Number Of Granulosa Cells Research Articles

Ascorbic acid and resveratrol improve the structural integrity of the extracellular matrix and enhance follicular survival in cultured bovine ovarian tissue.

This study aimed to investigate the changes induced by the culture system and the effect of ascorbic acid and resveratrol on collagen fibers, stromal cells, follicle growth and survival, as well as antioxidant enzyme activity in cultured bovine ovarian tissues. In experiment 1, bovine ovarian fragments were cultured in α-minimum essential medium (α-MEM+) for 6 days. Before and after culturing, the fragments were fixed and processed to assess follicular morphology and diameters, stromal cell survival, collagen fibers, and glycosaminoglycans (GAGs). Uncultured and cultured tissues were also used to measure mRNA expression for superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), and peroxiredoxin (PRDX). Thiol levels and activity of CAT, SOD, and GPX enzymes were also investigated. In experiment 2, bovine ovarian fragments were cultured in α-MEM+ alone or supplemented with 50μg/mL ascorbic acid or both 50μg/mL ascorbic acid and 20μM resveratrol for 6 days. In experiment 1, cultured tissues had higher percentages of growing follicles, but higher percentage of degenerated follicles than uncultured slices (P<0.05). Additionally, the collagen and GAGs network became disorganized, with reduced deposition around primordial and primary follicles (P<0.05). The number of stromal and granulosa cells, as well as follicular and oocyte diameters were reduced in both follicular categories compared to uncultured tissue (P<0.05). Expression of mRNA for CAT, SOD, GPX, and PRDX was downregulated in 6-day cultured tissues (P<0.05). Similarly, thiol levels and CAT activity were also reduced (P<0.05). In experiment 2, ascorbic acid or both ascorbic acid and resveratrol increased the rate of follicular diameters and survival, and the number of granulosa and stromal cells compared to tissues cultured in the control medium (P<0.05). Both ascorbic acid and resveratrol improved collagen density and preserved the GAG network, as well as increased thiol levels and CAT activity (P<0.05). In conclusion, in vitro culture of ovarian tissue favored follicular activation, but reduced the proportion of normal follicles, collagen, GAG network, stromal cell numbers, and tissue antioxidant protection. Ascorbic acid alone or in association with resveratrol improved the preservation of extracellular matrix components and enhanced follicular survival.

Transcriptome and metabolome revealed the effects of hypoxic environment on ovarian development of Tibetan sheep

Many studies on the adaptability of Tibetan sheep to hypoxia have been reported, but little attention has been paid to the reproduction of Tibetan sheep living at an altitude of more than 4000 m. In this study, the ovaries of Alpine Merino sheep (AM) living in middle-high altitude areas (2500 m) and the ovaries of Gangba Tibetan sheep (GB) and Huoba Tibetan sheep (HB) living in ultra-high altitude areas (4400 m or more) were collected. Through morphological, transcriptomics and metabolomics, the effects of ultra-high altitude areas on Tibetan sheep ovarian development and the molecular mechanism of sheep's adaptability to ultra-high altitude environment were explored. The results showed that the number of granulosa cells in AM was significantly higher than that in GB and HB. The transcriptome revealed several genes related to follicular development, such as DAPL1, IGFBP1, C5, GPR12, STRA6, BMPER, etc., which were mainly enriched in related pathways such as cell growth and development. Through metabolomics analysis, it was found that the differential metabolites between the three groups of sheep were mainly lipids and lipid-like small molecules, such as Glycerol 3-Phosphate, PC (16: 0 / 18: 3 (9Z, 12Z, 15Z)), mainly enriched in lipid metabolism and other related pathways. The results of combined analysis showed that Tryptophan metabolism and Steroid hormone biosynthesis may have a significant effect on Tibetan sheep follicular development. Some genes (including HSD17B7, CYP11A1, CYP19, HSD3B1, CYP17, etc.) and some metabolites (including Cortisone, 2-Methoxyestrone, etc.) are enriched in these pathways, regulating ovarian and follicular development by affecting estrogen, progesterone, etc.. The results further revealed the molecular mechanism of Tibetan sheep to adapt to the ultra-high altitude environment and maintain normal ovarian and follicular development through the regulation of genes and metabolites.

Trehalose Alleviates D-Galactose-Induced Aging-Related Granulosa Cell Death in Ovaries.

