Rat Ovarian Tissue Research Articles

Effect of berberine combined with metformin on autophagy in polycystic ovary syndrome by regulating AMPK/AKT/mTOR pathway.

The pathologic mechanism of polycystic ovary syndrome (PCOS) is related to increased autophagy of granulosa cells. Both berberine and metformin have been shown to improve PCOS, but whether the combination of berberine and metformin can better improve PCOS by inhibiting autophagy remains unclear. PCOS models were constructed by injecting dehydroepiandrosterone into rats, and berberine, metformin or berberine combined with metformin was administered to rats after modeling. Rats' body weight and ovarian weight were measured before and after modeling. Histopathological examination of ovarian tissue and estrous cycle analysis of rats were performed. Insulin resistance, hormone levels, oxidative stress, and lipid metabolism in PCOS rats were assessed. Expression of the AMPK/AKT/mTOR pathway and autophagy-related proteins was analyzed by Western blot assays. Granulosa cells were isolated from rat ovarian tissue and identified by immunofluorescence staining followed by transmission electron microscopy analysis. Berberine combined with metformin reduced the body weight and ovarian weight of PCOS rats, increased the number of primordial and primary follicles, decreased the number of secondary and atretic follicles, normalized the estrous cycle, and improved insulin resistance, androgen biosynthesis, oxidative stress and lipid metabolism disorders, and increased estrogen production. In addition, berberine combined with metformin reduced the number of autophagosomes in granulosa cells, which may be related to AMPK/AKT/mTOR pathway activation, decreased Beclin1 and LC3II/LC3I levels, and increased p62 expression. Berberine combined with metformin could inhibit autophagy by activating the AMPK/AKT/mTOR pathway in PCOS, indicating that berberine combined with metformin is a potential treatment strategy for PCOS.

Gentisic acid attenuates 5-fluorouracil-induced ovotoxicity in rats via modulating Nrf2 signalling: An experimental approach

5-Fluorouracil (5-FU) is the third most used chemotherapeutic in the world with its anticancer effect resulting from its potential to block DNA replication. Like other cytotoxic agents, 5-FU has side effects on healthy tissues, and the reproductive system is among the tissues most affected by these undesirable effects. Gentisic acid (GEA) is a secondary metabolite that is abundant in fruits, vegetables and spices and has antioxidant activity. This study was conducted to investigate the toxicity of 5-FU in rat ovarian tissue and to determine the therapeutic activity of GEA on ovotoxicity caused by 5-FU. The results showed that 5-FU caused histopathological findings by suppressing Nrf2 pathway and accordingly increasing oxidative stress, inflammation, endoplasmic reticulum stress and apoptosis. However, GEA treatments after 5-FU application ameliorated 5-FU-induced ovotoxicity dose-dependently through activation of Nrf2 pathway. All these findings provided strong evidence supporting the hypothesis that GEA treatment may have therapeutic effects against 5-FU-induced ovarian damage. However, the beneficial effect of GEA use in eliminating ovarian damage in women after 5-FU chemotherapy should continue to be investigated with more detailed molecular studies.

Omaveloxolone Prevents Polystyrene Microplastic-Induced Ovarian Granulosa Cell Apoptosis via the Keap1/Nrf2/HO-1 Pathway in Rats.

Microplastics (MPs) are persistent environmental pollutants that enter the circulatory system and subsequently reduce sperm quantity and quality. However, the influence of polystyrene MPs (PS-MPs) on the ovary and relevant mechanisms remain elusive. Herein, we aimed to examine the impact of PS-MPs on oxidative disorders in ovarian tissues and elucidate the underlying mechanisms. Healthy female rats were treated with different concentrations of 0.5µm PS-MPs (diluted in deionized H2O) for 90 days. Upon examination of hematoxylin-eosin-stained ovarian tissue sections, the number of growing follicles was reduced in PS-MP-treated rats when compared with that in control rats. Enzyme-linked immunosorbent assays revealed that PS-MP exposure markedly reduced anti-Müllerian hormone (AMH) levels. Treatment with PS-MPs downregulated superoxide dismutase, glutathione, and catalase activities in ovarian tissues while upregulating malondialdehyde levels. Furthermore, exposure to PS-MP blocked the Keap1/Nrf2/HO-1 signal transduction pathway. PS-MPs also triggered apoptosis in the ovarian tissue, as evidenced by increased TUNEL staining and expression levels of cleaved caspase-9, Bax, and Bcl-2. To reactivate the Keap1/Nrf2/HO-1 pathway, rats were co-administered PS-MPs and omaveloxolone (Oma), an Nrf2 activator, for 1week. We found that Oma could counteract the PS-MP-mediated effects on oxidative disorder, apoptosis, AMH production, and follicle number in rat ovarian tissues. To develop an in vitro model, granulosa cells (GCs) were treated with 10μM H2O2 for 12h to induce oxidative stress. H2O2-stimulated GCs exhibited attenuated cell growth and upregulated apoptosis and oxidative stress. Oma administration could ameliorate the H2O2-induced effects in terms of regulating cell viability, apoptosis, and oxidative stress in GCs. In summary, PS-MPs could induce apoptosis and oxidative stress via the Keap1/Nrf2/HO-1 signaling pathway in both rats and GCs.

