A novel regulatory sex-skewing method that inhibits testicular DPY30 expression to increase female rate of dairy goat offspring.

The demand for goat milk products has increased exponentially with the growth of the global population. The shortage of dairy products will be addressed extraordinarily by manipulating the female rate of goat offspring to expand the goat population and goat milk yield. No studies have reported bioinformatic analyses of X- and Y-bearing sperm of dairy goats, although this will contribute to exploring novel and applied sex-skewing technologies. Regulatory subunit of the histone methyltransferase complex (DPY30) was determined to be the key differentially expressed protein (DEP) among 15 DEPs identified in the present study. The spatiotemporal expression of DPY30 strongly suggested a functional involvement of the protein in spermatogenesis. DPY30 promoted meiosis via upregulating SYCP3, which played a crucial role in mediating sex ratio skewing in goats. Although DPY30 suppressed the self-renewal of spermatogonia stem cells through AKT/PLZF, DPY30 inhibition in the testis did not induce testicular dysgenesis. Based on the biosafety assessment in mice testes, lentivirus-mediated DPY30 knockdown in bucks' testes increased X-bearing sperm proportion and female kids' rate (22.8 percentage points) without affecting sperm quality, pregnancy rate, and kidding rate. This study provides the first evidence of the DEGs in the sexed sperm of dairy goats. DPY30 inhibition in the testes of bucks increased the female kids' rate without influencing reproductive performance. The present study provides evidence for expanding the female dairy goat population to address the concern of dairy product shortage.

Goat milk has high digestibility, high nutritional quality, low allergenicity, and potential nutraceutical properties so the valorization of goat milk into value-added products is becoming increasingly important. However, the goat's milk production was less than 20% of cow's milk. To increase production, we investigated the differentially expressed proteins in the X- and Y-bearing sperm of dairy goat to explore the new sex-skewing method. The results showed that inhibiting the expression of DPY30 in the testes of male goats significantly increased the female kids' rate (22.8 percentage points). As such, no adverse effects on sperm quality, pregnancy rate or kidding rate were observed. The DPY30 silence mediated sex-skewing was achieved by disrupting meiosis via targeting SYCP3. Our results provide new insights into the preliminary mechanisms of sex-skewing in dairy goats, which could also form the basis for the development of novel sex-skewing strategies in livestock.

Goat Milk Improves Glucose Metabolism in Type 2 Diabetic Mice and Protects Pancreatic ß-Cell Functions.

SCOPE: Consuming goat milk is known to benefit high-fat diet-fed and streptozocin (STZ)-induced diabetic rats, but the underlying mechanisms are unknown. This study is conducted to investigate the metabolic effects of a goat milk diet (a form of goat milk powder) on glucose homeostasis and pancreatic conditions in a mouse model of Type 2 diabetes mellitus (T2DM) induced by STZ. METHODS AND RESULTS: T2DM mice are fed with a goat-milk-based diet containing 10.3% w/w goat milk powder for 10 weeks for investigating the in vivo effects; a ß-cell line MIN6 cells are used to test the in vitro effects of digested goat milk (DGM). Goat milk diet improves the deleterious effects of STZ on fasting glucose levels and glucose tolerance, accelerates pancreatic structure recovery, and alters blood metabolites in mice. Based on the significant differences observed in metabolites, the key pathways, metabolite regulatory enzymes, metabolite molecular modules, and biochemical reactions are identified as critical integrated pathways. DGM promotes the cell activity, glucose transportation, and AKT activation in cultured STZ-treated MIN6 cells in vitro. CONCLUSIONS: Goat milk diet improves glucose homeostasis and pancreatic conditions of T2DM mice, in association with improved blood metabolite profiles and activation of pancreatic AKT pathway.

Lactoferrin alleviates spermatogenesis dysfunction caused by bisphenol A and cadmium via ameliorating disordered autophagy, apoptosis and oxidative stress.