Ovarian dysfunction caused by aging restricts female reproductive capacity and is accompanied by oxidative stress and impaired autophagy. Recent studies have shown that trehalose (Tre) can activate autophagy and have antioxidant effects. However, whether Tre can be used to attenuate ovarian aging remains unclear. Therefore, the anti-aging effects of Tre on the ovary were explored both in vivo and in vitro. D-galactose (D-gal) was administered i.p. daily (200 mg/kg body weight) for 8 weeks to establish the mouse ovarian aging model (n = 10). We found that Tre significantly reversed ovarian weight loss and reduced the number of TUNEL-positive granulosa cells caused by D-gal in mouse ovaries. Tre elevated the protein expression levels of LC3-II, Parkin, PINK1, Beclin1, and LAMP2 in ovaries. Mitochondrial-related proteins TOM20 and COX IV expression levels were increased by Tre administration. In vitro studies further supported these findings, showing that Tre treatment significantly reduced the number of SA-β-gal and PI-positive cells, and decreased ROS levels in cultured granulosa cells. Thus, Tre alleviates ovarian aging by activating mitophagy and reducing oxidative stress, suggesting its potential as an anti-aging agent for ovarian health.

Human umbilical cord mesenchymal stem cell-derived extracellular vesicles harboring IGF-1 improve ovarian function of mice with premature ovarian insufficiency through the Nrf2/HO-1 pathway

ObjectivePremature ovarian insufficiency (POI) is a disease with medical, psychological and reproductive implications, but its common therapies have limited efficacy and a likelihood of complications. This study delves into the therapeutic role of human umbilical cord mesenchymal stem cell-derived extracellular vesicles (hUC-MSCs-EVs) in POI mice through the insulin-like growth factor 1 (IGF-1)/nuclear factor E2 related factor 2 (Nrf2)/heme oxygenase-1 (HO-1)/autophagy pathway.MethodshUC-MSCs were transfected with lentiviral short hairpin RNA of IGF-1 before EV extraction. Cyclophosphamide (CTX)-induced POI mouse models were administrated with hUC-MSCs-EVs. Mouse ovarian granulosa cells (GCs) were induced with CTX, then treated with hUC-MSCs-EVs and ML385. Ovarian histopathological changes were observed, changes in follicle number at all levels were counted and serum sex hormones were evaluated, as well as LC3II/I and Beclin-1 expression. GCs were subject to detection of proliferation, deaths, oxidative stress, and Nrf2 nuclear translocation.ResultsAfter CTX exposure, mice showed thinner GCs layer in the ovary, reduced number of GCs and follicles at all levels, disturbed serum sex hormones, enhanced oxidative stress and autophagy, and downregulated ovarian IGF-1; whereas, hUC-MSCs-EVs upregulated IGF-1 to improve the ovarian function. hUC-MSCs-EVs carrying IGF-1 activated Nrf2/HO-1 signaling to inhibit CTX-induced excessive autophagy of GCs, but this ameliorative effect was partially weakened by inhibiting Nrf2/HO-1 signaling. hUC-MSCs-EVs inhibited excessive autophagy of GCs and improved ovarian function of CTX-induced mice through IGF-1/Nrf2/HO-1 pathway.ConclusionhUC-MSCs-EVs activate the Nrf2/HO-1 signaling by carrying IGF-1, which in turn inhibits excessive autophagy and damage of GCs, thus improving ovarian function in POI mice.

Electroacupuncture attenuates depressive-like behaviors in poststroke depression mice through promoting hippocampal neurogenesis and inhibiting TLR4/NF-κB/NLRP3 signaling pathway.

The aim of this study was to investigate the impact and underlying molecular mechanisms of electroacupuncture on mice with poststroke depression (PSD). Mice were randomly allocated into sham, PSD, and electroacupuncture groups. Mice in the PSD and electroacupuncture groups underwent middle cerebral artery occlusion (MCAO) surgery following with sedentary behavior. Electroacupuncture targeting Zusanli (ST36) acupoint was performed 24 h after MCAO for 4 weeks in electroacupuncture group. The sucrose preference test, forced swimming test, open field test, tail suspension test, elevated plus maze, Catwalk analysis, RNA sequencing, Nissl staining, Golgi staining, TUNEL staining, Edu labeling, and doublecortin staining were performed. Lymphocyte subsets in peripheral blood and the levels of IL-1β, IL-6, TNF-α, and expression of Iba1/CD86, Iba1/NLRP3, TLR4/p38/NF-κB/NLRP3 pathways in the hippocampus were detected. Electroacupuncture effectively protected against the development of depression-like symptoms. The number of granulosa cells and doublecortin-positive cells in the dentate gyrus (DG) were significantly decreased in PSD group, which were significantly upregulated ( P < 0.01) by electroacupuncture. Electroacupuncture also significantly reduced ( P < 0.05) TUNEL-positive cells in the DG and CA1. RNA-seq revealed that electroacupuncture may exert antidepressant effect by regulating the inflammation mediated by TLR4/NF-κB/NLRP3 pathway in hippocampus. Electroacupuncture remarkably elevated ( P < 0.01) the ratio of CD4+ to CD8+ T cells and percentage of CD3-CD49b+ cells in CD45+CD49b+ cells in the peripheral blood. Electroacupuncture significantly reduced ( P < 0.05) the high levels of IL-1β, IL-6, TNF-α, iba1, TLR4, p-p38, p-NF-κB, and NLRP3 and sedentary behavior. Electroacupuncture was observed to mitigate depression symptoms and increase hippocampal neurogenesis in mice with PSD, possibly by inhibiting TLR4/p38/NF-κB/NLRP3 pathways and improving the microglia-mediated inflammatory microenvironment in the hippocampus.

