Peri-implantation Stage Research Articles

A recessive deletion in the GlcNAc-1-phosphotransferase gene results in peri-implantation embryonic lethality.

Formation of the dolichol oligosaccharide precursor is essential for the production of asparagine- (N-) linked oligosaccharides (N-glycans) in eukaryotic cells. The first step in precursor biosynthesis requires the enzyme UDP-GlcNAc: dolichol phosphate N-acetylglucosamine-1-phosphate transferase (GPT). Without GPT activity, subsequent steps necessary in constructing the oligosaccharide precursor cannot occur. Inhibition of this biosynthetic step using tunicamycin, a GlcNAc analog, produces a deficiency in N-glycosylation in cell lines and embryonic lethality during preimplantation development in vitro, suggesting that N-glycan formation is essential in early embryogenesis. In exploring structure-function relationships among N-glycans, and since tunicamycin has various reported biochemical activities; we have generated a germline deletion in the mouse GPT gene. GPT mutant embryos were analyzed and the phenotypes obtained were compared with previous studies using tunicamycin. We find that embryos homozygous for a deletion in the GPT gene complete preimplantation development and also implant in the uterine epithelium, but die shortly thereafter between days 4-5 postfertilization with cell degeneration apparent among both embryonic and extraembryonic cell types. Of cells derived from these early embryos, neither trophoblast nor embryonic endodermal lineages are able to survive in culture in vitro. These results indicate that GPT function is essential in early embryogenesis and suggest that N-glycosylation is needed for the viability of cells comprising the peri-implantation stage embryo.

Effect of early luteal phase administration of mifepristone (RU486) on leukaemia inhibitory factor, transforming growth factor beta and vascular endothelial growth factor in the implantation stage endometrium of the rhesus monkey

A single, low-dose administration of a potent antiprogesterone such as mifepristone (RU486) in the early luteal phase results in inhibition of blastocyst implantation in primates. The aim of the present study was to examine the status of leukaemia inhibitory factor (LIF), transforming growth factor beta (TGF beta) and vascular endothelial growth factor (VEGF) in day 6 gestational endometrium of rhesus monkeys with or without exposure to a single dose (2 mg/kg body weight, s.c.) of mifepristone on day 2 after ovulation. Densitometric analyses of immunoblots of endometrial spent media revealed an increase (P < 0.01) in TGF beta pan (TGF beta 1, 2, 3 and 5) and a decrease (P < 0.01) in VEGF secretion from RU486-exposed endometrial samples compared with control samples. Secretory profiles for LIF, TGF beta 1 and TGF beta 1 LAP (latency associated peptide) remained unchanged in the two treatment groups. Morphometric analyses of immunohistochemical staining showed altered cell-specific distribution. TGF beta 1 (P < 0.01) and TGF beta pan (P < 0.02) were higher, while VEGF declined (P < 0.05) in endometrial glands of RU486-exposed endometria compared with control tissue samples. Stromal cell staining patterns for all experimental cytokines studied remained unchanged. In blood vessels, VEGF was found to be low (P < 0.05), while LIF (P < 0.05) and TGF beta 1 (P < 0.01) were higher in mifepristone-exposed endometrial samples compared with control tissue samples. Increased TGF beta secretion together with elevated levels of TGF beta in glandular epithelia and in blood vessels with no apparent change in stromal levels of TGF beta or in levels of TGF beta LAP in any endometrial compartment in the two treatment groups suggest an altered paracrine involvement of this cytokine and an enhanced activation of latent TGF beta in endometrium following mifepristone treatment. Higher levels of TGF beta in gland cells may result in dysregulated growth control and degenerative morphology. Also, higher levels of LIF and TGF beta together with lower levels of VEGF in the vascular compartment in mifepristone-exposed endometrium suggest that endometrial vascular physiology is a target of this anti-progestin during the peri-implantation stage. It is thus plausible that LIF, TGF beta and VEGF in the glandular and vascular compartments of implantation stage endometrium play important roles in rendering the endometrium receptive, and that early luteal phase treatment with an anti-progestin such as mifepristone affects the involvement of these cytokines resulting in endometrial contraception.

