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.

Comparative analyses of copy number variations between swamp buffaloes and river buffaloes.

As an important source of genomic variation, copy number variation (CNV) contributes to environmental adaptation in worldwide buffaloes. Despite this importance, CNV divergence between swamp buffaloes and river buffaloes has not been studied previously. Here, we report 21 152 CNV regions (CNVRs) in 141 buffaloes of 20 breeds detected through multiple CNV calling strategies. Only 248 CNVRs were shared between river buffalo and swamp buffalo, reflecting great variation of CNVRs between the two subspecies. Population structure analysis based on CNVs successfully separated the two buffalo subspecies. We further assessed CNV divergence by calculating FST for genome-wide CNVs. Totally, we identified 110 significantly divergent CNV segments and 44 putatively selected genes between river buffaloes and swamp buffaloes. In particular, LALBA, a key gene controlling milk production in cattle, presented a highly differentiated CNV in the promoter region, which makes it a strong functional candidate gene for differences between swamp buffaloes and river buffaloes in traits related to milk production. Our study provides useful information of CNVs in buffaloes, which may help explain the genetic differences between the two subspecies.

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.

Hypo-glycosylated hFSH drives ovarian follicular development more efficiently than fully-glycosylated hFSH: enhanced transcription and PI3K and MAPK signaling.

STUDY QUESTION: Does hypo-glycosylated human recombinant FSH (hFSH18/21) have greater in vivo bioactivity that drives follicle development in vivo compared to fully-glycosylated human recombinant FSH (hFSH24)? SUMMARY ANSWER: Compared with fully-glycosylated hFSH, hypo-glycosylated hFSH has greater bioactivity, enabling greater follicular health and growth in vivo, with enhanced transcriptional activity, greater activation of receptor tyrosine kinases (RTKs) and elevated phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) and Mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling. WHAT IS KNOWN ALREADY: Glycosylation of FSH is necessary for FSH to effectively activate the FSH receptor (FSHR) and promote preantral follicular growth and formation of antral follicles. In vitro studies demonstrate that compared to fully-glycosylated recombinant human FSH, hypo-glycosylated FSH has greater activity in receptor binding studies, and more effectively stimulates the PKA pathway and steroidogenesis in human granulosa cells. STUDY DESIGN, SIZE, DURATION: This is a cross-sectional study evaluating the actions of purified recombinant human FSH glycoforms on parameters of follicular development, gene expression and cell signaling in immature postnatal day (PND) 17 female CD-1 mice. To stimulate follicle development in vivo, PND 17 female CD-1 mice (n = 8-10/group) were treated with PBS (150 µl), hFSH18/21 (1 µg/150 µl PBS) or hFSH24 (1 µg/150 µl PBS) by intraperitoneal injection (i.p.) twice daily (8:00 a.m. and 6:00 p.m.) for 2 days. Follicle numbers, serum anti-Müllerian hormone (AMH) and estradiol levels, and follicle health were quantified. PND 17 female CD-1 mice were also treated acutely (2 h) in vivo with PBS, hFSH18/21 (1 µg) or hFSH24 (1 µg) (n = 3-4/group). One ovary from each mouse was processed for RNA sequencing analysis and the other ovary processed for signal transduction analysis. An in vitro ovary culture system was used to confirm the relative signaling pathways. PARTICIPANTS/MATERIALS, SETTING, METHODS: The purity of different recombinant hFSH glycoforms was analyzed using an automated western blot system. Follicle numbers were determined by counting serial sections of the mouse ovary. Real-time quantitative RT-PCR, western blot and immunofluorescence staining were used to determine growth and apoptosis markers related with follicle health. RNA sequencing and bioinformatics were used to identify pathways and processes associated with gene expression profiles induced by acute FSH glycoform treatment. Analysis of RTKs was used to determine potential FSH downstream signaling pathways in vivo. Western blot and in vitro ovarian culture system were used to validate the relative signaling pathways. MAIN RESULTS AND THE ROLE OF CHANCE: Our present study shows that both hypo- and fully-glycosylated recombinant human FSH can drive follicular growth in vivo. However, hFSH18/21 promoted development of significantly more large antral follicles compared to hFSH24 (P < 0.01). In addition, compared with hFSH24, hFSH18/21 also promoted greater indices of follicular health, as defined by lower BAX/BCL2 ratios and reduced cleaved Caspase 3. Following acute in vivo treatment with FSH glycoforms RNA-sequencing data revealed that both FSH glycoforms rapidly induced ovarian transcription in vivo, but hypo-glycosylated FSH more robustly stimulated Gαs and cAMP-mediated signaling and members of the AP-1 transcription factor complex. Moreover, hFSH18/21 treatment induced significantly greater activation of RTKs, PI3K/AKT and MAPK/ERK signaling compared to hFSH24. FSH-induced indices of follicle growth in vitro were blocked by inhibition of PI3K and MAPK. LARGE SCALE DATA: RNA sequencing of mouse ovaries. Data will be shared upon reasonable request to the corresponding author. LIMITATIONS, REASONS FOR CAUTION: The observations that hFSH glycoforms have different bioactivities in the present study employing a mouse model of follicle development should be verified in nonhuman primates. The gene expression studies reflect transcriptomes of whole ovaries. WIDER IMPLICATIONS OF THE FINDINGS: Commercially prepared recombinant human FSH used for ovarian stimulation in human ART is fully-glycosylated FSH. Our findings that hypo-glycosylated hFSH has greater bioactivity enabling greater follicular health and growth without exaggerated estradiol production in vivo, demonstrate the potential for its development for application in human ART. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by NIH 1P01 AG029531, NIH 1R01 HD 092263, VA I01 BX004272, and the Olson Center for Women's Health. JSD is the recipient of a VA Senior Research Career Scientist Award (1IK6 BX005797). This work was also partially supported by National Natural Science Foundation of China (No. 31872352). The authors declared there are no conflicts of interest.