P-682 Malic enzyme 1 in granulosa cells promotes ovarian aging through inhibiting the AMPK-FoXO1-IDH2 signaling pathway

Abstract Study question Reduced levels of oxidative phosphorylation in granulosa cells are a key factor in regulating ovarian aging, but the upstream regulatory mechanisms are unknown. Summary answer Our study revealed that elevated malic enzyme 1 in granulosa cells promotes ovarian aging by inhibiting oxidative phosphorylation levels through the AMPK-FoXO1-IDH2 signaling pathway. What is known already With ovarian aging, the ovarian granulosa cells appear hallmarks of high apoptosis activity and low-proliferative activity, which lead to a reduction in ovarian reserve and a decrease in hormone synthesis, resulting in female infertility and reduced quality of life. And the decline of isocitrate dehydrogenase, IDH, in non-human primate ovarian granulosa cells is a key causative factor for the decline in the level of oxidative phosphorylation in granulosa cells. Recently, researchers reported that malic enzyme 1, which encodes a cytosolic, NADP-dependent Enzyme that generates NADPH for fatty acid biosynthesis inhibits IDH2 expression in tumor cells. Study design, size, duration Utilizing clinical samples, cells and animal models, with the help of research tools such as biochemical experiments, gene editing and single-cell transcriptome sequencing, at different levels, including molecular, cellular, animal and human samples, we systematically analyzed the characteristic changes and key molecules in granulosa cells along with ovarian aging and reveal the intracellular molecular pathways through which ME1 and IDH2 regulate the process of ovarian aging for 1 year. Participants/materials, setting, methods We first apply single-cell transcriptome sequencing analysis of young and old mouse ovaries to reveal cellular subpopulation changes and transcriptomic hallmarks of ovarian granulosa cell with aging. And then we validate the biological function and revealing the molecular mechanism to regulate ovarian aging by using physiological ovarian aging mouse model, clinical ovarian granulosa cells from young and old female and human ovarian granulosa cell line KGN utilizing technologies of protein immunology, qPCR and CRISPR/Cas9. Main results and the role of chance Ovarian single-cell transcriptome sequencing data from young and old mice showed that ovarian aging was accompanied by a dramatic decrease in the number of granulosa cells and drastic changes in the proportions of different subpopulations. The proportion of mitotically active granulosa cells was significantly lower in aging ovaries than in young mouse ovaries, and GO enrichment analysis showed that granulosa cell cycle, antioxidant and hormone synthesis-related signaling pathways were significantly down-regulated in aging ovaries. Using immunoblotting, immunofluorescence and qPCR, we demonstrated that the expression of ME1 in granulosa cells of aging mice and human ovaries was significantly elevated, while the expression of IDH2 was significantly decreased. Performing the overexpression and knockdown of ME1 in human ovarian granulosa cell line KGN, which further demonstrated that ME1 could inhibit the transcriptionally regulated IDH2 expression of downstream genes, such as FoXO1, by suppressing the phosphorylation of AMPK. The decrease of IDH2 in mitochondria reduces the level of oxidative phosphorylation in granulosa cells, resulting in the increase of intracellular reactive oxygen species, activation of the intracellular p53-p21 signaling pathway, inhibition of the cell cycle, and promotion of apoptosis, which in turn impairs the function of granulosa cells to synthesize hormones and promotes ovarian aging. Limitations, reasons for caution Malic enzyme 1, which links the glycolytic cycle to the citric acid cycle, expressing in other ovarian cells including oocytes, and the regulation of its expression is complex. Future studies will focus on its biological functions in other ovarian cells and its molecular mechanism in regulating ovarian aging. Wider implications of the findings Our study suggests that granulosa cell ME1 and IDH2 co-regulate oxidative phosphorylation levels to regulate ovarian aging, and that ME1 and IDH2 may serve as potential targets for the finding and treatment of premature ovarian aging and delaying ovarian aging in women. Trial registration number not applicable

