NR4A1-mediated regulation of lipid droplets in progesterone synthesis in goat luteal cells†.

Nuclear receptor NR4A1 is a key factor in glycolipid metabolism and steroidogenesis, while lipid droplets serve as crucial dynamic organelles for lipid metabolism in luteal cells. To investigate the effects of NR4A1 on lipid droplet metabolism and progesterone (P4) synthesis in goat corpus luteum in vitro, luteal cells from the middle-cyclic corpus luteum were isolated and treated with Cytosporone B (CSNB, an agonist) or siRNA of NR4A1. Results showed that both low (1 µM) and high (50 µM) concentrations of CSNB promoted lipid droplet accumulation, while NR4A1 knockdown reduced lipid droplet content. CSNB increased while siNR4A1 decreased total cholesterol content; however, CSNB and siNR4A1 did not change triglyceride content. CSNB increased the expression of perilipins at mRNA and protein levels, also increased LDLR, SCARB1, SREBFs, and HMGCR mRNA abundance. Treatment with siNR4A1 revealed opposite results of CSNB, except for HMCGR and SREBF2. For steroidogenesis, 1 µM CSNB increased, but 50 µM CSNB inhibited P4 synthesis, NR4A1 knockdown also reduced the P4 level. Further analysis demonstrated that 1 µM CSNB increased the protein levels of StAR, HSD3B, and P-HSL, while 50 µM CSNB decreased StAR, HSD3B, and CYP11A1 protein levels. Moreover, 50 µM CSNB impaired active mitochondria, reduced the BCL2, and increased DRP1, Caspase 3, and cleaved-Caspase 3 protein levels. siNR4A1 consistently downregulated the P-HSL/HSL ratio and the steroidogenic protein levels. In conclusion, NR4A1-mediated lipid droplets are involved in the regulation of progesterone synthesis in goat luteal cells.

Follicle stimulating hormone controls granulosa cell glutamine synthesis to regulate ovulation.

Polycystic ovary syndrome (PCOS) is the leading cause of anovulatory infertility. Inadequate understanding of the ovulation drivers hinders PCOS intervention. Herein, we report that follicle stimulating hormone (FSH) controls follicular fluid (FF) glutamine levels to determine ovulation. Murine ovulation starts from FF-exposing granulosa cell (GC) apoptosis. FF glutamine, which decreases in pre-ovulation porcine FF, elevates in PCOS patients FF. High-glutamine chow to elevate FF glutamine inhibits mouse GC apoptosis and induces hormonal, metabolic, and morphologic PCOS traits. Mechanistically, follicle-development-driving FSH promotes GC glutamine synthesis to elevate FF glutamine, which maintain follicle wall integrity by inhibiting GC apoptosis through inactivating ASK1-JNK apoptotic pathway. FSH and glutamine inhibit the rapture of cultured murine follicles. Glutamine removal or ASK1-JNK pathway activation with metformin or AT-101 reversed PCOS traits in PCOS models that are induced with either glutamine or EsR1-KO. These suggest that glutamine, FSH, and ASK1-JNK pathway are targetable to alleviate PCOS.

Genistein ameliorates starvation-induced porcine follicular granulosa cell apoptosis.

In brief: Genistein contributes to granulosa cell (GC) survival by two routes: one is that genistein induced p-AMPK and inhibited p-mTOR, which induces LC3 activation and autophagy; the other is that genistein inhibited caspase-3 and its cleavage, which induces PARP1 activation and PARylation. Abstract: Genistein is an isoflavone which is beneficial for health, but little is known regarding its function on granulosa cell fate during follicular atresia. In the present study, we established an in vitro model of porcine follicular granulosa cell apoptosis by serum deprivation and showed that treatments with 1 µM and 10 µM genistein significantly reduced the apoptotic rate of granulosa cells compared to the blank control (P < 0.05). These results suggest that genistein at micromolar levels alleviates serum deprivation-induced granulosa cell apoptosis, and the ameliorative effect of genistein on granulosa cell apoptosis is likely to be able to inhibit nutrient depletion-induced follicular atresia. Further experimental results revealed that the expression of the autophagic marker protein LC3II in 100 nM-10 µM genistein treatment increased in a dose-dependent manner and was higher than the control (P < 0.05). Genistein also dose dependently promoted the phosphorylation of AMPK (adenosine 5'-monophosphate-activated protein kinase) in granulosa cells. Poly(ADP-ribose) (pADPr) formation in genistein-treated groups was also notably higher than in the controls (P < 0.05). Collectively, genistein alleviates serum deprivation-induced granulosa cells in vitro through enhancing autophagy, which involving AMPK activation and PARylation signaling. However, further study should be carried out to investigate the role of the aforementioned signaling on this process.

