Granulosa Cells Of Ovarian Follicles Research Articles

Transcriptome profiling reveals SLC5A5 regulates chicken ovarian follicle granulosa cell proliferation, apoptosis, and steroid hormone synthesis

The egg-laying performance of hens holds significant economic importance within the poultry industry. Broody inheritance of the parent stock of chickens can result in poor options for the improvement of egg production, and is a phenomenon influenced by multiple genetic factors. However, few studies have been conducted to delineate the molecular mechanism of ovarian regression in brooding chickens. Here, we explored the pivotal genes responsible for the regulation of ovarian follicles in laying hens, using RNA-sequencing analysis on the small ovarian follicles from broody and laying chickens. Sequencing data analysis revealed the differential expression of 200 genes, with a predominant enrichment in biological processes related to cell activation and metabolism. Among these genes, we focused on solute carrier family 5 member 5 (SLC5A5), which exhibited markedly higher RNA expression levels in follicles from laying compared with broody chickens. Subsequent cellular function studies with knockdown of SLC5A5 in chicken ovarian follicle granulosa cells (GCs) led to the down-regulation of genes associated with cell proliferation and steroid hormone synthesis, and concurrent promotion of gene expression linked to apoptosis. These findings indicated that SLC5A5 deficiency led to the inhibition of proliferation, steroid hormone synthesis and secretion, and promotion of apoptosis in chicken GCs. Our study demonstrated a pivotal role for SLC5A5 in the development and function of chicken GCs, shedding light on its potential significance in the broader context of chicken ovarian follicle development, and providing a prospective target to improve the egg-laying performance of chickens via molecular marker-assisted breeding technology.

Anti-Müllerian Hormone: A Predictor of Successful Intrauterine Insemination.

The anti-Müllerian hormone (AMH) produced by the granulosa cells of ovarian follicles has been shown to correlate with ovarian reserve and is often measured for fertility therapies. In this study, we evaluated the relationship between serum AMH values and the clinical pregnancy (CP) rates of female partners with unexplained infertility undergoing intrauterine insemination utilizing varying ovarian simulation protocols. This is a retrospective cohort study conducted among couples who underwent intrauterine insemination therapy over a period of four years at Charleston Area Medical Center, a tertiary care medical center in West Virginia, USA.Logistic regression was used to determine the best predictor of CP. A total of 509 intrauterine inseminations resulting in81 (15.9%) Cps were analyzed. The cycles with a CPhad higher mean AMH values (3.7+3.5 vs. 2.2+2.1; p<0.001).The majority of patients were nulliparous (77.0%) with a mean age of 33.6+5.0 years.After including only patients with unexplained infertility (the predominate infertility diagnosis; n=255 (50.1% of the cycles)) and stimulation cycles >10, the final sample size for the analysis was 245/509=48.1%.Following a receiver operating characteristic (ROC) curve analysis, the optimal AMH cut-off point was 2.1 ng/mL with an area under the curve (AUC) equal to 0.61 and 95% confidence intervals (CIs) of 0.55- 0.67 (p=0.002). The CP rate was significantly higher with the AMH >2.1 ng/mL (20.0%) compared to <2.1 ng/mL (10.0%; p=0.041). With Clomid/human gonadotropins/human chorionic gonadotropin (hCG) trigger treatment, the CP rate quadrupled (odds ratio (OR): 4.6; 95% CI: 2.1-9.7; p<0.001). This study indicates that higher AMH levels and a more aggressive ovarian stimulation protocol for intrauterine insemination therapy (IUI) have a better probability of resulting in CP.

Seasonal AMH variability implies a positive effect of UV exposure on the deterioration of ovarian follicles

Anti-Müllerian hormone (AMH) is produced exclusively by granulosa cells of ovarian follicles and is an indicator of ovarian reserve which declines with age. Seasonality in AMH levels have been reported to be correlated with variations in Vitamin D levels, which is dependent on sunlight exposure. However, the effects of age and its association with solar radiation intensity with respect to AMH was never studied before. In this study, we investigated the relationship between AMH levels with season and with solar radiation intensity in a cohort of 2235 women aged 19–40 years undergoing hormonal work-up over a four-year period. Our findings revealed that among women aged 20–29 years, there was no significant association between AMH levels and either season or solar radiation intensity. However, for women aged 30–40 years, a seasonal pattern was observed, with higher AMH levels during spring and autumn months characterized by moderate solar radiation intensity. Women in their declining ovarian reserve age were found to be more sensitive to the effects of moderate solar radiation. Moderate solar radiation exposure positively impacted AMH levels, whereas low and high intensity exposure had a negative effect. Our findings indicate that age and solar radiation intensity must be considered when assessing AMH levels and provide valuable insights into the intricate relationship between AMH, seasonality, and UVB exposure in the context of reproductive health.

