Dietary supplementation of osthole and icariin improves the production performance of laying hens by promoting follicular development.

Osthole (Ost) and icariin (Ica) are extracted from traditional Chinese medicine Cnidium monnieri and Epimedii Folium, respectively, and both exhibit estrogen-like biological activity. This study aimed to determine the efficacy and safety of combining Ost with Ica on the production performance of laying hens and to explore their possible mechanisms. The production performance, egg quality, residues of Ost and Ica in eggs, serum reproductive hormone levels, expression of ovarian reproductive hormone receptor, proliferation of granulosa cells in small yellow follicles (SYF), and progesterone secretion in large yellow follicles (LYF) related genes and proteins expression were detected. The results showed that adding 2 mg/kg Ost + 2 mg/kg Ica to the feed increased the laying rate, average egg weight, Haugh unit, and protein height of laying hens. Serum follicle-stimulating hormone (FSH), luteinizing hormone (LH), and progesterone (P4) levels increased, and the expression of ovarian estrogen receptor (ER), follicle-stimulating hormone receptor (FSHR), and progesterone receptor (PGR) mRNA was up-regulated. Additionally, the mRNA and protein levels of steroidogenesis acute regulatory protein (StAR), cytochrome P450 side-chain cleavage (P450scc), and 3ß-hydroxysteroid dehydrogenase (3ß-HSD) increased in LYF. Furthermore, mRNA and protein levels of proliferating cell nuclear antigen (PCNA), cyclin E1, and cyclin A2 were up-regulated in SYF. The residues of Ost and Ica in egg samples were not detected by high-performance liquid chromatography (HPLC). In conclusion, dietary supplementation of Ost and Ica increased granulosa cells proliferation in SYF and increased P4 secretion in granulosa cells of LYF, ultimately improving the production performance of laying hens.

Melatonin protects oogenesis from hypobaric hypoxia-induced fertility damage in mice.

Environmental hypoxia adversely affects reproductive health in humans and animals at high altitudes. Therefore, how to alleviate the follicle development disorder caused by hypoxia exposure and to improve the competence of fertility in plateau non-habituated female animals are important problems to be solved urgently. In this study, a hypobaric hypoxic chamber was used for 4 weeks to simulate hypoxic conditions in female mice, and the effects of hypoxia on follicle development, proliferation and apoptosis of granulosa cells, reactive oxygen species (ROS) levels in MII oocyte and 2-cell rate were evaluated. At the same time, the alleviating effect of melatonin on hypoxic exposure-induced oogenesis damage was evaluated by feeding appropriate amounts of melatonin daily under hypoxia for 4 weeks. The results showed that hypoxia exposure significantly increased the proportion of antral follicles in the ovary, the number of proliferation and apoptosis granulosa cells in the follicle, and the level of ROS in MII oocytes, eventually led to the decline of oocyte quality. However, these defects were alleviated when melatonin was fed under hypoxia conditions. Together, these findings suggest that hypoxia exposure impaired follicular development and reduced oocyte quality, and that melatonin supplementation alleviated the fertility reduction induced by hypoxia exposure.

Effectiveness of melatonin adjuvant treatment in cisplatin to prevent depletion of ovarian follicles in mice: systematic review†.

INTRODUCTION: Cisplatin-based chemotherapy is the standard cancer therapy; however, this treatment causes depletion of ovarian follicles in women of reproductive age. Adjuvant treatment with melatonin can protect the ovaries from oxidative stress, reducing the side effects of chemotherapy. The objective was to evaluate the effects of the use of melatonin on the ovarian follicles of mice treated with cisplatin. METHODOLOGY: A systematic review was performed. The search strategy used the terms: "cisplatin", "melatonin," and "ovarian". MEDLINE EMBASE, Cochrane Library, and gray literature (Google Scholar) were used as databases. The search was limited to experimental studies, performed on animals, with no language restrictions. RESULTS: The search identified 30 studies and 5 primary studies, published between 2016 and 2021, and met the inclusion criteria, with a total of 115 mice. For the p-FOX3a/FOXO3a pathway, the meta-analysis showed a standard mean difference (SMD) of -4.79 (95% CI -6.16 to -3.42; P < 0.00001, two studies, 38 mice; I2 = 0%). For the p-PTEN pathway, the meta-analysis showed an SMD of -1.65 (95% CI -2.71 to -0.59; P = 0.002, two studies, 38 mice; I2 = 47%). CONCLUSION: Melatonin variation in efficacy varies according to the dose used in mice previously exposed to cisplatin. However, melatonin was able to alter the p-PTEN and p-FOX3a/FOXO3a pathways.

