[Mechanism of Kuntai Capsules in treatment of polycystic ovary syndrome rat models based on AGE-RAGE signal pathway].

This study aims to investigate the impact of Kuntai Capsules(KTC) on polycystic ovarian syndrome(PCOS) rat models and explore the underlying mechanism. Fifty female SD rats were randomly divided into five groups(10 rats in each group), including control group, model group, low-, medium-, and high-dose KTC group. Except for the control group, the other groups were injected with dehydroepiandrosterone(DHEA) combined with a high-fat diet(HFD) to induce the PCOS rat model for 28 days. 0.315, 0.63, and 1.26 g·kg~(-1)·d~(-1) KTC was dissolved in the same amount of normal saline and given to low-, medium-, and high-dose KTC groups by gavage. Both control group and model group were given the same amount of normal saline for 15 days. After administration, fasting blood glucose(FBG) was measured by a glucose meter. Fasting insulin(FINS), luteinizing hormone(LH), testosterone(T), and follicle-stimulating hormone(FSH) were detected by enzyme-linked immunosorbent assay(ELISA), and LH/FSH ratio and insulin resistance index(HOMA-IR) were calculated. The pathological morphology of ovarian tissue was observed by hematoxylin-eosin(HE) staining. The expression levels of collagen α type Ⅲ 1 chain(COL3A1), apoptotic factors Bax, and Bcl-2 were detected using Western blot and immunofluorescence. The mRNA expressions of COL3A1, Bax, and Bcl-2 in ovarian tissue were performed by real-time PCR(RT-PCR). The results show that compared with the control group, the body weight, serum levels of FBG, FINS, LH, T, LH/FSH, and HOMA-IR are higher in model group(P<0.05 or P<0.01), and the level of FSH is lower(P<0.05). In model group, a large number of white blood cells are found in the vaginal exfoliated cells, mainly in the interictal phase. There are more cystic prominences on the surface of the ovary. The thickness of the granular cell layer is reduced, and oocytes are absent. COL3A1 and Bax protein expression levels are increased(P<0.01), while Bcl-2 protein expression levels are decreased(P<0.05) in the ovarian tissue COL3A1 and Bax mRNA expression levels are increased in ovarian tissue(P<0.05). Compared with the model group, the body weight, FBG, FINS, LH, T, LH/FSH, and HOMA-IR in low-, medium-, and high-dose KTC groups are decreased(P<0.05 or P<0.01), while the levels of FSH in medium-, and high-dose KTC groups are increased(P<0.05 or P<0.01). Low-, medium-, and high-dose KTC groups gradually show a stable interictal phase. The surface of the ovary is smooth. Oocytes and mature follicles can be seen in ovarian tissue, and the thickness of the granular cell layer is increased. The expression level of COL3A1 protein decreases in low-and medium-dose KTC groups(P<0.05 or P<0.01), and that of Bax protein decreases in low-dose KTC group(P<0.05 or P<0.01), and the expression level of Bcl-2 protein increases in low-dose KTC group(P<0.01). The expression levels of COL3A1 and Bax mRNA decreased in the low-dose KTC group(P<0.05), while the expression levels of Bcl-2 mRNA increased(P<0.05). In summary, KTC can inhibit ovarian granulosa cell apoptosis and reduce follicular atresia by regulating the AGE-RAGE signaling pathway. It can promote insulin secretion, reduce blood sugar and body weight, restore serum hormone levels, improve symptoms of PCOS, alleviate morphological damage of the ovary, and restore ovarian function, which is of great value in the treatment of PCOS.

Vitamin D3 promotes fish oocyte development by directly regulating gonadal steroid hormone synthesis†.

Vitamin D receptors and vitamin D3-metabolizing enzymes have been found to be highly expressed in the ovaries and spermatophores of fish. However, the role of vitamin D3 on fish gonadal development has rarely been reported. In this study, 2-month-old female zebrafish were fed with different concentrations of vitamin D3 diets (0, 700, 1400, and 11 200 IU/kg) to investigate the effects of vitamin D3 on ovarian development. The diet with 0 IU/kg vitamin D3 resulted in elevated interstitial spaces, follicular atresia, and reproductive toxicity in zebrafish ovaries. Supplementation with 700 and 1400 IU/kg of vitamin D3 significantly increased the oocyte maturation rate; upregulated ovarian gonadal steroid hormone synthesis capacity; and elevated plasma estradiol, testosterone, and ovarian vitellogenin levels. Furthermore, the current study identified a vitamin D response element in the cyp19a1a promoter and demonstrated that 1.25(OH)2D3-vitamin D response directly activated cyp19a1a production through activating the vitamin D response element. In conclusion, this study shows that an appropriate concentration of vitamin D3 can promote zebrafish ovarian development and affect vitellogenin synthesis through the vdr/cyp19a1a/er/vtg gene axis.

