Embryo-induced alterations in the molecular phenotype of primate endometrium.

Reproductive biomedicine has made significant advances in the area of assisted reproductive technologies in the last two and half decades. However, embryo implantation remains a major obstacle in securing high pregnancy rates. Various non-human primate models including rhesus, marmoset and baboon have been employed to elucidate in vivo mechanisms underlying the uterine events that initiate, sustain and complete implantation. This review collates the information available on the molecular profile of gestational endometrium in primates. Collectively, these studies reveal dynamic spatio-temporal changes in the expression of cytokines, growth factors, cell-adhesion molecules, cytoskeleton elements and other factors in the endometrium during the post-implantation phase of pregnancy. Considering that the endometrial events during the pre-implantation stages of pregnancy may dictate implantation success, we have developed a bonnet monkey (Macaca radiata) model where pregnancy can be detected at the pre-implantation stage. Using this model, we investigated some of the endometrial events that occur before the completion of implantation. Remarkable changes were observed in endometrial expression of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNFalpha), as well as expression of immunosuppressive factors such as transforming growth factor beta-2 (TGFbeta2), interleukin-6 (IL-6) and placental protein-14 (PP-14), even before the embryo starts invading the endometrium. This highlights the super-imposition of endometrial receptivity by embryonic stimuli, marked by differential expression and/or localization of the factors that regulate endometrial transformation for embryo survival, growth and development.

Differential expression of calreticulin, a reticuloplasmin in primate endometrium.

BACKGROUND: To our knowledge, there are no data on hormonal regulation of reticuloplasmins in primate endometrium. We report the presence and modulation of expression of three reticuloplasmins in endometrium of bonnet monkeys (Macaca radiata). METHODS: Receptive and non-receptive endometria obtained from vehicle-treated control and onapristone (antiprogestin)-treated animals, respectively, were compared for differentially expressed proteins by two-dimensional proteomics. Mass spectrometric analysis annotated two such proteins as calreticulin and protein disulfide-isomerase (PDI), known to be molecular chaperones in endoplasmic reticulum. We then investigated if endoplasmin, another reticuloplasmin is also differentially expressed. Expression of these reticuloplasmins was also investigated in the endometriuma during pregnancy in bonnet monkeys. Samples were analysed by immunohistochemistry and western blot (calreticulin in human endometrium), and calreticulin transcript levels in Ishikawa cell line were assessed by real time PCR. RESULTS: Immunohistochemical analysis of the functionalis region of non-receptive endometria in monkeys revealed higher expression of (i) calreticulin (P < 0.01) in glandular epithelium and (ii) PDI in stroma (P < 0.0001), but no change in endoplasmin in stroma or glands, compared with receptive endometria. Protein level of all three reticuloplasmins in the stromal region of endometrial functionalis was higher in pregnant than non-pregnant animals (P < 0.05). Human endometrial calreticulin protein was higher in the estrogen-dominant (proliferative) phase than progesterone-dominant (mid-secretory) phase of the cycle. Calreticulin mRNA in Ishikawa cells is up-regulated by estrogen (P < 0.05 versus control), with a trend towards down-regulation by progesterone. CONCLUSION: Our data suggest that endometrial reticuloplasmins are regulated by hormones and embryonic stimuli in a cell-type specific manner. These novel data open up new lines of investigation for elucidating the mechanisms by which hormones or embryonic stimuli influence the sub-cellular physiology of endometrium.

Protein repertoire of human uterine fluid during the mid-secretory phase of the menstrual cycle.

BACKGROUND: This study is an attempt to construct a repository of polypeptide species in human uterine fluid during the mid-secretory phase of menstrual cycle. This information is essential to generate alternative and less invasive tools for the assessment of uterine functions. METHODS: Two-dimensional polyacrylamide gel electrophoresis (2D PAGE) and mass spectrometric analysis were used to resolve and identify the major components of human uterine fluid. RESULTS: Uterine fluid collected during the mid-secretory phase (n = 6) demonstrated ca. 590 polypeptide spots in the linear range of pH 4-7 after 2D PAGE. Mass spectrometric analysis revealed the presence of heavy and light chains of immunoglobulins, alpha-1 anti-trypsin precursor, anti-chymotrypsin precursor, haptoglobin, apolipoprotein A4, apolipoprotein A1 fragment, beta-actin fragment, heat shock protein 27, hemopexin precursor and transferrin precursor. 2D protein profile of fluid collected during the proliferative phase (n = 5) revealed ca. 433 polypeptide spots, of which 279 could be paired with mid-secretory phase protein spots on the basis of their coordinates (isoelectric point and molecular weight) in 2D gels. Apolipoprotein A4, apolipoprotein A1 fragment and alpha-1 anti-trypsin precursor were 2-3-fold more abundant in uterine fluid collected during the mid-secretory phase as compared with that in the proliferative phase. Further, 86 uterine fluid polypeptides were conserved across species, being detected in human, rat and bonnet monkeys. CONCLUSIONS: The molecular repertoire of the mid-secretory phase human uterine fluid, when compared with that of the proliferative phase uterine fluid, is broadened due to differential expression of proteins. Further, some of the mid-secretory phase proteins were conserved across species.

