Atretic Follicles Research Articles

Mechanism Research of DHEA Treatment Improving Diminished Ovarian Reserve by Attenuating the AMPK-SIRT1 Signaling and Mitophagy.

This study aims to investigate the effect and mechanism of dehydroepiandrosterone (DHEA) on diminished ovarian reserve (DOR) by modulating the AMPK-SIRT1 signaling and mitophagy in rats. Three-month-old female Sprague-Dawley (SD) rats were randomized and injected intraperitoneally with sesame oil as the control or deoxyvinylcyclohexene (VCD) to induce DOR. The VCD-injected rats were randomized and injected subcutaneously with vehicle as the model group or with DHEA for 21days as the DHEA group. After being identified in proestrus, rat blood sampleswere collected to prepare serum samples, and their ovarian tissues were dissected. Compared with the controls, significantly lower serum estradiol (E2), anti-Müllerian hormone (AMH), and inhibin B (IHNB) and higher follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels were detected in the model group (DOR rats). The model group of rats displayed an increase in follicular atresia and a decrease in ovarian volume and the number of growing follicles and corpus luteum, accompanied by increased frequency of oocyte apoptosis and reduced levels of mitochondrial function. Furthermore, significantly higher levels of the AMPK-SIRT1 signaling and mitophagy were observed in the ovaries of rats in the model group. In contrast, treatment with DHEA significantly ameliorated the hormone disorder and morphological changes in the ovaries, reduced the frequency of apoptotic oocytes, and improved mitochondrial function in the ovaries of DOR rats. Mechanistically, DHEA treatment significantly attenuated the AMPK-SIRT1 signaling and mitophagy in the ovaries of DOR rats. DHEA treatment reduced the severity of DOR and enhanced ovarian reserve function by attenuating the AMPK-SIRT1 signaling and mitophagy in the ovaries of rats.

Ulipristal (UPA) effects on rat ovaries: Unraveling follicle dynamics, ovulation inhibition, and safety implications for prolonged use

Ulipristal (UPA), a selective progesterone receptor modulator, has both agonistic and antagonistic effects on progesterone receptors. UPA suppresses ovulation by inhibiting the luteinizing hormone (LH) surge from the pituitary gland; however, the direct effect of UPA on ovarian tissue remains poorly studied. In the present study, we examined the effects of UPA on the ovaries of rats. Rats were treated for 28 days with UPA, and the effects of UPA on ovarian tissue were examined histologically and the expression of antioxidant genes and cell death markers were also investigated. UPA treatment increased the number of primordial follicles at each treatment group, primordial follicles increased at all dose levels, but the size/magnitude of the effect decreased with the increasing dose. The number of primary and antral follicles tended to increase with increasing UPA levels. Furthermore, the decrease in primary follicle number could be attributed to the exhaustion of follicles, but the examination of proliferation markers, oxidative stress markers, and cell death markers revealed no remarkable toxic effects on ovarian tissues. These results suggest that UPA treatment promotes follicle development at each stage but inhibits ovulation by suppressing the LH surge, resulting in an increase in atretic follicles or unruptured luteinized cysts. These results suggest that UPA may not have both toxic effects on the ovary and a direct local effect on ovarian follicles, but we should be careful about the effects of prolonged UPA treatment in patients with uterine fibroids on their future fecundity.

TRIM28-Mediated Excessive Oxidative Stress Induces Cellular Senescence in Granulosa Cells and Contributes to Premature Ovarian Insufficiency In Vitro and In Vivo.

Premature ovarian insufficiency (POI) is a clinical syndrome of ovarian dysfunction characterized by the abnormal alteration of hormone levels such as FSH and E2. POI causes infertility, severe daily life disturbances, and long-term health risks. However, the underlying mechanism remains largely unknown. In this study, we found that POI is associated with the cellular senescence of ovarian granulosa cells, and TRIM28 mediates oxidative stress (OS)-induced cellular senescence in granulosa cells. Mechanistically, OS causes a decrease in TRIM28 protein levels in KGN cells. Subsequently, it triggers an increase in the levels of autophagy marker proteins ATG5 and LC3B-II, and the downregulation of P62. Abnormal autophagy induces an increase in the levels of cellular senescence markers γ-H2A.X, P16, and P21, provoking cellular senescence in vitro. The overexpression of ovarian TRIM28 through a microinjection of lentivirus attenuated autophagy, cellular senescence, and follicular atresia in the ovaries of POI mice and improved mouse fertility in vivo. Our study highlights the triggers for POI, where the reduction of TRIM28, which is regulated by reactive oxygen species, causes follicular atresia and POI via triggering autophagy and inducing granulosa cell senescence. Shedding light on TRIM28 may represent a potential intervention strategy for POI.