A screen of repurposed drugs identifies AMHR2/MISR2 agonists as potential contraceptives.

We identified the anti-Mullerian hormone (also known as Müllerian inhibiting substance or MIS) as an inhibitory hormone that induces long-term contraception in mammals. The type II receptor to this hormone, AMHR2 (also known as MISR2), represents a promising druggable target for the modulation of female reproduction with a mechanism of action distinct from steroidal contraceptives. We designed an in vitro platform to screen and validate small molecules that can activate MISR2 signaling and suppress ovarian folliculogenesis. Using a bone morphogenesis protein (BMP)­response element luciferase reporter cell­based assay, we screened 5,440 compounds from a repurposed drug library. Positive hits in this screen were tested for specificity and potency in luciferase dose­response assays, and biological activity was tested in ex vivo Mullerian duct regression bioassays. Selected candidates were further evaluated in ex vivo follicle/ovary culture assays and in vivo in mice and rats. Here, we report that SP600125, CYC-116, gandotinib, and ruxolitinib can specifically inhibit primordial follicle activation and repress folliculogenesis by stimulating the MISR2 pathway.

Progestogen supplementation during superovulation leads to higher embryo viability and TGFB1 gene expression in sheep.

This study aimed to compare the effect of the administration of either medroxyprogesterone acetate (MPA) or progesterone (P4) in superovulation (SOV) treatments applied during the first follicular wave on follicular development, embryo yield, and the expression of genes related to pluripotency maintenance, differentiation of the trophectoderm, cell growth and differentiation, apoptosis and energy metabolism in sheep embryos. The estrous cycle of 36 multiparous ewes was synchronized with a short protocol, and the animals were randomly allocated to three groups. At the beginning of SOV, 12 ewes per treatment received an intravaginal sponge impregnated with 60 mg of MPA (TMPA), or an intravaginal device containing 0.33 g of P4 (TP4), or received no progestogen treatment (CON). The device was kept until the fifth dose of FSH. Ewes were mated with five fertile rams. Gene expression was performed by RT-qPCR using grade I and II blastocysts. The numbers of corpora lutea, total structures and viable embryos recovered per ewe were similar (P > 0.05) among groups. However, the viability rate was higher in TP4 (71.9 ± 16.3%) compared to CON (24.4 ± 16.8%; P = 0.01) and similar to TMPA (49.9 ± 16.3%; P = 0.2). Similarly, when compared with CON, treatment with P4 or MPA positively regulated the TGFB1 transcript involved in cell proliferation and differentiation (P = 0.01 and P = 0.03, respectively). In conclusion, supplementation with P4 during the first follicular wave of the estrous cycle improves embryo viability and alters the expression of the TGFB1 gene.

Effect of retinol as antioxidant on the post-thaw viability and the expression of apoptosis and developmental competence-related genes of vitrified preantral follicles in buffalo (Bubalus bubalis).

The present study evaluated the effect of supplementation of retinol in the vitrification solution on the viability, apoptosis and development-related gene expression in vitrified buffalo preantral follicles. Preantral follicles isolated from cortical slices of ovaries were randomly assigned into three groups: Group1-Control fresh preantral follicles; Group 2-Vitrification treatment (Vitrification solution 1 (VS1) -TCM-199 + 25 mM HEPES + Foetal bovine serum (FBS) 10%, Ethylene glycol (EG): 10%, Dimethyl sulphoxide (DMSO): 10%, Sucrose-0.3 M for 4 min; VS2- TCM-199 + 25 mM HEPES + FBS10%, EG:25%, DMSO: 25%, Sucrose:0.3 M for 45 s); Group3-vitrification treatment +5 µM of Retinol. Preantral follicles were placed in corresponding vitrification medium and plunged into liquid nitrogen (-196°C). After a week, the follicles were thawed and analysed for follicular viability and gene expression. There was no significant difference in the viability rates among the Group 1(Fresh preantral follicles) (91.46 ± 2.39%), Group 2 (89.59 ± 2.46%) and Group 3 (87.19 ± 4.05%). There was a significantly (p < .05) higher mRNA expression of BCL2L1, GDF-9 and BMP-15 in the vitrification + retinol group compared with the control group. There was a significantly (p < .05) higher expression of Caspase-3 and Annexin-5 in the vitrification group and Vitrification + retinol group compared with control group of follicles. It is concluded that the supplementation of 5 µM of Retinol in Vitrification solution was an efficient vitrification procedure for the vitrification of buffalo preantral follicles.