Expression of genes regulating cell division in porcine follicular granulosa cells

BackgroundCell cycle regulation influences the proliferation of granulosa cells and affects many processes related to ovarian folliclular growth and ovulation. Abnormal regulation of the cell cycle can lead to many diseases within the ovary. The aim of this study was to describe the expression profile of genes within granulosa cells, which are related to the formation of the cytoskeleton, organization of cell organelles inside the cell, and regulation of cell division. Established in vitro primary cultures from porcine ovarian follicle granulosa cells were maintained for 48, 96, 144 h and evaluated via microarray expression analysis.ResultsAnalyzed genes were assigned to 12 gene ontology groups "actin cytoskeleton organization", "actin filament organization", "actin filament—based process", "cell—matrix adhesion", "cell—substrate adhesion", "chromosome segregation", "chromosome separation", "cytoskeleton organization", "DNA integrity checkpoint", "DNA replication initiation", "organelle fision", "organelle organization". Among the genes with significantly changed expression, those whose role in processes within the ovary are selected for consideration. Genes with increased expression include (ITGA11, CNN1, CCl2, TPM2, ACTN1, VCAM-1, COL3A1, GSN, FRMD6, PLK2). Genes with reduced expression inlcude (KIF14, TACC3, ESPL1, CDC45, TTK, CDC20, CDK1, FBXO5, NEK2—NIMA, CCNE2). For the results obtained by microarray expressions, quantitative validation by RT-qPCR was performed.ConclusionsThe results indicated expression profile of genes, which can be considered as new molecular markers of cellular processes involved in signaling, cell structure organization. The expression profile of selected genes brings new insight into regulation of physiological processes in porcine follicular granulosa cells during primary in vitro culture.

Evidence for the expression of vasorin in the human female reproductive tissues

Objective: To investigate the expression and localization of Vasorin (Vasn) in human female reproductive system. Methods: The presence of Vasorin was evaluated by RT-PCR and immunoblotting analyses in patient-derived endometrial, myometrial and granulosa cells (GCs) primary cultures. Immunostaining analyses were performed to detect Vasn localization in primary cultures and in ovarian and uterine tissues. Results: Vasn mRNA was detected in patient-derived endometrial, myometrial and GCs primary cultures without significant differences at the transcript level. Otherwise, immunoblotting analysis showed that Vasn protein levels were significantly higher in GCs than proliferative endometrial stromal cells (ESCs) and myometrial cells. Immunohistochemistry performed in ovarian tissues revealed that Vasn was expressed in the GCs of ovarian follicles at different stages of development with a higher immunostaining signal in mature ovarian follicles such as the antral follicle or on the surface of cumulus oophorus cells than in early-stage follicles. The immunostaining of uterine tissues showed that Vasn was expressed in the proliferative stroma endometrium while it was significantly less expressed in the secretory endometrium. Conversely, no protein immunoreactivity was revealed in health myometrial tissue. Conclusions: Our results revealed the presence of Vasn in the ovary and the endometrium. The pattern of Vasn expression and distribution suggests that this protein may have a role in the regulation of processes such as folliculogenesis, oocyte maturation, and endometrial proliferation.

Review: Roles of follicle-stimulating hormone in preantral folliculogenesis of domestic animals: what can we learn from model species and where do we go from here?