VCD-induced menopause mouse model reveals reprogramming of hepatic metabolism

ObjectiveMenopause adversely impacts systemic energy metabolism and increases the risk of metabolic disease(s) including hepatic steatosis, but the mechanisms are largely unknown. Dosing female mice with vinyl cyclohexene dioxide (VCD) selectively causes follicular atresia in ovaries, leading to a murine menopause-like phenotype. MethodsIn this study, we treated female C57BL6/J mice with VCD (160 mg/kg i.p. for 20 consecutive days followed by verification of the lack of estrous cycling) to investigate changes in body composition, energy expenditure (EE), hepatic mitochondrial function, and hepatic steatosis across different dietary conditions. ResultsVCD treatment induced ovarian follicular loss and increased follicle-stimulating hormone (FSH) levels in female mice, mimicking a menopause-like phenotype. VCD treatment did not affect body composition, or EE in mice on a low-fat diet (LFD) or in response to a short-term (1-week) high-fat, high sucrose diet (HFHS). However, the transition to a HFHS lowered cage activity in VCD mice. A chronic HFHS diet (16 weeks) significantly increased weight gain, fat mass, and hepatic steatosis in VCD-treated mice compared to HFHS-fed controls. In the liver, VCD mice showed suppressed hepatic mitochondrial respiration on LFD, while chronic HFHS resulted in compensatory increases in hepatic mitochondrial respiration. Also, liver RNA sequencing revealed that VCD promoted global upregulation of hepatic lipid/cholesterol synthesis pathways. ConclusionOur findings suggest that the VCD-induced menopause model compromises hepatic mitochondrial function and lipid/cholesterol homeostasis that sets the stage for HFHS diet-induced steatosis while also increasing susceptibility to obesity.

LncRNA S100PBP promotes proliferation and steroid hormone synthesis of granulosa cells by sponging MiR-2285bc-BMPR2 in bovine†.

In bovine follicular development, the proliferation of bovine granulosa cells affects follicular selection, atresia, and cystic follicle formation. When cystic follicles appear on the ovaries, granulosa cells stop proliferating, resulting in the reduction of granulosa cells layer. In our previous study, the whole transcriptome sequencing revealed that Bone morphogenetic protein receptor 2 (BMPR2) was differentially expressed between cystic and normal follicular granulosa cells. We speculated that long noncoding RNA may act as competing endogenous RNA targeting microRNAs and then regulating the expression of BMPR2 and the function of granulosa cells, thereby affecting follicular development and cyst formation. In this study, the results elucidated that long noncoding RNA S100PBP (NONBTAT011846.2) directly bound miR-2285bc, which targeted in the BMPR2 3'-UTR. miR-2285bc suppresses granulosa cells proliferation by downregulating BMPR2 expression. Furthermore, long noncoding RNA S100PBP was silenced by small interfering RNA, and long noncoding RNA S100PBP regulated BMPR2 expression by sponging miR-2285bc investigated through cross-verification. When small interfering RNA of long noncoding RNA S100PBP was transfected into granulosa cells, the results revealed similar molecular changes as those transfected with miR-2285bc mimics. Silencing long noncoding RNA S100PBP or overexpressing miR-2285bc altered the expressions of some follicular development-related genes, which could be related to follicular cyst occurrence. In conclusion, our findings support that long noncoding RNA S100PBP regulates the expression of BMPR2 through sponge miR-2285bc, promotes the proliferation of granulosa cells, inhibits their apoptosis, and increases the synthesis and secretion of follicular steroid hormones, thus promoting the development of bovine follicles.

Low levels of Cd2+ combined with procymidone may cause ovarian damage in mice via unfolded protein response.

