artificial insemination (AI) artificial pollination artificial selection asexual reproduction cloned gene cloning conception contraception embryo splitting embryo transfer embryonic stem cells enucleated fecundity gene therapy genetic engineering genetic screening genetic variation genetically modified organism (GMO) genome in-vitro fertilisation (IVF) multiple ovulation multiple ovulation and embryo transfer (MOET) multipotent oestrus oestrus synchronisation pluripotent reproductive cloning selective breeding semen sex selection sexual reproduction somatic stem cells stem cells telomeres therapeutic cloning totipotent transfected transformed transgenic organisms (TGOs) vectors
Questions
1 Making connections Construct a concept map using as many as possible of the key words above. 2 Applying your understanding a Identify one benefit of the use of artificial insemination (AI) in livestock management. b How has the development of successful techniques for freezing sperm affected the use of AI? c For what purpose is AI used in human reproduction? 3 Applying your understanding and making comparisons Compare cloning and artificial fertilisation in terms of the following. a source(s) of genetic material b sex of offspring c variability among the offspring 4 Analysing information and applying your understanding Suggest explanations for each of the following observations. a Four calves are identical to each other and to their female parent. b An outbreak of a bacterial disease caused the death of all animals in a domesticated herd but killed only small numbers of feral (wild) animals of the same species. c Over many generations, the average wool yield of Australian merino sheep has increased. d The genetic variability in a herd produced by AI using sperm from a single sire is less than that from natural matings involving several sires. e The mitochondrial DNA in the cloned sheep Dolly was found to be identical to that of the Scottish Blackface ewe that supplied the enucleated egg. f The milk produced by some female goats was found to contain a specific human protein.
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FIGURE 16.38 Cell membrane being penetrated by violet laser light
5 Applying your understanding in a new context Assume that in a cloning experiment the procedures included the following steps: i The nucleus of a skin cell from a male corgi dog was transferred to an enucleated egg of a labrador dog. The resulting cell was then implanted in the uterus of a German shepii herd dog. a What sex would the resulting clone be? b What phenotypic characteristics would the cloned offspring display those of a corgi, a labrador or a German shepherd? c Which of the three dogs can be identified as the surrogate female parent? 6 Applying your understanding a Identify the essential characteristic of a transgenic organism. b Of the following, which are examples of transgenic organisms or cells? i mammals produced by cloning ii bacteria containing a plant gene iii plants produced by artificial pollination iv wheat cells with a mouse gene v cow cells with a gene mutation 7 Applying and communicating your understanding Assume that a person has gene therapy involving his liver cells. Will this change in his liver cells be passed on to his children? Explain. 8 Applying and communicating your understanding A person claimed that, starting with just one body cell of a champion dairy cow, it would be possible to produce a small herd of identical clones. Explain whether this is possible. 9 Applying your understanding Identify one critical difference between the members of the following pairs. a Dolly and Polly b a TGO and a GMO c cloning by embryo splitting and cloning by nuclear transfer d transfection and transformation 10 Analysing information and communicating your understanding In 2005, Scottish scientists successfully demonstrated a laser-based technique for introducing foreign genes into mammalian cells. They showed that exposing a cell to violet light from a diode laser for 40 milliseconds perforated the cell membrane and allowed it to take up foreign genes (see figure 16.38). The membrane repaired itself after the process. To test their laser technique, the scientists used it to try to transfect some mammalian cells with an antibiotic resistant gene and with a red fluorescent protein. a Explain why this was done. b How would transfected cells be recognised? Applying your understanding At the opening of the Svalbard Global 11 Seed Vault in February 2008, the Norwegian Prime Minister said: The Svalbard Global Seed Vault is our insurance policy . . . It is the Noahs Ark for securing biological diversity for future generations. Briefly explain the meaning of this statement. 12 Discussion In October 2000, the report was published of the successful implantation of a cloned endangered species, known as the Asian gaur (the largest form of wild cattle in the world). The clone had been developed using the nucleus from a single skin cell that was taken from a gaur that had recently died. The clone was transferred to a surrogate cow mother. This was the first example of the cloning of an endangered species. (In this case, however, the gaur, named Noah, died 2 days after birth due to an infection.)
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Some people have welcomed the use of cloning for endangered species. One person said: One hundred species are lost every day . . . Now that we have the technology to reverse this, I think we have the responsibility to try. In contrast, another scientist saw dangers in cloning, arguing that the cloning could compete with efforts to preserve the habitats of endangered species. What issues are involved in this matter? What are your views on this matter? Are these similar to those of others in your class?
FIGURE 16.39 Noah, the cloned

