Induction Of Gametogenesis And Superovulation ^[3]

Comparative research on ovarian stimulation of non-human primates and domestic animals such as cattle, horses, sheep, and pigs raises interesting issues. Some work highlights the differences between the normal estrous cycles of animals such as cattle, and the cycles of human and non-human primates. Yet there are problems that are common among species in the stimulation of follicular development and the induction of mature oocytes. Ovarian stimulation and superovulation are exogenous interventions into a highly regulated physiological process. The normal physiology is not always easily manipulated. Developing strategies to circumvent the differences between normal and induced ovulation is a major goal of research proposed for this area.

Non-Human Primates Non-human primates exhibit a menstrual cycle that closely approximates that of humans. In monkeys, the hormonal events of the cycle include a geometric increase in serum estradiol, midcycle surges of luteinizing hormone (LH), follicle stimulating hormone (FSH), and a later increase in serum progesterone after LH and FSH decrease in concentration. These hormone levels are precisely synchronized in the normal cycle and lead to the development of one follicle, the dominant follicle, from which ovulation occurs. One of the purposes of superovulation is to encourage more than one follicle to develop fully, thereby producing multiple oocytes for in vitro fertilization. In monkeys, one of four chemicals is administered in combination with human chorionic gonadotropin to stimulate ovulation. These are human menopausal gonadotropin, human FSH, pregnant mare' s serum gonadotropin, or porcine FSH. However, monkeys sometimes produce antibodies that block the activity of hormones obtained from other species activity, and ovarian stimulation fails. Thus, there is a need to develop stores of monkey hormones to be used for ovarian stimulation in this species. The response to the administration of hormones to stimulate ovulation is also not uniform in non-human primates. In general, estradiol levels are higher than in a natural cycle, the LH surge is lacking, LH concentration remains high over a long period, FSH is low, and progesterone remains high in concentration. However, in some subjects there is a premature, spontaneous LH surge that is not synchronized with follicular maturation; this surge stops the follicles and oocytes from maturing. Even if such an LH surge does not occur, there is a wide range of responses to gonadotropin administration. It is impossible to predict the response of any individual animal. The LH surge and the heterogenous responses, however, can often be prevented by additional administration of gonadotropin releasing hormone. Another difficulty often observed with superovulation protocols is an overstimulation of prolactin secretion. These departures from the normal hormonal levels raise important questions relating to the efficacy and unknown effects of ovarian stimulation.

Despite the problems of ovarian stimulation in non-human primates, reasonable success rates have been achieved. Nevertheless, there are additional barriers to progress in this field. The supply of non-human primates is severely limited. In addition, these animals often cannot be used more than once for ovarian stimulation because of the immunological responses to human, equine, and porcine gonadotropins used in the stimulation procedures. Finally, eggs must be recovered from monkeys by either laparotomy or 1 copy, surgical techniques that are restricted by ethical considerations and formal legal constraints to a low number of repetitions per animal. Although these problems of availability, reuse, and the lack of non-human gonadotropins are more difficult to overcome, than the biological problems of overriding normal physiology, questions about the basic biological processes remain unanswered. Resolution of the problems associated with the use of non-human primates in this type of research could be facilitated by an increased ability to augment natural hormone and gonadotropin release, by improved resolution of ultrasound imaging to identify mature oocytes and guide their collection by non-surgical means, and by the development of cell lines that could produce larger quantities of non-human primate gonadotropins. Finally, it is important to note that many of the same questions remain unanswered regarding human ovarian stimulation and that particular non-human primate species serve as the best model for human reproductive physiology.

Domestic Animals Extensive use of artificial insemination in cattle has increased the genetic contribution of desirable bulls to the overall supply of cattle. However, there is little likelihood that the desirable characteristics of females will be further increased by augmenting the male genetic contributions. Superovulation, on the other hand, combined with embryo freezing and embryo transfer, has the potential to directly the gene pool of desirable female traits by increasing the number of offspring from a valuable cow. There are a number of differences between non-human primates and domestic animals in the ways ovarian stimulation is accomplished. Some of these differences arise from differences in the reproductive cycles of the two groups. In dairy and beef cattle, stimulation is accomplished with the administration of either pregnant mare's serum gonadotropin (PMSG) or porcine FSH. The latter is preferred to PMSG because PMSG has a long half-life and often results in asynchronous ovulations. Porcine FSH is not without problem either since it is contaminated by variably high levels of luteinizing hormone. Human chorionic gonadotropin, routinely used in non-human primates, is not used in the commercial practice of ovarian stimulation of cattle. To control the variability and lack of predictability in the estrous cycles of cattle, current protocols often include an injection of a specific type of prostaglandin (PGF-2 alpha). This treatment causes the regression of the corpus luteum, thereby artificially restarting the cycle.

The success rate of superovulation in cattle has been variable. This is caused by a number of factors including seasonal variability, breed differences, dose and timing of gonadotropin administration, and history of previous superovulations. Such differences contribute to difficulty in comparing studies performed on different breeds and, at times, in comparing studies in the same breeds, but done with varied techniques or in animals of varying age. It has been found that, even if all the factors possible are controlled, there remains a great deal of variability in the success rate of superovulation. A number of strategies to improve the success rate are currently being tried. One is an attempt to purify porcine FSH preparations to exclude the contaminating LH and, further, to determine the FSH to LH ratios that are optimum for successful superovulation. Another strategy is to try to neutralize PMSG with antibodies so that the complications that arise from the long half-life of PMSG can be prevented. Because the lack of normal coordination of endocrine events is a common occurrence in superovulation, investigators are also trying to define the sources of asynchrony and develop ways of normalizing a coordinated series of biological events.

Superovulation in domestic animals other than cattle also has had mixed results. In goats, the technique has been tremendously successful. Superovulation of sheep has also been largely successful, and a few innovative strategies have been applied in this species. Sheep are seasonal breeders and attempts have been made, with initial success, to induce ovulation in this species during times when they are normally anestrous. In order to bypass the use of exogenous gonadotropins, workers have employed antibodies to inhibin in sheep. Inhibin is a substance made in an ovarian follicle that suppresses FSH secretion. The blocking of inhibin with antibodies raises FSH levels, thereby increasing the natural stimulus for follicular development. Antibodies against steroids have also been tried in sheep to achieve the same end.

In horses, superovulation has proven to be difficult, and to require nonstandard protocols. The period of estrous or "heat" in mares is unusually long and variable. In addition, there are preprogrammed physiological mechanisms in mares to prevent the development of twins. These two aspects of equine reproductive physiology greatly complicate the successful application of ovarian stimulation techniques. Also of critical importance is the fact that PMSG, even in high doses, does not adequately stimulate the development of follicles in mares. Unlike other animals, where PMSG acts as both FSH and LH receptors, in mares PMSG has only LH activity. Some success has been achieved in mares with administration of porcine FSH, especially if combined with human chorionic gonadotropin. In general, however, progress in successful superovulation in horses lags well behind that in other domestic animals, except for the pig.