As no study about the combined effect of low levels of Cd2+ with procymidone (PCM) on organs and organisms, we investigated their actions on mouse-ovary in vivo and in vitro. Four-week mice were treated with corn oil for the control group, corn oil + 0.0045 mg/L Cd2+ (CdCl2 was dissolved in ultrapure water and freely consumed by mice) for Cd2+ group, 50 mg/kg/d PCM (suspended in corn oil and administered orally to mice) for PCM group, and 50 mg/kg/d PCM + 0.0015 (0.0045 and 0.0135) mg/L Cd2+ for L+ (M+ and H+) PCM group for 21 days. For in vitro experiment, the cultured ovaries were treated with acetone for the control group, 0.1% acetone + 8.4 μg/L Cd2+ for the Cd2+ group, 0.63 mg/L PCM (dissolved in acetone) for the PCM-group, and 0.63 mg/L PCM + 2.8 (8.4 and 25.2) μg/L Cd2+ for L+ (M+ and H+) PCM group for 7 days. Mouse body weight in each treatment group, the weight and volume of ovaries in all PCM groups were lower than the control. Both in vivo and in vitro, all-stage follicle numbers were lower in M+PCM and H+PCM groups, whereas the atretic follicles and CASPASE3/8 were higher; meanwhile, lower estradiol and progesterone and higher unfolded protein response (UPR) members in all PCM groups. L+, M+, and H+PCM groups had further ovarian damage and stronger UPR than PCM groups, as did M+PCM groups over Cd2+ groups. It is hypothesized low-level PCM and Cd2+ may mutually promote each other's triggered UPR and exacerbate ovarian damage.

Plasma-derived from human umbilical cord blood restores ovarian function and improves serum reproductive hormones levels in mice with premature ovarian insufficiency (POI) through cytokines and growth factors.

Premature ovarian insufficiency (POI) patients experience a decline in ovarian function and a reduction in serum reproductive hormones, leading to a significant impact on the outcomes of assisted reproductive technology. Despite the absence of an effective clinical treatment to restore fertility in POI patients, recent research has indicated that cord blood plasma (CBP) derived from human umbilical cord blood (hUCB) may offer therapeutic benefits for various degenerative diseases. The primary aim of this study is to explore approaches for enhancing ovarian function and serum reproductive hormones through the administration of CBP in a murine model. Initially, hUCB was utilized to obtain CBP (CBP), which was subsequently analyzed for cytokine and growth factor profiles in comparison to adult blood plasma (ABP) by use of flow cytometry. Subsequently, POI mouse models were established through the induction of 4-vinylcyclohexene diepoxide, followed by the injection of CBP into the tail. At 7, 14, and 21 days posttreatment, mouse ovaries and blood were collected, and their estrus cycle, body weight, and ovarian weights were evaluated using precise electronic balance. Finally, ovarian morphology and follicle number were assessed through HE staining, while serum levels of anti-Müllerian hormone (AMH), estradiol (E2) and follicle-stimulating hormone (FSH) were determined by ELISA. Our study revealed that individuals with CBP exhibited significantly lower concentrations of proinflammatory cytokines, including IL-β (p < 0.01) and IL-2 (p < 0.05), while displaying elevated levels of anti-inflammatory cytokines and chemokines, such as IL-2, IL-4, IL-6, IL-8, IL-12P70, IL-17A, IP-10, interferon-γ, and tumor necrosis factor-α (p < 0.01). Furthermore, CBP demonstrated remarkably higher levels of growth factors, including transforming growth factor-β1, vascular endothelial growth factor, and insulin-like growth factor-1 (p < 0.01) than ABP. Notably, our investigation also revealed that CBP restored the content of serum reproductive hormones, such as AMH, E2, and FSH (p < 0.05), and increased the number of primordial and primary follicles (p < 0.01) and decreased the number of luteal and atretic follicles (p < 0.01) in vivo. Our findings suggested that CBP-secreted cytokines and growth factors could be restored POI ovarian function, enhanced serum reproductive hormones and rescued follicular development in vivo. These findings further support the potential of CBP as a promising strategy in clinical applications for POI related infertility.

Differential analysis of ovarian tissue between high and low-yielded laying hens in the late laying stage and the effect of LECT2 gene on follicular granulosa cells proliferation.