Impact of Cadmium and Lead Exposure on Camel Testicular Function: Environmental Contamination and Reproductive Health.

The free grazing habits of camels from various sources may cause heavy metals to bioaccumulate in their tissues and organs, possibly resulting in higher amounts of these toxic substances in their bodies over time. The aim of this study was to assess the exposure impact of lead (Pb) and cadmium (Cd) on bull camels of the Lassi breed, aged 7 to 8 years, at a site near the industrial area and another two non-industrial sites, to analyze the presence of heavy metals. Samples from three sites were collected from thirty camels (n = 10/each), soil and water (n = 30), and five different plants (n = 15/each) for analysis. Testes were collected for atomic absorption spectrometry (AAS), and hematoxylin-eosin (HE) staining. Serum samples were obtained to measure testosterone levels by radioimmunoassay (RIA). Samples were obtained from plants, soil, water, blood, serum and urine for AAS. According to the results, the testes' weight, length, width, and volume significantly decreased at the industrial site compared with the other two sites as a result of exposure to Cd and Pb. Additionally, blood testosterone concentrations were considerably lower at the industrial site, indicating a detrimental impact on testicular steroidogenesis. The histological investigation of the industrial site indicated structural disturbances, including seminiferous tubule degeneration and shedding, cellular debris in seminiferous tubules, lining epithelium depletion, and vacuolation. Elevated amounts of Cd and Pb were found at the industrial site when analyzed using water, soil, plants, testes, serum, and urine. These findings demonstrate the adverse effects of Pb and Cd exposure on camel testicular function, including decreased weight and altered steroidogenesis. These findings are essential for understanding the impact of exposure to Pb and Cd on camel reproductive function and for developing successful prevention and management plans for these exposures in this species.

Dietary curcumin supplementation relieves hydrogen peroxide-induced testicular injury by antioxidant and anti-apoptotic effects in roosters.

This study was aimed to investigate the effects of dietary curcumin supplementation on the hydrogen peroxide (H2O2)-induced testicular oxidative damage in breeder roosters. Thirty-two 20-week roosters were randomly divided into four groups: (1) basal diet (CON); (2) basal diet with H2O2 challenge (H2O2); (3) basal diet with 200 mg/kg curcumin (CUR); (4) basal diet with 200 mg/kg curcumin and H2O2 challenge (CUR + H2O2). The trial lasted for 8 weeks, H2O2 challenged groups got an intraperitoneal injection of H2O2 at the 50 and 53 days, while the CON and CUR groups received an injection of saline. The results showed that dietary curcumin supplementation significantly decreased abnormal sperm rates in the semen, notably improved seminiferous tubules, increased testis scores, and serum testosterone levels. Curcumin supplementation could also ameliorate the redox damage caused by H2O2, by enhancing the capacities of antioxidant enzymes (CAT, GSH-Px, SOD, and T-AOC), and reducing MDA levels. In addition, curcumin normalized the H2O2-induced negative effects, which included downregulations in spermatogenesis-related genes (STAR, HSD3-ß1, SYCP3, AKT1) and antioxidant genes (HMOX-1, NQO-1), reduced protein expressions of Nrf2, PCNA, and Bcl-2, and increased protein expressions of Caspase 3 and Bax. Moreover, H2O2-induced decreased mRNA expressions of EIF2AK3, Caspase3, and BCL-2 were all reversed by dietary curcumin supplementation. In summary, dietary curcumin supplementation could relieve H2O2-induced oxidative damage and reproduction decline through the Nrf2 signaling pathway and anti-apoptotic effects in roosters.

Impact of adrenocorticotropin hormone administration on the endocrinology, estrus onset, and ovarian function of weaned sows.