The pituitary gonadotropin FSH is a glycoprotein critical for the development of ovarian follicles. Upon binding to its G protein-coupled membrane receptor located on the granulosa cells of ovarian follicles, FSH elicits a cascade of downstream intracellular responses to promote follicle growth, maturation and steroidogenic activity, leading to the acquisition of meiotic and developmental competence of the enclosed oocyte. The essential role of FSH for proper antral follicle development and fertility is indisputable; over the decades, increasing evidence has also pointed toward survival and growth-promoting effects elicited by FSH in earlier-stage preantral follicles, deeming these follicles FSH-responsive as opposed to the FSH-dependent antral follicles. Transgenic mouse models lacking GnRH1, Fshβ or Fshr clearly demonstrate this difference by showing that, morphologically, preantral follicles develop to the secondary stage without FSH signaling; however, exogenous expression or administration of FSH to hormone-deficient mice promotes preantral follicle development, with more pronounced effects seen in earlier stages (i.e., primary follicles). In hypophysectomized sheep, FSH administration also promotes the growth of primary-stage preantral follicles. However, in vivo studies in this area are more challenging to perform in domestic animals compared to rodents, and therefore most of the research to date has been done in vitro. Here, we present the existing evidence for a role of FSH in regulating the growth and survival of preantral follicles from data generated in rodents and domestic animals. We provide an overview of the process of folliculogenesis, FSH synthesis and cellular signaling, and the response to FSH by preantral follicles in vivo and in vitro, as well as interactions between FSH and other molecules to regulate preantral folliculogenesis. The widespread use of FSH in ovarian stimulation programs for assisted reproduction creates a real need for a better understanding of the effects of FSH beyond stimulation of antral follicle growth, and more research in this area could lead to the development of more effective fertility programs. In addition to its importance as an agricultural species, the cow provides a desirable model for humans regarding ovarian stimulation due to similar timing of folliculogenesis and follicle size, as well as similar ovarian architecture. The refinement of minimally invasive methods to allow the study of preantral folliculogenesis in live animals will be critical to understand the short- and long-term effects of FSH in ovarian folliculogenesis.

N-3 PUFA Promotes Ferroptosis in PCOS GCs by Inhibiting YAP1 through Activation of the Hippo Pathway.

Polycystic ovary syndrome (PCOS) is an endocrine disorder characterized by hyperandrogenemia with multiple suspended sinus follicles, thickened cortical tissue, and excessive proliferation of ovarian granulosa cells that severely affects the fertility and quality of life of women. The addition of n-3 PUFA to the diet may slightly reduce body weight and greatly alleviate disturbed blood hormone levels in PCOS mice. We treated KGN as a cell model for n-3 PUFA addition and showed that n-3 PUFA inhibited the proliferation of GCs and promoted ferroptosis in ovarian granulosa cells. We used CCK-8, fluorescence quantitative transmission electron microscopy experiments and ferroptosis marker gene detection and other methods. Furthermore, n-3 PUFA was found to promote YAP1 exocytosis by activating Hippo and weakening the cross-talk between YAP1 and Nrf2 by activating the Hippo signaling pathway. In this study, we found that n-3 PUFA inhibited the over proliferation of granulosa cells in ovarian follicles by activating Hippo, promoting YAP1 exocytosis, weakening the cross-talk between YAP1 and Nrf2, and ultimately activating the ferroptosis sensitivity of ovarian granulosa cells. We demonstrate that n-3 PUFA can alleviate the hormonal and estrous cycle disorder with PCOS by inhibiting the YAP1-Nrf2 crosstalk that suppresses over proliferating ovarian granulosa cells and promotes iron death in GCs. These findings reveal the molecular mechanisms by which n-3 PUFA attenuates PCOS and identify YAP1-Nrf2 as a potential therapeutic target for regulation granulosa cells in PCOS.

Monitoring of indicators of chromatin status in quails ovarian follicles granulosa cells of different directions of productivity

Relevance. Identification and analysis of the reasons for reducing egg productivity is an urgent problem in quail breeding. The aim of this study is to assess the destructive processes in the somatic cells in ovarian follicles (the level of apoptosis and pycnosis) of mature quail layers of different productivity in connection with egg production and quality indicators of eggs.Methods. In experiments, the level of apoptosis and pyknosis in granulosa cells of ovarian follicles of 3–8 mm females of Estonian, Manchurian, English white, tuxedo, English black and Pharaoh breeds was evaluated. The quails were kept in individual cages in monopreed pairs with a muted 14-hour light day. The age of the females was 160–180 days. The level of pycnotic cells was assessed by the Tarkowski cytological method, apoptotic processes were analyzed by staining with fluorochromes, propidium iodide and acridine orange (10 mcg/mlPI/AO).Results. chromatin. The results of the study of chromatin destruction using PI/AO showed a high average percentage of apoptotic cells in almost all breeds except thep &lt; 0.05). The maximum proportion of dead granulosa cells was tested in Tuxedo and English Black layers (52% and 43% respectively). At the same time, the English white breed of quails at the age of 183 days has one of the best indicators of egg production and the proportion of yolk in relation to egg weight, which indicates the possibility of prolonging the period of their use as laying hens.

