The FSH-mTOR-CNP signaling axis initiates follicular antrum formation by regulating tight junction, ion pumps, and aquaporins.

The initial formation of the follicular antrum (iFFA) serves as a dividing line between gonadotropin-independent and gonadotropin-dependent folliculogenesis, enabling the follicle to sensitively respond to gonadotropins for its further development. However, the mechanism underlying iFFA remains elusive. Herein, we reported that iFFA is characterized by enhanced fluid absorption, energy consumption, secretion, and proliferation and shares a regulatory mechanism with blastula cavity formation. By use of bioinformatics analysis, follicular culture, RNA interference, and other techniques, we further demonstrated that the tight junction, ion pumps, and aquaporins are essential for follicular fluid accumulation during iFFA, as a deficiency of any one of these negatively impacts fluid accumulation and antrum formation. The intraovarian mammalian target of rapamycin-C-type natriuretic peptide pathway, activated by follicle-stimulating hormone, initiated iFFA by activating tight junction, ion pumps, and aquaporins. Building on this, we promoted iFFA by transiently activating mammalian target of rapamycin in cultured follicles and significantly increased oocyte yield. These findings represent a significant advancement in iFFA research, further enhancing our understanding of folliculogenesis in mammals.

Regulatory impacts of PPARGC1A gene expression on milk production and cellular metabolism in buffalo mammary epithelial cells.

The PPARGC1A gene plays a fundamental role in regulating cellular energy metabolism, including adaptive thermogenesis, mitochondrial biogenesis, adipogenesis, gluconeogenesis, and glucose/fatty acid metabolism. In a previous study, our group investigated seven SNPs in Mediterranean buffalo associated with milk production traits, and the current study builds on this research by exploring the regulatory influences of the PPARGC1A gene in buffalo mammary epithelial cells (BuMECs). Our findings revealed that knockdown of PPARGC1A gene expression significantly affected the growth of BuMECs, including proliferation, cell cycle, and apoptosis. Additionally, we observed downregulated triglyceride secretion after PPARGC1A knockdown. Furthermore, the critical genes related to milk production, including the STATS, BAD, P53, SREBF1, and XDH genes were upregulated after RNAi, while the FABP3 gene, was downregulated. Moreover, Silencing the PPARGC1A gene led to a significant downregulation of ß-casein synthesis in BuMECs. Our study provides evidence of the importance of the PPARGC1A gene in regulating cell growth, lipid, and protein metabolism in the buffalo mammary gland. In light of our previous research, the current study underscores the potential of this gene for improving milk production efficiency and overall dairy productivity in buffalo populations.

Maternal exposure to DON during lactation induces testicular toxicity in pubertal and adult offspring mice.

Deoxynivalenol (DON), a type B trichothecene mycotoxin, commonly occurs in cereal grains, and poses significant health risks to humans and animals. Numerous studies reveal its obvious toxic effects on male reproductive performance as well as its ability to transfer from the lactating mother to the suckling offspring through colostrum and milk. The objective of this study was to evaluate the toxic effect of lactational DON exposure on testicular morphology, hormonal levels, inflammation, apoptosis and proliferation of germ cells, tight junction, and sperm quality in male offspring. Sixty-six male offspring mice from lactating dams exposed to DON were euthanized at PND 21 and PND 70 to investigate the reproductive toxicity. Our results indicated that maternal DON exposure had a significant impact on the weight and volume of the testes, caused testicular histopathology, and reduced testosterone levels by downregulating expressions of StAR, CYP11A1, and CYP17A1 in male offspring. We also found that maternal DON exposure led to testicular inflammation in male offspring, which was attributed to increased levels of inflammatory markers, including IL-1ß, IL-6, TNF-α, and IFN-γ. Maternal DON exposure resulted in impaired tight junctions of Sertoli cells in male offspring, as evidenced by decreased expressions of ZO-1, Occludin, and Claudin-3. In addition, maternal DON exposure caused a reduction in the number of Sertoli cells and germ cells, ultimately leading to decreased sperm count and quality in adult male offspring. Collectively, these findings provide compelling evidence that maternal exposure to DON during lactation causes testicular toxicity in both pubertal and adult male offspring.

Evolutionary analysis of buffalo sterol regulatory element-binding factor (SREBF) family genes and their affection on milk traits.