Chronic stress affects the reproductive health of mammals; however, the impact of adrenocorticotropin hormone (ACTH) level elevation during chronic stress on the reproduction of weaned sows remains unclear. In this study, nine weaned sows with the same parturition date were randomly divided into control group (n = 4) and ACTH group (n = 5). Each group received intravenous administration of ACTH three times daily for 7 days. Blood samples were collected every 3 h after injection. A radioimmunoassay was used to measure the concentrations of cortisol, luteinizing hormone (LH), follicle-stimulating hormone (FSH), progesterone (P4) and estradiol-17ß (E2) in the blood. Estrus was determined according to changes in the vulva and the boar contact test. The mRNA expressions of glucocorticoid receptor, FSH receptor, LH receptor (LHR) in the corpus luteum (CL) were detected by qRT-PCR. The results showed that ACTH administration substantially delayed the initiation of estrus and the pre-ovulatory LH peak. The sows of control group ovulated within 10 days and the ovulation rate was 100%, while it was 60% in the ACTH group. Two sows of ACTH group showed pseudo-estrus. The E2 concentrations significantly decreased in the ACTH group at 36 h, 42 h and 66 h of the experimental period. The P4 concentrations in the ACTH group significantly decreased at 132, 138, and 147 h of the experimental period. ACTH significantly reduced the LHR mRNA expression in CLs. In conclusion, long-term repeated ACTH administration affects the endocrinology, estrus onset, and ovarian function of weaned sows.

Dietary mulberry-leaf flavonoids improve the eggshell quality of aged breeder hens.

Eggshell quality is subject to a significant decline in the late laying period, which results in huge economic losses. The purpose of this study was to investigate the effects of dietary mulberry-leaf flavonoids (MF) on the eggshell quality of aged breeder hens. A total of 270 (60-week-old) Qiling breeder hens were randomly assigned to 3 treatments with supplemental dietary MF doses (0, 30, and 60 mg/kg). The results showed that dietary MF improved the eggshell thickness and strength, following the reduced broken egg ratio (P < 0.05). Histological analysis showed that dietary MF increased glandular density and luminal epithelium height in the shell gland (P < 0.05). MF treatment reduced the apoptotic index of the shell gland, following by improved antioxidant capacity (P < 0.05). The protein expression of Caspase 3 was down-regulated, and Nrf2 was up-regulated by dietary MF (P < 0.05). Furthermore, calcium (Ca) content in the serum and shell gland, as well as the activity of Ca2+-ATPase in the shell gland were increased by dietary MF (P < 0.05). Ca transport-related genes (ESRα, ESRß, KCNA1, OPN, CABP-28K and CDH6) in the shell gland were upregulated by dietary MF treatment (P < 0.05). In conclusion, dietary MF could ameliorate the eggshell quality of aged hens by improving antioxidative capability and Ca deposition in the shell gland of uterus.

Activation of Ovarian Taste Receptors Inhibits Progesterone Production Potentially via NO/cGMP and Apoptotic Signaling.

Taste receptors are not only expressed in the taste buds, but also in other nongustatory tissues, including the reproductive system. Taste receptors can be activated by various tastants, thereby exerting relatively physiologic functions. The aim of this study was to investigate the effects and potential mechanisms underlying ovarian taste receptor activation on progesterone production using saccharin sodium as the receptor agonist in a pseudopregnant rat model. Taste 1 receptor member 2 (TAS1R2) and taste 2 receptor member 31 (TAS2R31) were demonstrated to be abundantly expressed in the corpora lutea of rats, and intraperitoneal injection of saccharin sodium can activate both of them and initiate their downstream signaling cascades. The activation of these ovarian taste receptors promoted nitric oxide (NO) production via endothelial nitric oxide synthase (eNOS). NO production then increased ovarian cyclic guanosine 3',5'-monophosphate (cGMP) levels, which, in turn, decreased ovarian cyclic adenosine 3',5'-monophosphate levels. In addition, the activation of ovarian taste receptors induced apoptosis, possibly through NO and mitogen-activated protein kinase signaling. As a result, the activation of ovarian taste receptors reduced the protein expression of steroidogenesis-related factors, causing the inhibition of ovarian progesterone production. In summary, our data suggest that the activation of ovarian taste receptors inhibits progesterone production in pseudopregnant rats, potentially via NO/cGMP and apoptotic signaling.

Mesenchymal Stem Cell Therapy Using Human Umbilical Cord in a Rat Model of Autoimmune-Induced Premature Ovarian Failure.