Chromosome studies in human sperm nuclei using fluorescence in-situ hybridization (FISH).

The use of chromosome specific DNA probes labelled with fluorochromes and especially the combination of several probes has been used to indirectly study the chromosome constitution of decondensed sperm nuclei by fluorescence in-situ hybridization (FISH), and has allowed to include this test in the protocol of study of infertile males. Still, if the test is to be valid, several strict conditions must be met, and some specific characteristics have to be taken into account. This becomes evident when comparing earlier results with more recent ones. The basic technical factors to be taken into account are the methods of chromatin decondensation, the number of spermatozoa and of individuals to study, the use of internal controls, the scoring criteria, the specificity of the probes and the possible existence of polymorphisms that may interfere with the detection of fluorescent signals. In the last 7 or 8 years, a large number of papers has been published, describing the incidence of aneuploidies in controls, in individuals in whom a tendency to non-disjunction was suspected and in infertile males. Studies in controls have shown a considerable intra- and inter-individual variability in the frequency of aneuploidies, the tendency of some chromosomes to undergo non-disjunction (chromosome 21 and the sex chromosomes) and the importance of alpha-satellite polymorphisms when using centromere probes. In the control population, the frequency of aneuploidy per haploid set has been estimated at approximately 6%. The incidence of aneuploidies in sperm nuclei for some of the chromosomes more frequently involved in trisomies is considerably higher than the incidence of these trisomies established through epidemiological data using the global incidence of chromosome abnormalities during the peri-implantation stage. In infertile males and in males with sex-chromosome abnormalities (usually with very low numbers of spermatozoa) the results show an increased incidence of sex chromosome aneuploidies and diploid (multi-aneuploid?) sperm nuclei. The results could be related to the higher incidence of chromosome abnormalities (especially sex-chromosome aneuploidies) observed in children conceived by intracytoplasmic sperm injection (ICSI).

A programmed oxyradical burst causes hatching of mouse blastocysts.

The emergence of the mammalian blastocysts from their thick glycoprotein investment known as the zona pellucida is an important, but poorly understood, event in embryogenesis. In this paper, we demonstrate that peri-hatching blastocysts generate a considerably high quantum of an active oxyradical species for an extremely short period of time when compared to the pre-hatching (unhatched) and post-hatching (hatched) blastocysts. Hatching could be induced in pre-hatching blastocysts by exposing them to superoxide artificially generated to match the observed peri-implantation stage specific levels of superoxide, without impairing their viability. These observations suggest the operation of a superoxide-dependent hatching initiation in developing mammalian embryos.

Maternal and paternal genomes function independently in mouse ova in establishing expression of the imprinted genes Snrpn and Igf2r: no evidence for allelic trans-sensing and counting mechanisms.

It has often been suggested that the parental-specific expression of mammalian imprinted genes might be dependent on maternal-paternal intergenomic or interallelic interactions. Using quantitative allele-specific RT-PCR single nucleotide primer extension assays developed for two imprinted genes, Snrpn and Igf2r, we demonstrate: (i) No role for maternal-paternal allelic interactions: the modes of parental-specific expression of Snrpn and Igf2r in normal ova were unchanged in gynogenetic and androgenetic ova; the latter contain two maternal and two paternal genomes respectively, and cannot undergo maternal-paternal interactions. (ii) No role for allelic counting or exclusion mechanisms: in individual blastomeres of androgenetic ova, both paternal Snrpn alleles were active (Snrpn was not expressed in gynogenetic ova), and in individual gynogenetic and androgenetic blastomeres, both maternal and paternal Igf2r alleles, respectively, were active. (iii) No role for ploidy: the mode of parental-specific expression of Snrpn and Igf2r in normal diploid ova was unchanged in individual blastomeres of triploid and tetraploid ova. Thus, the maternal and paternal genomes function independently in establishing the pre-implantation mode of parental-specific expression of Snrpn and Igf2r, with no role for trans-allelic/genomic interaction phenomena. In addition, the results show that inactive and biallelic modes of expression of imprinted genes are potential mechanisms for the death of gynogenones and androgenones at the peri-implantation stage.

