Cellular localization and seasonal variation of GnRH and Bradykinin in the ovary of Heteropneustes fossilis (Bloch.) during its reproductive cycle.

The present study investigates the distribution and dynamics of gonadotropin-releasing hormone I (GnRH I) and bradykinin in the air-breathing catfish, Heteropneustes fossilis, in relation to the reproductive cycle. Changes in bradykinin, bradykinin B2-receptor, and ovarian GnRH I regulation were demonstrated during the reproductive cycle. The localization of GnRH I, bradykinin, and their respective receptors in the ovaries was investigated by immunohistochemistry, while their levels were quantified by slot/western blot followed by densitometry. GnRH I and its receptor were mainly localized in the cytoplasm of oocytes during the early previtellogenic phase. However, as the follicles grew larger, immunoreactivity was observed in the granulosa and theca cells of the late previtellogenic follicles. The ovaries showed significantly higher expression of GnRH I protein and its receptor during the early to mid-previtellogenic phase, suggesting their involvement in follicular development. Bradykinin and bradykinin B2-receptor showed a distribution pattern similar to that of GnRH I and its receptor. This study further suggested the possibility that bradykinin regulates GnRH I synthesis in the ovary. Thus, we show that the catfish ovary has a GnRH-bradykinin system and plays a role in follicular development and oocyte maturation in H. fossilis.

Asprosin promotes steroidogenesis and spermatogenesis with improved glucose metabolism in adult mice testis.

Asprosin is an orexigenic adipokine that regulates appetite and glucose homeostasis in mammals. To date, only fragmentary findings are reported regarding its role in testicular activities. In the current investigation, immunolocalization and direct action of asprosin in adult mice testis was evaluated. Immunohistochemical and immunoblot studies were performed to analyse the testicular expression of asprosin. Intratesticular treatment of asprosin (0.1 µg and 1.0 µg per testis) was given to evaluate its direct action on testicular functions. Sertoli and Leydig cells were found to be immuno-positive for asprosin. Intratesticular administration of asprosin resulted into a significant increase in glucose and lactate levels along with enhanced expression of asprosin receptor OLFR734, insulin receptor (IR), glucose transporter 8 (GLUT 8), lactate dehydrogenase (LDH) activity and monocorboxylate transporters (MCT2 and 4). In addition, asprosin administration increased the testicular expression of cell proliferation (proliferating cell nuclear antigen: PCNA), cell survival (B cell lymphoma 2: Bcl2) and decreased germ cell apoptosis (Cysteine aspartic acid protease 3: Caspase 3) leading to increased sperm counts. Further, asprosin treatment resulted into increased level of total cholesterol, testosterone and steroidogenic markers (steroidogenic acute regulatory protein: StAR; 3beta-hydroxysteroid dehydrogenases: 3ß HSD and 17beta-hydroxysteroid dehydrogenases: 17ß HSD). Asprosin treatment promotes testicular glucose uptake and lactate synthesis to provide energy for steroidogenesis and spermatogenesis. The significant correlation between the asprosin-induced increased IR expression and increased testosterone, glucose and lactate levels suggests its role in increased survival and proliferation but decrease in germ cell apoptosis. This study proposed asprosin's role as an autocrine/paracrine regulator of testicular functions in adult mice.

Central and peripheral neuropeptide RFRP-3: A bridge linking reproduction, nutrition, and stress response.

This article is an amalgamation of the current status of RFRP-3 (GnIH) in reproduction and its association with the nutrition and stress-mediated changes in the reproductive activities. GnIH has been demonstrated in the hypothalamus of all the vertebrates studied so far and is a well-known inhibitor of GnRH mediated reproduction. The RFRP-3 neurons interact with the other hypothalamic neurons and the hormonal signals from peripheral organs for coordinating the nutritional, stress, and environmental associated changes to regulate reproduction. RFRP-3 has also been shown to regulate puberty, reproductive cyclicity and senescence depending upon the nutritional status. A favourable nutritional status and the environmental cues which are permissive for the successful breeding and pregnancy outcome keep RFRP-3 level low, whereas unfavourable nutritional status and stressful conditions increase the expression of RFRP-3 which impairs the reproduction. Still our knowledge about RFRP-3 is incomplete regarding its therapeutic application for nutritional or stress-related reproductive disorders.

