Distinct regulation of gene expression by prostaglandin F(2alpha) (PGF(2alpha)) is associated with PGF(2alpha) resistance or susceptibility in human granulosa-luteal cells

The effects of human chorionic gonadotrophin (HCG) and prostaglandin F(2alpha) (PGF(2alpha)) on regulation of human granulosa-luteal cell (GLC) function at different stages of differentiation (day 2 versus day 8 of culture) were studied. Expression of LH receptor mRNA and biosynthesis of progesterone were HCG dependent in human GLC at all stages (n = 6, P < 0.05). Steady-state concentrations of mRNA encoding for FP (a specific high-affinity plasma membrane receptor for PGF(2alpha)) were not dependent on, but were stimulated by, addition of HCG (10 IU/ml) or 8-bromo-cAMP (0.5 mmol/l) (n = 6, P < 0.05). Treatment with PGF(2alpha) (100 nmol/l) decreased FP mRNA concentration, but had no effect on LH receptor and cyclo oxygenase-2 (COX-2) expression on day 2 of cultured GLC (n = 8). As a result, the progesterone biosynthesis by GLC was not affected. On day 8, PGF(2alpha) induced FP and PGHS-2 expression and at the same time decreased LH receptor expression, resulting in inhibition of progesterone output by GLC. Our data demonstrated that early stage GLC (day 2 of culture) are resistant to PGF(2alpha)-induced inhibition of progesterone synthesis but underwent further differentiation and acquired luteolytic capacity after 8 days culture in vitro. We conclude that, via distinct gene regulation at different stages of differentiation, human GLC may become resistant or susceptible to PGF(2alpha)-induced luteolysis.     

Steroidogenic enzyme expression in human corpora lutea in the presence and absence of exogenous human chorionic gonadotrophin (HCG).

In a human conception cycle, the expected decline in progesterone production by the corpus luteum during the late luteal phase is prevented by human chorionic gonadotrophin (HCG) secreted by the implanting blastocyst. This study investigated the expression of components of the synthetic pathway for progesterone in human corpora lutea in the presence and absence of HCG in vivo. Corpora lutea were obtained from: (i) normally cycling women at the time of hysterectomy and classified on the basis of the urinary luteinizing hormone (LH) surge as early (n = 3), mid- (n = 3), or late luteal (n = 3); or (ii) women who had received daily doubling doses of HCG (n = 3) to 'rescue' the corpus luteum. Expression patterns of steroidogenic acute regulatory protein (StAR), cytochrome P450 cholesterol side-chain cleavage (P450scc) and 3beta-hydroxysteroid dehydrogenase (3beta-HSD) were investigated by Northern blotting, in-situ hybridization and immunohistochemistry. Luteal 'rescue' with HCG was associated with the continued expression of these components. In the late luteal phase, in the absence of HCG, expression remained but was more variable. The expression of 3beta-HSD mRNA was significantly reduced during the luteal phase (P<0.01). In conclusion, during luteal 'rescue', HCG acts to maintain the steroidogenic pathway. In the absence of HCG, the decline in progesterone production begins in the presence of the main components of the steroidogenic pathway. While unlikely to initiate this decline, the altered expression levels of these components, particularly that of 3beta-HSD, may contribute to the continued reduction in progesterone production.     

The human corpus luteum: reduction in macrophages during simulated maternal recognition of pregnancy