Cloning and expression analyses of mouse dystroglycan gene: specific expression in maternal decidua at the peri-implantation stage.

While constructing a catalog of mouse cDNAs which are expressed in the maternal-fetal interface during the peri-implantation period, we encountered a 1.6 kb cDNA clone showing a strong sequence similarity to the 3' untranslated region of the human dystroglycan gene. We cloned an additional 1.7 kb cDNA by reverse transcriptase-PCR (RT-PCR) and confirmed that this is a true mouse homolog of human dystroglycan cDNA by sequence analyses, Southern blotting, and genetic mapping of this gene on the distal region of mouse chromosome 9. Although it is well established that dystroglycan, a transmembrane protein, plays an important role in muscle tissues by bridging intracellular dystrophin to the laminin in the extracellular matrix, its role in non-muscle tissues remains elusive. To further investigate the role of the dystroglycan gene at the peri-implantation stage, we analyzed the expression patterns of this gene by in situ hybridization, which revealed that this gene is specifically expressed in decidual cells, especially in the cells surrounding the implantation site at 6.5, 7.5, and 8.5 day post conception (p.c.) stages, but not expressed in non-pregnant endometrial cells of uterus nor in the decidua at 12.5 day p.c. Further analyses by RT-PCR confirmed that the amount of dystroglycan mRNA in 8.5 day p.c. decidua was indeed 100-fold higher than that of non-pregnant uterus and 12.5 day p.c. mature placenta. These results suggest that dystroglycan may work as a mediator for adhesion between decidual cells themselves or between decidual cells and trophoblast cells, and provide a structural and functional support for maintaining pregnancy at its early stage.

Effect of single-dose, early luteal phase administration of mifepristone (RU486) on implantation stage endometrium in the rhesus monkey.

The characteristics of implantation stage endometrium following a single-dose, early luteal phase application of mifepristone (RU486) in proven conception cycles has been examined in the rhesus monkey in an attempt to understand the physiological basis of the anti-implantation activity of the drug. Endometrial samples were collected from monkeys subjected to vehicle (group 1, n = 14) and RU486 (2 mg/kg body weight; group 2, n = 12) on day 2 after the presumed day of ovulation of successfully mated cycles. The average diameter of glands (P < 0.05), number of vacuolated cells (P < 0.01), number of supranuclear vacuolated cells (P < 0.05) in glandular epithelium and amount of glandular secretion (P < 0.05) were significantly lower in RU486-treated endometrium compared with control tissue samples. Additionally, 18% of glandular epithelial cells showed apoptotic and degenerative features in RU486-treated tissue samples. These data, together with the observed significant decreases in precipitate area (P < 0.02) and in the optical absorbance of alkaline phosphatase reaction end-product (P < 0.05), confirm that retardation in glandular differentiation in the upper functionalis is a likely target of antiprogestin action in implantation stage endometrium. An increased frequency of mitosis in stromal cells (P < 0.05) and a greater degree of extravasation (P < 0.05) were also observed after RU486 exposure. Despite an apparent indication of constriction and regression in few RU486-exposed endometria compared with controls, morphometric analyses did not show any changes in capillary structure. Whether endometrial vasculature in progesterone-exposed uterus is a target of antiprogestin action during the peri-implantation stage remains to be determined. Further studies are required to explain the observed increase (P < 0.02) in the area of precipitate of von Willebrand (vW) factor with no change in vW factor-positive vessels, and the apparent increase in collagen IV immunostain in subepithelial and perivascular basement membrane in implantation stage endometrium after early luteal phase RU486 treatment in monkeys.