FGF2/FGFR signaling promotes cumulus-oocyte complex maturation in vitro.

Fibroblast growth factor 2 (FGF2), a member of FGF family, binds with FGF receptors (FGFR) to initiate biological functions in various somatic cells. However, little is known regarding the role of FGF2/FGFR on oocyte meiosis. In this study, we investigated expression patterns and functions of FGF2/FGFR during in vitro maturation (IVM) of mouse cumulus-oocyte complexes (COCs). Among four FGFRs, Ffgr1 was the most abundant in COCs. The transcripts for Fgf2 and Ffgr1 in COCs increased during IVM. Ffgr1 was present in oocytes and cumulus cells, while Fgf2 was present in only cumulus cells. Treatment of COCs with the selective FGFR inhibitor SU5402 blocked oocyte meiotic progression and downregulated expression of Bmp15 and Gdf9. In contrast, supplement of FGF2 promoted oocyte meiotic progression and upregulated Bmp15 and Gdf9 expression. Inhibition of FGFR with SU5402 reduced cumulus expansion and expressions of Ptx3, Has2 and Tnfaip6. Treatment with FGF2 increased Ptx3 and Has2 expression. Inhibition of FGFR had no effect on meiotic progression of denuded oocytes (DOs). However, co-culture of DOs with COCs or supplementation with FGF2 promoted meiotic progression of DOs. Inhibition of FGF2/FGFR signaling also downregulated Ffgr1 expression, while supplemental FGF2 upregulated Fgfr1 expression. Furthermore, inhibition of FGFR in COCs interrupted the c-Mos/MAPK pathway and maturation-promoting factor (MPF), as indicated by downregulation of oocyte c-mos and Ccnb1 transcripts, respectively. Overall, this study suggests that FGF2 produced by cumulus cells, activates a FGF2/FGFR autocrine/paracrine loop within COCs to regulate cumulus expansion and oocyte meiosis. These findings reveal a novel role for FGF2/FGFR signaling during in vitro maturation of COCs.

YAP1-LATS2 feedback loop dictates senescent or malignant cell fate to maintain tissue homeostasis.