Protective effect of Huyang Yangkun Formula on ovarian function in premature ovarian insufficiency rats based on apoptotic mechanism.

ETHNOPHARMACOLOGICAL RELEVANCE: Huyang Yangkun Formula(HYF) is a traditional Chinese medicine formula based on the traditional theory of Yin and Yang. It could consolidate the Qi of Yin and Yang, adjust the balance of Qi and blood. It has shown clinical efficacy for patients with Premature Ovarian Insufficiency(POI). AIM OF THE STUDY: Aim to access the effect of Huyang Yangkun formula (HYF) on premature ovarian insufficiency rat model and explores the mechanism related to aquaporins(AQPs) and apoptosis. MATERIALS AND METHODS: Female SD rats were injected with 4-vinylcyclonhexenediepoxide(VCD, 160 mg/kg/day) for 15 days. Then, HYF (0.297 g/kg)/estradiol valerate (0.1 mg/kg) was administered for 105 days in the HYF/estradiol valerate treatment(EVT) group. Serum AMH, FSH and E2 were detected by ELISA, and the developing follicles were counted in each group.The TUNEL assay was used to detect positive apoptotic signals. IHC and western blots were used to verify differentially expressed AQPs and apoptosis-related regulators potentially associated with HYF. RESULTS: Total follicles were increased significantly in the HYF group. Serum AMH was increased in the HYF group compared with MOD group. Serum FSH and E2 showed no obvious difference between HYF group and MOD group. Apoptosis occurred in POI model was proved by TUNEL and Caspase3/9 and HYF could rescue this apoptosis, besides the anti-apoptotic effect may be better than EVT. Distribution of AQPs in rat ovaries based on developmental stages of follicle was observed, and AQP8 was obviously expressed in the developing follicles and corpus luteum, particularly in granulosa cells. Upregulation of AQP8 in the MOD group and downregulation by HYF were observed. BCL-XL was significantly upregulated in the HYF group and EVT group; BCL-2 was upregulated in the HYF group with no statistical difference; MCL-1 was downregulated in the HYF group. BAX and BIM were significantly upregulated in the MOD group. The ratio of BCL-2/BAX and BCL-XL/BAX were decreased in the MOD group, and BCL-XL/BAX was increased in the HYF group and EVT group. CONCLUSION: This study evaluated the treatment effect of HYF on POI in rats. It showed that HYF could promote the follicles development by regulating AQP8/Bcl-2 family-related mitochondrial apoptosis, which provides basic evidence for TCM as an alternative therapy for POI.

Effect of samul-tang on female fertility via RAS signaling pathway in ovaries of aged mice.

Samul-tang (SM), a traditional herbal medicine, is used to treat age-related human conditions, such as infertility and menstrual irregularities. The mechanism underlying the role of SM in ovary function needs elucidation. In this study, the influence of SM administration on the ovarian reserve of aged mice was investigated. Female BALB/c mice (8 and 40 weeks-old) were administered with distilled water (young or old group) or SM for 4 weeks. SM administration prevented age-related ovarian follicle loss in mice. Quality of oocytes and blastocysts were enhanced in SM-administrated mice compared to those of non-treated old mice. Further, SM administration increased the pregnancy rate and number of litters. SM triggered changes in aging-related genes that are linked to the RAS-mediated pathway. Thus, we demonstrate that SM can be used to increase the oocyte yield in aged women, potentially improving age-related cognitive decline in the ovarian reserve.

Postpartum nutrition affects the insulin-like growth factor system in dominant follicles and plasma of anestrous beef cows.

