This study aimed to: (i) characterize cultured granulosa cells (GCs) from different follicle sizes morphologically and molecularly; and (ii) select a suitable model according to follicular size that maintained GC function during culture. Buffalo ovaries were collected from a slaughterhouse and follicles were classified morphologically into: first group ≤ 4 mm, second group 5–8 mm, third group 9–15 mm and fourth group 16–20 mm diameter. GC pellets were divided into two portions. The first portion served as the control fresh pellet, and the secondwas used for 1 week for GC culture. Total RNA was isolated, and qRT-PCR was performed to test for follicle-stimulating hormone receptor (FSHR), cytochrome P450 19 (CYP19), luteinizing hormone/choriogonadotropin receptor (LHCGR), proliferating cell nuclear antigen (PCNA), apoptosis-related cysteine peptidase (CASP3), anti-Müllerian hormone (AMH), and phospholipase A2 group III (PLA2G3) mRNAs. Estradiol (E2) and progesterone (P4) levels in the culture supernatant and in follicular fluids were measured using enzyme-linked immunosorbent assay (ELISA). Basic DMEM-F12 medium maintained the morphological appearance of cultured GCs. The relative abundance of FSHR, CYP19, and LHCGR mRNAs was 0.001 ≤ P ≤ 0.01 and decreased at the end of culture compared with the fresh pellet. There was a fine balance between expression patterns of the proliferation marker gene (PCNA) and the proapoptotic marker gene (CASP3). AMH mRNA was significantly increased (P < 0.001) in cultured GCs from small follicles, while cultured GCs from other three categories (5–8 mm, 9–15 mm and 16–20 mm) showed a clear reduction (P < 0.001). Interestingly, the relative abundance of PLA2G3 mRNA was significantly (P < 0.001) increased in all cultured GCs. E2 and P4 concentrations were significantly (P < 0.001) decreased in all cultured groups. Primary cultured GCs from small follicles could be a good model for better understanding follicular development in Egyptian buffaloes.

We sought to examine whether rat maternal food restriction (MFR) affects the expression of steroidogenesis-related genes Cyp19, Cyp17a1, Insl3 and Gdf-9 in the ovaries of offspring from the first (FRG1) and second (FRG2) generations at pre-pubertal age (week 4) and during adulthood (week 8). At week 4, MFR significantly increased the expression of RNAs for all analyzed genes in both FRG1 and FRG2 females, which may indicate that MFR affects the onset of the reproductive lifespan, by inducing early pubertal onset. At week 8, the Cyp19 gene was still upregulated in MRF-subjected animals (Cyp19: P=0.0049 and P=0.0508 in FRG1 and FRG2, respectively), but MFR induced a significant decrease in Cyp17 and Gdf-9 gene expression in the offspring of both FRG1 and FRG2 females when compared with the controls (Cyp17: P=0.0018 and P=0.0016, respectively; Gdf-9: P=0.0047 and P=0.0023, respectively). This suggests that females at week 8, which should normally be in their optimal reproductive capacity, experience premature ovarian aging. At week 4, the activation of Cyp19 and Cyp17 was higher in the FRG1 ovaries than in the FRG2 ovaries, whereas the extent of Insl3 and Gdf-9 activation was lower in the FRG1 ovaries. This may indicate that FRG2 females were more vulnerable to MFR than their mothers (FRG1) and grandmothers, which is consistent with the ‘predictive adaptive response’ hypothesis. Our findings reveal that MFR may induce intergenerational ovarian changes as an adaptive response to ensure reproductive success before death.

The purpose of this study was to evaluate the effects of FSH and PI3K on the nuclear maturation, viability, steroidogenesis and embryo development of bovine cumulus–oocyte complexes (COCs). Oocyte maturation was achieved with MIV B, MIV B+100 µM LY294002, MIV B+10 ng/mL follicle stimulating hormone (FSH), or MIV B+10 ng/mL FSH+100 µM LY294002 treatments for 22–24 h. After the cultured COCs were denuded, oocytes were separated into those that extruded polar bodies (mature) and those that did not, and real-time polymerase chain reaction (PCR) for BAX, BCL2, LHR, FSHR, CYP11A1, CYP19A1 and HSD17B1 genes was performed. The culture medium was collected to determine the levels of 17β-estradiol (E2) and progesterone (P4). The trypan blue test was used to study COC viability, and embryo development was evaluated. FSH increased nuclear maturation and PI3K blocked the maturation but did not influence oocyte viability. BAX and BCL2 expression levels in the cumulus cells were only affected by FSH, and the BAX levels decreased after treatment with LY294002. FSH increased the levels of E2 and P4, however inhibition of PI3K decreased E2 levels. MIV B enhanced levels of LHR, FSHR, CYP11A1, CYP19A1 and HSD17B1, whereas LY294002 inhibited the expression levels of all genes. MIV B+FSH decreased the expression levels of all genes except CYP11A1. LY294002 did not demonstrate any effects in the presence of FSH. Embryo development was significantly decreased when the MIV B+FSH medium was used. In conclusion, FSH controls the steroidogenesis, viability and gene expression in COCs. PI3K plays essential roles in nuclear maturation, steroidogenesis and embryo development.

We aimed in this study to assess whether serum-decreased bovine cumulus–oocyte complex (COC) steroidogenesis during in vitro maturation (IVM) is caused by deficient androgen milieu. For this approach, bovine COCs were cultured in serum-supplemented IVM medium in the presence or absence of 1 μM androstenedione. After 24 h of culture, medium was collected and analyzed for its content of estradiol-17β (E2) and progesterone (P4). Medium E2 content markedly increased after incubation of COCs with androstenedione (17.52 ± 1.86 ng/ml to the androgen group; 0.32 ± 0.05 ng/ml to the non-androgen group). No significant difference in the P4 content was detected despite the presence of androstenedione (21.83 ± 1.61 ng/ml to the androgen group; 21.73 ± 1.67 ng/ml to the non-androgen group). Our data provide compelling evidence that bovine COCs steroidogenesis remains functional during culture in serum-supplemented medium and suggest that serum-induced decreased COCs estradiol secretion is caused by deficiency of an aromatizable androgen source.