Backgroud Before fertilization,spermatozoa undergo a crucial maturation step called capacitation,which is a unique event regulates the sperm’s ability for successful fertilization.The capacitation process takes place...Backgroud Before fertilization,spermatozoa undergo a crucial maturation step called capacitation,which is a unique event regulates the sperm’s ability for successful fertilization.The capacitation process takes place as the spermatozoa pass through the female reproductive tract(FRT).Dihydrolipoamide dehydrogenase(DLD)protein is a post-pyruvate metabolic enzyme,exhibiting reactive oxygen species(ROS)production which causes capacitation.Additionally,other vital functions of DLD in buffalo spermatozoa are hyperactivation and acrosome reaction.DLD produces the optimum amount of ROS required to induce capacitation process in FRT.Depending on physiological or patho-physiological conditions,DLD can either enhance or attenuate the production of reactive oxygen species(ROS).Aim of this study was to investigate whether changes in the production of ROS in sperm cells can impact their ability to fertilize by triggering the capacitation and acrosome reaction.Results In this study,abundance of DLD protein was quantified between high(n=5)and low fertile bull(n=5)sper-matozoa.It was found that compared to high-fertile(HF)bulls,low-fertile(LF)bulls exhibited significantly(P<0.05)higher DLD abundances.Herein,we optimised the MICA concentration to inhibit DLD function,spermatozoa were treated with MICA in time(0,1,2,3,4,and 5 h)and concentrations(1,2.5,5,and 10 mmol/L)dependent manner.Maximum DLD inhibition was found to be at 4 h in 10 mmol/L MICA concentration,which was used for further exper-imentation in HF and LF.Based on DLD inhibition it was seen that LF bull spermatozoa exhibited significantly(P<0.05)higher ROS production and acrosome reaction in comparison to the HF bull spermatozoa.The kinematic parameters of the spermatozoa such as percent total motility,velocity parameters(VCL,VSL,and VAP)and other parameters(BCF,STR,and LIN)were also decreased in MICA treated spermatozoa in comparison to the control(capacitated)spermatozoa.Conclusions The present study provides an initial evidence explaining the buffalo bull spermatozoa with higher DLD abundance undergo early capacitation,which subsequently reduces their capacity to fertilize.展开更多
Background： The Y chromosome in mammal is paternally inherited and harbors genes related to male fertility and spermatogenesis. The unique intra-chromosomal recombination pattern of Y chromosome and morphological dif...Background： The Y chromosome in mammal is paternally inherited and harbors genes related to male fertility and spermatogenesis. The unique intra-chromosomal recombination pattern of Y chromosome and morphological difference of this chromosome between Bos taurus and Bos indicus make it an ideal model for studying structural variation, especially in crossbred （Bos taurus x Bos indicus） bulls. Copy Number Variation （CNV） is a type of genomic structural variation that gives information complementary to SNP data. The purpose of this study was to find out copy number differences of four Y chromosomal spermatogenesis-related candidate genes in genomic DNA of crossbred and purebred Indicine bulls. Result： Four Y chromosomal candidate genes of spermatogenesis namely, sex determining gene on Ychromosome （SRY）, DEAD box po/ypeptide 3-Y chromosome （DDX3 Y）, Ubiquidn specific peptidase 9, Y-linked （ usPgY）, testis-specific protein on Y chromosome （TSPY） were evaluated. Absolute copy numbers of Y chromosomal genes were determined by standard curve-based quantitative real time PCR. Copy numbers of SRYand TSPYgenes per unit amount of genomic DNA are higher in crossbred than Indicine bulls. However, no difference was observed in DDX3Yand usPgYgene copy numbers between two groups. Conclusion： The present study demonstrates that the structural organization of Y chromosomes differs between crossbred and Indicine bulls which are reproductively healthy as observed from analysis of semen attributes. The absolute copy numbers of SRY and TSPY genes in unit mass of genomic DNA of crossbred bulls are significantly higher than Indicine bulls. No alteration in absolute copies ofDDX3Yand usPgYgene was found between the genome of crossbred and Indicine bulls. This study suggests that the DDX3Yand USPgYare likely to be single copy genes in the genome of crossbred and Indicine bulls and variation in Y chromosome length between crossbred and Indicine bulls may be due to the copy number variation of SRY gene and TSPYarray.展开更多