Menstrual blood-derived endometrial stem cell, a unique and promising alternative in the stem cell-based therapy for chemotherapy-induced premature ovarian insufficiency.

Chemotherapy can cause ovarian dysfunction and infertility since the ovary is extremely sensitive to chemotherapeutic drugs. Apart from the indispensable role of the ovary in the overall hormonal milieu, ovarian dysfunction also affects many other organ systems and functions including sexuality, bones, the cardiovascular system, and neurocognitive function. Although conventional hormone replacement therapy can partly relieve the adverse symptoms of premature ovarian insufficiency (POI), the treatment cannot fundamentally prevent deterioration of POI. Therefore, effective treatments to improve chemotherapy-induced POI are urgently needed, especially for patients desiring fertility preservation. Recently, mesenchymal stem cell (MSC)-based therapies have resulted in promising improvements in chemotherapy-induced ovary dysfunction by enhancing the anti-apoptotic capacity of ovarian cells, preventing ovarian follicular atresia, promoting angiogenesis and improving injured ovarian structure and the pregnancy rate. These improvements are mainly attributed to MSC-derived biological factors, functional RNAs, and even mitochondria, which are directly secreted or indirectly translocated with extracellular vesicles (microvesicles and exosomes) to repair ovarian dysfunction. Additionally, as a novel source of MSCs, menstrual blood-derived endometrial stem cells (MenSCs) have exhibited promising therapeutic effects in various diseases due to their comprehensive advantages, such as periodic and non-invasive sample collection, abundant sources, regular donation and autologous transplantation. Therefore, this review summarizes the efficacy of MSCs transplantation in improving chemotherapy-induced POI and analyzes the underlying mechanism, and further discusses the benefit and existing challenges in promoting the clinical application of MenSCs in chemotherapy-induced POI.

Attenuation of sodium arsenite mediated ovarian DNA damage, follicular atresia, and oxidative injury by combined application of vitamin E and C in post pubertal Wistar rats.

Arsenic being a toxic metalloid ubiquitously persists in environment and causes several health complications including female reproductive anomalies. Epidemiological studies documented birth anomalies due to arsenic exposure. Augmented reactive oxygen species (ROS) generation and quenched antioxidant pool are foremost consequences of arsenic threat. On the contrary, Vitamin E (VE) and C (VC) are persuasive antioxidants and conventionally used in toxicity management. Present study was designed to explore the extent of efficacy of combined VE and VC (VEC) against Sodium arsenite (NaAsO2) mediated ovarian damage. Thirty-six female Wistar rats were randomly divided into three groups (Grs) and treated for consecutive 30 days; Gr I (control) was vehicle fed, Gr II (treated) was gavaged with NaAsO2 (3 mg/kg/day), Gr III (supplement) was provided with VE (400 mg/kg/day) & VC (200 mg/kg/day) along with NaAsO2. Marked histological alterations were evidenced by disorganization in oocyte, granulosa cells and zona pellucida layers in treated group. Considerable reduction of different growing follicles along with increased atretic follicles was noted in treated group. Altered activities ofΔ5 3ß-Hydroxysteroid dehydrogenase and 17ß-Hydroxysteroid dehydrogenase accompanied by reduced luteinizing hormone, follicle-stimulating hormone and estradiol levels were observed in treated animals. Irregular estrous cyclicity pattern was also observed due to NaAsO2 threat. Surplus ROS production affected ovarian antioxidant strata as evidenced by altered oxidative stress markers. Provoked oxidative strain further affects DNA status of ovary. However, supplementation with VEC caused notable restoration from such disparaging effects of NaAsO2 toxicities. Antioxidant and antiapoptotic attributes of those vitamins might be liable for such restoration.

Protective effect of nano-selenium on mercury-induced prehierarchical follicular atresia in laying hens.