Premature ovarian failure (POF) is one of the principal causes of female infertility, and although its causes are complex and diverse, autoimmune deficiency may be involved. Human umbilical cord mesenchymal stem cells (UCMSCs) can be used for tissue regeneration and repair. Therefore, the present study was designed to determine the role of UCMSCs in immune factor-induced POF in rats. In this study, different concentrations of UCMSCs were injected into induced POF rats. Ovarian functions were examined by evaluating the estrus cycle, follicular morphology, hormonal secretion, and the proliferation and apoptosis of granulosa cells. Our results showed that the estrus cycle of rats returned to normal and follicular development was significantly improved after transplantation of UCMSCs. In addition, serum concentrations of 17-estradiol (E2), progesterone (P4), and anti-Müllerian hormone (AMH) increased significantly with treatment. Transplantation of UCMSCs also reduced the apoptosis of granulosa cells and promoted the proliferation of granulosa cells. All of these improvements were dose dependent. Furthermore, the results of related gene expression showed that transplanted human UCMSCs upregulated the expression of Bcl-2, AMH, and FSHR in the ovary of POF rats and downregulated the expression of caspase-3. These results further validated the potential mechanisms of promoting the release of cell growth factors and enhancing tissue regeneration and provide a theoretical basis for the clinical application of stem cells in the treatment of premature ovarian failure.

Sweet taste receptor agonists alter ovarian functions and ovarian cycles in aged mice.

Saccharine sodium and rebaudioside A are low-calorie sweeteners, and the biologic effects of these sweeteners in rat ovaries are related to the activity of sweet taste receptors. Data on the impact and regulatory mechanisms underlying such sweeteners on the reproduction of aged animals are currently lacking. In the present study we assessed how the consumption of sweeteners affects the ovarian cycle, ovulation, biochemical indices, and other biologic functions. Thirty-six 1-year-old mice were randomly divided into 3 groups: a control (C) group receiving regular water, a saccharin sodium group receiving a 7.5 mM solution, and the rebaudioside A group receiving a 2.5 mM solution for 30 days. We observed no significant changes in body weights in any group. However, uterine weight in the rebaudioside A group significantly increased in diestrus, and we recorded a significant increase in the percentage of abnormal estrous cycles and the number of corpora lutea in the treatment groups. TUNEL staining and Immunoreactivity for the apoptosis-inducing factor (AIF) confirmed apoptosis in granulosa cells, oocyte, and corpus luteum. Serum glucose increased significantly in both treatment groups and there was a significant increase in cholesterol in the rebaudioside A group. Furthermore, the saccharin sodium-treated group exhibited elevated serum progesterone levels compared with the other groups. In conclusion, sweeteners manifested deleterious effects on reproductive indices in aged mice.

Heat exposure affected the reproductive performance of pregnant mice: Enhancement of autophagy and alteration of subcellular structure in the corpus luteum.

To investigate whether autophagy and subcellular changes are involved in the corpus luteum after heat exposure, a total of 30 early pregnant mice were divided equally into heat stress (HS) and non-HS (NHS) groups (n = 15). Mice in the HS group were exposed to 40.5 ±â€¯0.2 ℃ for 7 consecutive days. Ovaries were collected for immunohistochemistry (IHC), western blot (WB) analysis and transmission electron microscopy (TEM). Serum was collected to determine progesterone by RIA and uteri were collected to count the implantation sites. Results showed that heat exposure increased rectal temperature, decreased body weight and number of implantation sites. WB analysis revealed that ovarian expression of LC3B and Atg7 was up-regulated, while p62 was down-regulated in the HS group. IHC results demonstrated that ovarian staining intensity of LC3B was more intense in the HS group than that of the NHS group. LC3B was mainly localized in the granulosa cells, oocytes and luteal steroidogenic cells of the HS group. TEM results revealed double-layered separated membranes indicative of autophagosomes in the luteal steroidogenic cells of the HS group. Moreover, TEM showed that the mitochondrial cristae became dearth, structure-less, swollen after HS. Additionally, the nucleus expanded and accumulation of lipid droplets increased after HS. Results also showed that heat exposure decreased serum progesterone level and ovarian P450scc expression. These results indicate that HS enhanced autophagy and altered the subcellular structure of luteal steroidogenic cells, which may contribute to interfering with the maintenance of luteal function in early pregnant mice.

Bisphenol A attenuates thyroxine-induced apoptosis in ovarian granulosa cells of pigs.

