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This study was aimed to address melatonin receptor expression, mRNA level of hypothalamus and hypophysis hormone receptors (GnRHR, FSHR, and LHR), steroidogenesis, cell cycle, apoptosis, and their regulatory factors after addition of melatonin for 24 hr in cultured buffalo granulosa cells (GCs). The results revealed that direct addition of different concentrations of melatonin (100 pM, 1 nM, and 100 nM) resulted in significant upregulation (p < 0.05) of mRNA level of melatonin receptor 1a (MT1) without affecting melatonin receptor 1b (MT2). Melatonin treatment significantly downregulated (p < 0.05) mRNA level of FSH and GnRH receptors, whereas 100 nM dose of melatonin significantly increased mRNA level of LH receptor. Treatment with 100 nM of melatonin significantly decreased the basal progesterone production with significant decrease (p < 0.05) in mRNA levels of StAR and p450ssc, and lower mRNA level of genes (Insig1, Lipe, and Scrab1) that affect cholesterol availability. Melatonin supplementation suppressed apoptosis (100 nM, p < 0.05) and enhanced G2/M phase (1 nM, 100 nM, p < 0.05) of cell cycle progression which was further corroborated by decrease in protein expression of caspase-3, p21, and p27 and increase in bcl2. Our results demonstrate that melatonin regulates gonadotrophin receptors and ovarian steroidogenesis through MT1. Furthermore, the notion of its incorporation in apoptosis and proliferation of buffalo GCs extends its role in buffalo ovaries., (© 2019 Japanese Society of Animal Science.)

The objective of the present study was to examine if administration of oestradiol benzoate (OEB) after removal of control internal drug releasing device (CIDR) could enhance oestrous intensity and pregnancy rate in anovular nulliparous and multiparous Nili-Ravi buffalo. For this, a total of 298 nulliparous (n=91) and multiparous (n=207) buffaloes received a CIDR on Day 0, and were administered PGF2α on Day 6 followed by removal of CIDR on Day 7. At Day 8, OEB was administered in approximately half of nulliparous (n=45/91) and multiparous (n=100/207) buffalo. All animals were fixed time inseminated 48 and 60h after CIDR removal, respectively. The results showed that administration of OEB but not the parity, improved oestrous intensity (3.15±0.05 vs 2.99±0.05; P=0.0026) compared to those not received OEB, respectively. However, OEB did not affect (46.2 vs 44.1; P=0.8) pregnancy per AI (P/AI). In addition, P/AI was greater (50.7 vs 39.6; P=0.036) in multiparous compared to nulliparous buffalo, respectively. The oestrous intensity (P=0.025) and response to OEB (P=0.0002) was greater in buffalo having a greater body condition (>3.0). Though, non significant, timing of ovulation was more synchronous (62.9±1.8 vs 72.4±3.6h; P>0.05) and ovulation rate was greater (91% vs 64%; P>0.05) in buffalo after OEB administration. It is concluded that administration of OEB in conjunction with the CIDR improves oestrous intensity without affecting P/AI in nulliparous and multiparous anovular buffalo., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Follistatin (FST), a local regulator of gonadal functions is a powerful inhibitor of follicle stimulating hormone (FSH) secretion. In the present study, the expression of FST was partially silenced at both transcriptional and translational levels by RNAi-Ready pSIREN-RetroQ-ZsGreen Vector mediated recombinant pshRNA vectors in bovine granulosa cells (bGCs). The results showed that transfection with FST-1 and FST-2 vectors significantly down-regulated mRNA and protein expressions of follistatin by 51% (P = 0.0093) and 72% (P = 0.0078) respectively. After down-regulation of FST in bGCs, cell cycle was arrested at S-phase (9.2 ± 0.6 vs 12.5 ± 0.2, P = 0.0055), and apoptosis was significantly (21.3 ± 2.7 vs 13.9 ± 2.5, P = 0.0051) increased. These findings were further verified by down-regulation of protein level of B-cell leukemia/lymphoma 2 (Bcl2, P = 0.0423), and up-regulation of caspase-3 (P = 0.0362), p21 (P = 0.0067) and mRNA levels of Bcl2-associated X protein (Bax, P = 0.041). Knockdown of FST in bGCs significantly increased activin A concentration in culture medium, while level of estradiol (E2) was suppressed without affecting progesterone