This study investigated the effect of nano-selenium (nano-Se) in protecting laying hens from mercury (Hg)-induced prehierarchical follicular atresia. Furthermore, the endoplasmic reticulum stress (ERS) was explored to reveal the molecular mechanism. In vivo, 720 Hyline-Brown laying hens were treated with Hg and nano-Se alone or in combination. In vitro, the prehierarchical follicles were treated with Hg, nano-Se and 4-phenyl butyric acid (4-PBA) alone or in combination (Control, 25 µM Hg group, 10 µM nano-Se group, 20 µM nano-Se group, 25 µM Hg + 10 µM nano-Se group, 25 µM Hg + 20 µM nano-Se group, 25 µM Hg + 4-PBA group, and 25 µM Hg + 20 µM nano-Se + 4-PBA group). The GCs were treated with Hg and nano-Se alone or in combination (Control, 15 µM Hg group, 6 µM nano-Se group, 12 µM nano-Se group, 15 µM Hg + 6 µM nano-Se group, 15 µM Hg + 12 µM nano-Se group). The results revealed that dietary Hg significantly reduced laying performance (P < 0.05) and egg quality (P < 0.05), whereas nano-Se addition prevented these reductions (P < 0.05). Hg exposure significantly induced the accumulation of Hg in PHFs (P < 0.05), prehierarchical follicular atresia (P < 0.05) and apoptosis in PHFs, whereas nano-Se addition significantly prevented these effects (P < 0.05). The levels of sex hormones (P < 0.05) were significantly decreased after Hg exposure in vivo and in vitro, while nano-Se addition prevented the reductions. Furthermore, the RNA-Seq results showed that the key factors of the ERS presented differential expression, including C/EBP homologous protein, protein kinase RNA-like endoplasmic reticulum kinase (PERK) and activating transcription factor 6 (ATF6) in GCs. Hg exposure significantly increased the key gene expression of endoplasmic reticulum stress in GCs, whereas nano-Se addition prevented the induction of expression of these genes. In addition, the protein levels of PERK, inositol requiring protein 1α (IRE1α) and ATF6 were significantly increased, whereas nano-Se addition prevented the enhancements of protein expression in GCs. In conclusion, this study shows that Hg exposure can reduce induce prehierarchical follicular atresia, whereas nano-Se can prevent these effects. Our results also elucidate a key role of ERS in these protective effects of nano-Se in laying hens.

Role of Endoplasmic Reticulum Stress in Nano-Selenium Alleviating Prehierarchical Follicular Atresia Induced by Mercury in Laying Hens.

This study investigated that the effect of nano-selenium (nano-Se) addition preventing prehierarchical follicular atresia induced by mercury (Hg) exposure in laying hens. Furthermore, endoplasmic reticulum (ER) stress pathway was explored to reveal the protective mechanism of nano-Se in vitro. The results revealed that Hg could significantly reduce laying performance (P < 0.05) and egg quality (P < 0.05), whereas nano-Se addition partially reversed the reductions. Besides, Hg significantly induced the deposition of Hg in prehierarchical follicles (P < 0.05) and prehierarchical follicular atresia (P < 0.05), whereas nano-Se addition could alleviate these toxicities in vitro. In addition, Hg exposure could significantly reduce cell viability (P < 0.05) and induce pyknotic nucleus in prehierarchical granulosa cells, while nano-Se addition reversed these effects. The levels of follicle-stimulating hormone (P < 0.05), luteinizing hormone (P < 0.05), progesterone (P < 0.05), and estradiol (P < 0.05) were significantly decreased after Hg exposure in vitro. However, nano-Se addition reversed the decreases of sex hormone levels. Furthermore, Hg exposure significantly increased the gene expressions of CHOP (P < 0.05), PERK (P < 0.05), ATF4 (P < 0.05), ATF6 (P < 0.05), ASK1 (P < 0.05), IRE1α (P < 0.05), TRAF2 (P < 0.05), caspase-9 (P < 0.05), caspase-3 (P < 0.05), and Bax/Bcl-2 (P < 0.05), whereas nano-Se addition reversed these increases of gene expressions in vitro. In summary, this study provides that Hg can induce prehierarchical follicular atresia, whereas nano-Se addition can ameliorate it, and elucidates an important role of ER stress in nano-Se alleviating prehierarchical follicular atresia induced by Hg in laying hens.

Stress associated ovarian dysfunctions in a seasonal breeder Funambulus pennanti: Role of glucocorticoids and possible amelioration by melatonin.