Adolescent exposure to low-dose Δ9-tetrahydrocannabinol depletes the ovarian reserve in female mice.

Cannabis use by adolescents is widespread, but its effects on the ovaries remain largely unknown. Δ9-tetrahydrocannabinol (THC) exerts its pharmacological effects by activating, and in some conditions hijacking, cannabinoid receptors (CBRs). We hypothesized that adolescent exposure to THC affects ovarian function in adulthood. Peripubertal female C57BL/6N mice were given THC (5 mg/kg) or its vehicle, once daily by intraperitoneal injection. Some mice received THC from postnatal day (PND) 30-33 and their ovaries were harvested PND34; other mice received THC from PND30-43, and their ovaries were harvested PND70. Adolescent treatment with THC depleted ovarian primordial follicle numbers by 50% at PND70, 4 weeks after the last dose. The treatment produced primordial follicle activation, which persisted until PND70. THC administration also caused DNA damage in primary follicles and increased PUMA protein expression in oocytes of primordial and primary follicles. Both CB1R and CB2R were expressed in oocytes and theca cells of ovarian follicles. Enzymes involved in the formation (N-acylphosphatidylethanolamine phospholipase D) or deactivation (fatty acid amide hydrolase) of the endocannabinoid anandamide were expressed in granulosa cells of ovarian follicles and interstitial cells. Levels of mRNA for CBR1 were significantly increased in ovaries after adolescent THC exposure, and upregulation persisted for at least 4 weeks. Our results support that adolescent exposure to THC may cause aberrant activation of the ovarian endocannabinoid system in female mice, resulting in substantial loss of ovarian reserve in adulthood. Relevance of these findings to women who frequently used cannabis during adolescence warrants investigation.

The Effect of Tamoxifen Citrate on Granulosa Cells of Ovarian Follicles in Adult Female Rats: Light Microscopic Study.

Tamoxifen is a drug that has been used extensively as a chemotherapeutic agent for breast cancer. It should be taken for a long period, from few weeks up to many years, so it can induce gynecological and nongynecological complications. Present study was conducted to clarify the histopathological effects of tamoxifen intake on the ovarian follicles of rats and evaluate the promising recovery after drug withdrawal. Adult female albino rats (n = 24) were randomly divided into four groups. Group I: Control rats without treatment. Group II: Rats received olive oil vehicle. Group III: Rats received 5 mg/kg daily of tamoxifen dissolved in olive oil by oral administration for 4 weeks. Group IV: Rats received tamoxifen as in Group III then will be kept for another 4 weeks without treatment for recovery. Then, the rats were anaesthetized and the ovaries were removed and prepared for histological assessment by light microscope. The ovarian histological findings in the ovary of Group III revealed an increase in atretic ovarian follicles, appearance of cystic ovarian follicles, and cystic corpus luteum. The granulosa cells of ovarian follicles were disorganized with vacuolation of their cytoplasm, increased number of pyknotic nuclei, fragmented nuclei, and apoptotic bodies. After the withdrawal of drug, the ovarian tissue showed slight improvement with the appearance of some atretic follicles with degenerated oocyte and stromal hyperplasia. Based on the results, tamoxifen induced marked histological changes in the ovary. If tamoxifen is mandatory for the prevention of breast cancer, frequent gynecological examination should be carried out to detect any side effects.