Dysfunction of the homeostasis-maintaining systems in specific cell types or tissues renders the organism susceptible to a range of diseases, including cancers. One of the emerging mechanisms for maintaining tissue homeostasis is cellular senescence. Here, we report that the Hippo pathway plays a critical role in controlling the fate of ovarian cells. Hyperactivation of Yes-associated protein 1 (YAP1), the major effector of the Hippo pathway, induces senescence in cultured primary human ovarian surface epithelial cells (hOSEs). Large tumor suppressor 2 (LATS2), the primary upstream negative regulator of YAP1, is elevated in both YAP1-induced and natural replicative-triggered senescence. Deletion of LATS2 in hOSEs prevents these cells from natural replicative and YAP1-induced senescence. Most importantly, loss of LATS2 switches ovarian cells from YAP-induced senescence to malignant transformation. Our results demonstrate that LATS2 and YAP1, two major components of the Hippo/YAP signaling pathway, form a negative feedback loop to control YAP1 activity and prevent ovarian cells from malignant transformation. Human cancer genomic data extracted from TCGA datasets further confirm the clinical relevance of our finding.

Evaluation of Ovsynch versus modified Ovsynch program on pregnancy rate in water buffaloes: a meta-analysis.

Ovsynch is a widely accepted estrus synchronization protocol for improving the reproductive performance of water buffaloes who manifest low reproductive efficiency. Recently, some modified protocols based on Ovsynch such as 2 injections of prostaglandin 14 days apart following the Ovsynch are also introduced to enhance the reproductive potential of this species. In the present study, a meta-analytical assessment was performed with the objective to evaluate the reproductive performance of water buffaloes synchronized with Ovsynch or modified Ovsynch programs. Meta-analysis of the fixed or random effects model was determined by the heterogeneity among the studies. Reproductive outcome of interest was pregnancy per artificial insemination (P/AI) measured on day 25 (25-100). A total of 32 articles including 4003 buffaloes using either Ovsynch or modified Ovsynch protocol were reviewed. In the random effects model for buffaloes, the overall proportion of P/AI was 42.55% [95% confidence interval (CI): 37.48-47.70; n = 3,089] and 46.44% (95% CI: 39.63-53.31; n = 914) on day 25 after AI for Ovsynch and modified Ovsynch, respectively. Results for P/AI were then categorized by ovarian activity, where P/AI was available for 3575 cyclic buffaloes and 320 non-cyclic buffaloes. For cyclic buffaloes, the overall proportion of P/AI was 47.54% (95% CI: 42.72-52.38; n = 2911) and 57.97% (95% CI: 54.12-61.77; n = 664) on day 25 after AI for Ovsynch and modified Ovsynch, respectively. In the fixed effects model for non-cyclic buffaloes, the overall proportion of P/AI was 19.68% (95% CI: 13.48-26.58; n = 167) and 33.01% (95% CI: 25.50-40.94; n = 153) on day 25 after AI for Ovsynch and modified Ovsynch, respectively. In conclusion, a benefit for P/AI is detected in buffaloes with the modified Ovsynch protocol. Besides, whichever estrus synchronization protocols (Ovsynch or modified Ovsynch), cyclic buffaloes have higher P/AI compared with non-cyclic buffaloes.

Timely expression and activation of YAP1 in granulosa cells is essential for ovarian follicle development.

Although the role of the Hippo signaling pathway in development and tumorigenesis has been extensively studied in multiple organs, its role in ovarian follicle development remains largely unknown. Here, we report that Yes-Associated Protein 1 (YAP1), the major effector of Hippo signaling, is spatiotemporally expressed in ovarian granulosa cells and plays a critical role in the regulation of follicle development. We found that the active form of YAP1 (nuclear YAP1) was predominantly expressed in proliferative granulosa cells, whereas the inactive form of YAP1 (cytoplasmic YAP1) was mainly detected in luteal cells (terminally differentiated granulosa cells). Pharmacological inhibition of YAP1 activity disrupted mouse ovarian follicle development in vitro and in vivo. Foxl2 promoter-driven knockout of Yap1 in ovarian granulosa cells resulted in increased apoptosis of granulosa cells, decreased number of corpora lutea, reduced ovarian size, and subfertility in transgenic mice. However, Cyp19a1 promoter-driven knockout of Yap1 in differentiated granulosa cells of preovulatory follicles and luteal cells of corpora lutea had no effect on ovarian morphology and fertility. Mechanistic studies demonstrated that YAP1 interacted with epidermal growth factor receptor and TGF-ß signaling pathways to regulate granulosa cell proliferation, differentiation, and survival. Results from this study identify YAP1 as a critical regulator of granulosa cell proliferation and differentiation. Balanced expression and activation of YAP1 is essential for follicle development and successful reproduction. YAP1 is a promising target for treatment of subfertility associated with abnormal granulosa cell function.-Lv, X., He, C., Huang, C., Wang, H., Hua, G., Wang, Z., Zhou, J., Chen, X., Ma, B., Timm, B. K., Maclin, V., Dong, J., Rueda, B. R., Davis, J. S., Wang, C. Timely expression and activation of YAP1 in granulosa cells is essential for ovarian follicle development.