Contaminants in food have severely threatened human health, and appropriate antioxidants derived from food could reduce impairment risk. Lactoferrin from milk could control iron concentration in the blood to ameliorate oxidative stress, which is also required for sperm maturation, but the underlying mechanisms remain unclear. The present study used mice with spermatogenetic dysfunction caused by bisphenol A (BPA) and cadmium (Cd) to evaluate the ameliorative effects of lactoferrin and milk (bioactive substances). BPA (50 mg/kg) and Cd (1.6 mg/kg) caused severe damage to testis, including globally decreased germ cell counts, poor sperm quality, disordered apoptosis, oxidative stress, and autophagy; however bioactive substances comprehensively ameliorated spermatogenetic dysfunction via mitigating the increased levels of BAX/BCL2, LC3II/LC3I, and P62. AMPK was involved in autophagic regulation, while ERK1/2 inhibition attenuated the protective effects of lactoferrin, including restimulating apoptosis, oxidative stress, and arrested autophagic flux. Notably, P62 was consistently stimulated with different ERK1/2 inhibitors, which was ubiquitin-dependent. The study provides evidence for the alleviative effects of lactoferrin and milk in mice with spermatogenetic dysfunction through ERK1/2 mediated the ubiquitin-dependent degradation of P62. The involved signals and molecules could be identified as novel therapeutic targets for male infertility, which contributes to expanding LF's interests in research and application.

Bisphenol A exposure causes testicular toxicity by targeting DPY30-mediated post-translational modification of PI3K/AKT signaling in mice.

Bisphenol A (BPA), one of the chemicals with the highest volume of production worldwide, has been demonstrated to cause testicular toxicity via different pathways. However, there is little evidence concerning the mechanism of BPA exposure induced histone modification alterations, especially regarding the effect on the histone H3 lysine 4 (H3K4) epigenetic modification. Our results demonstrated a new epigenetic regulation of BPA exposure on testicular damage using both cell culture and mouse models. With BPA treatment, disordered and shrunken seminiferous tubules and poor sperm quality were observed in vivo, and mouse spermatogonial germ cell proliferation was inhibited in vitro. BPA attenuated PI3K expression inducing phospho-AKT inhibition in vivo and in vitro. DPY30 was the only downregulated subunit in BPA and MEK2206 (AKT inhibitor) treated cells, which contributed to reducing H3K4me3 recruitment at the PIK3CA transcriptional start site (TSS) in BPA treated cells. The toxicity caused by BPA exposure was relieved after the transduction of adenoviruses expressing DPY30 transgenes, which resulted in the stimulation of PI3K/AKT with H3K4me3 enriched at the PI3KCA TSS. DPY30 promoted cell glycolysis via AMPK and proliferation through AKT/P21. DPY30 was mainly located in the round and elongated spermatids for energy accumulation in mature sperm in AD-DPY30-treated mice which showed higher sperm quality. Overall, our results indicated that BPA exposure causes testicular toxicity through a DPY30-mediated H3K4me3 epigenetic modification, which serves to regulate the PI3K/AKT/P21 pathway.

Overexpression of the Tuberous sclerosis complex 2 (TSC2) gene inhibits goat myoblasts proliferation and differentiation in understanding the underlying mechanism of muscle development.

The growth of animal skeletal muscle is mainly determined by the synthesis processes of total proteins in skeletal muscle cells, which has a significant impact on the postnatal growth of young animals. An increasing number of studies are focusing on the functions of Tuberous sclerosis complex 2 (TSC2) during the process of cell protein synthesis and growth. However, it is still unclear the effect of whether and how TSC2 on goat myoblasts proliferation and differentiation. Here, we found that TSC2 gene has opposite expression patterns in proliferation and differentiation of myoblasts. An expression vector containing goat TSC2 cDNA sequences linked with pcDNA3.1 plasmid was constructed. Myoblasts proliferation activity was significantly inhibited and cell cycle transition slowed down after the transfection of pcDNA3.1-TSC2 plasmid into goat primary myoblasts by EdU staining, CCK-8 and flow cytometry. Mechanically, we further confirmed that the overexpression TSC2 was able to down-regulate the mRNA and protein expression of mechanistic target of rapamycin (mTOR), p70 ribosomal S6 kinase 1 (p70S6K) and some cell cycle related genes. In addition, the expression of myogenic genes and myotube formation were attenuated. Collectively, all our results of the experiment demonstrate that TSC2 could regulate myoblasts cells proliferation and differentiation via the activation of the mTOR/p70S6K signaling pathway.

A novel role of SIRT2 in regulating gap junction communications via connexin-43 in bovine cumulus-oocyte complexes.