The most limited of all commercial applications of superovulation in animals has been in the pig. Although possible, the technique does not confer many of the advantages that it holds for other species. One reason is that pigs deliver litters as opposed to one offspring at a time. Therefore, the need to increase the offspring from one individual, while helpful in certain circumstances, is not as compelling in pigs as in other domestic animals. Nevertheless, superovulation and embryo transfer may be able to lower the frequency of diseases by increasing the hum of offspring from disease-free or disease-resistant populations. More frequent reasons for the use of embryo transfer in pigs are to obtain disease-free embryos from infected pigs² and to introduce new genetic material into specific pathogen-free herds. Thus, further research into the normal reproductive physiology of pigs will be useful.

To improve IVFET in domestic animals as well as in human and non-human primates, more needs to be known about the normal regulatory events of ovulation, including the physiology and biochemistry of the development of a dominant follicle. Improved ultrasonography has potential for increasing the success of superovulation in all species. As with monkeys, developing ways to bypass the use of exogenous agents like PMSG and porcine FSH would be of enormous use in domestic animals. One way of doing this may be by blocking inhibin as has been done in sheep. In order to improve the success rate of assisted conception in animals, many of these research areas must, nevertheless, be pursued in each species separately with realization of the inter-species differences. It is not always possible to predict when the results obtained from one species can contribute knowledge applicable to other species.

Biology And Maturation ^[4]

This section examines three topics relating to the biology and maturation of oocytes: 1) The study of membrane biochemistry, which has relevance to the complex membrane interactions that occur at fertilization; 2) the study of molecules that are important in maturation events, is also important to understanding the possible reasons for failure in IVFET; and 3) the biological and physical properties of eggs and embryos that are affected by preservation techniques such as freezing, known as cryopreservation.

Membrane Biochemistry The membranes of all cells of the body share certain standard structural characteristics. The basis of a cell membrane is called a bilayer, which is a two-layered collection of phospholipid molecules (lipids containing phosphorus). Phospholipid molecules are polar in that each has one end that is stable in water (hydrophilic), and one end that repels water (hydrophobic). In a bilayer, then, the lipid molecules line up so that the hydrophobic ends meet each other on the inside of the bilayer. The hydrophilic ends are thus oriented on the outside of the bilayer such that one group faces the intracellular space and the other faces the extracellular space. Such an arrangement makes sense when one considers that both the cytoplasm of a cell and the extracellular spaces are composed largely of water. It is, in fact, a quite natural response of lipids to form bilayers in an aqueous environment; such a process underlies soap bubble and oil droplet formations.

Proteins are part of the membrane also. Some proteins span the thickness of the membrane and act as hydrophilic channels for ions or participate in molecular signal transduction mechanisms. Other proteins are partly embedded in the lipid bilayer and partly exposed to the extracellular space. Attached to such proteins are long, often branching, chains of carbohydrate molecules that also extend into the extracellular space. Finally, large extracellular proteins often associate with or stick to the most external parts of the carbohydrate molecules. Thus, the cell membrane, even in its standard form, is a complex structure made up of different types of molecules arranged in regions that each exhibit special pries. The possible interactions of membrane molecules are immense since the various molecules are mobile within the bilayer. These interactions underly the equally immense range of biological responses of cells including cell-cell interactions, receptor-mediated signal processing, and transmembrane transport of nutrients or toxins.

Reproductive events in which membrane biochemistry is of particular importance include sperm capacitation and fusion of sperm and egg membranes for fertilization. Basic aspects of membrane biochemistry are most often studied using membranes from cells such as red blood cells or membranes manufactured in the laboratory, called liposomes. Such membranes are simpler than the membranes from eggs or sperm and, therefore, easier to study. However, data obtained from these model membranes must be extrapolated to eggs and sperm with great caution.

Mammalian Oocyte Maturation In a normal follicle there is communication between the oocytes and the surrounding nurse cells, called granulosa cells, through specialized junctions (gap junctions) through which small molecules pass from one cell to another. Developing oocytes undergo a type of cell division in which only half the normal number of are retained. This process is known as meiosis and has a number of stages. Until just before maturation, however, the growing oocytes remain in a state of suspended or arrested meiosis. It is thought that the granulosa cells help to maintain this arrest of meiosis by producing substances that enter the oocyte through the gap junctions. It is also possible that substances that induce final maturation of the oocytes are produced in the surrounding cells and are transmitted to the oocytes in the same manner. Since the ability to mature oocytes in vitro would be of enormous value in IVFET, the understanding of the substances that either maintain or abolish meiotic arrest is particularly desirable. It is important to note that many of the studies summarized in this section done with mouse oocytes and these data were compared to those obtained from other mammals including rabbits and rats. Investigators have also studied frog oocytes, but the mouse is the most common animal model. Eventually, of course, certain critical experiments will have to be replicated using oocytes from human or non-human primates.

Several substances are thought to maintain meiotic arrest. One, cyclic adenosine monophosphate (cyclic AMP), is a molecule present ubiquitously in the body. Another, hypoxanthine, is common in the body and belongs to a group of molecules known as purines. Guanosine, another purine, is also a potent inhibitor of meiosis and the purine, adenosine, has been shown to augment the activity of hypoxanthine. By examining the effects of some of these substances on each other, a hypothesis has arisen which suggests that hypoxanthine and other purines may increase the level of cyclic AMP, which then inhibits meiosis. Future research, focused upon the molecules of the cyclic AMP pathway inhibiting meiosis, may elucidate ways in which oocytes can be maintained in meiotic arrest in vitro.

While knowledge regarding the inhibition of oocyte maturation is important, knowing how to mature oocytes in culture would have many applications that are described later. In vivo, oocyte maturation is produced by the surge of LH immediately prior to ovulation. The mechanism of action of LH is unclear, however. One might assume that, if cyclic AMP maintains meiotic arrest, then LH somehow causes a decrease in cyclic AMP in the oocytes or the surrounding nurse cells. It has been shown in vivo that administration of LH in combination with human menopausal gonadotropin reduces cyclic AMP in the oocyte, but not in the nurse cells. This effect is puzzling since the gap junctions between the oocytes and the other cells still appear to be functional, so cyclic AMP should pass through these junctions easily. An alternative hypothesis is that LH acts indirectly by causing some maturation-inducing substance to be produced in the surrcunding cells that decreases the cyclic AMP in the oocyte. Since an increase in intracellular calcium has been shown to be essential in somatic cell division, it is possible that a maturation-inducing substance operates in the oocytes by causing an increase in intracellular calcium. The search for these substances and their mechanism of action is a major area for future research.

The study of oocyte maturation has led to extremely successful methods for maturing mouse oocytes in vitro. Such work has shown that the in vitro culture of oocytes and their surrounding cells can be accomplished in ways that allow subsequent fertilization, implantation, and delivery of normal young to occur. Successful in vitro fertilization of oocytes from other species, however, depends upon continued basic research on oocyte nutrition and metabolism and on the factors or substances that control differentiation and maturation. The potential applications of an increased ability to mature oocytes in vitro are numerous. As discussed earlier, such an ability would be of enormous value in the production of agriculturally important animals. These techniques also have important applications in attempts to preserve endangered species. For example, it barn been demonstrated that oocytes removed from mice up to six hours after death can be matured in vitro (Lazarus effect). Yet another application can be found in basic science research into genetic diseases through the use of transgenic mice. Such mice are produced by introducing known gene fragments into the genome of a mouse. These gene fragments have been inserted by microinjection into eggs. Alternatively, the fragments, incorporated into a harmless virus, have been injected into neonatal female mice. In this way, such fragments are incorporated into the DNA of developing oocytes in these females. It would be extremely helpful to be able to transfer gene fragments by vital infection of oocytes, which could then be matured in vitro for subsequent fertilization. Finally, the ability to mature human oocytes may have some application for human IVFET, since matured oocytes from donors would obviate the need for hormonal stimulation therapy and permit synchronization of the developmental state of the embryo with the state of the uterine lining. The degree to which data obtained from experiments with mouse oocytes can be applied to humans is not known. Therefore, for such applications to be realized in human IVFET, it is probably necessary that basic research be done using oocytes from human ovaries removed for justified clinical reasons and/or oocytes from non-human primates.

