Butyrolactone-I (BL-I) and roscovitine (ROSC) are selective inhibitors of the cyclin-dependent kinases, and both have been shown to reversibly inhibit meiotic resumption in cattle oocytes for 24 hr without having a negative affect on subsequent development to the blastocyst stage. The aim of the present study was to describe the morphological changes occurring in fully grown immature and in vitro matured bovine oocytes following exposure to either BL-I or ROSC for 24 hr at concentrations known to be consistent with normal development. Immature bovine cumulus oocyte complexes, recovered from the ovaries of slaughtered heifers, were incubated for 24 hr in the presence of one of the inhibitors. They were then either fixed immediately and processed for transmission electron microscopy (TEM), or cultured for a further 24 hr in the absence of the inhibitor, in conditions permissive to maturation, and subsequently processed for TEM. A control group of oocytes were processed for TEM immediately upon recovery (0 hr) or following in vitro maturation (IVM) for 24 hr. In general, incubation with either inhibitor disrupted the integrity of the surrounding cumulus cells and affected their subsequent expansion during IVM. Within the oocyte cytoplasm, swelling of the mitochondrial cristae was immediately noticeable following meiotic inhibition in the presence of ROSC, while an increased population of pleomorphic mitochondria and mitochondria with electron lucent matrices following BL-I treatment was not observed until after the subsequent IVM period. Both inhibitors caused degeneration of the cortical granules, effectively reducing the population, most noticeably following IVM. At the level of the nucleus, both inhibitory treatments caused convolution of the nuclear membrane, furthermore, aberrant structures were observed within the nucleoplasm of ROSC-treated cumulus oocyte complexes (COCs). In conclusion, while it has been shown that inhibition of meiotic resumption using specific cdk inhibitors is possible and that such oocytes are capable of undergoing maturation, fertilization, and early embryo development, there is as yet no definitive proof that oocytes treated in this way can ultimately give rise to normal offspring. We have shown here that some modifications are induced in the oocytes at the ultrastructural level. Whether or not these modifications are compatible with normal gestation and the birth of a live calf remain to be elucidated.

The sterol 4,4-dimethyl-5-cholesta-8,14,24-trien-3-ol (follicular fluid meiosis-activating sterol [FF-MAS]) isolated from human follicular fluid induces resumption of meiosis in mouse oocytes cultured in vitro. The purpose of this study was to examine the hypothesis that differential signal transduction mechanisms exist for FF-MAS-induced and spontaneous in vitro resumption of meiosis in mouse oocytes. Mouse oocytes were dissected from ovaries originating from mice primed with FSH 48 h before oocyte collection. Mechanically denuded germinal vesicle (GV) oocytes were in vitro matured in medium supplemented with hypoxanthine and FF-MAS or allowed to mature spontaneously; both groups were exposed to individual compounds known to inhibit specific targets in the cell. After 20-22 h of in vitro maturation, resumption of meiosis was assessed as the frequency of oocytes in GV breakdown (GVBD) stage. Pertussis toxin (2.5 microg/ml) did not influence resumption of meiosis in either group. Dibutyryl cyclic GMP (320 microM) inhibited FF-MAS-induced GVBD, but not spontaneous GVBD, whereas the subtype 5 phosphodiesterase-inhibitor zaprinast (50 microM) inhibited GVBD in both groups. Microinjection of the catalytic subunit of cAMP-dependent protein kinase into oocytes inhibited spontaneous GVBD, but not FF-MAS-induced GVBD. An inhibitor of cytoplasmic polyadenylation, cordycepin (80 microM), inhibited or retarded spontaneous GVBD to a further extent than it did FF-MAS-induced GVBD. Spontaneous GVBD was more sensitive to the histone H1 kinase-inhibitor olomoucine (250 microM) than was FF-MAS-induced GVBD. Addition of the mitogen-activated protein kinase (MAPK)-inhibitor PD 98059 (50 microM), phospholipase C-inhibitor U-73122 (10 microM), p21(ras)-inhibitor lovastatine (250 microM), and the src-like kinase inhibitor PP2 (20 microg/ml) inhibited FF-MAS-induced GVBD, but not spontaneous GVBD. Both MAPKs, extracellular regulated kinase (ERK) 1 and ERK2, were phosphorylated under FF-MAS-induced meiotic resumption, in contrast to spontaneous meiotic resumption, in which ERK1 and ERK2 phosphorylation occurred 2 h after GVBD. In the present study, we show that FF-MAS acts through an MAPK-dependent pathway, and we suggest that src-like kinase, p21(ras), and phosphoinositide signaling lie upstream of MAPK in the FF-MAS-activated signaling pathway. Clearly, striking pathway differences are present between spontaneous versus FF-MAS-induced meiotic resumption.

No abstract

Follicular fluid meiosis-activating sterol (FF-MAS) has been isolated from the follicular fluid (FF) of several species including man. FF-MAS increases the quality of in vitro oocyte maturation, and thus the developmental potential of oocytes exposed to FF-MAS during in vitro maturation is improved. The aim of the present study was to investigate the effects of FF-MAS on porcine oocyte maturation and pronucleus formation in vitro. Porcine cumulus-oocyte complexes (COCs) were isolated from abattoir ovaries and in vitro matured for 48 h in NCSU 37 medium supplemented with 1 mg/l cysteine, 10 ng/ml epidermal growth factor and 50 microM 2-mercaptoethanol with or without 10% porcine follicular fluid (pFF). For the first 22 h, 1 mM db-cAMP and 10 I.E PMSG/hCG was added. The medium was supplemented with 1 microM, 3 microM, 10 microM, 30 microM or 100 microM FF-MAS dissolved in ethanol. After maturation the COCs were denuded mechanically using a fine glass pipette under constant pH and in vitro fertilized with fresh semen (5 x 10(5) spermatozoa/ml). The presumptive zygotes were evaluated 18 h after fertilization. The addition of pFF increased the monospermic as well as the polyspermic penetration of oocytes. In the absence of pFF, the addition of FF-MAS decreased the polyspermic penetration rate, whereas FF-MAS in combination with pFF decreased monospermic and increased polyspermic penetration. The degeneration rate of ova decreased in the presence of FF-MAS irrespective of the presence or absence of pFF. In the absence of pFF, FF-MAS at 3-10 microM increased the number of zygotes with advanced maternal pronuclear stages. In supraphysiological doses, i.e. 30-100 microM, FF-MAS dose-dependently and reversibly inhibited nuclear maturation in the absence of pFF.

