Critical role of hyaluronidase-2 during preimplantation embryo development

Abstract

Biological functions of hyaluronan (HA) depend on its molecular size. Small size HAs are known to regulate cell proliferation; however, the expression of HA synthases (HASs) and hyaluronidase-2 (HYAL2) and their role during early embryo development have not been previously identified. In this paper, we have shown by immunostaining that HA is produced by bovine in vitro-produced embryos at all stages of early development to blastocyst. Addition of HA-synthesis inhibitor (4-methylumbelliferone; 4MU) to in vitro embryo culture inhibited blastocyst formation. HASs HAS2 and HAS3 mRNA were expressed at all stages of embryo development; however, relative mRNA expression of HAS2 was significantly reduced as the embryos develop to the blastocyst stage. HAS1 was detected during 2- and 4-cell stages but was barely detectable in subsequent stages. HYAL2 mRNA expression was detected in oviducts at the early luteal phase but was only detected in the embryos at morula and blastocyst stages (Day 6 and 7 post-fertilization). Addition of HYAL2 to embryo culture media at Day 2 post-fertilization increased phosphorylated mitogen-activated protein kinases (MAPK1 and 3) in the embryos and improved development to the blastocyst stage and increased embryo cell numbers. Addition of an anti-CD44 antibody or an MAPK inhibitor (U0126) abrogated the positive effects of HYAL2 on blastocyst rates. In conclusion, we demonstrate that the expression of different HAS genes and HYAL2 in bovine embryos varies according to the stage of development and that the supplementation of HYAL2 in vitro mimics oviductal conditions and is shown to improve the blastocyst rate and embryo quality, an effect which requires CD44 activity and MAPK signalling.

Hyaluronan and hyaluronidase, which is better for embryo development?

“…In cattle, we have demonstrated that HAS2 and HAS3 are expressed at all stages of early embryo development (from 2-cell to blastocyst stage). We have found that HAS2 mRNA expression tends to decrease with the progression to the blastocyst stage, whereas level of HAS3 expression did not change (Marei, et al 2013). …”

“…The presence of HA and its turnover in the vicinity of the oocyte during fertilization and the developing embryo suggest a biological role of HA in regulating these processes; however, this role is not well defined. We have demonstrated that in vitro supplementation of cleaved bovine embryos with Hyal2 significantly increased their development to blastocyst stage and increased embryonic cell numbers (Marei, et al 2013). However, the differential effects of HA and its fragments during early embryo development have not been previously studied.…”

“…We have recently shown that the bovine oviductal epithelial cells (OECs) express Hyal2 and the level of expression in the isthmus is much higher than in the ampulla (Marei, et al 2013) suggesting their dependence on the oviductal secretions for endogenous HA breakdown. The presence of HA and its turnover in the vicinity of the oocyte during fertilization and the developing embryo suggest a biological role of HA in regulating these processes; however, this role is not well defined.…”

Localisation and endocrine control of hyaluronan synthase (HAS) 2, HAS3 and CD44 expression in sheep granulosa cells

“…For example, HAS was detected using immunohistochemistry in pig ovary (Miyake et al 2009), ovine cervix (Perry et al 2012) and uterus (Raheem et al 2013) and human endometrium (Nykopp et al 2010). In addition, the HA receptor (CD44) was immunolocalised in ovine cervix (Perry et al 2010) and uterus (Raheem et al 2013), pig ovary (Miyake et al 2006) and oviduct (Tienthai et al 2003), bovine oocytes, embryos and oviduct (Ulbrich et al 2004;Marei et al 2012;Marei et al 2013), cumulus and mural granulosa (Ohta et al 1999;Marei et al 2012) and cervical cells (El Maradny et al 1997). …”

In vivo and in vitro studies of MUC1 regulation in sheep endometrium

“…The amplicons were 166 visualized by electrophoresis on 1% agarose gels. The amplicon bands for MUC1 were quantified 167 with AlfaEase software as reported in earlier study [32] and expressed as fold change compared to the 168 control after initial normalisation with GAPDH. …”