Time Of Blastocyst Implantation Research Articles

P-547 Confined placental mosaicism for inherited segmental aneuploidies in miscarriages traces early karyotype self-correction events

Abstract Study question Does tissue-specific compartmentalization of segmental aneuploidies in first-trimester miscarriages reflect their postzygotic de novo origin or correction of inherited copy number variants (CNV)? Summary answer Most of mosaic CNVs (84%) in spontaneous abortions with normal karyotype arose de novo, but 16% resulted from postzygotic tissue-specific correction of maternally inherited variants. What is known already Chromosomal mosaicism is a hallmark of early human embryogenesis affecting significantly developmental potential of blastocysts, it’s implantation and delivery rates. Most of the currently available data are related to mosaic aneuploidies for a whole chromosome in IVF cycles, whereas little is known about origin and fate of mosaic segmental CNVs due to inherent limitations of NGS and aCGH for detection of copy number changes less for 5-10 Mb in size after single-cell whole genome amplification. Study design, size, duration Chorionic villi (CV) and extraembryonic mesoderm (EM) of miscarriages as well as maternal and paternal peripheral blood DNA samples were evaluated by aCGH+SNP arrays to detect the prevalence and tissue-specific distribution of segmental aneuploidies. As far as CV and EM are derivatives of trophectoderm and inner cell mass lineages, comparative analysis of both tissues allows to investigate the origin of mosaic CNVs by postzygotic de novo appearance or correction of the inherited variants. Participants/materials, setting, methods Eleven trios (mother, father, CV and EM samples from spontaneous abortion with normal karyotype by G-banding) were included in the study. Mean gestational age was 7.9 weeks. Mean maternal and paternal ages were 30.8 and 30.5 years, respectively. aCGH+SNP analysis was performed by SurePrint G3 Human CGH + SNP 4 × 180K microarrays (Agilent Technologies) with 13 kb median aCGH probes spacing. Main results and the role of chance Altogether 116 CNVs were detected in CV and EM samples from 11 abortions. Fifty four (46.6%) variants were present in both tissues, while 29 (25%) were confined by CV and 33 (28.4%) by EM. Most of tissue-specific segmental aneuploidies in CV (90%) and EM (79%) arose de novo, whereas others were inherited from parents. It is important that 5 maternally inherited microduplications in some clinically significant regions like 1p21.1 (103 kb in size), 2p21 (3.56 kb), 3p22.3 (128 kb), 7q36.3 (42.7 kb), and Xq28 (182 kb) were confined by EM, whereas two at 4p16.3 (22.3 kb) and 19q13.43 (2.2 kb) by CV. No cases of tissue-specific maternally inherited microdeletions or paternal CNVs were observed in our study. One can suggest that confined placental mosaicism for inherited maternal microduplications arisen by trisomy rescue mechanism with loss of additional maternal chromosome copy in CV or EM after its divergence around the time of blastocyst implantation. This hypothesis is confirmed by comparative SNP-haplotype analysis of DNA samples from both CV and EM tissues as well as parental samples in the subchromosomal regions of microduplications and adjusted loci. Limitations, reasons for caution Control group of induced (social) abortions of the same developmental terms are required to estimate the prevalence and distribution of mosaic segmental aneuploidies in embryonic and extraembrionic tissues during normal human embryo development. Wider implications of the findings Tracing of tissue-specific mosaic segmental aneuploidies in first-trimester miscarriages allows to estimate the level of karyotype instability and self-correction during human embryo development. This study was supported by the Russian Science Foundation, № 21-65-00017, https://rscf.ru/project/21-65-00017/ Trial registration number not applicable

Prominin-2 Prevents the Formation of Caveolae in Normal and Ovarian Hyperstimulated Pregnancy.