Role of 14-3-3ß protein on ovarian folliculogenesis, steroidogenesis and its correlation in the pathogenesis of PCOS in mice.

This study was designed to assess for the first time the circulating and ovarian level of 14-3-3ß protein in the PCOS mice and the possible correlation between 14-3-3ß protein with PCOS related increase in testosterone (HA), insulin levels (HI) and reduced insulin sensitivity in the ovary. PCOS was induced in mice using treatment of letrozole (by oral gavage) for 21 days. Immunohistochemical study showed increased expression of 14-3-3ß protein in PCOS ovary compared to the control ovary. The circulating testosterone and insulin levels, together with circulating and ovarian levels of 14-3-3ß protein also showed significant increase in PCOS mice compared to the control mice. An increase in 14-3-3ß protein was observed positively correlated with circulating testosterone and insulin levels but showed a negative correlation with ovarian expression of insulin receptor protein in PCOS mice. The treatment of 14-3-3ß protein in vitro to the normal ovary showed a significant increase in testosterone synthesis but a significant decline in insulin receptor protein expression compared to the vehicle-treated ovary of adult mice. The present study showed the direct role of 14-3-3ß protein in increasing testosterone synthesis along with decreasing insulin sensitivity. Thus, 14-3-3ß protein may be playing possible role in PCOS pathogenesis.

Adiponectin/AdipoRs signaling as a key player in testicular aging and associated metabolic disorders.

Aging undergoes serious worsening of peripheral organs and vital physiological processes including reproductive performances. Altered white adipose tissue and adipocyte functioning during aging results in ectopic lipid storage/obesity or metabolic derangements, leading to insulin resistance state. Eventually, accelerating cellular senescence thereby enhancing the high risk of age-associated metabolic alterations. Such alterations may cause derangement of numerous physiologically active obesity hormones, known as "adipokines." Specifically, adiponectin exhibits insulin sensitizing action causing anti-aging and anti-obesity effects via activation of adiponectin receptors (AdipoRs). The male reproductive physiology from reproductive mature stage to advanced senescent stage undergoes insidious detrimental changes. The mechanisms by which testicular functions decline with aging remain largely speculative. Adiponectin has also recently been shown to regulate metabolism and longevity signaling thus prolonging lifespan. Therefore, the strategy for activating adiponectin/AdipoRs signaling pathways are expected to provide a solid basis for the prevention and treatment of aging and obesity-associated reproductive dysfunctions, as well as for ensuring healthy reproductive longevity in humans.

RF-amide related peptide-3 (RFRP-3): a novel neuroendocrine regulator of energy homeostasis, metabolism, and reproduction.

A hypothalamic neuropeptide, RF-amide related peptide-3 (RFRP-3), the mammalian ortholog of the avian gonadotropin-inhibitory hormone (GnIH) has inhibitory signals for reproductive axis via G-protein coupled receptor 147 in mammals. Moreover, RFRP-3 has orexigenic action but the mechanism involved in energy homeostasis and glucose metabolism is not yet known. Though, the RFRP-3 modulates orexigenic action in co-operation with other neuropeptides, which regulates metabolic cues in the hypothalamus. Administration of GnIH/RFRP-3 suppresses plasma luteinizing hormone, at the same time stimulates feeding behavior in birds and mammals. Likewise, in the metabolically deficient conditions, its expression is up-regulated suggests that RFRP-3 contributes to the integration of energy balance and reproduction. However, in many other metabolic conditions like induced diabetes and high-fat diet obesity, etc. its role is still not clear while, RFRP-3 induces the glucose homeostasis by adipocytes is reported. The physiological role of RFRP-3 in metabolic homeostasis and the metabolic effects of RFRP-3 signaling in pharmacological studies need a detailed discussion. Further studies are required to find out whether RFRP-3 is associated with restricted neuroendocrine function observed in type II diabetes mellitus, aging, or sub-fertility. In this context, the current review is focused on the role of RFRP-3 in the above-mentioned mechanisms. Studies from search engines including PubMed, Google Scholar, and science.gov are included after following set inclusion/exclusion criteria. As a developing field few mechanisms are still inconclusive, however, based on the available information RFRP-3 seems to be a putative tool in future treatment strategies towards metabolic disease.

Protective role of adiponectin against testicular impairment in high-fat diet/streptozotocin-induced type 2 diabetic mice.