It has been shown that immune cells, particularly macrophages, accumulate in the corpus luteum during luteolysis. This study aimed to investigate the effect of maternal recognition of pregnancy on the localization and numbers of macrophages in the human corpus luteum. Corpora lutea (n = 12) were obtained from normally cycling women at the time of hysterectomy and were dated on the basis of serial urinary luteinizing hormone (LH) estimation. In addition, corpora lutea (n = 4) were collected from women who had received daily doubling doses of human chorionic gonadotrophin (HCG) to mimic the hormonal changes of early pregnancy. Macrophages were localized by immunohistochemistry using an anti-CD68 antibody. Steroidogenic cells, steroidogenic cells of thecal origin and endothelial cells were identified on serial sections by immunohistochemistry for 3beta-hydroxysteroid dehydrogenase, 17alpha-hydroxylase and von Willebrand factor, respectively. The luteal cells capable of responding directly to HCG were identified by isotopic in-situ hybridization for messenger RNA encoding LH/HCG receptors. Macrophages were localized primarily to the vascular connective tissue and theca-lutein areas of the corpus luteum, although some were found in the granulosa-lutein cell layer. Macrophage numbers increased throughout the luteal phase to a maximum in the late- luteal phase (P < 0.05). Luteal 'rescue' with HCG was associated with a marked reduction in the numbers of tissue macrophages when compared with those of the late-luteal phase (P < 0.001). One of the effects of HCG during maternal recognition of pregnancy is to prevent the normal influx of macrophages into the corpus luteum. As LH/HCG receptors localized to the steroidogenic cells, this implies a fundamental role for steroidogenic cell products in the control of macrophage influx into the human corpus luteum.      

The newly formed corpora lutea of normal cycling rats exhibit drastic changes in steroidogenic and luteolytic gene expressions

In normal estrous cycling rats, corpora lutea (CL) regress over several cycles; however, the period during which they secrete progesterone (P4) is strictly limited. In the present study, we clarified the function of CL in normal cycling rats. We especially focused on expression levels of four steroidogenic and two luteolytic genes in the two different populations of the CL (new and old CL) at each estrous stage. The ovaries of female rats at each estrous cycle were collected, and new and old CL were separated with laser microdissection and analyzed for mRNA expression. In the new CL, the expressions of scavenger receptor class B type I (SR-BI), steroidogenic acute regulatory protein (StAR), and P450 cholesterol side-chain cleavage (P450scc) mRNA reached their highest levels at metestrus, and 3β-hydroxysteroid dehydrogenase (3β-HSD) mRNA gradually increased from estrus to diestrus. Meanwhile, 20α-hydroxysteroid dehydrogenase (20α-HSD) and prostaglandin F2 alpha receptor (PGF2α-R) mRNA levels were remarkably low from estrus to metestrus and gradually increased thereafter. These gene levels in new CL corresponded to serum P4 levels during the estrous cycle. In the old CL, all steroidogenic and luteolytic gene levels were consistently high throughout the estrous cycle. These results provide clear evidence that new CL at metestrus have strong steroidogenic activity and through inhibition of luteolysis, maintain P4 production in normal cycling rats. The elevation of 20α-HSD and PGF2α-R levels in new CL at diestrus may be a trigger of functional luteolysis.     