Effects of nutrition on insulin-like growth factor-I (IGF-I), IGF binding proteins (IGFBP), and insulin in plasma and dominant follicles were evaluated at day 72 and 56 (Exp. 1, n = 12 and Exp. 2, n = 28, respectively) postpartum in anovulatory primiparous beef cows. Cows were stratified based on body condition score at calving and randomly assigned to nutritional treatments: maintain (M), 2.27 kg of a 40 % CP supplement per day and ad libitum hay; or gain (G), ad libitum access to a 50 % concentrate diet and ad libitum hay. Blood samples were collected twice weekly starting 30 days postpartum. Ovarian follicles were evaluated using ultrasonography commencing 42 (Exp. 1) or 30 (Exp. 2) days postpartum. Body weight and condition score were greater (P < 0.05) for cows of G than M groups and postpartum interval to luteal function was longer for cows of the M than G group. Insulin and IGF-I concentrations in follicular fluid (FF) and plasma were greater (P < 0.05) for cows of the G than M group at follicular aspiration. Plasma and FF IGFBP4 and IGFBP5 concentrations were greater (P <  0.05) in Exp. 2, and IGFBP5 was greater in Exp. 1 for cows of the G than M group. Treatment did not affect FF steroid concentrations or granulosal cell CYP19A1, PAPPA, IGFBP4, and IGFBP5 mRNA abundance. These results indicate concentrations of IGF-I, insulin, IGFBP4, and IGFBP5 in FF and plasma are affected by nutritional intake and may be related to follicular function.

Alpha Lipoic Acid Supplementation Improves Ovarian Tissue Vitrification Outcome: An Alternative to Preserve the Ovarian Function of Morada Nova Ewe.

This study evaluated the effect of adding alpha lipoic acid (ALA) to the vitrification solution of sheep ovarian tissue on 7 days of in vitro culture or 15 days of xenotransplantion. ALA was used at two different concentrations (100 µM: ALA100 and 150 µM: ALA150). Ovarian tissue was evaluated by classical histology (follicular morphology, development, and stromal cell density); immunohistochemistry for forkhead box O3a (FOXO3a); Ki67 (cell proliferation); cluster of differentiation 31 (CD31); and alpha smooth muscle actin (α-SMA). Reactive oxygen species (ROS) levels in ovarian tissue, as well as malondialdehyde (MDA) and nitrite levels in the culture medium, were assessed. Similar percentage of morphologically normal follicles was found in the vitrified ovarian tissue in the presence of ALA100 or ALA150 after in vitro culture or xenotransplantation. Follicular development from all treatments was higher (P < 0.05) than the control group. Moreover, an activation of primordial follicles was observed by FOXO3a. Stromal cell density and immunostaining for Ki67 and CD31 were significantly higher (P < 0.05) in ALA150 vitrified tissue. No difference (P > 0.05) was found in α-SMA between ALA concentrations after in vitro culture or xenograft. ROS levels in the ovarian tissue were similar (P > 0.05) in all treatments, as well as MDA and nitrite levels after 7 days of culture. We concluded that the addition of ALA 150 is able to better preserve the stromal cell density favoring granulosa cell proliferation and neovascularization.

Melatonin delays ovarian aging in mice by slowing down the exhaustion of ovarian reserve.

Studies have shown that melatonin (MLT) can delay ovarian aging, but the mechanism has not been fully elucidated. Here we show that granulosa cells isolated from mice follicles can synthesize MLT; the addition of MLT in ovary culture system inhibited follicle activation and growth; In vivo experiments indicated that injections of MLT to mice during the follicle activation phase can reduce the number of activated follicles by inhibiting the PI3K-AKT-FOXO3 pathway; during the early follicle growth phase, MLT administration suppressed follicle growth and atresia, and multiple pathways involved in folliculogenesis, including PI3K-AKT, were suppressed; MLT deficiency in mice increased follicle activation and atresia, and eventually accelerated age-related fertility decline; finally, we demonstrated that prolonged high-dose MLT intake had no obvious adverse effect. This study presents more insight into the roles of MLT in reproductive regulation that endogenous MLT delays ovarian aging by inhibiting follicle activation, growth and atresia.

Gallic acid promotes the in vitro development of sheep secondary isolated follicles involving the phosphatidylinositol 3-kinase pathway.