Type 2 diabetes (T2D) is the most common endocrine and metabolic disorder, leading to reproductive impairments and infertility in male. Our recent study showed crucial role of adiponectin in the regulation of testicular functions, and the circulating level of adiponectin declines in diabetes. The current study thus aimed to examine the efficacy of adiponectin in improving testicular dysfunction in high-fat diet/streptozotocin-induced T2D mice. T2D was induced in pre-pubertal mice fed with high-fat diet for ∼10 weeks followed by a single dose of streptozotocin. T2D mice showed presence of increased body mass, hyperglycemia, hyperinsulinemia, insulin resistance, increased oxidative stress, and declined serum testosterone compared to vehicle-treated control mice. The spermatogenic, steroidogenic, metabolic, and antioxidative parameters were evaluated in T2D mice treated with adiponectin for both two and four weeks. The exogenous administration of adiponectin to T2D mice showed enhanced serum testosterone and expression of testicular steroidogenic markers proteins, insulin receptor and GLUT8 proteins, increase in intra-testicular concentrations of glucose and lactate and activity of LDH and antioxidant enzymes compared to the levels in untreated T2D mice. This suggests that treatment of adiponectin effectively improves testicular functions by increasing expression of insulin receptor-mediated increased transport of energy substrate (glucose and lactate) and a marked reduction in oxidative stress are the possible mechanism by which adiponectin effectively improves testicular function in T2D mice.

Role of putrescine in ovary and embryo development in fruit bat Cynopterus sphinx during embryonic diapause.

The aim of this study was to evaluate the effect of putrescine on ovarian activity and the rate of embryonic development in Cynopterus sphinx during delayed development. The result showed the presence of a rate-limiting enzyme, ornithine decarboxylase-1, in both ovary and utero-embryonic unit of C. sphinx suggests a synthesis of putrescine in these sites. The corpus luteum showed increased, whereas utero-embryonic unit showed decreased production of putrescine during delayed development as compared with the normal development. The bat treated in vivo with putrescine during delayed development showed increase in progesterone and estradiol synthesis, correlated with increased expression of luteinizing hormone receptor, steroidogenic acute receptor protein, and 3ß-hydroxysteroid dehydrogenase through extracellular signal-regulated kinase (ERK1/2)-mediated pathway in the ovary; but showed increase in the weight and expression of progesterone receptor (PR), B-cell lymphoma 2, proliferating cell nucleus antigen, and vascular endothelial growth factor proteins in utero-embryonic unit. The in vitro treatment of putrescine showed stimulatory whereas treatment with an inhibitor of putrescine, 2-difluoromethylornithine caused an inhibitory effect on ovarian progesterone synthesis and cell proliferation, and cell survival in the utero-embryonic unit. In conclusion, the putrescine showed two separate roles during embryonic diapause, high concentration of putrescine in the ovary may support corpus luteum and basal synthesis of progesterone, whereas a low level of putrescine causes retarded embryonic development by inhibiting cell proliferation in the utero-embryonic unit. The bat treated with putrescine either directly promotes cell proliferation, cell survival, and angiogenic activities or acts indirectly increasing PR on utero-embryonic unit thereby activating development in delayed embryo in C. sphinx.

Immunohistochemical localization and possible functions of nesfatin-1 in the testis of mice during pubertal development and sexual maturation.

The study was aimed to address the role of nesfatin-1 on the sexual maturation of testis during the pubertal transition. The immunostaining of testis suggested nesfatin-1 is expressed in Leydig cells with pubertal maturation. The pre-pubertal mice for in vivo study were randomly divided in three groups; (a) control-saline (b) treated with low (0.25 nM) dose of nesfatin-1/gbw/day and (c) treated with high (1.25 nM) dose nesfatin-1/gbw/day. Histological analysis showed that nesfatin-1 loaded mice showed facilitated maturation of testis. Western blot analysis on various protein expressions upon injection of nesfatin-1 into pre-pubertal mice suggested that expressions of proteins involving steroid hormone production, spermatogenic markers (PCNA, Bcl2, AR), glucose uptake-related proteins (GLUT8 and insulin receptor) and GnRH-R and GPR-54 proteins were facilitated. Both of lactose dehydrogenase activity and lactate levels were increased. The treatment with nesfatin-1 also reduced oxidative stress, which further facilitates testicular functions during puberty. The treatment of nesfatin-1 on cultured testis also supports in vivo findings as evident by the increased testosterone production and StAR protein expression as well as increased glucose and lactate production. In sum, our data report for the first time the accelerative role of nesfatin-1 on spermatogenesis and steroidogenesis of pre-pubertal male mice by directly acting on the testis coupled with the advancement of puberty.

