Control Of Ovarian Function Research Articles

Assessment of Epiregulin Effect and its Combination with Gonadotropins on Proliferation, Apoptosis, and Secretory Activity by Human Ovarian Cells.

The release of epidermal growth factor ligand epiregulin (EREG) by human ovarian granulosa cells, its direct action on basic ovarian cell functions, and interrelationships with gonadotropins were investigated. We examined (1) the ovarian production of EREG (the time-dependent accumulation of EREG in the medium incubated with human ovarian granulosa cells, and (2) the effect of the addition of EREG (0, 1, 10, and 100ng.ml-1) given alone or in combination with FSH or LH (100ng.ml-1) on basic granulosa cells functions. Viability, proliferation (accumulation of PCNA and cyclin B1) and apoptosis (accumulation of bax and caspase 3), the release of steroid hormones (progesterone, testosterone, and estradiol), and prostaglandin E2 (PGE2) were analyzed by using the Trypan blue exclusion test, quantitative immunocytochemistry, and ELISA. A significant time-dependent accumulation of EREG in a medium cultured with human granulosa cells with a peak at 3 and 4days was observed. The addition of EREG alone increased cell viability, proliferation, progesterone, testosterone, and estradiol release, decreased apoptosis, bud did not affect PGE2 release. The addition of either FSH or LH alone increased cell viability, proliferation, progesterone, testosterone, estradiol, and PGE2 release and decreased apoptosis. Furthermore, both FSH and LH mostly promoted the stimulatory action of EREG on granulosa cell functions. These results demonstrated, that EREG produced by ovarian cells can be an autocrine/paracrine stimulator of human ovarian cell functions. Furthermore, they demonstrate the functional interrelationship between EREG and gonadotropins in the control of ovarian functions.

Involvement of microRNA miR-125b in the control of porcine ovarian cell functions

The existing knowledge of the involvement of miR-125b in the control of ovarian functions is insufficient. To evaluate the role of miR-125b in the control of basic porcine ovarian granulosa cell functions, we examined the upregulation (using miR-125b mimics) and downregulation (using miR-125b inhibitor) of this miR-125b. Expression levels of miR-125b, viability, proliferation (expression and accumulation of PCNA and cyclin B1), the proportion of proliferative active cells, apoptosis (expression and accumulation of bax and caspase 3), the proportion of cells containing DNA fragmentation, steroid hormones, IGF-I, oxytocin, and prostaglandin E2 release were analysed by RT-qPCR, Trypan blue exclusion test, quantitative immunocytochemistry, XTT and TUNEL assays, and ELISA. Transfection of cells with miR-125b mimics decreased cell viability, proliferation, and the release of progesterone, testosterone, estradiol, and oxytocin, but stimulated apoptosis and prostaglandin E2 output. Transfection of cells with miR-125b inhibitor had the opposite effect. Moreover, it prevented the effects of miR-125b mimics. Our observations suggest that miR-125b is a potent physiological inhibitor of granulosa ovarian cell functions – cell cycle, apoptosis, and secretory activity.

Apelin and Apelin Receptor in Follicular Granulosa Cells of Buffalo Ovaries: Expression and Regulation of Steroidogenesis.

Apelin (APLN), as a ligand for APJ (an orphan G-protein-coupled receptor), is an adipokine with pleiotropic effects in many physiological processes of the body. It has an important role in the control of reproduction particularly in females (mainly in control of ovarian function). This study was carried out to investigate the mRNA and protein amounts of APLN/APJ in granulose cells (GCs) of ovarian follicles with small (SF), medium (MF), and large (LF) sizes of buffalo (Bubalus bubalis) and the effect of IGF1 and follicle-stimulating hormone (FSH) on the expression levels of APLN/APJ. In addition, we evaluated the effect of various doses of APLN (isoforms -13 and -17) singly or in combination with IGF1 and FSH on estradiol (E2) and progesterone (P4) secretion in GCs. The mRNA and protein abundance of APLN was the highest in GCs of LF while the APJ expression enhanced with follicle enlargement in GCs (p-value <0.01). IGF1 and FSH elevated the mRNA and protein amounts of APLN and FSH, and IGF1 increased the expression of APJ in buffalo GCs (p-value <0.01). Both isoforms of APLN (-13/-17) singly or in the presence of IGF1 or FSH increased the secretion of E2 and P4 with or without preincubation of cells with APJ antagonist (ML221 10 µM), although we had some variation in the effects. Concurrently, APLN-13/-17 significantly increased the mRNA and protein expression of CYP19A1 and StAR (p-value <0.01). ML221 substantially diminished the secretion of E2 and P4 and also the expression of CY19A1 and StAR in buffalo GCs (p-value <0.01). We also revealed that APLN-13/-17 (10-9 M), singly or in response to IGF1 and FSH, increased the production of E2 and P4 in different times of stimulation. In conclusion, APLN may play a crucial role in steroidogenesis and follicular development in ovarian GCs of buffalo.

