Volume 6, Issue S2
July 1984, pages 1-126
pp 1-6 July 1984
Hormonal requirement for ovum implantation varies among the species of animals. The methods attempting to clarify the requirement in each species may be classified as follows:
hormonal replacement therapy after removal of the pituitary or/and the ovaries
hormonal treatment after reduction of specific hormones by its antiserumin vivo
close observation of hormone secretion pattern in early pregnancy
examination of physiological conditions where implantation is delayed and analyze the hormone levels and the receptivity of the target tissues
examination of effects on hormone levels and the receptivity of target tissues of drugs which interfere with implantation. The reported results indicate that both progesterone and estrogen are needed for implantation in rats, mice, and Mongolian gerbils; in other species of animals progesterone alone may be sufficient to induce implantation, although synergistic effect of estrogen appears to be seen in some species such as in the rabbit. It remains to be determined whether the blastocysts of those animals that need only progesterone for implantation have greater ability to produce estrogen than the blastocysts of the animals that need both progesterone and estrogen. Control mechanism of secretion of progesterone and estrogen for inducing implantation may be different in various species. It has been suggested that both leutropin and follicle stimulating hormone are needed for pre-implantation estrogen secretion in the rat, whereas only follicle stimulating hormone is needed in the mouse. In the species where the obligatory delay in implantation is observed, neuroendocrine mechanisms are reported to be involved in controlling the pituitary-ovarian function that causes a delay in implantation.
pp 7-10 July 1984
Attempting to analyse the role of the ovarian hormones upon the onset, magnitude and loss of uterine receptivity/sensitivity, particular emphasis is given to uterine vascular changes. Information concerning the modulation by hormones of uterine micro-circulation appears essential for the understanding of the receptivity/sensitivity uterine changes. The generation, storage and release of vasoactive mediators and prostaglandins appear involved. As shown in the rat recently, the onset of uterine receptivity/sensitivity is temporarily correlated with the appearance of endometrial PGE binding sites under hormonal control. On the other hand catecholamines may also modulate the uterine vascular functions. Endometrial monoamine oxidase and catecholO-methyltransferase two enzymatic activities involved in catecholamine deactivation show hormone dependant changes parallel to the manifestation of uterine receptivity/sensitivity. The precise role of these phenomena is discussed.
pp 11-21 July 1984
In response to the ovarian secretion of progesterone and estrogen during early pregnancy, the mammalian uterus develops the capacity to perform complex cellular activities which occur before and after blastocyst implantation. Luminal epithelial cells participate in regulation of the metabolism of the blastocyst through the control of its humoral environment, provide an appropriate matrix for changes to occur at the interface between trophoblast and epithelium, and appear to transmit information from the blastocyst to the underlying stroma to initiate decidualization. With the completion of these functions during implantation in rodents, the epithelial cells self-destruct and are removed by phagocytic activity of the trophoblast. Control of both the endocytotic and secretory activity of luminal epithelial cells and their eventual self-destruction would require regulation of the Golgi-endoplasmic reticulum-lysosomes system within these cells. Progesterone secretion during early pseudo-pregnancy increases levels of cathepsin D, a lysosomal proteinase, in luminal epithelial cells by increasing the rate of enzyme synthesis. Progesterone pretreatment of ovariectomized rats followed by estradiol treatment results in the development of uterine sensitivity to deciduogenic stimuli. The number of proteins which are synthesized by luminal epithelial cells in response to estradiol to achieve this sensitivity has been determined. Epithelial cytosol proteins from rats treated with medroxyprogesterone acetate (3.5 mg sc) or medroxyprogesterone acetate plus estradiol (200 ng sc) were separated by two dimensional polyacrylamide gel electrophoresis. The synthesis of two proteins increased after 8 h of estradiol treatment and the synthesis of another three was increased by 12 h. The increased synthesis of these proteins could be related to changes in the capacity of the luminal epithelial cell for prostaglandin synthesis. The epithelial capacity for prostaglandin synthesis increases during pseudopregnancy to maximum levels at the time of maximum sensitivity to deciduogenic stimuli. Epithelial prostaglandin synthetic capacity may also depend upon the accumulation of prostaglandin precursors within these cells. Estradiol treatment of medroxyprogesterone acetate pretreated ovariectomized rats increased the arachidonic acid content and composition of epithelial phosphatidyl choline but the increases were not statistically significant. These changes in protein and lipid synthesis controlled by progesterone and estrogen would appear to contribute to the cellular activities of the luminal epithelium during early pregnancy.