The sterol regulatory element-binding factor (SREBF) genes are a vital group of proteins binding to the sterol regulatory element 1 (SRE-1) regulating the synthesis of fatty acid. Two potential candidate genes (SREBF1 and SREBF2) have been identified as affecting milk traits. This study aims to identify the SREBF family of genes and find candidate markers or SREBF genes influencing lactation production in buffalo. A genome-wide study was performed and identified seven SREBF genes randomly distributed on 7 chromosomes and 24 protein isoforms in buffalos. The SREBF family of genes were also characterized in cattle, goat, sheep and horse, and using these all-protein sequences, a phylogenetic tree was built. The SREBF family genes were homologous between each other in the five livestock. Eight single nucleotide polymorphisms (SNPs) within or near the SREBF genes in the buffalo genome were identified and at least one milk production trait was associated with three of the SNP. The expression of SREBF genes at different lactation stages in buffalo and cattle from published data were compared and the SREBF genes retained a high expression throughout lactation with the trend being the same for buffalo and cattle. These results provide valuable information for clarifying the evolutionary relationship of the SREBF family genes and determining the role of SREBF genes in the regulation of milk production in buffalo.

Lactational exposure to Deoxynivalenol causes mammary gland injury via inducing inflammatory response and impairing blood-milk barrier integrity in mice.

Lactation is a unique physiological process to produce and secrete milk. Deoxynivalenol (DON) exposure during lactation has been demonstrated to affect adversely the growth development of offspring. However, the effects and potential mechanism of DON on maternal mammary glands remain largely unknown. In this study, we found the length and area of mammary glands were significantly reduced after DON exposure on lactation day (LD) 7 and LD 21. RNA-seq analysis results showed that the differentially expressed genes (DEGs) were significantly enriched in acute inflammatory response and HIF-1 signaling pathway, which led to an increase of myeloperoxidase activity and inflammatory cytokines. Furthermore, lactational DON exposure increased blood-milk barrier permeability by reducing the expression of ZO-1 and Occludin, promoted cell apoptosis by upregulating the expression of Bax and cleaved Caspase-3 and downregulating the expression of Bcl-2 and PCNA. Additionally, lactational DON exposure significantly decreased serum concentration of prolactin, estrogen, and progesterone. All these alterations eventually resulted in a decrease of ß-casein expression on LD 7 and LD 21. In summary, our findings indicated that lactational exposure to DON caused lactation-related hormone disorder and mammary gland injury induced by inflammatory response and blood-milk barrier integrity impairment, ultimately resulting in lower production of ß-casein.

Curcumin alleviates bisphenol AF-induced oxidative stress and apoptosis in caprine endometrial epithelial cells via the Nrf2 signaling pathway.

Bisphenol AF (BPAF), a BPA-substitute, has been widely used in industrial compounds throughout the world. Several studies have shown that BPAF has endocrine interference and reproductive toxicity. However, the toxic effects of BPAF on pregnancy and placenta of goats are still unclear. Therefore, the objective of this study was to reveal the toxic effect of BPAF by using an in vitro culture model of caprine endometrial epithelial cells (EECs) and further attempted to alleviate the toxicity by curcumin pretreatment. The results showed that BPAF induces significant effects on EECs, including decreased cell viability and mitochondrial membrane potential (△ψm), elevating intracellular reactive oxygen species (ROS), promoting cell apoptosis through upregulating the expression of Bax, Cytochrome c, and downregulating the expression of Bcl-2. Meanwhile, BPAF induced dysregulation of oxidative stress by increasing the levels of malondialdehyde (MDA) and glutathione peroxidase (GSH-Px) but decreasing the activities of superoxide dismutase (SOD). However, curcumin pretreatment could significantly attenuate BPAF-induced toxic effects in EECs. Further study revealed that BPAF treatment could activate mitogen-activated protein kinase (MAPK) pathway and nuclear factor-erythroid 2-related factor 2 (Nrf2) expression, but curcumin pretreatment significantly inhibited the activation of MAPK signal pathway and Nrf2 expression induced by BPAF. Overall, this study indicated that curcumin could prevent BPAF-induced EECs cytotoxicity, which provides a potential therapeutic strategy for female infertility associated with BPAF exposure.

High-dose synthetic phenolic antioxidant propyl gallate impairs mouse oocyte meiotic maturation through inducing mitochondrial dysfunction and DNA damage.