Studies have shown that stress caused by lack of physical activity disrupts the normal pattern of glucocorticoid secretion which adversely affects the reproductive axis. We studied the effect of chronic movement restriction on ovarian responses in the Indian Palm Squirrel Funambulus pennanti, a highly active diurnal rodent. Physical restraint of squirrels induced stress that led to a significant increase in plasma cortisol, corticosterone and decreased 17ß-estradiol level leading to follicular atresia. Ovarian Reactive Oxygen Species (ROS) content, lipid peroxidation (LPO), activities of superoxide dismutase (SOD) and catalase (CAT) enzymes increased in restrained squirrels. Elevated ROS increased the oxidative load that led to ovarian cell death as evidenced by increased Bax and decreased Bcl2 expression causing further decline in Aromatase and ERα proteins. To elaborate the mechanism(s) involved in stress induced glucocorticoid mediated oxidative damages to the ovary we extended our study by exposing ovaries in vitro to the synthetic glucocorticoid dexamethasone (200 µM). We observed that glucocorticoid receptor (GR) expression was significantly increased in dexamethasone treated ovaries in vitro with a decrease in expression of Nrf2 and HO-1 proteins. Melatonin supplementation (10 nM) along with dexamethasone significantly decreased ovarian ROS production, lipid peroxidation and increased antioxidant enzyme activities by improving the expression of Nrf2 and HO-1, reinstating the cellular redox homeostasis. Therefore, it can be suggested that physical restraint induced glucocorticoid and its receptor activation interfered with the ovarian antioxidant defense mechanism. Melatonin via its receptor MT1 significantly alleviated ovarian damages acting as a cytoprotective agent.

Epigallocatechin gallate and theaflavins independently alleviate cyclophosphamide-induced ovarian damage by inhibiting the overactivation of primordial follicles and follicular atresia.

BACKGROUND: Cyclophosphamide (CTX), which has been used to treat common female cancers for several years, often causes ovarian damage, early menopause and infertility. However, strategies for the effective prevention and treatment of CTX-induced ovarian damage are still lacking. Epigallocatechin gallate (EGCG) and theaflavins (TFs), key molecules derived from green tea or black tea, have been shown to exert preventive effects on many ageing-related diseases. PURPOSE: We aimed to explore the potential preventive and protective effects of EGCG and TFs on CTX-induced ovarian damage and compare the two compounds. STUDY DESIGN: Six-week-old female mice were administered a low or high dose of EGCG or TFs. The low dose was equivalent to the average daily amount of tea consumed by a drinker. METHODS: We determined the oestrous cycle and serum hormone levels to evaluate ovarian endocrine function, and we performed mating tests for reproductivity. We also assessed the follicle count and AMH level to evaluate ovarian reserve, and we performed Masson's trichrome and Sirius red staining to evaluate ovarian fibrosis. We conducted γ-H2AX and TUNEL analyses to evaluate DNA damage, and we also measured the relevant indicators of oxidative stress and follicular activation, including NRF2, HO-1, SOD2, AKT, mTOR and RPS6. RESULTS: EGCG and TFs treatment independently improved the ovarian endocrine function and reproductivity of mice that were administered CTX. EGCG and TFs also increased the ovarian reserve of these animals. Furthermore, EGCG and TFs alleviated oxidation-induced damage to ovarian DNA in mice by activating the NRF2/HO-1 and SOD2 pathways and reducing the apoptosis of growing follicles. At the same time, EGCG and TFs reduced the overactivation of primordial follicles by inhibiting the AKT/mTOR/RPS6 pathway. CONCLUSION: The present study showed that EGCG and TFs independently improved ovarian function in mice with CTX-induced ovarian damage, thereby providing useful information for designing a potential clinical strategy that will protect against chemotherapy-induced ovarian damage.

Apoptotic mechanism of premature ovarian failure and rescue effect of Traditional Chinese Medicine: a review.

Social and environmental factors render premature ovarian failure (POF) as a major cause of decline or loss of female fertility. The natural pregnancy rate of patients with POF is only 5%-10%. Follicular atresia is the main factor in the pathogenesis of POF. Due to the unique ovarian physiological environment and follicular developmental processes, the apoptosis of ovarian granulosa cells and oocytes together cause follicular atresia, which involves the apoptosis-related internal and external pathways. Furthermore, during POF, apoptosis and oxidative stress forms a ""vicious circle"", which involves a variety of changes between the molecules. The existing pharmaceutical preparations such as gonadal hormones are the basic methods for the treatment of POF, and the curative effect was affirmative; however, it was ineffective after withdrawn, while the long-term application led to adverse reactions. Traditional Chinese Medicine (TCM) has a history of treating gynecological diseases and infertility and has gained increasing attention. Studies have shown that compounds isolated from herbal medicine exerted a positive effect on follicular atresia caused by cell apoptosis that also improved the POF. The present study reviewed the mechanisms underlying the apoptosis in POF and elaborated the internal mechanism of TCM in the treatment of the condition.

MAPK, AKT/FoxO3a and mTOR pathways are involved in cadmium regulating the cell cycle, proliferation and apoptosis of chicken follicular granulosa cells.

