Effect of Oral Applied Lead Acetate on the Expression of Caspase-3 on Antral Granulosa Cells and Histopathology of Ovary in Female Wistar Rat (Rattus Norvegicus) Ovaries

Background: Lead exposure affects several human organs, including the reproductive organ. Aims: This research aims to prove the effect of oral applied lead acetate on the expression of caspase-3 in antral granulosa cells, the diameter of the tertiary follicle, and the amount of follicle atresia inside ovaries. Methods: Twenty-four female Wistar rats (Rattus norvegicus) are classified into 4 groups. Group 1 consists of 6 rats acting as control groups. Group 2, 3, and 4 each consist of 6 rats receiving daily oral lead acetate of 30 ppm, 100 ppm, and 300 ppm in dose, respectively. The experiment will be conducted in 30 days. The rats are then dissected, and the weight of ovaries are measured. The expression of caspase-3 is assessed using immunohistochemistry, while the diameter of tertiary follicles and the amount of follicle atresia are both observed using Hematoxylin-Eosin stain. Results: Oral administration of lead acetate significantly decreased the weight of ovaries. Oral exposure of lead enhances the expression of caspase-3 in antral granulosa cells of all experiment groups, especially in the 300 ppm group. It significantly shrinks tertiary follicles' diameter in rats' ovaries to 100 ppm and 300 ppm groups. It also increases the amount of follicle atresia in the 300 ppm group. Conclusion: Oral exposure of lead enhances the expression of caspase 3 in antral granulosa cells at 300 ppm, shrinks the diameter of tertiary follicles at 100 ppm and 300 ppm doses, and increases the amount of follicle atresia at 300 ppm dose.

Matrix Metalloproteinases (MMPs) and Inhibitors of MMPs in the Avian Reproductive System: An Overview

Many matrix metalloproteinases (MMPs) are produced in the mammalian reproductive system and participate in the regulation of its functions. In birds, the limited information available thus far indicates that MMPs are significant regulators of avian ovarian and oviductal functions, too. Some MMPs and inhibitors of MMPs are present in the hen reproductive tissues and their abundances and/or activities change according to the physiological state. The intraovarian role of MMPs likely includes the remodeling of the extracellular matrix (ECM) during folliculogenesis, follicle atresia, and postovulatory regression. In the oviduct, MMPs are also involved in ECM turnover during oviduct development and regression. This study provides a review of the current knowledge on the presence, activity, and regulation of MMPs in the female reproductive system of birds.

Ovarian follicles are resistant to monocyte perturbations—implications for ovarian health with immune disruption

Monocytes and macrophages are the most abundant immune cell populations in the adult ovary, with well-known roles in ovulation and corpus luteum formation and regression. They are activated and proliferate in response to immune challenge and are suppressed by anti-inflammatory treatments. It is also likely they have a functional role in the healthy ovary in supporting the maturing follicle from the primordial through to the later stages, however this role has been unexplored until now. Here we utilised a Cx3cr1-Dtr transgenic Wistar rat model that allows a conditional depletion of circulating monocytes, to investigate their role in ovarian follicle health. Our findings show that circulating monocyte depletion leads to a significant depletion of ovarian monocytes and monocyte-derived macrophages. Depletion of monocytes was associated with a transient reduction in circulating anti-Müllerian hormone (AMH) at 5 days post-depletion. However, the 50–60% ovarian monocyte/macrophage depletion had no effect on ovarian follicle numbers, follicle atresia or apoptosis, within 5 to 21 days post-depletion. These data reveal that the healthy adult ovary is remarkably resistant to perturbations of circulating and ovarian monocytes despite acute changes in AMH. These data suggest that short-term anti-inflammatory therapies that transiently impact on circulating monocytes are unlikely to disrupt ovarian follicle health, findings that have significant implications for fertility planning relative to the experience of an immune challenge or immunosuppression.

New Anti-Müllerian Hormone Target Genes Involved in Granulosa Cell Survival in Women With Polycystic Ovary Syndrome

Purpose A protective effect of anti-Müllerian hormone (AMH) on follicle atresia was recently demonstrated using long-term treatments, but this effect has never been supported by mechanistic studies. This work aimed to gain an insight into the mechanism of action of AMH on follicle atresia and on how this could account for the increased follicle pool observed in women with polycystic ovary syndrome (PCOS). Methods In vivo and in vitro experiments were performed to study the effects of AMH on follicle atresia and on the proliferation and apoptosis of granulosa cells (GCs). RNA-sequencing was carried out to identify new AMH target genes in GCs. The expression of some of these genes in GCs from control and PCOS women was compared using microfluidic real time quantitative RT-PCR. Results A short-term AMH treatment prevented follicle atresia in prepubertal mice. Consistent with this result, AMH inhibited apoptosis and promoted proliferation of different models of GCs. Moreover, integrative biology analyses of 965 AMH target genes identified in 1 of these GC models, confirmed that AMH had initiated a gene expression program favoring cell survival and proliferation. Finally, on 43 genes selected among the most up- and down-regulated AMH targets, 8 were up-regulated in GCs isolated from PCOS women, of which 5 are involved in cell survival. Main conclusions Our results provide for the first time cellular and molecular evidence that AMH protects follicles from atresia by controlling GC survival and suggest that AMH could participate in the increased follicle pool of PCOS patients.