The ovaries of high-yield laying hens exhibited signs of aging beyond 400 days of age, subsequently resulting in a decline in both egg production and egg quality. Oxidative stress, characterized by an increase in the production of reactive oxygen species (ROS), stands as one of the principal processes contributing to ovarian aging. Elevated ROS levels are implicated in the induction of apoptosis in granulosa cells (GCs), provoking mitochondrial impairment, and diminishing the capacity of the antioxidant defense system. This investigation stratified laying hens into two distinct groups, predicated upon their egg production levels: high-yield hens (HH) and low-yield hens (LL). The study focused on evaluating oxidative stress markers and identifying differentially expressed genes between these two groups. The findings revealed that the LL group exhibited follicular atresia, mitochondrial disruptions, and apoptotic occurrences in ovarian GCs. Notably, ROS levels, Malondialdehyde (MDA) concentrations, and 8-hydroxy-2'-deoxyguanosine (8-OHdG) concentrations in ovarian tissue and follicular GCs were substantially higher in the HH group. Furthermore, the RNA-sequencing results unveiled differential expression of the LECT2 gene between the HH and LL groups. Consequently, an overexpression vector for the LECT2 gene was successfully constructed and introduced into GCs. The quantitative polymerase chain reaction (QPCR) analysis exhibited significant downregulation (p<0.01) of key apoptotic genes such as Caspase-3 and C-myc and significant upregulation (p<0.01) of BCL2 following the overexpression of the LECT2 gene in GCs. In conclusion, oxidative stress emerges as a pivotal factor influencing the laying traits of both high and low-yield laying hens. The accumulation of reactive oxygen species (ROS) within the ovaries precipitates apoptosis in GCs, subsequently leading to follicular atresia and a reduction in egg production. Furthermore, we employed RNA sequencing technology to examine the ovarian matrix tissue in high and low laying hens during the late phase of egg laying. Our analysis revealed a substantial upregulation of the LECT2 gene in the ovarian matrix tissue of high laying hens. This observation implies that the LECT2 gene exerts a pivotal influence on driving the proliferation and differentiation of follicular GCs, thereby exerting a crucial regulatory role in follicular development.

Ameliorative effect of salidroside on the cyclophosphamide-induced premature ovarian failure in a rat model

Background Oxidative stress injury is an important pathological factor of premature ovarian failure (POF). Salidroside, extracted from the Chinese herb-Rhodiola rosea, has advantages in antioxidant characteristics. However, their therapeutic efficacy and mechanisms in POF have not been explored. Purpose This study aims to assess the therapeutic effects of salidroside in chemotherapy-induced ovarian failure rats. Methods A POF rat model was established by injection of cyclophosphamide, followed by treatment with salidroside. The therapeutic effect of salidroside was evaluated based on hormone levels, follicle count, and reproductive ability. Oxidative stress injury was assessed by the detection of SOD enzyme activity and MDA levels. Differential gene expression of Keap1, Nrf2, HMOX1, NQO1, AMH, BMP15, and GDF9, were identified by qRT‑PCR. The protein expression of Keap1, Nrf2, P53, and Bcl-2 were detected by western blot. Results Salidroside treatment markedly restored FSH, E2, and AMH hormone secretion levels, reduced follicular atresia, and increased antral follicle numbers in POF rats. In addition, salidroside improves fertility in POF rats, activates the Nrf2 signaling pathway, and reduces the level of oxidative stress. The recovery function of high dose salidroside (50 mg/kg) in a reproductive assay was significantly improved than that of lower dose salidroside (25 mg/kg). Meanwhile, the safety evaluation of salidroside treatment in rats showed that salidroside was safe for POF rats at doses of 25–50 mg/kg. Conclusions Salidroside therapy improved premature ovarian failure significantly through antioxidant function and activating Nrf2 signaling.

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

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

Activation of interleukin 33-NFκB axis in granulosa cells during atresia and its role in disposal of atretic follicles†.

