Tirtha Kumar Datta

Polymorphism of BMPR1B, BMP15 and GDF9 fecundity genes in prolific Garole sheep

Mutation studies in different prolific sheep breeds have shown that the transforming growth factor beta super family ligands viz. the growth differentiation factor 9 (GDF9/FecG), bone morphogenetic protein 15 (BMP15/FecX) and associated type I receptors, bone morphogenetic protein receptor (BMPR1B/FecB), are major determinant of ovulation rate and consequent increase in litter size. The Garole sheep is a highly prolific sheep breed of India. Characterization of fecundity genes in these animals could substantially improvise the breeding programme in these animals as well as other sheep breeds of the region. The present study was therefore designed with the objective of polymorphism study of fecundity genes in these prolific microsheep. A total of 11 point mutations were detected by polymerase chain reaction (PCR)-based method. A competitive technique called tetra-primer amplification refractory mutation system-PCR was adapted to type a total of ten points of two ovine fecundity genes (GDF9 and BMP15). The FecB locus of the BMPR1B gene and G1 locus of GDF9 gene were found to be polymorphic. In FecB locus, two genotypes, wild type (FecB+) and mutant (FecBB), were detected with allele frequencies of 0.39 and 0.61, respectively. At G1 locus, two genotypes, mutant (A) and wild types (G) were detected with allele frequencies of 0.18 and 0.82, respectively. This study reports Garole sheep as the fourth sheep breed after Belclare/Cambridge, Lacaune and Small-tailed Han sheep, where coexisting polymorphism has been found in two different fecundity genes (BMPRIB and GDF9 genes).

Tissue-specific promoter methylation and histone modification regulate CYP19 gene expression during folliculogenesis and luteinization in buffalo ovary.

Aromatase, the key enzyme of estrogen biosynthesis, is encoded by the CYP19 gene. The expression of CYP19 gene is regulated in species- and tissue-specific manner by alternate use of different promoters. We have previously, cloned and characterized the tissue-specific promoter and tissue-specific transcripts in preovulatory (granulosa cells) and postovulatory (corpus luteum) structure of buffalo ovary. The present study was aimed to understand if epigenetic gene regulation through DNA methylation and histone modifications is involved in tissue-specific CYP19 gene regulation during folliculogenesis and luteinization in buffalo ovary. Methylation analysis of five CpG dinucleotides of ovary specific proximal promoter II showed hypo-methylation in large follicle while hyper-methylation in corpus luteum. However, PI.1, the exclusive promoter responsible for residual CYP19 gene expression in corpus luteum, was found to be hypermethylated. Analysis of histone modifications using ChIP assay revealed that the distal promoter (PI.1) of CYP19 gene is ~40-fold more enriched with acetylated Histone H3 in corpus luteum than in the large follicle. This indicates that PI.1 chromatin was more accessible for transcription in corpus luteum as compared to large follicles. The chromatin accessibility for the proximal promoter (PII) in the preovulatory stage tends to be higher than the luteal tissue. However, the difference was not found to be significant. In vitro experiments showed the similar results. In conclusion, results of the present study suggests that tissue-specific methylation status of PII and chromatin remodeling through histone modifications of PI.1, coincides with the changes in expression of CYP19 gene and thus are the regulatory mechanism controlling its tissue-specific expression and promoter activity during folliculogenesis and luteinization.

Characterization of serum-free buffalo granulosa cell culture and analysis of genes involved in terminal differentiation from FSH- to LH-responsive phenotype

In the present study, buffalo granulosa cells were cultured under serum-free conditions and characterized to study the changes in gene expression associated with the transition of granulosa cells from estrogen- to progesterone-secreting phenotype. The cells were cultured in vitro under completely serum-free conditions for 8 d. Gene expression and hormone analysis showed that on day 4 granulosa cells exhibit FSH responsiveness with preovulatory phenotype having highest CYP19 gene expression and 17β-estradiol production, whereas a significant increase in transcript abundance of STAR, CYP11, and HSD3B genes accompanied with an increase in progesterone production was observed on day 8. Cells treated with LH on day 4 followed by gene expression analysis at 1, 2, 4, 6, 18, and 24 h showed significant increase in transcripts of LH-responsive genes. In conclusion, culture condition used in the present study showed that granulosa cells were FSH responsive and attained attributes of granulosa cells of dominant follicles at day 4 with highest CYP19 and LHR gene expression beyond which they acquired the ability to luteinize and thus were more LH responsive. In addition, after LH treatment, analysis of early LH-responsive genes (EGR2, RUNX1, and NR4A1) on day 4 showed that granulosa cells at this stage in culture exhibits phenotype similar to that of preovulatory follicles before LH surge in vivo and corresponds to the in vivo transition of well-orchestrated gene expression profile after LH surge. The characterized culture conditions represent a suitable in vitro model for analysis of genes involved in terminal differentiation of granulosa cells from FSH- to LH-responsive phenotype during folliculogenesis in buffalo.

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.