Protective effect of thyroid and restores of ovarian function of Buzhong Yiqi granule on experimental autoimmune thyroiditis in female rats.

To observe the effects of Buzhong Yiqi granule on thyroid function and ovarian function in rats with experimental autoimmune thyroiditis (EAT). EAT model was replicate by using the method of mixing and injecting porcine thyroglobulin with Freund's adjuvant and high iodine. Rats were randomly divided into normal control (NC) group, EAT model (EAT) group, selenium yeast (PC) group, low dose Buzhong Yiqi (BZYQ-L) group, medium dose Buzhong Yiqi (BZYQ-M) group and high dose Buzhong Yiqi (BZYQ-H) group. After two months of drug intervention according to dosage, enzyme-linked immunosorbent assay (ELISA) was used to measure the levels of free triiodothyronine (FT3), free thyroxine (FT4), thyroid-stimulating hormone (TSH), anti-thyroid peroxidase antibody (TPOAb), thyroglobulin antibody (TGAb) in peripheral blood of rats. The pathological changes of rat thyroid tissues were observed under light microscope with HE staining; ELISA was used to determine estradiol (E2), follicle-stimulating hormone (FSH), luteinizing hormone (LH), testosterone (T), anti-müllerian hormone (AMH), and the pathological changes of rat ovarian tissues were observed under light microscope with hematoxylin and eosin staining. Compared with the NC group, BZYQ granule improved the thyroid and ovarian tissue morphology, and the levels of TPOAb, TGAb and TSH in the model group rats significantly increased (P < 0.05), the thyroid tissue was severely destroyed, the levels of E2, FSH, LH, T, AMH significantly increased (P < 0.05), and the ovary exhibited polycystic changes; Compared with the model group, TSH level in the BZYQ-L group rats decreased (P < 0.05), FSH, T, AMH levels decreased (P < 0.05), in the BZYQ-M group TPOAb, TSH levels decreased (P < 0.05), FSH, LH, T, AMH levels significantly decreased (P < 0.05), BZYQ-H group TPOAb, TGAb, TSH levels significantly decreased (P < 0.05), FSH, LH, T, AMH levels significantly decreased (P < 0.05), with the greatest improvement and significantly better than selenium yeast group (P < 0.05). BZYQ granule could regulate the thyroid function of EAT rats, reduce thyroid antibody titers, then act on the ovarian function, regulate hormone disorders, and alleviate the pathological damage of rat's ovarian tissues. The effect of high dose Buzhong Yiqi granule is the best.

Assessment of the Effects of Two Different Doses of Methotrexate in Rat Ovarian Tissue: A Histological Study

Objective: The use of methotrexate (MTH), a drug commonly prescribed to treat rheumatoid arthritis, cancer, and certain autoimmune diseases, has been linked to early ovarian failure and infertility in women when used in varying amounts and durations. In this study, the possible effects of MTH exposure at different doses on rat ovary were investigated histologically and immunohistochemically. Materials and Methods: A total of 21 female rats, Wistar albino, were utilized in the investigation. The control group (n = 7) was not subjected to any treatment. The 10 mg/kg MTH group (n = 7) and the 20 mg/kg MTH group (n = 7) were given intraperitoneal injections of 10 mg/kg and 20 mg/kg methotrexate, respectively. Five days after injection, the ovarian tissues were taken out of the anesthetized rats and subjected to routine histological tissue processing. The histological evaluation was performed with hematoxylin and eosin (H &amp; E) and Masson's trichrome (MT) on paraffin block sections. Additionally, immunohistochemical staining was performed to determine AMH immunoreactivity. Results: In both MTH groups, histopathological changes were observed, particularly in the 20 mg/kg group, and these included the presence of areas of edema as well as dilated or congested blood vessels. Furthermore, the 20 mg/kg MTH group exhibited increased fibrotic tissue in the ovarian medulla. The number of follicles and AMH immunoreactivity were observed to be reduced in the MTH groups, with the reduction reaching a statistically significant level in the 20 mg/kg MTH group ovary. In terms of AMH immunoreactivity, it is demonstrated that there is no statistical significance between the control and 10 mg/kg MTH groups. Conclusion: The dose of MTH should be considered carefully, as increasing the dose may lead to deterioration of ovarian histopathology and a reduction in the reserve of the ovary.