Bisphenol A (BPA) is a chemical of high production volume that is used widely in many industries and is known as a xenooestrogen and anti-thyroid hormone endocrine disrupter. There is little information regarding the effects of BPA in the presence of thyroid hormone on porcine granulosa cell development. Thus, the primary granulosa cells were treated with thyroxine (T4, 10 nM), BPA (10 µM) or T4 plus BPA; we subsequently evaluated the effects of T4 or BPA on 17ß-estradiol synthesis, cellular proliferation and apoptosis. Our data showed that BPA significantly increased the accumulation of 17ß-estradiol and promoted granulosa cell proliferation, whereas T4 significantly decreased 17ß-estradiol and had no effect on cellular proliferation. In addition, it was noteworthy that T4 treatment induced apoptosis in porcine granulosa cells and BPA co-incubation attenuated T4-induced apoptosis as shown from flow cytometric assay analysis. We hypothesized that BPA attenuates T4-induced apoptosis by regulating 17ß-estradiol accumulation and oestrogen receptor-mediated signalling pathways. In conclusion, our results demonstrated that T4 affected 17ß-estradiol accumulation and induced cellular apoptosis, but did not affect granulosa cell proliferation. Exposure to BPA increased 17ß-estradiol accumulation, promoted granulosa cell proliferation and attenuated T4-induced apoptosis in porcine granulosa cells in vitro.

Roles of poly (ADP-ribose) polymerase 1 activation and cleavage in induction of multi-oocyte ovarian follicles in the mouse by 3-nitropropionic acid.

3-nitropropionic acid (3-NPA) is known to be a mitochondrial toxin produced by plants and fungi, which may produce DNA damage in cells. However, studies of its reproductive toxicology are lacking. We know that poly(ADP-ribose) polymerase (PARP) plays an important role in a large variety of physiological processes and is involved in DNA repair pathways. The present study was therefore aimed at exploring the involvement of PARP-1 activation and cleavage after 3-NPA stimulation in female mice. We observed an increased number of atretic follicles and multi-oocyte follicles (MOFs) after treatment with 3-NPA and serum concentrations of 17ß-oestradiol and progesterone were significantly reduced. Our results provide evidence that PARP-1 cleavage and activational signals are involved in pathological ovarian processes stimulated by 3-NPA. In addition, total superoxide dismutase, glutathione peroxidase and catalase activities were significantly increased, whereas succinate dehydrogenase was decreased in a dose-dependent manner. Results from our in vitro study similarly indicated that 3-NPA inhibited the proliferation of mouse granulosa cells and increased apoptosis in a dose-dependent manner. In summary, 3-NPA induces granulosa cell apoptosis, follicle atresia and MOFs in the ovaries of female mice and causes oxidative stress so as to disrupt endogenous hormonal systems, possibly acting through PARP-1 signalling.

The role of active immunization against inhibin α-subunit on testicular development, testosterone concentration and relevant genes expressions in testis, hypothalamus and pituitary glands in Yangzhou goose ganders.

The present study was designed to investigate the potential role of immunization against inhibin on testicular development, plasma testosterone concentration and expression of relevant genes in hypothalamus, pituitary, Leydig and Sertoli cells in Yangzhou ganders. For this purpose, Yangzhou ganders, n = 30 were divided into groups A and B. Group B ganders were actively immunized against inhibin α-subunit, while group A ganders were immunized with bovine serum albumin (BSA), which served as control. Immunization against inhibin elevated testes weights. In addition, immunization against inhibin elevated GnRH, StAR, CYP11A1 and AMH mRNA transcription expressions as depicted by qRT-PCR. Furthermore, hypothalamic GnRH-I mRNA expressions were up regulated, while GnIH mRNA transcription expression showed reciprocal expression on day 227. LH-ß mRNA transcription expression remained unaffected. In conclusion, our findings suggest that active immunization against inhibin affect spermatogenesis and testicular development through regulations of hypothalamic, pituitary and testicular genes expressions.

Sodium Arsenite Injection Induces Ovarian Oxidative Stress and Affects Steroidogenesis in Rats.