This study was conducted to evaluate the effect of addition of gallic acid as the single antioxidant to the base medium for in vitro culture of sheep secondary follicles and if the phosphatidylinositol 3-kinase (PI3K) pathway is involved in the action of gallic acid. Secondary follicles were isolated and cultured for 12 days in α-MEM supplemented with bovine serum albumin (BSA), insulin, glutamine, hypoxanthine, transferrin, selenium, and ascorbic acid (control medium: α-MEM+) or in α-MEM supplemented with BSA, insulin, glutamine, hypoxanthine and different concentrations of gallic acid (25, 50 or 100 µM), thus replacing transferrin, selenium and ascorbic acid in the medium. Follicle morphology, glutathione (GSH), and mitochondrial activity, and meiotic resumption were evaluated. Furthermore, inhibition of PI3K pathway was performed by pretreatment with LY294002. After 12 days of culture, the follicle survival in a medium containing 100 µM gallic acid was similar (P > 0.05) to α-MEM+ and greater (P < 0.05) compared with other gallic acid concentrations. Antrum formation, follicle diameter, GSH, and mitochondrial activity, and meiotic resumption, however, were greater (P < 0.05) when 100 µM gallic acid was included in the α-MEM+ culture medium compared with the control medium. Furthermore, LY294002 inhibited (P < 0.05) follicle survival, development, and meiotic resumption stimulated by 100 µM gallic acid. In conclusion, concentration of 100 µM of gallic acid can be a substitute for transferrin, selenium, and ascorbic acid in the base medium during in vitro culture of sheep secondary follicles, inducing follicle development likely through the PI3K pathway.

Diosmin Mitigates Cyclophosphamide Induced Premature Ovarian Insufficiency in Rat Model.

The current study was designed to investigate the protective role of diosmin against cyclophosphamide-induced premature ovarian insufficiency (POI). Female Swiss albino rats received a single intraperitoneal dose of cyclophosphamide (200 mg/kg) followed by 8 mg/kg/day for the next 15 consecutive days either alone or in combination with oral diosmin at 50 or 100 mg/kg. Histopathological examination of ovarian tissues, hormonal assays for follicle stimulating hormone (FSH), estradiol (E2), and anti-Mullerian hormone (AMH), assessment of the oxidative stress status, as well as measurement of the relative expression of miRNA-145 and its target genes [vascular endothelial growth factor B (VEGF-B) and regulator of cell cycle (RGC32)] were performed. Diosmin treatment ameliorated the levels of E2, AMH, and oxidative stress markers. Additionally, both low and high diosmin doses significantly reduced the histopathological alterations and nearly preserved the normal ovarian reserve. MiRNA-145 expression was upregulated after treatment with diosmin high dose. miRNA-145 target genes were over-expressed after both low and high diosmin administration. Based on our findings, diosmin has a dose-dependent protective effect against cyclophosphamide-induced ovarian toxicity in rats.

Evaluation of expression and serum concentration of anti-Mullerian hormone as a follicle growth marker following consumption of fennel and flaxseed extract in first-generation mice pups.

BACKGROUND: The aim of the present study was to assess the expression and serum level of AMH in first-generation female mice pups following fennel and flaxseed consumption. METHODS: Twenty pregnant NMRI mice were allocated into four groups including control (CTL), fennel (FV), flaxseed (LU) and FV+ LU. Sixty-four female offsprings after lactation period, received the same regimen as their mothers for 56 and 240 days. The ovarian follicles development, serum concentration of AMH, as well as gene and protein expression of AMH were evaluated in the female offsprings at post-natal day 56 (PND56) and 240 (PND240). RESULTS: The number of total growing follicles were raised in the FV group in compression to the all experimental groups. In contrast, LU group showed a marked decrease in their numbers. The highest level of serum AMH was seen in the FV-diet mice, whereas LU negatively affected it. The expression level of AMH also increased in the FV and FV + LU groups, while a reduction was observed in the LU group. As well, IHC data showed that the number of AMH-positive cells in almost ovarian follicles of FV and FV + LU-treated mice was in compared to those of the LU group. CONCLUSIONS: The overall effect of fennel treatment (alone and in combination with flaxseed) on ovary might be maintain primordial follicle storage through increased expression and serum level of AMH.

Effect of Melatonin on Expression of Apoptosis Regulator Proteins Bcl-2 and Bad in Ovarian Follicular Apparatus after High Temperature Exposure.

