Expression and Characterization of Constitutive Heat Shock Protein 70.1 (HSPA‐1A) Gene in In Vitro Produced and In Vivo‐Derived Buffalo (Bubalus bubalis) Embryos

Abstract

Cells are blessed with a group of stress protector molecules known as heat shock proteins (HSPs), amongst them HSP70, encoded by HSPA-1A gene, is most abundant and highly conserved protein. Variety of stresses hampers the developmental competence of embryos under in vivo and in vitro conditions. Present work was designed to study the quantitative expression of HSPA-1A mRNA in immature oocytes (IMO), matured oocytes (MO), in vitro produced (IVP) and in vivo-derived (IVD) buffalo embryos to assess the level of stress to which embryos are exposed under in vivo and in vitro culture conditions. Further, HSPA-1A gene sequence was analysed to determine its homology with other mammalian sequences. The mRNA expression analysis was carried out on 72 oocytes (40 IMO; 32 MO), 76 IVP and 55 IVD buffalo embryos. Expression of HSPA-1A was found in oocytes and throughout the developmental stages of embryos examined irrespective of the embryo source; however, higher (p < 0.05) expression was observed in 8-16 cell, morula and blastocyst stages of IVP embryos as compared to IVD embryos. Phylogenetic analysis of bubaline HSPA-1A revealed that it shares 91-98% identity with other mammalian sequences. It can be concluded that higher level of HSPA-1A mRNA in IVP embryos in comparison with in vivo-derived embryos is an indicator of cellular stress in IVP system. This study suggests need for further optimization of in vitro culture system in which HSPA-1A gene could be used as a stress biomarker during pre-implantation development.

Reference Gene Selection for Gene Expression Analysis of Oocytes Collected from Dairy Cattle and Buffaloes during Winter and Summer

“…Approximately 10% of the viable COCs were randomly used for molecular analyses whereas the remaining 90% of the COCs were used for in vitro embryo production, with the results published previously [33]. Oocytes used for molecular analyses were mechanically separated from cumulus cells by vortexing (3 min at maximum speed) followed by three thorough washes in PBS with 0.1% polyvinyl-pyrrolidone (PVP) to completely remove cumulus cells [16], [25], [30], [34]–[38]. Expression of cumulus-specific genes was evaluated in denuded oocytes to validate the protocol used for removal of cumulus cells (File S1).…”

“…Additionally, two non-reference target genes (Table S1), HSPA1AB (which corresponds to amplification of both isoforms HSPA1A and HSPA1B ) and HSP90AA1 were used to further understand the effect of reference gene selection on final gene expression data. As members of the heat shock protein family, expression of these two target genes is known to be affected by heat stress [25], [43].…”

“…However, the environmental effects on buffalo fertility does not appear to be due to a failure on follicular development, but due to an impairment of oocyte developmental competence [10], [11]. The causes behind this climate-dependent developmental impairment remain widely unknown and studies of the effects of seasonal temperature oscillations on oocyte gene expression are of value in the understanding of the low developmental potential of bovine and buffalo oocytes during summer [14]–[16], [21], [24], [25].…”

Developmental competence and expression profile of genes in buffalo (Bubalus bubalis) oocytes and embryos collected under different environmental stress

“…Studies of the effects of seasonal temperature fluctuations on embryo gene expression are of importance in understanding the mechanism behind season influenced variation in developmental potential of embryos (Rutldge et al 1999;Gendelman and Roth 2012b;Sakatani et al 2012;Sharma et al 2012). Therefore, the present study investigated the effect of season (winter and summer) in Indian water buffalo ovarian follicle-population, oocyte recovery, quality, in vitro developmental competence and expression of candidate genes related to developmental competence, heat stress, oxidative stress and apoptosis in cumulus oocyte complexes (COCs), in vitro matured oocytes and embryos.…”

Studies on Gene Expression and Developmental Competence of Bovine Embryos Produced Under Different Conditions of Heat Stress

“…The HSPA14 confers thermotolerance against variety of stressors. Thus, the preferential maintenance of HSPA14 gene expression would allow this gene product to help maintain cellular function by acting as molecular chaperones to stabilize or refold proteins damaged by heat, and by blocking apoptosis [16]. There are many ways in which cells communicate with other cells, and gap junctional communication is one of these ways.…”