Objective:To understand the level of progesterone (P4) in different quality of buffalo cumulus oocyte complexes (COCs) and further to evaluate the effect of exogenous P4 supplementation on maturation and subsequent de...Objective:To understand the level of progesterone (P4) in different quality of buffalo cumulus oocyte complexes (COCs) and further to evaluate the effect of exogenous P4 supplementation on maturation and subsequent developmental ability of poor quality brilliant cresyl blue (BCB-)COCs.Methods: Progesterone secreted by different quality of buffalo oocytes was estimated by enzyme linked immunosorbent assay and the concentration differences were translated into P4 doses to be incorporated in the maturation medium of BCB-ve COCs followed by expression analysis of genes involved in the cumulus expansion, extracellular matrix disintegration and progesterone receptor signalling. In addition, the study also evaluated the effect of exogenous P4 on sperm-cumulus interaction. Results: More than 10-fold up-regulated expression of progesterone receptor in P4 supplemented oocytes signified that P4 might be acting predominantly through this receptor. Also, exogenous P4 supplementation had significant effect on transcatheter arterial chemoembolization protease regulated by P4-progesterone receptor pathway which in turn had an important role in extracellular matrix disintegration. On the contrary, cumulus expansion genesHAS2,TNFAIP6,AREG were not altered upon P4 supplementation. Also, it was observed that P4 addition did facilitate passage of significantly more number of spermatozoa through P4 treated cumulus cells. Further, incorporation of different doses of P4 did not improve significantly the cleavage and blastocyst rates of BCB-ve COCs.Conclusions: Different qualities of buffalo COCs secrete substantially diverse levels of P4, and its supplementation has a role in oocyte maturation via modulation of cumulus characteristics but perhaps not fertilization.展开更多
Objective:To assess the stability of 10 candidate internal control genes(ICGs),namely GAPDH,ACTB,RPL23,RPS15A,ATPSF1,GLUT5,HMBS,ATP2B4,PPIA,and BRP to normalize the transcriptional data from testes samples of Zebu and...Objective:To assess the stability of 10 candidate internal control genes(ICGs),namely GAPDH,ACTB,RPL23,RPS15A,ATPSF1,GLUT5,HMBS,ATP2B4,PPIA,and BRP to normalize the transcriptional data from testes samples of Zebu and crossbred bulls.Methods:Total RNA was isolated from testicular tissue of Zebu and crossbred bulls(n=6 each)between 2-8 years of age.cDNA was synthesized,and the quantitative real-time polymerase chain reaction(PCR)was performed.The cycle threshold values were used for the analysis of the stability of ICGs.Four different statistical algorithms:geNorm,Normfinder,BestKeeper,and RefFinder,were used to assess the stability of these genes.Results:ATPSF1,HMBS,PPIA,and RPS15A were the most reliable and stable ICGs for Zebu testes,and ATPSF1,RPL23,and PPIA for crossbred testes.Conclusions:A panel of stable ICGs(ATPSF1,HMBS,PPIA,RPS15A for Zebu and ATPSF1,RPL23,and PPIA for crossbred)for normalization of gene expression data in testes samples can be helpful for researchers to conduct functional genomics studies at the testicular level in cattle bulls.展开更多
基金Bill&Melinda Gates Foundation(Grant number OPP1154401).
文摘Backgroud Before fertilization,spermatozoa undergo a crucial maturation step called capacitation,which is a unique event regulates the sperm’s ability for successful fertilization.The capacitation process takes place as the spermatozoa pass through the female reproductive tract(FRT).Dihydrolipoamide dehydrogenase(DLD)protein is a post-pyruvate metabolic enzyme,exhibiting reactive oxygen species(ROS)production which causes capacitation.Additionally,other vital functions of DLD in buffalo spermatozoa are hyperactivation and acrosome reaction.DLD produces the optimum amount of ROS required to induce capacitation process in FRT.Depending on physiological or patho-physiological conditions,DLD can either enhance or attenuate the production of reactive oxygen species(ROS).Aim of this study was to investigate whether changes in the production of ROS in sperm cells can impact their ability to fertilize by triggering the capacitation and acrosome reaction.Results In this study,abundance of DLD protein was quantified between high(n=5)and low fertile bull(n=5)sper-matozoa.It was found that compared to high-fertile(HF)bulls,low-fertile(LF)bulls exhibited significantly(P<0.05)higher DLD abundances.Herein,we optimised the MICA concentration to inhibit DLD function,spermatozoa were treated with MICA in time(0,1,2,3,4,and 5 h)and concentrations(1,2.5,5,and 10 mmol/L)dependent manner.Maximum DLD inhibition was found to be at 4 h in 10 mmol/L MICA concentration,which was used for further exper-imentation in HF and LF.Based on DLD inhibition it was seen that LF bull spermatozoa exhibited significantly(P<0.05)higher ROS production and acrosome reaction in comparison to the HF bull spermatozoa.The kinematic parameters of the spermatozoa such as percent total motility,velocity parameters(VCL,VSL,and VAP)and other parameters(BCF,STR,and LIN)were also decreased in MICA treated spermatozoa in comparison to the control(capacitated)spermatozoa.Conclusions The present study provides an initial evidence explaining the buffalo bull spermatozoa with higher DLD abundance undergo early capacitation,which subsequently reduces their capacity to fertilize.