5 Contraceptive vaccines

The basic principle of a contraceptive (or anti-fertility) vaccine is to use the body's own immune defence mechanisms to provide protection against an unplanned pregnancy. Factors such as: economic production, convenience of use, relatively long-lasting but reversible action, low failure rate, and the avoidance of mechanical devices or exogenous hormones make this approach a potentially attractive option for family planning programmes in both developing and developed countries. The major efforts in research and development have involved the prospect of active immunization against specific antigens of sperm, ovum, zygote and early embryo, and the pregnancy hormone hCG. Several anti-hCG vaccines have entered clinical trials and show promising results. These vaccines operate by preventing or interrupting pregnancy at the peri-implantation stage probably by neutralizing the luteotrophic action of hCG. The most refined vaccine is one directed against the unique C-terminal peptide on the beta-subunit of hCG. This vaccine provokes antibodies that are specific for hCG and do not cross react with hLH. Future research efforts aim to optimize the anti hCG approach, utilize new vaccine delivery systems, and broaden the spectrum of target antigens of potential utility for contraceptive vaccines.

Embryotoxicity as a marker for recurrent pregnancy loss.

Some cases of unexplained recurrent pregnancy loss may be due to embryotoxic factors present in the mother's serum. In vitro assays have been devised to study the effect of these factors on pre-implantation, peri-implantation, and somite stage rodent embryos. Embryotoxic activity has been found in a high percentage of recurrent aborters but is also present in women with endometriosis and unexplained infertility, suggesting that there may be a range of factors acting at different stages of development. Embryotoxic activity may be predictive of pregnancy outcome and has been shown to be decreased after hormone and immunotherapy. However, its true value can only be assessed when the nature of the toxic factors is understood. Preliminary characterisation studies suggest that toxic activity may due to both immunological (antibodies or cytokines) and nutritional factors.

Interference with uterine blood flow by clomiphene citrate in women with unexplained infertility

To investigate the effect of clomiphene citrate on the uterine blood flow in women with unexplained infertility. Twenty-one women who had normal uterine blood flow (pulsatility index [PI] less than 3 from the LH peak to the middle luteal phase of a natural ovulatory cycle) were enrolled in this study. Patients were given clomiphene citrate (100 mg/day) from days 5-9 of the next ovulatory cycle. The PI values measured in the natural and clomiphene citrate-treated cycles were compared using repeated measures analysis of variance. Their correlations with the estradiol (E2) and progesterone concentrations in the treatment cycle were calculated using the Pearson correlation coefficient. In the natural cycles of those with normal uterine blood flow, the highest impedance of uterine blood flow was detected at the late follicular phase, then blood flow increased during the luteal phase. In the clomiphene citrate-treated cycles, a statistically significant decrease in uterine blood flow also occurred during the early luteal phase (P < .05). No significant correlation of E2 or progesterone serum concentrations with PI values of the uterine artery could be found in either natural or clomiphene citrate-treated cycles. The use of clomiphene citrate decreases the uterine blood flow during the early luteal phase, a periimplantation stage.

Pentoxifylline improves sperm capacitation and in vitro fertilization of oocytes in the golden hamster

Pentoxifylline (PF) is used to improve motility of spermatozoa from subfertile or nonfertile males to accomplish in vitro fertilization in humans. The possible adverse effect of PF on pre- and peri-implantation stage embryo development in a suitable rodent model, such as the golden hamster, is yet to be determined. In this study, hamster cauda epididymal spermatozoa were exposed to different concentrations (0.23 to 3.6 mM) of PF, and their quantitative [percentage of motility] and qualitative [Score 0 to 5] motility were assessed and values expressed as the sperm motility index. Upon addition of spermatozoa to dishes containing PF, an immediate increase in sperm motility and sperm motility index was evident, which increased up to 4 to 6 h and then declined. The sperm motility index increase by PF was dose-dependant, and ≥ 1.8 mM PF was detrimental after 4 h. The optimum dose of PF was found to be 0.45 mM. To assess the fertilizing ability of PF-treated spermatozoa, in vitro fertilization was carried out. Fertilization rates for spermatozoa treated with 3.6 mM PF were lower (53.8 ± 7.8) than for the controls (69.5 ± 10.2), whereas treatment with 0.45 mM PF increased the rates (91.6 ± 4.3) compared with that of the controls (80.2 ± 5.9). In conclusion, low concentrations (0.23 to 0.45 mM) of PF improve sperm capacitation and fertilization of oocytes in vitro in the golden hamster.

Stem cell defects in parthenogenetic peri-implantation embryos.