Interactions between Nutrition and the "Ram Effect" in the Control of Ovarian Function in the Merino Ewe.

Simple SummaryClean, green, and ethical management of sheep flocks involves the use of socio-sexual stimuli (the “ram effect”) to coordinate nutritional inputs into reproductive success. However, the value of the “ram effect” is limited by three factors: (i) the proportion of the ewe flock that ovulates; (ii) ovulation rate in ewes that respond to the ram stimulus; and (iii) luteal failure after the first ram-induced ovulation, leading to short luteal phases. We tested whether these problems could be overcome by using a brief nutritional supplement (500 g lupin grain per head daily for 6 days) to stimulate ovarian activity. Lupin supplementation before ram introduction did not improve the percentage of the ewe flock that ovulates or reduce the frequency of short cycles (so will not improve the synchrony of lambing). However, lupin supplementation after ram introduction should be used to increase prolificacy.We tested whether short-term nutritional supplementation (500 g lupin grain per head daily) would affect the response of ewes to the ram effect. Experiment 1 (end of non-breeding season): ewes were supplemented for either Days −6 to −1 relative to ram introduction (n = 24) or for 12 days after ram introduction (Days 11 to 22 of the ram-induced cycle; n = 29). Controls (n = 30) were not supplemented. Across all groups, 94–100% of ewes ovulated. Supplementation before ram introduction did not affect ovulation rate at the ram-induced ovulation but increased it during the ram-induced cycle (Control 1.37; supplemented 1.66; p < 0.05). Experiment 2 (the middle of non-breeding season): the supplement was fed for Days −5 to −1 relative to ram introduction. Again, supplementation did not increase number ovulating (Control 16/29; Supplemented 10/29) but it did increase ovulation rate at the ram-induced ovulation (Control 1.31; Supplemented 1.68; p < 0.05). In neither experiment did supplementation affect the frequency of short cycles. Supplementation before ram introduction did not improve the percentage of ewes ovulating or reduce the frequency of short cycles (so will not improve the synchrony of lambing). However, supplementation after ram introduction can increase prolificacy.

Interrelationships between amphiregulin, kisspeptin, FSH and FSH receptor in promotion of human ovarian cell functions.

The aim of this study was to investigate: (1) the ability of granulosa cells to produce amphiregulin (AREG), kisspeptin (KISS) and FSH receptor (FSHR); (2) the role of AREG and KISS in the control of ovarian functions; (3) the effect of FSH and KISS on AREG; and (4) the ability of KISS to affect FSHR and to modify FSH action on AREG output by human ovarian granulosa cells. We examined: (1) time-dependent accumulation of AREG; (2) effects of AREG (0, 1, 10, 100ng/mL) and KISS (0, 1, 10, 100ng/mL) on granulosa cell functions; and (3) the effects of KISS (0, 1, 10, 100ng/mL), FSH (0, 1, 10, 100ng/mL), and their combinations on AREG release. Viability, markers of proliferation [accumulation ofproliferating cell nuclear antigen (PCNA) cyclin B1 and sodium 3'-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy6-nitro)benzene sulfonic acid hydrate (XTT formazan)] and apoptosis (accumulation of bax, caspase 3 and terminal deoxynucleotidyl transferase dUTP nick-end labelling), accumulation of KISS, FSHR and steroid hormones, and AREG release were analysed by Trypan blue exclusion test, quantitative immunocytochemistry, XTT, terminal deoxynucleotidyl transferase dUTP nick-end labelling assays and enzyme-linked immunosorbent assay. AREG promoted cell viability, proliferation and steroid hormone output, and inhibited apoptosis. KISS (1 and 10ng/mL) stimulated viability, proliferation, steroid hormone release and occurrence of FSHR and suppressed apoptosis and AREG output; KISS (100ng/mL) had the opposite effect. FSH stimulated AREG release, whilst addition of KISS reversed this FSH effect. FSH mimicked and promoted the inhibitory effect of KISS on AREG release. These results suggest an intra-ovarian production and a functional interrelationship between AREG, KISS, FSH and FSHR in direct regulation of basic ovarian cell functions.