pp 23-31 July 1984
An interaction between the blastocyst and the uterus is essential for establishment of pregnancy. Because maternal estrogen is not an absolute requirement, estrogen of embryonic origin has been implicated in this process in the pig and the rabbit. Furthermore, estrogen forming capacity has been documented in the blastocyst of these species. However, while the complete machinery for steroid synthesis in the pig balstocyst has been demonstrated, the issue is still unresolved for the rabbit blastocyst. In the present communication we have shown that 17α-hydroxylase and C17–20-lyase, enzymes involved in the formation of androgens (C19-steroids) from C21-steroids (progestins), are present in day-6 rabbit blastocysts. C17–20-lyase activity was undetectable to low in day-5 and increased in day-6 balstocysts. The activity was further increased in day-6 blastocysts cultured for 24 h. Because prostaglandins have been implicated in uterine vascular changes at about the time of implantation and pregnancy establishment, and because catechol estrogens are more potent than phenolic estrogens in stimulating prostaglandin synthesis in the blastocyst and the uterus, we determined catechol estrogen forming capacity in the rabbit and pig blastocyst. Catechol estrogen forming capacity (estrogen-2/4-hydroxylase) in the pig blastocyst appears on day 10 of pregnancy, peaks on day 12 and then declines. Our preliminary experiments also indicate that day-6 rabbit blastocysts have catechol estrogen forming capacity. On the basis of our present findings and of others, we propose that catechol estrogens of embryonic origin mediate the stimulatory effect of estrogens on prostaglandin synthesis in the embryo and/or the uterus and thus participate in the process of establishment of pregnancy.
pp 33-42 July 1984
The ability of preimplantation mouse embryos to utilize glucose oxidatively is controlled, in part at least, at the level of glycolysis. Various experimental observations are reviewed that indicate the regulatory mechanism in delayed implanting blastocysts involves the classic negative allosteric feedback of high levels of ATP on phosphofructokinase while the situation in 2-cell embryos appears to be more complicated. That is, in addition to the usual negative effect of ATP and citrate on phosphofructokinase, there appears to be a modification of hexokinase that prevents phosphorylation of adequate amounts of glucose and results in low levels of fructose-6-phosphate at the 2-cell stage and consequently there is a failure to release the inhibition of phosphofructokinase even if ATP and citrate levels decrease. Although both types of embryos have limited glycolytic activity, they do have adequate capacity for citric acid cycle activity and oxidative phosphorylation, and are able to maintain a high ATP : ADP. It is argued, therefore, that the reduced levels of macromolecular synthesis characteristic of 2-cell and delayed implanting blastocysts are not due to restricted energy substrates or regulatory controls on glycolysis and a subsequent low energy state. On the contrary, it seems that the reduction in oxidative utilization of glucose in these situations is a result of diminished energy demand because of the low level of synthetic activity. The potential significance of this relationship between energy production and utilization in terms of potential regulatory mechanisms in preimplantation embryos is discussed.
pp 43-52 July 1984
Mouse blastocyst attaches on the antimesometrial side of the uterus through mural trophoblasts. Later the polar trophoblasts begin proliferation, and rapid multiplication towards the mesometrial side of the uterus occurs resulting in the formation of an excrescence designated as ectoplacental cone. The morphogenesis of ectoplacental cone, viewedin utero, initiates on day 6post-coitum when microvilli of the trophoblast and the uterine epithelial cells are lost and as a result of this opposing membranes appear interlocked with each other. Soon following the invasion by surrounding trophoblasts the necrosis of the epithelial cells starts. Mitochondriae of the epithelial cells, at this stage, are shrunken and lack well defined cristae. Several leucocytes are seen at the site and few electron dense structures appear wedged between the trophoblasts and epithelial cells. At places the cell membrane is studded with the basement membrane of the uterine epithelium giving an impression of a bristle coated membrane. By day 7post-coitum the basement membrane has almost disappeared leaving trophoblast cells to develop close contact with stromal cells. Collagen fibres appeared between the trophoblasts and the stromal cells, many large inclusions of high electron density representing engulfed necrotic epithelial cells are discernible. On day 8post-coitum the ectoplacental cone is fully developed. Four types of trophoblast cells can be identified in it: (i) basal cells lying on the base of the cone, are polyhedral and compactly arranged. They have a large nucleus and well developed nucleoli, (ii) central cells forming the middle area of the cone are of two types; one contained several osmiophilic granules enclosing translucent area (eccentric) and a well developed golgi complex around the nucleus, while the other has many heterophagosomes, vacuoles and residual bodies and (iii) peripheral cells contained several pleomorphic structures resembling secondary lysosomes. Minute dense granules and band of microfibrils on the apical region of these cells are seen. Dense granules probably release lytic proteins at the site and microfibrils help in forming cytoplasmic projections.
