Marwa S. Faheem

Atresia is a process in ovarian follicles that is regulated by hormone-induced apoptosis. During atresia, granulosa cell (GC) apoptosis is a key mechanism orchestrated through diverse signaling pathways. Cocaine- and amphetamine-regulated transcript (CART) signaling within ovarian GCs has been demonstrated to play a key role in the regulation of follicular atresia in cattle, pigs, and sheep. The present work aimed to investigate the potential local regulatory role of CART in GC apoptosis-induced follicular atresia in buffalo, focusing on the modulation of the AKT/GSK3β/β-catenin signaling pathways, which are the intracellular signaling pathways involved in cell viability. Our findings revealed increased expression of CARTPT and BAX and decreased levels of AKT, β-catenin, and CYP19A1 genes in atretic follicles compared to healthy follicles. Subsequently, CART treatment in the presence of FSH inhibited the FSH-induced increase in GC viability by reducing estradiol production and increasing apoptosis. This change was accompanied by an increase in the gene expression levels of both CARTPT and BAX. At the protein level, treatment with CART in the presence of FSH negatively affected the activity of AKT, β-catenin, and LEF1, while the activity of GSK3β was enhanced. In conclusion, our study shows how CART negatively influences buffalo GC viability, underlying the modulation of the AKT/GSK3β/β-catenin pathway and promoting apoptosis—a key factor in follicular atresia.

The impact of antioxidant supplementation of cysteamine during in vitro culture of buffalo granulosa cells (GCs) was evaluated in relation to their viability and enzymatic activities under hydrogen peroxide (H2O2)-induced oxidative stress. Mural GCs were harvested, cultured for 24 h of confluence at 37.5ºC and then exposed to 500 µM H2O2 for 30 min followed by cysteamine supplementation (0, 100, 200, 300 and 400 µM) for further 24 h of culture and their counterpart-control groups were supplemented with cysteamine without H2O2 pre-treatment. Granulosa cell viability was assessed using neutral red assay, while the culture conditioned media were subjected to enzymatic analyses. Our results revealed that GCs viability rate was decreased by H2O2 pre-treatment; being 63.0±14.0, 61.8±14.1, and 61.8±10.3 % for 100, 300, and 400 µM of cysteamine, respectively, except increasing viability for 200µM cysteamine (80.0±10.0 %) as compared to control (65.2±4.0). However, the viability was insignificantly enhanced by cysteamine levels under normal physiological conditions. For enzymatic analyses, comparable GSH-Px activity was monitored for all groups of the study. Catalase activity was consistent in 100 and 200 µM of cysteamine under H2O2 or normal physiological condition. While a significant reduction was observed in 300 µM of cysteamine under H2O2 pre-treatment, there were no significant differences between all groups of the study for malondialdehyde (MDA) levels. In conclusion, H2O2 negatively affected GCs viability; however, 200 µM of cysteamine ameliorated cells viability under oxidative stressful condition. The antioxidant enzymes GSH-Px/CAT acted in synergistic dynamics against oxidative stress.

The steroidogenesis capacity and adaptive response of follicular granulosa cells (GCs) to heat stress were assessed together with the underlying regulating molecular mechanisms in Egyptian buffalo. In vitro cultured GCs were exposed to heat stress treatments at 39.5, 40.5, or 41.5 °C for the final 24 h of the culture period (7 days), while the control group was kept under normal conditions (37 °C). Comparable viability was observed between the control and heat-treated GCs at 39.5 and 40.5 °C. A higher release of E2, P4 and IGF-1 was observed in the 40.5 °C group compared with the 39.5 or 41.5 °C groups. The total antioxidant capacity was higher in response to heat stress at 39.5 °C. At 40.5 °C, a significant upregulation pattern was found in the expression of the stress resistance transcripts (SOD2 and NFE2L2) and of CPT2. The relative abundance of ATP5F1A was significantly downregulated for all heat-treated groups compared to the control, while TNFα was downregulated in GCs at 39.5 °C. Expression analyses of stress-related miRNAs (miR-1246, miR-181a and miR-27b) exhibited a significant downregulation in the 40.5 °C group compared to the control, whereas miR-708 was upregulated in the 39.5 and 40.5 °C groups. In conclusion, buffalo GCs exhibited different adaptive responses, to the different heat stress conditions. The integration mechanism between the molecular and secretory actions of the GCs cultured at 40.5 °C might provide possible insights into the biological mechanism through which buffalo GCs react to heat stress.

Gene expression is required for understanding bovine oocytes meiotic maturation as well as the potential of embryonic development. In the present study a s

Summary: Immature bovine oocytes were vitrified using the cryotop method and their post-warming survivability and capability to undergoin vitromaturation, fertilization and subsequent embryonic development were evaluated. In addition throughout the embryonic 2-cell, 4-cell, morula and blastocyst stages, the expression of four developmentally important genes (Cx43, CDH1, DNMT1andHSPA14) was analysed using the real-time polymerase chain reaction (PCR). Immature oocytes (n= 550) were randomly assigned to non-vitrified (fresh) or cryotop vitrification groups using ethylene glycol (EG) with 1,2 propanediol (PROH) or dimethylsulphoxide (DMSO). After warming, oocytes survivability, embryo cleavage and embryonic developmental rates were not statistically different between the two cryoprotectants groups. However, the DMSO group had a lower (P< 0.05) oocyte maturation rate compared with the fresh and PROH groups. For morula and blastocyst rates, the DMSO group achieved a lower (P< 0.05) morula rate compared with the fresh group, while at the blastocyst stage, there were no differences between fresh and both cryoprotectants groups. For molecular analysis, at the 4-cell stage, most studied genes showed an inconsistent pattern of expression either from the PROH or DMSO groups. Noteworthily, these differences were limited at the morula and blastocyst stages. In conclusion, the cryotop method is sufficient for vitrification of immature bovine oocytes, both for embryonic developmental competence and at the molecular level. Moreover, PROH showed some advantage over DMSO as a cryoprotectant.