Liraglutide modulates adipokine expression during adipogenesis, ameliorating obesity, and polycystic ovary syndrome in mice.

PURPOSE: The incidence of obesity is increasing among all age groups throughout the world and it is highly associated with numerous other metabolic disorders, such as insulin resistance, polycystic ovarian syndrome (PCOS) etc. METHODS AND RESULTS: Using in vitro and in vivo approach, this study investigated the adipokine profile after liraglutide on differentiated murine 3T3-L1 pre-adipocytes. Effect of liraglutide on DHEA-induced PCOS mice were investigated. This study showed Liraglutide treatment resulted in up-regulation of adiponectin and IL-6 along with down-regulation of ICAM 1 in differentiated 3T3-L1 cells. Liraglutide in absence of other differentiating factors, significantly increased glucose, lipid uptake and PPARγ, C/EBPα expression in the adipocytes suggesting its ability to solely promote pre-adipocyte differentiation into mature adipocyte. Liraglutide treatment showed increased adiponectin expression and decreased number of cystic follicles, body weight, circulating glucose, triglyceride and testosterone levels in comparison to the PCOS induced mice. CONCLUSION: This study suggests that adiponectin may act as a link between metabolic disorders and PCOS and that liraglutide might be a promising therapeutic agent for the treatment of PCOS in addition to obesity and insulin resistance.

Direct actions of adiponectin on changes in reproductive, metabolic, and anti-oxidative enzymes status in the testis of adult mice.

Obesity is a major health problem that is linked to decreased sperm count. It is hypothesized that an obesity-associated reduction in adiponectin secretion may be responsible for impairment of spermatogenesis. Therefore, the aim of the study was to evaluate the direct role of adiponectin in spermatogenesis and steroid synthesis in adult mice. This study showed that adiponectin receptors (AdipoR1 and AdipoR2) were localized in Leydig cells and seminiferous tubules in the testis of adult mice. The result of the in vitro study showed the direct action of adiponectin on spermatogenesis by stimulating cell proliferation (PCNA) and survival (Bcl2) and by suppressing cell apoptosis. Treatment of testis with adiponectin also enhanced transport of the energetic substrates glucose and lactate to protect cells from undergoing apoptosis. Adiponectin treatment further showed a significant reduction in oxidative stress and nitric oxide. Our findings suggest that adiponectin effectively facilitates cell survival and proliferation, as well as protects from apoptosis. Thus, adiponectin treatment may be responsible for enhancing sperm counts. Interestingly, this study showed the stimulatory effect of adiponectin in spermatogenesis but showed an inhibitory effect on testosterone and estradiol synthesis in the testes. Based on the present study, it is hypothesized that systemic adiponectin treatment may be a promising therapeutic strategy for the improvement of spermatogenesis and sperm count.

Direct effects of neuropeptide nesfatin-1 on testicular spermatogenesis and steroidogenesis of the adult mice.

Recent studies have revealed nesfatin-1 as a hypothalamic neuropeptide, regulating food intake, energy expenditure and reproduction primarily by acting on the hypothalamic-pituitary-gonadal axis. Nesfatin-1 is also localized in several peripheral tissues including testes. However, functional significance of nesfatin-1 in testicular activities is not yet well documented in mammals. Therefore, this study was aimed to elucidate the direct effects of nesfatin-1 on testicular markers for steroid productions, spermatogenesis, metabolic changes and oxidative stress. The results revealed the expression of both protein and mRNA of nesfatin-1 in the testes of adult mice. The testes treated in vitro with nesfatin-1 showed significant increase in testosterone production, which correlated significantly with increased expression of steroidogenic markers and insulin receptor proteins in the testes. Furthermore, the in vitro treatment with nesfatin-1 showed stimulatory effects on spermatogenesis by promoting cell proliferation (PCNA) and survival (Bcl2), while inhibiting apoptosis (caspase-3) in the testes. The nesfatin-1 treatment in vitro further increased the expression of insulin receptor and GLUT8 proteins, in parallel with increase in the intra-testicular transport of glucose and production of lactate. This nesfatin-1 induced enhanced transport of energy substrate (glucose and lactate) may be responsible for promoting spermatogenesis and steroidogenesis. Nesfatin-1 significantly reduced oxidative stress and nitric oxide, which may also be responsible for stimulatory effects on testicular activities. The present finding suggests that nesfatin-1 acts via paracrine manner to increase sperm count and fertility, thus promoting the testicular function.

