GnRH-a-Induced Perimenopausal Rat Modeling and Black Cohosh Preparations’ Effect on Rat’s Reproductive Endocrine

BackgroundEndometriosis (EMS) is an estrogen-dependent disease, which easily recurs after operation. Gonadotropin-releasing hormone agonist (GnRH-a), an estrogen-inhibiting drug, can effectively inhibit the secretion of gonadotropin by pituitary gland, so as to significantly decrease the ovarian hormone level and facilitate the atrophy of ectopic endometrium, playing a positive role in preventing postoperative recurrence. The application of GnRH-a can lead to the secondary low estrogen symptoms, namely the perimenopausal symptoms, and is a main reason for patients to give up further treatment. The add-back therapy based on sex hormones can well address the perimenopausal symptoms, but long-term use of hormones may cause the recurrence of EMS, as well as liver function damage, venous embolism, breast cancer and other risks, which has long been a heated topic in the industry. Therefore, it is necessary to find effective and safe anti-additive drugs soon. Studies at home and abroad show that, as a plant extract, isopropanolic extract of cimicifuga racemosa (ICR) can well relieve the perimenopausal symptoms caused by natural menopause. Some studies have preliminarily confirmed that black cohosh preparations can antagonize perimenopausal symptoms of EMS patients treated with GnRH-a after operation.ObjectiveTo establish a rat model of perimenopausal symptoms induced by GnRH-a injection, for the purposes of laying a foundation for further research and preliminarily exploring the effect of black cohosh preparations on reproductive endocrine of the rat model.MethodThe rat model of perimenopausal symptoms was established by GnRH-a injection, and normal saline (NS injection) was used as the control. The rats were randomly divided into four groups according to different modeling methods and drug intervention schemes. GnRH-a injection + normal saline intervention group (GnRH-a + NS), normal saline injection control + normal saline intervention group (NS + NS), GnRH-a injection + estradiol intervention group (GnRH-a + E2), and GnRH-a injection + black cohosh preparations intervention group (GnRH-a + ICR). After modelling was assessed to be successful with the vaginal smear method, the corresponding drugs were given for intervention for 28d. In the process of rat modeling and drug intervention, the skin temperature and anus temperature of the rat tails were measured every other day, the body weights of the rats were measured every other day, and the dosage was adjusted according to the body weight. After the intervention was over, the serum sex hormone level, the uterine weight, the uterine index, and the endometrial histomorphology changes, as well as the ovarian weight, the ovarian index, and the morphological changes of ovarian tissues of each group were measured.Results(1) The vaginal cell smears of the control group (NS + NS) showed estrous cycle changes, while other model rats had no estrous cycle of vaginal cells. (2) The body weight gains of the GnRH-a + NS, GnRH-a + E2 and GnRH-a + ICR groups were significantly higher than that of the NS + NS control group. The intervention with E2 and ICR could delay the weight gain trend of rats induced by GnRH-A. (3) After GnRH-a injection, the temperature of the tail and anus of rats showed an overall upward trend, and the intervention with E2 and ICR could effectively improve such temperature change. (4) The E2, FSH, and LH levels in the GnRH-a + NS, GnRH-a + E2, and GnRH-a + ICR groups were significantly lower than those in the NS + NS group (P < 0.01). The E2 level was significantly higher and the LH level was significantly lower in the GnRH-a + E2 group than those in the GnRH-a + NS and GnRH-a + ICR groups (P < 0.05). Compared with those of the GnRH-a + NS and GnRH-a + ICR groups, the FSH level of the GnRH-a + E2 group showed a slight downward trend, but the difference was not statistically significant (P > 0.05). There was no significant difference in the levels of sex hormones between the GnRH-a + NS group and GnRH-a + ICR group (P > 0.05). (5) Compared with those of the NS + NS group, the uterine weight and uterine index of the GnRH-a + NS, GnRH-a + E2 and GnRH-a + ICR groups significantly decreased (P < 0.01). In a comparison between the groups, the uterine weight and uterine index in the GnRH-a + NS and GnRH-a + ICR groups were significantly lower than those in the GnRH-a + E2 group (P < 0.01). There was a statistical difference in the uterine weight and uterine index between the GnRH-a + NS group and GnRH-a + ICR group (P > 0.05). (6) Compared with those of the NS + NS group, the ovarian weight and ovarian index of the GnRH-a + NS, GnRH-a + E2 and GnRH-a + ICR groups significantly decreased (P < 0.01). There was no statistical difference in the ovarian weight and ovarian index among the GnRH-a + E2, GnRH-a + NS and GnRH-a + ICR groups (P > 0.05). (7) Compared with those in the NS + NS group, the number of primordial follicles increased significantly, while the number of growing follicles and mature follicles decreased significantly in the GnRH-a + NS, GnRH-a + E2, and GnRH-a + ICR groups (P < 0.01), but there was a statistical difference in the total number of follicles among the four groups (P > 0.05).ConclusionsThe GnRH-a injection could achieve the desired effect. The animal model successfully achieved a significant decrease in the E2, FSH, and LH levels in rats, and could cause the rats to have rising body surface temperature similar to hot flashes in the perimenopausal period. The intervention with E2 and ICR could effectively relieve such “perimenopausal symptoms”, and ICR had no obvious effect on the serum sex hormone level in rats.