Deoxynivalenol induces ovarian apoptosis in peripubertal rats

Puberty, governed by the endocrine system, marks the onset of reproductive functions in animals and humans through a series of physiological and biological transformations. Although the mycotoxin DON can disrupt hormonal balance and cause reproductive abnormalities, its impact on puberty-associated reproductive changes remains understudied. Considering the increased exposure of children and adolescents to DON, our study aimed to elucidate its influence on follicular integrity and the expression of pro-apoptotic proteins (BAX and Caspase-3) and anti-apoptotic protein (BCL-2) in juvenile rat ovarian tissue. We divided ten 28-day-old prepubertal Wistar rats into two dietary groups for 28 days: a control group with a mycotoxin-free diet and a DON group with a diet containing 10 mg DON/Kg. After the experiment, ovaries and uterus weights were recorded, and the ovaries underwent morphometric and immunohistochemical analysis. DON exposure led to significant reductions in both ovarian and uterine weights. Although DON intake did not change the number of ovarian follicles across developmental stages, we observed an increased expression of BAX and Caspase-3 and a decreased BCL-2 expression in most follicular stages and corpora lutea. In summary, DON exposure during puberty can interfere with apoptotic processes in diverse ovarian cell populations during early adulthood.

Effects of vitrification solution supplemented with platelet-rich plasma in rat ovarian tissue cryopreservation.

The subject of this study was to investigate the utility of platelet-rich plasma (PRP) in the cryopreservation process to reduce cryodamage and increase tissue viability. Twenty-one female Wistar rats were randomly allocated to three groups. In Group 1 (G1), rats were not subjected to vitrification (n = 7). Group 2 (G2) was the vitrification group in which PRP was added to the basic vitrification solution (n = 7). Group 3 (G3) was the vitrification group in which fetal bovine serum was added to the basic vitrification solution (n = 7). Warmed tissues were evaluated with histochemical (HC) and immunohistochemical (IHC) staining, the TUNEL method, immunofluorescence (IF) staining, and biochemical analyses. The percentages of IHC staining, TUNEL method positivity, and IF staining were significantly higher in G2 compared to both G1 and G3 (P < 0.05). G2 ovaries exhibited a significant increase in both malondialdehyde and catalase values in comparison to G1 (P < 0.05). In HC staining, degenerations in primary and secondary follicles and in ovarian tissue were more common in the PRP-supplemented group. The calcium used in PRP activation was suspected to have increased the degeneration and prevented the possible positive effects of PRP. To the best of our knowledge, PRP-supplemented vitrification solution was used for the first time in the literature in this study in whole rat ovarian tissue vitrification. If PRP is to be used as a component in vitrification solution for rat ovarian tissue, the use of lower amounts of calcium or different methods in PRP activation, or the use of nonactivated PRP, should be considered from the beginning.

Mechanisms of DHEA-induced AMH increase in the ovarian tissues of PCOS rat

The present study aimed to build a DHEA-induced polycystic ovary syndrome (PCOS) rat model to evaluate the potential mechanism of DHEA-induced AMH rise in these rat ovarian tissues. A total of 36 female 3-week-old rats were allocated into two groups at random. The control group received merely the same amount of sesame oil for 20 days while the experimental group received 0.2 mL of sesame oil Plus DHEA 6 mg/100 g daily. Both groups' vaginal opening times were noted, and vaginal smears were taken. By using RT-qPCR and Western blot, the mRNA and protein expression of AMH, GATA4, SF1, and SOX9 in the ovarian tissues of the two groups was investigated.The rats in the experimental group appeared to have obvious disorders of the estrus cycle, as evidenced by the ratio of estrus being significantly higher than that in the control group (P < 0.05); HE staining revealed that the ovarian volume, follicular vacuoles, and follicular lumen of the rats in the experimental group increased significantly.The ELISA results revealed that T and AMH in the experimental group were higher than those in the control group at day 15 and 20. AMH、GATA4 and SF1 mRNA and protein expression were higher in the experimental group than in the control group on day 15 and 20 (P < 0.05). On day 20, the experimental group outperformed the control group (P < 0.05). In the DHEA-induced PCOS rat model, androgen may have enhanced AMH expression via increasing the expression of genes associated to the AMH promoter binding site (GATA4, SF1, SOX9).