Role of adiponectin as a modulator of testicular function during aging in mice.

The mechanisms by which testicular functions decline with aging remain largely speculative. Our recent finding showed the importance of adiponectin in the regulation of testicular functions, whereas its concentration declines during male infertility. Thus, the aim of present study was to explore the potential role of adiponectin during aging. The changes in adiponectin, adiponectin-receptors, and insulin receptor proteins expression in the testis were evaluated and compared with the testicular parameters, mass, and testosterone level in the mice from early post-natal to late senescence period. Further, the current study has examined the effect of exogenous adiponectin treatment on testicular functions in aged mice. The results showed a significant decline in adiponectin/adiponectin-receptors expression simultaneously with a significant decline in testicular mass, insulin receptor expression and testosterone synthesis in the testis of aged mice. Exogenous treatment of adiponectin to aged mice resulted in marked improvements in testicular mass, histological features (cells proliferation), insulin receptor expression, testicular glucose uptake, anti-oxidative enzymes activity and testosterone synthesis as compared with the control. Based on these findings, it may be concluded that a marked decline in adiponectin synthesis and action results in decreased insulin sensitivity (development of insulin resistance) and increased oxidative stress which consequently suppresses testicular functions during aging. This study further showed that treatment with adiponectin ameliorates reduced testicular functions by enhanced expression of insulin receptor in the testis of senescent mice. It is thus hypothesized that systemic adiponectin treatment could be a promising therapeutic strategy for improvement of testosterone production and sperm counts during aging.

Comparative Effects of Estrogen and Phytoestrogen, Genistein on Testicular Activities of Streptozotocin-Induced Type 2 Diabetic Mice.

The aim of this study was to compare the effect of synthetic estrogen (E2) with a phytoestrogen and genistein in ameliorating type 2 diabetes mellitus (T2D)-mediated testicular dysfunction in mice. The streptozotocin (STZ)-induced type 2 diabetic mice were treated exogenously with either E2 or genistein for 2 durations and compared their effects on testicular activities, serum glucose, and insulin level. Type 2 diabetic mice treated with E2 for only short term (14 days) improved regressive changes in the testicular histology by increasing testosterone synthesis and improving insulin sensitivity, whereas those treated for longer duration (28 days) failed to improve testicular dysfunctions. On the other hand, genistein treated for both short- and long term was useful in improving T2D-induced adverse effects on testicular functions. This study further suggests that treatment with genistein improves spermatogenesis in type 2 diabetic mice by increasing insulin-induced formation of lactate and antioxidative enzymes, which contributes to prevent germ cell apoptosis. Thus, genistein can be used to ameliorate T2D-induced testicular dysfunction.

Adiponectin and Chemerin: Contrary Adipokines in Regulating Reproduction and Metabolic Disorders.

Metabolic disorders such as obesity and type 2 diabetes are one of the most familiar risk factors in the present time among every age-group. It is associated with altered levels of adipokines such as adiponectin, chemerin, leptin, resistin, visfatin, and so on. Adiponectin is one of the adipocyte-specific protein with novel applications pertaining to metabolism by promoting insulin sensitivity and regulating glucose and fatty acid catabolism, while chemerin is considered as an inhibitor of insulin signaling and glucose catabolism. Other than these established functions, both the adipokines are intimately involved in coordinating reproductive activities, but they exhibit contrary functions. This review is an amalgamation of recent information related to adiponectin and chemerin in male and female reproduction and further its association with metabolism-related reproductive disorders. The direct effect of adiponectin and chemerin on various reproductive parameters has been investigated, but there was a rampant failure to account for in vivo data which gives a broad outlook on the regulatory mechanism of both adiponectin and chemerin related to male and female reproductive functions. Adiponectin is known to promote gonadal activities, while chemerin exerts antigonadal actions. Recent research suggests that high chemerin/low adiponectin ratio plays a vital role in causing dyslipidemia and metabolic syndrome in patients. The dysregulated ratio of adiponectin to chemerin during various metabolic disorders makes it really worthy in relation to an application for therapeutics. Still, a lot regarding both the adipokines has to be explored and brought forward in order to deal with therapeutics of metabolism-related reproductive disorders.