The occurrence of cadmium (Cd) in feed is a major problem in animal health and production. Studies have confirmed that Cd depresses egg production of laying hens, which is closely related to follicular atresia. This study aimed to assess the toxic impacts of Cd on the ovarian tissue, and to examine the mechanism of Cd-induced granulosa cell proliferation and apoptosis. Results from the nitric oxide (NO) and malondialdehyde (MDA) content, total superoxide dismutase (T-SOD), glutathione peroxide (GSH-Px), total nitric oxide synthase (T-NOS) and adenosine triphosphatase (ATPase) activities, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay, and hematoxylin-eosin (H & E) staining indicated that excess Cd induced oxidative stress, granulosa cell apoptosis and follicular atresia in the layer ovary. Low-dose Cd exposure (1 µM) induced the granulosa cell proliferation, upregulated the mRNA levels of RSK1 and RHEB, activated FoxO3a, AKT, ERK1/2, mTOR and p70S6K1 phosphorylation, and promoted cell cycle progression from phase G1 to S. However, high-dose Cd exposure (15 µM) induced reactive oxygen species (ROS) generation and cell apoptosis, upregulated the mRNA levels of the inflammatory factors, ASK1, JNK, p38 and TAK1, downregulated the expressions of RSK1 and RHEB genes, and inhibited the phosphorylation of ERK1/2, mTOR and p70S6K1 proteins, and the cell cycle progression. Rapamycin pre-treatment completely blocked the phosphorylation of mTOR and p70S6K1 proteins, and the cell cycle progression induced by 1 µM Cd, and accelerated 15 µM Cd-induced cell apoptosis and cell cycle arrest. The microRNA sequencing result showed that 15 µM Cd induced differential expression of microRNA genes, which may regulate AKT, ERK1/2 and mTOR signaling and cell cycle progression by regulating the activity of G proteins and cell cycle-related proteins. Conclusively, these results indicated that Cd can cause the ovarian damage and follicular atresia, and regulate cell cycle, cell proliferation or apoptosis of granulosa cells through MAPK, AKT/FoxO3a and mTOR pathways in laying hens.

Gut microbial metabolism of dietary fibre protects against high energy feeding induced ovarian follicular atresia in a pig model.

To investigate the effects of dietary fibre on follicular atresia in pigs fed a high-fat diet, we fed thirty-two prepubescent gilts a basal diet (CON) or a CON diet supplemented with 300 g/d dietary fibre (fibre), 240 g/d soya oil (SO) or both (fibre + SO). At the 19th day of the 4th oestrus cycle, gilts fed the SO diet showed 112 % more atretic follicles and greater expression of the apoptotic markers, Bax and caspase-3, and these effects were reversed by the fibre diet. The abundance of SCFA-producing microbes was decreased by the SO diet, but this effect was reversed by fibre treatment. Concentrations of serotonin and melatonin in the serum and follicular fluid were increased by the fibre diet. Overall, dietary fibre protected against high fat feeding-induced follicular atresia at least partly via gut microbiota-related serotonin-melatonin synthesis. These results provide insight into preventing negative effects on fertility in humans consuming a high-energy diet.

Metformin Prevents Follicular Atresia in Aging Laying Chickens through Activation of PI3K/AKT and Calcium Signaling Pathways.

Increased follicular atresia occurs with aging and results in reduced fecundity in laying chickens. Therefore, relieving follicular atresia of aging poultry is a crucial measure to maintain sustained high laying performance. As an antiaging agent, metformin was reported to play important roles in preventing aging in diverse animals. In this study, the physiological state of the prehierarchical follicles in the peak-laying hens (D280) and aged hens (D580) was compared, followed with exploration for the possible capacity of metformin in delaying atresia of the prehierarchical follicles in the aged D580 hens. Results showed that the capacity of yolk deposition within follicles declined with aging, and the point of endoplasmic reticulum- (ER-) mitochondrion contact decreased in the ultrastructure of the follicular cells. Meanwhile, the expression of apoptosis signaling genes was increased in the atretic small white follicles. Subsequently, the H2O2-induced follicular atresia model was established to evaluate the enhancing capacity of metformin on yolk deposition and inhibition of apoptosis in the atretic small white follicles. Metformin inhibited apoptosis through regulating cooperation of the mitochondrion-associated ER membranes and the insulin (PI3K/AKT) signaling pathway. Furthermore, metformin regulated calcium ion homeostasis to relieve ER-stress and inhibited release of mitochondrion apoptosis factors (BAD and caspase). Additionally, metformin activated PI3K/AKT that suppressed activation of BAD (downstream of the insulin signaling pathway) in the atretic follicles. Further, serum estrogen level and liver estrogen receptor-α expression were increased after dietary metformin supplementation in D580 hens. These results indicated that administration of dietary metformin activated the PI3K/AKT and calcium signaling pathway and enhanced yolk deposition to prevent chicken follicular atresia.

