Dialogue between blastocyst hCG and endometrial LH/hCG receptor: which role in implantation?

The specific interaction of blastocyst-derived human chorionic gonadotropin (hCG) and endometrial LH/hCG-R constitutes a fundamental component of the molecular dialogue at the materno-fetal interface. From our observations and studies from other groups, hCG was indeed shown to play a significant role in implantation and tolerance of the embryo, decidual differentiation and remodeling, as well as in placentation. The profile pattern of LH/hCG-R expression by endometrial epithelium correlates with the theoretical timing of the implantation window. Studies are currently being conducted in assisted medical procreation and in an animal model of implantation to establish the index of LH/hCG-R expression as a new biomarker of uterine receptivity for embryo implantation.

[What's new at the maternal-foetal interface: role of the hCG/LH-hCG receptor couple during embryo implantation]. / Du nouveau à l'interface materno-foetale: rô1e du couple hCG/récepteur LH-hCG dans l'implantation embryonnaire.

Implantation of the embryo into the maternal endometrium represents a unique biological process, combining an immunological (tolerance of an allograft) and biological (adhesion of two epitheliums) paradox. The success of implantation depends on a receptive endometrium, a functionally normal blastocyst and a synchronized cross-talk between embryonic and maternal tissues. Though sexual steroids control the process, a cascade of growth factors or cytokines are the prime paracrine mediators of the dialogue at the maternal-embryonic interface. HCG is one of the molecules most precociously produced by the embryo and is the most specific marker of its presence. HCG is a luteotropic factor which relays the inadequate support provided by the reduced rates of LH, but also influences the pregnancy on a paracrine mode by a local action on implantation process, probably by interacting with its receptor, the LH/hCG-R that we have evidenced on endometrial epithelium. We demonstrate that embryo actively participate into its implantation, tolerance and placentation.

Human chorionic gonadotropin and growth factors at the embryonic-endometrial interface control leukemia inhibitory factor (LIF) and interleukin 6 (IL-6) secretion by human endometrial epithelium.

BACKGROUND: The elucidation of the molecular mechanisms by which the embryo contributes to its implantation is an area of extensive research. The main objective of this study was to investigate the pattern of leukemia inhibitory factor (LIF) and interleukin-6 (IL-6) secretion by human endometrial epithelium, and their regulation by human chorionic gonadotropin (hCG) and other growth factors present at the embryonic-endometrial interface. METHODS: Endometrial epithelial cells (EEC) were isolated from biopsies collected at both proliferative and secretory phases of fertile women. RESULTS: HCG (1-50 IU/ml) increased LIF secretion by EEC cultures derived from follicular phase (up to 285+/-75%) or from secretory phase (up to 212+/-16%). In contrast, hCG reduced IL-6 secretion by EEC in both phases. The hCG/LH receptor gene was transcribed by EEC as evidenced by RT-PCR. Insulin-like growth factors 1 and 2 increased LIF secretion by EEC. Transforming growth factor beta1 stimulated LIF and reduced IL-6 secretion. CONCLUSIONS: Through hCG, the blastocyst may be involved in the control of its implantation (via an increase of proimplantatory LIF) and tolerance (via an inhibition of proinflammatory IL-6). Other growth factors present at the embryonic-endometrial interface are also involved in the control of LIF and IL-6 endometrial secretion.

[The implantation window]. / La fenêtre implantatoire.

BACKGROUND: Embryo implantation is a complex event involving apposition followed by adhesion of the blastocyst to the maternal endometrium, and finally invasion of this endometrium. Though implantation could occur in any human tissue, the endometrium is the only tissue where embryo implantation cannot occur except during a restricted period called the implantation window. During this window, the endometrium is highly receptive to the embryo. MATERIAL: and methods. We reviewed the literature concerning the different factors involved in improved endometrial receptivity and implantation. RESULTS: Maternal - embryo crosstalk is favored by the implantation window. Endometrial receptivity results from the acquisition of ligands or receptors facilitating apposition, then adhesion of the embryo, or from the loss of components preventing it. The molecular basis of the implantation window remains to be defined. CONCLUSION: Despite progress in assisted reproduction technologies, the lack of control of implantation remains a major obstacle to successful pregnancy. It is of prime importance to determine the characteristic features of a receptive endometrium and, among the many markers proposed by in vitro studies, to analyze in humans those demonstrated by knock-out experiments to play a crucial role in mice.