Adiposity associated changes in serum glucose and adiponectin levels modulate ovarian steroidogenesis during delayed embryonic development in the fruit bat, Cynopterus sphinx.

The aim of the present study was to evaluate the mechanism by which embryonic development in Cynopterus sphinx is impaired during the period of increased accumulation of white adipose tissue during winter scarcity of food. The change in the mass of white adipose tissue during adipogenesis showed significant positive correlation with the circulating glucose level. But increase in circulating glucose level during the adipogenesis showed negative correlation with circulating progesterone and adiponectin levels. The in vivo study showed increased glucose uptake by the adipose tissue during adipogenesis due to increased expression of insulin receptor (IR) and glucose transporter (GLUT) 4 proteins. This study showed decline in the adiponectin level during fat accumulation. In the in vitro study, ovary treated with high doses of glucose showed impaired progesterone synthesis. This is due to decreased glucose uptake mediated decrease in the expression of luteinizing hormone-receptor, steroidogenic acute regulatory protein, IR, GLUT4 and AdipoR1 proteins. But the ovary treated with adiponectin either alone or with higher concentration of glucose showed improvement in progesterone synthesis due to increased expression of IR, GLUT4 and AdipoR1 mediated increased glucose uptake. In conclusion, increased circulating glucose level prior to winter dormancy preferably transported to white adipose tissue for fat accumulation diverting glucose away from the ovary. Consequently the decreased availability of adiponectin and glucose to the ovary and utero-embryonic unit may be responsible for impaired progesterone synthesis and delayed embryonic development. The delayed embryonic development in Cynopterus sphinx may have evolved, in part, as a mechanism to prevent pregnancy loss during the period of decreased energy availability.

Systemic adiponectin treatment reverses polycystic ovary syndrome-like features in an animal model.

The present study examined the efficacy of adiponectin for regulating the reproductive, metabolic and fertility status of mice with polycystic ovary syndrome (PCOS). PCOS was induced in prepubertal (21- to 22-day-old) mice using dehydroepiandrosterone (6mg 100g-1day-1 for 25days), after which mice were administered either a low or high dose of adiponectin (5 or 15µgmL-1, s.c., respectively). PCOS mice exhibited typical features, including the presence of numerous cystic follicles, increased circulating androgens, increased body mass, altered steroidogenesis, decreased insulin receptor expression and increased serum triglycerides, serum glucose, Toll-like receptor (TLR)-4 (a marker of inflammation) and vascular endothelial growth factor (VEGF; a marker of angiogenesis). These parameters were significantly correlated with a reduction in adiponectin in PCOS mice compared with vehicle-treated control mice. Exogenous adiponectin treatment of PCOS mice restored body mass and circulating androgen, triglyceride and glucose levels. Adiponectin also restored ovarian expression of steroidogenic markers (LH receptors, steroidogenic acute regulatory protein and 3ß-hydroxysteroid dehydrogenase), insulin receptor, TLR-4 and VEGF levels in control mice. Adiponectin restored ovulation in PCOS mice, as indicated by the presence of a corpus luteum and attainment of pregnancy. These findings suggest that adiponectin effectively facilitates fertility in anovulatory PCOS. We hypothesise that systemic adiponectin treatment may be a promising therapeutic strategy for the management of PCOS.

Kisspeptin regulates ovarian steroidogenesis during delayed embryonic development in the fruit bat, Cynopterus sphinx.