During early pregnancy, uterine epithelial cells (UECs) become less adherent to the underlying basal lamina and are subsequently removed so the blastocyst can invade the underlying stroma. This process involves the removal of focal adhesions from the basal plasma membrane of UECs. These focal adhesions are thought to be internalized by caveolae, which significantly increase in abundance at the time of blastocyst implantation. A recent in vitro study indicated that prominin-2 prevents the formation of caveolae by sequestering membrane cholesterol. The present study examines whether prominin-2 affects the formation of caveolae and loss of focal adhesions in UECs during normal and ovarian hyperstimulation (OH) pregnancy in the rat. At the time of fertilization during normal pregnancy, prominin-2 is distributed throughout the basolateral plasma membrane. However, at the time of implantation and coincident with an increase in caveolae, prominin-2 is lost from the basal plasma membrane. In contrast, prominin-2 remains in the basolateral plasma membrane throughout OH pregnancy. Transmission electron microscopy showed that this membrane contained few caveolae throughout OH pregnancy. Our results indicate that prominin-2 prevents the formation of caveolae. We suggest the retention of prominin-2 in the basal plasma membrane during OH pregnancy prevents the formation of caveolae and is responsible for the retention of focal adhesions in this membrane, thereby contributing to the reduced implantation rate observed after such treatments.

Inhibition of mTOR induces a paused pluripotent state.

Cultured pluripotent stem cells are a cornerstone of regenerative medicine owing to their ability to give rise to all cell types of the body. Although pluripotent stem cells can be propagated indefinitely in vitro, pluripotency is paradoxically a transient state in vivo, lasting 2-3 days around the time of blastocyst implantation. The exception to this rule is embryonic diapause, a reversible state of suspended development triggered by unfavourable conditions. Diapause is a physiological reproductive strategy widely employed across the animal kingdom, including in mammals, but its regulation remains poorly understood. Here we report that the partial inhibition of mechanistic target of rapamycin (mTOR), a major nutrient sensor and promoter of growth, induces reversible pausing of mouse blastocyst development and allows their prolonged culture ex vivo. Paused blastocysts remain pluripotent and competent-able to give rise to embryonic stem (ES) cells and live, fertile mice. We show that both naturally diapaused blastocysts in vivo and paused blastocysts ex vivo display pronounced reductions in mTOR activity, translation, histone modifications associated with gene activity and transcription. Pausing can be induced directly in cultured ES cells and sustained for weeks without appreciable cell death or deviations from cell cycle distributions. We show that paused ES cells display a remarkable global suppression of transcription, maintain a gene expression signature of diapaused blastocysts and remain pluripotent. These results uncover a new pluripotent stem cell state corresponding to the epiblast of the diapaused blastocyst and indicate that mTOR regulates developmental timing at the peri-implantation stage. Our findings have implications in the fields of assisted reproduction, regenerative medicine, cancer, metabolic disorders and ageing.

The adherens junction is lost during normal pregnancy but not during ovarian hyperstimulated pregnancy

During early pregnancy in the rat, the luminal uterine epithelial cells (UECs) must transform to a receptive state to permit blastocyst attachment and implantation. The implantation process involves penetration of the epithelial barrier, so it is expected that the transformation of UECs includes alterations in the lateral junctional complex. Previous studies have demonstrated a deepening of the tight junction (zonula occludens) and a reduction in the number of desmosomes (macula adherens) in UECs at the time of implantation. However, the adherens junction (zonula adherens), which is primarily responsible for cell–cell adhesion, has been little studied during early pregnancy.This study investigated the adherens junction in rat UECs during the early stages of normal pregnancy and ovarian hyperstimulated (OH) pregnancy using transmission electron microscopy. The adherens junction is present in UECs at the time of fertilisation, but is lost at the time of blastocyst implantation during normal pregnancy. Interestingly, at the time of implantation after OH, adherens junctions are retained and may impede blastocyst penetration of the epithelium.The adherens junction anchors the actin-based terminal web, which is known to be disrupted in UECs during early pregnancy. However, artificial disruption of the terminal web, using cytochalasin D, did not cause removal of the adherens junction in UECs.This study revealed that adherens junction disassembly occurs during early pregnancy, but that this process does not occur during OH pregnancy. Such disassembly does not appear to depend on the disruption of the terminal web.