N-acetyl-l-cysteine mediated regulation of DNA fragmentation, an apoptotic event, against methoxychlor toxicity in the granulosa cells of ovarian antral follicles.

Methoxychlor (MXC), an organo-chlorine insecticide, is a reproductive toxicant in females, causing apoptosis-mediated follicular atresia. To elucidate the potentials of Methoxychlor as a geno-toxicant, granulosa cells of healthy antral follicles, exposed to MXC and antioxidant, N-acetyl-l-cysteine, were studied by the terminal deoxynucleotidyltransferase-dUTP nick end-labelling and single-cell gel electrophoresis (comet) assays. MXC caused DNA fragmentation, as revealed by the increased incidence of dark brown condensed TUNEL positive cells in contrast with lightly brown TUNEL negative cells with maximum TUNEL positive cells were observed in 100 µg/mL MXC treated groups. Quantitatively, maximum geno-toxicity was exhibited at highest MXC treatment with percent tail DNA as 17.87 ± 0.85, 41.16 ± 3.94, and 47.73 ± 3.71 in comparison with control (0.65 ± 0.03, 2.91 ± 0.27, and 7.16 ± 1.39) after 24, 48 and 72 h exposure duration, respectively. MXC treated groups exhibited Type 1-Type 3 comets as compared to Type 0 comets in control groups. Supplementation of NAC led to significant (p < 0.05) decline in geno-toxicity in MXC treated groups with maximum amelioration observed at 5 and 10 mM. Consequently, increased DNA damage attributed to the granulosa cells apoptosis in response to Methoxychlor exposure was significantly combated by NAC supplementation, preventing the geno-toxicity induced cyto-toxicity in GCs.

Roles of vitamin D and its receptor in the proliferation and apoptosis of luteinised granulosa cells in the goat.

The objective of this study was to investigate the dose-dependent effect of 1α,25-(OH)2VD3 (Vit D3) on invitro proliferation of goat luteinised granulosa cells (LGCs) and to determine the underlying mechanisms of its action by overexpressing and silencing vitamin D receptor (VDR) in LGCs. Results showed that VDR was prominently localised in GCs and theca cells (TCs) and its expression increased with follicle diameter, but was lower in atretic follicles than in healthy follicles. The proliferation rate of LGCs was significantly higher in the Vit D3-treated groups than in the control group, with the highest proliferation rate observed in the 10nM group; this was accompanied by changes in the expression of cell cycle-related genes. These data indicate that Vit D3 affects LGC proliferation in a dose-dependent manner. Contrary to the VDR knockdown effects, its overexpression upregulated and downregulated cell cycle- and apoptosis-related genes respectively; moreover, supplementation with 10nM of Vit D3 significantly enhanced these effects. These results suggest that changes in VDR expression patterns in LGCs may be associated with follicular development by regulation of cell proliferation and apoptosis. These findings will enhance the understanding of the roles of Vit D3 and VDR in goat ovarian follicular development.

Anti-hypercholesterolemic impacts of barley and date palm fruits on the ovary of Wistar albino rats and their offspring.

A high cholesterol diet is related to ovarian dysfunction and infertility which has been increased among young ages consuming processed food products. The present study was conducted to evaluate the role of a high cholesterol diet on the ovaries of young female rats via assessments of histopathology, immunohistochemistry, oxidative stress and apoptic markers. Also, mating of hypercholesterolemic female rats was carried out to measure the fertility and numbers of their offspring. At the same time, phytotherapy was carried out through supplementing the diet with barley and/ or date palm fruits (10%) during the experiment to assess the phyto-therapeutic impacts in attenuation of drastic hypercholesterolemic effects. Hypercholesterolemic diet-fed rats exhibited damage of the ovarian follicles and increased follicular atresia. Furthermore, expression of cleaved caspase-3 was upregulated, while PCNA was downregulated in granulosa, theca and stroma cells. Hypercholesterolemic female rats showed marked depletion of antioxidative enzymes, increased lipid peroxidation and apoptotic markers. Alterations to the female serum hormones were detected. Offspring maternally fed on hypercholesterolemic diet showed a significant decrease of body weight and altered sex ratio. However, concomitant supplementation of barley and or date fruits to hypercholesterolemic groups revealed marked improvement of ovarian structure and function. On the basis of these evidences, it is believed that the enhanced synergistic effects of barley and/or date palm fruits in the amelioration of ovarian structure and functions were elicited by the potential antioxidant activity of their phytomicronutrients, polyphenols, ß-glucan and trace elements. These materials scavenge free radicals from inflamed cells that can be used to establish an effective and novel therapeutic strategy for activating ovarian cell regeneration.