It has been previously shown that the cytokine interleukin 33 is required for two processes, i.e., autophagic digestion of granulosa cells and recruitment of macrophages into atretic follicles, for full disposal of atretic follicles. Now, this study shows that activation of interleukin 33-suppression of tumorigenicity 2-Nuclear Factor ĸB (NFκB) axis in granulosa in early atretic follicles may regulate those two events. Injection of human chorionic gonadotropin has been shown to induce a transient peak of interleukin 33 expression with synchronized atresia. In this model, interleukin 33-independent expression of suppression of tumorigenicity 2 in granulosa cells was detected in early atretic follicles before macrophage invasion. The activation of NFκB pathway in ovaries was further demonstrated in vivo in Tg mice with luciferase-reporter for NFκB activation; the activation was microscopically localized to granulosa cells in early atretic follicles. Importantly, antibody blockage of interleukin 33 or interleukin 33 Knock-out (KO) (Il33-/-) not only inhibited NFκB activity in ovaries, but it also altered expression of two key genes, i.e., reduction in proinflammatory interleukin6 (IL6) expression, and a surge of potential autophagy-inhibitory mammalian target of rapamycin (mTOR) expression in atretic follicles. By contrast, apoptosis and other genes, such as interleukin1β (IL1β) were not affected. In conclusion, in parallel to apoptosis, atresia signals also trigger activation of the interleukin 33-suppression of tumorigenicity 2-NFκB pathway in granulosa, which leads to (1) down-regulated expression of mTOR that is a negative regulator of autophagy and (2) up-regulated expression of proinflammatory IL6.

Protective effect of Luffa cylindrica fermentation liquid on cyclophosphamide-induced premature ovarian failure in female mice by attenuating oxidative stress, inflammation and apoptosis

Premature ovarian failure (POF) is a leading cause of women’s infertility without effective treatment. The purpose of this study was to investigate the protective effects of Luffa cylindrica fermentation liquid (LF) on cyclophosphamide (CTX) -induced POF in mice and to preliminarily investigate the underlying mechanisms. Thirty-two Balb/c mice were divided into four groups randomly. One group served as the control, while the other three received CTX injections to establish POF models. A 14-day gavage of either 5 or 10 μL/g LF was administered to two LF pretreatment groups. To analyze the effects of LF, the ovarian index, follicle number, the levels of serum sex hormones, superoxide dismutase (SOD), glutathione (GSH), malondialdehyde (MDA), inflammatory factors, and apoptosis of the ovarian cells were measured. The effects of LF pretreatment on the expression of TLR4/NF-κB and apoptosis pathways were also evaluated. We found that LF pretreatment increased the ovarian index and the number of primordial and antral follicles while decreasing those of atretic follicles. LF pretreatment also increased the serum levels of estradiol (E2) and anti-Müllerian hormone (AMH), while decreasing those of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Furthermore, LF pretreatment increased the levels of SOD and GSH in the ovaries, while decreasing those of MDA, tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). LF administration reduced the amount of TUNEL+ ovarian cells and the levels of TLR4 and NF-κB P65 protein expression. In conclusion, LF has antioxidant, anti-inflammatory as well as anti-apoptotic effects against CTX-induced POF, and the inhibition of TLR4/NF-κB and apoptosis pathways may be involved in its mechanisms.Graphical

Background and common lesions in the female reproductive organs of giant anteaters (Myrmecophaga tridactyla).

The giant anteater (Myrmecophaga tridactyla) is a vulnerable species in South America and is considered endangered or near extinction in Central America. Therefore, studies describing the reproductive characteristics of this species are pivotal for its conservation. Thus, this study aimed to provide a morphological description of the female reproductive tissues of this species. We collected tissue samples from six female giant anteaters and performed gross, morphological, and histochemical analyses. Five adult subjects and one juvenile were included in the study. In the ovary, classifications were made according to the follicle and oocyte sizes: primordial, primary, secondary, early antral, or antral. Typical follicles with a single oocyte surrounded by a simple or stratified layer of cubic epithelium, atretic follicles, corpora lutea, corpora albicans, and ovarian cysts were also observed. No ovarian lesions were observed. By contrast, endometritis, metritis, mucometra, and endometrial cysts were identified in the uterus. Uterine alterations in these subjects were frequent and could affect reproduction.

Sinapic acid modulates oxidative stress and metabolic disturbances to attenuate ovarian fibrosis in letrozole-induced polycystic ovary syndrome SD rats.