Cryopreservation of Oocytes and Embryos Cryobiology is the study of how living tissues can be frozen and stored for later revival. There are a number of uses for cryopreservation of oocytes and embryos in connection with IVFET and other reproductive technologies. Cryopreservation of human and cattle sperm has been a routine and successful procedure for many years. It is thought that, properly stored, frozen embryos can be preserved for many decades before thawing and transfer to the uterine cavity. To date, embryo cryopreservation has been successful in many mammalian species, including mouse, sheep, cow, human, and baboon. Rare mutant animals have also been preserved for later study.

The Mouse Embryo Banks at the Jackson Laboratory in Maine contain many examples of cryopreserved mutant mouse embryos. Such mutants are valuable for research into genetic and other diseases. Cryopreservation allows easy transportation of frozen embryos by researchers who move frozen mice embryos from one lab to another, and for the national and international transportation of cattle embryos.

If proved safe for use in clinical practice, routine cryopreservation of eggs or embryos could reduce substantially the number of times a would have to undergo hormone stimulation, because the eggs harvested could be frozen for later fertilization or fertilized in vitro and the extra embryos stored for future transfer. These stored embryos could be used if the first pregnancy failed or used at a later time so that an infertile couple could have more than one child from one stimulation/IVFET procedure. thus, such preservation techniques could also minimize the waste of extra eggs and extra embryos produced by superovulation and other IVFET procedures.

Although the specific methods of cryopreservation vary, the basic steps involved are the same. The first is chemical protection of the tissue against damage from freezing. The tissue is then sometimes cooled to -196ºc long-term storage. The procedures used for warming and cooling are variable. After rewarming, the chemical protective solutions are removed by rinsing. Protection from damage by freezing is critical to the survival of an embryo because, without protection, the formation of ice crystals causes tissue damage that can not be repaired. Cryoprotective solutions, which are made from chemicals such as glyoerol or dimethyl sulfoxide, function in a manner similar to antifreeze in a car. They replace the water in the embryo.

Because of the importance of this step in cryopreservation, much basic research is being conducted to find appropriate cryoprotective solutions. Certain specific properties of the solutions are important determinants of the survival rate of eggs and embryos. For example, some chemicals, such as sucrose, cannot enter cells and, therefore, do not protect from freezing damage in the same way, or with the same efficiancy, as other molecules that can enter cells. Glycerol, on the other hand, can permeate a cell, partially replace the water, and impart the cryoprotection necessary. The osmotic characteristics of particular solutions are important also. In the case of glycerol, if one exposes a cell to a solution of water and glycerol, glycerol moves into the cell by osmosis, a process by which molecules in a high concentration will move across a semi-permeable barrier, such as a cell membrane, into a region of low concentration of that molecule. Likewise, water moves out of the cell by osmosis into the surrounding fluid. Eventually, the concentration of glycerol will equilibrate and be the same on both the inside and outside of the cell. Since glycerol moves into the cell more slowly than water moves cut of the cell, the cryoprotection procedure must be carefully controlled so that the cell does not lose water so quickly as to shrink and die.

It is especially important to control the movement of molecules across the cell membrane when thawing ova and embryos. After thawing, it is important to remove the glycerol from the cells and to return the intracellular contents to normal. However, a cell protected with glycerol, if suddenly exposed to a normal saline solution, will take up a great deal of water very quickly. This influx of water causes the cell to swell and the membrane may burst. Although this problem can be avoided by exposing the cells to solutions of decreasing concentration of glycerol in a stepwise fashion, this is not always easy to accomplish. Research has shown that adding the impermeable solute, sucrose, to the saline solution results in a more even exchange of glycerol and water across the membrane and obviates the rapid influx of water into the cell. This one-step method has proven to be of particular use in the application of these techniques under field conditions such as in work with endangered species.

The method used to freeze and thaw the ova and embryos is also important. Until recently, the tissue was first cooled to a few below zero so that ice formation could be controlled. The tissue was then cooled to some intermediate subzero temperature before being rapidly cooled to the storage temperature of -196º C. Research with mouse ova showed that the cooling procedure used could affect the survival rate in important ways. For example, it was demonstrated that, if ova lose a considerable amount of their original volume during cooling, the survival rate is greater. Cells that retain most of their original volume usually do not survive. This is probably because a cell is primarily composed of water from which the damaging ice crystals form. The method used to freeze the tissue also determines the subsequent thawing procedure. Thawing is performed stepwise in reverse order to the freezing steps. New freezing protocols have been developed that simplify the multiple step methods. For example, mouse embryos have been successfully frozen in one step after incubation in glycerol and sucrose. This method uses the glycerol to cryoprotect the tissue and the sucrose to dehydrate it before freezing, eliminating the need to slowly cool the tissue before freezing. Variations of this method have also been successful. One novel method is called vitrification, which means freezing without crystal formation. Sometimes, this can be accomplished by extremely rapid freezing. However, in a variation of this approach, embryos are exposed to a solution of chemicals that form a glassy solid substance when cooled. No ice crystals form in the embryos exposed to this solution so freezing can be rapid and done in one step. These new freezing procedures are still being developed, but they have the potential to increase the success rate of ova and embryo freezing.

Improvements in cryobiology would have significant impact. Improved preservation and storage of embryos from valuable cattle and other agriculturally important animals would greatly increase the yield from superovulation and IVFET procedures. The preservation of endangered species and rare genetic breeds of laboratory animals would be enhanced. Finally, successful storage of human embryos that result from IVFET techniques can increase the chances of an infertile couple to have a baby.

Intratesticular And Intraovarian Paracrine Control ^[5]

Development of male and female gametes occurs over varied periods of time in special body compartments, the ovaries and the testes. In this section, the biochemical control mechanisms that affect gamete development will be discussed. In contrast to hormonal control mechanisms in which bioactive substances are released into the bloodstream and act at distant sites, paracrine control mechanisms involve chemical interactions between neighboring cells. For example, one cell can release an active substance into the extracellular space, the substance then diffuses around other cells in the region and affects the activity of these other cells in some way. These effects most often are mediated by cell surface receptors for the substance released.