RNA-seq identifies differentially expressed genes involved in csal1 overexpression in granulosa cells of prehierarchical follicles in Chinese Dagu hens

The transcription factor csal1 is an important molecule that plays a critical regulatory function in ovarian follicle development, as confirmed by our previous data. However, the candidate genes of csal1 and its regulatory mechanism remain poorly understood in the granulosa cells (GCs) of chicken prehierarchical follicles (PFs). Six transcriptomes of csal1 and empty vector were analyzed in Chinese Dagu hens by RNA sequencing. Six cDNA libraries were constructed, with more than 42 million clean reads and 16,779 unigenes. Of these 16,779 unigenes, 2,762 differentially expressed genes (DEGs) were found in GCs, including 1,605 upregulated and 1,157 downregulated unigenes. Fourteen genes, including BMP5, TACR2, AMH, PLAG1, MYOD1, BOP1, SIPA1, NOTCH1, BCL2L1, SOX9, ADGRA2, WNT5A, SLC7A11, and GATAD2B, were related to GC proliferation and differentiation, hormone production, ovarian follicular development, regulation of reproductive processes, and signaling pathways in the PFs. Further analysis demonstrated the DEGs in GCs of ovarian follicles were enriched in neuroactive ligand-receptor interaction, cell adhesion molecules, and pathways related to cytochrome P450, indicating a critical function for csal1 in the generation of egg-laying features by controlling ovarian follicle development. For the first time, the current study represents the transcriptome analysis with ectopic csal1 expression. These findings provide significant evidence for investigating the molecular mechanism by which csal1 controls PF development in the hen ovary.

MicroRNA-27a-3p targeting Vangl1 and Vangl2 inhibits cell proliferation in mouse granulosa cells

BackgroundMammalian folliculogenesis is the complex process through which primordial follicles develop into preovulatory follicles. The chief function of ovarian follicle granulosa cells is to play a vital role in the growth, development and atresia of ovarian follicles via gap junctions. Increasing evidence suggests that microRNAs (miRNAs) are essential regulators of granulosa cell apoptosis or proliferation. MethodsThe expression level of miR-27a-3p, myogenic differentiation (MyoD), Vangl1 and Vangl2 was investigated by Real-time quantitative PCR (RT-qPCR) and Western blot. Luciferase reporter assay, bioinformatics analysis and ChIP-PCR was used to detect the binding sites between miR-27a-3p, transcription factor and target genes. KEGG pathway analyses were performed to reveal the predicted targets of miR-27a-3p. Ethynyl deoxyuridine (EdU) proliferation assay was used to measure cell proliferation. ResultsTo explore the underlying mechanisms of the miR-27a-3p function in the development of mouse granulosa cells (mGCs), we screened for the target genes of miR-27a-3p, confirmed its interaction with Vangl1 and Vangl2 and elucidated their roles in mGCs. MiR-27a-3p inhibited the proliferation of mGCs, whereas target genes Vangl1 and Vangl2 had the opposite effect. In addition, the transcription factor MYOD bound to and activated the promoter of miR-27a-3p. MiR-27a-3p suppressed Vangl1 and Vangl2 expression by targeting their 3′-untranslated region (3′-UTR). Furthermore, Vangl1 and Vangl2 suppressed the Wnt pathway by reducing the expression of β-catenin and B-cell lymphoma/leukemia-2 (Bcl-2). ConclusionThese findings indicate a pro-survival mechanism of the MyoD/miR-27a-3p/Vangl1/Vangl2 axis for granulosa cell proliferation and suggest a novel target for the improvement of female fertility.

Molecular Genomic Study of Inhibin Molecule Production through Granulosa Cell Gene Expression in Inhibin-Deficient Mice.

Inhibin is a molecule that belongs to peptide hormones and is excreted through pituitary gonadotropins stimulation action on the granulosa cells of the ovaries. However, the differential regulation of inhibin and follicle-stimulating hormone (FSH) on granulosa cell tumor growth in mice inhibin-deficient females is not yet well understood. The objective of this study was to evaluate the role of inhibin and FSH on the granulosa cells of ovarian follicles at the premature antral stage. This study stimulated immature wild-type (WT) and Inhibin-α knockout (Inha−/−) female mice with human chorionic gonadotropin (hCG) and examined hCG-induced gene expression changes in granulosa cells. Also, screening of differentially expressed genes (DEGs) was performed in the two groups under study. In addition, related modules to external traits and key gene drivers were determined through Weighted Gene Co-Expression Network Analysis (WGCNA) algorithm. The results identified a number of 1074 and 931 DEGs and 343 overlapping DEGs (ODEGs) were shared in the two groups. Some 341 ODEGs had high relevance and consistent expression direction, with a significant correlation coefficient (r2 = 0.9145). Additionally, the gene co-expression network of selected 153 genes showed 122 nodes enriched to 21 GO biological processes (BP) and reproduction and 3 genes related to genomic pathways. By using principal component analysis (PCA), the 14 genes in the regulatory network were fixed and the cumulative proportion of fitted top three principal components was 94.64%. In conclusion, this study revealed the novelty of using ODEGs for investigating the inhibin and FSH hormone pathways that might open the way toward gene therapy for granulosa cell tumors. Also, these genes could be used as biomarkers for tracking the changes in inhibin and FSH hormone from the changes in the nutrition pattern.