Propyl gallate (PG) is one of the most widely used antioxidants in food products, cosmetics and pharmaceutical industries. Increased research has suggested that exposure to PG influences reproductive health in humans and animals. However, until now, it has not yet been confirmed whether PG would impact oocyte quality. In this study, the hazardous effects of PG on oocyte meiotic maturation were investigated in mice. The findings showed that PG exposure compromises oocyte meiosis by inducing mitochondrial stress which activates apoptosis to trigger oocyte demise. Moreover, DNA damage was significantly induced in PG-treated oocytes, which might be another cause of oocyte developmental arrest and degeneration. Besides, the level of histone methylation (H3K27me2 and H3K27me3) in oocyte was also significantly increased by PG exposure. Furthermore, PG-induced oxidative stress was validated by the increased level of reactive oxygen species (ROS), which might be the underlying reason for these abnormities. In conclusion, the foregoing findings suggested that PG exposure impaired oocyte meiotic maturation by yielding mitochondrial stress to activate apoptosis, inducing DNA damage and oxidative stress, and altering histone methylation level.

Novel Insight into the Role of Squalene Epoxidase (SQLE) Gene in Determining Milk Production Traits in Buffalo.

Understanding the genetic mechanisms underlying milk production traits contribute to improving the production potential of dairy animals. Squalene epoxidase (SQLE) is one of the rate-limiting enzymes for cholesterol biosynthesis and was highly expressed in the buffalo mammary. The objectives of the present study were to detect the polymorphisms within SQLE in buffalo, the genetic effects of these mutations on milk production traits, and to understand the gene regulatory effects on buffalo mammary epithelial cells (BuMECs). A total of five SNPs were identified by sequencing, g.18858G > A loci were significantly associated with fat yield, and g.22834C > T loci were significantly associated with peak milk yield, milk yield, fat yield, and protein yield. Notably, linkage disequilibrium analysis indicated that 2 SNPs (g.18858G > A and g.22834C > T) formed one haplotype block, which was found to be significantly associated with milk fat yield, fat percentage, and protein yield. Furthermore, expression of SQLE was measured in different tissues of buffalo and was found to be higher in the mammary. Knockdown of SQLE gene expression significantly affected the growth of BuMECs, including proliferation, cell cycle, and apoptosis, and significantly downregulated the expression of related genes MYC, PCNA, and P21. In addition, knockdown of the SQLE gene significantly reduces triglyceride concentrations and the signal intensity of oil red O staining. In addition, silencing of SQLE was also found to regulate the synthesis and secretion of ß-casein and κ-casein negatively. Furthermore, SQLE knockdown is accompanied by the downregulation of critical genes (RPS6KB1, JAK2, eIF4E, and SREBP1) related to milk fat and protein synthesis. The current study showed the potential of the SQLE gene as a candidate for buffalo milk production traits. It provides a new understanding of the physiological mechanisms underlying buffalo milk production regulation.

Novel functional mutation of the PDIA3 gene affects milk composition traits in Chinese Holstein cattle.

Protein disulfide isomerase family A member 3 (PDIA3) is a multifunctional protein, and it plays a vital role in modulating various cell biological functions under physiological and pathological conditions. Our previous study on Mediterranean buffalo demonstrated that PDIA3 is a potential candidate gene associated with milk yield based on genome-wide association study analysis. However, the genetic effects of the PDIA3 gene on milk performance in dairy cattle and the corresponding mechanism have not been documented. This study aims to explore the genetic effects of PDIA3 polymorphisms on milk production traits in 362 Chinese Holstein cattle. The results showed that 4 SNPs were identified from the 5' untranslated region of the PDIA3 gene in the studied population, of which 2 SNPs (g.-1713 C>T and g.-934 G>A) were confirmed to be significantly associated with milk protein percentage, whereas g.-434 C>T was significantly associated with milk fat percentage. Notably, linkage disequilibrium analysis indicated that 3 SNPs (g.-1713 C>T, g.-934 G>A, and g.-695 A>C) formed one haplotype block, which was found to be significantly associated with milk protein percentage. The luciferase assay demonstrated that allele C of g.-434 C>T exhibited a higher promotor activity compared with allele T, suggesting that g.-434 C>T might be a potential functional mutation affecting PDIA3 expression. Furthermore, overexpression of the PDIA3 gene was found to induce higher levels of triglyceride and BODIPY fluorescence intensity. In addition, PDIA3 overexpression was also found to positively regulate the synthesis and secretion of α-casein, ß-casein, and κ-casein, whereas knockdown of this gene showed the opposite effects. In summary, our findings revealed significant genetic effects of PDIA3 on milk composition traits, and the identified SNP and the haplotype block might be used as genetic markers for dairy cow selected breeding.