Intragonadal Control of Testis Function The testes are composed of a number of different cell types, including germ cells in their various developmental stages (spermatogonia, spermatocytes, spermatids, and mature sperm) each of which displays unique characteristics. Other cell types include Sertoli cells, which are nurse cells for the developing spermatogonia and spermatocytes, and Leydig cells, which release the hormone testosterone, required for sperm maturation. Many bioactive hormones and peptides have also been found in the testis and research has begun to determine the cell sources of these chemicals and their function in spermatogenesis. Some of the molecules found in the testis have been shown to affect cell cycles and cell differentiation. For example, somatomedin C (also called insulin-like growth factor, IGF) and epidermal growth factor stimulate cells to begin DNA synthesis for mitotic cell division. Fibroblast growth factor stimulates quiescent cells to reenter the cell cycle in order to replicate and divide. Various hormones and growth factors act as stimulators to cell differentiation, thus causing the cells to exit permanently from the cycle of chromosome replication and mitotic division. Cells are inhibited from exiting the cell cycle by interleukin-l, which is a molecule made by cells of the immune system. The functions of most of the hormones and growth factors in the testis are not known. For a few, however, certain functions are beginning to be described.

In the rat, a contain animal model for investigations of sperm development, Sertoli cells start to divide and proliferate around the 22nd day of embryonic life, and complete this proliferation by 20 days after birth. Leydig cells, in contrast, do not start to divide until 2 or 3 days after birth and continue to proliferate until 35 days after birth. By the end of these processes, almost equivalent numbers of Sertoli and Leydig cells are found in the testis, despite very different developmental timetables. A hypothesis has been proposed that these cells may regulate each other by complicated interactions during development. Fibroblast factor stimulates replication of Sertoli cells by causing an increase in the number of receptors for follicle stimulating hormone (FSH). As this occurs, the numbers of Sertoli cells increase and release somatomedin C in greater and greater amounts. Somatomedin C increases the number of receptors for luteinizing hormone (LH), which causes the Leydig cells to divide and increase in number. The increasing numbers of Leydig cells leads to an increase in beta-endorphin released by the Leydig cells. A negative feedback occurs such that beta-endorphin decreases the proliferation of Sertoli cells.

Such paracrine interactions are probably not unusual and may occur between other types of cells in the testis. In addition, non-paracrine mechanisms of communication probably occur, including cell to cell adhesion and communication through gap junctions between cells. Very little is known regarding the mechanisms of interaction between developing sperm cells and their nurse cells, the Sertoli cells. Cell adhesion molecules have been found on spermatocytes that, some suggest, promote continued meiosis of the spermatocytes bound to the Sertoli cells. Experiments done in culture have show that sperm cells synthesize more DNA and RNA when they are cultured with Sertoli cells (especially if FSH is in the culture medium), than if the sperm cells are cultured alone, or alone with FSH. Such data suggest that the Sertoli cells are important to sperm cell development.

Investigators have looked for proteins that can be related to specific stages of sperm cell development. The stages of sperm cell development are quite complex, but can be generally grouped into four main stages. The primordial germ cells are called spermatogonia, and undergo six mitotic divisions before becoming spermatocytes. Meiosis begins in spermatocytes. Further development forms spermatids, which are immature germ cells in which meiosis is arrested. Spermatogonia, spermatocytes, and early spermatids are in close apposition to Sertoli cells in the long tubular structures of the testis where sperm are formed, called the seminiferous tubules. The seminiferous tubules open into other tubes through which sperm are transported. As spermatids mature and differentiate into early sperm cells, they are released from the Sertoli cells into the seminferous tubule. An amazing feature of the seminiferous tubule in some species is that, in any given segment of the tubule, the germ cells form a similar pattern of stages. While this arrangement argues for some kind of cellular control of developmental stage, it also provides a definable tissue state with which to experiment.

Scientists have isolated specific segments of seminiferous tubules and extracted the proteins contained in them. Maps of these proteins have shown that the different stages are associated with different proteins. By isolating some of these proteins and producing monoclonal antibodies to them, it has also been possible to determine which cells contain the protein of interest. One such protein has been found in Sertoli cells, and its synthesis and secretion have been shown to be stage-specific. These experiments have also found that such proteins diffuse into the extracellular spaces of the seminiferous tubules, but because of the slow flow rate within the tubules, remain in spaces near developing sperm cells for quite a long time before being degraded by proteolytic enzymes. Therefore, there seems to be strong evidence that Sertoli cell products greatly influence the development of sperm cells.

Continued research into the molecular events of male gamete development would fill some of the large gaps in knowledge about the normal development of sperm cells and would further the search for causes and cures of male infertility.

Paracrine Control of the Ovary Although it is generally agreed that paracrine control is important to the development of follicles in the ovary, much of the evidence is circumstantial. This state of affairs derives from the nature of autocrine/paracrine relation. It has proved difficult to manipulate experimentally systems in which the cell of origin of an observed signal is the same as or adjacent to the target cell.

Early experiments to examine the importance of intraovarian chemical control involved removing the pituitaries of rats. Without the pituitary hormones (especially LH and FSH), which normally promote follicular development, investigators could then look at the effects of a number of substances on ovarian follicles. Such experiments found that estrogen, which is synthesized and released from cells in the ovary, was needed for follicular development and that other steroids produced inhibitory effects. Further, it was demonstrated that estrogen could act in concert with or synergistically with LH and FSH to differentiate follicular cells. Later experiments used granulosa cells from pig follicles in culture to show various effects of estrogen. Following a brief inhibitory effect, estrogens stimulated progesterone synthesis in the granulosa cells in a manner that was time-dependent and blockable by pharmacological inhibitors to estrogen. Continued research demonstrated that estrogen action was exerted after the generation of cyclic AMP (see earlier section on oocyte maturation for other actions of cyclic AMP).

There are several chemical forms of estrogen that are made by enzyme modification of a basic structure. One type, catecholestrogen, is found in high concentrations in the walls of large ovarian follicles in the pig and probably enhances, by paracrine mechanisms, the effects of FSH in the follicle. This concept arose from culture experiments which found that catecholestrogen with FSH stimulates progesterone synthesis and production of cyclic AMP. However, antiestrogens do not block this effect. Thus, while experiments have shown that estrogen plays an important role in the control of follicular development, its true role in the natural state remains to be determined.

Estrogen is only one of the substances under investigation in terms of paracrine regulation in the ovary. Tissue culture experiments have shown a number of peptides and growth factors to cause proliferation of granulosa cells. For some of these factors, it is known that they have effects on follicular cells and that they are present in the ovary in vivo. However, whether or not the factors are necessary for follicular development and normal ovarian physiology is not known. The most research has been done on insulin-like growth factors (IGFs). IGFs are synthesized by many tissues and have been found to be a powerful local mediator of growth and differentiation. They seem to stimulate all kinds of effects in granulosa cells including increasing enzyme levels, cell numbers, protein synthesis, glucose utilization, and various secretions. Insulin itself and the IGFs seem essential for FSH induction of receptors for LH, for generation of cyclic AMP, and for FSH stimulation of steroids. IGFs also interact with other growth factors in the ovary to increase cell number. Since most of these findings have been from in vitro studies, it is important to demonstrate the presence of IGFs in follicles in vivo. In fact, IGFs have been shown to be present in follicular fluid in in vivo concentrations similar to those that produce effects in culture. Furthermore, the concentration of IGFs increases during follicular growth and under the influence of FSH, LH, and growth hormone. This is important because any substance active in follicular development and local regulation in the ovary would be expected to change in concentration in response to pituitary hormones.