pp 53-61 July 1984
Examination of plastic-embedded rhesus monkey and baboon blastocysts through the implantation period has provided information on normal differentiation and development. The blastocysts show many features in common with non-primate laboratory animals, including differentiation of endoderm and its extension beyond the inner cell mass prior to implantation. However, there appears to be more cell death, and more aberrations in development. Implantation involves the adherence of trophoblast to healthy uterine luminal epithelial cells, and intrusion of syncytial trophoblast between these cells, followed by lateral expansion of the site of invasion prior to penetration of the uterine epithelial basal lamina. An amnionic cavity is formed within the inner cell mass, and is preceded by establishment of cell polarity. The definitive yolk sac is formed by an aggregation of endodermal cells adjacent to the inner cell mass. The trophoblast does not give rise to mesodermal cells, but some of these cells may be formed from endoderm prior to primitive streak formation. In both rhesus monkey and baboon, syncytial trophoblast taps the maternal vascular system relatively rapidly. In the baboon in particular large blood-filled spaces elevate the implantation site from the level of the endometrium at the stage of primary and secondary villus formation.
pp 63-74 July 1984
In pigs, the blastocyst begins to elongate from a sphere to a long filamentous thread around day 10.5 of pregnancy. At about this time the endometrium secretes large quantities of protein into the uterine lumen. The synthesis of this material which is believed to be required for nutritional support of the conceptus is under the control of progesterone. The release of secretory protein appears to be triggered by the production of estrogens by the elongating blastocyst. Blastocyst estrogens are also involved in the phenomenon of maternal recognition of pregnancy in swine, and their interaction with the maternal system, by a mechanism as yet unknown, prevents a return to reproductive cyclicity. Maternal recognition of pregnancy in the sheep and cow occurs at around the time of blastocyst elongation. Here estrogens do not appear to be involved, and protein products secreted by the conceptus have been implicated. One product of the sheep, ovine trophoblast protein-1, which is produced only during a brief period (days 13–21) of pregnancy, has been purified. It appears to be a hormone whose target tissue is the uterine endometrium.
pp 75-82 July 1984
The effect of super-ovulatory dose of pregnant mare serum gonadotropin and human chorionic gonadotropin on ovulation, advancement of ovulation, subsequent embryo development and implantation were studied in the hamster. Groups of hamsters received pregnant mare serum gonadotropin injection on day 1 of the estrous cycle followed by human chorionic gonadotropin injection either at 56 or 76 h later, pregnant mare serum gonadotropin alone on day 1 or human chorionic gonadotropin alone on day 3.
The combination therapy (pregnant mare serum gonadotropin and human chorionic gonadotropin) resulted in super-ovulation (an average of 40 mature ova/animal) while human chorionic gonadotropin alone yielded an average of 10 mature ova/animal. Ovulation was advanced by 24 h by giving human chorionic gonadotropin at 56 h instead of 76 h after pregnant mare serum gonadotropin. Subsequent embryo development and implantation occurring under different hormonal regimens were studied. The ova obtained by giving human chorionic gonadotropin injection at 56 h were poorly fertilizablein vivo and hence the pregnancy rate was low (6 %). These ova however, were fertilizablein vitro, suggesting that the low fertilization rate and developmental failure may be due to inhibition of sperm capacitation/transport because of premature human chorionic gonadotropin administration. In the group receiving human chorionic gonadotropin alone on day 3 there was fertilization and cleavage, but no implantation occurred due to failure of functional corpora lutea. However, administration of progesterone and estrone from day 2 of gestation resulted in 80% implantation and sustenance of pregnancy. On the other hand, the pregnant mare serum gonadotropin and human chorionic gonadotropin combination therapy resulted in super-pregnancy. The number of fetuses present at term was higher in the group receiving pregnant mare serum gonadotropin alone than in the group receiving the combination therapy. Embryo resorption however was higher (37%) in the latter group compared with the former (9.5%). However, preimplantation embryos were found to be viable as evidenced by fluorescein diacetate staining.
pp 83-91 July 1984
Nonhuman primates represent a strong model for examining the chromosomal, biochemical, and temporal normality of embryos produced byin vitro fertilization. Morein vitro fertilized embryos from the squirrel monkey(Saimiri sciureus) have been produced and examined than with all other primate species combined. In studies over a 13 year period a fertilization rate approximating 60 % has been developed in this species with 30% of these embryos proceeding to the two cell stage and 50% of these to the three-four cell stage. Chromosomal abnormalities (primarily missing or extra chromosomes) at a level of nine to 16% have been found, a value corresponding to that found inin vivo mating andin vitro fertilization in other species. An incidence of triploidy of 16.7% was observed. RNA and protein synthetic rates appear comparable with those of laboratory species subjected toin vitro fertilization and indicate the initial stages of metabolic activity of the newly formed embryo. Similarly, increases in estrogen incorporation appear after fertilization but no effect is observed in progesterone incorporation. Utilizing 2-deoxy-glucose and insulin, it was determined that the glucose requirement as an energy source for early preimplantationin vitro fertilized primate embryos is very low.