Kisspeptin as autocrine/paracrine regulator of human ovarian cell functions: Possible interrelationships with FSH and its receptor

The present study aims to examine the role of kisspeptin (KP), FSH, and its receptor (FSHR), and their interrelationships in the control of basic human ovarian granulosa cells functions. We investigated: (1) the ability of granulosa cells to produce KP and FSHR, (2) the role of KP in the control of ovarian functions, and (3) the ability of KP to affect FSHR and to modify the FSH action on ovarian functions. The effects of KP alone (0, 10 and 100 ng/mL); or of KP (10 and 100 ng/mL) in combination with FSH (10 ng/mL) on cultured human granulosa cells were assessed. Viability, markers of proliferation (PCNA and cyclin B1) and apoptosis (bax and caspase 3), as well as accumulation of KP, FSHR, and steroid hormones, IGF-I, oxytocin (OT), and prostaglandin E2 (PGE2) release were analyzed by the Trypan blue exclusion test, quantitative immunocytochemistry, and ELISA. KP given at a low dose (10 ng/mL) stimulated viability, proliferation, inhibited apoptosis, promoted the release of progesterone (P4), estradiol (E2), IGF-I, OT, and PGE2, the accumulation of FSHR, but not testosterone (T) release. KP given at a high dose (100 ng/mL) had the opposite, inhibitory effect. FSH stimulated cell viability, proliferation and inhibited apoptosis, promoted P4, T, E2, IGF-I, and OT, but not PGE2 release. Furthermore, KP at a low dose promoted the stimulatory effect of FSH on viability, proliferation, P4, E2, and OT release, promoted its inhibitory action on apoptosis, but did not modify its action on T, IGF-I, and PGE2 output. KP at a high dose prevented and inverted FSH action. These results suggest an intra-ovarian production and a functional interrelationship between KP and FSH/FSHR in direct regulation of basic ovarian cell functions (viability, proliferation, apoptosis, and hormones release). The capability of KP to stimulate FSHR, the ability of FSH to promote ovarian functions, as well as the similarity of KP (10 ng/mL) and FSH action on granulosa cells’ viability, proliferation, apoptosis, steroid hormones, IGF-I, OT, and PGE2 release, suggest that FSH influence these cells could be mediated by KP. Moreover, the capability of KP (100 ng/mL) to decrease FSHR accumulation, basal and FSH-induced ovarian parameters, suggest that KP can suppress some ovarian granulosa cell functions via down-regulation of FSHR. These observations propose the existence of the FSH-KP axis up-regulating human ovarian cell functions.

Uterine and ovarian countercurrentpathways in the control of ovarian function in the pig

Uterine and ovarian countercurrentpathways in the control of ovarian function in the pig

FSH, oxytocin and IGF-I regulate the expression of sirtuin 1 in porcine ovarian granulosa cells.

The involvement of the mTOR system/enzyme sirtuin 1 (SIRT1) intracellular signaling system in the control of ovarian functions and its role in mediating hormonal action on the ovary has been proposed, but this hypothesis should be supported by a demonstrated influence of hormones on mTOR/SIRT1. Therefore, the aim of our in vitro experiments was to examine the effect of the known hormonal regulators of ovarian functions, such as follicle-stimulating hormone (FSH), oxytocin (OT) and insulin-like growth factor I (IGF-I), on mTOR/SIRT1. The accumulation of SIRT1 in porcine ovarian granulosa cells cultured with and without these hormones (at doses of 1, 10 or 100 ng.ml-1) was evaluated using immunocytochemistry. It was observed that the addition of FSH (at 10 ng.ml-1 but not at 1 or 100 ng/ml) and OT (at all tested doses) increased the expression of SIRT1 in ovarian cells. In addition, 100 ng.ml-1, but not at 1 or 10 ng.ml-1, of IGF-I decreased SIRT1 accumulation. Our observations are the first demonstration that hormones can directly regulate the ovarian mTOR/SIRT1 system and that this system could mediate the action of hormonal regulators on the ovary.

