Arpana Verma

Genome-wide profiling of sperm DNA methylation in relation to buffalo (Bubalus bubalis) bull fertility

The DNA methylation pattern in spermatozoa of buffalo bulls of different fertility status was investigated. Spermatozoa isolated DNA from two groups of buffalo bulls (n = 5), selected based on their artificial insemination-generated conception rate data followed by IVF efficiency, were studied for global methylation changes using a custom-designed 180 K buffalo (Bubalus bubalis) CpG island/promoter microarray. A total of 96 individual genes with another 55 genes covered under CpG islands were found differentially methylated in sperm of high-fertile and subfertile buffalo bulls. Important genes associated with biological processes, cellular components, and functions were identified to be differentially methylated in buffalo bulls with differential fertility status. The identified differentially methylated genes were found to be involved in germ cell development, spermatogenesis, capacitation, and embryonic development. The observations hint that methylation defects of sperm DNA may play a crucial role in determining the fertility of breeding bulls. This growing field of sperm epigenetics will be of great benefit in understanding the graded fertility conditions of breeding bulls in commercial livestock production system.

Effect of varying glucose concentrations during in vitro maturation and embryo culture on efficiency of in vitro embryo production in buffalo.

This study was aimed to optimize glucose level at different stages of buffalo in vitro embryo production procedure. Three glucose levels (1.5, 5.6 and 10 mm) along with a control (0 mm) were used at three phases of in vitro fertilisation (IVF) procedure viz. in vitro maturation (IVM), in vitro culture (IVC-I) (12-72 hpi) and IVC-II (72 hpi to 7 dpi). Maturation rate of oocytes was found different under different glucose concentrations, and significantly more number of oocytes reached to MII under 5.6 mm glucose. The glucose levels at each phase (IVM, IVC-I and IVC-II) individually had significant effect on blastocyst rate, and the level used at one phase had significant effect on the outcome of next phase. Complete withdrawal of glucose from any of these stages irrespective of concentrations used at subsequent stage/s resulted in significantly lower number of blastocysts. However, the changing levels of glucose had differential effects during different phases of IVF steps. The most prominent effect of glucose level was observed during IVM. The presence of 5.6 mm glucose at all stages was most effective to yield highest blastocyst rate in buffalo IVF system.

Embryonic genome activation events in buffalo (Bubalus bubalis) preimplantation embryos

Embryonic genome activation (EGA) is the first major step towards successful initiation of preimplantation development, which culminates in the formation of implantation‐competent embryos. EGA occurs at species‐specific embryonic cell stages. In the present work, EGA was identified for buffalo embryos by studying the development rate of embryos in normal as well as imposed transcription block conditions, analyzing bromo‐uridine triphosphate (BrUTP) incorporation rates as evidence of de novo transcription initiation, and studying the expression status of eukaryotic translation initiation factor 1A (eIF1A), U2 auxiliary splicing factor (U2AF), and polyadenylate polymerase (PAP) genes at different embryonic cell stages. Under normal, in vitro fertilization and culture conditions, about 26% and 17% of oocytes could reach morula and blastocyst stages, respectively, but no embryos could progress beyond 8‐cell stages in presence of α‐amanitin. Culturing embryos in the presence of BrUTP revealed a marked increase in its incorporation between 4‐ and 8‐cell stages. All genes studied displayed an abrupt increase in expression between 4‐ and 8‐cell stages; PAP expression was upregulated earlier from 2‐ to 4‐cell stages. About 65% of PAP transcripts from the 4‐cell stage and more than 70% of eIF1A, U2AF, and PAP transcripts at 8‐cell stage embryos were found to be synthesized de novo. Together, these data suggest that a minor EGA in buffalo embryos happens from 2‐ to 4‐cell stages, while the major EGA takes place from 4‐ to 8‐cell stage transition. Mol. Reprod. Dev. 79: 321–328, 2012.

Identification of some unknown transcripts from SSH cDNA library of buffalo follicular oocytes.