Four and a Half LIM Domains 2 (FHL2) Contribute to the Epithelial Ovarian Cancer Carcinogenesis.

Epithelial ovarian cancer (EOC) is one of the most lethal gynecologic malignancies. To date, the etiology of this deadly disease remains elusive. FHL2, a member of the four and a half LIM domain family, has been shown to serve either as an oncoprotein or as a tumor suppressor in various cancers. Our previous study showed that FHL2 plays a critical role in the initiation and progression of ovarian granulosa cell tumor via regulating AKT1 transcription. However, direct and systematic evidence of FHL2 in the initiation and progression of EOC remains unclear. In the present study, immunohistochemical analysis from EOC patient tissues showed that positivity and intensity of FHL2 immunosignal were up-regulated in the EOC tissues compared with normal ovary tissues. Knockdown of FHL2 in SKOV-3 cell line reduced cell growth and cell viability, blocked cell cycle progression, and inhibited cell migration. Ectopic expression of FHL2 in IGROV-1 cells which have low endogenous FHL2, promoted cell growth, improved cell viability and enhanced cell migration. Additionally, knock down of FHL2 in the SKOV-3 cell line significantly inhibited anchorage-independent growth indicated by the soft agar assay. In comparison, overexpression of FHL2 in IGROV-1 cell improved the colonies growth in soft agar. Western blot data showed that knockdown of FHL2 downregulated AKT expression level, and upregulated apoptosis related proteins such as cleaved PARP, and cleaved-lamin A. Finally, by employing stable SKOV-3/FHL2 stable knock down cell line, our data clearly showed that knockdown of FHL2 inhibited EOC xenograft initiation in vivo. Taken together, our results showed that FHL2, via regulating cell proliferation, cell cycle, and adhesion, has a critical role in regulating EOC initiation and progression. These results indicate that FHL2 could be a potential target for the therapeutic drugs against EOC.

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.

RNAi-mediated knockdown of INHBB increases apoptosis and inhibits steroidogenesis in mouse granulosa cells.

Inhibins are members of the TGFß superfamily and act as suppressors of follicle stimulating hormone (FSH) secretion from pituitary glands via a negative feedback mechanism to regulate folliculogenesis. In this study, the INHBB gene was knocked down by three RNAi-Ready pSIREN-RetroQ-ZsGreen vector- mediated recombinant plasmids to explore the effects of INHBB silencing on granulosa cell (GC) cell cycle, apoptosis and steroid production in vitro. Quantitative real-time polymerase chain reaction, Western blot, flow cytometry and ELISA were performed to evaluate the role of INHBB in the mouse GC cell cycle, apoptosis and steroid production in vitro. The results showed that the relative mRNA and protein expression of INHBB in mouse GCs can be significantly reduced by RNAi with pshRNA-B1, pshRNA-B2 and pshRNA-B3 plasmids, with pshRNA-B3 having the best knockdown efficiency. Downregulation of the expression of INHBB significantly arrests cells in the G1 phase of the cell cycle and increases the apoptosis rate in GCs. This was further confirmed by downregulation of the protein expressions of Cyclin D1, Cyclin E and Bcl2, while the protein expression of Bax was upregulated. In addition, specific downregulation of INHBB markedly decreased the concentration of estradiol and progesterone, which was further validated by the decrease in the mRNA levels of CYP19A1 and CYP11A1. These findings suggest that inhibin ßB is important in the regulation of apoptosis and cell cycle progression in granulosa cells. Furthermore, the inhibin ßB subunit has a role in the regulation of steroid hormone biosynthesis. Evidence is accumulating to support the concept that inhibin ßB is physiologically essential for early folliculogenesis in the mouse.