Liquid nitrogen improves the decellularization effectiveness of whole-ovary

BACKGROUND: Ovarian tissue cryopreservation for fertility preservation carries a risk of malignant cell re-seeding. Artificial ovary is a promising method to solve such a problem. However, ovary decellularization protocols are limited. Hence, further studies are necessary to get better ovarian decellularization techniques for the construction of artificial ovary scaffolds. OBJECTIVE: To establish an innovative decellularization technique for whole porcine ovaries by integrating liquid nitrogen with chemical agents to reduce the contact time between the scaffolds and chemical reagents. MATERIALS AND METHODS: Porcine ovaries were randomly assigned to three groups: novel decellularized group, conventional decellularized group and fresh group. The ovaries in the novel decellularized group underwent three cycles of freezing by liquid nitrogen and thawing at temperatures around 37??C before decellularization. The efficiency of the decellularization procedure was assessed through histological staining and DNA content analysis. The maintenance of ovarian decellularized extracellular matrix(ODECM) constituents was determined by analyzing the content of matrix proteins. Additionally, we evaluated the biocompatibility of the decellularized extracellular matrix(dECM) by observing the growth of granulosa cells on the ODECM scaffold in vitro.RESULTS: Hematoxylin and eosin staining, DAPI staining and DNA quantification techniques collectively confirm the success of the novel decellularization methods in removing cellular and nuclear components from ovarian tissue. Moreover, quantitative assessments of ODECM contents revealed that the novel decellularization technique preserved more collagen and glycosaminoglycan compared to the conventional decellularized group (P&lt;0.05). Additionally, the novel decellularized scaffold exhibited a significantly higher number of granulosa cells than the conventional scaffold during in vitro co-culture (P&lt;0.05). CONCLUSION: The novel decellularized method demonstrated high efficacy in eliminating DNA and cellular structures while effectively preserving the extracellular matrix. As a result, the novel decellularized method holds significant promise as a viable technique for ovarian decellularization in forthcoming studies.

Moringa oleifera extract affects the diameter of the Graafian follicles in female Mus musculus

HIGHLIGHTS This study unveils a dose-dependent relationship between Moringa leaf extract and increased Graafian follicle size in female mice. The significant positive effect of Moringa leaf extract on Graafian follicles suggests promising implications for fertility interventions, offering hope for individuals seeking natural treatment to address infertility challenges. ABSTRACT Objective: This study aimed to determine the effect of Moringa leaf extract (Moringa oleifera Lam.) on the diameter of Graafian follicles in female mice (Mus musculus). Materials and Methods: This study used experimental design, employing a cohort of 24 female mice of 20-25 grams in weight, aged between 2-3 months, and in good health. These subjects were divided into three treatment groups and subjected to oral doses of Moringa leaf extract at 300 mg/kg BW, 400 mg/kg BW, and 500 mg/kg BW over a 14-day period. The study procedures involved the preparation of the experimental animals, preparation of Moringa leaf extract, treatments administration, and the preparation of histological specimens. Subsequently, the diameters of Graafian follicles within each treatment group were measured. Data analysis were performed using the ANOVA test (p &lt;0.05) followed with the Least Significance Difference (LSD) test utilizing the IBM SPSS software. Results: There were variations in Graafian follicle diameters across the experimental groups. The average diameters were 180.944 µm in the control group, 239.942 µm in treatment group 1, 315.006 µm in treatment group 2, and 396.650 µm in treatment group 3. This indicated that dose administration starting from 300 mg/kg, 400 mg/kg, and 500 mg/kg had an effect on the size of the follicle and antrum diameter. The number of granulosa cells was found to increase, as well as the size of the ovum. Conclusion: Moringa leaf extract in different doses has a significant positive effect on increasing the diameter of the Graafian follicles in female mice.

Local effect of allopregnanolone in rat ovarian steroidogenesis, follicular and corpora lutea development

Allopregnanolone (ALLO) is a known neurosteroid and a progesterone metabolite synthesized in the ovary, CNS, PNS, adrenals and placenta. Its role in the neuroendocrine control of ovarian physiology has been studied, but its in situ ovarian effects are still largely unknown. The aims of this work were to characterize the effects of intrabursal ALLO administration on different ovarian parameters, and the probable mechanism of action. ALLO administration increased serum progesterone concentration and ovarian 3β-HSD2 while decreasing 20α-HSD mRNA expression. ALLO increased the number of atretic follicles and the number of positive TUNEL granulosa and theca cells, while decreasing positive PCNA immunostaining. On the other hand, there was an increase in corpora lutea diameter and PCNA immunostaining, whereas the count of TUNEL-positive luteal cells decreased. Ovarian angiogenesis and the immunohistochemical expression of GABAA receptor increased after ALLO treatment. To evaluate if the ovarian GABAA receptor was involved in these effects, we conducted a functional experiment with a specific antagonist, bicuculline. The administration of bicuculline restored the number of atretic follicles and the diameter of corpora lutea to normal values. These results show the actions of ALLO on the ovarian physiology of the female rat during the follicular phase, some of them through the GABAA receptor. Intrabursal ALLO administration alters several processes of the ovarian morpho-physiology of the female rat, related to fertility and oocyte quality.

