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Isorhamnetin is a natural flavonoid widely distributed in fruits and vegetables. However, the roles of isorhamnetin involved in steroidogenesis, proliferation, and apoptosis in ovarian granulosa cells (GCs) are poorly understood. We found that isorhamnetin promoted the secretion of estrogen and inhibited the secretion of progesterone and testosterone by modulating steroidogenesis-associated proteins and mRNA such as CYP19A1, StAR, and 3β-HSD in ovarian GCs. Mechanistically, isorhamnetin stimulated the expression of the proliferating cell nuclear antigen and C-myc and promoted the proliferation of GCs via the PI3K/Akt signaling pathway. Furthermore, isorhamnetin increased the protein expression of CyclinB, CyclinD, CyclinE, and CyclinA, thereby raising the ratio of S-phase cells in response to GC proliferation. Changes in the expression of apoptosis-associated proteins (Bcl2, Bax, and cytochrome c) and intracellular reactive oxygen species levels showed that isorhamnetin inhibited GC apoptosis. Collectively, these findings indicate that isorhamnetin regulates steroidogenesis through the activation of PI3K/Akt, which promotes proliferation, inhibits apoptosis, and alleviates oxidative stress.

Background: Zearalenone (ZEA) is widely derived from moldy cereal grain, which has adverse effects on animal reproduction. In particular, pigs are more sensitive to ZEA-induced toxicity than other animals. Isorhamnetin, a flavonoid has extensive of pharmacological activities. However, little is known about the effects of isorhamnetin on reproduction. Thus, it would be interesting to clarify the effect and the underlying molecular mechanism of isorhamnetin involvement in ZEA-induced cytotoxicity in porcine granulosa cells.Results: Our findings showed that isorhamnetin suppressed ZEA-induced apoptosis by regulating Bcl-2 and Bax protein changes. Changes in intracellular Ca2+ levels and CHOP, ATF6, GRP78 indicated that isorhamnetin rescued ZEA-induced endoplasmic reticulum stress (ERS). Furthermore, isorhamnetin prevented ZEA-induced reactive oxygen species (ROS) via P38 signaling pathway. Mechanistically, isorhamnetin stimulated the expression of PCNA and CyclinD, thereby raising the ratio of S phases cells in response to ZEA-induced apoptosis via PI3K/Akt signaling pathway. Isorhamnetin also recovered ZEA-induced steroidogenesis disorder by regulating steroidogenic enzyme gene and proteins (FSH-R, CYP19A1). Conclusions: Collectively, these findings show that isorhamnetin protects granulosa cells from ZEA-induced damage via the PI3K/Akt signaling pathway, which promotes proliferation, alleviates steroidogenesis disorder, ERS and oxidative stress.

Estrogen and its analogues are ubiquitous in agricultural environments, with large biological functions of oocyte development. Gap junction intercellular communications (GJICs) are the structural basis in cumulus-oocyte complexes (COCs) and regulate oocyte maturation and developmental material transport through a number of pathways. This study mainly determines the effect and potential mechanism of estrogen (17β-estradiol) in regulating GJICs in porcine COCs. In our study, 17β-estradiol increased porcine nuclear maturation in a time-dependent manner. The analysis revealed that 17β-estradiol upregulated the autophagy in COCs during in vitro maturation. In contrast with the control, 17β-estradiol decreased GJICs in a time-dependent manner between cumulus cells and oocytes, while it was consistent with the control group at 24 h. Carbenoxolone (CBX) blocks GJICs as a negative control group used in our system. Autophagy inhibitor autophinib decreased oocyte maturation, and the reduced nuclear maturation treated with autophinib was abolished by 17β-estradiol. Besides, the upregulation effect of autophinib on GJICs and transzonal projections (TZPs) was decreased by 17β-estradiol. 17β-Estradiol could reduce serine 368 phosphorylation of connexin 43 (Cx43) protein by autophinib in porcine COCs. These results were dependent upon the MEK/ERK signaling pathway. Furthermore, 17β-estradiol-induced GJICs and Cx43 phosphorylation were inhibited by autophinib or the MEK/ERK pathway inhibitors (Trametinib and FR 180204), indicating that 17β-estradiol regulated GJICs through the MEK/ERK signaling pathway. In conclusion, 17β-estradiol improves the autophagy-mediated nuclear maturation with downregulating GJICs and TZPs in porcine COCs. Such an effect occurs by phosphorylation of Cx43, which was regulated via the MEK/ERK signaling pathway.