Sinapic acid (SA) is renowned for its many pharmacological activities as a polyphenolic compound. The cause of polycystic ovary syndrome (PCOS), a commonly encountered array of metabolic and hormonal abnormalities in females, has yet to be determined. The present experiment was performed to evaluate the antifibrotic properties of SA in rats with letrozole-induced PCOS-related ovarian fibrosis. SA treatment successfully mitigated the changes induced by letrozole in body weight (BW) (p < .01) and relative ovary weight (p < .05). Histological observation revealed that SA reduced the number of atretic and cystic follicles (AFs) and (CFs) (p < .01), as well as ovarian fibrosis, in PCOS rats. Additionally, SA treatment impacted the serum levels of sex hormones in PCOS rats. Luteinizing hormone (LH) and testosterone (T) levels were decreased (p < .01, p < .05), and follicle-stimulating hormone (FSH) levels were increased (p < .05). SA administration also decreased triglyceride (TG) (p < .01) and total cholesterol (TC) levels (p < .05) and increased high-density lipoprotein cholesterol (HDL-C) levels (p < .01), thereby alleviating letrozole-induced metabolic dysfunction in PCOS rats. Furthermore, SA treatment targeted insulin resistance (IR) and increased the messenger RNA (mRNA) levels of antioxidant enzymes in the ovaries of PCOS rats. Finally, SA treatment enhanced the activity of peroxisome proliferator-activated receptor-γ (PPAR-γ), reduced the activation of transforming growth factor-β1 (TGF-β1)/Smads, and decreased collagen I, α-smooth muscle actin (α-SMA), and connective tissue growth factor (CTGF) levels in the ovaries of PCOS rats. These observations suggest that SA significantly ameliorates metabolic dysfunction and oxidative stress and ultimately reduces ovarian fibrosis in rats with letrozole-induced PCOS.

Altered expression of angiogenic factors in dominant preovulatory follicles of dairy cattle treated with ACTH

Studies in cows have reported that ovulation, steroidogenesis and angiogenesis are affected by stress and consequently fertility decreases. The purpose of this study was to evaluate the effects of ACTH administration during the preovulatory period on the expression of growth factors (CD-31, PDGF-A, PDGF-B, VEGFA-164, VEGFA-164b, VEGF-R1 and VEGF-R2) associated with the angiogenic process by immunohistochemistry in cows (n = 14). Results evidenced the expression of these growth factors in theca and granulosa cells from antral, atretic and dominant preovulatory follicles of ACTH-treated cows, suggesting that, under stress conditions, their expression continues to be required. VEGFA-164, VEGF-R1 and VEGF-R2 expression was greater in theca cells of dominant preovulatory follicles of the ACTH-treated group than in those of the control group. CD-31 protein expression was lower in the dominant preovulatory follicles of the ACTH-treated group than in those of the control group. PDGF-A and PDGF-B expression did not differ between groups, either in granulosa or in theca cells. These results suggest that VEGFA-164, its receptors and CD-31 are actors in the normal cycle of the ovaries and could have greater pathophysiological importance in the altered angiogenic process and other events that occur during anovulation and stress conditions. This dysregulation reinforces the importance of the angiogenic process in the pathophysiology of cystic ovarian disease in cows. This is the first report on the expression and localization of components of the VEGF and PDGF systems and CD-31 in cells from dominant preovulatory follicles after ACTH administration.

Comparative effects of the antioxidant glutathione with metformin and Diane-35 on hormonal, metabolic, and inflammatory indicators in a DHEA-induced PCOS rat model