[Explore microcosmic connection between autophagy mechanism and follicular development based on "kidney governing reproduction" theory].

In the theory of traditional Chinese medicine(TCM) that "kidney storing essence and governing reproduction", reproductive essence is an important part of the kidney essence and acts as the original material of offspring embryos. Sperm, oocyte and zygote should be all included in the range of reproductive essence. Ovum is the essence of reproduction from inborn. The follicles maturation depends on the quality of oocyte and the vigor of kidney essence. Meanwhile, discharge of mature ovum relies on the stimulation and promotion by kidney Qi. Autophagy almost exists in different cells stages and all various of mammalian cells. Many studies have found that autophagy not only participates in the formation of follicles, but also in every phase of the follicles development, and is involved in the occurrence and development of ovarian diseases. Recently, more and more scholars believe that autophagy is a new field to explore the microcosmic relationship between autophagy and TCM. Kidney-nourishing TCM could promote follicular growth and improve variety clinical symptoms by inhibiting the apoptosis of ovarian granulosa cells and reducing follicular atresia. Meanwhile, apoptosis of ovarian granulosa cells is closely related to autophagy of ovarian granulosa cells. In order to provide some theoretical foundation for kidney-nourishing therapy's promoting effect on follicular growth and improving effect on ovarian function, also to further explore the molecular mechanism of kidney-nourishing medicine in promoting follicular development, this paper would explain the microcosmic relationship between autophagy and follicular development based on the theory of "kidney governing reproduction". All of these would be of great significance to prevent and intervene the diseases of reproductive system timely and effectively.

Silica nanoparticle exposure inducing granulosa cell apoptosis and follicular atresia in female Balb/c mice.

Given that the effects of ultrafine fractions (< 0.1 µm) on reproductive diseases are gaining attention, this study aimed to explore the influence of silica nanoparticle (SiNP)-induced female reproductive dysfunction. In this study, 80 female mice were randomly divided into four groups including a control group and three concentrations of SiNP groups (7, 21, 35 mg/kg). Mice were exposed to the vehicle control and silica nanoparticles by tracheal perfusion every 3 days a total of five times in 15 days. Then, half of the mice in each group were sacrificed on 15 and 30 days after the first dose, respectively. Our findings indicated that SiNPs can result in ovarian damage, cause an imbalance of sex hormones, increase the number of atretic and primary follicles, and induce oxidative stress and DNA strand breaks in ovary by day 15. The protein expressions of ATM, CHK-2, P53, E2F1, P73, BAX, Caspase-9, and Caspase-3 were significantly increased, while expressions of RAD51 were down-regulated after SiNP exposure by days 15. Estradiol increased, while progesterone increased in low dose and decreased in high dose after SiNP exposure by 15 days. However, these changes were recovered by 30 days. The results suggest that SiNPs can cause reversible damage to follicles in mice. SiNPs could primarily cause DNA damage and DNA damage response through oxidative stress, while DNA damage repair failure because of severe DNA damage activated the mitochondrial apoptosis pathway and therefore resulted in apoptosis of granulosa cell. In addition, the disorder of reproductive endocrine function caused by SiNPs could be another reason for SiNP-induced reproductive dysfunction in mice. These events in turn induce the follicles to undergo atresia.

The effect of DHEA on apoptosis and cohesin levels in oocytes in aged mice.