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

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

YTHDF2 as a Mediator in BDNF-Induced Proliferation of Porcine Follicular Granulosa Cells.

In female mammals, the proliferation and apoptosis of granulosa cells (GCs) are critical in determining the fate of follicles and are influenced by various factors, including brain-derived neurotrophic factor (BDNF). Previous research has shown that BDNF primarily regulates GC proliferation through the PI3K/AKT, NF-kB, and CREB tumour pathways; however, the role of other molecular mechanisms in mediating BDNF-induced GC proliferation remains unclear. In this study, we investigated the involvement of the m6A reader YTH domain-containing family member 2 (YTHDF2) in BDNF-stimulated GC proliferation and its underlying mechanism. GCs were cultured in DMEM medium supplemented with varying BDNF concentrations (0, 10, 30, 75, and 150 ng/mL) for 24 h. The viability, number, and cell cycle of GCs were assessed using the CCK-8 assay, cell counting, and flow cytometry, respectively. Further exploration into YTHDF2's role in BDNF-stimulated GC proliferation was conducted using RT-qPCR, Western blotting, and sequencing. Our findings indicate that YTHDF2 mediates the effect of BDNF on GC proliferation. Additionally, this study suggests for the first time that BDNF promotes YTHDF2 expression by increasing the phosphorylation level of the ERK1/2 signalling pathway. This study offers a new perspective and foundation for further elucidating the mechanism by which BDNF regulates GC proliferation.

ВЛИЯНИЕ КРАТКОСРОЧНОГО АНТИОРТОСТАТИЧЕСКОГО ВЫВЕШИВАНИЯ НА ОВАРИАЛЬНО-ГИПОФИЗАРНУЮ ОСЬ У МЫШЕЙ

With the increasing number of women-participants in piloted space programs, investigations of spaceflight effects on the female reproductive system are becoming vital. Purpose of the work is to determine thickness of the granulosa cells layer relative to the content of the luteinizing hormone (LH) receptor in antral follicles of the ovary, LH and a relative content of cytoskeletal proteins in the hypophysis of mice following suspension during the estrous cycle. SDS-PAGE and immunoblotting were used to define the relative proteins content. mRNAs were counted using RT-PCR. IHA was performed to measure thickness of the granulosa cells layer in ovarian sections. The results demonstrate an increase in the number of granulosa cells in the antral follicles of mice after 96-hr suspension, as well as in the relative content of the LH receptor and its mRNAs. However, the LH level in the hypophysis did not alter, even though we observed some changes in the content of cytoskeletal proteins, namely, an increase in the actin-binding proteins (alpha actinin 1 and 4) and a decrease in alpha tubulin and its acetylated form suggesting, probably, modification of the intracellular LH transport and release into blood.

The effect of Coenzyme Q10 on mitochondrial biogenesis in mouse ovarian follicles during in vitro culture.

The aim of this research was to investigate the effect of Coenzyme Q10 (CoQ10) on the expression of the Transcription Factor A Mitochondrial (Tfam) gene and mtDNA copy number in preantral follicles (PFs) of mice during in vitro culture. To conduct this experimental study, PFs were isolated from 14-day-old National Medical Research Institute mice and cultured in the presence of 50 µm CoQ10 for 12 days. On the 12th day, human chorionic gonadotropin was added to stimulate ovulation. The fundamental parameters, including preantral follicle developmental rate and oocyte maturation, were evaluated. Additionally, the Tfam gene expression and mtDNA copy number of granulosa cells and oocytes were assessed using the real-time polymerase chain reaction. The results revealed that CoQ10 significantly increased the diameter of PFs, survival rate, antrum formation, and metaphase II (MII) oocytes (P < 0.05). Moreover, in the CoQ10-treated groups, the Tfam gene expression in granulosa cells and oocytes increased considerably compared with the control group. The mtDNA copy number of granulosa cells and oocytes cultured in the presence of CoQ10 was substantially higher compared with the control groups (P < 0.05). The addition of CoQ10 to the culture medium enhances the developmental competence of PFs during in vitro culture by upregulating Tfam gene expression and increasing mtDNA copy number in oocyte and granulosa cells.

MFN2 regulates progesterone biosynthesis and proliferation of granulosa cells during follicle selection in hens.