Responsiveness of the cheetah (Acinonyx jubatus) ovary to exogenous gonadotropins after preemptive oral progestin treatment

Control of ovarian function in cheetahs is sub-optimal, which currently limits the integration of assisted reproductive techniques into the genetic management of that endangered species. The objective of this study was to determine the effect of preemptive progestin treatment on the quality of ovarian responses after exogenous gonadotropin stimulation in cheetahs. Adult females received either 1) 200 IU equine chorionic gonadotropin (eCG) followed with 3,000 IU porcine luteinizing hormone (pLH) (intramuscular route) (n = 5; control group) or 2) similar eCG/pLH administration preceded by a 7-day treatment with oral progestin (0.1 mg/kg altrenogest; ALT group; n = 7). At 42 h post-pLH administration, a series of metrics was assessed via laparoscopy (number of follicles ≥ 2 mm, number of corpora lutea, oviduct and uterine cornua diameter and overall vascularization). Concentrations of fecal estradiol, progesterone and glucocorticoid metabolites (FEM, FPM, and FGM, respectively) were measured by enzyme immunoassay for 3 wk before ALT treatment (Period 1), 7 d during ovarian suppression period (Period 2), throughout eCG/LH treatment and laparoscopy (Period 3), and 6 wk following laparoscopy (Period 4). Overall, nine out of 12 cheetahs (4/5 in control and 5/7 ALT group) had freshly-formed corpora lutea at the time of laparoscopy. Mean follicle and corpora lutea numbers in the control versus ALT group were not different (P > 0.05). Overall measurements and vascularization scores also did not differ (P > 0.05) among groups. FEM average concentrations increased (P ≤ 0.05) in response to eCG for the ALT-treated females between Periods 2 and 3 and were sustained during Period 4. However, FEM average concentrations did not vary (P > 0.05) for control females throughout Periods 1–4. Post-ovulatory FPM average concentrations (Period 4) did not differ (P > 0.05) between the ALT-treated females and controls. FPM average concentration from both groups increased in Period 4 compared to Periods 1–3 (P ≤ 0.05). Females receiving the ALT treatment also had lower (P ≤ 0.05) FGM metabolite average concentrations than control females during ovarian suppression (suggesting adrenal suppression). Collective results suggest that ovarian response to gonadotropin treatment in the cheetah was improved following oral progestin administration due to the normative increase in estradiol following stimulation for these females compared with control. This treatment should lead to more effective timed assisted reproduction procedures for this species.

The role of transcription factors in control of ovarian functions

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Molecular Aspects and Clinical Relevance of GDF9 and BMP15 in Ovarian Function.

Growth and differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are oocyte-secreted factors with a leading role in the control of ovarian function in female reproduction, modulating both the cell fate of the somatic granulosa cells and the quality and developmental competence of the egg. This short review aims to consolidate the molecular aspects of GDF9 and BMP15 and their integral actions in female fertility to understand particularly their effects on oocyte quality and fetal growth. The significant consequences of mutations in the GDF9 and BMP15 genes in women with dizygotic twins as well as the clinical relevance of these oocyte factors in the pathogenesis of primary ovarian insufficiency and polycystic ovary syndrome are also addressed.

Interrelationships between sirtuin 1 and transcription factors p53 and NF-κB (p50/p65) in the control of ovarian cell apoptosis and proliferation.