312. THE DISTRIBUTION OF PROMININ-1 IN THE RAT UTERUS DURING EARLY PREGNANCY

Prominin-1 is a recently discovered pentaspan membrane protein present in characteristic cholesterol-based vesicles and associated with microvilli. These vesicles are used to deliver prominin-1 to the apical plasma membrane in a number of cell types. Previous work on uterine epithelial cells has demonstrated a loss of microvilli and the presence of large, cholesterol-based vesicles at the time of implantation. Thus this study aims to determine a role for prominin-1 in rat uterine epithelial cells during early pregnancy. Immunofluorescence microscopy reveals punctate and diffuse prominin-1 staining below the apical plasma membrane on day 1 of pregnancy. At the time of blastocyst implantation (day 6) however, prominin-1 appears concentrated at the apical surface of the cell. Western blotting of isolated uterine epithelial cell lysate revealed a change in prominin-1 glycosylation during early pregnancy. Prominin-1 was determined to be glycosylated on day 1 of pregnancy, but these carbohydrate side chains were lost by the time of attachment. Results seen in the present study indicate that prominin-containing vesicles may be prevented from reaching the apical plasma membrane by the terminal web on day 1 of pregnancy. On day 6, the loss of the terminal web may allow the vesicles to approach and incorporate into the apical plasma membrane, as seen with other uterine vesicles. The deglycosylation of prominin-1 at this time is suggested to allow the protein to bind its ligand and activate downstream signalling pathways that permit implantation. This study constitutes the first reported observation of prominin in endometrial lumenal epithelial cells. These preliminary results, in consideration with previous reports of prominin expression in trophoblast cells, suggest an important role for this protein in early pregnancy.

Blocking LIF action in the uterus by using a PEGylated antagonist prevents implantation: A nonhormonal contraceptive strategy

Blastocyst implantation is a critical stage in the establishment of pregnancy. Leukemia inhibitory factor (LIF) is essential for mouse blastocyst implantation and also plays a role in human pregnancy. We examined the effect of a potent LIF antagonist (LA) on mouse implantation. In mice, LIF expression peaks on day 3.5 of pregnancy (D3.5) (D0.5 = day of mating plug detection) in the uterine glandular epithelium. LA (7 mg/kg per day) administered from D2.5 to D4.5 via four hourly i.p. injections plus continuous administration via miniosmotic pump resulted in complete implantation failure. To improve its pharmacokinetic properties, we conjugated LA to polyethylene glycol (PEG), achieving a significant increase in serum levels. PEGylated LA (PEGLA) (37.5 mg/kg per day) administered via three i.p. injections between D2.5 and D3.5 also resulted in complete implantation failure. PEGLA immunolocalized to the uterine luminal epithelium at the time of blastocyst implantation. Both LA and PEGLA reduced phosphorylation of the downstream signaling molecule STAT3 in luminal epithelial cells on D3.5. The effects of PEGLA were found to be endometrial, with no embryo-lethal effects observed. These data demonstrate that administration of a PEGylated LIF antagonist is an effective method of targeting LIF signaling in the endometrium and a promising novel approach in the development of nonhormonal contraceptives for women.

Stage-specific expression of leukaemia inhibitory factor and its receptor in rabbit pre-implantation embryo and uterine epithelium during early pregnancy.