Another growth factor found in the ovary is a type of transforming growth factor called TGF-alpha, which is a molecule related to epidermal growth factor. TGF-alpha increases the rate of cell division of ovarian cells in vitro and has a negative effect on the secretion of estrogen from granulosa cells. It also seems to decrease the effect of FSH on LH receptors, although this effect varies. TGF-alpha has been found in significant concentrations in follicular fluid and is in highest concentration in small follicles. Based on these findings, it has been proposed that TGF-alpha promotes cell replication in the follicles and inhibits differentiation. The other type of TGF, TGF-beta, is a molecule very similar to inhibin and Mullerian-inhibiting substance, both of which have effects in the ovary. In general, studies have suggested that TGF-beta augments the actions of FSH to increase estrogen secretion, LH receptor activity, and progesterone synthesis. These effects seem to be dependent on both the concentration of TGF-beta and FSH and seem to vary across species.

Fibroblast growth factor is another factor being investigated for its possible role in regulation of ovarian function. This growth factor is also present in many tissues and has been shown to be particularly potent in stimulating the formation of new blood vessels. In vitro, fibroblast factor has effects on cells of the corpus luteum and it stimulates granulosa cells to divide. In addition, the factor seems to support the growth of blood vessels in the corpus luteum. Its effects on synthesis of estrogen and progesterone are probably inhibitory. There are many other peptides that have been found in the ovary, but research on these substances is sparse. Many of them have not even been purified enough to allow analysis of their structure and examination of their effects in vitro or in vivo.

This complex area of research promises to provide useful knowledge about regulation in the ovary and could have far-reaching implications for both IVFET and natural reproductive biology. In order to demonstrate that a particular substance has effects in the ovary, the substance must be shown to be secreted by ovarian cells into the local extracellular space and to produce changes in the activity of neighboring cells. Such minimum criteria have been met for some of the factors discussed above; however, elucidation of the mechanisms involved is critical. Especially important for future research is to determine the biochemical regulation of cell replication and differentiation in the ovary. Also, it is necessary to determine how some of these factors are produced by the cells. For example, are there large precursor molecules produced that are modified inside the cells for specific needs? The interaction of these factors with known hormones is also important and may be of special application in evaluating the effects of superovulation. Strategies to block the activity of these factors could help to determine if any of them are absolutely necessary to normal ovarian function as this is entirely unknown. Finally, it is of utmost importance to relate the findings from in vitro studies to what is actually occurring in the natural state.

Assessment Of Gametes ^[6]

There are many reasons why IVFET techniques in human clinical practice do not result in ongoing pregnancies one hundred percent of the time. Certainly, some of the failure rate is attributable to such things as the varied skill levels of the practitioners, the physiological problems of the infertile male or female, or the difficulty in synchronizing the stage of the embryo with the stage of the uterine wall. Nevertheless, a significant contribution to the failure of IVFET derives from poorly understood physiological responses of oocytes and early embryos. Between 20 and 40 ere of apparently mature eggs fail to be fertilized by IVFET methods. Of the 60 to 80 percent that are fertilized, most of the zygotes produced fail to establish a pregnancy after transfer to the uterus. Such early reproductive failure raises critical questions for scientists interested in both improving IVFET and understanding the normal physiology of early reproduction.

Experimental strategies have been developed that are beginning to shed light on some of these problems. One such strategy is to examine the degree of chromosomal abnormality in preovulatory oocytes in an attempt to determine whether these abnormalities contribute to either failure to fertilize or failure to develop after fertilization. There has been a variety of studies assessing the number of eggs obtained from women who had undergone ovarian stimulation, that exhibit a particular type of chromosome abnormality called aneuploidy. A normal oocyte contains 23 chromosomes after meiosis, half of the full human complement of 46 chromosomes. Aneuploid oocytes contain less or more than 23 chromosomes. Comparison between the studies is difficult because the results range from 11 to 65 percent of the oocytes being aneuploid. However, the most exhaustive studies have indicated that the best estimates are likely to be between 20 and 25 percent. The interpretation of such studies is also difficult since the oocytes examined were those that failed to be fertilized. Thus, the possibility exists that the chromosome abnormality is simply an indicator of some other developmental problem. In addition, there are no data on the percentages of normal, unstimulated oocytes which are aneuploid, so how reach of the observed abnormality is caused by hormonal stimulation is not known. When human oocytes were matured in vitro, one study found only 1.5 percent to exhibit it the abnormality. This suggests that hormonal stimulation may increase the number of aneuploid oocytes. Finally, in a study of 163 women, a very high percentage of abnormal chromosomes was found in oocytes obtained from particular individual women. The possibility exists, therefore, that some women are especially likely to produce oocytes with abnormalities.

The extent to which chromosomal abnormalities affect fertilization and pre-or early post-implantation failures is important to know. This is especially true when one considers that sometimes aneuploid eggs are fertilized and the resulting embryos develop to term. Also, findings to date suggest that a significant number of embryos produced by IVFET exhibit same type of chromosomal abnormality. One way to approach these important questions is to develop a focused, multi-center research program to analyze eggs that either failed to fertilize in vitro, or that were produced in excess of the number needed for IVFET methods.

The use of fluorescent stains for chromosomal DNA in such egg could reveal a variety of abnormalities and answer a variety of questions. Such an approach could determine more precisely the frequency of abnormalities in stimulated cycles. Various stimulation methods could be compared to find ones with the lowest rate of chromosome perturbation. In addition, associations of chromosomal abnormalities with particular patients, or with patients of certain ages, could be made.

While the analysis of oocytes and eggs is important, it is equally necessary to analyze the processes that cause fertilized eggs to develop to varying stages before finally failing to develop. It has been estimated that only 10 to 15 percent of all fertilized eggs develop past a certain stage, and these failures occur at similar frequencies despite differences in ovarian stimulation or in vitro culture conditions. Thus, failures are probably due to processes within the eggs or embryos themselves.

In contrast to the analysis of oocytes, analysis of embryos is an extremely difficult endeavor. Between fertilization and implantation, a myriad of cellular and molecular changes take place in an embryo that have to be coordinated precisely and synchronized both spatially and temporally. Research, then, must focus upon individual stages beginning at the earliest stage of fertilization.

It has been found that 5 to 10 percent of human eggs that appear to be unfertilized actually do contain one or more sperm in the region just outside the egg membrane when viewed microscopically. At higher magnifications with electron microscopy, such oocytes exhibit an absence of binding between the sperm and the egg. Usually the membranes of the sperm form protrusions, called microvilli, which seem to be important for sperm binding to an egg. Sperm associated with these seemingly unfertilized eggs lack this structural feature. Although the reasons for this failure in sperm/egg binding are unknown, a number of possible explanations are likely. For example, cell surface molecules, which function as cell recognition markers, could be absent. Another possible cause is an abnormal organization of specific proteins, called microfilaments, that form the structural framework necessary for microvilli. This possibility is attractive because eggs have been found that lack microvilli except in small restricted regions of their membranes. Since the abnormality has been observed in mature, preovulatory oocytes, it is not likely to be the result of culture conditions. Further, the lack of microvilli is also typical of oocytes from women with a history of failed fertilization by in vitro methods. It may well be necessary to analyze immature but fully grown oocytes from such women to determine if stimulation protocols affect the frequency of this problem. Other research to determine the molecular changes in the egg membrane necessary for fertilization would also be extremely helpful.