Ginsenoside Rg1 attenuates premature ovarian failure of D-gal induced POF mice through downregulating p16INK4a and upregulating SIRT1 expression

Background: Premature ovarian failure (POF) refers to pathological amenorrhea before 40 years. Aim/Objective: To explore regulatory effect of Rg1 on POF and clarify associated mechanisms. Materials and methods: POF mice were induced by injecting with D-galactose (D-gal, 200 mg/kg/day). Mice were divided into phosphate buffered saline (PBS), D-gal (POF mice), D-gal/Rg1 group (POF mice administrating D-gal/Rg1). Weight growth rate and ovarian weight coefficient were measured. Serum estradiol (E2), follicle stimulating hormone (FSH), luteinizing hormone (LH), superoxide dismutase (SOD), catalase (CAT) levels were examined using ELISA. Status of follicle and corpus luteum was determined using hematoxylin-eosin (HE) staining. P16INK4a and silent-mating type information regulation-2 homolog-1 (SIRT1) was determined using western blotting and RT-PCR. Findings: Weight growth rate and ovarian weight coefficient of mice in D-gal group were significantly decreased than PBS group (p<0.05). Serum E2, LH, SOD, CAT levels were significantly decreased, FSH levels were remarkably increased in D-gal group than PBS group (p<0.05). Rg1 (D-gal/Rg1 group) significantly increased weight growth rate and ovarian weight coefficient, enhanced E2, LH, SOD, CAT levels and decreased FSH levels than D-gal group (p<0.05). HE staining demonstrated normal follicle morphology/structure of mice in PBS group and decreased number of follicles, obvious vacuolation of corpus luteum and increased atretic follicles of mice in D-gal group. Compared with D-gal group, number of follicles was increased, luteal follicles was decreased of mice in D-gal/Rg1 group (p<0.05). Rg1 significantly (D-gal/Rg1) downregulated p16INK4a and upregulated SIRT1 expression in ovarian tissues of mice compared to D-gal group (p<0.05). Conclusions: Rg1 could delay premature ovarian failure in D-gal induced POF mouse model through downregulating p16INK4a and upregulating SIRT1 expression.

A novel long-acting, follicle stimulating hormone-Fc fusion protein displays Gonal-f-like function in vitro and in vivo

Purpose To explore whether X002 can enhance bioactivity and has a long half-time in vitro and in vivo. Methods For the in vitro study, GVBD rate and COC expansion were applied. In the GVBD test, 21–24-day-old female KM mice were stimulated with PMSG for 46 h, and the naked oocytes of ovaries were then collected. Four hours after X002 treatment at 37℃, the GVBD rates of naked oocytes were counted. In COC expansion, COCs were collected from mice stimulated with PMSG. After coculture of COCs and X002 for 14 h, COC diameter was measured, and the expression of genes involved in COC expansion was also determined by RT-qPCR. For the in vivo study, 6–8-week-old female SD rats were administered a subcutaneous injection of X002 for pharmacokinetics. Serum was assayed by ELISA at different time points. For pharmacodynamics, 26-day-old female SD rats were used in ovarian weight and superovulation assay. To assay ovarian weight, rats were stimulated with hCG 84 h after X002 treatment. At 12 h after hCG injection, ovaries were weighted and E2 or P4 in serum was quantified. To assay superovulation, rats were treated with the above methods. At 108 h after X002 treatment, oocytes were counted from fallopian tubes. Results X002 promoted GVBD and COC expansion in vitro and effectively promoted significant ovarian weight gain and superovulation, similar to Gonal-f; Meanwhile, it had a longer T1/2. Conclusions X002 is a long-acting FSH-liked agent which has good bioactivity, similar to Gonal-f.