Objective Comparison of hormonal, metabolic and inflammatory markers of glutathione with metformin and Diane-35 in a rat model of PCOS induced by dehydroepiandrosterone. Methods Twenty-five female rats were randomized into four groups. Group 1 was administered a subcutaneous dose of 0.2 ml saline/day. Group 2 was given 0.2 ml of 1% carboxymethyl cellulose (CMC)/day orally for 28 days. A PCOS model was established with DHEA in rats. Group 3 was given 4.5 mg/kg/day of Diane-35 orally dissolved in 1% CMC for 28 days. Group 4 was given 300 mg/kg/day of metformin orally dissolved in 1 ml of saline for 28 days, and Group 5 was administered 100 mg/kg of glutathione intraperitoneally on days 35, 42, and 49. On day 56, the rats were sacrificed. Serum markers and follicle count were examined. Results Serum IL-6, hs-CRP, insulin, testosterone, SHBG, and MDA values were significantly lower in the glutathione group than in the PCOS group (p = 0.0006, p = 0.023, p = 0.0082, p = 0.0007, p = 0.0048, and p < 0.0001, respectively). The number of all follicles was similar between the control and glutathione groups (p < 0.05). When we compared the other groups with the PCOS group, the number of primary, secondary, atretic, and cystic follicles was significantly lower in the metformin and glutathione groups. The number of primordial and antral follicles was significantly higher than in the PCOS group. Conclusions Glutathione plays anti-inflammatory and antioxidant roles, similar to metformin, by lowering serum IL-6, insulin, testosterone, CRP, and MDA levels; decreasing atretic/cystic follicle count; and improving antral follicle count and folliculogenesis in PCOS patients.

Allium ampeloprasum var. Porrum (Alliaceae) Improves Metabolic and Reproductive Disorders Associated with Polycystic Ovary Syndrome in Wistar Rats.

To provide scientific evidence of the efficacy of Allium ampeloprasum against female infertility, the effects of the aqueous extract of the said plant (AE) were evaluated in rats with letrozole-induced polycystic ovary syndrome (PCOS). AE was administered orally to PCOS rats at doses of 192, 384, and 768 mg/kg. The positive control was co-treated with clomiphene citrate (1 mg/kg) and metformin (200 mg/kg). Normal and negative controls received distilled water. The vaginal contents of rats were examined daily under a microscope before (7 days) and during treatment. Treatments were administered orally for 15 days, and then, 6 rats from each group were sacrificed for biochemical and histological analyses. The remaining rats were mated with males of proven fertility for 5 days. The daily examination of vaginal smears allowed the evaluation of fertility index. After parturition, additional fertility parameters were determined. Results showed that in PCOS rats, AE decreased body weight (p < 0.001), abdominal fat weight (p < 0.001), serum levels of LH (p < 0.001), testosterone (p < 0.001), total cholesterol (p < 0.05), and LDL cholesterol (p < 0.01). HDL cholesterol increased and atherogenic indices decreased (p < 0.001). The number of Graafian follicles and corpora lutea increased, while cystic (p < 0.001) and atretic (p < 0.05) follicles decreased. AE also decreased oxidative stress in the ovaries, restored the estrous cycle, induced uterine epithelial cell hypertrophy, and improved fertility. These effects were attributed to phenols, flavonoids, terpenoids, and anthocyanins present in AE. The overall results justify the traditional use of A. ampeloprasum against female infertility and suggest its potential use as a dietary supplement for PCOS patients.

SIRT1 overexpression by melatonin and resveratrol combined treatment attenuates premature ovarian failure through activation of SIRT1/FOXO3a/BCL2 pathway

AimTo evaluate the synergistic effect of combined treatment with melatonin (MEL) and resveratrol (RES) in cisplatin (CIS)-induced premature ovarian failure (POF) model in rats and to elucidate the molecular mechanism of this therapeutic effect. Material & methodsFemale Sprague Dawley rats were divided into 7 experimental groups as follows; CONT (Control), CIS, MEL, RES, POF + MEL, POF + RES, and POF + MEL + RES. H&E staining was performed to evaluate follicular cell vacuolization/degeneration, vascular congestion/hemorrhage, and inflammation, by using an ordinal scale from 0 to 4 to grade the severity of observed changes (0 = normal, 1 = mild, 2 = moderate, 3 = severe, 4 = very severe). Zona pellucida integrity and connective tissue amount in the ovarian tissue were detected using PAS & Masson Trichrome staining. The immunofluorescence method was used to determine the immune localizations of pH2Ax, SIRT1, FOXO3a, and BCL2. The connective tissue amounts and immunoreactivity staining intensities were measured using ImageJ. The gene expression of SIRT1, FOXO3a, and BCL2 was determined using RT-PCR. Serum estrogen hormone levels were measured by ELISA. Statistically, Bonferroni correction was performed, and p < 0.002 were considered significant. ResultsA significant difference was observed in the POF group compared to the CONT group in all parameters except tertiary follicle count and hemorrhage. The decrease in the number of atretic follicles in the POF + MEL + RES group was found significant compared to both POF + MEL and POF + RES groups. The expression of pH2Ax, SIRT1, FOXO3a, and BCL2 at the protein level and SIRT1 and BCL2 at the mRNA level were significant in the POF + MEL + RES group compared to the POF group. Between the single and combination treatment groups, the difference in protein level was found in pH2Ax, SIRT1, FOXO3a, and BCL2 expression. The POF + MEL + RES group exhibited significantly higher SIRT1 mRNA expression compared to the groups receiving single treatments. ConclusionThe present study provides evidence that MEL and RES have synergistic effects in preventing the decrease in follicle reserve and increase in DNA break (pH2Ax) and follicle atresia in POF ovaries. This therapeutic effect is mediated by SIRT1 overexpression and activation of the SIRT1/FOXO3a/BCL2 pathway.