Deoxynivalenol exposure inhibits biosynthesis of milk fat and protein by impairing tight junction in bovine mammary epithelial cells.

Deoxynivalenol (DON) is one of the most common feed contaminants, and it poses a serious threat to the health of dairy cows. The existing studies of biological toxicity of DON mainly focus on the proliferation, oxidative stress, and inflammation in bovine mammary epithelial cells, while its toxicity on the biosynthesis of milk components has not been well documented. Hence, we investigated the toxic effects and the underlying mechanism of DON on the bovine mammary alveolar cells (MAC-T). Our results showed that exposure to various concentrations of DON significantly inhibited cell proliferation, induced apoptosis, and altered the cell morphology which was manifested by cell distortion and shrinkage. Moreover, the transepithelial electrical resistance (TEER) values of MAC-T cells exposed to DON were gradually decreased in a time- and concentration- dependent manner, but lactate dehydrogenase (LDH) leakage was significantly increased with the maximum increase of 2.4-fold, indicating the cell membrane and tight junctions were damaged by DON. Importantly, DON significantly reduced the synthesis of ß-casein and lipid droplets, along with the significantly decreases of phospho-mTOR, phospho-4EBP1, phospho-JAK2, and phospho-STAT5. Gene expression profiles showed that the expressions of several genes related to lipid synthesis and metabolism were changed, including acyl-CoA synthetase short-chain family member 2 (ACSS2), fatty acid binding protein 3 (FABP3), 3-hydroxy-3-methylglutaryl-CoA synthase 1 (HMGCS1), and insulin-induced gene 1 (INSIG1). GO and KEGG enrichment analyses revealed that the differentially expressed genes (DEGs) were significantly enriched in ribosome, glutathione metabolism, and lipid biosynthetic process, which play important roles in the toxicological process induced by DON. Taken together, DON affects the proliferation and functional differentiation of MAC-T cells, which might be related to the cell junction disruption and morphological alteration. Our data provide new insights into functional differentiation and transcriptomic alterations of MAC-T cells after DON exposure, which contributes to a comprehensive understanding of DON-induced toxicity mechanism.

Zinc pyrithione exposure compromises oocyte maturation through involving in spindle assembly and zinc accumulation.

Zinc Pyrithione (ZPT), a Food and Drug Administration (FDA) approved chemical, is widely used for topical antimicrobials and cosmetic consumer products, including anti-dandruff shampoos. ZPT and its degraded byproducts have detected in large quantities in the environment, and identified to pose healthy risks on aquatic organisms and human. However, so far, knowledge about ZPT effects on female reproduction, particularly oocyte maturation and quality, is limited. Herein, we investigated the adverse impact of ZPT on mouse oocyte maturation and quality in vitro and found exposure to ZPT significantly compromises oocyte maturation. The results revealed that ZPT disturbed the meiotic cell cycle by impairing cytoskeletal dynamics, kinetochore-microtubule attachment (K-MT), and causing spindle assembly checkpoints (SAC) continuous activation. Further, we observed the microtubule-organizing centers (MTOCs) associated proteins p-MAPK and Aurora-A were disrupted in ZPT-treated oocytes, signified by decreased expression and abnormal localization, responsible for the severe cytoskeletal defects. In addition, ZPT exposure induced a significant increase in the levels of H3K9me2, H3K9me3, H3K27me1, and H3K27me3, suggesting the alterations of epigenetic modifications. Moreover, the accumulation of zinc ions (Zn2+) was observed in ZPT-treated oocytes, which was detrimental because overmuch intracellular Zn2+ disrupted oocyte meiosis. Finally, these above alterations impaired spindle organization and chromosome alignment in metaphase-II (MII) oocytes, indicative of damaged oocytes quality. In conclusion, ZPT exposure influenced oocyte maturation and quality via involvement in MTOCs-associated proteins mediated spindle defects, altered epigenetic modifications and zinc accumulation.

Bisphenol F exposure affects mouse oocyte in vitro maturation through inducing oxidative stress and DNA damage.