SIRT2, the predominantly cytosolic sirtuin, plays important role in multiple biological processes, including metabolism, stress response, and aging. However, the function of SIRT2 in gap junction intercellular communications (GJICs) of cumulus-oocyte complexes (COCs) is not yet known. The purpose of the present study was to evaluate the effect and underlining mechanism of SIRT2 on GJICs in COCs. Here, we found that treatment with SIRT2 inhibitors (SirReal2 or TM) inhibited bovine oocyte nuclear maturation. Further analysis revealed that SIRT2 inactivation disturbed the GJICs of COCs during in vitro maturation. Correspondingly, both the Cx43 phosphorylation levels and MEK/MER signaling pathways were induced by SIRT2 inhibition. Importantly, SIRT2-mediated Cx43 phosphorylation was completely abolished by treatment with MEK1/2 inhibitor (Trametinib). Furthermore, treatment with SIRT2 inhibitors resulted in the high levels of MEK1/2 acetylation. Functionally, downregulating the MER/ERK pathways with inhibitors (Trametinib or SCH772984) could attenuate the closure of GJICs caused by SIRT2 inactivation in partly. In addition, inhibition of SIRT2 activity significantly decreased the membrane and zona pellucida localization of Cx43 by upregulating the levels of Cx43 acetylation. Taken together, these results demonstrated a novel role that SIRT2 regulates GJICs via modulating the phosphorylation and deacetylation of Cx43 in COCs.

Phospholipase C inhibits apoptosis of porcine primary granulosa cells cultured in vitro.

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 PLCß1, protein kinase C ß (PKCß), 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 functions in aging, autophagy, and apoptosis in post-maturation bovine oocytes.

AIMS: Sirtuins have been implicated in the aging process, however, the functions of SIRT2 in post-maturation aging of oocytes are not fully understood. The purpose of the present investigation was to assess the roles of SIRT2 in aged oocytes and mechanisms involved. MAIN METHODS: The fresh MII oocytes were aging in vitro, and treated with SIRT2 inhibitor (SirReal2), autophagy activator (Rapamycin), and autophagy inhibitor (3-Ma) for 24 h, respectively. Oocyte activation, cytoplasmic fragmentation, and spindle defects, mitochondrial distribution, ROS levels, ATP production, mitochondrial membrane potential, and early apoptosis were investigated. Western blotting was performed to determine LC3-II accumulation, SQSTM1 degradation, and caspase-3 activity. KEY FINDINGS: SIRT2 expression gradually decreased in a time-dependent manner during oocyte aging. Treatment with SirReal2 significantly increased the rates of oocyte activation, cytoplasmic fragmentation, and spindle defects. In particular, the high ROS levels, abnormal mitochondrial distribution, low ATP production, and lost ΔΨm were observed in SirReal2-exposed oocytes. Further analysis revealed that LC3-II accumulation and SQSTM1 degradation were induced by SIRT2 inhibition. By performing early apoptosis analysis showed that oocyte aging was accompanied with cellular apoptosis, and SIRT2 inhibition increased apoptosis rates of aged oocytes. Importantly, upregulating autophagy with Rapamycin could mimic the effects of SIRT2 inhibition on apoptosis by increasing caspase-3 activation, whereas downregulating autophagy with 3-MA could abolish those effects by blocking caspase-3 activation. SIGNIFICANCE: Our results suggest that SIRT2 inactivation is a key mechanism underlying of cellular aging, and SIRT2 inhibition contributes to autophagy-dependent cellular apoptosis in post-maturation oocytes.

Raf-ERK1/2 signalling pathways mediate steroid hormone synthesis in bovine ovarian 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.

SIRT2 plays a novel role on progesterone, estradiol and testosterone synthesis via PPARs/LXRα pathways in bovine ovarian granular cells.

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.

Combined effect of apigenin and ferulic acid on frozen-thawed boar sperm quality.

The main aim of the present study was to evaluate the cryoprotective effect of apigenin (AP) and ferulic acid (FA) on boar sperm during cryopreservation. AP and FA were both demonstrated to be high-efficiency antioxidants and had not previously been used to protect sperm from cryodamage. As boar sperm is sensitive to oxidative stress, suitable antioxidants are still needed for improving frozen-thawed sperm quality. With this purpose, semen samples coming from five boars were used in this study. Ejaculates of five boars were mixed and split into 16 aliquots, in which different doses of AP and FA were added separately or together. The motility, the plasma membrane integrity, the mitochondrial activity, the acrosomal integrity, the antioxidase activities and the malondialdehyde concentration of the frozen-thawed boar sperm were assessed. The results suggested that both AP and FA significantly improved the frozen-thawed boar sperm quality in all these aspects when they were added to the freezing extender separately, while the highest improvement was recorded when the extender was supplemented with 0.1 mmol/L AP plus 0.15 mmol/L FA. These findings demonstrated that supplementation of freezing extender with both AP and FA had a combined, beneficial effect on frozen-thawed boar sperm.