基金supported by World Bank funded National Agricultural Innovation Project(C4/C30015)
文摘Background： The Y chromosome in mammal is paternally inherited and harbors genes related to male fertility and spermatogenesis. The unique intra-chromosomal recombination pattern of Y chromosome and morphological difference of this chromosome between Bos taurus and Bos indicus make it an ideal model for studying structural variation, especially in crossbred （Bos taurus x Bos indicus） bulls. Copy Number Variation （CNV） is a type of genomic structural variation that gives information complementary to SNP data. The purpose of this study was to find out copy number differences of four Y chromosomal spermatogenesis-related candidate genes in genomic DNA of crossbred and purebred Indicine bulls. Result： Four Y chromosomal candidate genes of spermatogenesis namely, sex determining gene on Ychromosome （SRY）, DEAD box po/ypeptide 3-Y chromosome （DDX3 Y）, Ubiquidn specific peptidase 9, Y-linked （ usPgY）, testis-specific protein on Y chromosome （TSPY） were evaluated. Absolute copy numbers of Y chromosomal genes were determined by standard curve-based quantitative real time PCR. Copy numbers of SRYand TSPYgenes per unit amount of genomic DNA are higher in crossbred than Indicine bulls. However, no difference was observed in DDX3Yand usPgYgene copy numbers between two groups. Conclusion： The present study demonstrates that the structural organization of Y chromosomes differs between crossbred and Indicine bulls which are reproductively healthy as observed from analysis of semen attributes. The absolute copy numbers of SRY and TSPY genes in unit mass of genomic DNA of crossbred bulls are significantly higher than Indicine bulls. No alteration in absolute copies ofDDX3Yand usPgYgene was found between the genome of crossbred and Indicine bulls. This study suggests that the DDX3Yand USPgYare likely to be single copy genes in the genome of crossbred and Indicine bulls and variation in Y chromosome length between crossbred and Indicine bulls may be due to the copy number variation of SRY gene and TSPYarray.
文摘Objective:To understand the level of progesterone (P4) in different quality of buffalo cumulus oocyte complexes (COCs) and further to evaluate the effect of exogenous P4 supplementation on maturation and subsequent developmental ability of poor quality brilliant cresyl blue (BCB-)COCs.Methods: Progesterone secreted by different quality of buffalo oocytes was estimated by enzyme linked immunosorbent assay and the concentration differences were translated into P4 doses to be incorporated in the maturation medium of BCB-ve COCs followed by expression analysis of genes involved in the cumulus expansion, extracellular matrix disintegration and progesterone receptor signalling. In addition, the study also evaluated the effect of exogenous P4 on sperm-cumulus interaction. Results: More than 10-fold up-regulated expression of progesterone receptor in P4 supplemented oocytes signified that P4 might be acting predominantly through this receptor. Also, exogenous P4 supplementation had significant effect on transcatheter arterial chemoembolization protease regulated by P4-progesterone receptor pathway which in turn had an important role in extracellular matrix disintegration. On the contrary, cumulus expansion genesHAS2,TNFAIP6,AREG were not altered upon P4 supplementation. Also, it was observed that P4 addition did facilitate passage of significantly more number of spermatozoa through P4 treated cumulus cells. Further, incorporation of different doses of P4 did not improve significantly the cleavage and blastocyst rates of BCB-ve COCs.Conclusions: Different qualities of buffalo COCs secrete substantially diverse levels of P4, and its supplementation has a role in oocyte maturation via modulation of cumulus characteristics but perhaps not fertilization.
基金The present study was carried out under the project“Molecular markers for improving reproduction in cattle and buffaloes”under the funding of Bill and Melinda Gates Foundation,USA and Indian Council of Agricultural Research-National Dairy Research Institute.
文摘Objective:To assess the stability of 10 candidate internal control genes(ICGs),namely GAPDH,ACTB,RPL23,RPS15A,ATPSF1,GLUT5,HMBS,ATP2B4,PPIA,and BRP to normalize the transcriptional data from testes samples of Zebu and crossbred bulls.Methods:Total RNA was isolated from testicular tissue of Zebu and crossbred bulls(n=6 each)between 2-8 years of age.cDNA was synthesized,and the quantitative real-time polymerase chain reaction(PCR)was performed.The cycle threshold values were used for the analysis of the stability of ICGs.Four different statistical algorithms:geNorm,Normfinder,BestKeeper,and RefFinder,were used to assess the stability of these genes.Results:ATPSF1,HMBS,PPIA,and RPS15A were the most reliable and stable ICGs for Zebu testes,and ATPSF1,RPL23,and PPIA for crossbred testes.Conclusions:A panel of stable ICGs(ATPSF1,HMBS,PPIA,RPS15A for Zebu and ATPSF1,RPL23,and PPIA for crossbred)for normalization of gene expression data in testes samples can be helpful for researchers to conduct functional genomics studies at the testicular level in cattle bulls.