Bisphenol F (BPF), a substitute for bisphenol A (BPA), is progressively used to manufacture various consumer products. Despite the established reproductive toxicity of BPF, the underlying mechanisms remain to elucidate. This in-vitro study deep in sighted the BPF toxicity on mouse oocyte meiotic maturation and quality. After treating oocytes with BPF (300 µM), the oocyte meiotic progression was blocked, accentuated by a reduced rate in the first polar body extrusion (PBE). Next, we illustrated that BPF induced α-tubulin hyper-acetylation disrupted the spindle assembly and chromosome alignment. Concurrently, BPF resulted in severe oxidative stress and DNA damage, which triggered the early apoptosis in mouse oocytes. Further, altered epigenetic modifications following BPF exposure were proved by increased H3K27me3 levels. Concerning the toxic effects on spindle structure, oxidative stress, and DNA damage in mouse oocytes, BPF toxicity was less severe to oocyte maturation and spindle structure than BPA and induced low oxidative stress. However, compared with BPA, oocytes treated with BPF were more prone to DNA damage, indicating not less intense or even more severe toxic effects of BPF than BPA on some aspects of oocytes maturation. In brief, the present study established that like wise to BPA, BPF could inhibit meiotic maturation and reduce oocyte quality, suggesting it is not a safe substitute for BPA.

Novel Insight into the Potential Role of Acylglycerophosphate Acyltransferases Family Members on Triacylglycerols Synthesis in Buffalo.

Acylglycerophosphate acyltransferases (AGPATs) are the rate-limiting enzymes for the de novo pathway of triacylglycerols (TAG) synthesis. Although AGPATs have been extensively explored by evolution, expression and functional studies, little is known on functional characterization of how many members of the AGPAT family are involved in TAG synthesis and their impact on the cell proliferation and apoptosis. Here, 13 AGPAT genes in buffalo were identified, of which 12 AGPAT gene pairs were orthologous between buffalo and cattle. Comparative transcriptomic analysis and real-time quantitative reverse transcription PCR (qRT-PCR) further showed that both AGPAT1 and AGPAT6 were highly expressed in milk samples of buffalo and cattle during lactation. Knockdown of AGPAT1 or AGPAT6 significantly decreased the TAG content of buffalo mammary epithelial cells (BuMECs) and bovine mammary epithelial cells (BoMECs) by regulating lipogenic gene expression (p < 0.05). Knockdown of AGPAT1 or AGPAT6 inhibited proliferation and apoptosis of BuMECs through the expression of marker genes associated with the proliferation and apoptosis (p < 0.05). Our data confirmed that both AGPAT1 and AGPAT6 could regulate TAG synthesis and growth of mammary epithelial cells in buffalo. These findings will have important implications for understanding the role of the AGPAT gene in buffalo milk performance.

Cathepsin B Regulates Mice Granulosa Cells' Apoptosis and Proliferation In Vitro.

Cathepsin B (CTSB), a lysosomal cysteine protease's high expression and activity, has been reported to cause poor-quality embryos in porcine and bovine. Nevertheless, CTSB functions in mice granulosa cells remain to explore. To discuss the CTSB functional role in follicular dynamics, we studied apoptosis, proliferation, cell cycle progression, and related signaling pathways in primary mouse granulosa cells transfected with small interference RNA specific to CTSB (siCTSB) for 48 h. Further, mRNA and protein expression of cell proliferation regulators (Myc and cyclin D2), apoptosis regulators (caspase 3, caspase 8, TNF-α, and Bcl2), steroidogenesis-related genes (FSHR and CYP11A1), and autophagy markers (LC3-I and ATG5) were investigated. In addition, the effect of CTSB on steroidogenesis and autophagy was also examined. Flow cytometry analysis assay displayed that silencing of CTSB decreased the early and total apoptosis rate by downregulating TNF-α, caspase 8, and caspase 3, and upregulating Bcl2. By regulating Myc and cyclin D2 expression and activating the p-Akt and p-ERK pathways, CTSB knockdown increased GC proliferation and number. A significant decline in estradiol and progesterone concentrations was observed parallel to a significant decrease in autophagy-related markers LC3-I and ATG5 compared to the control group. Herein, we demonstrated that CTSB serves as a proapoptotic agent and plays a critical role in folliculogenesis in female mice by mediating apoptosis, autophagy, proliferation, and steroidogenesis. Hence, CTSB could be a potential prognostic agent for female infertility.

