Dynamic Changes in Meiotic Progression: DISCUSSION
Numerous studies have examined the effects of FSH, LH, and EGF on oocyte maturation, but very few have investigated their influence on the dynamics of meiotic progression in relation to cytoplasmic maturation. This may result from the lack of a suitable IVC system. Some of the terms used to describe effects on nuclear maturation are confusing. In the present study, we use stimulate for effects on the accumulated number of oocytes reaching nuclear maturation at a given time point (i.e., the effective rate of maturation), accelerate for identified effects on the speed of the nuclear maturation process, and enhance for effects on cytoplasmic maturation only. With both conventional and CHX-synchronized culture systems, the present study demonstrates that FSH and EGF, either alone or together, stimulate nuclear maturation in oocytes of both gilts and sows. However, whereas FSH and EGF have an additive effect on gilt oocytes, either FSH or EGF alone is sufficient to stimulate nuclear maturation in sow oocytes. Additionally, LH has no effect on meiotic resumption and does not interact with FSH or EGF, although it may influence later stages in CHX-pretreated oocytes (Fig. 4). Both FSH and EGF stimulate nuclear maturation, but their mechanisms apparently are different: FSH slows meiotic progression but supports maturation and even stimulates nuclear maturation by reducing its speed; EGF in the presence of FSH cannot significantly influence the speed of nuclear maturation. www.canadian-familypharmacy.com
The gonadotropin surge (LH/FSH) induces meiotic resumption of oocytes in preovulatory follicles in vivo. For this reason, these hormones commonly are supplemented in the maturation medium, either alone or in combination. Earlier studies indicated that FSH and LH both stimulate nuclear maturation in pig oocytes, with medium supplementation for only the first half (20-22 h) of culture being enough to improve maturation rates considerably. This approach has been adopted in most subsequent studies. In contrast, the present study shows that whereas FSH stimulates nuclear maturation, LH has virtually no effect (experiment 1). Nevertheless, this observation is not inconsistent with a maturational role for the gonadotropin surge in vivo, because this consists of an LH rise either coincident with or followed by a smaller FSH rise (for review, see ). Recent evidence shows very few, if any, LH receptors on porcine cumulus cells, at least in middle-sized follicles, although their formation may be stimulated by FSH. Although the mechanisms resulting in resumption of meiosis in oocytes have yet to be defined in mammals, in vitro studies suggest that FSH, rather than LH, induces meiotic resumption. Early preparations of LH likely were less pure than those used in the present study and may have contained FSH. In contrast, the stimulatory effect of FSH on oocyte maturation is confirmed.
Because of their general stimulation of nuclear maturation, EGF and FSH would be expected to accelerate meiotic progression. However, this was not the case: EGF treatment caused no difference in the rate of maturation to MI as measured at 14 h after CHX pretreatment, and oocytes already had matured at maximal rates to MII by 24 h (experiment 2). As shown previously, pretreatment with CHX and culture in medium supplemented with LH did not alter the pace of nuclear maturation: Maturation remained highly synchronized and predicable at 36 h for GVI oocytes to mature to MII and at 24 h for those undergoing GVBD (at or beyond GVII) to mature to MII. Unexpectedly, FSH slowed meiotic progression and supported nuclear maturation. This effect of FSH also has been noted recently by Schoevers et al., who observed that FSH retarded GVBD during the first half (20 h) of the culture period. However, their rates of maturation were not consistent among different experiments (no pretreatment for synchronization), and they did not investigate the interactions of FSH with other putative stimulators, such as EGF or LH. Whether FSH just postpones meiotic resumption (GVBD) or slows every phase of nuclear maturation remains to be investigated.
It is notable that FSH slows meiotic progression but stimulates the rate of nuclear maturation. This suggests an effect on cytoplasmic maturation that may be significant for subsequent embryo development. Because FSH can increase the concentration of cAMP in COCs, it may take longer for the oocyte to achieve the critical reduction in the concentration of cAMP that is necessary for meiotic resumption. Additionally, FSH might act directly through receptors on the oocyte. Positive effects of FSH on oocyte cytoplasmic maturation have been reported in many other studies. The present results suggest that improved cytoplasmic maturation may be associated with a decrease in the speed of meiotic progression and be reflected in a significant increase in the rate of cleavage after IVF. A decrease in the speed of nuclear maturation may facilitate cytoplasmic maturation by allowing the synthesis of specific proteins.
The present study demonstrates, to our knowledge for the first time, that pig oocytes pretreated with CHX to arrest meiosis temporarily can be fertilized successfully and will develop to the blastocyst stage at a higher frequency than untreated oocytes (experiment 3). This suggests that such pretreatment improves oocyte quality. It is possible that blastocyst yields from CHX-pretreated oocytes could be further increased by improvements to culture conditions: The development rate of untreated control oocytes was relatively low (10%) in the present study compared with those of other conventional cultures (e.g., 14-23% as reported by Marchal et al. ). The maturation medium influences subsequent embryo development (e.g., NCSU 23 may be better than Medium 199 ), and blastocyst yield is affected by features of the IVF protocol, such as boar identity, availability of frozen sperm, and ratio of medium volume or sperm to the number of oocytes.
Bovine oocytes treated with CHX before maturation retain full-term developmental competence, as demonstrated by the birth of calves. However, to our knowledge, no associated improvement in blastocyst yield has been observed, suggesting species-specific differences or a need for different levels or periods of exposure to CHX. Development to the blastocyst stage was compromised when bovine oocytes were pretreated with CHX at 10 ^g/ml for 24 h, although fertilization and cleavage appeared to be normal. However, the development of CHX-pretreated oocytes was as good as that of untreated ones when maturation medium was supplemented with hormones, the period of pretreatment was reduced (<18 h), or the period of maturation after pretreatment was prolonged (>24 h).
The present results agree with those of Funahashi et al., who showed that synchronization of nuclear maturation (by dbcAMP or by omission of hormones) improved subsequent embryo development. The improvement in pig oocyte developmental competence brought about by CHX may result from synchronized nuclear maturation, because asynchronous maturation is associated with poly-spermic fertilization resulting from suboptimal timing of insemination. Pretreatment with CHX has been reported to reduce the rate of polyspermy in bovine oocytes. In the present study, CHX pretreatment had no effect on this fertilization parameter when tested across a range of IVF protocols. More likely, CHX acts on oocytes either directly or through cumulus cells and improves meiotic and developmental competence during the blockage of GVBD: Almost all treated oocytes underwent GVBD on release from CHX, even without any further putative stimulators (experiment 2). Surprisingly, CHX pretreatment appeared to eliminate any intrinsic difference between the oocytes of gilts and sows not only in their meiotic competence but also in their subsequent development.
In conclusion, pig oocytes meiotically arrested by CHX before maturation not only retain but also improve their developmental competence. Supplementation with FSH stimulates the nuclear maturation rate but slows meiotic progression, implying an enhanced cytoplasmic maturation. Combining CHX-prematuration treatment and FSH supplementation of maturation medium results in an excellent blastocyst yield and has potential as a routine procedure both to improve pig IVP and to facilitate the application of novel animal biotechnologies.