Di-isononyl phthalate induces apoptosis and autophagy of mouse ovarian granulosa cells via oxidative stress

Di-isononyl phthalate (DINP) has been widely utilized in industrial, commercial and medical applications for the past few years. Therefore, more attention should be paid to the toxicity of DINP. DINP can cause damage to female reproductive system; however, the potential mechanism remains to be further investigated. In this study, female mice were orally administered with 0, 2, 20 and 200 mg DINP/kg/day for 14 days. We found that DINP significantly affected the arrangement of granulosa cells in ovarian follicles. In addition, DINP could induce apoptosis, autophagy and oxidative stress of the ovary tissue. Meanwhile, the serum estradiol concentration distinctly decreased in the 20 and 200 mg/kg DINP-treated groups, suggesting that DINP might affect the function of ovarian granulosa cells. Primary mouse ovarian granulosa cells were utilized for further investigation after the cells were treated with 0, 100, 200, 400 μM DINP for 24 h. Similar to the in vivo experiment, DINP could also induce apoptosis and autophagy of ovarian granulosa cells, as well as oxidative stress; while inhibition of oxidative stress by NAC could alleviate DINP-induced apoptosis and autophagy. Furthermore, inhibition of autophagy by 3-MA could also rescue the induction of apoptosis by DINP. Taken together, these results indicated that DINP induced apoptosis and autophagy of mouse ovarian granulosa cells via oxidative stress, and autophagy played a cytotoxic role in DINP-induced apoptosis.

Generation of an Ihh-mKate2-Dre knock-in mouse line.

Indian hedgehog (Ihh), a member of the Hh family, plays important roles in vertebrate development and homeostasis. To improve our understanding of the function of Ihh-expressing cells and their progeny as well, we generate an Ihh-mKate2tomm20 -Dre knock-in mouse line that can label Ihh-positive cells with a fluorescence protein mKate2 and trace Ihh-positive cells and their progeny via Dre-mediated recombination. Consistent with previous reports, we verified Ihh expression in hypertrophic chondrocytes of growth plate and granulosa cells of ovarian follicles by mKate2 immunostaining, and meanwhile confirmed Dre activity in these cells via a Dre reporter mouse line Rosa26-confetti2. We also found, for the first time, that Ihh can mark some cell types, including retinal ganglion cells, Purkinje cells, and gallbladder epithelial cells. Taken together, the Ihh-mKate2tomm20 -Dre mouse is a genetic tool for examining the precise expression profile of Ihh and tracing Ihh-expressing cells and their progeny.

Potential long-term developmental toxicity of in utero and lactational exposure to Triclocarban (TCC) in hampering ovarian folliculogenesis in rat offspring

Triclocarban (TCC), an antimicrobial compound commonly added to a wide range of household and personal hygiene care products, is one of the most prevalent endocrine-disrupting substances (EDS). This study was conducted to elucidate whether in utero and lactational exposure to TCC could adversely affect folliculogenesis and the onset of puberty in female rat offspring. Twenty pregnant Sprague Dawley rats were equally divided into Control and TCC dam groups (supplemented daily with drinking water enriched with 0.5 mg/L of TCC) from gestational day5 to postnatal day21 (PND21). Female offspring, 20 from control and 20 from TCC dams, were subdivided into 4 subgroups (PND21, PND28, PND35 & PND42). The day of vaginal opening and first estrous cycle were determined. Ovarian sections of the offspring were processed for H&E staining and for immunohistochemical expression of Ki67, Caspase-3 and androgen receptors (AR) on the granulosa cells of ovarian follicles. Follicular count and atretic index were assessed besides, serum estradiol, progesterone, FSH and LH, C-reactive protein (CRP), malondialdehyde (MDA) and total antioxidant capacity (TAC) were measured. TCC offspring exhibited a significant delay in the onset of puberty and impedance of normal transition of the primordial follicles to more developed ones with altered cyctoarchitecture. Also, TCC decreased follicular count, proliferation and gonado-somatic index while it increased atretic index, apoptosis and AR of the granulosa cells along with disturbance of the feminine hormonal profile and oxidant/antioxidant balance. This study highlighted the potential long-term consequences of in utero and lactational exposure to TCC on the postnatal development of the ovary in rat offspring.