Mouse embryos containing only maternal chromosomes (parthenotes) develop abnormally in vivo, usually failing at the peri-implantation stage. We have analyzed the development of parthenote embryos by using an inner cell mass (ICM) outgrowth assay that mimics peri-implantation development. ICMs from normal embryos maintained undifferentiated stem cells positive for stage-specific embryonic antigen-1 and Rex-1 while differentiating into a variety of cell types, including visceral endoderm-like cells and parietal endoderm cells. In contrast, ICMs from parthenotes failed to maintain undifferentiated stem cells and differentiated almost exclusively into parietal endoderm. This suggests that parthenote ICMs have a defect that leads to differentiation, rather than maintenance, of the stem cells, and a defect that leads to a parietal endoderm fate for the stem cells. To test the hypothesis that the ICM population is not maintained owing to a lack of proliferation of the stem cells, we investigated whether mitogenic agents were able to maintain the ICM population in parthenotes. When parthenote blastocysts were supplied with the insulin-like growth factor-1 receptor (Igf-1r) and insulin-like growth factor-2 (Igf-2), two genes not detectable in parthenote blastocysts by in situ hybridization, the ICM population was maintained. Similarly, culture of parthenote blastocysts in medium conditioned by embryonic fibroblasts and supplemented with the maternal factor leukemia inhibitory factor maintained the ICM population. However, once this growth factor-rich medium was removed, the parthenote ICM cells still differentiated predominantly into parietal endoderm.(ABSTRACT TRUNCATED AT 250 WORDS)

Transition from lacunar to villous stage of implantation in the macaque, including establishment of the trophoblastic shell.

Adhesion of trophoblast of the blastocyst to and penetration into the uterine epithelium, invasion into the maternal vessels and endometrial stroma, and establishment of the basic organization of the placenta all occur within the first week following the initiation of implantation in the human, macaques and several other primates. The cellular rearrangements and interactions of trophoblast with endometrial epithelial cells and stroma were studied during this preiimplantation stage in macaques. At the early lacunar stage, 1-2 days after the initiation of implantation, both cytotrophoblast and syncytial trophoblast can be found at the maternal surface of lacunae. As the lacunar stage advances, both syncytial trophoblast and cytotrophoblast are found throughout the implantation site including in septae partitioning lacunae, but syncytial trophoblast lines most of the lacunae and forms the confluence with the maternal vessels. Indentations of fetal mesenchyme and accumulation of cytotrophoblast cells within the septae occur rapidly. Over a period of about 2 days, clusters of cytotrophoblast cells pass beyond the syncytial trophoblast at the maternal surface as the anchoring villi, establishing the trophoblastic shell. The bypassing of epithelial plaque cells by cytotrophoblast cells and elimination of stromal matrix between such clusters verifies that this is a progressive invasion rather than simply superficial growth. Concomitant with establishment of the trophoblastic shell there is extravasation of blood, necrosis of plaque cells, and massive invasion of vessels by cytotrophoblast resulting in a necrotic zone between the trophoblastic shell and endometrium. It is concluded that only in the brief period of time of establishment of the trophoblastic shell is the full invasive potential of cytotrophoblast realized and that only at this stage does cytotrophoblast demonstrate the type of invasive and migratory behavior which has been achieved with isolated human cytotrophoblast cells in vitro.

Attachment, outgrowth, invasion and formation of the egg cylinder in mouse half embryos in vitro.