Characteristics of Circular RNA Expression Profiles of Porcine Granulosa Cells in Healthy and Atretic Antral Follicles

Circular RNAs (circRNAs) are thought to play essential roles in multiple biological processes, including apoptosis, an important process in antral follicle atresia. We aimed to investigate the potential involvement of circRNAs in granulosa cell apoptosis and thus antral follicle atresia. CircRNA expression profiles were generated from porcine granulosa cells isolated from healthy antral (HA) and atretic antral (AA) follicles. Over 9632 circRNAs were identified, of which 62 circRNAs were differentially expressed (DE-circRNAs). Back-splicing, RNase R resistance, and stability of DE-circRNAs were validated, and miRNA binding sites and related target genes were predicted. Two exonic circRNAs with low false discovery rate (FDR) high fold change, miRNA binding sites, and relevant biological functions—circ_CBFA2T2 and circ_KIF16B—were selected for further characterization. qRT-PCR and linear regression analysis confirmed expression and correlation of the targeted genes—the antioxidant gene GCLC (potential target of circ_CBFA2T2) and the apoptotic gene TP53 (potential target of circ_KIF16B). Increased mRNA content of TP53 in granulosa cells of AA follicles was further confirmed by strong immunostaining of both p53 and its downstream target pleckstrin homology like domain family a member 3 (PHLDA3) in AA follicles compared to negligible staining in granulosa cells of HA follicles. Therefore, we concluded that aberrantly expressed circRNAs presumably play a potential role in antral follicular atresia.

MIR-361-5P Mediates TGF-β Signalling to Promote Granulosa Cell Apoptosis Through Vegfa During Porcine Follicle Atresia

Background: Follicular atresia is an inevitable degenerative process of ovarian follicles in mammals. The molecular events involved in atresia, particularly in granulosa cell apoptosis, have always attracted researchers’ attention. It is known that VEGFA isdownregulated during follicular atresia in pig ovaries and serves as an apoptosis inhibitor of granulosa cells. Besides, the TGF-β signalling has been considered as a central trigger in granulosa cell apoptosis. However, the link between TGF-β signalling and VEGFA is missing.Results:We proved that miR-361-5p significantly up-regulated during the atresia process andenhances GC apoptosis by direct targeting to VEGFA 3’UTR. In addition,we revealed that miR-361-5p coding geneMIR361was significant downregulation bySMAD4, the central intracellular mediator of TGF-β signalling,through promoter binding.Conclusions: Our findingsenriched the knowledge of VEGFA post-transcriptional regulation and completed the TGF-β/miR-361-5p/VEGFAregulatory networkin ovarian granulosa cell apoptosis. It offereduseful references for follicular atresia and ovarian physiological function studies.

Effect of follicle size and atresia grade on mitochondrial membrane potential and steroidogenic acute regulatory protein expression in bovine granulosa cells

SummaryDuring follicular development, granulosa cells undergo functional and structural changes affecting their steroidogenic activity. Oestrogen synthesis mainly occurs in the endoplasmic reticulum and relies on aromatase activity to convert androgens that arise from theca cells. In the present study, indicators of mitochondria-related steroidogenic capacity, as steroidogenic acute regulatory (StAR) protein expression and mitochondrial membrane potential (MMP), have been evaluated in bovine granulosa cells (GCs) and related to follicle growth and atresia. Atresia was estimated by morphological examination of follicle walls and cumulus–oocyte complexes (COC) and assessed by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay for apoptosis detection. Bovine ovarian follicles were macroscopically classified according to their atresia grade and grouped into small, medium or large follicles. After follicle opening, the COCs were morphologically classified for follicle atresia and the GCs were collected. Granulosa cells were fixed for immunofluorescence (IF) and TUNEL assay, frozen for western blotting (WB) or freshly maintained for MMP analyses. StAR protein expression was assessed using both IF and WB analyses. The follicle atresia grade could be efficiently discriminated based on either follicle wall or COC morphological evaluations. Granulosa cells collected from small non-atretic follicles showed a higher (P <0.01) MMP and WB-based StAR protein expression than small atretic follicles. For IF analysis, StAR protein expression in large atretic follicles was higher (P <0.05) than that in large non-atretic follicles. These results suggest a role played by mitochondria in GC steroidogenic activity, which declines in healthy follicles along with their growth. In large follicles, steroidogenic activity increases with atresia and is possibly associated with progesterone production.