Rab coupling protein (RCP): a novel target of progesterone action in primate endometrium.

Acquisition of functional receptivity by the endometrium is assumed to be effected by progesterone-dependent expression and repression of several genes during the implantation window in a menstrual cycle. In the present study, we employed differential display (DD) reverse transcription-polymerase chain reaction (RT-PCR) to identify progesterone-dependent gene/gene fragments that are differentially expressed during the peri-implantation phase in receptive and nonreceptive endometria, obtained from fertile and infertile bonnet monkeys respectively. Receptive endometria were obtained from regularly cycling (n=5) fertile female bonnet monkeys. Endometrial nonreceptivity was induced by treating bonnet monkeys with either 2.5 mg (n=5) or 5.0 mg (n=5) onapristone (ZK 98.299), an antiprogestin, on every third day for one cycle. Ovulation, levels of circulatory hormones (estradiol and progesterone) and menstrual cycle length did not change in treated animals; however, endometrial growth was retarded. DD2, one of the differentially expressed cDNA fragments, showed higher representation in nonreceptive endometria than in receptive endometria. The DD2 sequence was found to be homologous to the sequence of the carboxyl terminal region of Rab coupling protein (RCP), a recently discovered protein involved in intracellular vesicular trafficking. To confirm the identity of DD2 as RCP, RT-PCR studies were carried out with a forward primer deduced from the RCP sequence and a reverse primer from the DD2 sequence. The product (DDRCP) obtained, when sequenced, revealed 95% homology with the nucleotide number 1196-1757 of human RCP cDNA. Furthermore, the pattern of DDRCP expression at transcript level was found to be similar to that shown by DD2; that is, it was higher in nonreceptive endometrium. Northern analysis using labeled DD2 or DDRCP cDNA fragments identified two transcripts of 6.0 and 4.0 kb in human endometrium. In situ hybridization studies using digoxigenin-labeled DD2 revealed significantly higher (P < 0.05) localization of endometrial RCP transcripts in the proliferative phase than in the peri-implantation phase in control animals. The localization was also significantly (P < 0.01) higher in peri-implantation-phase endometria from antiprogestin-treated animals than in control animals. These antiprogestin-treated animals, however, did not demonstrate any concomitant increase in the levels of immunoreactive endometrial Rab4 and Rab11 during the peri-implantation phase. A similar pattern of cycle-dependent RCP expression was observed in human endometrial biopsies. Furthermore, significantly higher (P < 0.05) levels of RCP transcripts were detected during the peri-implantation phase in women with unexplained infertility (n=3) than in fertile women (n=3). This is the first report indicating the endometrial expression of RCP and its hormonal regulation.

Expression profiles of endometrial leukemia inhibitory factor, transforming growth factor beta2 (TGFbeta2), and TGFbeta2 receptor in infertile bonnet monkeys.

The expression profiles of leukemia inhibitory factor (LIF), transforming growth factor beta2 (TGFbeta2), and transforming growth factor beta2 receptor (TGFbeta2R) were analyzed during the peri-implantation period in regularly menstruating, fertile bonnet monkeys and in animals in which endometrial nonreceptivity was induced by administering an antiprogestin, onapristone. Based on our previous experiences, a dose of 2.5 or 5 mg of onapristone was administered s.c. every third day during the menstrual cycle, because these dosages impair endometrial development without upsetting the normal gonadal endocrine profiles. Endometrial biopsy specimens were collected during the proliferative phase (estradiol levels about 200 pg/ml, n = 5) and peri-implantation period (Day 8 after midcycle peak in estradiol levels, n = 5) from normal ovulatory animals and during the peri-implantation period from onapristone-treated animals (n = 10). The biopsy specimens were processed to determine the expression patterns of LIF, TGFbeta2, and TGFbeta2R by immunohistochemical and reverse transcription-polymerase chain reaction (RT-PCR) methods. Levels of both protein and mRNA for LIF, TGFbeta2, and TGFbeta2R (analyzed by immunohistochemistry and RT-PCR, respectively) were greater in the endometrial samples collected during the peri-implantation period compared to samples collected during the proliferative phase in control animals. Treatment with either of the two doses (2.5 or 5 mg) of onapristone caused a significant (P < 0.05) down-regulation in the expression of LIF in the peri-implantation endometria. The endometrial expressions of TGFbeta2 and TGFbeta2R mRNAs were reduced significantly in animals treated with 5 mg of onapristone, but not in those treated with the lower dose. However, immunoreactive TGFbeta2 and TGFbeta2R proteins were significantly (P < 0.05) down-regulated in the endometrial samples from both the 2.5- and 5-mg-treated groups. The alterations observed in the expression patterns of LIF, TGFbeta2, and TGFbeta2R were specific, because the expression levels of epidermal growth factor receptor remained unaffected in the endometria from the treated groups. The present study demonstrates derangement in the expression profiles of LIF, TGFbeta2, and TGFbeta2R during the peri-implantation period in infertile bonnet monkeys. It may be hypothesized that TGFbeta2 function is one of the early steps in the regulation of the progesterone-driven cascade of events leading to endometrial receptivity, and that any aberration in this step may adversely affect the subsequent molecular events (i.e., expression of LIF). These data also suggest that potential aberrations in the functional network of locally produced cytokines and growth factors even may occur in an endometrium exposed to the optimal peripheral hormonal levels.