[Observation of curative effect of hemorrhoids lotion on pain, edema and bleeding after anorectal surgery].

The purpose of this study was to evaluate the value of Chinese herbal fumigation in the postoperative anal disease. The authors randomly divided 348 patients into treatment group and control group with 174 cases in each group. The treatment group was given to the Chinese herbal medicine hemorrhoids lotion for fumigation based on conventional anti infective therapy, routine dressing change and relaxing bowel. The control group was given to 1 000 mL 1: 5 000 potassium permanganate solution for sitz bath, fumigation based on conventional anti infective therapy, routine dressing change and relaxing bowel. The pain score, edema score, bleeding score, granulation tissue growth score and wound healing time of two groups were compared after operation. The results showed that the postoperative 6 h pain scores were higher in the two groups, the postoperative 3,5,7 d pain scores gradually decreased, the difference was statistically significant (P < 0.05). The difference of postoperative 6 h pain scores was no significant difference between the two groups, while postoperative 3,5,7 d pain scores in the treatment group were significantly lower than those in the control group (P < 0.05). 7 days after operation, anal margin of edema score and blood in the stool score in the treatment group were lower than those in control group, meat medicine growth score was higher than that of the control group, the difference had statistical meaning (P < 0.05). The healing time of two groups was respectively (13.89 + 2.78), (18.45 + 1.65) d (P < 0.05). This study suggested that Chinese herbal fumigation and washing could reduce the pain degree of patients, the anal margin of edema, and the blood in the stool, also could promote granulation tissue growth and shorten the time of wound healing, deserve the clinical expansion.

Benefits of using Double-Ovsynch versus presynch-ovsynch are affected by environmental heat in primiparous holstein lactating cows.

Optimized reproduction management enhances fertility of dairy cows, and thus improves their milk production efficiency. Comparing different synchronization protocols under variable ambient conditions would be conducive to protocol selection and production efficiency improvement. Here, 9538 primiparous Holstein lactating cows were enrolled to either Double-Ovsynch (DO) or Presynch-Ovsynch (PO) to determine the outcomes under different ambiences. We found that averaged THI of 21-days before the first service (THI-b) was the best indicators in a total of 12 environmental indexes to explain changes in conception rate. And the conception rate decreased linearly in DO treated cows when THI-b was over 73, whereas the threshold was 64 in cows subjected to PO. Compared with PO treated cows, DO increased conception rate by 6%, 13% and 19%, when THI-b was lower than 64, from 64 to 73, and over 73, respectively. Furthermore, employing treatment of PO would lead greater risk for cows staying open compared with DO when THI-b below 64 (hazard ratio, 1.3) and over 73 (hazard ratio, 1.4). Most importantly, calving intervals were 15 days shorter in DO treated cows compared PO when THI-b over 73, while no difference was detected when THI-b below 64. In conclusion, our results supported that, fertility of primiparous Holstein cows could be improved by employing DO, especially in hot weather (THI-b ≥ 73), and the benefits of DO protocol were abated under cool conditions (THI-b < 64). Considering the impacts of environmental heat load is necessary to determine reproductive protocols for commercial dairy farm.

Assessing the relationship between somatic cell count and the milk mid-infrared spectrum in Chinese Holstein cows.

BACKGROUND: Milk produced by dairy cows is a complex combination of many components, but the effect of mastitis has only been investigated for a few of these components. Milk mid-infrared (MIR) spectra can reflect the global composition of milk, and this study aimed to detect the relationships between milk MIR spectral wavenumbers and milk somatic cell count (SCC)-a sensitive biomarker for mastitis. METHODS: Pearson correlation analysis was used to calculate the correlation coefficient between somatic count score (SCS) and spectral wavenumbers. A general linear mixed model was applied to investigate the effect of three different classes of SCC (low, middle and high) on spectral wavenumbers. RESULTS: The mean correlation coefficient between the 'fingerprint region' (wavenumbers 925-1582 cm-1 ) and the SCS was higher than that for other regions of the MIR spectrum, and the specific wavenumber with the strongest correlation with the SCS was within the 'fingerprint region'. SCC class had a significant (p < 0.05) effect on 639 spectral wavenumbers. In particular, some spectral wavenumbers within the 'fingerprint region' were highly affected by the SCC class. LIMITATION: The data were collected from only one province in China, so the generalisability of the findings may be limited. CONCLUSION: SCC had close relationships with milk spectral wavenumbers related to important milk components or chemical bonds.