ScRNA-seq of ovarian follicle granulosa cells from different fertility goats reveals distinct expression patterns.

The new technology of high-throughput single-cell RNA sequencing (10×scRNA-seq) was developed recently with many advantages. However, it was not commonly used in farm animal research. There are few reports for the gene expression of goat ovarian follicle granulosa cells (GCs) during different developmental stages. In the current investigation, the gene expression of follicle GCs at different stages from two populations of Ji'ning grey goats: high litter size (HL; ≥3/L; 2L) and low litter size (LL; ≤2 /L; 2L) were analysed by scRNA-seq. Many GC marker genes were identified, and the pseudo-time showed that GCs developed during the time course which reflected the follicular development and differentiation trajectory. Moreover, the gene expression difference between the two populations HL versus LL was very clear at different developmental stages. Many marker genes differentially expressed at different developmental stages. ASIP and ASPN were found to be highly expressed in the early stage of GCs, INHA, INHBA, MFGE8 and HSD17B1 were identified to be highly expressed in the growing stage of GCs, while IGFBP2, IGFBP5 and CYP11A1 were found to be highly expressed in late stage. These marker genes could be used as reference genes of goat follicle GC development. This investigation for the first time discovered the gene expression patterns in goat follicle GCs in high- or low-fertility populations (based on litter size) by scRNA-seq which may be useful for uncovering the oocyte development potential.

Expression of PGC-1ɑ in Ovarian Tissue of PCOS Rat Model

To investigate the expression of peroxisome proliferator-activated receptor γ co-activator 1α (PGC-1α) in the ovarian tissue of polycystic ovary syndrome (PCOS) rat model and obese PCOS rat model, and the possible mechanism of PCOS. Thirty SD rats were randomly divided into control group, PCOS rat model group and obese PCOS rat model group. DHEA dissolved in 0.2 mL soybean oil was injected daily into the rats of two PCOS groups for 21 d. Rats in obese PCOS model group were added with high-fat diet based on DHEA modeling, and each group had 10 rats. Body mass were measured before and on the 22 nd day after modeling. The serum testosterone (T) levels were measured by abdominal aortic blood, and the ovarian tissues of rats were taken for histological changes were observed by HE staining, immunohistochemical staining and Western blot to detect PGC-1ɑ protein expression. The body mass of rats in each group increased after modeling, and the body mass of rats in PCOS group and obese PCOS group increased significantly ( P<0.05). The serum T concentration of two PCOS model groups was higher than that of control group ( P<0.01). The serum T concentration in obese PCOS model group was higher than that in the PCOS group ( P<0.05). The results of HE staining of rat ovarian tissue showed that there were follicles and a small amount of corpus luteum at different developmental stages in the control group, and the granulosa cells were arranged in 4-6 layers. The number of immature small follicles in the two PCOS groups was significantly increased. The granulosa cells were arranged in 1-3 layers, relatively looser, and some follicles were atresia. In the obese PCOS group, the diameter of ovarian atretic follicles increased, the number of granulocyte layers decreased, and oocytes disappeared more obviously. Immunohistochemical staining showed that the PGC-1ɑ protein was mainly expressed in the cumulus and granulosa cells of ovarian tissue in the control group. The mean gray level of PGC-1ɑ protein expression in PCOS group (0.53±0.06) and obese PCOS group (0.36±0.03) was lower than that of the control group (0.75±0.03), with the statistical difference ( P<0.05). PGC-1ɑ expression in the obese PCOS group was lower than that in the PCOS group ( P<0.01). The results of Western blot were consistent with those of immunohistochemical staining. PGC-1ɑ is associated with damage of ovarian granulosa cells in high-fat environment. The decrease of PGC-1ɑ expression in granulosa cells of ovarian follicles may be an important cause of PCOS development.