Leukaemia inhibitory factor (LIF) has been shown to play an important role in the development and implantation of blastocysts in mice. In the current study, the reverse transcription-polymerase chain reaction (RT-PCR) was employed to examine the expression patterns of LIF and its receptor (LIFR) genes in rabbit embryos during pre-implantation development, and the uterine expression of LIF and LIFR was also evaluated by Western blotting. Transcripts for LIFR were detected within morula and blastocyst-stage embryos, while the LIF mRNA was only found in blastocysts (from early to fully expanded blastocoel cavities), indicating that embryo-derived LIF can act in an autocrine manner on the process of blastocyst formation. The expression levels of LIF and LIFR in uterine epithelium were gradually increased during pre-implantation period and reached their highest levels on days 6.5 of pregnancy, just before the time of blastocyst implantation, suggest that paracrine LIF circuit should exist between the endometrium and the early embryos, which may be involved in the embryo-maternal dialogue and important for the blastocyst implantation. The data present here show the stage-specific and dynamic expression patterns of LIF and LIFR, both in embryos and endometrium, during early pregnancy in rabbits, which indicated that LIF might play an important role in the pre-implantation development and subsequent implantation of rabbit embryos.

Heparin-binding EGF-like growth factor is seen on the extracellular surface of uterine epithelial cells only after the initial stages of blastocyst attachment.

The presence and distribution of heparin-binding epidermal growth factor in rat uterine epithelial cells was determined immunohistochemically and localized ultrastructurally. Rat uterine tissue was examined on days 1, 3, 6 and 8 of pregnancy and it was found that while presence of this growth factor was evident from day 1, spatial reorganization occurred by the time of blastocyst implantation. Strong apical staining was evident from day 6 to day 8, day 6 being the approximate time of blastocyst implantation. Electron microscopy further revealed that this growth factor while shown to be expressed very strongly apically from day 6, actually localized on the plasma membrane only after attachment of the blastocyst. This suggests that heparin-binding epidermal growth factor is not involved in the initial stages of implantation but is more likely involved in the post attachment stages of pregnancy.

Junctional plaque proteins shift to the apical surface of uterine epithelial cells during early pregnancy in the rat

Antibodies against the cytoplasmic plaque molecules, plectin and plakoglobin, and cytokeratin, the molecular component of intermediate filaments (IFs), were used to examine the distribution of these molecules in rat uterine epithelial cells during early pregnancy including the period of blastocyst implantation. On day 1 of pregnancy plectin was detected in concentrated bands along the apical and basal plasma membranes, and diffusely throughout the cytoplasm. Plakoglobin was found along the entire lateral plasma membrane on day 1. By day 6, the time of blastocyst implantation, plectin was localised along the apical and basal membranes and reduced in the basal cytoplasm, and plakoglobin was seen exclusively at the apical-most quarter of the lateral plasma membrane. Cytokeratin was detected throughout the cytoplasm on day 1, but by day 6, was localised to the apical region of the cytoplasm only. These results show a redistribution of plectin, plakoglobin and cytokeratin away from the basal region of the uterine epithelial cells. The change in distribution of these molecules may contribute to the adhesion of the blastocyst to the apical and lateral surfaces of uterine epithelial cells and the subsequent detachment of the uterine epithelium from the basal lamina.

Scanning electron microscopic and morphometric analysis of the guinea pig uterine luminal surface: cyclic and ovarian steroid-induced modifications.

A morphometric and scanning electron microscopic analysis of endometrial, luminal topographical changes of cyclic and ovariectomized, ovarian steroid-treated guinea pigs was undertaken to determine the effects of cyclic endocrine influences on the uterine surface features recognized to be involved in the process of interstitial blastocyst implantation. Estrogenic stimulation of the uterine epithelial layer induced microvillus growth and closed the endometrial gland openings on the luminal surface. Progestins antagonized these parameters, as indicated by short, sparse microvillus formation over a flat luminal uterine surface on which gland openings were both numerous and prominent. The combined effects of estrogen and progesterone therapies induced luminal epithelial changes resembling those characterizing the cyclic uterine surface at the time of blastocyst implantation. These data indicate that the cyclic alterations in the uterine luminal surface are directly regulated by ovarian steroid hormones, and that these changes in surface structure are synchronized and directly involved in the preparation for blastocyst-uterine interaction during the nidatory process in this species.