Luteolysis: a neuroendocrine-mediated event

In many nonprimate mammalian species, cyclical regression of the corpus luteum (luteolysis) is caused by the episodic pulsatile secretion of uterine PGF2alpha, which acts either locally on the corpus luteum by a countercurrent mechanism or, in some species, via the systemic circulation. Hysterectomy in these nonprimate species causes maintenance of the corpora lutea, whereas in primates, removal of the uterus does not influence the cyclical regression of the corpus luteum. In several nonprimate species, the episodic pattern of uterine PGF2alpha secretion appears to be controlled indirectly by the ovarian steroid hormones estradiol-17beta and progesterone. It is proposed that, toward the end of the luteal phase, loss of progesterone action occurs both centrally in the hypothalamus and in the uterus due to the catalytic reduction (downregulation) of progesterone receptors by progesterone. Loss of progesterone action may permit the return of estrogen action, both centrally in the hypothalamus and peripherally in the uterus. Return of central estrogen action appears to cause the hypothalamic oxytocin pulse generator to alter its frequency and produce a series of intermittent episodes of oxytocin secretion. In the uterus, returning estrogen action concomitantly upregulates endometrial oxytocin receptors. The interaction of neurohypophysial oxytocin with oxytocin receptors in the endometrium evokes the secretion of luteolytic pulses of uterine PGF2alpha. Thus the uterus can be regarded as a transducer that converts intermittent neural signals from the hypothalamus, in the form of episodic oxytocin secretion, into luteolytic pulses of uterine PGF2alpha. In ruminants, portions of a finite store of luteal oxytocin are released synchronously by uterine PGF2alpha pulses. Luteal oxytocin in ruminants may thus serve to amplify neural oxytocin signals that are transduced by the uterus into pulses of PGF2alpha. Whether such amplification of episodic PGF2alpha pulses by luteal oxytocin is a necessary requirement for luteolysis in ruminants remains to be determined. Recently, oxytocin has been reported to be produced by the endometrium and myometrium of the sow, mare, and rat. It is possible that uterine production of oxytocin may act as a supplemental source of oxytocin during luteolysis in these species. In primates, oxytocin and its receptor and PGF2alpha and its receptor have been identified in the corpus luteum and/or ovary. Therefore, it is possible that oxytocin signals of ovarian and/or neural origin may be transduced locally at the ovarian level, thus explaining why luteolysis and ovarian cyclicity can proceed in the absence of the uterus in primates. However, it remains to be established whether the intraovarian process of luteolysis is mediated by arachidonic acid and/or its metabolite PGF2alpha and whether the central oxytocin pulse generator identified in nonprimate species plays a mediatory role during luteolysis in primates. Regardless of the mechanism, intraovarian luteolysis in primates (progesterone withdrawal) appears to be the primary stimulus for the subsequent production of endometrial prostaglandins associated with menstruation. In contrast, luteolysis in nonprimate species appears to depend on the prior production of endometrial prostaglandins. In primates, uterine prostaglandin production may reflect a vestigial mechanism that has been retained during evolution from an earlier dependence on uterine prostaglandin production for luteolysis.     

Dynamic expression of mRNAs and proteins for matrix metalloproteinases and their tissue inhibitors in the primate corpus luteum during the menstrual cycle

Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) may be involved in tissue remodelling in the primate corpus luteum (CL). MMP/TIMP mRNA and protein patterns were examined using real-time PCR and immunohistochemistry in the early, mid-, mid-late, late and very late CL of rhesus monkeys. MMP-1 (interstitial collagenase) mRNA expression peaked (by >7-fold) in the early CL. MMP-9 (gelatinase B) mRNA expression was low in the early CL, but increased 41-fold by the very late stage. MMP-2 (gelatinase A) mRNA expression tended to increase in late CL. TIMP-1 mRNA was highly expressed in the CL, until declining 21-fold by the very late stage. TIMP-2 mRNA expression was high through the mid-luteal phase. MMP-1 protein was detected by immunocytochemistry in early steroidogenic cells. MMP-2 protein was prominent in late, but not early CL microvasculature. MMP-9 protein was noted in early CL and labelling increased in later stage steroidogenic cells. TIMP-1 and -2 proteins were detected in steroidogenic cells at all stages. Thus, MMPs and TIMPs are dynamically expressed in a cell-specific manner in the primate CL. Early expression of MMP-1 is suggestive of a role in tissue remodelling associated with luteinization, whereas MMP-2 and -9 may contribute to later stage luteolysis. TIMP expression may control MMP activity, until declining at luteolysis.     

Localization of plasminogen activator and inhibitor, LH and androgen receptors and inhibin subunits in monkey epididymis