The efficacy and mechanism of Angelica sinensis (Oliv.) Diels root aqueous extract based on RNA sequencing and 16S rDNA sequencing in alleviating polycystic ovary syndrome

BackgroundPolycystic ovary syndrome (PCOS) leads to persistent anovulation, hyperandrogenism, insulin resistance, and polycystic ovary, and is mainly characterized by menstrual disorders, and reproductive dysfunction. Angelica sinensis (Oliv.) Diels root has been used in many classical formulas of traditional Chinese medicine, and is commonly used to treat various gynecological diseases. PurposeTo investigate the protective effect of water extract of A. sinensis root (WEA) on PCOS rats, and the mechanism by RNA sequencing, and 16S rDNA sequencing. MethodsThe PCOS rat model was established by letrozole combined with high-fat diet (gavage; 2 months), and treated with WEA (gavage; 2 g/kg, 4 g/kg or 8 g/kg; 1 month). To evaluate the therapeutic effect of WEA on PCOS rats, vaginal smear, hematoxylin-eosin staining, and biochemical indicators detection were performed. The rat ovarian tissue was analyzed by RNA sequencing, and the results were verified by qRT-PCR, and Western blot. 16S rDNA sequencing was used to analyze the gut microbiota of rats. ResultsThe results of the vaginal smear, and hematoxylin-eosin staining showed that WEA improved estrous cycle disorder, and ovarian tissue lesions. WEA (4 g/kg or 8 g/kg; 1 months) alleviated hormone disorders, insulin resistance, and dyslipidemia. RNA sequencing showed that WEA intervention significantly changed the expressions of 2756 genes, which were enriched in phosphatidylinositol3-kinase/phosphorylated protein kinase B (PI3K/AKT), peroxisome proliferator-activated receptor (PPAR), mitogen-activated protein kinase (MAPK), AMP-activated protein kinase (AMPK), and insulin signaling pathways. 16S rDNA sequencing found that WEA increased the species diversity of gut microbiota, and regulated the abundance of some microbiota (genus level: Dubosiella, Bifidobacterium, Coriobacteriaceae (UCG-002), and Treponema; species level: Bifidobacterium animalis, Lactobacillus murinus, and Lactobacillus johnsonii). ConclusionWEA regulated hormone, and glycolipid metabolism disorders, thereby relieving the PCOS induced by letrozole combined with high-fat diet. The mechanism was related to the regulation of PI3K/AKT, PPAR, MAPK, AMPK, and insulin signaling pathways in ovarian tissues, and the maintenance of gut microbiota homeostasis. Clarifying the efficacy and mechanism of WEA in alleviating PCOS based on RNA sequencing and 16S rDNA sequencing will guide the more reasonable clinical use of WEA.

Phthalate exposure and risk of ovarian dysfunction in endometriosis: human and animal data.

Objective: We aimed to explore the correlations between and possible mechanisms of common environmental endocrine disruptors, phthalates, and ovarian dysfunction in endometriosis. Methods: Subjects were included in the case group (n = 107) who were diagnosed with endometriosis by postoperative pathology in Fujian Maternal and Child Hospital from February 2018 to February 2021, and the women who were excluded from endometriosis by surgery were as the control group (n = 70). The demographic information of the subjects were evaluated by questionnaire, and the clinical characteristics were evaluated by medical records and 3-year follow-up results. Gas chromatography‒mass spectrometry was used to quantify 10 metabolites of phthalates, including dimethyl ortho-phthalate (DMP), mono-n-methyl phthalate (MMP), dioctyl ortho-phthalate (DEP), mono-ethyl phthalate (MEP), di-n-butyl ortho-phthalate (DBP), mono-butyl phthalate (MBP), benzylbutyl phthalate (BBzP), mono-benzyl; phthalate (MBzP), diethylhexyl phthalate (DEHP) and mono-ethylhexyl phthalate (MEHP), in the urine samples of the subjects. Furthermore, a total of 54 SD rats were exposed to DEHP 0, 5, 50, 100, 250, 500, 1,000, 2000, and 3,000mg/kg/day for 2weeks. The SD rats' body weight, oestrus cycle changes, and serum anti-mullerian hormone (AMH) levels were evaluated. After sacrifice, the mass index of the rat uterus and bilateral ovaries were calculated. Finally, bioinformatics analysis of rat ovarian tissues was performed to explore the possible mechanism. SPSS 24.0 (IBM, United States) was used for data analysis. p-value <0.05 was considered statistically significant. Results: The human urinary levels of DMP (p < 0.001), MMP (p = 0.001), DEP (p = 0.003), MEP (p = 0.002), DBP (p = 0.041), MBP (p < 0.001), BBzP (p = 0.009), DEHP (p < 0.001), and MEHP (p < 0.001) were significantly higher in women with endometriosis than in controls. Notably, DEHP was a significant risk factor for endometriosis (OR: 11.0, 95% CI: 5.4-22.6). The area under the ROC curve increased when multiple phthalates were diagnosed jointly, reaching 0.974 as the highest value, which was helpful for the diagnosis of endometriosis. In vivo experiments showed that after DEHP exposure in rats, the mass index of the ovary and uterus decreased in a dose-dependent manner; the oestrus cycle of SD rats was irregularly prolonged and disordered; and the serum AMH level was negatively correlated with the DEHP exposure dose (Rho = -0.8, p < 0.001). Bioinformatics analysis of rat ovarian tissues showed that seven genes involved in the steroid biosynthesis pathway were upregulated and may play a negative role in ovarian function. Conclusion: Exposure to phthalates, especially DEHP, is associated with the occurrence of endometriosis and affects women's reproductive prognosis and ovarian function. The steroid biosynthesis pathway may be related to ovarian dysfunction. The detection of phthalate in urine may become a new biological target for the diagnosis of endometriosis.