The roles of the mTOR system enzyme sirtuin 1 (SIRT1), the transcription factor p53 and the nuclear factor kappaB (NF-κB) and their interrelationships in the control of ovarian function have not been well studied. We examine, in vitro, the involvement of SIRT1, p53 and the p65 and p50 subunits of NFκB and their interrelationships in the control of the apoptosis and proliferation of porcine ovarian granulosa cells. Monolayers of primary granulosa cells were transfected with cDNA constructs encoding SIRT1, p53, p65 or p50 alone or were co-transfected with gene constructs for SIRT1 together with p53, p65 or p50. The accumulation of SIRT1, markers of proliferation (mitogen-activated protein kinase or extracellular-signal-regulated kinases 1,2) and a marker of apoptosis (caspase 3) was detected by immunocytochemistry. Transfection of cells with a SIRT1 gene construct alone promoted the accumulation of SIRT1 and decreased the accumulation of proliferation markers but did not affect the marker of apoptosis. Transfection of cells with gene constructs encoding p53, p50 or p65 decreased the expression of proliferation markers but not the apoptosis marker. Co-transfection of cells with SIRT1 cDNA changed the action of p65 on cell proliferation from inhibitory to stimulatory. SIRT1 overexpression induced the pro-apoptotic action of p53 and p50 but not of p65 constructs. Thus, SIRT1, p53 and NF-κB are involved in the control of both the proliferation and the apoptosis of ovarian cells. These novel data on the cross-talk between the mTOR/SIRT1 system and the transcription factors p53 and NF-κB show both the inhibitory (proliferation) and stimulatory (apoptosis) influences of SIRT1 on transcription factor action in ovarian cells.

Using sheep lines with mutations in single genes to better understand ovarian function

Livestock populations have been subjected to strong selection pressure to improve reproductive success, and this has led to the identification of lines of animals with increased fecundity. These animals provide a rich biological resource for discovery of genes and regulatory mechanisms that underpin improved reproductive success. To date, three genes, all related to the transforming growth factor β pathway, have been identified as having mutations that lead to alterations in ovulation in sheep. In addition, several other sheep lines have been identified with putative mutations in single genes with major effects on ovulation rate. This review is focused on the identification of the mutations affecting ovulation rate and how these discoveries have provided new insights into control of ovarian function.

Tumor Necrosis Factor Alpha May Act as an Intraovarian Mediator of Luteinizing Hormone-Induced Oocyte Maturation in Trout1

In fish, like in other vertebrates, luteinizing hormone (Lh) is an essential hormone for the completion of oocyte maturation. In salmonid fish (i.e., salmon and trout), oocyte maturation is induced by Lh through its stimulation of the production of the maturation-inducing steroid, 17alpha,20beta-dihydroxy-4-pregnen-3-one (17,20beta-P). In mammals, several factors, including ovarian cytokines and growth factors, have been reported to contribute to the regulation of oocyte maturation. In fish, growing evidence suggests that tumor necrosis factor alpha (hereafter referred to as Tnf) could play multiple physiological roles in the control of ovarian function. In the present study, we have investigated the possible involvement of Tnf in the regulation of oocyte maturation in brown trout (Salmo trutta). Our results show that in vitro treatment of brown trout preovulatory follicles with coho salmon (Oncorhynchus kisutch) Lh (sLh) significantly increased oocyte maturation, as assessed by germinal vesicle breakdown (GVBD), and that this effect was blocked by TAPI-1 (an inhibitor of Tnf-converting enzyme or Tace/Adam17). Furthermore, treatment of preovulatory follicles with sLh increased the expression of tnf and tace/adam17 as well as the secretion of the Tnf protein. Importantly, recombinant trout Tnf (rtTnf) significantly increased GVBD in vitro. Our results also show that the stimulatory effects of rtTnf on oocyte maturation may be the result of the direct involvement of rtTnf in stimulating the production of the maturation-inducing steroid as evidenced, first, by the stimulatory effects of rtTnf on 17,20beta-P production in vitro and on the expression of cholesterol side-chain cleavage P450 cytochrome (p450scc) and 20beta-hydroxysteroid dehydrogenase/carbonyl reductase 1 (cbr1), the enzyme responsible for the production of 17,20beta-P, and, second, by the ability of TAPI-1 to block the stimulatory effects of sLh on 17,20beta-P production and cbr1 expression. Furthermore, sLh and rtTnf increased the expression of the Lh receptor (lhr) and decreased the expression of aromatase (cyp19a1), and TAPI-1 completely blocked the effects of sLh. These results strongly suggest that Tnf may contribute to the regulation of oocyte maturation by Lh in trout.

