Association between HSP70 genotypes and oocytes development on in vitro maturation/fertilization in pig

ABSTRACT : This study was performed to clarify whether the variation of stress related heat shock protein 70 (HSP70) (GenBank X68213) gene was associated with the nuclear morphological change of in vitro maturation and in vitro capacitation in oocytes of pig ovaries obtained at the slaughterhouse. The nucleic acid substitution of C to G at the 483rd position was found out in HSP70 K1 (290-512) from X68213. The ovaries were categorized into CC, CG, and GG genotypes using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) (BsiHKA I). After the second in vitro maturation of immature fresh oocytes, the relation of nuclear morphological change in oocytes with the genotype of HSP70 K1 gene was such that the MII ratios of the genotype GG and CG (46.93% and 42.20%, respectively) were significantly higher than that of the CC genotype (10.71%) (p<0.05). With respect to in vitro maturation of frozen-thawed oocytes by an open pulled straw (OPS) method, the percentage of oocytes matured to MII stage of the CG genotype showed a higher trend than CC and GG genotypes. After the in vitro maturation of immature fresh oocytes and frozen-thawed oocytes by the OPS method, the relation of the pronuclei change in oocytes matured in vitro with HSP70 genotype was assessed, and the result showed that the enlarged sperm heads (ESH) of matured fresh oocytes and frozen-thawed oocytes were 80.0% and 60.0% in the CC genotype, respectively. The CC genotype group had a significantly higher rate of ESH than the CG and the GG genotype group (p<0.05). The ratios of polyspermic invasion were not different among HSP70 of the three genotypes. It was considered that the rate of in vitro maturation of fertilized oocytes was expected to differ according to genotype of the stress related gene. (Key Words : Fertilization, Maturation, Porcine Oocytes, HSP70 Genotypes)

INTRODUCTION

Porcine HSP reacts on cell growth substantially under stressful conditions caused by the change of temperature, particularly, in response to sudden elevation of the external temperature, HSP is very important for the maintenance of the cell viability of fetus or neonates (Dezeure et al., 1993; Lindquist, 1986). Although its importance on the protection of reproductive cells from temperature stress has not been elucidated yet, several HSP gene groups have been reported to mediate great effects on the development of normal reproductive cells (Allen et al., 1988; Zakeri et al., 1990). HSP70 gene is involved in proteins secreted by rising and dropping temperature, and pigs which were inherited DNA variation of physiological in adaptability to temperature changes are speculated to cause the deterioration of the productibility and breeding capacity of pigs (Huang et al., 2000, 2002). In addition, HSP70 that is a group of proteins expressed in response to stress in pigs has the function of heat tolerance and immune reaction. HSP70 gene is located on chromosome 7, and it is a protein classified as porcine major histocompatibility complex (MHC) class III group (Rothschild and Ruvinsky, 1998).

If oocytes are directly exposed to high heat during in vitro maturation, the phenomenon of nuclear maturation disrupts spindle formation at the metaphase I in mice (Baumgarner and Chrisman, 1981; Davis, 1985), and reduces the progress to metaphase II and the fertilization rate in mice and cattle (Baumgartner and Chrisman, 1981; Lenz et al., 1983). In addition, abnormalities of that chromosome and cytoplasm could decrease matured oocytes in porcine. This phenomenon mediates an important effect on the development of fertilized oocytes caused by the change of the temperature (Ju and Tseng, 2004). Particularly, porcine HSP70 has been reported to be a key molecule for the viability of oocytes during in vitro maturation of follicular oocytes (Neuer et al., 1999) and the protection of cells from heat shock stress (Ananthan et al., 1986; Lindquist, 1986; Welch, 1992; Hansen, 1999). Hendrey and Kola (1991) have reported that injection of a small amount of HSP70 mRNA showed the increase of the resistance from heat shock in rat oocytes. During the development of in vivo and in vitro fertilized oocytes, elevation of temperature may cause the impairment of the expression of specific genes and protein synthesis in cytoplasm at the molecular level within the cell, and the embryo development rate may be decreased. In addition, it is speculated that heat shock stress effects on the early embryo directly, and it also influences the development to the late embryo (Alliston et al., 1965; Ealy et al., 1993; Edwards and Hansen, 1997). The failure of heat shock protein induction after receiving heat shock also could be observed in bovine oocytes, and immature bovine oocytes could exhibit the reaction of heat shock cognate (HSC70) and HSP70 induction associated protein, nonetheless, the reaction rate of between these two proteins are not changed by heat shock (Edwards and Hansen, 1996). King et al. (2000) have reported that after the blocking stage, the necessity of HSP60 and HSP72 protein were increased for the development of porcine fertilized oocytes. This phenomenon proved that the HSP gene group is essential for the development of early porcine embryos. During the in vitro maturation of porcine follicular oocytes, the heat reaction of HSP70 gene functioned as an important factor of the in vitro fertilization and development (Fukui, 1991). However, Kawarsky and King (2001) have reported that the induction of HSP70 was not influenced by the exposure to high temperature in immature or mature oocytes, and this protein was closely associated with spindles formation during meiosis. Thus, it mediates an effect of the stability of the structure of bovine cytoplasmic micro-organisms (Kawarsky and King, 2001).