Follicle selection in hens refers to a biological process that only one small yellow follicle (SYF) is selected daily or near-daily for following hierarchical development (from F5/F6 to F1) until ovulation. MFN2 is a kind of GTPases located on the mitochondrial outer membrane, which plays a crucial role in mitochondrial fusion. This study aimed to elucidate the role of MFN2 in proliferation and progesterone biosynthesis of granulosa cells (GCs) during follicle selection in hens. The results showed that GCs began to produce progesterone (P4) after follicle selection, accompanied with changes from multi-layer with flat cells to single layer with cubic cells. MFN2 was detected in GCs of follicles from SYF to F1. After follicle selection, the expression level of MFN2 in GCs upregulated significantly, accompanied with increases in P4 biosynthesis, ATP production, mitochondrial DNA (mtDNA) copy numbers of granulosa cells. FSH (80 ng/mL) facilitated the effects of P4 biosynthesis and secretion, ATP production, mtDNA copy numbers, cell proliferation and the MFN2 transcription of granulosa cells from F5 (F5G) in vitro. However, FSH treatment did not promote P4 secretion in granulosa cells from SYF (SYFG) in vitro. Meanwhile, we observed that change fold of MFN2 transcription, ATP production, mtDNA copy numbers and cell proliferation rate in F5G after treatment with FSH were greater than those in SYFG. Furthermore, expression levels of MFN2 protein and messenger RNA in F5G were significantly higher than those in SYFG after treatment with FSH. P4 biosynthesis, ATP production, mtDNA copy numbers as well as cell proliferation reduced significantly in F5G with MFN2 knockdown. Oppositely, P4 biosynthesis, ATP production, mtDNA copy numbers and cell proliferation increased significantly in SYFG after the overexpression of MFN2. Our results suggest that the upregulation of MFN2 may be involved in the initiation of P4 biosynthesis, and promotion of GCs proliferation during follicle selection.

Spatial Transcriptomics of Aging Rat Ovaries Reveals Unexplored Cell Subpopulations with Reduced Antioxidative Defense

Introduction: Ovarian aging is characterized by a gradual decline in quantity and quality of oocytes and lower chance of fertility. Better understanding the genetic modulation during ovarian aging can further address available treatment options for aging-related ovarian diseases and fertility preservation. Methods: A novel technique spatial transcriptomics (ST) was used to investigate the spatial transcriptome features of rat ovaries. Transcriptomes from ST spots in the young and aged ovaries were clustered using differentially expressed genes. These data were analyzed to determine the spatial organization of age-induced heterogeneity and potential mechanisms underlying ovarian aging. Results: In this study, ST technology was applied to profile the comprehensive spatial imaging in young and aged rat ovary. Fifteen ovarian cell clusters with distinct gene-expression signatures were identified. The gene expression dynamics of granulosa cell clusters revealed three sub-types with sequential developmental stages. Aged ovary showed a significant decrease in the number of granulosa cells from the antral follicle. Besides, a remarkable rearrangement of interstitial gland cells was detected in aging ovary. Further analysis of aging-associated transcriptional changes revealed that the disturbance of oxidative pathway was a crucial factor in ovarian aging. Conclusions: This study firstly described an aging-related spatial transcriptome changes in ovary and identified the potential targets for prevention of ovarian aging. These data may provide the basis for further investigations of the diagnosis and treatment of aging-related ovarian disorders.

Early milk-feeding regimes in calves exert long-term effects on the development of ovarian granulosa cells

BackgroundNutrition has not only an impact on the general wellbeing of an animal but can also affect reproductive processes. In cattle, feeding regimes can influence the age of puberty onset and alter gonadal development. We analyzed effects of different milk replacer (MR) feeding regimes during rearing on ovarian physiology with specific emphasis on the numbers as well as gene expression characteristics of granulosa cells (GCs) at the age of puberty onset. Two groups of calves received either 10% or 20% of bodyweight MR per day during their first 8 weeks. After weaning, both groups were fed the same mixed ration ad libitum until slaughter at 8 months.ResultsAnimals of the 20% feeding group had a significantly higher body weight, but the proportion of animals having a corpus luteum at the time of slaughter was not different between groups, suggesting a similar onset of puberty. Calves of the 10% group showed a constant GC count regardless of the number of follicles (r = 0.23) whereas in the 20% group increasing numbers of GCs were detected with a higher follicle count (r = 0.71). As a first effort to find a possible molecular explanation for this unexpected limitation of GC numbers in the 10% group, we comparatively analyzed GC transcriptomes in both diet groups. The mRNA microarray analysis revealed a total of 557 differentially expressed genes comparing both groups (fold change > |1.5| and p < 0.05). OAS1X, MX2 and OAS1Z were among the top downregulated genes in the 20% vs. the 10% group, whereas top upregulated genes comprised BOLA and XCL1. All of these genes are known to be regulated by interferon. Subsequent signaling pathway analysis revealed the involvement of several immune response mechanisms in accordance with a number of interferons as upstream regulators.ConclusionsThe results indicate that the plane of MR feeding in early life has an impact on the number and physiology of GCs later in life. This might influence the overall reproductive life initiated by the onset of puberty in cattle. In addition, the observed alterations in GCs of calves fed less MR might be a consequence of interferon regulated immunological pathways.