To investigate the effect of the decrease in the number of embryonic cells on the development and morphogenesis of mouse embryos at the peri-implantation stage, half embryos which developed from bisected morulae were morphologically classified into eu-blastocysts (eu-blasts), pseudo-blastocysts (pseudo-blasts), trophectodermal vesicles (TVs) and non-integrated forms (NIFs) and then cultured on plastic substratum, uterine-epithelial-cell monolayer and type I collagen gel. When half embryos were cultured on plastic substratum and cell monolayer, the rates of attachment and trophoblast outgrowth in the eu- and pseudo-blasts were not significantly different from those of the controls. The TVs and NIFs showed significantly lower rates of outgrowth than the controls (P < 0.01). When half embryos were cultured on type I collagen gel, no significant difference was observed in the rate of primitive endoderm formation between the eu-blasts and controls after 36 hr of culture. In the eu-blasts, however, the developmental rate to the egg cylinder at stage 8 was significantly lower than in the controls after 72 hr (P < 0.05). The pseudo-blasts revealed significantly lower rates of endoderm formation and development to the egg cylinder than the controls after 36 and 72 hr, respectively (P < 0.05). In the TVs and NIFs, the rates of outgrowth were significantly lower than those of the controls (P < 0.05) and no egg cylinder was observed. The invasion of type I collagen gel by the cytoplasmic protrusions of trophoblast cells was observed regardless of the type and developmental stage of the embryos. The results show that a decrease in the number of embryonic cells affects the formation of the primitive endoderm and the development to the egg cylinder in vitro.

12 Contraception

The principle of vaccination for the purposes of fertility regulation is scientifically elegant and socially compelling. Factors such as economic production, convenience of use, relatively long-lasting but reversible protection, low failure rate and the avoidance of mechanical devices or exogenous hormones make this approach a potentially attractive option for family planning programmes in both developing and developed countries. The major efforts in research and development have involved the prospect of active immunization against specific antigens of sperm, oocyte, zygote and early embryo, and the pregnancy hormone human chorionic gonadotrophin (hCG). Several anti-hCG vaccines have entered clinical trials. They operate by preventing or interrupting pregnancy at the peri-implantation stage probably by neutralizing the luteotrophic effect of hCG. The most refined vaccine is one directed against the unique C-terminal peptide on the beta-subunit of hCG. This vaccine provokes antibodies that are specific for hCG and do not cross-react with human luteinizing hormone (hLH). Preclinical studies in baboons and data from a phase I human trial indicate that this method is free of side-effects and provides the promise of a duration of effectiveness of up to 12 months. Future research will optimize the anti-hCG approach, utilize new vaccine delivery systems and broaden the spectrum of target antigens of potential utility for contraceptive vaccines.

The mechanism of the antifertility effects of the luteinizing hormone-releasing hormone (LH-RH) and its agonist

Research results demonstrated that repeated dosages of luteinizing hormone-releasing hormone (LH-RH) or its agonists (LH-RH A) terminate pregnancy when given at the periimplantation stage in female rats. Several scientists proposed that the mechanism for this action directly inhibits metabolism and development of the uterus. An endocrinologist at the Institute of Zoology of Academia Sinica in Beijing China experimented with female rats to identify LH-RH receptors in endometrial tissue. Very specific LH-RH binding occurred and binding was especially great on day 3 of pregnancy. Other research revealed that 250 mcg/day of LH-RH A administered to rats and 300 mcg/day of LH-RH A administered to rabbits delayed implantation for 2 days. LH-RH A did not affect blastocyst development. It did inhibit protein synthesis of the rabbit uterus particularly uteroglobin however. 50 mcg/2ml culture medium of LH-RH inhibited secretory protein (50000-60000 MW) synthesis in human trimester trophoblast. 300 mg/kg/day of LH-RH A was administered for 4 8 and 11 days to rabbits to examine the effect LH-RH A has on endometrial polysomal mRNA. The amount of this mRNA in pregnant rabbits was consistently lower than that in the nonpregnant rabbits. Administration of LH-RH A also reduced the translation activity of endometrial polysomal mRNA in the reticulolysate system. The endometrial polysomal mRNA in pregnant rabbits caused 2 important uterine proteins with MWs varying from 22000 to 69000 to almost disappear. These results suggested that LH-RH A inhibits polysomal mRNA and thus plays a role in the regulation of uterine protein synthesis.