Involvement of the oestrogenic receptors in superior mesenteric ganglion on the ovarian steroidogenesis in rat

Oestradiol (E(2)) is a key hormone in the regulation of reproductive processes. The aims of this work were a) to examine the distributions of oestrogen receptor α (ERα) and ERβ in the neurons of the superior mesenteric ganglion (SMG) in the oestrus stage by immunohistochemistry, b) to demonstrate whether E(2) in the SMG modifies progesterone (P(4)), androstenedione (A(2)) and nitrite release in the ovarian compartment on oestrus day and c) to demonstrate whether E(2) in the ganglion modifies the activity and gene expression in the ovary of the steroidogenic enzymes 3β-hydroxysteroid dehydrogenase (3β-HSD) and 20α-hydroxysteroid dehydrogenase (20α-HSD). The ex vivo SMG-ovarian nervous plexus-ovary system was used. E(2), tamoxifen (Txf) and E(2) plus Txf were added in the ganglion to measure ovarian P(4) release, while E(2) alone was added to measure ovarian A(2) and nitrites release. Immunohistochemistry revealed cytoplasmic ERα immunoreactivity only in the neural somas in the SMG. E(2) increased ovarian P(4) and A(2) release at 15, 30 and 60 min but decreased nitrites. The activity and gene expression of 3β-HSD increased, while the activity and gene expression of 20α-HSD did not show changes with respect to the control. Txf in the ganglion diminished P(4) release only at 60 min. E(2) plus Txf in the ganglion reverted the effect of E(2) alone and the inhibitory effect of Txf. The results of this study demonstrate that ERα activation in the SMG has an impact on ovarian steroidogenesis in rats, thus providing evidence for the critical role of peripheral system neurons in the control of ovarian functions under normal and pathological conditions.

The Expression of Thrombopoietin and its Receptor During Different Physiological Stages in the Bovine Ovary

Thrombopoietin (TPO) is known to be involved in megakaryocytopoiesis, but its role in the control of ovarian function is unknown in cattle. The aims of this study were to demonstrate the expression of TPO and its receptor (c-MPL) in detail in bovine corpus luteum (CL) obtained from different stages of the oestrous cycle and during pregnancy--and to demonstrate that TPO/c-MPL system is expressed clearly in bovine follicles. Real-time RT-PCR (qPCR) and ELISA were applied to investigate mRNA expression of examined factors and TPO protein, respectively. In this investigation, increases in the concentrations of TPO protein and the mRNA expression of TPO and c-MPL were noticed during both early luteal stage and late luteal stage of the oestrous cycle. Furthermore, the expression of TPO/c-MPL system does not show any significant regulation in the CL throughout pregnancy. Highest co-expression of TPO/c-MPL system in both theca interna (TI) and granulosa cells (GC) in small follicles (<10 mm in diameter) was observed in this study that may suggest the possible role of TPO/c-MPL system in proliferation of TI and GC cells. To conclude, the results demonstrate the possible involvement of locally produced TPO/c-MPL system as a 'physiological filter' in bovine ovary where they may promote cell selection by inducing proliferation of viable cells and scavenging non-viable cells and thereby may play an important role in modulation of ovarian function.

The expression of leptin and its receptor during different physiological stages in the bovine ovary

Leptin, the hormonal product of the obese (ob) gene, circulates in the blood at levels paralleling those of fat reserves and regulates satiety. In cattle, leptin has also been implicated in the control of ovarian function, but its local production in the ovary and role in the control of ovarian function in autocrine/paracrine manner is unknown. The aims of this study were to document the expression of leptin and its receptor (Ob-R) in detail in bovine corpus luteum (CL) obtained from different stages of the oestrous cycle and during pregnancy-and to determine if the leptin/Ob-R system is expressed clearly in bovine follicles during final growth to preovulatory follicles. Real-time RT-PCR (qPCR) was applied to investigate mRNA expression of examined factors. In general, we demonstrated leptin and its receptor transcripts and leptin protein are consistent with in vivo luteinisation of bovine CL and decline coincidental with luteal regression. The highest co-expression of leptin/Ob-R system was observed in TI and GC of the smallest follicles with E2 concentration <0.5 ng/ml followed by significant down regulation in growing follicles with the increase of follicular size and E2 content in the follicular fluid. Furthermore, expression of the leptin/Ob-R system does not show any significant variation in the CL throughout pregnancy. To conclude, our results are the first to demonstrate the possible involvement of locally produced leptin/Ob-R system in the bovine ovary, suggesting roles in the function and/or development of the CL and growth of small follicles in an autocrine/paracrine fashion.