Granulosa cells play important roles in the regulation of ovarian functions. Phospholipase C is crucial in several signalling pathways and could participate in the molecular mechanisms of cell proliferation, differentiation and ageing. The objective of this study was to identify the effects of phospholipase C on the steroidogenesis of oestradiol and progesterone in porcine granulosa cells cultured in vitro. Inhibitor U73122 or activator m-3M3FBS of phospholipase C was added to the in vitro medium of porcine granulosa cells, respectively. The secretion of oestradiol decreased after 2 hr, 8 hr, 12 hr, 24 hr and 48 hr of treatment with 500 nM U73122 (p < .05) and decreased after 2 hr of treatment in the 500 nM m-3M3FBS addition group (p < .05). The secretion of progesterone increased after 4 hr of treatment with 500 nM U73122 (p < .05) and increased after 2 hr and 8 hr of treatment in the 500 nM m-3M3FBS addition group (p < .05). The ratio of oestradiol to progesterone decreased at each time point, except 8 hr after the addition of 500 nM U73122 (p < .05). The ratio of oestradiol to progesterone decreased after 2 hr (p < .05) of treatment with 500 nM m-3M3FBS. In genes that regulate the synthesis of oestradiol or progesterone, the mRNA expression of CYP11A1 was markedly increased (p < .05), and the mRNA expression of other genes did not change significantly in the U73122 treatment group, while the addition of m-3M3FBS did not change those genes significantly despite the contrary trend. Our results demonstrated that phospholipase C can be a potential target to stimulate the secretion of oestradiol and suppress progesterone secretion in porcine granulosa cells cultured in vitro, which shed light on a novel biological function of phospholipase C in porcine granulosa cells.

Steroid hormones are required for normal reproductive function of female. The aim of this study was to investigate the role of Raf-ERK1/2 on steroid hormone synthesis in bovine ovarian granulosa cells. Immunohistochemistry assay showed that both B-Raf and C-Raf were expressed in granulosa cells, theca cells and Sertoli cells. The protein expression of Raf or ERK1/2 was clearly decreased by Raf inhibitor GSK2118436 or ERK1/2 inhibitor SCH772984, respectively (p < 0.05). In addition, western blotting was performed for investigating the crosstalk between Raf and ERK1/2, the data showed that Raf positively regulated ERK1/2, whereas ERK1/2 had a negative feedback effect on Raf. The biosynthesis of oestradiol or testosterone was significantly decreased by treatment with GSK2118436 or SCH772984 (p < 0.05). Conversely, the progesterone biosynthesis was clearly increased by treatment with those inhibitors (p < 0.05). Furthermore, the mRNA expression of STAR, aromatase and CYP17 was blocked by Raf-ERK1/2 signalling inhibition, which oppositely induced the mRNA expression of CYP11. Together, these findings suggested that Raf-ERK1/2 signalling pathways mediate steroid hormone synthesis via affecting the expression of steroidogenic enzymes.

Phospholipase C (PLC) can participate in cell proliferation, differentiation and aging. However, whether it has a function in apoptosis in porcine primary granulosa cells is largely uncertain. The objective of this study was to examine the effects of PLC on apoptosis of porcine primary granulosa cells cultured in vitro. The mRNA expression of BAK, BAX and CASP3, were upregulated in the cells treated with U73122 (the PLC inhibitor). The abundance of BCL2 mRNA, was upregulated, while BAX and CASP3 mRNA expression was decreased after treatment with m-3M3FBS (the PLC activator). Both the early and late apoptosis rate were maximized with 0.5 μM U73122 for 4 h. The rate of early apoptosis was the highest at 4 h and the rate of late apoptosis was the highest at 12 h in the m-3M3FBS group. The protein abundance of PLCβ1, protein kinase C β (PKCβ), calmodulin-dependent protein kinaseII α (CAMKIIα) and calcineurinA (CalnA) were decreased by U73122, and CAMKIIα protein abundance was increased by m-3M3FBS. The mRNA expression of several downstream genes (CDC42, NFATc1, and NFκB) was upregulated by PLC. Our results demonstrated that apoptosis can be inhibited by altering PLC signaling in porcine primary granulosa cells cultured in vitro, and several calcium−sensitive targets and several downstream genes might take part in the processes.

SIRT2, a member of the sirtuin family, has been recently shown to exert important effects on mitosis and/or metabolism. However, its roles in oocyte maturation have not been fully clarified. In this study, SIRT2, located in the cytoplasm and nucleus, was found in abundance in the meiotic stage, and its expression gradually decreased until the blastocyst stage. Treatment with SIRT2 inhibitors resulted in the prevention of oocyte maturation and the formation of poor-quality oocytes. By performing confocal scanning and quantitative analysis, the results showed that SIRT2 inhibition induced prominent defects in spindle/chromosome morphology, and led to the hyperacetylation of &alpha, tubulin and H4K16. In particular, SIRT2 inhibition impeded cytoplasmic maturation by disturbing the normal distribution of cortical granules, endoplasmic reticulum, and mitochondria during oocyte meiosis. Meanwhile, exposure to SirReal2 led to elevated intracellular reactive oxygen species (ROS) accumulation, low ATP production, and reduced mitochondrial membrane potential in oocytes. Further analysis revealed that SIRT2 inhibition modulated mitochondrial biogenesis and dynamics via the downregulation of TFAM and Mfn2, and the upregulation of DRP1. Mechanistically, SIRT2 inhibition blocked the nuclear translocation of FoxO3a by increasing FoxO3a acetylation, thereby downregulating the expression of FoxO3a-dependent antioxidant genes SOD2 and Cat. These results provide insights into the potential mechanisms by which SIRT2-dependent deacetylation activity exerts its effects on oocyte quality.