To explore the possible signaling pathways of meiosis-activating sterol (MAS)-induced oocyte maturation and to elucidate whether the MAS pathway involves transcription or translation, arrested immature mouse oocytes were cultured with either the protein synthesis inhibitor cycloheximide or the heteronuclear RNA inhibitors alpha-amanitin or actinomycin D, respectively. Moreover, the possible involvement of a G protein-coupled receptor mechanism in MAS-mediated oocyte maturation was explored by influencing oocyte maturation with cholera toxin (CT). MAS-induced oocyte maturation was completely blocked by the addition of 50 microg/ml cycloheximide 4 h before the addition of MAS. Simultaneous addition of MAS and the protein synthesis inhibitor also significantly reduced the meiotic resumption compared to that in MAS-treated controls. In contrast, neither of the treatment regimens to inhibit transcription of DNA to RNA was observed to have any effect on the MAS-induced resumption of meiosis. CT was observed to inhibit MAS-induced, but not spontaneous, oocyte maturation in vitro, suggesting a putative involvement of G protein-coupled receptor mechanism in the MAS mode of action. In conclusion, protein synthesis was found to be an essential requirement for maintaining the oocytes' responsiveness to MAS-induced resumption of meiosis, in contrast to transcription.

A rapid increase in FF-MAS level occurred in vivo in the rabbit ovary in response to LH, and clear differences were seen in the cAMP pattern during spontaneous and induced oocyte maturation in mice.

Various components of the ovarian follicle as well as different chemicals can suppress the resumption of meiosis in cumulus-oocyte complexes (COCs). In this study the nuclear ultrastructure of bovine COCs was assessed after 8 h of meiotic inhibition with 50 microM roscovitine (ROSC), 50 microM butyrolactone (BL-I), 2 mM 6-DMAP, 2 microM cycloheximide (CX), or a theca cell monolayer (TC). COCs were recovered according to standard in vitro methods, cultured in a simple and defined medium, and processed for transmission electron microscopy. Control COCs were processed before onset of culture and multiple oocytes were evaluated for each treatment. In all groups, the oocyte nucleus presented a dense fibrillar nucleolus consisting of a fibrillar sphere with a fibrillar center. In TC and 6-DMAP inhibited COCs condensed chromatin adhered to the nucleolus while in all other groups the perinuclear chromatin was separated from the nucleolus. In ROSC inhibited COCs, the nuclear envelope presented only slight small amplitude undulation. The BL-I-inhibited COCs presented an intermediate level of low amplitude undulation of the NE. In CX, 6-DMAP, and TC inhibited COCs the nuclear envelope presented extensively low amplitude undulations. In ROSC inhibited COCs, electron-dense granules formed ring-shaped structures. In some of the BL-I inhibited COCs multiple stellate crystal-like structures were found, and in these COCs the nuclear envelope and the perinuclear cisternae appeared less distinct than in the other BL-I inhibited COCs. In 6-DMAP inhibited COCs interchromatin-like granule clusters were present. In conclusion, the oocyte nuclei in all COCs presented a dense fibrillar nucleolus resembling that in control COCs. However, variations were observed in 1) the nuclear envelope morphology; 2) the chromatin location in relation to the nucleolus; and 3) the presence of different populations of intranuclear granules. Although all treatments inhibited oocyte nucleus breakdown, the mechanisms underlying these effects are different and require further characterization. Mol. Reprod. Dev. 59: 459-467, 2001.

The sterol, 4,4-dimethyl-5alpha-cholesta-8,14,24-trien-3beta-ol (FF-MAS), isolated from human follicular fluid, can induce resumption of meiosis in denuded and cumulus-enclosed mouse oocytes inhibited by hypoxanthine, IBMX, or dibutyric cyclic adenosine monophosphate. In this study the distribution of FF-MAS binding sites in denuded oocytes and in cumulus-oocyte complexes (COCs) was studied using light microscopic (LM) and transmission electron microscopic (TEM) autoradiography in marmoset, cow, and mouse oocytes. Denuded (n = 39) and cumulus-enclosed (n = 28) marmoset, cow, and mouse oocytes were cultured in the presence of [3H]FF-MAS with and without excess of unlabeled FF-MAS, respectively. In denuded oocytes LM autoradiography demonstrated specific binding to the oolemma and zona pellucida and, to some extent, the cytoplasm. In the nucleus, no specific binding of [3H]FF-MAS was demonstrated. In some COCs the labeling was dispersed throughout the zona pellucida, the oolemma, and the cytoplasm as well as the cumulus cells; whereas in others, only the outer part of the cumulus cells were labeled. TEM autoradiograms of denuded cow oocytes (n = 6) demonstrated that specific [3H]FF-MAS binding was closely related to the oolemma and that a low level of [3H]FF-MAS binding to cumulus cell remnants was present. In conclusion, specific binding of FF-MAS is predominant at the oolemma of denuded oocytes, suggesting the existence of a plasma membrane-associated molecule with affinity for FF-MAS (i.e., a putative FF-MAS receptor).