Dynamic Changes in Meiotic Progression: INTRODUCTION
Pig oocytes have been successfully matured and fertilized in vitro (IVF), and in vitro culture (IVC) conditions that support development to the blastocyst stage have been established. Recent work has focused on improving the culture conditions for in vitro maturation (IVM) to improve the efficiency of in vitro production (IVP) of embryos. In early studies of pig IVM/IVF, nuclear maturation was achieved, but problems existed with poor male pronuclear (MPN) formation and polyspermic fertilization. This showed that cytoplasmic maturation as well as nuclear maturation was necessary for the IVP of viable embryos. Poor MPN formation is avoided by supplementing the maturation medium with cysteine to increase the concentration of glutathione in matured oocytes, but the problem of polyspermy remains. Further modification of the maturation medium is required to improve oocyte developmental competence and blastocyst yield. Despite numerous attempts, few laboratories achieve rates of blastocyst development greater than 30%. buy zyrtec online
A better understanding of the inter- and intracellular processes underlying oocyte maturation is essential to improve pig IVP. Many factors are beneficial for oocyte maturation and subsequent embryo development, but their mechanisms of action are poorly understood. In particular, it is not known precisely how FSH and/or LH influence oocyte maturation. These hormones commonly are added to maturation medium, because in vivo, the gonadotropin surge induces oocyte maturation in preovulatory follicles. However, oocytes removed from follicles will mature spontaneously in the absence of FSH and LH. Epidermal growth factor (EGF) has an effect on oocyte IVM similar to that of gonadotropins and may be used in the medium, but it may have differential effects on nuclear and cytoplasmic maturation. Previous studies concerning the influence of these treatments on nuclear maturation investigated only the accumulated rate of attainment of metaphase II (MII), ignoring the dynamics of meiotic progression and its relationship to cytoplasmic maturation and developmental competence. In addition, most previous studies used complex maturation media containing follicular cell constituents, follicular fluid or serum, in which unspecified hormones and growth factors may have been present.
It is possible that aspirated oocytes may have already acquired developmental competence, with only limited potential for change during conventional culture. Oocytes from larger follicles usually are more competent than those from smaller ones, and those from sows develop better than those from prepubertal gilts. The heterogeneity of oocytes from different sources leads to asynchronous meiotic progression during IVM, especially because pig oocytes need a longer culture period than those of other species. Although oocytes usually are ‘‘fully grown’’ in middle- or large-sized follicles, they are less developmentally competent when matured in vitro than when they are matured in vivo. This may be caused by removal of a final growth or maturation phase. Reducing nuclear morphological variation (meiotic synchronization) before maturation, by preincubation without gonadotropins or with dibutyryl cAMP (dbcAMP), appears to enhance pig oocyte developmental potential. However, the latter treatment does not improve the development of embryos reconstructed by somatic nuclear transfer.
In vivo administration of FSH (‘‘coasting’’ for 2 days) and LH before oocyte aspiration by ovum pickup results in approximately 80% of bovine oocytes developing to blastocysts following IVPs. To allow a similar treatment in vitro, meiosis needs to be reversibly arrested. This can be achieved physiologically by coculture with different follicle components, such as theca cells, but the identity of the meiosis inhibitor is unknown. Recently, bu-tyrolactone I and roscovitine, which are specific inhibitors of Cdc2 (a universal G2/M-phase regulator in eukaryotic cells), have been found to arrest meiosis in vitro. These drugs reversibly block meiotic resumption and may be used to synchronize subsequent nuclear maturation. However, there is little evidence to suggest any significant improvement in oocyte developmental competence, and to our knowledge, no proof of full-term development in any species has been reported.
Protein synthesis is essential for meiotic resumption of oocytes in vitro in the pig, as in some other mammals. Cycloheximide (CHX), a nonspecific protein-synthesis inhibitor, can reversibly block meiotic resumption in porcine and bovine oocytes. The CHX-pretreated and matured bovine oocytes can be successfully fertilized, and they develop to the blastocyst stage at a rate similar to that of untreated ones. The birth of live calves from CHX-pretreated oocytes has confirmed that the effect of CHX truly is reversible.
A reliable culture system in a relatively simple, defined maturation medium, together with a highly predictable meiotic progression, is necessary to study the effects of any given factor on oocyte maturation and to assess developmental capacity. We have shown that nuclear maturation of pig oocytes can be efficiently synchronized at the germinal vesicle (GV) stage by pretreatment with CHX. Le Beux et al. have reported that CHX is more effective than butylactone or roscovitine for achieving reversible meiotic arrest of porcine oocytes. However, it remains unknown whether CHX treatment can affect fertilization or subsequent embryo development. The present study examined the effects of FSH, LH, and eGf on the nuclear dynamics of pig oocyte maturation and evaluated the feasibility of producing pig embryos from CHX-pretreated oocytes.