Oxidative stress is involved in the regulation of mammalian reproduction. The present study was conducted to detect the sodium arsenite-induced oxidative stress and alterations in the structure and steroidogenesis in rat ovary. Twenty female adult rats were injected i.p. with sodium arsenite (8 mg/kg BW, T) or 0.9% saline (C) for 16 days. The oxidative stress indexes and morphology of the liver, kidney, and ovary were detected using commercial kits and HE staining, respectively. The serum progesterone and estradiol were detected by RIA, and the ovarian steroidogenic gene expressions were detected by real-time PCR. Results showed that the ovarian activities of SOD and GSH-PX decreased (P < 0.05), while the ROS activity and MDA level increased (P < 0.05) in the T group. HE staining results showed that treatment with sodium arsenite damaged the ovarian morphology, resulting in reduced large and medium follicles and increased atretic follicles. Nonetheless, neither the liver nor kidney showed evident changes in the oxidative stress indexes or morphology after sodium arsenite treatment. The serum progesterone and estradiol levels decreased (P < 0.05) with the reduced expressions in the ovarian steroidogenic genes (StAR, P450scc, and 3ß-HSD) (P < 0.05). In conclusion, sodium arsenite injection can induce ovarian oxidative stress in rats which set up an appropriate model for future studies of ovarian diseases as well as the toxic mechanism of arsenic in the reproduction.

Neonatal genistein exposure disrupts ovarian and uterine development in the mouse by inhibiting cellular proliferation.

Soy-based formula contains high concentrations of the isoflavone genistein. Genistein possesses estrogenic and tyrosine kinase inhibitory activity and interferes with cellular proliferation and development. To date, the acute and chronic effects of genistein on ovarian and uterine development have not been fully elucidated. In this study, mice at postnatal day 1 were subcutaneously injected with 100 mg/kg genistein for 10 consecutive days, and then their ovaries and uteri were collected on days 10, 21, and 90. Histological evaluation was performed after hematoxylin and eosin staining. The proliferating activity was indicated by the proliferating indicator protein Ki67. Results showed that the subcutaneous injection of genistein to neonatal mice induced the formation of multi-oocyte follicles and delayed the primordial follicle assembly in the ovaries. Genistein significantly enlarged the cross-sectional area of the uterine cavity and wall and disrupted the regularity between the uterine stroma and myometrium. Genistein exposure inhibited proliferative activity because fewer Ki67-positive nuclei were detected in ovarian and uterine cell populations than in the control. Furthermore, most ovaries from adult mice given neonatal genistein were without corpora lutea, and there appeared to be cystic follicles and hypertrophy of the theca, and cortical and medullary layers. Considering the high concentration of isoflavone in soy-based infant formulas and livestock feed, we suggest that the use of isoflavone-rich diets in humans and livestock receive closer examination.

Melatonin mitigates bisphenol A-induced estradiol production and proliferation by porcine ovarian granulosa cells in vitro.

Melatonin plays a crucial role in the amelioration of reproductive toxicity induced by endocrine-disrupting chemicals. However, very few studies have investigated the mitigating effects of melatonin on BPA-induced dysfunction in porcine granulosa cells. In the present study, primary granulosa cells were cultured in serum-low conditions with bisphenol A (BPA) (10 µM) with or without melatonin (100 µM), followed by evaluation of estradiol synthesis and cell proliferation. Our results showed that BPA significantly increased estradiol concentration and granulosa cell proliferation. Interestingly, melatonin co-incubation reduced the high levels of estradiol in porcine ovarian granulosa cells induced by BPA stimulation. Furthermore, melatonin co-incubation also attenuated BPA-induced proliferation as shown by a decline in the Ki67-positive cell ratio and PCNA expression level. However, treatment with melatonin-alone did not dramatically reduce estradiol levels or expression of proliferative regulatory protein markers (Ki67, PCNA). We hypothesize that the regulation by melatonin of estradiol biosynthesis and cellular proliferation is highly correlated with BPA stimulation. In conclusion, this study first showed that melatonin mitigated BPA-induced estradiol increase and proliferation in porcine ovarian granulosa cells in vitro. Our results suggest that melatonin may be a promising pharmacologic agent for preventing the potential reproductive toxicity caused by endocrine-disrupting chemicals.

Expression patterns of claudin-5 and its related signals during luteal regression in pseudopregnant rats: The enhanced effect of additional PGF treatment.