Apolipoprotein J/Clusterin Expression Defines Distinct Stages of Blastocyst Implantation in the Mouse Uterus1

The endometrium is a dynamic tissue that responds to hormonal cues and growth factors to accommodate, regulate, and nurture developing embryos. To provide clues about the molecular mechanisms underlying the responsiveness of this tissue, we have begun to identify genes that are expressed at specific stages of early pregnancy. One such gene, apolipoprotein J (apoJ), encodes a secretory glycoprotein capable of binding lipids and membrane-active proteins. Uterine apoJ gene activity was not detected immediately following fertilization, but glandular epithelial expression of apoJ mRNA appeared just before the time of blastocyst implantation and persisted postimplantation. During implantation, uterine luminal epithelial cells also expressed apoJ, but expression was excluded from luminal cells adjacent to the sites of attached blastocysts. ApoJ protein accumulated in the glandular and uterine lumens in proximity to the epithelial cells that expressed apoJ mRNA. We suggest that apoJ expression is a marker of uterine receptivity to blastocyst implantation. Subsequent expression of apoJ message in uterine stromal cell types and in circular muscle myocytes coincided with the onset of decidualization. During this period the myocytes of the longitudinal muscle layer showed no evidence of apoJ mRNA. ApoJ protein was localized to nondecidualized tissue but was not evident in decidualized cells. In contrast, the protein was dispersed throughout both the circular and longitudinal myometrium. In the uteri of hormone-treated females stimulated with oil, apoJ was also expressed during decidualization in stromal cells and in circular myocytes, indicating that signals specifically transmitted from the embryo itself are not responsible for apoJ mRNA accumulation.

Modulation of the levels of interleukin-8 messenger ribonucleic acid and interleukin-8 protein synthesis in human endometrial stromal cells by transforming growth factor-beta 1.

Selected functions of uterine endometrium of ovulatory women before and during pregnancy appear to be modulated by cytokines and other paracrine-acting factors. Some of these functions are regulated, in turn, by cyclic changes in ovarian steroid secretion or by pregnancy-induced endocrine and paracrine factors. The recruitment of specific types and numbers of bone marrow-derived cells into the endometrium occurs in a predictable manner with hormonal changes of the ovarian cycle, during the process of endometrial decidualization, at the time of blastocyst implantation, and during pregnancy, parturition, and the puerperium. As part of an investigation of the regulation of the leukocyte population of endometrium/decidua, this study was conducted to evaluate further the regulation of interleukin-8 (IL-8) gene expression by transforming growth factor-beta (TGF beta). IL-8 is a neutrophil chemoattractant/activating and T cell chemotactic factor as well as a chemotactic factor for fibroblasts. IL-8 is produced by mesenchymal cells of many tissues, including human endometrial stromal cells in culture. The level of IL-8 messenger ribonucleic acid (mRNA) in endometrial stromal cells and the accumulation of immunoreactive IL-8 in medium are increased by TGF beta 1 treatment of these cells. This response to TGF beta 1 is attributable primarily to an increase in the stability of IL-8 mRNA through a process that is dependent on protein synthesis. Transcription of the IL-8 gene in endometrial stromal cells is not increased, but, rather, is slightly decreased, by treatment with TGF beta 1. The findings of this study indicate that TGF beta may act in endometrial stroma to modulate the stability of IL-8 mRNA.

Hydrolysis of estrone sulfate in uterine minces of the 6-days pregnant rat.

