Discovery Of Developmental,identification And Application Of Genes Associated With Follicle Development In Buffalo

Buffalo has characteristics such as good environmental adaptability and resistance to adversity,which are economically and biologically important for the development of tropical and subtropical regions.As the second largest milk-producing livestock in the world,buffalo play an important role in the agricultural economies of many developing countries by providing milk,meat,and animal power.However,the low reproductive efficiency of buffalo severely restricts the healthy development of the industry.Therefore,it is particularly important to improve the reproductive efficiency of buffalo.The process of follicle development,maturation,and ovulation is crucial for normal conception in females,while the proliferation and differentiation of granulosa cells are important prerequisites for primordial follicle formation and development and can regulate the growth and development of follicles during the anagenesis phase and follicular atresia at a later stage through the receptor pathway,thus playing a key regulatory function in follicle development.Therefore,the identification of novel regulatory molecules with the potential to regulate ovarian granulosa cell function is essential for a deeper understanding of follicular development.In this study,buffalo follicle development-related genes were analyzed at three levels of discovery,identification,and application,and the main findings are as follows:1.Analysis of the function of buffalo APOA1 gene in regulating reproductive traits in buffalo based on apolipoprotein（APO）family theory.In this experiment,buffalo APO genes and proteins were systematically studied at the genome-wide level,and a total of five buffalo APO genes and ten APO protein sequences were identified and classified into three subfamilies based on phylogenetics,and genes of the same subfamily were found to contain similar motifs and structural domains.The evolutionary trees of APO proteins from buffalo,horse,goat,sheep,and cow were constructed and the evolutionary relationships were analyzed.Chromosomal co-localization and covariance analysis indicated a high degree of homology between APO genes of buffalo and dairy cattle.In addition,four SNP loci of the APOA1 gene（g.-1643C>T,g.-1544A>G,g.-1456A>G,and g.-454C>A）were screened and identified in buffalo,and the association analysis showed that the screened SNPs and different haplotype combinations were significantly associated with calving interval and age at first calving in buffalo（P<0.05）.2.The effect of APOA1 on the growth of ovarian granulosa cells and steroid hormone secretion in buffalo and its mechanism was studied.In this study,we applied RNAi,CCK-8,flow cytometry,RT-q PCR,WB,ELISA and transcriptome sequencing to investigate the effects of interfering with the expression of APOA1 gene on the growth and steroid hormone synthesis and secretion of buffalo granulosa cells.The results showed that knockdown of APOA1 expression significantly inhibited the cell viability and the number of buffalo granulosa cells by down-regulating the expression of proliferation-related genes（Myc,Cyclin D1）（P<0.05）;significantly promoted the early and total apoptosis ratio of buffalo granulosa by up-regulating the expression of apoptosis-related genes（FAS,Caspase8 and Cytochrome C）（P<0.05）;significantly inhibited the synthesis and secretion of steroid hormone synthesis substrates（cholesterol and triglycerides）by down-regulating the expression of related genes（HMGCR and SREBF2）（P<0.05）;and significantly inhibited the synthesis and secretion of E₂ and P₄ by down-regulating the expression of steroid hormone synthesis related genes（st AR,CYP11A1,and LHcgr）（P<0.05）.Transcriptome sequencing results screened yielded 747 differentially expressed genes,which contained431 down-regulated genes and 316 up-regulated genes;the results of KEGG enrichment analysis showed that the highest enrichment of differential genes was in the ovarian steroid hormesis signaling pathway,with down-regulated expression of ARTISt,EPOX,St AR,CYP11A1,and CYP17 in the pathway;AC,SR-B1,17β-HSD,and BMP-6 were upregulated.3.Study the function and regulatory mechanism of Cathepsin B（CTSB）in granulosa cells.To investigate the functional role of CTSB in follicle development dynamics,RNAi,CCK-8,flow cytometry,RT-q PCR,WB,and ELISA were applied to study its effects on granulosa cell growth and steroidogenesis.The results showed that the knockdown of CTSB expression significantly increased the cell viability and the number of granulosa cells by upregulating the expression of proliferation-related genes（Myc and Cyclin D2）and activating the P-Akt and P-ERK pathways（P<0.05）;significantly affected the expression of apoptosis-related genes（TNF-α,Caspase 8,Caspase 3 and Bcl2）by reduced the early and total apoptosis rate（P<0.05）;significantly inhibited the synthesis and secretion of E₂and P₄ by downregulating the expression of steroid synthesis-related genes（FSHR and CYP11A1）;and the expression of autophagy markers LC3-I and ATG5 was also significantly downregulated（P<0.05）.4.Effects of AMH-INH-RFRP gene immunization on follicle development as well as reproductive performance in buffalo.To investigate the role and mechanism of AMH,INH,and RFRP genes on follicle development and ovulation in buffalo,this study applied gene immunization,simultaneous estrus,ultrasound detection,and ELISA to investigate the effects of this vaccine on follicle development and reproductive performance of buffalo.The results showed that both immunization methods（nasal spray and intramuscular injection）of AMH-INH-RFRP gene vaccine-induced cellular and humoral immune responses in buffaloes;significantly increased the concentration of E2 in buffalo serum（P<0.05）.Also,the vaccine significantly increased the diameter of preovulatory follicles and accelerated the growth of dominant follicles in buffaloes（P<0.05）.In addition,it significantly increased the rate of estrus,ovulation,and conception in buffaloes（P<0.05）.Following pregnant buffaloes until calving,calving data revealed no significant effect of the vaccine on calf weight and body size（height,length,and girth）（P>0.05）.In conclusion,family-based and association analyses showed that APOA1 has potential as a candidate gene for the selection of reproductive traits in buffalo;APOA1 and CTSB genes play important roles in the growth of granulosa cells and the synthesis and secretion of steroid hormones,and play a key role in the regulation of folliculogenesis;AMH-INH-RFRP co-expression gene vaccine immunization in buffalo can promote follicular The AMH-INH-RFRP co-expression gene vaccine can promote follicular development and improve the reproductive efficiency of buffalo without affecting the growth of calves.