Age-related Changes in Humanin Expression in the Ovarian Tissue of Rat.

This study examined humanin expression in rat ovarian tissue, its cellular localization, and its correlation with rat age under physiological conditions. A total of 40 Sprague-Dawley rats of various ages (2, 12, 30, and 60 days old and 1 year old) were grouped by age. Immunofluorescence and immunohistochemical techniques were used to observe humanin expression and cellular location in the ovarian tissues of rats from each age group. In addition, Western blotting and Real-time quantitative reverse transcription PCR (qRT-PCR) techniques were used to measure humanin expression level in the ovarian tissues of rats from each age group. The immunofluorescence and immunohistochemical results confirmed that humanin was expressed in rat ovarian tissues. In addition, cellular localization analysis showed that humanin was expressed in the cytoplasm of oocytes, interstitial cells, granulosa cells and theca cells in all levels of follicles after the primary follicles, and in the corpus luteum. The qRT-PCR results revealed that the level of humanin expression in the ovarian tissues of 12-day-old rats was non-significantly higher than that in the ovarian tissues of 2-day-old rats (P>0.05), whereas the levels of humanin expression in the ovarian tissues of 30-day-old, 60-day-old, and 1-year-old rats were significantly lower than that in the ovarian tissues of 2-day-old rats (P<0.05). The Western blotting results demonstrated that the levels of humanin protein expression in the ovarian tissues of 60-day-old and 1-year-old rats were significantly lower than those of 2-day-old rats (P<0.01), whereas there was no significant difference in the level of humanin protein expression between the ovarian tissues of 12-day-old and 30-day-old rats. This study confirmed that humanin is expressed in the cytoplasm of various cells in rat ovarian tissues. Moreover, the level of humanin expression was highest in the ovarian tissues of 12-day-old rats, and it subsequently decreased with age. The changes in the expression of humanin in the ovary of rats at different ages will lay the foundation for the role of humanin in ovarian aging. The effect of humanin on ovarian function is worthy of further study in the future.

The immunoexpression of valosin-containing protein and small VCP-interacting protein in rat ovaries.

Numerous cellular processes are controlled by the ubiquitin-proteasome-mediated degradation pathway, involve the 97-kDa valosin-containing protein (p97/VCP). Small p97/VCP-interacting protein (SVIP) was first discovered as one of the novel androgen-responsive genes as well as one of the many cofactors controlling p97/VCP. The aim of the study was to investigate localization and immunoexpression of p97/VCP and SVIP in rat ovarian tissue. The histomorphological examination of rat ovarian tissue was performed by using haematoxylin-eosin (HE) staining. Using the immunohistochemical technique, cellular location and expression of p97/VCP and SVIP in rat ovarian tissue were examined. The nuclear and cytoplasmic immunoexpression of p97/VCP and SVIP was observed in the different stages of ovarian follicles and corpus luteum in the rat ovaries. The immunolocalization of SVIP and VCP in the rat ovaries suggest that they may be involved in the oogenesis. Further studies should be performed about the function of the VCP and SVIP in the female reproductive tract.

Androgen increases klotho expression via the androgen receptor-mediated pathway to induce GCs apoptosis

BackgroundMany epidemiological studies have shown that anovulatory polycystic ovary syndrome (PCOS) is accompanied by hyperandrogenism. However, the exact mechanism of hyperandrogen-induced anovulation remains to be elucidated. In this study, we aimed to investigate the potential mechanism of anovulation in PCOS. To investigate the role of klotho as a key factor in the androgen receptor (AR)-mediated development of PCOS, we investigated the effects of testosterone on ovarian klotho expression in vivo and in vitro.ResultsTestosterone propionate (TP)-induced rats showed cycle irregularity, hyperandrogenism, polycystic ovarian changes, dyslipidemia. However, inhibition of AR expression could relieve PCOS traits. We also found that AR and klotho showed relatively high expression in PCOS rat ovarian tissue and in TP-induced granulosa cells (GCs), which was inhibited by the addition of flutamide. TP-induced GCs apoptosis was suppressed by AR antagonist, as well as silencing klotho expression in human GCs. Chromatin immunoprecipitation assay demonstrated that AR indirectly binds to the klotho promoter.ConclusionsOur results demonstrated TP mediates the expression of klotho via androgen receptor and klotho alterations could be a reason for ovarian dysfunction in PCOS.