In view of the possible participation of estrogen 3-sulfoconjugates in reproductive events, such as reproductive cycles, gestation and parturition, an experiment was carried out where the conversion of labeled estrone sulfate ([3H]E1S) to estrone ([3H]E1) was measured during in vitro incubation with minced uterine tissues representing implantation sites (IS) and non-implanted areas (NIS), from pregnant rats at the time of blastocyst implantation. Significant hydrolysis of the 3-sulfate, by the action of the estrogen sulfatase, was found in both tissues being less in IS than in NIS, when expressed either as pmol of E1 formed/mg wet tissue/h (238 +/- 37 vs 337 +/- 15, respectively) or as pmol of E1 formed/mg protein/h (1278 +/- 198 vs 1773 +/- 81). Both differences are statistically significant at the 0.001 level. The results obtained here suggest that E1S present in uterine fluids may be taken up and hydrolyzed by the sulfatase present in both intrauterine tissues of the 6-days pregnant rat. However, the decreased formation of E1 found in IS suggests that rat blastocyst is able to regulate the local concentration of unconjugated estrogens required at IS by modulating the activity of the estrogen sulfatase.

Reorganization of the apical cytoskeleton of uterine epithelial cells during early pregnancy in the rat: a study with myosin subfragment 1

Actin filaments were identified in the epithelial cells of rat uterus following detergent extraction and decoration of microfilaments (MF) with myosin subfragment 1 (SI). MF connections with cytoplasmic organelles and the apical plasma membrane are also described. Transmission electron microscopy revealed that the regular microvilli of non-pregnant, oestrous animals contain several decorated MF with rootlets descending into a densely filamentous terminal web. Following mating, the actin cytoskeleton was examined on days 1, 3 and 6 of pregnancy. In this period, the irregular projections that replace MV assumed an underlying, dense network of decorated MF, whilst smoother surfaces displayed few cytoplasmic filaments. At the time of blastocyst implantation, a structured terminal web was no longer present. Structural details were revealed concerning the contents of large, bleb-like projections found on the apical surface.

The effect of leukaemia inhibitory factor (LIF) on embryogenesis.

Leukaemia inhibitory factor (LIF) was originally identified as a haemopoetic factor that induced the differentiation of certain myeloid leukaemia cell lines. In contrast to this action, LIF was subsequently shown to inhibit the spontaneous differentiation of murine embryonic stem cells in culture, thus maintaining their pluripotency and ability to contribute to the germline of chimaeric mice. In the mouse, mRNA for LIF is expressed by the endometrial glands of the uterus coincident with the time of blastocyst implantation and receptors have been found on the preimplantation blastocyst. The signal for LIF expression appears to be of maternal origin, perhaps regulated by oestradiol. Recombinant LIF improves the development of murine and ovine blastocysts in culture although there is some species specificity with respect to the type of LIF that is bioactive. It is proposed here that LIF acts on the trophectoderm of the rapidly expanding blastocyst and improves the implantation rate of otherwise compromised embryos. Further studies in livestock should elicit therapeutic uses for LIF in embryo culture, embryo transfer and embryo survival in vivo.

Uterine estrogen sulfatase activity at the time of blastocyst implantation in the rat

The conversion of estrone sulfate (E 1S) to estrone (E 1) was measured during the in vitro incubation of the labeled sulfoconjugate with implantation sites (IS) and nonimplanted regions (NIS) of uterine horns from 6-day pregnant rats. Extensive metabolism of E 1S occurred in both tissues, being noticeably less (29.31%) in IS than in NIS. Estrogen sulfatase activity present in the uterus of ovariectomized virgin rats was found to be higher than in both uterine regions of the pregnant rats. We suggest that E 1S present in uterine fluids may be accessible to be metabolized into unconjugated estrogens by both intrauterine tissues of 6-day pregnant rats. This metabolism could be locally modulated in IS through the participation of the estrogen sulfatase, the activity of which is in turn controlled by the presence of free estrogens, possibly synthesized and/or secreted by the embryo, which has been shown to inhibit the sulfohydrolase activity.