Characterization of the insulin-like growth factor axis in the human thymus.

The components of the insulin-like growth factor (IGF) axis have been investigated in the normal human thymus. Using ribonuclease protection assays (RPA), IGF-II transcripts were detected in the normal human thymus. By reverse transcriptase polymerase chain reaction (RT-PCR) analyses, promoters P3 and P4 were found to be active in the transcription of IGF2 gene within human thymic epithelial cells (TEC). No IGF-II mRNA could be detected in human lymphoid Jurkat T cells with 30 cycles of RT-PCR. By Northern blot analyses, IGFBP-2 to -6 (but not IGFBP-1) were found to be expressed in TEC with a predominance of IGFBP-4. Interestingly, Jurkat T cells only express IGFBP-2 but at high levels. The type 1 IGF receptor was detected in Jurkat T cells but not in human TEC. The identification of the components of the IGF axis within separate compartments of the human thymus adds further evidence for a role of this axis in the control of T-cell development. The precise influence of thymic IGF axis upon T-cell differentiation and immunological self-tolerance however needs to be further investigated.

The thymic repertoire of neuroendocrine-related self antigens: biological role in T-cell selection and pharmacological implications.

Thymic epithelium, including nurse cells (TEC/TNC), as well as other thymic stromal cells (macrophages and dentritic cells), express a repertoire of polypeptide belonging to various neuroendocrine protein families (such as the neurophypophysial, tachykinin, neurotensin and insulin families). A hierarchy of dominance exists in the organization of the thymic repertoire of neuroendocrine precursors. Oxytocin (OT) is more expressed in the TEC/TNC than vasopressin (VP); insulin-like growth factor 2 (IGF-2) thymic expression predominates over IGF-1, and much more over (pro)insulin. Thus, OT was proposed to be the self antigen of the neurohypophysial family, and IGF-2 the self antigen precursor of the insulin family. The dual role of the thymus in T-cell life and death is recapitulated at the level of the thymic neuroendocrine protein repertoire. Indeed, thymic polypeptides behave as accessory signals involved in T-cell development and positive selection according to the cryptocrine model of signaling. Moreover, thymic neuroendocrine polypeptides are the source of self antigens presented by thymic MHC molecules to developing pre-T cells. This presentation might induce the negative selection of T cells bearing a randomly rearranged antigen receptor (TCR) oriented against neuroendocrine families. Using an animal model of autoimmune type 1 diabetes (BB rat), we have shown a defect in intrathymic expression of the self antigen of the insulin family (IGF-2) and in IGF-2-mediated T-cell education to recognize and tolerate the insulin family. Altogether these studies have enlightened the crucial role played by the thymus in the induction of the central self tolerance of neuroendocrine families. The tolerogenic properties of thymic self peptides could be used in a novel type of vaccination for the prevention of autoimmune diseases.

Neurohypophysial peptides stimulate the phosphorylation of pre-T cell focal adhesion kinases.

Thymic oxytocin (OT) behaves as a cryptocrine signal targeted at the outer surface of thymic epithelial cell plasma membrane from where OT is able to interact with neurohypophysial peptide receptors expressed by pre-T cells. Immature T cells bear a receptor of the V1 subtype, while OT receptors are predominantly expressed by cytotoxic CD8+ lymphocytes. In both T cell types, neurohypophysial peptide receptors transduce OT via the phosphoinositide pathway. Protein tyrosine phosphorylation is an early event of T cell activation. Western blots of murine pre-T cells (RL12-NP line) proteins probed with anti-phosphotyrosine (PY-20) revealed a great number of proteins the phosphorylation of which increased either with OT or vasopressin treatment. Two were immunoprecipitated with anti-focal adhesion kinase (FAK) mAb 2A7 and were identified one as p125FAK and the other as a coprecipitating 130-kDa protein. The p125FAK is connected to the Ras/MAPK pathway and is also implicated in TCR/CD3 signalling in T cell. Another protein phosphorylated by OT in RL12-NP was identified as paxillin, a 68-kDa protein localised at focal adhesion sites and associated with p 125FAK. These results indicate that phosphorylation of focal adhesion kinase may be induced in pre-T cell by thymic OT.

Effects of a novel gonadotropin-releasing hormone antagonist (ORG 30850) on gonadotropin and prolactin secretion by rat pituitary cells in culture.