Effects of follicle-stimulating hormone, insulin-like growth factor 1, fibroblast growth factor 2, and fibroblast growth factor 9 on sirtuins expression and histone deacetylase activity in bovine granulosa cells

Granulosa cells (GC) are critical regulators of fertility. During the process of ovarian folliculogenesis, these cells undergo profound changes while producing steroid hormones that are important to control follicular growth, oocyte maturation, and ovulation. Sirtuins are enzymes that regulate several biological processes and have been associated with control of GC function. However, how sirtuins are regulated in GC during ovarian folliculogenesis remains to be unveiled. The present study was designed to investigate effects of hormones that control GC proliferation, differentiation, and steroidogenesis on expression of the seven members of the mammalian sirtuins family (SIRT1-7) and on histone deacetylase activity of nuclear sirtuins (SIRT1, 6, and 7) in GC. Bovine granulosa cells were isolated from small antral follicles (1–5 mm) and were treated with or without follicle-stimulating hormone (FSH), insulin-like growth factor 1 (IGF-1), and fibroblast growth factors 2 (FGF2) and 9 (FGF9). Following treatments, cell proliferation was determined via a cell analyzer, estradiol synthesis and histone deacetylase activity were determined via ELISA, and sirtuins mRNA expression was determined via qPCR. Treatments with FSH and IGF-1 stimulated cell proliferation while addition of FGF2 or FGF9 suppressed estradiol production stimulated by FSH plus IGF-1. In terms of treatments that regulated sirtuins expression in GC, fibroblast growth factors were the most impactful: FGF2 alone increased SIRT1 mRNA expression in comparison to several treatments and increased mRNA abundance of SIRT2 and SIRT7 when added to the combination of FSH and IGF-1; the addition of FGF9 to the combination of FSH and IGF-1 increased mRNA expression of SIRT2, SIRT3, SIRT4, SIRT6, and SIRT7 and increased mRNA expression of SIRT5 in comparison to the negative control group that received no treatment. Also, FGF2 alone increased histone deacetylase activity of sirtuins in comparison to all treatments that contained FSH and/or IGF-1. Furthermore, several correlations were observed between treatments and sirtuins expression and activity, between estradiol or GC numbers and sirtuins expression, and between expression of sirtuins. As FGF2 and FGF9 are considered anti-differentiation factors of GC that stimulate GC proliferation while suppressing estradiol production in combination with FSH and IGF-1, data of this study suggest that sirtuins are associated with control of differentiation of bovine GC.

Acupuncture regulates the apoptosis of ovarian granulosa cells in polycystic ovarian syndrome-related abnormal follicular development through LncMEG3-mediated inhibition of miR-21-3p

BackgroundThe main features of polycystic ovary syndrome (PCOS) are abnormal follicular development and ovulatory dysfunction, which are caused by excessive apoptosis of ovarian granulosa cells. Acupuncture has been shown to improve follicular development abnormalities in patients with PCOS, but its mechanism is unknown. This study hypothesized that the mechanism of acupuncture on follicular development abnormalities in PCOS patients is the inhibition of granulosa cell apoptosis through LncMEG3-mediated regulation of miR-21-3p.MethodsA PCOS-like rat model was established using subcutaneous injection of dehydroepiandrosterone (DHEA). Acupuncture was performed on rats for 15 d (CV-4, RN-3, CV-6, SP-6 and EX-CA 1). Ovarian morphology was observed by HE staining, and sex hormone and AMH levels were detected by ELISA. Primary granulosa cells were isolated from each group of rats to assess the association of acupuncture treatment, LncMEG3, miR-21-3p, and granulosa cell apoptosis in rats with PCOS.ResultsLncMEG3 and miR-21-3p were highly expressed in the ovarian granulosa cells of rats with PCOS, and LncMEG3-mediated regulation of miR-21-3p was involved in the development of PCOS in rats. Silencing of MEG3 attenuated sex hormone dysregulation and ovarian histopathological changes in PCOS rats and promoted follicle cell development and maturation. In addition, silencing MEG3 increased the viability and number of granulosa cells. In addition, silencing MEG3 further inhibited early and late apoptosis of ovarian granulosa cells in PCOS rats. Acupuncture improved polycystic ovarian morphology and sex hormone levels in PCOS rats. Acupuncture intervention increased the viability and number of granulosa cells. Acupuncture intervention inhibited early and late apoptosis of ovarian granulosa cells in PCOS rats by targeting miR-21-3p via LncMEG3.ConclusionThese results suggest that acupuncture can downregulate LncMEG3, thereby targeting and regulating miR-21-3p to suppress early and late granulosa cell apoptosis and normalize their proliferation. These factors ultimately compensate for abnormal follicular development. These findings shed light on the clinical potential of acupuncture as a safe treatment for follicular developmental abnormalities in PCOS.