Higher intake of β-glucan impairs reproduction in a female teleost, Tor putitora (Hamilton, 1822).

Although the immuno-modulatory and stress-relieving properties of β-glucan is well elucidated in humans and other animal models, including fish, its role as a dietary supplement on reproduction is extremely scarce. Therefore, in this study, adult female fish were fed one of four test diets having 0 (control), 0.5, 1, and 1.5% β-D-glucan for 130 days and its effect on reproductive performance, ovarian and liver histology, sex hormones, and transcript abundance of selected reproduction-related genes was assessed. Low dietary intake of β-glucan improved fertilization and hatching rates (p<0.05). The relative fecundity and percentage of spawning females were higher (non-significant) in 0.5% β-glucan-fed groups. Surprisingly, even after 130 days, spawning did not occur in 1.5% β-glucan-fed individuals. Irrespective of β-glucan intake, all the brooders recorded similar plasma 17β-estradiol and maturation-inducing hormone (p>0.05). Higher intake of β-glucan (1.5%) upregulated aromatase genes without a parallel increase in 17β-estradiol. However, plasma vitellogenin increased with increasing β-glucan up to 1.0% then declined at 1.5% (p<0.05). The fish that received control, 0.5, and 1.5% β-glucan recorded similar vitellogenin levels in their plasma. Significantly higher plasma cortisol was evidenced in 1.5% β-glucan fed brooders (p<0.05). Histologically, higher follicular atresia and leaking of yolk material was evidenced in 1.5% β-glucan-fed group. Liver histology revealed the highest nutrient/lipid accumulation in fish that received 1.0% and 1.5% β-glucan. This study demonstrated the stimulatory effect of β-glucan intake at a lower dose (0.5%) on reproduction. However, higher intake (1.5%) could perturb normal reproductive function in a fish model and caused an increased number of atretic follicles leading to spawning/reproductive failure.

Subtotal hysterectomy causes fewer long-term detrimental effects on ovary tissues than total hysterectomy.

Hysterectomy is the basic surgical procedure of gynecological surgery. Traditionally, it is divided into total hysterectomy (TH) and subtotal hysterectomy (STH) according to the scope of surgery. The ovary is a dynamic organ appended with the uterus, and the uterus provides vascular supply to the developing ovary. However, the long-term impacts of TH and STH on ovary tissues need to be evaluated. In this study, rabbit models of different ranges of hysterectomy were successfully created. The estrous cycle of animals was determined by vaginal exfoliated cell smear 4 months after the operation. The apoptosis rate of ovarian cells in each group was determined by flow cytometry, and the morphology of ovarian tissue and granulosa cells in the control group, triangular hysterectomy group and total hysterectomy group were observed under microscope and electron microscope, respectively. After total hysterectomy, the apoptotic events in ovarian tissues were significantly increased when compared to the sham and triangle hysterectomy group. Increased apoptosis was accompanied with the morphological changes and disrupted organelle structures in ovarian granulosa cells. The follicles in the ovarian tissue were dysfunctional and immature, with more atretic follicles being observed. In contrast, ovary tissues in triangular hysterectomy groups showed no obvious defects on the morphology of ovarian tissue and granulosa cells. Our data suggest that subtotal hysterectomy may serve as an alternative to total hysterectomy, with fewer long-term detrimental effects on ovary tissues.