Increase in Lysosomal Numbers and Activity in the Rat Uterine Luminal and Glandular Epithelium during Early Pregnancy: A Histochemical Study

Lysosomal acid phosphatase was studied at both the light and electron microscope level in the rat uterine luminal and glandular epithelium, at oestrous, late dioestrous and day 6 of pregnancy. At the light-microscopic level lysosomal numbers were quantified and statistically analysed. A morphological study was also carried out on the lysosomes at the electron-microscopic level in the above-mentioned stages. Quantification of lysosomal numbers found day 6 of pregnancy to have a significantly higher lysosomal population in the luminal and glandular epithelium compared to non-pregnant states. At the electron-microscopic level the luminal epithelial lysosomes were frequently observed in an invaginated or vesiculated form whereas these characteristics were rarely observed during late dioestrous and were non-existent during oestrous. Generally, the lysosomes appeared more active in the luminal epithelium at day 6 of pregnancy compared to the non-pregnant state. The findings are discussed in reference to the role of the lysosome at the time of blastocyst implantation.

Expression of cellCAM-105 in the apical surface of rat uterine epithelium is controlled by ovarian steroid hormones

Affinity-purified antibodies to cellCAM-105, an adhesive cell surface glycoprotein, were used in immunohistochemical investigations of rat uteri at various functional stages: (i) the oestrous, pro-oestrous, metoestrous, and dioestrous stages of the oestrous cycle, (ii) Days 1-8 of normal pregnancy, (iii) delayed implantation, (iv) 18 h after oestrogen reactivation from delay of implantation, and (v) juvenile rats, and normal ovariectomized adults, respectively, before and after experimental injection of progesterone and/or oestrogen. CellCAM-105 was present in the apical zones of the luminal and glandular epithelium cells in a stage-specific and hormone-dependent manner. The results indicate that: (1) steroid hormones are essential for the expression of cellCAM-105 in the uterine epithelial cells; (2) progesterone induces cellCAM-105 expression in the glandular epithelium, and oestrogen induces cellCAM-105 expression in the luminal epithelium; (3) progesterone induces down-regulation of cellCAM-105 from the surface of the uterine luminal epithelium of juvenile rats; (4) cellCAM-105 is absent in the luminal epithelial cells but present in the glandular epithelial cells of the rat uterus at the time of blastocyst implantation.

Oestrogen and progestogen receptors in endometrium and myometrium at the time of blastocyst implantation in pregnant diabetic rats.

A suitable hormonal environment is a prerequisite for blastocyst implantation. Experimental diabetes was previously shown to modify the hormonal milieu and produce alterations in oestrogen receptor kinetics in the uterine tissue. In the present work, oestrogen and progestogen receptor levels were measured on the morning of day 6 of pregnancy in normal and in streptozotocin-induced diabetic rats, both in implantation sites and in interembryonic segments of endometrium and myometrium. Receptor levels were different in the implantation sites compared to the interembryonic segments of endometrium, both in the control and in the diabetic animals. Indeed, implantation sites were characterized by lower oestrogen receptor levels in cytosol and higher progestogen receptor levels in cytosol and nuclei. However, compared to the control rats, the diabetic rats had lower oestrogen receptor levels in implantation sites, both in cytosol and nuclei. In the myometrium, the differences between sites or between types of rats were minimal. Plasma levels of oestradiol were lower in diabetic rats than in control animals, whereas progesterone levels were similar. A 20% lower implantation rate was found in diabetic rats, compared to normal rats. These results show that the specific distribution of oestrogen and progestogen receptors between implantation sites and interembryonic segments was preserved in the diabetic rats; however the absolute level of oestrogen receptor was lower. This abnormal endocrine milieu might arise from a lower oestradiol level and a decreased oestradiol/progesterone ratio in the circulating blood. Whether the lower implantation rate in diabetic rats might be a consequence of the overall disturbed hormonal status remains to be elucidated.

Delayed implantation in lactating bank voles, Clethrionomys glareolus

The time of blastocyst implantation in lactating and non-lactating female bank voles was studied. In primiparous females implantation started 4 days and 9-11 h after mating. A delayed implantation of at least 1 1/2 days was found in lactating, multiparous females when compared to non-lactating, multiparous and primiparous females.