Integrated transcriptomic analysis on small yellow follicles reveals that sosondowah ankyrin repeat domain family member A inhibits chicken follicle selection.

ObjectiveFollicle selection is an important process in chicken egg laying. Among several small yellow (SY) follicles, the one exhibiting the highest expression of follicle stimulation hormone receptor (FSHR) will be selected to become a hierarchal follicle. The role of lncRNA, miRNA and other non-coding RNA in chicken follicle selection is unclear.MethodsIn this study, the whole transcriptome sequencing of SY follicles with different expression levels of FSHR in Jining Bairi hens was performed, and the expression of 30 randomly selected mRNAs, lncRNAs and miRNAs was validated by quantitative real-time polymerase chain reaction. Preliminary studies and bioinformatics analysis were performed on the selected mRNA, lncRNA, miRNA and their target genes. The effect of identified gene was examined in the granulosa cells of chicken follicles.ResultsIntegrated transcriptomic analysis on chicken SY follicles differing in FSHR expression revealed 467 differentially expressed mRNA genes, 134 differentially expressed lncRNA genes and 34 differentially expressed miRNA genes, and sosondowah ankyrin repeat domain family member A (SOWAHA) was the common target gene of three miRNAs and one lncRNA. SOWAHA was mainly expressed in small white (SW) and SY follicles and was affected by follicle stimulation hormone (FSH) treatment in the granulosa cells. Knockdown of SOWAHA inhibited the expression of Wnt family member 4 (Wnt4) and steroidogenic acute regulatory protein (StAR) in the granulosa cells of prehierarchal follicles, while stimulated Wnt4 in hierarchal follicles. Overexpression of SOWAHA increased the expression of Wnt4 in the granulosa cells of prehierarchal follicles, decreased that of StAR and cytochrome P450 family 11 subfamily A member 1 in the granulosa cells of hierarchal follicles and inhibited the proliferation of granulosa cells.ConclusionIntegrated analysis of chicken SY follicle transcriptomes identified SOWAHA as a network gene that is affected by FSH in granulosa cells of ovarian follicles. SOWAHA affected the expression of genes involved in chicken follicle selection and inhibited the proliferation of granulosa cells, suggesting an inhibitory role in chicken follicle selection.

Оценка деструктивных процессов хроматина гранулезных клеток овариальных фолликулов коров и функциональный статус ооцита

Abstract. Currently, there is the possibility of more detailed studies to the study of oocytes and somatic cells (granulosa cells). The possibility to develop successful models of maturation of female gametes defines the possibility to improve existing methods for the selection of donor eggs and the search for new donor eggs. Oocyte maturation in vivo occurs with the participation of structural follicle elements and follicular fluid [3, 4, 6]. Granulosa cells are widely used in bovine oocyte maturation systems and used in cloning and transgenesis technologies [8, 13]. Purpose of this study: to perform destructive changes granulosa cells in ovarian follicles of bovines (Ø 3–5 mm),which contain growing(ВСВ–) or completed the growth phase of oocytes (ВСВ+). Methods: Functional testing of oocytes wascarried out using the vital dye BCB (brillant cresyl blue – diamond crystal blue) [10]. Viability indices in granulosa cells isolated from follicles that contain oocyte s that grow or complete the growth phase were determined by flow cytometry. The result. It was found, that cellsof granulosa from cow follicles are characterized by different indicators of apoptosis levels depending on the status of oocytes (completed growth and growing) isolated from these follicles. The proportion of apoptotic granulosa cells in bovine follicles of containing oocytes that completed the growth phase, exceeded that in follicles containing growing oocytes by 11 % (29 % vs. 18 %, c:dP &lt; 0.05). The scientific novelty: The data obtained using flow cytometry, allow us to evaluate the level of apoptosis in granulosa cells of bovine ovarian follicle as indicator of functional status of developing oocytes (growing or completed growth phases). This indicator can be used in prognosis of competencies for maturation of bovine oocytes.