Endometrial contraception: modulation of molecular determinants of uterine receptivity.

Modulation of endometrial receptivity is a promising approach for fertility regulation since it allows a contraceptive to act specifically at the endometrium. This was corroborated by our previous observations that treatment with low doses of a pure progesterone antagonist (PA, antiprogestin), onapristone (ZK 98299), in bonnet monkeys inhibited fertility by selectively retarding endometrial development, without affecting the hypophyseal-hypothalamic function. In the present study, further investigations, undertaken to analyze the molecular repertoire of a nonreceptive primate endometrium, determined expression of: steroid hormone receptors, i.e. progesterone receptor (PR) and estrogen receptor (ER); cytokines, i.e. leukemia inhibitory factor (LIF): transforming growth factor beta (TGFbeta) and its receptor (TGFbetaR); and cell adhesion molecules, i.e. integrins (alpha(v)beta(3), alpha(1)beta(1)). These studies were conducted during the different phases of the normal menstrual cycle and following treatment with different doses of onapristone (2.5 mg, 5 mg, or 10 mg every third day for one cycle) in bonnet monkeys. The molecules were analysed collectively to explore the possibility of a correlation between expression of these markers and endometrial receptivity and to investigate whether there exists a regulatory link between expression of these molecules under in vivo conditions. Three types of expression patterns of endometrial factors were observed during the peri-implantation period following onapristone treatment: 1) LIF, alpha(v)beta(3), and alpha(1)beta(1) showed significant (P < 0.02) down regulation in glandular epithelium of endometria in animals treated with all three doses of onapristone as compared to the control group. This was indicative of their critical role in the progesterone-driven cascade leading to implantation. 2) PR, TGFbeta, and TGFbetaR remained unaffected in the endometria from 2.5 mg treated animals and showed down regulation in animals treated with 5 and 10 mg onapristone as compared to the control group, thereby suggesting that the expression of these markers may not truely reflect endometrial receptivity per se. However, their facilitatory role in preparing the endometrium for implantation can not be ruled out since continued perturbation in the expression of these molecules may affect endometrial growth, remodelling, and differentiation, which in turn may render the endometrium nonreceptive; 3) ER remained unaltered in endometria of animals rendered infertile with 2.5, 5, and 10 mg onapristone. This observation indirectly suggests that onapristone-induced endometrial changes are mediated via some specific mechanisms. The present study clearly demonstrates that endometrial non-receptivity induced at low doses of onapristone is associated with changes in the expression pattern of specific molecular markers. However, no direct correlation was observed between in vivo expression of TGFbeta, LIF, and integrins, thereby lending support to the concept that there exists redundancy or multiple pathways which regulate implantation events.

Evaluation of the antifertility effect of magainin-A in rabbits: in vitro and in vivo studies.

OBJECTIVE: To evaluate the safety and contraceptive potential of magainin-A in rabbits. DESIGN: Controlled laboratory study. SETTING: Department of Immunology, Institute for Research in Reproduction, Mumbai, Parel, India. ANIMAL(S): Forty-eight female New Zealand white rabbits. INTERVENTION(S): The effect of magainin-A on sperm motility (in vitro and in vivo studies) and on vaginal epithelium (histologic study) was assessed along with serum and liver biochemical profiles. MAIN OUTCOME MEASURE(S): Suitability of magainin-A for contraceptive use. RESULT(S): Magainin-A arrested sperm motility, and 1 mg of magainin-A administered intravaginally blocked conception. No histopathologic abnormalities in the vaginal tissue or any changes in serum biochemical profiles were observed. CONCLUSION(S): Magainin-A may be used as an effective and safe intravaginal contraceptive compound that also has antibacterial properties.