Ameliorative effects of Withania coagulans and Metformin on Ovarian morphology in Polycystic ovarian disease induced rats

Background: Various medicinal herb plants are being used in place of metformin for treatment of polycystic ovarian disease for their less harmful effects. Withania coagulans (WC) is a herb known for its insulin sensitizing and weight reducing properties. The present study was done to determine the influence of aqueous extract of Withania coagulans (aqWC) and metformin on ovarian weight and ovarian folliculogenesis in polycystic ovarian disease-induced rats. Material and Methods: An experimental animal study was carried out at the Physiology Department of Islamic International Medical College, Rawalpindi from April 2016 to March 2017. Forty female Sprague Dawley rats were divided initially into two groups. Group A (Normal Control) and Disease induced group. Standardized laboratory diet was fed to Group A while the disease induced group was given standardized laboratory diet and letrozole solution orally (1mg/kg) for 21 days to induce Polycystic ovary syndrome, which was established by observing estrous cycle of rats. Disease induced group was then split into group B (PCOS control), C (Aqueous Withania coagulans treated) and D (Metformin treated) and observed after 14 days. Groups A and B underwent ovarian dissection after 21 days and groups C and D underwent dissection at the end of experiment (after 35 days). Independent sample t-test was used for the comparison between the control and disease induced group. Results: Group B showed a significant increase in ovarian weight in comparison to group A rats (P < .05). Treatment with Withania coagulans and metformin significantly decreased ovarian weight (P < .05) and increased primary, Graafian, antral follicular count and corpus luteum along with reduction in cystic follicular count in letrozole-induced polycystic ovarian disease rats. Improved folliculogenesis was also observed in the same groups (groups C & D). Conclusions: Withania coagulans can be a substitute for improvement of ovarian follicular development in polycystic ovarian disease.

Resveratrol (3,4ʹ,5-trihydroxy-trans-stilbene) or caffeic acid phenethyl ester supplementation prevents premature ovarian insufficiency due to chemotherapy

IntroductionThis study was planned to investigate the protective effect of resveratrol (RSV), caffeic acid phenethyl ester (CAPE), and coenzyme Q10 (CoQ10) on chemotherapy-induced premature ovarian insufficiency (POI).Material and methodsTwenty-eight female rats were divided into four groups with 7 rats in each group. The groups consisted of a vehicle-treated control group (group 1), rats treated with chemotherapy followed by intraperitoneal RSV injection (group 2) or rats treated with chemotherapy followed by CoQ10 (group 3), or rats treated with chemotherapy followed by intraperitoneal CAPE injection (group 4). Cisplatin was administered intraperitoneally once daily at doses of 2.0 mg/kg for 10 days. Rats in groups 2, 3, and 4 were given RSV (100 mg/kg, i.p.), CoQ10 (20 mg/kg, oral), and CAPE (10 mg/kg, i.p.) respectively. Five animals in the sham group underwent laparotomy for ovarian weight measurements on the first day of cisplatin injection. Animals in the treatment and control groups were sacrificed two weeks later, and their ovaries were excised for histopathological analysis. Serum levels of anti-Mullerian hormone (AMH) were also analyzed.ResultsRats in groups 2 and 4 showed vaginal smears under estrogenic effect. At the end of the second week, the total body weight and ovarian weight of the animals in group 1 increased. Significantly higher mean follicle count was detected in groups 2 and group 4 compared to group 3. The ovaries of the rats in the control group showed follicles in all stages of development. While the ovaries of the rats in the RSV or CAPE showed significantly decreased numbers of primordial follicles they showed increased numbers of early growing follicles.ConclusionsBoth RSV and CAPE administration might improve follicle survival and AMH production in rats with chemotherapy-induced POI but further research is necessary.