To study the expression patterns of claudin-5 and its related signals during luteal regression in rats, a sequential PMSG/hCG treatment paradigm was used to obtain a single, well-defined generation of corpus luteum (CL). A total of 35 rats were treated with one PGF or two PGF at an interval of 24 h from day 7 of pseudopregnancy to induce CL regression. Serum and ovaries were collected at 0, 2, 4, 8 or 24 h after one PGF injection (1 PGF), 2 or 24 h after two PGF injections (2 PGF). The serum progesterone level was detected by RIA; the ovarian expression of claudin-5, the phosphorylations of STAT3 (p-STAT3), Akt (p-Akt), ERK1/2 (p-ERK) and p38 MAPK (p-p38) were detected by western blot, real-time PCR and IHC. Results showed that serum progesterone (P4) decreased after PGF treatment. Claudin-5 mRNA decreased at 4 h and 8 h after 1 PGF and 2 h after 2 PGF, and claudin-5 protein decreased at 4 h after 1 PGF. p-STAT3 increased at 4 h after 1 PGF and 2 h after 2 PGF. p-ERK increased at 2 h after 2 PGF. The level of p-Akt decreased at 4 h after 1 PGF. PGF treatment did not alter the phosphorylation of p38 MAPK at any time points in this study. IHC results revealed that claudin-5 was expressed in the nuclei and cytoplasm of steroidogenic cells and in the vessels, while PGF induced-p-STAT3 was expressed uniformly in the cytoplasm of luteal steroidogenic cells. In conclusion, PGF treatment decreased the expression of claudin-5 and the additional PGF treatment enhanced the decrease in claudin-5 mRNA expression and the increases in ERK1/2 and STAT3 phosphorylation in the corpus luteum of pseudopregnant rats, which will contribute new information to the further study of molecular mechanism of luteal regression.

Thyroid hormones alter estrous cyclicity and antioxidative status in the ovaries of rats.

To expand our understanding of the roles of thyroid hormones on female reproduction, we induced hypo- and hyper-T rat models to investigate the roles of thyroid hormones on estrous cyclicity, as well as the antioxidative status in the ovaries of rats. In the current study, our data show that hypothyroidism (hypo-T) and hyperthyroidism (hyper-T) led to significantly reduced body weights and ovarain weights and delayed vaginal opening day. For hyper-T, thyroxine (T4), tri-iodothyronine (T3), progesterone (P4) and follicle-stimulating hormone (FSH) were significantly increased, while estradiol (E2) and luteinizing hormone (LH) were significantly decreased. For hypo-T rats, serum levels of total T4 and T3, E2, P4, FSH and LH were significantly increased, while concentrations of E2 and LH were significantly decreased. For ovary morphology, the numbers of secondary and antral follicles were significantly decreased with more atretic antral follicles and less corpora lutea in both hyper- and hypo-T groups. Both hyper-T and hypo-T treatment significantly decreased the expressions of thyroid hormone receptor α1 in the ovary. Hypo-T significantly reduced nitric oxide (NO), total NO synthase (tNOS), inducible NOS and constitutive NOS activities, but hyper-T increased them. For antioxidative parameters, hypo-T and hyper-T treatment significantly increased malondialdehyde (MDA) contents. The activities of both glutathione peroxidase (GSH-Px) and catalase (CAT) significantly decreased in the hypo-T group but increased in the hyper-T group. Total superoxide dismutase (T-SOD) activity was significantly increased in the hyper-T group. In summary, thyroid hormones alter estrous cyclicity and antioxidative status in the ovary of the rat may act through the NOS signaling pathway.

Effects of daily exposure to saccharin sodium and rebaudioside A on the ovarian cycle and steroidogenesis in rats.

Saccharin sodium and rebaudioside A are widely used as non-caloric sweeteners in our daily life; however, the impacts and regulatory mechanisms of such sweeteners on reproduction remain unclear. In the present study, we used rats as animal models to evaluate the effects of daily exposure to saccharin sodium and rebaudioside A on ovarian biologic functions. Weanling rats were distributed into five experimental groups receiving normal water, 1.5 or 7.5 mM saccharin sodium solution, or 0.5 or 2.5 mM rebaudioside A solution for 48 days of exposure. The results showed an increased percentage of abnormal estrous cycles, augmented number of ovarian cysts, elevated serum progesterone levels, and increased expression of steroidogenesis-related factors in saccharin sodium-treated groups. Conversely, rebaudioside A-treated groups showed decreased serum progesterone levels. Our findings suggest that saccharin sodium exerts adverse biologic effects on ovaries, and rebaudioside A is a potential steroidogenic disruptor in female rats.