production. In addition, mRNA levels of all activin receptor subtypes [activin receptor types I (ACRI) and II (ACRIIA and ACRIIB)] and inhibin α-subunit were augmented (P < 0.05) without altering both inhibin β-subunits. These findings suggest that follistatin may participate in caspase3-dependent apoptosis through Bcl2/Bax gene family in bovine GCs, whereas, activin and its receptors are associated with its regulation. Activin-induced up-regulation of inhibin-α subunit in bGCs seems to be involved in the regulation of steroidogenesis., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Cultured ovarian granulosa cells (GCs) are essential models to study molecular mechanisms of gene regulation during folliculogenesis. CCAAT enhancer binding proteins β (CEBPβ) has been identified in the ovary and is critical for follicular growth, ovulation and luteinization in mice. In the present study, hormonal treatment indicated that luteinizing hormone (LH) and exogenous human chorionic gonadotropins (hCG) significantly increased the expression of CEBPβ in porcine GCs. By RNAi-Ready pSIREN-RetroQ-ZsGreen Vector mediated recombinant pshRNA vectors, CEBPβ gene was successfully knocked down in porcine GCs, confirmed by mRNA and protein level analyzed by real time PCR and western blot, respectively. We further found that knockdown of CEBPβ significantly increased the expression of p-ERK1/2. Furthermore, CEBPβ knockdown arrested the GCs at S phase of cell cycle, but had no effects on cell apoptosis. More importantly, it markedly down regulated the concentration of estradiol (E2) and progesterone (P4) in the culture medium. To uncover the regulatory mechanism of CEBPβ knockdown on cell cycle and steroids synthesis, we found that the mRNA expression of bcl-2 (anti-apoptosis), StAR and Runx2 (steroid hormone synthesis) was up-regulated, while genes related to apoptosis (Caspase-3 and p53), hormonal synthesis (CYP11A1) and cell cycle (cyclinA1, cyclinB1, cyclinD1) were down-regulated, suggesting that knockdown of CEBPβ may inhibit apoptosis, regulate cell cycle and hormone secretions at the transcriptional level in porcine GCs. Furthermore, knockdown of CEBPβ significantly increased the expression of PTGS2 and decreased the expression of IGFBP4, Has2 and PTGFR which are important for folliculogenesis in porcine GCs. In conclusion, this study reveals that CEBPβ is a key regulator of porcine GCs through modulation of cell cycle, apoptosis, steroid synthesis, and other regulators of folliculogenesis., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The current study aimed to define the serum profile of anti-mullerian hormone (AMH), testosterone and estradiol (E2) concentrations in Beetal goat kids from 10 days of age to puberty (175±12.6 days). The study was conducted from July 2018 to January 2019. Blood samples were collected from male kids (n=26) at 10 days interval from 10 days of age to puberty for measurement of AMH, testosterone and E2 concentrations. The body weight of all kids was measured at 10 days of age and then on monthly basis till puberty. The results showed that the mean body weight at 10 d after birth was 7.04±0.86 kg, and significantly increased till the onset of puberty (36.4±4.49 kg). The minimal plasma AMH concentration was seen at 10 d, gradually increased to its peak level (P<0.05) at 40 d and 100 d and then declined steadily till puberty. The serum testosterone level increased non-significantly from day 10 to day 50 and then gradually increased to its peak level at 170 and 180d (P<0.05). The serum E2 concentration was low at 10 days of age (5.76±0.78 pg/ml), increased to its peak level at 60d (74.3±6.26 pg/ml; P<0.05 compared with 10d), then declined till puberty. The serum AMH at early pre-pubertal age (40, 50, 70 and 80d) was positively correlated with pubertal age around 180d (r=0.99, 0.95, 0.99 and 0.94), whereas testosterone concentration was negatively correlated with serum AMH level at 60d, while positively correlated at 150 and 180d. In conclusion, goat kids exhibit a characteristic serum AMH profile along with testosterone and E2 during pre-pubertal life, such that serum AMH at an early pre-pubertal age could be used as a biomarker for predicting initiation of sexual behavior and onset of puberty. [ABSTRACT FROM AUTHOR]