Michelle Myers

HCG up-regulates hypoxia inducible factor-1 alpha in luteinized granulosa cells: implications for the hormonal regulation of vascular endothelial growth factor A in the human corpus luteum

Vascular endothelial growth factor (VEGF)-dependent angiogenesis is essential for normal luteal development. Although it is believed that hypoxia is the primary inducer of VEGF, in the corpus luteum it is up-regulated by human chorionic gonadotrophin (hCG). As hypoxia-inducible factor (HIF)1A has been shown to regulate VEGFA under ligand-stimulated conditions, we hypothesized that the effect of hCG on luteal VEGFA was mediated through HIF1A. We studied the effect of hCG on VEGFA and HIF1A expression in human luteinized granulosa cells in vitro and in human corpora lutea in vivo. HCG up-regulated VEGFA (P < 0.05) and HIF1A (P < 0.001) in vitro and VEGFA (P < 0.05) and HIF1A (P < 0.05) in vivo. There was a correlation between HIF1A and VEGFA in vivo (P < 0.005) and in vitro (P < 0.05). Nuclear HIF1A in granulosa-lutein cells was highest during luteal formation and absent from the fully functional corpus luteum (P < 0.05). Both VEGFA (P < 0.001) and HIF1A (P < 0.01) were up-regulated by dibutyryl-cAMP, through a PKA pathway. Hypoxia increased VEGFA (P < 0.001) and HIF1A (P < 0.05) expression and hCG further augmented VEGFA (P < 0.001) and HIF1A (P < 0.01) under hypoxic conditions. However, progesterone increased hCG-stimulated VEGFA but had no effect on HIF1A expression. The expression of HIF1A is therefore hormonally regulated in luteal cells in vitro and in vivo and may regulate VEGFA expression under normoxic and hypoxic conditions. However, the differential effects of progesterone suggest that not all regulation of VEGFA is associated with an up-regulation of HIF1A.

The primordial follicle reserve is not renewed after chemical or γ-irradiation mediated depletion

Reports indicate that germ-line stem cells present in adult mice can rapidly generate new oocytes and contribute to the primordial follicle reserve following conditions of ovotoxic stress. We further investigated the hypothesis that adult mice have the capacity to generate new oocytes by monitoring primordial follicle numbers throughout postnatal life and following depletion of the primordial follicle reserve by exposure to doxorubicin (DXR), trichostatin A (TSA), or whole-body γ-irradiation. We show that primordial follicle number remains stable in adult C57BL/6 mice between the ages of 25 and 100 days. However, within 2 days of treatment with DXR or TSA, primordial follicle numbers had declined to 65 and 51% respectively (P<0.05-0.01 when compared to untreated controls), with no restoration of follicle numbers evident after 7 days for either treatment. Furthermore, ovaries from mice subjected to sterilizing doses of γ-irradiation (0.45 or 4.5 Gy) revealed complete ablation of all primordial follicles 5 days after treatment, with no indication of follicular renewal. We conclude that neo-folliculogenesis does not occur following chemical or γ-irradiation mediated depletion of the primordial follicle reserve.

Endometrial Inhibin/Activin β-B Subunit Expression Is Related to Decidualization and Is Reduced in Tubal Ectopic Pregnancy

CONTEXT Ectopic pregnancy is common but remains difficult to diagnose accurately. There is no serum test to differentiate ectopic from intrauterine gestation. OBJECTIVE Our objective was to investigate differential gene expression in decidualized endometrium of ectopic pregnancy. DESIGN Tissue and serum analysis informed by microarray study was performed. SETTING The study was performed at a large United Kingdom teaching hospital. PATIENTS OR OTHER PARTICIPANTS Women undergoing surgical termination of pregnancy (n = 8), evacuation of uterus for miscarriage (n = 6), and surgery for tubal ectopic pregnancy (n = 11) were included in the study. Endometrium was collected from normally cycling women undergoing hysterectomy. INTERVENTIONS Decidualized endometrium was subjected to microarray analysis, morphological assessment, and immunohistochemistry. Endometrial stromal fibroblasts were cultured in the presence of decidualizing stimuli. MAIN OUTCOME MEASURES Differential expression of potentially secreted molecules was calculated. RESULTS Inhibin/activin beta-B expression was lower in decidualized endometrium from ectopic pregnancies when compared with that of ongoing pregnancies (P < 0.01) or miscarriages (P < 0.01). The localization of the beta-B subunit was more marked in decidualized than nondecidualized stroma. Decidualization of stromal fibroblasts in vitro was associated with increased beta-B expression (P < 0.05). Endometrial stroma of ectopic pregnancies was less decidualized morphologically (P < 0.05), with lower prolactin (P < 0.01) and IGF binding protein-1 (P < 0.005) expression. Serum activin B was lower in ectopic pregnancies (P < 0.005) than in intrauterine pregnancies, whereas there was no difference in progesterone concentrations. CONCLUSIONS Despite similar concentrations of progesterone, the endometrium of ectopic pregnancies is less decidualized than intrauterine pregnancies. Expression of the beta-B subunit is related to decidualization and can be detected in the circulation as activin B. Serum activin B concentrations are lower in ectopic pregnancy.

Activin A reduces luteinisation of human luteinised granulosa cells and has opposing effects to human chorionic gonadotropin in vitro.