HUMSCs Transplantation Regulates AMPK/NR4A1 Signaling Axis to Inhibit Ovarian Fibrosis in POI Rats.

The mechanism of human Umbilical Cord Mesenchymal Stem Cells (hUMSCs) transplantation to improve ovarian function in the rats with Premature Ovarian Insufficiency (POI) is still unclear. The aim of this study is to investigate the signal axis mechanism that is involved in the ovarian function recovery of POI rats following hUMSCs transplantation. The rat model with POI was established by intraperitoneal injection of cisplatin. The hUMSCs were transplanted by caudal vein injection into POI rats.Hematoxylin-eosin (H&E) staining was performed to examine the morphology of rat ovarian tissue.Masson staining, Sirus red staining and immunofluorescence were used to observe the fibrosis extent of ovarian tissue. The levels of serum sex hormones and the expression of fibrosis related markers in ovarian tissueswere measured by enzyme-linked immunosorbent assay (ELISA).The expression of NR4A1, Phospho-NR4A1 and AMP-activated protein kinase (AMPK) signaling in rat ovarian tissues was measured by immunohistochemistry and immunofluorescence. The role of AMPK/NR4A1 signaling axis in the regulation of ovarian function recovery in POI rats following hUMSCs transplantation was further investigated by adenovirus and siRNA intervention in isolated stromal cells. The results showed that the hUMSCs transplantation significantly inhibited ovarian tissue fibrosis and restored the ovarian function in POI rats.The level of NR4A1 and AMPK expression in ovarian tissue of POI rats after hUMSCs transplantation was significantly increased compared with the control group.In the cultured ovarian stromal cells, the similar results were obtained on the expression of NR4A1 and its regulation on fibrosis related molecular markers in Cisplatin (CDDP) damaged stromal cells following hUMSCs supernatant treatment. Both hUMSCs supernatant treatment and the addition of AMPK inhibitors increased NR4A1 expression in stromal cells. And after NR4A1 molecular intervention, fibrosis-related indicators in stromal cells changed. The data suggests that the AMPK/NR4A1 signaling axis is involved in the ovarian function changes in POI rats following hUMSCs transplantation. The data from this study indicate that the inhibition of tissue fibrosis and recovery of ovarian function is regulated by AMPK/NR4A1 signaling axis in POI rats following hUMSCs transplantation.

Immunofluorescent Evidence for Nuclear Localization of Aromatase in Astrocytes in the Rat Central Nervous System.

Estrogens regulate a variety of neuroendocrine, reproductive and also non-reproductive brain functions. Estradiol biosynthesis in the central nervous system (CNS) is catalyzed by the enzyme aromatase, which is expressed in several brain regions by neurons, astrocytes and microglia. In this study, we performed a complex fluorescent immunocytochemical analysis which revealed that aromatase is colocalized with the nuclear stain in glial fibrillary acidic protein (GFAP) positive astrocytes in cell cultures. Confocal immunofluorescent Z-stack scanning analysis confirmed the colocalization of aromatase with the nuclear DAPI signal. Nuclear aromatase was also detectable in the S100β positive astrocyte subpopulation. When the nuclear aromatase signal was present, estrogen receptor alpha was also abundant in the nucleus. Immunostaining of frozen brain tissue sections showed that the nuclear colocalization of the enzyme in GFAP-positive astrocytes is also detectable in the adult rat brain. CD11b/c labelled microglial cells express aromatase, but the immunopositive signal was distributed only in the cytoplasm both in the ramified and amoeboid microglial forms. Immunostaining of rat ovarian tissue sections and human granulosa cells revealed that aromatase was present only in the cytoplasm. This novel observation suggests a new unique mechanism in astrocytes that may regulate certain CNS functions via estradiol production.

Astragaloside IV regulates autophagy-mediated proliferation and apoptosis in a rat model of PCOS by activating the PPARγ pathway.