PSVII-20 Heat stress alters reproductive phenotypic impacts of Zearalenone in pre-pubertal gilts

Zearalenone (ZEA) is an estrogenic mycotoxin produced by strains of Fusarium and is often inadvertently consumed via feed contamination. Heat stress (HS) occurs when heat accumulation exceeds heat dissipation resulting in increased body temperature. Independently, HS and ZEA cause swine reproductive dysfunction such as delayed puberty onset, altered circulating steroid hormones and irregular estrous cycles. During HS, gilts become hyperinsulinemic and insulin regulates hepatic and ovarian chemical metabolism. We hypothesized that during HS, ZEA-induced alterations to reproductive parameters are heightened such that HS is additive to ZEA-induced reproductive toxicity. Prepubertal crossbred gilts (n = 38) were randomly assigned to six treatment groups: thermal neutral (TN) ad libitum fed controls (TNCT; n = 6); TN + ZEA (2 ppm; TNZ; n = 6); pair-fed (PF) control (PFCT; n = 6); PF + ZEA (2 ppm; PFZ; n = 6); cyclical HS control (HSCT; n = 7); and HS + ZEA (2 ppm; HSZ; n = 7) for 7 d. Ovarian and uterine weights (g) were measured, and nipple and vulva diameters (l × w; mm2) were assessed by digital calipers. Ovarian weight was decreased (P = 0.04) in the PFZ relative to PFCT, but ZEA did not affect ovarian weight in the other groups (P &gt; 0.73). There was no impact of ZEA exposure on uterine weight (P &gt; 0.22) or nipple diameter (P &gt; 0.51) in any treatment groups, respectively. There was no effect of ZEA on vulva size in either of the TN groups; however, vulva diameter increased (P = 0.04) in the HSZ relative to HSCT. These data suggest that HS exaggerates some ZEA-induced phenotypic effects in prepubertal gilts. This project was supported by the Iowa Pork Producers Association.

Inimical Effects of Sodium Fluoride on Ovarian Weight and Relative Tissue Weight Index of Adult Albino Rats

Objective: To determine the impacts of sodium fluoride on ovarian weight and relative tissue weight index (RTWI) of adult Wistar albino rats. Study Design: Comparative study. Place and Duration of Study: This experimental study was performed at the Department of Anatomy, at Shaikh Zayed Postgraduate Medical Institute, Lahore from 25th November to 24th December 2016. Materials and Methods: Forty-four adult female albino Wistar rats were selected randomly for this study. They were segregated into 4 groups, each comprised of eleven rats. Group A was control, group B was low dose experimental, group C was medium dose experimental and group D was high dose experimental. The control group received distilled water whereas low, medium and high dose experimental groups received 100mg/L, 200mg/L and 300mg/L sodium fluoride solution respectively. The animals were weighed before and after experiment. At 31st and 32nd day, dissection was done, ovaries were removed and evaluated for ovarian tissue weight and RTWI. Results: The mean weight and RTWI of paired ovaries among the experimental groups were decreased. The differences among groups were statistically significant. Conclusion: The present study confirms that sodium fluoride has detrimental dose dependent effects on ovarian weight and RTWI of adult albino rats.

Mangiferin ameliorates insulin resistance in a rat model of polycystic ovary syndrome via inhibition of inflammation

Purpose: To examine the effect of mangiferin on insulin resistance (IR) in a rat polycystic ovary syndrome (PCOS) model.Methods: The rat PCOS model was established via subcutaneous injection of 6 mg/kg of dehydroepiandrosterone (DHEA), and mangiferin was orally administered. Body and ovarian weights were recorded. Serum levels of glucose, insulin, and related inflammatory cytokines were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay, while the expression levels of key proteins were analyzed by western blotting.Results: DHEA significantly increased ovarian weight and the ratio of ovarian weight/body weight (p <0.001), while mangiferin treatment decreased them (p < 0.001). Mangiferin also lowered DHEA-induced enhancements in serum glucose and insulin levels (p < 0.001). The mRNA and, expression and concentrations of inflammatory cytokines (interleukin-6(IL-6), interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α)) were also significantly reduced by mangiferin treatment (p < 0.001). Furthermore, mangiferin suppressed phosphorylation of nuclear factor-kappa B (NF-κB) but increased the phosphorylation of protein kinase B (AKT, p < 0.001).Conclusion: These results reveal that mangiferin not only decreases inflammatory cytokine levels by regulating NF-κB signaling pathway but also ameliorates IR in a rat PCOS model via regulating AKT signaling pathway. Thus, mangiferin is a potential therapeutic strategy for the management of PCOS. Keywords: Polycystic ovary syndrome, Mangiferin, Inflammation, Insulin resistance, NF-κB, AKT

Factors Affecting Ovarian Activity in Donkeys: The Effect of Season, Age and Body Condition Score (BCS)