POLYMORPHISM OF FECUNDITY GENES (FECB, FECX, AND FECG) IN THE INDIAN BONPALA SHEEP

The present study was designed for screening polymorphism of known fecundity genes in prolific Indian Bonpala sheep. Employing tetra-primer amplification refractory mutation system PCR, 11-point mutations of BMP1B, BMP15, and GDF9 genes of 97 Bonpala ewes were genotyped. The FecB locus of the BMPR1B gene and two loci (G1 and G4) of GDF9 gene were found to be polymorphic. In FecB locus, three genotypes, namely, wild type (Fec++, 0.02), heterozygous (FecB+, 0.23), and mutant (FecBB, 0.75) were detected. At G1 locus of GDF9 gene, three genotypes, namely, wild type (GG, 0.89), heterozygous (GA, 0.10), and mutant (AA, 0.01) were detected. At G4 locus of GDF9 gene, three genotypes, namely, wild type (AA, 0.01), heterozygous (AG, 0.14), and mutant (GG, 0.85) were detected. Statistically no significant correlation of polymorphism of FecB, G1, and G4 loci and litter size was found in this breed. All five loci of BMP15 and three loci of GDF 9 genes were monomorphic. This study reports Bonpala sheep as the first sheep breed where concurrent polymorphism at three important loci (FecB, G1, and G4) of two different fecundity genes (BMPR1B and GDF9) has been found.

Involvement of transcription factor GATA-4 in regulation of CYP19 gene during folliculogenesis and luteinization in buffalo ovary.

CYP19 gene encode aromatase, the key enzyme of estrogen biosynthesis, is regulated in species- and tissue-specific manner by alternate use of different promoters. Previously, we have reported the cloning and characterization of tissue-specific promoter and transcripts in buffalo ovary and placenta. In human and rat ovary, FSH induces the phosphorylation of transcription factor CREB (cAMP response element binding protein) through PKA pathway which binds to cAMP response element like sequence (CLS) in CYP19 gene ovarian promoter. However, in buffalo as well as in bovine, in silico analysis of ovary specific promoter sequence identified a single base pair deletion in CLS site and is designated as CLS-like sequence. To understand if CLS with a point mutation is still a functional cis-element and is involved in FSH stimulated transactivation of CYP19 gene in buffalo ovary, the present study was thus aimed to functionally characterize the role of buffalo CLS in CYP19 gene transactivation. We also studied the involvement of GATA-4, having consensus binding sites in CYP19 gene ovarian promoter in the vicinity of CLS during different stages of the buffalo estrus cycle. Reporter construct analyses and EMSA results showed that CLS is playing no significant role in CYP19 gene regulation in buffalo ovary. Real time absolute quantification of GATA-4 showed the differential expression of GATA-4 mRNA during folliculogenesis and luteinization with significantly higher transcript abundance in large follicle in comparison to other tissues. Western blot analysis of granulosa cells nuclear protein isolated from different stage of follicular development (small and large follicles) and differentiation (corpus luteum) showed that abundance of phosphorylated GATA-4 (Ser261) was significantly higher in granulosa cell nuclear protein of large follicles as compared to small follicles and corpora lutea. Interestingly, binding studies using ChIP showed significantly enhanced binding to the CYP19 gene promoter in large follicle which was seen to be declined in the luteal tissue. Similar results were obtained in the in vitro experiments as well. Finally, RNAi experiments were performed to validate the involvement of GATA-4 in CYP19 gene regulation. Results of RNAi showed that knockdown of GATA-4 mRNA significantly declined CYP19 gene mRNA as well as 17β-estradiol contents. In conclusion, result of the present study indicated that that in the absence of consensus CRE (cAMP response element); GATA-4 could be a downstream effector of cAMP/PKA pathway in regulation of CYP19 gene during folliculogenesis and luteinization.

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.

Absolute copy number differences of Y chromosomal genes between crossbred (Bos taurus × Bos indicus) and Indicine bulls

BackgroundThe 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 × 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.ResultFour Y chromosomal candidate genes of spermatogenesis namely, sex determining gene on Y chromosome (SRY), DEAD box polypeptide 3-Y chromosome (DDX3Y), Ubiquitin specific peptidase 9, Y-linked (USP9Y), 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 SRY and TSPY genes per unit amount of genomic DNA are higher in crossbred than Indicine bulls. However, no difference was observed in DDX3Y and USP9Y gene copy numbers between two groups.ConclusionThe 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 of DDX3Y and USP9Y gene was found between the genome of crossbred and Indicine bulls. This study suggests that the DDX3Y and USP9Y are 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 TSPY array.

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.

Diversity, Antimicrobial Action and Structure-Activity Relationship of Buffalo Cathelicidins.

Cathelicidins are an ancient class of antimicrobial peptides (AMPs) with broad spectrum bactericidal activities. In this study, we investigated the diversity and biological activity of cathelicidins of buffalo, a species known for its disease resistance. A series of new homologs of cathelicidin4 (CATHL4), which were structurally diverse in their antimicrobial domain, was identified in buffalo. AMPs of newly identified buffalo CATHL4s (buCATHL4s) displayed potent antimicrobial activity against selected Gram positive (G+) and Gram negative (G-) bacteria. These peptides were prompt to disrupt the membrane integrity of bacteria and induced specific changes such as blebing, budding, and pore like structure formation on bacterial membrane. The peptides assumed different secondary structure conformations in aqueous and membrane-mimicking environments. Simulation studies suggested that the amphipathic design of buCATHL4 was crucial for water permeation following membrane disruption. A great diversity, broad-spectrum antimicrobial action, and ability to induce an inflammatory response indicated the pleiotropic role of cathelicidins in innate immunity of buffalo. This study suggests short buffalo cathelicidin peptides with potent bactericidal properties and low cytotoxicity have potential translational applications for the development of novel antibiotics and antimicrobial peptidomimetics.