Epididymis is a site of sperm maturation and storage. Limited and directed-proteolysis regulated by plasminogen activator (PA), plasminogen activator inhibitor type-1 (PAI-1) and other related factors may play an essential role in these processes. Our previous studies have demonstrated that rat epididymis expressed luteinizing hormone receptor (LHR), tissue type (t) and urokinase type (u)PA, mRNAs, and tPA activity was stimulated in vitro by human chorionic gonoadotrophin (HCG). In the present study we further examined localization of mRNAs for tPA, uPA, LHR, androgen receptor (AR), as well as inhibin subunits alpha, betaA and betaB in rhesus monkey epididymis. Using in-situ hybridization with digoxygenin-labelled cRNA probes, we have demonstrated that tPA and PAI-1 mRNAs were localized in epithelial cells of adult monkey epididymis. uPA mRNA was localized in the same areas, but to a much smaller extent. tPA, uPA and PAI-1 mRNAs were greatly expressed in the caput and corpus of adult epididymis than in other regions. In-vitro experiments showed that both tPA and uPA activities in epididymal cells were dramatically stimulated by HCG, but not by follicle stimulating hormone (FSH). LHR (but not FSH receptor) and AR mRNAs were localized in the epithelial cells of the epididymis. However, LHR mRNA was detected in both adult and immature infant monkeys, whereas AR was found only in the adult. Inhibin alpha, betaA and betaB mRNAs were also detected in this organ, betaA mRNA being more strongly expressed in the caput than in other regions of the epididymis. We suggest that LH and androgen may be the key hormones in coordination with the PA-PAI-1 system in regulating epididymal differentiation and sperm maturation.      

Bone Morphogenetic Protein‐2 (BMP‐2) Increases Gene Expression of FSH Receptor and Aromatase and Decreases Gene Expression of LH Receptor and StAR in Human Granulosa Cells

Citation Shi J, Yoshino O, Osuga Y, Koga K, Hirota Y, Nose E, Nishii O, Yano T, Taketani Y. Bone morphogenetic protein‐2 (BMP‐2) increases gene expression of FSH receptor and aromatase and decreases gene expression of LH receptor and stAR in human granulosa Cells. Am J Reprod Immunol 2011; 65: 421–427 Problem A growing body of evidence indicates that bone morphogenetic protein (BMP) cytokines play a key role in female fertility in mammals. BMP‐2 is known to be expressed in the ovary of many species. In the present study, we examined the expression and function of BMP‐2 in the human ovary. Method of Study BMP‐2 mRNA expression in the human ovary was evaluated by in situ hybridization. Human granulosa cells were obtained from in vitro fertilization patients. Human granulosa cells were cultured with recombinant BMP‐2 or human chorionic gonadotrophin (HCG), followed by RNA extraction. Results BMP‐2 expression was detected in granulosa cells of antral follicles but not of corpus luteum. The in vitro study showed that BMP‐2 induced follicular stimulating hormone (FSH) receptor and aromatase expression, while decreasing luteinizing hormone (LH) receptor and steroidogenic acute regulatory protein expression in human granulosa cells. HCG decreased gene expression of BMP‐2 and increased BMP and activin membrane‐bound inhibitor (BAMBI), an antagonist of BMP‐2. Conclusion Expression and disappearance of BMP‐2 might contribute to folliculogenesis and luteinization by regulating gonadotropin receptor expression in human granulosa cells. HCG can modulate BMP‐2 function by controlling BMP‐2 and BAMBI expression.     

HCG concentration and receptor gene expression in placental tissue from trisomy 18 and 21

Trisomy 21 is associated with high maternal serum concentrationsof intact human chorionic gonadotrophin (HCG) and free ß-HCGwhereas these concentrations are markedly decreased in trisomy18. In this study, we investigated the effect of trisomy 21and 18 on endogenous HCG concentrations and luteinizing hormone(LH)/HCG receptor expression in placental villous tissue ineight trisomy 21, six trisomy 18 and 42 chromosomally normalsamples, collected at 12–16 weeks gestation. The tissueconcentrations of intact HCG, free -HCG and free ß-HCGsubunits were measured using solid-phase two-site immunoradiometricassay. LH/HCG receptor expression was evaluated with immunohistochemistryand in-situ hybridization. Villous tissue in trisomy 21 containedhigher ß-HCG concentrations than the controls (P <0.05). In trisomy 18 cases, the ß-HCG concentration waslower than in the control group (P < 0.01). Both immunocytochemistryand in-situ hybridization demonstrated a more intense stainingof the trophoblast in cases of trisomy 21 and 18, compared withcontrols with the strongest signal in cases of trisomy 18 (P< 0.01). We concluded that in trisomy 21 the high tissueHCG concentration and expression of LH/HCG receptor in the trophoblastmay reflect the relative immaturity of the trophoblastic tissuewhereas in trisomy 18, the very low concentration of endogenousHCG, associated with an over-expression of LH/HCG receptor inthe trophoblast, is probably secondary to the poor differentiationof the cytotrophoblast. HCG/placenta/pregnancy/receptors/trisomy Notes ⁴ To whom correspondence should be addressed at: Academic Departmentof Obstetrics and Gynaecology, University College London MedicalSchool, 86–96 Chenies Mews, London WC1E 6HX, UK     