Female fertility declines with age as the number of ovarian follicles decreases and aneuploidy increases. Degradation of the cohesin complex might be responsible for age-related aneuploidy. Dehydroepiandrosterone (DHEA) can improve the ovarian reserve and reduce the rate of aneuploidy, but the relationship between DHEA and cohesin levels in oocytes is still unknown. The aim of the current study was to evaluate the effect of the supplement DHEA on ovarian function, including the number of follicles and cohesin levels in oocytes. C57BL/6J mice at 3 weeks, 6 weeks, 12 weeks, 6 months, and 10 months of age were used to obtain a systematic view into follicle apoptosis and cohesin levels in oocytes. Nine-month-old C57BL/6J mice were administered saline (n = 5), 17ß-estradiol (100 µg/kg per day, n = 5), or DHEA (5mg/Kg per day, n = 5). After 4 weeks, aged mice were weighed and sacrificed, and ovarian tissue samples were prepared. Anti-VASA staining and HE staining were used to count the number of follicles. Anti-γH2AX staining and TUNEL were used to measure follicle apoptosis and immunofluorescent staining was used to detect the levels of three oocyte cohesin subunits: REC8, SMC1ß, and SMC3. Administration of the supplements 17ß-estradiol and DHEA to aged mice increased the number of primordial and primary follicles and decreased the age-related apoptosis of follicles. Levels of the cohesin subunits REC8 and SMC1ß declined with age, but DHEA and 17ß-estradiol tended to delay that decline. The supplement DHEA increased the number of primordial and primary follicles in aged mice by inhibiting follicle apoptosis and tended to delay the decrease in cohesin levels in oocytes.

Effects of melatonin on follicular atresia and granulosa cell apoptosis in the porcine.

The accumulation of reactive oxygen species is detrimental to the health of the ovarian follicle. The protective, antioxidant properties of melatonin, an endogenous component of porcine follicular fluid, on apoptosis of granulosa cells were evaluated in this study. Porcine granulosa cells from medium-sized (3-5 mm), healthy follicles were cultured in serum-free conditions with melatonin (0, 0.01, 0.1, 1.0, 10, and 100 ng/mL) with or without its receptor antagonist, luzindole, followed by evaluation of apoptotic markers in the treated cells. Results revealed that endogenous, intrafollicular melatonin concentration decreased as follicular atresia progressed, whereas the percentage of apoptotic granulosa cells increased. Spontaneous apoptosis of granulosa cells, triggered by serum deprivation in vitro, was remarkably blocked by melatonin (1.0 ng/mL melatonin, 32.7 ± 0.5%, vs. control, 47.0 ± 1.0%; P < 0.05). Treatment with 1.0 ng/mL of melatonin also significantly elevated MT2, SOD1, and GPX4 while lowering FASL, CHOP, and GRP78 mRNA abundance compared to the untreated control. The anti-apoptotic effect and some changes of apoptotic-relevant genes in granulosa cells invoked by melatonin supplementation were markedly blocked by luzindole, suggesting that melatonin could prevent the apoptosis of porcine granulosa cells during follicular atresia via its membrane receptors and its free-radical-scavenging activity. These findings provide new insights into the regulatory mechanism of melatonin in follicular atresia-related functions. Mol. Reprod. Dev. 83: 692-700, 2016 © 2016 Wiley Periodicals, Inc.

Pregnenolone co-treatment partially restores steroidogenesis, but does not prevent growth inhibition and increased atresia in mouse ovarian antral follicles treated with mono-hydroxy methoxychlor.

Mono-hydroxy methoxychlor (mono-OH MXC) is a metabolite of the pesticide, methoxychlor (MXC). Although MXC is known to decrease antral follicle numbers, and increase follicle death in rodents, not much is known about the ovarian effects of mono-OH MXC. Previous studies indicate that mono-OH MXC inhibits mouse antral follicle growth, increases follicle death, and inhibits steroidogenesis in vitro. Further, previous studies indicate that CYP11A1 expression and production of progesterone (P4) may be the early targets of mono-OH MXC in the steroidogenic pathway. Thus, this study tested whether supplementing pregnenolone, the precursor of progesterone and the substrate for HSD3B, would prevent decreased steroidogenesis, inhibited follicle growth, and increased follicle atresia in mono-OH MXC-treated follicles. Mouse antral follicles were exposed to vehicle (dimethylsulfoxide), mono-OH MXC (10 µg/mL), pregnenolone (1 µg/mL), or mono-OH MXC and pregnenolone together for 96 h. Levels of P4, androstenedione (A), testosterone (T), estrone (E1), and 17ß-estradiol (E2) in media were determined, and follicles were processed for histological evaluation of atresia. Pregnenolone treatment alone stimulated production of all steroid hormones except E2. Mono-OH MXC-treated follicles had decreased sex steroids, but when given pregnenolone, produced levels of P4, A, T, and E1 that were comparable to those in vehicle-treated follicles. Pregnenolone treatment did not prevent growth inhibition and increased atresia in mono-OH MXC-treated follicles. Collectively, these data support the idea that the most upstream effect of mono-OH MXC on steroidogenesis is by reducing the availability of pregnenolone, and that adding pregnenolone may not be sufficient to prevent inhibited follicle growth and survival.