Sandeep K. Rajput

Evidence Supporting a Role for SMAD2/3 in Bovine Early Embryonic Development: Potential Implications for Embryotropic Actions of Follistatin

ABSTRACT The TGF-beta-SMAD signaling pathway is involved in regulation of various aspects of female reproduction. However, the intrinsic functional role of SMADs in early embryogenesis remains poorly understood. Previously, we demonstrated that treatment with follistatin, an activin (TGF-beta superfamily ligand)-binding protein, is beneficial for bovine early embryogenesis and specific embryotropic actions of follistatin are dependent on SMAD4. Because SMAD4 is a common SMAD that can bind both SMAD2/3 and SMAD1/5, the objective of this study was to further determine the intrinsic role of SMAD2/3 in the control of early embryogenesis and delineate if embryotropic actions of follistatin in early embryos are SMAD2/3 dependent. By using a combination of pharmacological and small interfering RNA-mediated inhibition of SMAD2/3 signaling in the presence or absence of follistatin treatment, our results indicate that SMAD2 and SMAD3 are both required for bovine early embryonic development and stimulatory actions of follistatin on 8- to 16-cell and that blastocyst rates, but not early cleavage, are muted when SMAD2/3 signaling is inhibited. SMAD2 deficiency also results in reduced expression of the bovine trophectoderm cell-specific gene CTGF. In conclusion, the present work provides evidence supporting a functional role of SMAD2/3 in bovine early embryogenesis and that specific stimulatory actions of follistatin are not observed in the absence of SMAD2/3 signaling.

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.

Expression of TGFβ superfamily components and other markers of oocyte quality in oocytes selected by brilliant cresyl blue staining: relevance to early embryonic development.

Brilliant cresyl blue (BCB) is a super‐vital stain that has been used to select competent oocytes in different species. One objective of the present study was to assess the relationship between BCB staining, which correlates with an oocytes developmental potential, and the transcript abundance for select TGFβ‐superfamily components, SMAD2/3 and SMAD1/5 phosphorylation levels, and oocyte (JY1) and cumulus‐cell (CTSB, CTSK, CTSS, and CTSZ) transcript markers in bovine oocytes and/or adjacent cumulus cells. The capacity of exogenous follistatin or JY1 supplementation or cathepsin inhibitor treatment to enhance development of embryos derived from low‐quality oocytes, based on BCB staining, was also determined. Cumulus‐oocyte complexes (COCs) from abattoir‐derived ovaries were subjected to BCB staining, and germinal‐vesicle‐stage oocytes and cumulus cells were harvested from control, BCB+, and BCB− (low‐quality oocyte) groups for real‐time PCR or Western‐blot analysis. Remaining COCs underwent in vitro maturation, in vitro fertilization, and embryo culture in the presence or absence of the above exogenous supplements. Levels of FST, JY1, BMP15, and SMAD1, 2, 3, and 5 transcripts were higher in BCB+ oocytes whereas CTSB, CTSK, CTSS, and CTSZ mRNA abundance was higher in cumulus cells surrounding BCB− oocytes. Western‐blot analysis revealed higher SMAD1/5 and SMAD2/3 phosphorylation in BCB+ than BCB− oocytes. Embryo‐culture studies demonstrated that follistatin and cathepsin inhibitor treatment, but not JY‐1 treatment, improve the developmental competence of BCB− oocytes. These results contribute to a better understanding of molecular indices of oocyte competence. Mol. Reprod. Dev. 82: 251–264, 2015.

Embryotropic actions of follistatin: paracrine and autocrine mediators of oocyte competence and embryo developmental progression

Despite several decades since the birth of the first test tube baby and the first calf derived from an in vitro-fertilised embryo, the efficiency of assisted reproductive technologies remains less than ideal. Poor oocyte competence is a major factor limiting the efficiency of in vitro embryo production. Developmental competence obtained during oocyte growth and maturation establishes the foundation for successful fertilisation and preimplantation embryonic development. Regulation of molecular and cellular events during fertilisation and embryo development is mediated, in part, by oocyte-derived factors acquired during oocyte growth and maturation and programmed by factors of follicular somatic cell origin. The available evidence supports an important intrinsic role for oocyte-derived follistatin and JY-1 proteins in mediating embryo developmental progression after fertilisation, and suggests that the paracrine and autocrine actions of oocyte-derived growth differentiation factor 9, bone morphogenetic protein 15 and follicular somatic cell-derived members of the fibroblast growth factor family impact oocyte competence and subsequent embryo developmental progression after fertilisation. An increased understanding of the molecular mechanisms mediating oocyte competence and stage-specific developmental events during early embryogenesis is crucial for further improvements in assisted reproductive technologies.

Regulation and regulatory role of WNT signaling in potentiating FSH action during bovine dominant follicle selection.

Follicular development occurs in wave like patterns in monotocous species such as cattle and humans and is regulated by a complex interaction of gonadotropins with local intrafollicular regulatory molecules. To further elucidate potential mechanisms controlling dominant follicle selection, granulosa cell RNA harvested from F1 (largest) and F2 (second largest) follicles isolated at predeviation (PD) and onset of diameter deviation (OD) stages of the first follicular wave was subjected to preliminary RNA transcriptome analysis. Expression of numerous WNT system components was observed. Hence experiments were performed to test the hypothesis that WNT signaling modulates FSH action on granulosa cells during follicular waves. Abundance of mRNA for WNT pathway members was evaluated in granulosa cells harvested from follicles at emergence (EM), PD, OD and early dominance (ED) stages of the first follicular wave. In F1 follicles, abundance of CTNNB1 and DVL1 mRNAs was higher and AXIN2 mRNA was lower at ED versus EM stages and DVL1 and FZD6 mRNAs were higher and AXIN2 mRNA was lower in F1 versus F2 follicle at the ED stage. Bovine granulosa cells were treated in vitro with increasing doses of the WNT inhibitor IWR-1+/− maximal stimulatory dose of FSH. IWR-1 treatment blocked the FSH-induced increase in granulosa cell numbers and reduced the FSH-induced increase in estradiol. Granulosa cells were also cultured in the presence or absence of FSH +/− IWR-1 and hormonal regulation of mRNA for WNT pathway members and known FSH targets determined. FSH treatment increased CYP19A1, CCND2, CTNNB1, AXIN2 and FZD6 mRNAs and the stimulatory effect on CYP19A1 mRNA was reduced by IWR-1. In contrast, FSH reduced CARTPT mRNA and IWR-1 partially reversed the inhibitory effect of FSH. Results support temporal and hormonal regulation and a potential role for WNT signaling in potentiating FSH action during dominant follicle selection.

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.