During the period of year (representing spring, summer, autumn, and winter) Jennies at Giza Zoo abattoir were checked for body condition score (from 1 to 5), age (1 to 7 years; 8 to 14 years; and >15 years old). After culling, ovaries of 377 Jennies were collected, two ovaries were weighed, and the number and size of ovarian follicles were recorded. Cumulus-Oocyte Complexes (COCs) were collected after the ovarian follicles have been sliced and scraped, and COCs number and quality were determined. Data indicated that right and left ovaries and overall ovarian weight were significantly increased (P<0.05) in summer than the other seasons. The number of small size ovarian follicles was decreased (P<0.05) in spring than other seasons, while oocytes number and quality were not significantly affected by season. Jennies of 1 to 7 years old showed a significant (P<0.05) smaller left ovary and total ovarian weight than the two other groups. Jennies of 8-14 years old) have a significant high (P<0.05) small size and a whole number of ovarian follicles than the other groups. Jennies with BCS-3 possess a significantly (P<0.05) higher total ovarian weight, total number of ovarian follicles and the total number of COCs recovered than BCS-1 or BCS-4. While the BCS-4 group showed the lowest (P<0.05) number of ovarian follicles and produced less (P<0.05) number of COCs than the other groups. Conclusion: In Egyptian Jennies, ovarian activity was affected by season, BCS and age of the animal, while, COCs yield and quality were more affected by BCS.

127 Chemokine receptor 2 (CCR2) is expressed in growing oocytes, and its deficiency affects follicular activation and long-term female fertility in mice

Chemokine receptor 2 (CCR2) has important functions in several biological processes, including activation of PI3K/Akt/mTOR signalling, a key pathway in follicular activation. However, there is no report about the role of CCR2 in ovarian follicular physiology. The objectives of this study were (1) to immunolocalize CCR2 in mice ovaries and (2) to evaluate the effects of CCR2 deficiency on follicular growth during adult life and aging. A total of 74 C57Bl/6 (wild type, WT) and 68 B6.129S4-Ccr2tm1Ifc/J (CCR2−/− (knockout)) female mice were used. For objective 1, ovaries were collected from WT mice at 1.5 months old (m.o.), fixed in 4% PFA, embedded in paraffin, and used for immunoperoxidase staining with an anti-CCR2 antibody [EPR19698] (ab222496; 1:200) and using anti-rabbit IgG (Ab6721, 1:100) as a secondary antibody. Also, MII oocytes from oviducts of superovulated WT mice were processed with the same primary antibody for immunofluorescence. For objective 2, body and ovarian weight were evaluated. Follicle populations were assessed in WT and CCR2−/− mice at 1.5, 2.5, 6, 10, and 12m.o., by serial sectioning; the total follicle population was counted in every third section in the whole ovary. Additionally, ovarian total RNA isolation was performed from WT and CCR2−/−. Real-time PCR was used to evaluate differential gene expression according to standard protocols, using primers for Bax, Casp3, Bcl2, Fshr, and B-actin (endogenous control). The data were analysed using the GraphPad Prism Software, using t-test, and a P-value of 0.05 was considered as significant. Localization of CCR2 was observed exclusively on the membrane and cytoplasm of growing oocytes in primary, secondary, antral, and atretic follicles, as well as on ovulated MII oocytes membrane and cytoplasm. Body and ovarian weight were similar between WT and CCR2−/− mice. At 1.5m.o., CCR2−/− mice had more primordial follicles and fewer primary and secondary follicles compared with WT (P&lt;0.05), whereas there was no difference in the antral follicle populations. Follicular activation (primordial to primary transition) and atresia rates were decreased in CCR2−/− (P&lt;0.05) at 1.5m.o. A downregulation of pro-apoptotic genes (Bax and Casp3) was observed on CCR2−/− (P&lt;0.05), while anti-apoptotic Bcl2 was upregulated (P&lt;0.05) compared with WT in 1.5-m.o. animals. A larger ovarian follicular reserve at 1.5, 2.5, and 6m.o., but not at 10 or 12m.o., was observed in CCR2−/− mice. At 6 to 12m.o., CCR2−/− ovulated more (P&lt;0.05) MII oocytes than WT. Altogether, these data may suggest that CCR2 plays an important role in the regulation of ovarian follicular reserve mobilization, potentially affecting reproductive lifespan. Financial support was provided by Fapemig and CNPq.