There are two other conditions associated with early failure at fertilization. In one condition, the DNA of the sperm fails to decondense. Decondensation is a necessary prior condition to the combining of the male and female chromosomes. While it is possible that there is abnormal packaging of the sperm DNA, it is more likely that there is a problem in the egg. Animal studies have suggested that chemicals in the egg cytoplasm are responsible for causing the sperm DNA to decondense. Clearly, more needs to be known about the process.

Another critical stage in fertilization that can go awry is the migration, juxtaposition, and fusion of the male and female pronuclei, each of which contains the respective chromosomal contribution to the resulting embryo. Electron microscopic analysis has shown that failure here is associated with failure of the membranes between the two pronuclei to dissolve. Therefore, the two pronuclei cannot fuse. Very preliminary data using fluorescent DNA stains have shown that, in these cases, DNA replication is incomplete or absent. It is possible that DNA replication is required for membrane dissolution. Research into this problem could proceed in two ways. First, analysis of fertilized eggs that have arrested development at this stage could be done to further knowledge about the mechanisms of such failure. Second, the mouse could provide an animal model with which to study this problem. It has already demonstrated in the mouse that chemicals, which specifically inhibit DNA replication, do not affect the formation of pronuclei, their migration, or their juxtaposition. Such chemicals do, however, prevent the dissolution of membranes between the pronuclei. These results also occur when DNA replication is prevented by other means, so probably are not simply a side effect of the inhibiting chemicals.

While this discussion has centered upon very early reproductive failures, there remain many other conditions that cause the demise of early embryos. For example, human development is sometimes arrested when the embryo reaches the four-cell stage. This may be due to a failure in the expression of embryonic genes that results in a lack of proteins required for further development. At the 12-16 cell stage, some normally fertilized eggs develop multiple nuclei. Finally, during the first mitotic division of the zygote to form two cells, a chromosome pair sometimes does not separate or disjoin. This results in different numbers of chromosomes in different cell lines of a developing embryo. These are called ''mosaics''. Research into the causes of these problems can be of benefit to the clinical practice of IVFET as well as to the understanding of human reproduction in general. While animal models, like the mouse, have great usefulness in this research, the use of developmentally arrested zygotes and embryos produced by IVFET is also necessary. Such zygotes and embryos no longer have the potential to develop into a fetus but may, nevertheless, contain information critical to a better understanding of reproductive success and failure.

Metabolic Substrates And Pathways

Metabolism encompasses the processes by which cells extract energy from their environment and synthesize the necessary building blocks to form large molecules. Thus, metabolism is an exceedingly complex network of biochemical reactions that can be viewed as intersecting sets of interactions. The sets overlap at key points that regulate which set of reactions will be more or less active, depending on the needs of the cell at the time. The regulators at these key points are enzyme proteins. In most cascades of biochemical reactions, one enzyme's activity will be more important than any other's. Such an enzyme is called a rate-limiting enzyme and is often positioned at the key points of overlap between different pathways of metabolism.

In organisms dependent upon oxygen, glucose is used as a basic fuel for metabolism. There are three linked systems that subserve glucose metabolism—glycolysis, the citric acid cycle, and the oxidative respiratory chain. In glycolysis, glucose is chemically changed in a series of enzyme-catalyzed reactions to pyruvate. In the process, molecules of adenosine triphosphate (ATP) are produced. ATP is the energy source for vast numbers of biochemical reactions. The citric acid cycle uses pyruvate, formed by glycolysis, as a substrate for another series of reactions to produce carbon dioxide and molecules (NADH and FADH) that are required for a particular type of chemical reaction also important in biological systems. Other products of the citric acid cycle are precursors for the synthesis of other molecules. For example, certain amino acids, which are the building blocks for proteins, are formed from the citric acid cycle. Finally, the NADH and FADH from the citric acid cycle are used in the oxidative respiratory chain to transfer electrons to molecular oxygen and form additional molecules of ATP.

Glucose is also necessary in two other systems that synthesize important biological chemicals. Both of these systems intersect with the glycolytic pathway. The first reaction of glycolysis is the conversion of glucose into glucose-6-phosphate (G-6-P) by the enzyme, hexokinase. The G-6-P produced can be diverted when necessary to enter the pentose-phosphate path or to be used to synthesize glycogen. In the pentose-phosphate path, G-6-P is converted to another kind of sugar by a series of reactions and, in the process, molecules of NADPH are formed. NADPH functions as a hydrogen and electron donor in biosynthethic reactions. In glycogen synthesis, the G-6-P is converted to glycogen, which is the way cells store glucose for later use. When cells need ATP, for example, glycogen can be broken down to form G-6-P, which can then continue in the glycolytic pathway. Eventually, through the citric acid cycle and oxidative respiratory chain, every G-6-P molecule leads to the production of 37 ATP molecules. For many years, glucose metabolism was not studied in eggs and early embryos, because it was generally assumed that results of studies from other cells would also apply to eggs. With the advent of tissue culture, it became apparent that eggs and early embryos required unexpected nutrients in their culture media, which seemed to violate this assumption. Specifically, it was shown that, for 2-cell mouse embryos, glucose alone would not promote survival in culture. Rather, lactate was critical for these embryos. Lactate is formed from pyruvate when there is little oxygen in the tissue. Exercised muscles, for example, produce a great deal of lactate from pyruvate. Lactate can also be converted to pyruvate, which may then be used in the citric acid cycle. Thus, the finding of a lactate requirement of these embryos suggested that certain pathways of metabolism were restricted, especially the glycolytic pathway. It was later found that maturing oocytes and embryos up to the 8-cell stage needed pyruvate and other citric acid cycle precursors to survive. While the exact requirements have been shown to differ from one animal species to another, it is interesting that pyruvate is often used in culture media for human and monkey eggs and early embryos despite the lack of experimental data to justify its use.

The nutrient studies described above are not the only evidence that metabolism is restricted in oocytes and early embryos. Morphological studies have demonstrated that the intracellular organelles in which the oxidative respiratory chain takes place are not the same in early cells and embryos of 8 or more cells. These organelles are called mitochondria and are small membrane bound packages of enzymes and other molecules. Inside each mitochondrion is a complex labyrinth of membranes called cristae. It has been known for a long time that the arrangement of the cristae differ among cell types depending on the function of the cell and the metabolic state of the cell. Studies of eggs and early embryos showed a pattern of cristae that included a concentric and transverse arrangement. However, the concentric cristae disappeared by the 8-cell stage in most species. This change in structure correlated well with an observed increase in oxygen consumption by these 8-cell embryos.

Other studies have explored which substrates were taken up by cells. These studies also showed that eggs and early embryos took up pyruvate rather than glucose until they reached the 8-cell stage. To assess whether the cells actually used the pyruvate, studies were done to measure the carbon dioxide produced by metabolism of pyruvate compared to glucose. This is done by labeling the pyruvate or glucose with radioactive tags which, if the pyruvate is metabolized to carbon dioxide, results in the formation of radioactively tagged carbon dioxide. As might be expected, most carbon dioxide was produced from breakdown of pyruvate in early (1 to 2 cell) embryos. At 8 cells, the carbon dioxide was mostly from glucose metabolism.