Expression of proapoptotic Bad and antiapoptotic Bcl-2 proteins in ovarian follicular apparatus of Wistar rats was evaluated on days 3, 7, and 14 after single experimental hyperthermia (EH) followed by therapeutic correction with subcutaneous melatonin (0.1 mg) dissolved in 0.2 ml physiological saline (PS) injected daily for 3 days. Duration of EH was less than 17 min; it was terminated when the rectal temperature attained 43.5°C. In acute posthyperthermia period (on experimental day 3), Bcl-2 and Bad expression area in folliculocytes of the experimental (EH+melatonin) and reference rats simultaneously increased in comparison with the corresponding values in control rats. At this, Bcl-2/Bad ratio of expression areas in experimental and reference rats did not differ from the control level. During the recovery period (on posthyperthermia day 7), Bad protein expression area significantly decreased in experimental rats resulting in elevation of Bcl-2/Bad ratio in comparison with control and reference groups on days 3 and 7. On day 14, Bcl-2 and Bad expression areas and Bcl-2/Bad ratio restored in experimental animals to the corresponding values assessed in control and reference groups. Thus, melatonin produced no effect on Bcl-2/Bad protein expression ratio during acute posthyperthermia period, but reduced the expression area of proapoptotic Bad protein and increased Bcl-2/Bad ratio on posthyperthermia day 7. Thus, melatonin can inhibit apoptosis via mitochondrial pathway in ovarian follicles on posthyperthermia day 7.

Zihuai recipe alleviates cyclophosphamide-induced diminished ovarian reserve via suppressing PI3K/AKT-mediated apoptosis.

ETHNOPHARMACOLOGICAL RELEVANCE: Zihuai recipe (ZHR), a Chinese herbal prescription, is widely used for the clinical treatment of Diminished ovarian reserve (DOR) infertility. However, little is known regarding its underlying mechanisms of DOR treatment. AIM OF THE STUDY: This study aimed to investigate the beneficial effects of ZHR on the treatment of DOR and to reveal the underlying mechanisms. MATERIALS AND METHODS: Sixty female 8-week-old Sprague-Dawley rats were randomly divided into the following six groups (n=10 per group): control, DOR, low-dose(2.7 g/kg/day) ZHR (L-ZHR), medium-dose(5.4 g/kg/day), ZHR (M-ZHR), high-dose(10.8 g/kg/day) ZHR (H-ZHR), and hormone replacement therapy (HRT) treatment groups. The DOR model was established in all the groups, except the control group, by a single intraperitoneal injection of 90 mg/kg cyclophosphamide. After the induction of the DOR model, rats were weighed and administered either the relevant dose of ZHR or an equal volume of saline solution (in the control and DOR groups). Rats in the HRT group received estradiol valerate tablets (0.16 mg/kg/day), and with medroxyprogesterone acetate tablets (0.86 mg/kg/day) added on day 4. After 32 days of treatment, the rats were euthanized and the ovaries were collected for sampling. Ovarian morphology was observed by hematoxylin and eosin staining and the number of follicles was counted under a microscope. The serum levels of anti-Müllerian hormone (AMH), gonadotropin-releasing hormone (GnRH), follicle-stimulating hormone (FSH), luteinizing hormone (LH), and estradiol (E2) were quantified by ELISA. A TUNEL assay was used to analyze the level of apoptosis of the ovarian cells. The protein expressions of p-PI3K, p-AKT, PI3K, AKT, cleaved caspase-3, BAX, and Bcl-2 were measured by western blotting and immunohistochemistry. Data analysis was performed with SPSS 20.0 software. RESULTS: ZHR administration increased the ovarian index and the serum levels of AMH, GnRH, and E2, while lowering those of FSH and LH. ZHR treatment also increased the number of primordial, primary, secondary, and antral follicles, as well as the number of corpora lutea, but decreased the number of atretic follicles. Furthermore, ZHR administration decreased the percentage of TUNEL-positive ovarian cells. After treatment with ZHR, the protein expression levels of p-PI3K/PI3K, p-AKT/AKT, cleaved caspase-3 and BAX were decreased, whereas the level of Bcl-2 was increased. CONCLUSIONS: ZHR improved the ovarian reserve in CTX-induced DOR rats. The mechanisms of ZHR on DOR may be mediated through the regulation of gonadal hormones of the hypothalamic-pituitary-ovarian axis (HPOA), and the inhibition of PI3K/AKT-mediated apoptosis in granulosa cells.