Orexin-A Regulates Follicular Growth, Proliferation, Cell Cycle and Apoptosis in Mouse Primary Granulosa Cells via the AKT/ERK Signaling Pathway.

Granulosa cells (GCs) are essential for follicular growth, development, and atresia. The orexin-A (OXA) neuropeptide is widely involved in the regulation of various biological functions. OXA selectively binds to orexin receptor type 1 (OX1R) and mediates all its biological actions via OX1R. This study aimed to explore the expression of OXA and OX1R and their regulatory role in GCs proliferation, cell cycle progression, apoptosis, oocyte maturation, and underlying molecular mechanisms of these processes and elucidate its novel signaling pathway. Western blotting and RT-qPCR showed that OXA and OX1R were expressed during different developmental stages of GCs, and siRNA transfection successfully inhibited the expression of OX1R at the translational and transcriptional levels. Flow cytometry revealed that OX1R knockdown upregulated GCs apoptosis and triggered S-phase arrest in cell cycle progression. RT-qPCR and Western blotting showed significantly reduced expression of Bcl-2 and elevated expression of Bax, caspase-3, TNF-α, and P21 in OX1R-silenced GCs. Furthermore, the CCK-8 assay showed that knockdown of OX1R suppressed GCs proliferation by downregulating the expression of PCNA, a proliferation marker gene, at the translational and transcriptional levels. Western blotting revealed that knockdown of OX1R resulted in a considerable decrease of the phosphorylation level of the AKT and ERK1/2 proteins, indicating that the AKT/ERK1/2 pathway is involved in regulating GCs proliferation and apoptosis. In addition, OX1R silencing enhanced the mRNA expression of GDF9 and suppressed the mRNA expression of BMP15 in mouse GCs. Collectively, these results reveal a novel regulatory role of OXA in the development of GCs and folliculogenesis by regulating proliferation, apoptosis, and cell cycle progression. Therefore, OXA can be a promising therapeutic agent for female infertility.

Genome-wide association study for buffalo mammary gland morphology.

This research communication describes a genome-wide association study for Italian buffalo mammary gland morphology. Three single nucleotide polymorphisms (AX-85117983, AX-8509475 and AX-85117518) were identified to be significantly associated with buffalo anterior teat length, posterior teat length and distance between anterior and posterior teat, respectively. Two significant signals for buffalo mammary gland morphology were observed in two genomic regions on the chromosome 10, and chromosome 20. One of the regions located on the chromosome 10 has the most likely candidate genes ACTC1 and GJD2, both of which have putative roles in the regulation of mammary gland development. This study provides new insights into the genetic variants of buffalo mammary gland morphology and may be beneficial for understanding of the genetic regulation of mammary growth.

Yes-associated protein 1 is required for proliferation and function of bovine granulosa cells in vitro†.

Yes-associated protein 1 (YAP1) is a major component of the Hippo signaling pathway. Although the exact extracellular signals that control the Hippo pathway are currently unknown, increasing evidence supports a critical role for the Hippo pathway in embryonic development, regulation of organ size, and carcinogenesis. Granulosa cells (GCs) within the ovarian follicle proliferate and produce steroids and growth factors, which facilitate the growth of follicle and maturation of the oocyte. We hypothesize that YAP1 plays a role in proliferation and estrogen secretion of GCs. In the current study, we examined the expression of the Hippo signaling pathway in bovine ovaries and determined whether it was important for GC proliferation and estrogen production. Mammalian STE20-like protein kinase 1 (MST1) and large tumor suppressor kinase 2 (LATS2) were identified as prominent upstream components of the Hippo pathway expressed in granulosa and theca cells of the follicle and large and small cells of the corpus luteum. Immunohistochemistry revealed that YAP1 was localized to the nucleus of growing follicles. In vitro, nuclear localization of the downstream Hippo signaling effector proteins YAP1 and transcriptional co-activator with PDZ-binding motif (TAZ) was inversely correlated with GC density, with greater nuclear localization under conditions of low cell density. Treatment with verteporfin and siRNA targeting YAP1 or TAZ revealed a critical role for these transcriptional co-activators in GC proliferation. Furthermore, knockdown of YAP1 in GCs inhibited follicle-stimulating hormone (FSH)-induced estradiol biosynthesis. The data indicate that Hippo pathway transcription co-activators YAP1/TAZ play an important role in GC proliferation and estradiol synthesis, two processes necessary for maintaining normal follicle development.