gaur
13 Discussion A company was set up in the United States for the sole purpose of cloning dead domestic pets. This commercial venture is generating debate around many issues because of the low success rates of this cloning process, its still-experimental nature, the associated high costs and the number of abandoned pet animals that are ultimately destroyed annually. Discuss with your group the appropriateness of this commercial venture. 14 In April 2005, scientists at Stanford University announced that they had been able to make fetal brain stem cells develop into insulin-producing islet cells. (It is the islet cells that are defective in type I diabetes.) While this research raises future possibilities for treatment of diabetes, these stem cell-based therapies require significant further research before their potential use can be put into action. Use the Stem cells weblink for this chapter in your eBookPLUS to answer the following questions. a List five of the conditions that might potentially be treated using stem cells. b Suggest the differentiation path along which stem cells would need to develop if they were to be used in the treatment of burns. c Examine the left-hand side of the figure shown at this website. Briefly describe the potential use of stem cells shown in this figure. d This website lists six steps that need to be taken before stem cells could be successfully used in clinical treatments. List the six steps. e Research on therapeutic cloning of stem cells (refer back to pages 6478) aims to address one of these steps. Which step is this? f The possible use of stem cells in the treatment of various conditions is known as regenerative medicine. Suggest why this term is used.
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16 Human Intervention in Evolution

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16 Human Intervention in Evolution

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CHAPTER 16 HUMAN INTERVENTION IN EVOLUTION

NATURE OF BIOLOGY BOOK 2

Selective breeding in action

Selective breeding: Australian sheep

Data from the National Wool Museum (Geelong, Victoria)

HUMAn InTERVEnTIOn In EVOLUTIOn

(Data from NSW DPI Prime Facts 579, March 2007)

NATURE OF BIOLOGY BOOK 2

Selective breeding: Australian cattle

eLesson eles-1567 Ancient resurrection

HUMAn InTERVEnTIOn In EVOLUTIOn

Technologies in selective breeding

Artificial insemination (AI)

Embryo transfer in livestock (MOET)

NATURE OF BIOLOGY BOOK 2

Sex selection through sperm sorting

HUMAn InTERVEnTIOn In EVOLUTIOn

(b) beef cattle

FIGURE 16.7 (a) Dairy cattle, and

eLesson eles-1568 Sexual selection

Manipulating breeding cycles

NATURE OF BIOLOGY BOOK 2

Artificial pollination in plants

Creating new species

HUMAn InTERVEnTIOn In EVOLUTIOn

Fertile hybrid (Triticosecale) WWRR

Genetic impact of selective breeding

NATURE OF BIOLOGY BOOK 2

Does loss of genetic variation matter?

Saving genetic variation

HUMAn InTERVEnTIOn In EVOLUTIOn

SWEDEN Spitsbergen (Norway) NORWAY

NATURE OF BIOLOGY BOOK 2

GENE BANkS fOR cROP GENETIc RESOURcES

FIGURE 16.15 Part of the 2 C storage area at the Australian

HUMAn InTERVEnTIOn In EVOLUTIOn

FIGURE 16.16 (a) Dr Bob Redden, Curator, showing some of

NATURE OF BIOLOGY BOOK 2

No sex at all! Cloning

(a) Enucleating a cell

Embryo splitting: identical twins, triplets, quads or quins

Egg cell with nucleus

Embryo splitting occurs when the cells of an

(b) Nuclear transfer

(c) Cell fusion

Cloning using somatic cell nuclear transfer techniques

HUMAn InTERVEnTIOn In EVOLUTIOn

Nucleus extracted from unfertilised egg

Donor cell fuses with enucleated cell by an electric pulse.

Embryo develops as in a fertilised egg.

FIGURE 16.19 (a) Megan and Morag, two

. . . and then came Dolly!

NATURE OF BIOLOGY BOOK 2

eLesson eles-1569 The Shine brothers

Remove udder cell from FinnDorset ewe

After Dolly what next?

Use electricity to fuse cells Single cell

Culture containing early embryo Implant in surrogate

Dolly, a clone of FinnDorset mother

FIGURE 16.21 Technique of animal cloning by nuclear transfer

HUMAn InTERVEnTIOn In EVOLUTIOn

Cloning: the downside

NATURE OF BIOLOGY BOOK 2

HUMAn InTERVEnTIOn In EVOLUTIOn