The effect of a new GnRH antagonist (ORG 30850 ANT) on FSH, LH, and PRL secretion was studied using male rat pituitary cells in monolayer cell culture. In the absence of GnRH, ORG 30850 ANT did not alter spontaneous FSH and LH secretion into culture medium or the cell content of these hormones. In the presence of GnRH (10(-8) mol/l), ORG 30850 ANT significantly and dose-dependently inhibited FSH and LH secretion into culture medium while increasing their cell content. Conversely, in the presence of a single dose of ORG 30850 ANT, FSH and LH secretion rose significantly when subjected to increasing amounts of GnRH, whereas the hormonal cell content diminished. Furthermore, inhibition of GnRH-induced FSH and LH release by ORG 30850 ANT was not changed by pre-incubation with the GnRH antagonist regardless of the pre-incubation time. The inhibitory effect of the GnRH antagonist was observed early, with its peak occurring within 6 h of culture. These short-term studies indicate that ORG 30850 ANT specifically inhibits GnRH-induced gonadotropin release into culture medium, exerts no effect on the rate of gonadotropin production in the presence or absence of GnRH, competitively and reversibly inhibits the binding of natural GnRH to its receptors, and does not lead to any modifications in PRL secretion.

Correlation between follicular fluid content and the results of in vitro fertilization and embryo transfer. II. Inhibin and aromatase inhibitor activity.

The inhibin content and aromatase inhibitor activity (AIA) of 72 follicular fluids (FF) obtained from 42 women undergoing in vitro fertilization (IVF) and embryo transfer (ET) were studied as a function of IVF ET outcome. Inhibin levels were determined by bioassay (BA) and RIA; AIA was measured by BA. The inhibin content of follicles characterized as immature by their estradiol (E2) levels and E2/progesterone (P) ratios was significantly lower (P less than 0.05) than that of mature follicles (i.e. leading to pregnancy). The mean AIA for mature follicles were significantly lower than AIA in groups where pregnancy was not obtained. AIA for follicles from which a pregnancy was obtained for each ET was also significantly lower than that in FF characterized as immature of hypermature. The highest E2/AIA and inhibin BA/AIA ratios were associated with the highest incidence of successful IVF ET outcome. No correlation was found between AIA and inhibin, on the one hand, and E2, delta 4-androstenedione, E2/P, and PRL, on the other. However, a positive correlation was found between inhibin (RIA and BA) and P, reflecting the production of inhibin by granulosa cells during luteinization. These studies allowed us to conclude that FF inhibin levels do not differ according to IVF ET outcome, but are an index of follicular maturation. AIA not only constitutes an index of follicular maturation and granulosa cell luteinization, but is of predictive value for IVF ET outcome as E2/AIA and inhibin BA/AIA ratios.

Inhibin and related peptides: mechanisms of action and regulation of secretion.

The structure of inhibin is known; it consists of a heterodimer composed of one alpha and one beta subunit. The homodimer of beta A (beta A-beta A) and the heterodimer beta A-beta B, called activin A and B, respectively, stimulate the release and synthesis of FSH by gonadotrophs. Inhibin exerts effects at the hypophyseal, hypothalamic, and gonadal levels. Produced by granulosa cells in the female and by Sertoli cells in the male, inhibin synthesis is stimulated by FSH and reduced by hypophysectomy and progesterone. At present, there is no evidence for a signal from germinal cells to modify inhibin production. Inhibin secretion evolves in parallel with follicular maturation and aromatase activity, whereas luteinization arrests its production. Nevertheless, important differences in the regulation of inhibin secretion seem to exist from one species to another. Sperm inhibin levels can be correlated with spermatozoa number. Administration of inhibin to sheep induces either anovulation or an increase in the rate of ovulation depending on the scheme of treatment.

Inhibin and related peptides. Mechanisms of action and regulation of secretion.

The structure of inhibin is known: it consists in a heterodimer constituted by one alpha and one beta subunits. The homodimer of beta A or the heterodimer beta A or the heterodimer beta A-beta B called activin A and B stimulates the release and the synthesis of FSH by gonadotrophs. Inhibin displays actions at hypophyseal, hypothalamic and gonadal levels. Produced by granulosa cells in female and by Sertoli cells in male, inhibin synthesis is stimulated by FSH, and reduced by hypophysectomy and progesterone. At the present time, there is no evidence for a signal from germinal cells to modify inhibin production. Inhibin secretion evolves with follicular maturation as aromatase activity whereas luteinization arrests its production. Nevertheless it seems to exist large difference in the regulation of inhibin secretion from one species to the other. Sperm inhibin levels are correlated with spermatozoa number. Its administration to the sheep induce either an anovulation or an increase of ovulation rate according to the scheme of treatment.