Novel insights into the genetic basis of buffalo reproductive performance.

BACKGROUND: Fertility is a complex trait that has a major impact on the development of the buffalo industry. Genome-wide association study (GWAS) has increased the ability to detect genes influencing complex traits, and many important genes related to reproductive traits have been identified in ruminants. However, reproductive traits are influenced by many factors. The development of the follicle is one of the most important internal processes affecting fertility. Genes found by GWAS to be associated with follicular development may directly affect fertility. The present study combined GWAS and RNA-seq of follicular granulosa cells to identify important genes which may affect fertility in the buffalo. RESULTS: The 90 K Affymetrix Axiom Buffalo SNP Array was used to identify the SNPs, genomic regions, and genes that were associated with reproductive traits. A total of 40 suggestive loci (related to 28 genes) were identified to be associated with six reproductive traits (first, second and third calving age, calving interval, the number of services per conception and open days). Interestingly, the mRNA expressions of 25 of these genes were also observed in buffalo follicular granulosa cells. The IGFBP7 gene showed high level of expression during whole antral follicle growth. The knockdown of IGFBP7 in buffalo granulosa cells promoted cell apoptosis and hindered cell proliferation, and increased the production of progesterone and estradiol. Furthermore, a notable signal was detected at 2.3-2.7 Mb on the equivalent of bovine chromosome 5 associated with age at second calving, calving interval, and open days. CONCLUSIONS: The genes associated with buffalo reproductive traits in this study may have effect on fertility by regulating of follicular growth. These results may have important implications for improving buffalo breeding programs through application of genomic information.

DGAT1 polymorphism in Riverine buffalo, Swamp buffalo and crossbred buffalo.

This Research Communication describes the polymorphisms in the coding region of DGAT1 gene in Riverine buffalo, Swamp buffalo and crossbred buffalo, and associations between polymorphisms and milk production performance in Riverine buffalo. Two polymorphisms of DGAT1were identified, located in exon 13 and exon 17, respectively. The distribution of the genotypes of the two SNP loci in different buffalo population varied, especially the polymorphism located in exon 13 which was not found in the Swamp buffalo. Moreover, SNP located in exon 17 was a nonsynonymous switch resulting in the animo acid sequence changed from an arginine (Arg) to a histidine (His) at position 484. Both SNPs were in Hardy-Weinberg equilibrium, and the polymorphism of g.8330T>C in the exon 13 was significantly associated with peak milk yield, total milk yield and protein percentage. The C variant was associated with an increase in milk yield and peak yield but less in protein percentage compared with the T variant. The polymorphisms of g.9046T>C in exon 17 were significantly associated with fat percentage, in that the buffaloes with TT genotype had a significantly higher fat percentage than those with CC genotype. These findings reveal the difference in the genetic evolution of the DGAT1 between Riverine buffalo and Swamp buffalo, and provide evidence that the DGAT1 gene has potential effects for Riverine buffalo milk production traits, which can be used as a candidate gene for marker-assisted selection in buffalo breeding.

An association analysis between PRL genotype and milk production traits in Italian Mediterranean river buffalo.

This Research Communication describes the association between genetic variation within the prolactin (PRL) gene and the milk production traits of Italian Mediterranean river buffalo (Bufala mediterranea Italiana). High resolution melting (HRM) techniques were developed for genotyping 465 buffaloes. The association of genetic polymorphism with milk production traits was performed and subsequently the effects of parity and calving season were evaluated. Single nucleotide polymorphisms (SNPs) were identified at exons 2 and 5 and at introns 1 and 2. All the SNPs were in Hardy-Weinberg equilibrium, and statistical analysis showed that the polymorphism of intron1 was significantly (P < 0·05) associated with milk yield, milk protein content and peak milk yield. The average contribution of the intron1 genotype (r 2 intron1) to total phenotypic variance in milk production traits was 0·09, and the TT genotype showed lower values than CC and CT genotypes. A nonsynonymous SNP was identified in exon 2, which resulted in an amino acid change from arginine to cysteine. Moreover, the polymorphism of exon 2 was associated significantly with milk fat content (P < 0·05), and the buffaloes with TT genotype showed higher total fat content than the buffaloes with CT genotype. These findings provide evidence that polymorphisms of the buffalo PRL gene are associated with milk production traits and PRL can be used as a candidate gene for marker-assisted selection in Italian Mediterranean river buffalo breeding.