Effects of food-borne cholesterol supplementation on lead-induced neurodevelopmental impairments of rats based on BDNF signaling pathway and cholesterol metabolism

Despite the ubiquity and prevalence of lead (Pb) in the environment and industry, the mechanism of lead-induced neurotoxicity in the brain remains unclear, let alone its prevention and treatment. In this study, we hypothesized that exogenous cholesterol supplementation acts as an effective remedy for lead-induced neurodevelopmental impairments caused by lead. Forty 21-day-old male rats were randomly divided into four groups and administered 0.1 % lead water and/or 2 % cholesterol-containing feed for 30 d. Ultimately, rats in the lead group lost weight, accompanied by spatial learning and memory impairments as verified by the Morris water maze test, in which the escape latency of rats was prolonged, and the number of crossings in the target platform and the residence time in the target quadrant were significantly diminished compared to the control group. Hematoxylin-Eosin (H&E) staining and Nissl staining illustrated that typical pathological morphology occurred in the brain tissue of the lead group, where the tissue structure was loose, the number of hippocampal neurons and granulosa cells decreased significantly and were arranged loosely, along with enlarged intercellular space, light matrix staining, and decline in Nissl bodies. In addition, inflammatory response and oxidative stress were significantly induced by lead. Immunofluorescence experiments showed apparent activation of astrocytes and microglia, followed by the enhancement of TNF-α and IL-β levels. Moreover, the MDA content in the lead group was elevated dramatically, whereas the activities of SOD and GSH were significantly inhibited. As for the mechanism, western blot and qRT-PCR experiments were performed, where lead could significantly inhibit the BDNF-TrkB signaling pathway, lowering the protein expression of BDNF and TrkB. Cholesterol metabolism was also affected by lead exposure, in which cholesterol metabolism-related protein expression and gene transcription, including SREBP2, HMGCR, and LDLR, were downregulated. However, cholesterol supplementation efficiently detoxified the negative effects of lead-induced neurotoxicity, reversing the inflammatory response, oxidative stress, inactivation of the BDNF signaling pathway, and imbalance of cholesterol metabolism, thus improving the learning and memory ability of rats. In brief, our study demonstrated that cholesterol supplementation could ameliorate the deficiency of learning and memory induced by lead, which is closely associated with the initiation of the BDNF/TrkB signaling pathway and regulation of cholesterol metabolism.

Preantral follicle numbers and size in heifers carrying Trio, a bovine high fecundity allele.

The bovine high fecundity allele, Trio, results in the occurrence of multiple ovulations and is characterized by antral follicles that develop slower and acquire ovulatory capacity at smaller sizes. This study provides novel information on the effect of the Trio allele on early folliculogenesis. The bovine high fecundity allele, Trio, causes overexpression in granulosa cells (GCs) of SMAD6, an inhibitor of BMP15-activated SMAD signalling. Furthermore, the Trio allele results in antral follicles that develop slower, acquire ovulatory capacity at smaller sizes, and have three-fold greater ovulation rate compared to half-sib non-carriers. The present study was designed to determine preantral follicle numbers and size in Trio carrier and non-carrier cattle testing the hypothesis that inhibition of SMAD signalling would alter preantral follicle activation and/or growth. Ovarian tissues from Trio carrier (n = 12) and non-carrier (n = 12) heifers were obtained by laparotomy after follicle wave synchronization. Follicle numbers and dimensions were determined for each stage of development (primordial, transitional, primary, and secondary) from paraffin-embedded sections. There were no differences in the number of primordial, transitional, or secondary follicles or in antral follicle count, circulating AMH, or ovarian volume between carriers and non-carriers. Trio carriers had ~2.5-fold greater (P < 0.01) number of primary follicles than non-carriers, and transitional and primary follicles were larger (~1.2-fold; P < 0.1) in Trio carriers. Oocyte volume of primordial and transitional follicles was not different between genotypes; however, oocytes were larger (P < 0.05) in primary (~1.3-fold) and secondary (~1.8-fold) follicles for Trio carriers. Granulosa cell numbers were not different (P > 0.3) between carriers and non-carriers, irrespective of the stage of development. These results suggest that, after primordial follicle activation, follicles in Trio carrier cattle have slower progression through the primary stage, hence the larger oocyte and greater number of primary follicles.