The transition of the dominant follicle into the corpus luteum is of fundamental reproductive importance. Luteinisation involves disparate changes in the gene expression of follicular granulosa cells that differentiate into the granulosa-lutein cells of the corpus luteum after the gonadotrophin surge. We have shown that activin and human chorionic gonadotropin (hCG) have opposing effects during luteolysis. Therefore, we hypothesised that activin A was an inhibitor of luteinisation that was blocked during the pre-ovulatory gonadotrophin surge. Ovarian tissue and cells were collected from women with regular cycles having hysterectomy and women undergoing oocyte retrieval for assisted conception. Genes that changes during luteinisation were investigated in primary cultures of luteinised granulosa cells exposed to activin A and hCG in vitro. hCG promotes a luteinised granulosa cell phenotype, while activin A promotes a more follicular phenotype in luteinised cells by upregulating granulosa cells markers such as FSHR, HSD11B2 and downregulating LHCGR. In addition, activin A blocked hCG upregulation of STAR, HSD3B1 and HSD11B1 and downregulation of oestrogen receptor alpha. Activin A antagonised hCG effects in a dose-dependent manner and could block the hCG-stimulated molecular inhibitors of activin action (inhibin alpha-subunit, follistatin and TGFBR3). These studies show that hCG and activin A have opposing effects on luteinised granulosa cells and some effects of activin are seen only in the presence of hCG. While hCG can inhibit activin action in granulosa cells to facilitate luteinisation, activin A can promote an unluteinised phenotype in luteinised granulosa cells. This confirms the importance of adequate activin withdrawal during luteinisation in women.

A new ‘total’ activin B enzyme-linked immunosorbent assay (ELISA): development and validation for human samples

Background and objective  There are currently no sensitive and specific assays for activin B that could be utilized to study human biological fluids. The aim of this project was to develop and validate a ‘total’ activin B ELISA for use with human biological fluids and establish concentrations of activin B in the circulation and fluids from the reproductive organs.

A new 'total' activin B enzyme-linked immunosorbent assay (ELISA)

Background and objective  There are currently no sensitive and specific assays for activin B that could be utilized to study human biological fluids. The aim of this project was to develop and validate a ‘total’ activin B ELISA for use with human biological fluids and establish concentrations of activin B in the circulation and fluids from the reproductive organs.

Expression and regulation of oestrogen receptors in the human corpus luteum

The molecular mechanisms underlying the control of corpus luteum lifespan in women are not fully understood. Oestradiol has various luteolytic, or luteotrophic, functions in some species, and as it is synthesised within the human corpus luteum, it is an excellent candidate molecule to be a paracrine regulator of luteal function. This study aimed to comprehensively investigate the expression, regulation and effects of oestrogen receptors (ER) in human luteal cells. Genomic oestrogen receptors ERalpha, ERbeta1 and ERbeta2 were immunolocalised in human corpora lutea from throughout the luteal phase. mRNA expression was investigated throughout the luteal phase and after luteal rescue with exogenous human chorionic gonadotrophin (hCG). The regulation of ER expression and oestradiol action was investigated in cultures of luteinised granulosa cells. ER subtypes ERbeta1 and ERbeta2 were localised throughout the luteal phase to steroidogenic cells in the human corpus luteum and cells of the surrounding stroma. Unlike follicular granulosa cells, steroidogenic cells in the corpus luteum showed minimal ERalpha immunostaining. The presence of endothelial cells in the granulosa cell layer with ERbeta1 and ERbeta2 positive nuclei was noted. ERbeta1 and ERbeta2 were differentially regulated across the luteal phase with ERbeta1 maximally expressed in the mid-luteal phase, while ERbeta2 expression was maximal in the early luteal phase. In vivo and in vitro, hCG had no long-term effect on ER expression, although in vitro hCG and oestradiol acutely down-regulated ERs. Treatment with oestradiol in vitro down-regulated 11beta-hydroxysteroid dehydrogenase type 1 and inhibin betaA subunit confirming a functional oestradiol response. These data highlight functional and differentially regulated oestradiol reception in human luteal cells.

The hypogonadal (hpg) mouse as a model to investigate the estrogenic regulation of spermatogenesis

The hypogonadal (hpg) mouse is an excellent animal model in which to investigate the mechanism of action of estrogens on spermatogenesis because it has arrested reproductive development without the need for surgical, endocrine, pharmacological or immunological intervention. Hpg mice are hypogonadotrophic and fail to show normal postnatal testicular development due to the congenital inability to synthesize gonadotropin-releasing hormone in the hypothalamus. The hpg testis remains responsive to gonadotropins and androgens in that fertility can be induced by treatment with these hormones. Surprisingly, chronic treatment with low concentrations of estradiol alone induces qualitatively normal spermatogenesis. The induction of testicular development by estradiol in hpg mice is accompanied by a paradoxical increase in FSH production. The actions of estradiol in hpg mice appear to be via genomic estrogen receptors, as concurrent treatment with estrogen-receptor antagonist ICI182,780 completely blocks these pituitary and testis responses. Concurrent treatment with the androgen receptor antagonist bicalutamide does not affect the estradiol-induced increase in pituitary FSH content, but markedly attenuates the estradiol-induced increase in testicular weight. Western blot analyses and immunohistochemistry provide evidence for estrogen-receptor α and β expression in both pituitary gland and testis of the hpg mouse. Estradiol may therefore exert direct actions within the testes and/or indirect neuroendocrine actions via the release of FSH or other hormones from the pituitary gland, but its actions are dependent upon the availability of low levels of androgen within the testis.