A buffalo oocyte-specific subtracted cDNA library was constructed to identify exclusively or preferentially oocyte-expressed genes. The library represented an enriched population of transcripts obtained from oocytes of diverse ovarian follicular origin and at different stages of in vitro maturation. A total of 1173 high-quality sequences of oocyte-specific genes were clustered into 645 unique sequences, out of which 65.76% were represented as singlets and 34.26% as contig expressed sequence tags (ESTs; clusters). Analysis of sequences revealed that 498 of these sequences were identified as a known sequence in mammalian species including buffalo, 103 as uncharacterized ESTs and 44 unknown sequences including 1 novel EST, so far not reported in any species. Gene ontology annotation classified these sequences into functional categories of cellular events and biological processes associated with oocyte competence. Expression status of the isolated unknown ESTs confirmed that many of these are expressed in oocytes exclusively and in others preferentially, some in excess of 80-fold greater in comparison with a variety of somatic tissues. The isolated novel EST was detected to be expressed exclusively in oocytes and testicular cells only. To our knowledge, this is the first report giving a detailed transcriptome account of oocyte-expressed genes in buffalo. This study will provide important information on the physiological control of oocyte development, as well as many questions yet to be addressed on the reproductive process of buffalo.

Differential Histone Modification Status of Spermatozoa in Relation to Fertility of Buffalo Bulls

In this study genome‐wide di‐methylated H3K4 (H3K4me2) and tri‐methylated H3K27 (H3K27me3) modification profiles were analyzed in spermatozoa of buffalo bulls having wide fertility differences. The custom designed 4 × 180 K buffalo (Bubalus bubalis) ChIP‐on‐chip array was fabricated by employing array‐based sequential hybridization using bovine and buffalo genomic DNA for comparative hybridization. The buffalo specific array developed had 177,440 features assembled from Coding sequences, Promoter and CpG regions comprising 2967 unique genes. A total of 84 genes for H3K4me2 and 80 genes for H3K27me3 were found differentially enriched in mature sperm of high and sub‐fertile buffalo bulls. Gene Ontology analysis of these genes revealed their association with different cellular functions and biological processes. Genes identified as differentially enriched between high and sub‐fertile bulls were found to be involved in the processes of germ cell development, spermatogenesis and embryonic development. This study presents the first genome‐wide H3K4me2 and H3K27me3 profiling of buffalo bull sperm. Results provide a list of specific genes which could be made responsible for differential bull fertility. J. Cell. Biochem. 116: 743–753, 2015.

Expression pattern of glucose metabolism genes in relation to development rate of buffalo (Bubalus bubalis) oocytes and in vitro-produced embryos

The expression pattern of glucose metabolism genes (hexokinase, phosphofructokinase, glucose-6-phosphate dehydrogenase [G6PDH], lactate dehydrogenase [LDH], and pyruvate dehydrogenase [PDH]) were studied in buffalo in vitro-matured oocytes and in vitro-produced embryos cultured under different glucose concentrations (0 mM, 1.5 mM, 5.6 mM, and 10 mM) during in vitro maturation of oocytes and culture of IVF produced embryos. The expression of the genes varied significantly over the cleavage stages under different glucose concentrations. Developmental rate of embryos was highest under a constant glucose level (5.6 mM) throughout during maturation of oocytes and embryo culture. Expression pattern of glucose metabolism genes under optimum glucose level (5.6 mM) indicated that glycolysis is the major pathway of glucose metabolism during oocyte maturation and early embryonic stages (pre-maternal to zygotic transition [MZT]) and shifts to oxidative phosphorylation during post-MZT stages in buffalo embryos. Higher glucose level (10 mM) caused abrupt changes in gene expression and resulted in shifting toward anaerobic metabolism of glucose during post-MZT stages. This resulted in decreased development rate of embryos during post-MZT stages. High expression of LDH and PDH in the control groups (0 mM glucose) indicated that in absence of glucose, embryos try to use available pyruvate and lactate sources, but succumb to handle the post-MZT energy requirement, resulting to poor development rate. Expression pattern of G6PDH during oocyte maturation as well early embryonic development was found predictive of quality and development competence of oocytes/ embryos.

Prediction of Structure and Functions from Full Length Coding Sequence of SRY Gene in Crossbred and Indicine Bulls

Sex-determining Region on Y chromosome (SRY) is testis determining factor in mammalian species. It encodes a protein that possess high mobility group (HMG) DNA-binding domain and acts as an architectural transcription factor. Mutation in this gene causes sex-reversal in many mammalian species. Detailed structure and exact biological functions of this protein in bovine species are not known. In the present study the full length coding sequences of the gene from crosssbred and indicine bulls were amplified, translated in silico and 3D model of the protein were predicted from the amino acid sequences. Predicted structure-activity relationship of the protein states that the protein is subcellular, essential, N-glycosylated in nature and is involved in sex determination and neuroendocrine functioning in central and peripheral nervous systems. The proteins have also plausible role in RNA metabolism, transition-metal and calmodulin binding. Overall this structural and functional information about the protein in bovine species will reinforce the understanding of domain-specific role of this protein and development of diagnostics for sex-determination in bovine species.