Successful Piglet Production in a Chemically Defined System for In-vitro Production of Porcine Embryos: Dibutyryl Cyclic AMP and Epidermal Growth Factor-family Peptides Support In-vitro Maturation of Oocytes in the Absence of Gonadotropins

Abstract

Abstract.To induce meiotic resumption of porcine oocytes, it is thought to be necessary to expose the cumulus-oocyte complexes (COCs) to gonadotropins during in-vitro maturation (IVM). However, the detailed mechanism of meiotic resumption by gonadotropins is still unknown, and successful piglet production has not been reported by using oocytes matured in gonadotropin-free media and fertilized in vitro. The present study was undertaken to examine the combinational effects of epidermal growth factor (EGF)-family members and dibutyryl cyclic AMP (cAMP) in a chemically defined medium on IVM of porcine oocytes and the developmental competence following in vitro fertilization (IVF). The basic IVM medium was a chemically defined medium, modified porcine oocyte medium (mPOM). Supplementation of the IVM medium with 10 or 1000 ng/ml EGF, amphiregulin and betacellulin during the whole IVM period, except for 10 ng/ml amphiregulin, increased the percentage of oocytes maturing to the metaphase-II stage. When COCs were exposed to both dibutyryl cAMP and EGF-family members during the first 20-h of IVM and then culture was continued in the absence of EGF-family members and dibutyryl cAMP, the incidence of metaphase-II oocytes was significantly increased and was not different from that of oocytes cultured in a standard IVM system with gonadotropins. The developmental competence of the oocytes to the blastocyst stage following IVF was no different from that of control oocytes matured with gonadotropins. When these blastocysts were transferred into the uterine horn of three recipients, all of gilts became pregnant and delivered a total of 11 piglets. These observations indicate that supplementation of a chemically defined maturation medium with EGF-family members and dibutyryl cAMP during the first 20 h of IVM can support well the meiotic progress and developmental competence of porcine oocytes. Key words: Cyclic AMP (cAMP), Epidermal growth factor (EGF), In vitro maturation, Oocyte, Pig (J. Reprod. Dev. 55: [446][447][448][449][450][451][452][453] 2009) uccessful in-vitro production of porcine embryos has the potential to provide an effective system for mass production of embryos for better understanding of the physiology of embryonic development and animal reproductive technologies, including production of transgenic and/or cloned animals [1,2]. Relatively efficient systems for in-vitro production of porcine embryos have already been developed, but they contain materials derived from organisms, such as porcine follicular fluid and bovine serum albumin, in the culture media [3,4]. Chemically defined media, which only known chemicals, but none derived from animals, yeast or plants, should be useful in clarifying the mechanism of interactive communication between the oocyte and the surrounding cumulus cells during meiosis, the oocyte reaction following sperm penetration and early development. Furthermore, application of a defined system for in-vitro embryo production would be good for a quality control against the risk of viral c...

Effect of epidermal growth factor-like peptides on pig cumulus cell expansion, oocyte maturation, and acquisition of developmental competence in vitro: comparison with gonadotropins

“…Consequently, the differences between FSH and the EGF-like peptides in stimulating maturation to MII stage may reflect the relative degree of meiotic resumption. Correspondingly with the results of our study, the EGF-like peptides were shown to stimulate maturation of pig COCs in vitro but they were less efficient than gonadotropins (eCGChCG; Akaki et al 2009). A reason of the lower potential of the EGF-like peptides to initiate resumption of meiosis in isolated pig COCs cannot be specified yet due to incomplete information about mechanisms involved in this process.…”

“…We assume that the reason may lie in low level of cAMP in the AREG-and EREG-stimulated COCs, which may adversely affect function of the cumulus cells and also communication between the oocyte and the cumulus compartment. This assumption is supported by the finding that combination of the EGF-like peptides with dbcAMP during first 20 h of culture improved the results of maturation to the level observed with gonadotropins and IVF, and culture of such oocytes yielded blastocysts with full developmental competence (Akaki et al 2009). In mouse preantral follicle culture model, EREG-induced incomplete mucification and expansion of the cumulus cells had a significantly lower effect than hCG/EGF on meiotic resumption and progesterone production (Romero & Smitz 2009).…”

“…The explanation of these questions requires further studies about the mechanisms driving synthesis of EGF-like peptides in granulosa/cumulus cell and their engagement in a series of post-transcriptional events leading to resumption of meiosis in mammalian oocytes. Nevertheless, the blastocysts originated from AREG-treated COCs and transferred to recipients supported development to term (Akaki et al 2009), indicating the capability of exogenous EGF-like peptides to induce a physiological mechanism leading to completion of meiosis and acquisition of full oocyte developmental competence.…”

Signaling pathways regulating FSH- and amphiregulin-induced meiotic resumption and cumulus cell expansion in the pig

“…The EGF-like peptides are also produced in cumulus cells of in vitro cultured cumulus-oocyte complexes (COCs) upon stimulation by FSH and trigger resumption of meiosis (Downs & Chen 2008). The data published by our and other laboratories strongly suggest that similar mechanisms work also in large animal species (Prochazka et al 2000, Akaki et al 2009, Yamashita et al 2009. We have recently shown that FSH induces expression of AREG and EREG but not BTC in cultured pig COCs and that these peptides stimulate expansion of cumulus cells, maturation of oocyte, and acquisition of the oocyte developmental competence.…”

In-vitro Culture with a Tilting Device in Chemically Defined Media During Meiotic Maturation and Early Development Improves the Quality of Blastocysts Derived from In-vitro Matured and Fertilized Porcine Oocytes

“…Although many piglets have been produced following IVM, in vitro fertilization (IVF) and in vitro culture (IVC) [2,3], the quality of embryos produced in vitro is still poor, and the developmental competence remains an area requiring further research [3,4]. Furthermore, a successful IVM-IVF-IVC system utilizing chemically defined media has been recently developed to produce blastocysts and piglets [5,6]. Development of an efficient system for in-vitro production of embryos in chemically defined media would have an advantage in research to clarify the mechanism or effects of various materials during IVM, IVF and/or IVC.…”