Effect of mouse epidermal growth factor on DNA and protein synthesis, progesterone and inhibin production by bovine granulosa cells in culture.

The effect of mouse epidermal growth factor (EGF) was investigated on DNA and protein synthesis, progesterone and inhibin production by bovine antral granulosa cells. When incubated for the whole period of culture, EGF inhibited inhibin production the second day of culture, progesterone the third and the fourth days whereas it stimulated DNA and protein synthesis only the fourth day of culture. Inhibition of progesterone and stimulation of DNA and protein were dose-dependent when treatment with EGF (pre-incubation) is followed by 24 h without EGF, a stimulatory effect on DNA and protein synthesis was observed after 48 and 72-h pre-incubation. Progesterone was reduced after 3 day pre-incubation and inhibin only after 2-day pre-incubation. Effects observed after 3-day pre-incubation were dose-dependent. These experiments demonstrated the stimulatory effect of EGF on growth of granulosa cells and its inhibitory action on hormonal production by these cells in vitro. The inhibitory effect on progesterone and inhibin production is more precocious than stimulatory effect on DNA and protein synthesis. The inhibitory action of EGF on granulosa cell production of progesterone and inhibin could thus be not directly dependent on its stimulatory action on DNA synthesis.

Effect of follicular atresia on inhibin production by bovine granulosa cells in vitro and inhibin concentrations in the follicular fluid.

The ability of bovine granulosa cells to produce inhibin and to synthesize oestradiol-17 beta increased with increasing follicle size in healthy but not atretic follicles. Granulosa cells from small (less than or equal to 5 mm diam.) healthy follicles were indistinguishable from cells of atretic follicles in terms of their ability to produce inhibin and to aromatize androgen. However, granulosa cells from healthy and atretic follicles, irrespective of size, differed markedly in their morphological appearance after culture for 24 h. Testosterone (1 microgram/ml) stimulated inhibin production by granulosa cells from healthy and atretic follicles while FSH (100 ng/ml) stimulated inhibin production by granulosa cells from healthy follicles only. The relative ability of granulosa cells from different sizes of healthy and atretic follicles to produce inhibin in vitro was reflected in inhibin concentrations in follicular fluid. There was a significant positive correlation between inhibin concentration in follicular fluid and the number of granulosa cells per follicle. There was also a significant positive correlation between follicular diameter and inhibin concentration in follicular fluid, but only in healthy follicles. These findings show that both follicular size and atresia influence follicular inhibin production.

Effect of neuraminidase on inhibin activity in vivo and in vitro.

Matrex gel red A purified follicular fluid has been used to study whether or not this material contains sialic acid residues and their importance in maintaining the biological activity of inhibin both in vitro and in vivo. It appears that sialic acid is present in these preparations and can be released either by neuraminidase treatment of acid hydrolysis. The addition of intact and desialylated inhibin-containing material to isolated rat pituitary cells in culture gives similar inhibition of LHRH-induced FSH release of these cells indicating that sialic acid is not required for inhibin activity in vitro. The injection of intact inhibin preparations leads to a reduction of the uterine weight increase seen in immature female mice primed with human chorionic gonadotropin. By contrast, the inhibition of this uterine weight increment by 80% desialylated inhibin-containing material is significantly reduced, suggesting that sialic acid residues play an important role in maintaining the biological activity of inhibin in vivo.

[Intracellular mechanism for the action of inhibin on the secretion of the follicular-stimulating hormone and luteinizing hormone induced by LH-RH in vitro]. / Un mécanisme intracellulaire de l'action de l'inhibine sur la sécrétion de l'hormone folliculo-stimulante et de l'hormone lutéinisante induite par le LH-RH in vitro.

The FSH secretion-inhibiting action of inhibin in vitro under basal conditions and also in the presence of LH-RH is suppressed by the addition of MIX, a phosphodiesterase inhibitor. In the presence of LH-RH, inhibin reduces significantly the intracellular level of cAMP in isolated pituitary cells. In contrast, the simultaneous addition of MIX and inhibin raises the cAMP level, and this stimulation is comparable to the increase observed when MIX is added alone. These observations suggest that one mode of action of inhibin could be mediated by a reduction in cAMP within the pituitary gonadotropic cell.