Objective(s):Astragaloside IV (AS-IV) is a bioactive saponin with a wide range of pharmacological effects. This study was aimed at investigating its potential effect on polycystic ovary syndrome (PCOS). Materials and Methods:Female Sprague-Dawley rats were randomly divided into five groups (control, PCOS, PCOS+AS-IV 20 mg/kg, PCOS+AS-IV 40 mg/kg, and PCOS+AS-IV 80 mg/kg). The pathological injury level of rat ovary was observed with hematoxylin-eosin (H&E) staining; enzyme-linked immunosorbent assay (ELISA) kit was utilized to measure the levels of luteinizing hormone (LH), follicle-stimulating hormone (FSH), and testosterone in rat serum; western blot detected autophagy-associated or peroxisome proliferator-activated receptor γ (PPARγ) pathway-related protein expression; immunofluorescence was performed to observe LC3 level in rat ovarian tissue. After co-treatment with AS-IV and PPARγ inhibitor, the proliferation in ovarian granulosa cell line KGN was examined employing cell counting kit-8 (CCK-8), EdU staining, and colony formation; cell apoptosis was observed with TdT-mediated dUTP nick-end labeling (TUNEL); apoptosis-related protein expression was assayed by western blot. Results:Treatment with AS-IV inhibited the ovarian pathological damage in PCOS rats. It also promoted the level of autophagy and activated PPARγ signaling in the rat PCOS model. In KGN cells, the level of autophagy and expression of PPARγ-related proteins were also elevated by AS-IV treatment. Furthermore, AS-IV facilitated autophagy, thus inhibiting KGN cell proliferation and promoting its apoptosis, through activating the PPARγ signaling pathway. Conclusion:AS-IV-activated PPARγ inhibits proliferation and promotes the apoptosis of ovarian granulosa cells, enhancing ovarian function in rats with PCOS.

P-686 Molecular mechanism of Zishen Yutai Pills protecting ovarian reserve in premature ovarian insufficiency: a multi-omics analysis

Abstract Study question What is the potential molecular mechanism of Zishen Yutai Pills when compared to Hormone Replacement Therapies in treating premature ovarian insufficiency through protecting ovarian reserve? Summary answer Compared to estrogen, the cAMP signaling pathway and steroid hormone biosynthesis may be the potential molecular mechanisms for Zishen Yutai Pills preserving ovarian function. What is known already Premature ovarian insufficiency (POI) is a major reproductive disease that greatly affects ovarian function. It is a complex disorder with multiple pathogenic factors and high etiological heterogeneity. The conventional treatment for POI is hormone replacement therapies (HRT), which could alleviate the low estrogen syndromes. Compared to HRT, Zishen Yutai Pills (ZSYTP), a patented formula that is widely used for treating POI, can induce follicle development and stimulate ovulation. The combination of omics, including proteomics, metabolomics and transcriptomics, could promote the comprehensive understanding of the potential molecular mechanism involved in both effective treatments for POI in the rat model. Study design, size, duration 20 female Sprague-Dawley rats were randomized to 4 groups: the normal control group (NC), POI model group (POI), ZSYTP group and E2 Gavage group(E2). Intraperitoneally injected with cyclophosphamide for 15 days to establish a POI rat model, follow up with intragastric administration with ZSYTP or E2 daily for 3 weeks. The combined analysis of the proteomics, metabolomics and transcriptomics of rat ovarian tissues shows the potential pathways and molecular targets involved in ZSYTP and E2. Participants/materials, setting, methods All protocols of animal experiments were approved by the Institutional Animal Care and Ethics Committee of Zhuhai BestTest (IAC (S) 202108005). Rat ovary tissues were weighed and quickly frozen after dissection. Muti-omics analysis was performed based on the results from proteomics, metabolomics and transcriptomics analysis, in which Correlation and KEGG pathway analysis were conducted. Statistical analyses were performed with SPSS; statistical significance was set to P &amp;lt; 0.05. Main results and the role of chance Both the E2 and ZSYTP groups show elevated serum estrogen levels compared with the POI group(P&amp;lt;0.05). In the histological assessment, a significantly elevated amount of primary follicles was observed in the ZSYTP group compared to the E2 POI group, indicating that ZSYTP improves ovarian follicle development. Under electron microscopy, fewer necrosis cells were observed in the ZSYTP group, which shows that ZSYTP could improve ovarian repair function and reserve. A combined analysis of ovary proteomics, metabolomics and transcriptomics in ZSYTP vs. E2 shows that both interventions share molecular functions, including steroid binding, protein digestion and absorption, and carbon metabolism. In the KEGG pathway enrichment analysis, the cAMP signaling pathway and steroid hormone biosynthesis were enriched. Limitations, reasons for caution A larger sample size of animal experiments is needed for future study. Quantitative expression analyses of specific genes disclosed in this multi-omics analysis are the next stage of the study. Wider implications of the findings This is the first study to conjoint analyses multi-omics to explore the two different treatments on POI. Zishen Yutai Pills could alleviate low estrogen syndrome and preserve ovarian function through regulating the cAMP signaling pathway and steroid hormone biosynthesis. Zishen Yutai Pills could serve as an effective treatment for POI. Trial registration number Not applicable

Melatonin protects against ovarian damage by inhibiting autophagy in granulosa cells in rats