Effects of ovariohysterectomy on prostaglandin F2alpha-induced nesting behaviour in pigs

Exogenously administered prostaglandin (PG) F2alpha induces behaviour similar to prepartum nest building in pregnant, pseudopregnant and nonpregnant female postpubescent pigs (Sus scrofa). These effects may be regulated by PGF2alpha-induced endocrine changes within the reproductive tract, such as those that initiate luteolysis. This study investigated the short-term effects of ovariohysterectomy on PGF2alpha-induced nesting behaviour in nonpregnant females. Cyclic 9-month-old virgin female pigs (gilts) received an oral dose (20 mg/day) of a synthetic progestogen (altrenogest; Regumate porcine, Hoechst, Milton Keynes, UK) for 18-21 days to synchronize oestrus. The gilts were then ovariohysterectomized (n=8) or sham-operated (n=7) on Days 3-8 after oestrus. They were housed individually and initially subjected to a series of control behavioural tests to establish the effect of ovariohysterectomy on their responses to the experimenters, novel objects, straw bedding and space restriction. Ovariohysterectomized gilts had a shorter latency to approach the experimenters than sham-operated animals, but there were no differences in their responses to a novel object, straw bedding or space restriction. Twelve to 16 days after oestrus, corresponding to the midluteal phase in sham-operated gilts, they were treated intramuscularly with 15 mg PGF2alpha (0.12 mg/kg, dinoprost; Lutalyse, Upjohn, Crawley, UK). PGF2alpha treatment induced a significant increase in straw gathering in ovariohysterectomized but not in sham-operated gilts. Other nesting behaviours, including rooting and pawing at straw, were induced in all animals. These results show that the uterus and ovaries are not required for the expression of PGF2alpha-induced nesting behaviour and the removal of the reproductive tract appears to have facilitated increased levels of gathering. This suggests that PGF2alpha induces luteolysis and nest building separately, and that PGF2alpha or a metabolite, may act centrally to mediate directly its effects on prepartum nest building in the pig.     

GnRH receptor mRNA expression by in-situ hybridization in the primate pituitary and ovary

Gonadotrophin-releasing hormone (GnRH) receptors are presenton the ovary as well as in the anterior pituitary gland. GnRHanalogues may exert their actions in part via these ovarianreceptors. However, in the primate ovary, GnRH receptors areof low affinity and their significance is questionable. Theaim of the present study was to compare pituitary and ovarianexpression of the GnRH receptor mRNA by in-situ hybridizationto gain further information on the possible significance ofthe ovarian receptor. Pituitaries and ovaries were obtainedfrom two stump-tailed macaque monkeys and three marmoset monkeysat the mid-luteal phase of the ovulatory cycle. Human corporalutea were obtained during the early and mid-luteal phase andafter ‘rescue’ by human chorionic gonadotrophin(HCG) and a whole ovary obtained during the late luteal phase(n=1 per group). Frozen tissue sections were incubated witha 33P-labelled probe to the human GnRH receptor and exposedfor 4 weeks. All pituitary glands exhibited intense silver grainsin the anterior pituitary gland. In the ovaries, grains werepresent at low levels in the granulosa cells of antral follicles,just above tissue background in corpora lutea and indistinguishablefrom tissue background in the remaining ovarian compartments.These results demonstrate that the GnRH receptor mRNA in theprimate pituitary is present in sufficient quantities to beclearly detectable in the anterior pituitary gland by in-situhybridization. In contrast, in the human and monkey, ovary levelsof mRNA appear to be very low. corpus luteum/follicle/GnRH mRNA/in-situ hybridization     

Primary pigmented nodular adrenocortical disease (PPNAD): immunohistochemical and in situ hybridization analysis of steroidogenic enzymes in eight cases.