Identification of a putative inhibitor of early pregnancy factor in mice

Previous studies have indicated that early pregnancy factor (EPF) produced in the pre- and peri-implantation stage of pregnancy appears to consist of inactive components which combine to produce the active species. This is in contrast with EPF produced later in gestation which appears to consist of a single active species. The original studies on ammonium sulphate fractionation of mouse serum and in-vitro culture of mouse ovaries and oviducts have been repeated but tested in the bioassay for EPF, the rosette inhibition test, over an extended range of dilutions. This revealed that the two components in early pregnancy can be understood as EPF and an inhibitor(s). Once this inhibitor is removed, the active fractions in both early and late pregnancy sera exhibit similar behaviour in the above assay. It was shown also that the ovary alone is the source of activity but that this is modulated by an inhibitory substance(s) from the oviduct. Reversed-phase HPLC studies on purified 'early' EPF confirm that active and inhibitory components are present and demonstrate that the active component exhibits an identical elution pattern to 'late' EPF. Thus as pregnancy proceeds, it is not EPF that alters but rather the inhibitor(s), which disappears from the circulation soon after implantation. This substance(s) is under hormonal control, being present during oestrus as well as the early stages of pregnancy; it may be an important biological regulator of EPF. Its action in the rosette inhibition test has profound implications for further study using this bioassay.

EGF receptor activities in mammalian development

The receptor for epidermal growth factor (EGF) and its analog transforming growth factor alpha (TGF alpha) is ubiquitous, implying quite general roles for EGF/TGF alpha in cell viability and tissue maintenance in adult tissues. There is also evidence that the EGF receptor is active in promoting wound healing and tissue regeneration in adult organs, such as skin, liver, and intestinal epithelium. It is likely that EGF receptors have more specific roles during the gestation period. For example, we have detected EGF receptors on the 3.5-day blastocyst (trophectoderm) surface and since TGF alpha-like mRNA sequences and peptides have been detected at this time (Rappolee et al., Science 241:1823, 1988), there is a strong implication for autocrine stimulation in pre- and peri-implantation stage embryos. Paracrine stimulation between the embryo and maternal tissues is also likely since both receptors and TGF alpha are present in decidual cells. Therefore EGF receptors may take part in growth regulation of the early embryo and in the process of implantation. Other examples where EGF receptors may play specific roles during embryonic development are discussed.

Estradiol and progesterone levels in the endometrium and the reproductive tract luminal washing during the pre-implantation stages of gestation in the rhesus monkey

To obtain an understanding of the sex steroid hormonal milieu during early gestation, the concentrations of estradiol-17 beta and progesterone were measured in endometria and reproductive tract luminal fluids collected during the post-ovulatory period (days 2 to 6) of conception, and of non-mated menstrual cycles of the rhesus monkey (Macaca mulatta). Estradiol content was found to be higher (P less than 0.05) in endometria obtained from conception cycles; day-wise analysis revealed higher (P less than 0.05) level of tissue estradiol concentration on day 6 of post-ovulation as compared with the normal menstrual cycle. Endometrial progesterone content remained, however, unaltered in both groups of monkeys and thus relatively lower (P less than 0.05) endometrial progesterone to estradiol ratios were seen on days 4, 5 and 6 of gestation compared with normal menstrual cycle. There were no significant changes in the profiles and concentrations of estradiol and progesterone in luminal washings. The typical sex steroid hormonal milieu observed in the endometrium during the peri-implantation stage of gestation in conception cycle may be related to the endometrial differentiation towards implantation in the rhesus monkey.

Isolation and characterization of the specific peptides from rabbit blastocyst at peri-implantation stage with emphasis on their physiological significance relevant to the mechanism of implantation.

The secretory materials from the uterine flushings of the pregnant, non-pregnant and pseudopregnant rabbits were analysed by the method of SDS-polyacrylamide gel electrophoresis. The results demonstrated that there are three specific peptides in the pregnant uterine flushings with molecular weights of 3000, 4500 and 6000. No correspondent peptides were found in non-pregnant and pseudopregnant uterine flushings. The peptides were also isolated and identified from the blastocystic fluid. At least 4 peaks of small peptides were shown by high pressure liquid chromatography. The range of their isoelectrophoretic points is between pH 4.0-5.5 Concavalin A (Con A) affinitive electrophoresis indicated that they are glycopeptides in nature. The LH-RH A is able to exert inhibitory effects on the synthesis of these peptides. Their possible physiological function has been discussed.