ObjectivesThis study sought to further verify the protective mechanism of Melatonin (MT) against ovarian damage through animal model experiments and to lay a theoretical and experimental foundation for exploring new approaches for ovarian damage treatment. MethodThe wet weight and ovarian index of rat ovaries were weighted, and the morphology of ovarian tissues and the number of follicles in the pathological sections of collected ovarian tissues were recorded. And the serum sex hormone levels, the key proteins of the autophagy pathway (PI3K, AKT, mTOR, LC3II, LC3I, and Agt5) in rat ovarian tissues, as well as the viability and mortality of ovarian granulosa cells in each group were measured by ELISA, western blotting, CCK8 kit and LDH kit, respectively. ResultsThe results showed that MT increased ovarian weight and improved the ovarian index in ovarian damage rats. Also, MT could improve autophagy-induced ovarian tissue injury, increase the number of primordial follicles, primary follicles, and sinus follicles, and decrease the number of atretic follicles. Furthermore, MT upregulated serum AMH, INH-B, and E2 levels downregulated serum FSH and LH levels in ovarian damage rats and activated the PI3K/AKT/mTOR signaling pathway. Besides, MT inhibited autophagic apoptosis of ovarian granulosa cells and repressed the expression of key proteins in the autophagic pathway and reduced the expression levels of Agt5 and LC3II/I. ConclusionsMT inhibits granulosa cell autophagy by activating the PI3K/Akt/mTOR signaling pathway, thereby exerting a protective effect against ovarian damage.

The Effect of Chrysin on Oxidative Stress in the Ovarian Tissue of Aspirin Administered Rats

Aspirin is a medication with anti inflammatory and antioxidant properties. Chrysin is a natural flavonoid that has been extensively investigated for its significant biological impacts. In this study, we aimed to investigate the protective effect of chrysin against aspirin induced oxidative damage in rat ovarian tissue. Forty female albino rats were used in the study. Rats were randomly divided into 5 groups. Group1 (control group): No medication was administered to the rats in this group. Group 2 (ASA group): 1 mg/kg aspirin was administered to rats in this group by oral gavage for 28 days. Group 3 (ASA+CH): 1 mg/kg aspirin and 50 mg/kg CH were administered to rats in this group by oral gavage for 28 days. Group 4 (CH): 50 mg/kg CH was administered by oral gavage to the rats in this group for 28 days. Group 5 (olive oil): 1 ml/kg of olive oil was administered orally to the rats in this group. At the end of the 28 day trial, ovarian tissues were taken under anesthesia, after the rats fasted for one night. The supernatant was obtained by homogenizing the ovarian tissues, and Malondialdehyde (MDA), Advanced Oxidation Protein Products (AOPP), Total sulfhydryl (T SH) level, and catalase (CAT) activity were quantified spectrometrically from the supernatant. Ovarian tissue MDA and AOPP levels in the ASA group were determined to be significantly higher than in the control group (p&lt;0.05). Ovarian tissue MDA level in the ASA+CH group was lower compared to the ASA group, but the difference between the two groups was not significant (p&gt;0.05). The AOPP level of the ASA+CH group was found to be significantly lower than that of the ASA group (p&lt;0.05). T SH level of the ASA group was significantly lower than the control group (p&lt;0.05). No significant difference was determined between T SH levels of the ASA and AS+CH group (p&gt;0.05). CAT activity in the ASA group was lower than in the control group, but the decrease was not significant (p&gt;0.05). Ovarian tissue CAT activity was found to be significantly higher in the ASA+CH group, compared to the control and ASA groups (p&lt;0.05). ASA administration causes an increase in oxidative stress markers from the ovarian tissue, whereas it causes a decrease in antioxidants. By reducing this effect of aspirin, CH could increase antioxidant levels and reduce oxidative stress Keywords: Aspirin, Chrysin, Ovarian tissue, oxidative stress, antioxidant Special Issue of Health Sciences DOI: 10.7176/JSTR/7-08-05

The effects of prenatal and postnatal exposure to electromagnetic field on rat ovarian tissue.

Exposure to an electromagnetic field (EMF) can have adverse effects on many organs and tissues, including the reproductive system. This study aimed to investigate the effects of EMF exposure during prenatal and postnatal periods on ovarian development in rat offspring. In this study, rat pups born from eight pregnant rats were used. EMF exposure was initiated on the first day of pregnancy and continued until the 42nd postnatal day. The blood and ovarian tissue samples of female offspring in sham and EMF groups were collected when they reached the age of 42 days. Follicle-stimulating hormone levels were significantly higher in the EMF group than in the sham group. Estradiol levels were significantly lower in the EMF group than in the sham group. Tissue-inducible nitric oxide synthase (iNOS) levels and expression were significantly greater in the EMF group than in the sham group. In the EMF group, congestion, bleeding areas, and degeneration of follicle structures were observed in ovarian tissue. The findings suggest that exposure to 50-Hz, 3-mT EMF used in this study during prenatal and postnatal periods may lead to impaired ovarian structure and function in female offspring. EMF may affect ovarian physiology by increasing iNOS levels and may lead to fertility disorders.