Primary pigmented nodular adrenocortical disease (PPNAD) is a rare but an interesting adrenocortical disorder associated with ACTH-independent hypercortisolism. We have studied eight cases of the adrenals with PPNAD by immunohistochemistry of all steroidogenic enzymes involved in cortisol biosynthesis (P-45scc, 3 beta-HSD, P-450c21, P-45017 alpha, and P-45011 beta) and also by performing in situ hybridization of P-45017 alpha in seven cases in order to localize the sites of specific steroidogenesis in this unique disorder. Immunoreactivity of all the enzymes examined was intense in almost all of the cells in adrenocortical nodules, especially the cells with abundant eosinophilic cytoplasm in all the cases examined. The internodular cortex, which demonstrated atrophy in five cases, normal appearance in two cases and hyperplasia in one case, was negative for the enzymes with an exception of 3 beta-HSD. Hybridization signals of P-45017 alpha were condensed over the nodules in in situ hybridization study, suggestive of an increased production of the enzyme itself in cortical cells of the nodules. These results may be consistent with autonomous cortisol production by the nodular cells and indicate that almost all of the cells in the nodules produce cortisol, which can also explain the presence of hypercortisolism despite small sizes of adrenals in PPNAD. Immunoreactivity of steroidogenic enzymes is observed in a small cluster of cortical cells with abundant eosinophilic cytoplasm located at the zona reticularis but not in adjacent non-nodular cortex, which may support an abnormal development of the zona reticularis as a possible pathogenesis of this disorder.     

In Situ Hybridization Method Reveals (Pro)renin Receptor Expressing Cells in the Pituitary Gland of Rats: Correlation with Anterior Pituitary Hormones

Expression of (pro)renin receptor ((P)RR), a specific receptor for renin and prorenin, was studied in rat pituitary gland. In situ hybridization showed that cells expressing (P)RR mRNA were widely distributed in the anterior lobe and intermediate lobe of the pituitary gland. Double-staining using in situ hybridization for (P)RR mRNA and immunohistochemistry for the pituitary hormones showed that (P)RR mRNA was expressed in most of the GH cells and ACTH cells in the anterior lobe. (P)RR mRNA was also expressed in a few prolactin cells and TSH cells, but not in LH cells. The present study has shown for the first time the distribution of (P)RR mRNA expressing cells in the rat pituitary gland. These findings suggest that (P)RR plays physiological roles in the pituitary gland, such as the modulation of the pituitary hormone secretion.     

Alternative splicing of the human luteal LH receptor during luteolysis and maternal recognition of pregnancy

Deletion of exon 10 of the human LH receptor impairs LH but not hCG action. Other splice variants of the LH receptor impair both LH and hCG action in other species. We hypothesized that alternatively spliced LH receptors are involved in luteolysis and luteal rescue with hCG in women. mRNA was extracted from human luteinized granulosa cells and from corpora lutea from across the luteal phase and after luteal rescue in vivo with exogenous hCG. Splice variants were detected by RT-PCR using carefully designed primer pairs. Products were visualized on agarose gels, extracted, purified and sequenced. Three splice variants of the human LH receptor were detected and characterized. These demonstrate a region of multiple splicing between exons 8 and 11 of the receptor. A naturally occurring splice variant with exon 10 alone removed was not identified. There was no obvious change in the pattern of splice variants across the luteal phase in the presence or absence of hCG. These data do not support the hypothesis that qualitative changes in LH receptor splicing have a role in luteolysis or that a naturally occurring LH receptor lacking exon 10 has a role in maternal recognition of pregnancy.