Of very great importance is the temporal relationship of the development ofin vitro fertilized squirrel monkey embryos compared with similar development in other primates (including humans) afterin vivo andin vitro fertilization. An analysis of over a decade of work with the squirrel monkey embryos demonstrates a pattern of temporal development that is comparable with all other primate species that have been examined (including the human) and comparable with development afterin vivo fertilization.
pp 93-96 July 1984
An attempt has been made in this paper to review our present understanding of luteal function during the periimplantation period and in particular hormonal requirement for implantation and maintenance of early pregnancy in the non-human primate.
In a fertile cycle thecorpus luteum is apparently rescued from luteolysis by chorionic gonadotropin secreted by the implanted blastocyst, In the bonnet monkey the serum progesterone titers during the luteal phase of a fertile cycle seems higher compared to that of nonmated cycling monkeys. This suggested that thecorpus luteum is receiving some stimulatory signal from the blastocyst even prior to implantation. The recent demonstration that human blastocyst in culture secretes into the medium human chorionic gonadotropin essentially support the above assumption. However, attempts to extend the luteal phase of cycling unmated monkeys with exogenous human chorionic gonadotropin injection has hitherto not met with complete success suggesting that there could be other than chorionic gonadotropin, additional luteal stimulatory factors the unimplanted blastocyst is secreting.
Corpus luteum is the principle source of both progesterone and estrogen produced during the periimplantation period and dysruption of luteal function, brought about by either lutectomy or ovariectomy or luteinizing hormone antiserum treatment, followed by progesterone supplementation leads to maintenance of pregnancy. This has lead to questioning the need for estrogen in the maintenance of early pregnancy. Recent work using Zuclomiphene, an antiestrogen during days 5–11 of cycle in rhesus monkeys mated between day 9–14, has however, suggested that estrogen may be required for implantation. Further work is needed to arrive at an unequivocal decision regarding the need for estrogen in maintenance of early pregnancy in the primate.
pp 97-106 July 1984
The regulation of secretion of chorionic gonadotropin in primates has been studied using bothin vivo andin vitro models.In vivo studies using the pregnant bonnet monkey revealed that at the doses tested, the administration of progesterone or estradiol 17Β in combination or alone did not result in any appreciable change in the duration or magnitude of serum chorionic gonadotropin levels. However, administration of lutropin-releasing hormone by intravenous route resulted in significant increase in chorionic gonadotropin levels within 30–60 min and the extent of stimulation seemed to depend on the state of pregnancy. Forin vitro studies, explants or cells prepared from first trimester human placenta has been used. The functional integrity of these cells has been established by demonstrating the binding of [125I]-labelled human chorionic gonadotropin antibody to the cells as well as the synthesis of [3H]-labelled human chorionic gonadotropin.In vitro studies using the cells revealed that addition of lutropin-releasing hormone caused a significant increase in chorionic gonadotropin and estradiol 17Β secreted into the medium. Thus bothin vivo andin vitro results suggest that lutropin-releasing hormone could be one of the factors involved in regulation of chorionic gonadotropin secretion in primates.
pp 107-119 July 1984
The importance of developing of drugs which could be taken post-coitally or used once-a-month in the case of a delay in the onset of the menses is well recognized. The availability of such technology would limit exposure to fertility regulating agents to such occasions where there is coital exposure or possibility of pregnancy.
Methods of post-coital contraception used so far include IUD’s inserted post-coitally, estrogens, and combinations of estrogens and gestagens. These are reserved primarily for emergency situations to protect women from unwanted pregnancy resulting from rape or unprotected coitus. Levonorgestrel has shown satisfactory results in terms of contraceptive efficacy and is being further evaluated clinically. A number of problems inherent in the development of post-coital contraception are discussed.
Menstrual regulation could be achieved by a number of approaches: (a) block progesterone receptors and interfere with the preparation of the endometrium for implantation; (b) luteolysis leading to decreased progesterone levels and interruption of implantation; and (c) termination of early pregnancy by prostaglandins. A number of progesterone antagonists have been evaluated. One of the compounds, RU38486 is being evaluated clinically for termination of very early pregnancy.
Deglycosylated derivatives of human chorionic gonadotropin have been shown to antagonize the action of human chorionic gonadotropin and interfere with established pregnancy in rats. Appropriate methods of delivery, immunogenicity and alternate methods for production of human chorionic gonadotropin need to be considered before evaluation of the derivatives for clinical use.
In vitro and invivo models need to be developed for evaluation of the teratogenicity and embryotoxicity of post-coital and menses inducing agents.
There are a number of gaps in the knowledge of the processes regulating implantation which should be investigated in rodents and in different non-human primate species.
pp 121-126 July 1984