Guizhi Fuling Wan reduces autophagy of granulosa cell in rats with polycystic ovary syndrome via restoring the PI3K/AKT/mTOR signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Guizhi Fuling Wan (GFW) is a traditional Chinese medicine used to remove blood stasis and dissipate phlegm for treating gynecological diseases that was invented by Zhang Zhongjing in the Eastern Han dynasty. In recent years, GFW has been widely used to treat patients with polycystic ovary syndrome (PCOS). Clinical and animal studies have shown that it is effective in the treatment of PCOS, but its mechanism is unknown. Generally, it works by regulating autophagy via the PI3K/AKT/mTOR signaling pathway. AIM OF THE STUDY: This study investigated the effects and mechanism of GFW in PCOS rats with insulin resistance (IR) in order to provide better understanding of its observed clinical effects and a theoretical basis for the study of traditional Chinese medicine. MATERIALS AND METHODS: Eighty-four female Sprague-Dawley rats were randomly divided into seven groups (n = 12 per group): 1) control, 2) PCOS model, 3) low-dose GFW, 4) medium-dose GFW, 5) high-dose GFW, 6) metformin, and 7) medium-dose GFW plus LY294002. In all non-control groups, we induced PCOS through daily letrozole combined with intragastric high-fat emulsion for 21 days. After treatment, rats were sacrificed and serum follicle-stimulating hormone (FSH), testosterone (T), progesterone, luteinizing hormone (LH), 17ß-estradiol, fasting insulin (FINS), and fasting plasma glucose levels were measured by enzyme-linked immunosorbent assay (ELISA). The LH/FSH ratios and HOMA-IR values were calculated. Ovarian morphology was observed by hematoxylin and eosin staining, and all follicles were counted under a microscope. MDC-positive vesicles were used as markers to detect autophagy, and the expression levels of p62, Beclin1, and LC3-II were examined by immunostaining. Western blotting was used to measure PI3K/AKT/mTOR pathway activation, granulosa cell apoptosis, and autophagy. RESULTS: Compared with the PCOS model group, GFW-treated rats had less atretic and cystic follicles, and more mature follicles and corpus lutea. The GFW-treated rats had lower serum T, LH, and FINS levels than the PCOS model group, as well as lower LH/FSH ratios and HOMA-IR values. GFW treatment resulted in significantly reduced levels of cleaved-Caspase-3, cleaved-Caspase-9, BAX, Beclin1, Atg5, and LC3-II. Phosphorylation of PI3K, AKT, and mTOR was significantly higher in GFW-treated rats compared with the PCOS model group. The phosphorylation of PI3K, AKT, and mTOR was decreased with the use of a PI3K antagonist. CONCLUSIONS: Our results indicate that GFW inhibited granulosa cell autophagy and promoted follicular development to attenuate ovulation disorder in PCOS-IR rats. This was associated with activation of the PI3K/AKT/mTOR signaling pathway.

Lack of Efect of Short-term Lupin Grain Feeding on Ovulation Rate in Non-prolific Polish Mountain Ewes during the Breeding Season: Ultrasonographic and Endoscopic Assessment of Ovarian Activity.

The specific changes in antral follicle numbers and wave-like development have remained unrevealed in cyclic ewes fed high-protein, high-energy lupin grain for 6 days during the luteal phase of the estrous cycle (i.e., short-term nutritional flushing). This study was mainly conducted to determine ovarian effects of the 6-day lupin grain feeding in non-prolific Polish Mountain ewes, using transrectal ovarian ultrasonography and abdominal videoendoscopy. Estrus and ovulations were synchronized in 24 ewes with progestin-releasing intravaginal sponges for 12 days during the middle portion of the breeding season (September-October; 50.0458&deg;N, 19.8406&deg;E). Twenty-four ewes were assigned to three equal groups (n=8 each), including the Control group being fed the maintenance diet (i.e., hay-only), Treatment 1 receiving 500 g of lupin grain once a day, and Treatment 2 receiving 250 g of lupin grain twice a day, from days 9-14 of the synchronized estrous cycle (day 0=first ovulation of the interovulatory period studied). No differences were observed in the mean ovulation rate among the three groups of Polish Mountain ewes (P&gt;0.05). Ovarian antral follicles emerging in the penultimate wave of the estrous cycle in Treatment 2 ewes had a longer growth phase (p &lt;0.05) and attained a greater diameter (p &lt;0.05) before ovulation, in comparison to those in the other two groups. A final wave of the interovulatory interval emerged ~1 day earlier in Treatment 2 than in Treatment 1 ewes (p &lt;0.05). Nutritional supplementation with lupin grain increased the number of 3-mm follicles in Treatment 2 ewes (p &lt;0.05). The results of this study indicated that short-term nutritional flushing with lupin grain from mid- to late luteal phase did not consistently enhance ovulatory responses in non-prolific genotypes of ewes. Although the administration of lupins altered the timing of wave emergence, ovulatory follicle diameter, or duration of different stages of the follicular lifespan, it failed to increase the number of ovulatory follicles emerging in the penultimate and final waves of the estrous cycle in non-prolific Polish Mountain sheep.