A large-scale association study to assess the impact of known variants of the human INHA gene on premature ovarian failure

Three variants of the human INHA gene have been reported to be associated with premature ovarian failure (POF) in case-control studies involving a small number of patients and controls. Since inhibin has a fundamental role in the control of ovarian function, it is important to establish the relevance of the reported variants for disease risk. Three independent POF cohorts, recruited in Northern and Central Italy and in Germany consisting of a total of 611 patients and 1084 matched controls, were genotyped for the three variants: -16C > T, -124A > G and 769G > A. No significant difference was detected between allelic frequencies of the INHA promoter variants between POF patients and controls. The rare allele in the coding variant appeared to be more frequent among the control populations. The association between the INHA promoter variants and POF could not be replicated, and our results suggest that this discrepancy is likely to be due to the small sample size of previous studies. The rare allele of the coding variant seems to exert a protective effect against loss of ovarian function, which should be confirmed in additional large and ethnically diverse cohorts.

Delayed Puberty in Spontaneously Hypertensive Rats Involves a Primary Ovarian Failure Independent of the Hypothalamic KiSS-1/GPR54/GnRH System

Spontaneously hypertensive (SH) rats, extensively used as experimental models of essential human hypertension, display important alterations in the neuroendocrine reproductive axis, which manifest as markedly delayed puberty onset in females but whose basis remains largely unknown. We analyze herein in female SH rats: 1) possible alterations in the expression and function of KiSS-1/GPR54 and GnRH/GnRH-receptor systems, 2) the integrity of feedback mechanisms governing the hypothalamic-pituitary-ovarian axis, and 3) the control of ovarian function by gonadotropins. Our data demonstrate that, despite overtly delayed puberty, no significant decrease in hypothalamic KiSS-1, GPR54, or GnRH mRNA levels was detected in this strain. Likewise, in vivo gonadotropin responses to ovariectomy and systemic kisspeptin-10 or GnRH administration, as well as in vitro gonadotropin responses to GnRH, were fully preserved in SH rats. Moreover, circulating LH levels were grossly conserved during prepubertal maturation, whereas FSH levels were even enhanced from d 20 postpartum onwards. In striking contrast, ovarian weight and hormone (progesterone and testosterone) responses to human chorionic gonadotropin (CG) in vitro were profoundly decreased in SH rats, with impaired follicular development and delayed ovulation at puberty. Such reduced hormonal responses to human CG could not be attributed to changes in LH/CG or FSH-receptor mRNA expression but might be linked to blunted P450scc, 3beta-hydroxy steroid dehydrogenase, and aromatase mRNA levels in ovaries from SH rats. In conclusion, our results indicate that the expression and function of KiSS-1/GPR54 and GnRH/GnRH-receptor systems is normal in SH rats, whereas ovarian development, steroidogenesis, and responsiveness to gonadotropins are strongly compromised.

Leptin in the bovine corpus luteum: Receptor expression and effects on progesterone production

In cattle, leptin has been implicated in the control of ovarian function and has been shown to modulate steroid production by theca and granulosa cells in a number of species. However, a direct effect of leptin on bovine luteal function has not been demonstrated. This study was conducted to determine if the leptin receptor (OB-R) is expressed in the bovine corpus luteum (CL), and to examine the effects of leptin on progesterone production by dispersed luteal cells in vitro. RT-PCR was used to detect the presence of OB-R and, more specifically, the long, biologically active isoform (OB-Rb), in CL, collected on days 2-18 of the oestrous cycle (n=18). The effects of leptin on progesterone production were investigated in dispersed luteal cells prepared from CL collected on days 5 and 8 (n=14) of the cycle. The dispersed luteal cells were cultured for 24 hr with recombinant human leptin and/or LR3-IGF-1 and/or LH. OB-Rs, in particular, OB-Rb, were expressed in the CL at all stages of development. Progesterone production by luteal cells was increased (P<0.001) by treatment with LH (10 ng/ml) but treatment with leptin alone had no effect. However, in the presence of IGF-1 (100 ng/ml), leptin (10 ng/ml) caused a significant (P<0.005) increase in progesterone production. In conclusion, we have shown that the leptin receptor is expressed in the bovine CL and have demonstrated a modulatory effect of leptin on luteal progesterone production in vitro.