Comparative Genomic Analysis of the Thiolase Family and Functional Characterization of the Acetyl-Coenzyme A Acyltransferase-1 Gene for Milk Biosynthesis and Production of Buffalo and Cattle.

Cattle and buffalo served as the first and second largest dairy animals, respectively, providing 96% milk products worldwide. Understanding the mechanisms underlying milk synthesis is critical to develop the technique to improve milk production. Thiolases, also known as acetyl-coenzyme A acetyltransferases (ACAT), are an enzyme family that plays vital roles in lipid metabolism, including ACAT1, ACAT2, ACAA1, ACAA2, and HADHB. Our present study showed that these five members were orthologous in six livestock species including buffalo and cattle. Transcriptomic data analyses derived from different lactations stages showed that ACAA1 displayed different expression patterns between buffalo and cattle. Immunohistochemistry staining revealed that ACAA1 were dominantly located in the mammary epithelial cells of these two dairy animals. Knockdown of ACAA1 inhibited mammary epithelial cell proliferation and triglyceride and ß-casein secretion by regulating related gene expressions in cattle and buffalo. In contrast, ACAA1 overexpression promoted cell proliferation and triglyceride secretion. Finally, three novel SNPs (g.-681A>T, g.-23117C>T, and g.-24348G>T) were detected and showed significant association with milk production traits of Mediterranean buffaloes. In addition, g.-681A>T mutation located in the promoter region changed transcriptional activity significantly. Our findings suggested that ACAA1 play a key role in regulating buffalo and cattle milk synthesis and provided basic information to further understand the dairy animal lactation physiology.

FHL2 deficiency impairs follicular development and fertility by attenuating EGF/EGFR/YAP signaling in ovarian granulosa cells.

Female subfertility is an increasing reproductive issue worldwide, which is partially related to abnormal ovarian follicular development. Granulosa cells (GCs), by providing the necessary physical support and microenvironment for follicular development, play critical roles in maintaining female fertility. We previously showed that ectopic expression of four and a half LIM domains 2 (FHL2) promoted ovarian granulosa cell tumor progression. However, its function in follicular development and fertility remains unknown. Here, we confirmed that FHL2 is highly expressed in human and mouse ovaries. FHL2 immunosignals were predominantly expressed in ovarian GCs. A Fhl2 knockout (KO) mouse model was generated to examine its roles in follicular development and fertility. Compared with wildtype, knockout of Fhl2 significantly decreased female litter size and offspring number. Furthermore, Fhl2 deficiency reduced ovarian size and impaired follicular development. RNA-sequencing analysis of GCs isolated from either KO or WT mice revealed that, Fhl2 deletion impaired multiple biological functions and signaling pathways, such as Ovarian Putative Early Atresia Granulosa Cell, ErbB, Hippo/YAP, etc. In vitro studies confirmed that FHL2 silencing suppressed GCs growth and EGF-induced GCs proliferation, while its overexpression promoted GC proliferation and decreased apoptosis. Mechanistic studies indicated that FHL2, via forming complexes with transcriptional factors AP-1 or NF-κB, regulated Egf and Egfr expression, respectively. Besides, FHL2 depletion decreased YAP1 expression, especially the active form of YAP1 (nuclear YAP1) in GCs of growing follicles. EGF, serving as an autocrine/paracrine factor, not only induced FHL2 expression and nuclear accumulation, but also stimulated YAP1 expression and activation. Collectively, our study suggests that FHL2 interacts with EGFR and Hippo/YAP signaling to regulate follicular development and maintain fertility. This study illuminates a novel mechanism for follicular development and a potential therapeutic target to address subfertility.