Ovulation induction with high progesterone levels may be more suitable for elderly patients with low ovarian response.

BACKGROUND: The objective of this study was to explore the outcomes of using the progestin-primed ovarian stimulation (PPOS) protocol in aged infertile women. The patients recruited in the study had displayed a poor ovarian response (POR) in the first IVF/ICSI-ET cycles with the ultra-short gonadotropin-releasing hormone agonist (GnRH-a) protocols. MATERIALS AND METHODS: A self-controlled retrospective study was conducted to investigate the clinical outcomes of 117 aged infertile women who met the inclusion criteria. The patients were grouped into two; group B included patients who had displayed a poor ovarian response (POR) in the first IVF/ICSI-ET cycle with the ultra-short GnRH-a protocol. Group A was made up of patients who underwent the PPOS protocol in the second cycle. The study was done between January 2015 to May 2018 in the reproductive and genetic centre of integrated traditional and western medicine, Affiliated hospital of Shandong University of traditional Chinese medicine. Reproduction-related clinical outcomes in the two groups were compared. RESULTS: There were no statistically significant differences in the serum levels of LH, E2, and P on the trigger day between group A and group B (P＞0.05). The number of follicles with a diameter > 14 mm was significantly higher in the PPOS protocol patients than in the ultra-short GnRH-a protocol group (4.83 ± 2.82 vs. 3.25 ± 2.53, P < 0.01). The duration and total dosage of gonadotropin of the PPOS protocol group were less than in the previous ultra-short GnRH-a protocol, although the statistical differences were not significant (P > 0.05). The number of eggs obtained in the PPOS group was significantly higher than that of the previous one (4.29 ± 3.11 vs. 2.76 ± 2.33, P < 0.05). The numbers of MII eggs, cleavage, 2 P N, transplantable embryos, and high quality embryos were higher in the PPOS protocol group than that in the ultra-short protocol group. However, the differences between the two groups in all the above parameters were not statistically significant (P > 0.05). The rate of high-quality embryos was significantly higher in the PPOS protocol group than in the ultra-short protocol group (38.61(100/259) vs. 32.02(65/203), P < 0.05). Although not statistically significant (P > 0.05), the abortion rate of the PPOS protocol group was higher than that of the ultra-short protocol group. The clinical pregnancy and live birth rates were significantly higher in the PPOS protocol group than in the ultra-short protocol group (p < 0.05). The clinical pregnancy rates in the PPOS protocol group and the ultra-short protocol group were 32.35 % and 25.53 % respectively while the live birth rates were 27.45 % and 21.28 % respectively. CONCLUSION: Compared with the ultra-short protocol, the PPOS protocol improves the number of follicles, the number of eggs, clinical pregnancy, and live birth rates in POR patients. The PPOS protocol could, therefore, provide a novel treatment strategy for inducing ovulation in POR patients.

Effects of Prepubertal Exposure to Aroclor-1221 on Reproductive Development and Transcriptional Gene Expression in Female Rats.

Polychlorinated biphenyls (PCBs), as persistent organic pollutants, are environmental endocrine-disrupting chemicals (EDCs). We aim to investigate the effects of prepubertal exposure to PCBs on the reproductive development and expression and regulation of related genes in rats. Female rats were treated with Aroclor-1221 (A-1221) (4 mg/kg/day, 0.4 mg/kg/day) or castor oil daily from postnatal day (PND) 28 for 2 weeks by gavage. Morphological, histological, hormonal, and biochemical parameters were studied. Lower weight and relative weight of hypothalamus, earlier puberty onset, a longer length of the estrous cycle, lower serum estradiol and progesterone levels, accelerated ovarian folliculogenesis, and higher apoptotic index in the ovary were found. The in vitro fertilization study showed a lower fertilization rate and cleavage rate. The genetic study revealed higher expression of Kiss-1 mRNA and lower expression of GnRH mRNA in the hypothalamus and higher expression of AMH mRNA and lower expression of C-myc mRNA in the ovary. These confirmed the reproductive damage of A-1221 in rats.