Cynopterus sphinx, a fruit bat, undergoes delayed embryonic development during the winter months, a period that corresponds to low levels of progesterone and estradiol synthesis by the ovary. Kisspeptins (KPs) are a group of neuropeptide hormones that act via G-protein coupled receptor 54 (GPR54) to stimulate hypothalamic secretion of Gonadotropin-releasing hormone, thereby regulating ovarian steroidogenesis, folliculogenesis, and ovulation. GPR54 is also expressed in the ovary, suggesting a direct role for KPs in ovarian steroidogenesis. The aim of present study was to determine if a low serum level of KP is responsible for reduced progesterone and estradiol levels during the period of delayed embryonic development in C. sphinx. Indeed, low serum KP abundance corresponded to reduced expression of GPR54 in ovarian luteal cells during the period of delayed development compared to normal development. In vitro and in vivo treatment with KP increased GPR54 abundance, via Extracellular signal regulated kinase and its downstream mediators, leading to increased progesterone synthesis in the ovary during delayed embryonic development. KP treatment also increased cholesterol uptake and elevated expression of Luteinizing hormone receptor and Steroid acute regulatory protein in the ovary, suggesting that elevation in circulating KP during delayed embryonic development may reactivate luteal activity. KPs may also enhance cell survival (BCL-2, reduced Caspase 3 activity) and angiogenesis (Vascular endothelium growth factor) during this period. The findings of this study thus demonstrate a regulatory role for KPs in the maintenance of luteal steroidogenesis during pregnancy in C. sphinx.

Direct effects of RFRP-1, a mammalian GnIH ortholog, on ovarian activities of the cyclic mouse.

Arg(R)-Phe(F)-amide related peptide-1 (RFRP-1) and -3 (RFRP-3) are known as mammalian orthologs of gonadotropin-inhibitory hormone (GnIH). In mammals, these RFRPs are expressed not only in the hypothalamus and but also in gonads. Inhibitory roles of the hypothalamic and gonadal RFRP-3 in reproduction have been documented in mammals. However, functional roles of the hypothalamic and gonadal RFRP-1 in reproduction are still unclear in mammals. Therefore, in vitro studies were conducted to elucidate the direct effect of RFRP-1, a mammalian GnIH ortholog, on ovarian activities, such as steroidogenesis, apoptosis, cell proliferation and metabolism in the cyclic mouse. The ovaries collected from the proestrus mice were cultured in vitro with different doses (Control, 1ng/ml, 10ng/ml and 100ng/ml) of RFRP-1 for 24h at 37°C. A significant dose-dependent increase in estradiol release from the ovary was detected after the treatment of RFRP-1. Therefore, changes in the ovarian activities, such as steroidogenic markers (luteinizing hormone receptors; LH-R and 3ß-hydroxysteroid dehydrogenase; 3ß-HSD), apoptotic markers [Poly(ADP-ribose) polymerase-1; PARP-1 and cysteine-aspartic protease; caspase-3], a cell proliferation marker (proliferating cell nuclear antigen; PCNA) and metabolic markers (GLUT-4; glucose uptake) were assessed by the treatment of RFRP-1 in the proestrus ovary. The densitometry analysis showed the treatment of RFRP-1 significantly increased the expressions of LH-R and 3ß-HSD, steroidogenic markers. In contrast, the expressions of PCNA, a cell proliferation maker; PARP-1 and caspase-3, apoptotic markers were significantly decreased. Interestingly, RFRP-1 treatment further increases significantly glucose uptake and GLUT-4 receptor expression. These findings indicate that RFRP-1 possesses a stimulatory effect on ovarian steroidogenesis in the proestrus mouse. This is the first evidence showing the direct action of RFRP-1 on steroidogenesis in any vertebrate. In addition, RFRP-1 may also act directly on ovarian folliculogenesis as an inhibitory factor.

Direct action of adiponectin ameliorates increased androgen synthesis and reduces insulin receptor expression in the polycystic ovary.

Polycystic ovary syndrome (PCOS) is a common endocrine and metabolic disorder of reproductive age females, leading to infertility. Despite extensive research, the exact pathophysiology and treatment are lacking. The aim of the current study was to evaluate the direct role of adiponectin either alone or together with luteinizing hormone (LH) on sex hormone synthesis and changes in insulin receptor (IR) in the ovary of dehydroepiandrosterone (DHEA)-treated PCOS-mice. Immunohistochemical study showed decreased expression of adiponectin receptor (AdipoR1) in PCOS-ovary which correlated with increased synthesis of androgen and decreased expression of IR. The treatment with adiponectin with or without LH to PCOS-ovary in vitro caused significant decline in androgen synthesis and increase in IR. This study showed the direct role of adiponectin in ameliorating hyperandrogenism and reducing IR in the ovary of PCOS-mice. Thus adiponectin treatment may be a novel therapeutic strategy for combating PCOS.