While the results of these studies seem clear, it is important to note some possible confounding factors in these experiments. The most important of these is that, since much of the work has been done with cells in culture, the differences between the amount of oxygen available in culture and the amount available in the natural state my have affected the results. The amount of oxygen available can, by itself, cause metabolism to be switched from one pathway to another. It is also important to determine if the enzymes responsible for glycolysis are present, and if glucose is being used in a pathway other than glycolysis. Various studies have compared the metabolic processes at play by using the strategy of starvation and refeeding of cells with the substances of interest. It was found that, if one starves embryos of glucose at any stage, the level of G-6-P decreases dramatically. Upon refeeding with glucose, the G-6-P levels increase in all stages also. This suggests that transport of glucose into cells and the first enzymatic reaction by hexokinase to form G-6-P are both normal across different stages of embryonic development. In contrast, the levels of fructose 1,6, biphosphate (an intermediate molecule formed in glycolysis) do not change with starvation of glucose at any embryonic stage. However, the levels of this intermediate increase substantially with refeeding of glucose in late embryos, but not early ones. Such data provide strong evidence that, like all cells, the rate-limiting enzyme for glycolysis (phosphofructokinase) is the one that catalyzes the formation of fructose 1,6, biphosphate. Moreover, the results suggest that this enzyme operates at a very low level in early embryos and does not reach its normal activity level until after the 8-cell stage. A similar starvation/refeeding experiment has suggested that the presence of pyruvate inhibits the formation of fructose 1,6, biphosphate from glucose and that the addition of glucose resulted in no increase in the metabolites of the citric acid cycle, whereas pyruvate did result in an increase in these metabolites at all stages of embryonic development.

There is much that should be known regarding the control of glycolysis in oocytes and early embryos. It is important to continue to investigate the rate-limiting enzymes at the intersection points between different metabolic pathways. Since the energy charge of a cell determines to a great extent the pathway chosen, it is also necessary to consider changes in the total energy charge of oocytes as they proceed through maturation and embryonic development. The energy charge can be expressed as the amount of ATP molecules available. Often, it is measured by determining the ratio of ATP to adenosine diphosphate, ADP, which is a lower energy state form. It is well known that if the energy charge of a cell is high, the pentose-phosphate and glycogen synthesis pathways are favored. This is because more energy in the form of ATP molecules is not needed. If there are not enough ATP molecules, the energy level is low and glycolysis, citric acid cycle, and oxidative respiratory chains are the favored routes. It is known that, in the mouse, zygotes and early embryos have a high energy charge that begins to fall as development proceeds. Such a finding is consistent with the evidence that these early embryos exhibit a restricted metabolism in the glycolytic and citric acid pathways. It is important to know the metabolic requirements of oocytes and early embryos because these requirements are critical to successful maintenance of these cells in culture.

Gene Expression In Early Embryonic Development

The determination of when embryonic genes begin to be expressed for the synthesis of new proteins is important in the detection of genetic diseases in embryos. Of the general population, two percent are at risk to produce children with genetically transmitted diseases. For some the risk is as high as one chance in two. With the development of IVFET techniques, it may become possible to fertilize eggs from high risk couples in vitro to allow for early diagnoses of genetic diseases. In theory, one cell from a multicelled embryo could be removed, analyzed for disease and, if found to be affected, a decision could be made not to implant that embryo. As an alternative, naturally fertilized eggs could be removed from the uterus by flushing, biopsied, and then returned via embryo transfer. For such diagnostic procedures to be realized, however, certain prerequisites are necessary.

First, there must be a realistic chance of maintaining a pregnancy after embryo transfer. Embryo biopsy may adversely affect the chance of a successful transfer. Second, the embryo should not be damaged by biopsy. In cattle, embryo biopsy is a relatively common technique and has been done with embryos at various stages. The optimum stage for biopsy is, nevertheless, important to determine. For example, techniques using 8-cell embryos involve removing the zona pellucida and removing one cell. An advantage of biopsy at this stage is that the embryo can be transferred to the mother when it is well synchronized with the state of the uterine wall. There are some disadvantages at this stage, howler. These include the fact that there is only one cell with which to work, and the fact that embryos lacking zonae can stick together during culture or transfer, and can develop into a mosaic or chimera that, at best, is undesirable and, at worst, is fatal to the embryo. In addition to these problems, research has suggested that zona-free embryos do not exhibit normal cleavage and often exhibit cells that vary greatly in size. In later embryonic stages, it is possible to nick the zona and slice off the 5 to 10 cells that herniate out the nick. This approach will likely result in a lower implantation rate, because the optimum time for implantation would be lost. However, the embryos could be frozen for later implantation. Clearly, much more needs to be known about these changes and their effects.

The third prerequisite for preimplantation diagnoses is to have reliable probes with which to identify the presence of genetic diseases. Ideally these probes would make the identification at the level of the genome or DNA, as is possible with new technologies, such as Polymerase Chain Reaction techniques. In situ hybridization is a technique whereby a strand of DNA is made that matches with the strand of DNA one is looking for. To use in situ hybridization, however, the sequence of the gene in question must be known. Some of the sequences for human genetic diseases are known, especially the sex-linked diseases. The problem, however, is that many in situ probes are linked to the Y sex chromosome. This causes an unacceptable rate of false negative readings. A procedure with greater potential is to look at the defective gene products, like hemoglobin, as an indication of genetic blood diseases, or like hypoxanthine phosphoribosyl transferase (HPRT) as an indication of Lesch-Nyhan disease. The last prerequisite is absolutely critical, and that is that the probes must assess the activity of embryonic genes not maternal genes.

Studies in the mouse have suggested that the embryo c genome turns on between the 2- and 4-cell stage. Before this stage, intracellular proteins are determined by RNA which was stored in the oocyte. Investigators at the Medical Research Council Unit of Mammalian Development in London have developed a miry for the presence of in embryos that, applied in mouse embryos, has begun to answer some of the questions about the time of appearance of embryonic gene activity. is an enzyme that is lacking in children born with the sex-linked Lesch-Nyhan disease. The hallmark of the disease is self-mutilation and such children also exhibit strange motor behavior, are often mentally retarded, and usually die by the age of 10. Using the microassay for HPRT, workers have found that the enzyme increases in concentration in 8-cell mouse embryos suggesting that the embryonic genes have become active. So, it seems fairly clear that, in the mouse, gene activity begins at this point. However, before preimplantation diagnoses can be achieved in humans, one must know the timing of human embryonic gene activity.