Follicle stimulating hormone (FSH), composed of FSHα and FSHβ subunits, is essential for female follicle development and male spermatogenesis. The recombinant human FSH (rhFSH) products on the market are mainly generated from mammalian cells and are expensive. Large animal mammary gland bioreactors are urgently needed to produce large amounts of rhFSH. However, there are currently no effective methods to prepare rhFSH by large animals mainly due to the fact that excessive accumulation of FSH might cause many adverse effects in animals. We herein report the development and characterization of functional self-assembled rhFSH produced in goat mammary epithelial cells (GMECs). FSHα and FSHβ stably expressed in Chinese hamster ovary (CHO) cell lines were secreted into culture medium and well glycosylated. Importantly, FSHα and FSHβ expressed apart were able to assemble into functional FSH. We next inserted human FSHα or FSHβ gene separately into goat β-Lactoglobulin locus in GMECs by CRISPR/Cas9. Inactive FSHα and FSHβ subunits expressed from GMECs assembled into rhFSH as analyzed by His-tag pull down assay. Functional assessment of rhFSH by cAMP induction assay, mouse ovulation induction and rat ovarian weight gain experiments showed that the bioactivity of self-assembled rhFSH expressed by GMECs was comparable to that of Gonal-F both in vitro and in vivo. Our study demonstrated that FSHα and FSHβ can be separately expressed and assembled into functional rhFSH, and provided the basis for future preparing FSH by goat mammary gland bioreactor with less health problems on the producing animals.

SIRT2 has been shown to possess NAD+-dependent deacetylase and desuccinylase enzymatic activities, it also regulates metabolism homeostasis in mammals. Previous data has suggested that resveratrol, a potential activator of Sirtuins, played a stimulation role in steroidogenesis. Unfortunately, to date, the physiological roles of SIRT2 in ovarian granular cells (GCs) are largely unknown. Here, we studied the function and molecular mechanisms of SIRT2 on steroid hormone synthesis in GCs from Qinchuan cattle. Immunohistochemistry and western blotting showed that SIRT2 was expressed not only in GCs and cumulus cells, but also in oocytes and theca cells. We found that the secretion of progesterone was induced, whereas that of estrogen and testosterone secretion was suppressed by treatment with the SIRT2 inhibitor (Thiomyristoyl or SirReal2) or siRNA. Additionally, the PPARs/LXRα signaling pathways were suppressed by SIRT2 siRNA or inhibitors. The mRNA expression of CYP17, aromatase and StAR was suppressed, but the abundance of CYP11A1 mRNA was induced by SIRT2 inhibition. Furthermore, the PPARα agonist or PPARγ antagonist could mimic the effects of SIRT2 inhibition on hormones levels and gene expression associated with steroid hormone biosynthesis. In turn, those effects were abolished by the LXRα agonist (LXR-623). Together, these data support the hypothesis that SIRT2 regulates steroid hormone synthesis via the PPARs/LXRα pathways in GCs.

Wingless-int (WNT) signaling pathway is vital to modulate life processes, including cell fate determination, cell differentiation, cell proliferation, cell apoptosis and embryogenesis. To demonstrate the uncertain effect of the canonical WNT signaling pathway on oocyte maturation, immature porcine oocytes were collected and cultured in vitro with the WNT/β-catenin inhibitor FH535. The concentrations of FH535 were selected as 0.00, 0.01, 0.10, 1.00 and 10.00 μmol/L. The results showed that the optimum concentration of FH535 on oocyte maturation was 1.00 μmol/L. In this concentration, the proportion of MII oocytes increased (P 0.05). The rate of cleavage was the same with the control (P 0.05), while the rate of blastocysts in the 1.00 μmol/L FH535 treated group was higher than that of control (P 0.01). Additionally, the average number of nuclei in blastocysts raised significantly (P 0.05). The inhibition of WNT could regulate expression of maturation-related genes, including Cdc-2, Bmp-15, Gdf-9 and Mos. In the 1.00 μmol/L FH535 treated group, the messenger RNA level of β-catenin showed no significant change compared to the control (P 0.05), but the protein abundance was decreased (P 0.05). This study revealed that the inhibition of FH535 on the WNT signaling pathway could promote the maturation of porcine oocytes and altered gene expressions in vitro.