In Great Britain, research on human embryos is allowed under the control of the Voluntary Licensing Authority. In the Cambridge University clinic, all patients are asked to donate their excess eggs and embryos for research purposes. Of 300 patients, only two refused permission for the research, and these two were still afforded all the clinical services available. Investigators at this clinic have thus conducted a number of studies on human embryos. In a series of experiments, radioactive methionine was added to the culture media of cultured embryos at various stages of development. Methionine is taken up by cells and incorporated into newly synthesized proteins and so is a qualitative measure of protein synthesis. Researchers found new proteins that contained the radioactively tagged methionine were synthesized for the first time between at the 4- and 8-cell stage. In another experiment, when chemicals, which blocked transcription of messenger RNA, were added to the culture medium, they had no effect on unfertilized oocytes or early embryos and development proceeded normally. However, the blockade of RNA, or protein synthesis, from the 4-cell stage caused the embryos to stop developing. While the results suggested that embryonic gene activity, which directs synthesis of new proteins, is important at the 4- to 8-cell transition period, the data are complicated by the fact that embryos have a tendency in vitro to stop development spontaneously at this stage. There is, however, further support for the idea that the embryonic genes are active at this stage. Since earlier experiments had looked at the pattern of proteins present in early and late embryos and found that the pattern changed quite clearly, the protein patterns were measured with and without the blockade of messenger RNA. These results demonstrated that, when RNA synthesis was blocked, the changes in protein pattern characteristic of the 8-cell stage and beyond did not occur, but the pattern remained in the early state.

Because the objective of such research was to develop preimplantation diagnostic techniques, the presence of HPRT was also assayed from human embryos. Unlike the findings in the mouse, there was a large variation in HPRT levels in human embryos, and the massive rise in HPRT at the 8-cell stage Characteristic of the onset of gene activity in the mouse was not seen in the human. The reasons for these differences and for discrepancies between these data and that of others are not clear. It could be that HPRT is synthesized by the human embryo later than in the mouse embryo, or that the HPRT from the maternal genes is being broken down as rapidly as new HPRT is produced so that no net increase can bee seen. It is also possible that there are simply unexplained differences in storage, culture conditions, or other variables. Whatever the reasons, the application of HPRT assays to preimplantation diagnosis of Lesch-Nyhan Syndrome in the human has been disappointing. The situation does point out, however, the dangers of extrapolating from animal studies and underscores the need to conduct some research with human embryos that, otherwise, would be wasted.

Regulative Potential Of Micromanipulated Embryos

Many of the classic studies on embryonic development involved manipulation of certain parts of animal embryos and subsequent analysis of the effects of that manipulation on final development. Such manipulations included removal of certain parts (limb buds, for example, to study regenerative capacity), transplantation or exchange of parts (as in exchanging upper spinal cord regions with lower spinal cord to see if nerve outgrowth would follow the limb buds in the normal way), and grafting of parts of one embryo to another. Many new technologies allow micro-manipulation of embryos at the level of cells and, sometimes, even individual molecules.

It is clear that the developmental potential of cells gradually as development proceeds and that embryos have remarkable capabilities to readjust to disturbance. Yet little is known about what governs the point at which readjustment can no longer occur because the potential of a given embryonic region has been irreversibly determined. Further, it is expected that that point of detention will vary according to the specific region in question and according to the species involved.

It has long been thought that prior to blastulation an embryo is totipotent, meaning that no cell of the developing embryo is committed to any particular developmental fate. In other words, the cells are undifferentiated and uncommitted. However, it is becoming evident that embryos of different species, even though exhibiting the same morphological state and the same number of cells, may differ in the of determination and potential of each cell. Such findings call into question the long-held assumptions of totipotentiality. Since an embryonic cell is totipotent only if it can develop into an entire organism, experiments on embryo splitting can answer questions regarding the real potential of embryonic cells at different stages and in different species.

If a 2-cell stage embryo of a laboratory mouse is bisected, development proceeds normally. Up to the 8-cell stage, individual blastomeres can be dissected from an embryo and aggregated with embryo successfully. However, such isolated blastomeres cannot develop or organize into viable fetuses on their own. The findings in the rat are similar. In contrast, rabbit blastomeres isolated from 4- and 8-cell stage embryos have been shown to be totipotent. Other experiments have examined the effects of bisecting mouse embryos exactly in half. While 65 percent of half embryos survive from the 2-cell stage, only about 45 percent survive from later stages. The success at later stages may not mean that all the cells are totipotent; rather, it may mean that each half embryo contained the required number and type of cell to complete development.

Information on domestic species is derived mostly from experiments with sheep and cattle embryos. In sheep, isolated blastomeres have been shown to develop normally from embryos up to the 8-cell stage. However, for unknown reasons, only some blastomeres are capable of such development. One case was reported in which an 8-cell embryo was divided into four equal parts. Transfer of these split embryos into ewes resulted in the birth of four lambs. Sheep embryos, which have been halved, seem to survive well from the 2-cell to blastocyst stage. In cattle, similar success has been achieved with quartered and halved embryos from the 8-cell stage to the blast O cyst stage. Compared to the mouse, sheep and cattle exhibit blastulation at a later cleavage stage. Therefore, the number of cells is higher at blastulation for sheep and cattle. It is possible that a higher total cell number may allow for greater flexibility in embryonic adjustment to manipulations such as splitting.

Beyond questions of immediate survival of micromanipulated embryos, there are other questions that have been addressed by investigators. Some studies have assessed the size of embryos after splitting and the birth weight of resulting offspring. While one study in mice found no differences in these measurements between control and halved embryos, others have reported differences at various times of development. An examination was made of blastocyst formation following 2-cell stage embryos that had been bisected. One half of the embryo was discarded and the other was cultured, returned to a synchronized female, or placed in an immature oviduct. Those placed into culture conditions exhibited delayed development. A higher percentage of the half embryos developed normally when returned to a synchronized female or an immature oviduct. Such findings indicate that placing the split embryos in vitro compromises their development. However, it is possible that the number of degenerated half embryos were underestimated in the in vivo conditions, since it is more difficult to recover and identify degenerated embryo from the uterus or oviducts.

These studies further showed that viability was correlated with cell number such that the lower the cell number achieved in the first few days after bisection, the lower the chances of survival. Many of the half embryos failed after implantation, indicating that the bisection did not result in a failure to implant. One of the most clear suggestions from these studies is that the cell number at blastulation (which occurs at a particular time, independent of the cell number) is critical and sometimes blastulation results in an embryo with a low number of inner cell mass cells. A reduction in the inner cell mass may then cause the demise of some half embryos. This idea was tested directly by another group of studies that used differential stains to compare the number of cells in the inner cell mass to the total number of cells. Embryos with a low cell number at blastulation had a lower inner cell mass ratio and exhibited a lower viability than embryos with a higher cell number at blastulation. These experiments further showed that maintaining the half embryos in vivo, even in a nonpregnant uterus, increased the formation of the inner cell mass, resulting in a higher inner cell mass ratio and higher viability than that achieved in vitro. In a study examining the long-term effects of embryo splitting, genetically identical embryos were either bisected and immediately placed into foster mothers or were left intact and placed into foster mothers. The half embryos were generally less successful and those born had a higher rate of neonatal mortality than did the controls. However, some of the neonatal mortality might have been because the half embryos were born from smaller litters than the controls. Small litters usually result in longer pregnancies, which are also associated with higher neonatal mortality rates. Comparison of other phenotypic characteristics of the neonates born revealed no significant differences between half and whole embryos in measures such as the age at eye opening or growth curves. Thus, by birth, some type of regulation has taken place. The studies taken together argue strongly for the existence of crisis points for half embryos including blastulation and implantation. Further study of the mechanisms of regulation in micromanipulated embryos not only can add to our basic knowledge of reproductive biology, but can also elucidate important requirements for successful splitting of embryos in different species.