In higher eukaryotes, cell cycle progression is controlled by cyclin dependent kinases (Cdks) complexed with cyclins. A-type cyclins are involved at both G1/S and G2/M transitions of the cell cycle. Cyclin A2 activates cdc2 (Cdk1) on passage into mitosis and Cdk2 at the G1/S transition. Antisense constructs, or antibodies directed against cyclin A2 block cultured mammalian cells at both of these transitions. In contrast, overexpression of cyclin A2 appears to advance S phase entry and confer anchorage-independent growth, and can lead to apoptosis. A second A-type cyclin, cyclin A1 has been described recently which, in the mouse, is expressed in germ cells but not somatic tissues. To address the possible redundancy between different cyclins in vivo and also the control of early embryonic cell cycles, we undertook the targeted deletion of the murine cyclin A2 gene. The homozygous null mutant is embryonically lethal, demonstrating that the cyclin A2 gene is essential. Surprisingly, homozygous null mutant embryos develop normally until post-implantation, around day 5.5 p.c. This observation may be explained by the persistence of a maternal pool of cyclin A2 protein until at least the blastocyst stage, or an unexpected role for cyclin A1 during early embryo development.

In unfertilized eggs from vertebrates, the cell cycle is arrested in metaphase of the second meiotic division (metaphase II) until fertilization or activation. Maintenance of the long‐term meiotic metaphase arrest requires mechanisms preventing the destruction of the maturation promoting factor (MPF) and the migration of the chromosomes. In frog oocytes, arrest in metaphase II (M II) is achieved by cytostatic factor (CSF) that stabilizes MPF, a heterodimer formed of cdc2 kinase and cyclin. At the metaphase/anaphase transition, a rapid proteolysis of cyclin is associated with MPF inactivation. In Drosophila, oocytes are arrested in metaphase I (M I); however, only mechanical forces generated by the chiasmata seem to prevent chromosome separation. Thus, entirely different mechanisms may be involved in the meiotic arrests in various species. We report here that in mouse oocytes a CSF‐like activity is involved in the M II arrest (as observed in hybrids composed of fragments of metaphase II‐arrested oocytes and activated mitotic mouse oocytes) and that the high activity of MPF is maintained through a continuous equilibrium between cyclin B synthesis and degradation. In addition, the presence of an intact metaphase spindle is required for cyclin B degradation. Finally, MPF activity is preferentially associated with the spindle after bisection of the oocyte. Taken together, these observations suggest that the mechanism maintaining the metaphase arrest in mouse oocytes involves an equilibrium between cyclin synthesis and degradation, probably controlled by CSF, and which is also dependent upon the three‐dimensional organization of the spindle.

FSH and IGF-I synergistically stimulate gonadal steroid production; conversely, silencing the FSH or the IGF-I genes leads to infertility and hypogonadism. To determine the molecular link between these hormones, we examined the signaling cross talk downstream of their receptors. In human and rodent granulosa cells (GCs), IGF-I potentiated the stimulatory effects of FSH and cAMP on the expression of steroidogenic genes. In contrast, inhibition of IGF-I receptor (IGF-IR) activity or expression using pharmacological, genetic, or biochemical approaches prevented the FSH- and cAMP-induced expression of steroidogenic genes and estradiol production. In vivo experiments demonstrated that IGF-IR inactivation reduces the stimulation of steroidogenic genes and follicle growth by gonadotropins. FSH or IGF-I alone stimulated protein kinase B (PKB), which is also known as AKT and in combination synergistically increased AKT phosphorylation. Remarkably, blocking IGF-IR expression or activity decreased AKT basal activity and abolished AKT activation by FSH. In GCs lacking IGF-IR activity, FSH stimulation of Cyp19 expression was rescued by overexpression of constitutively active AKT. Our findings demonstrate, for the first time, that in human, mouse, and rat GCs, the well-known stimulatory effect of FSH on Cyp19 and AKT depends on IGF-I and on the expression and activation of the IGF-IR.

The cumulus cell response to FSH resembles the differentiation of preantral to preovulatory granulosa cells. This differentiation program requires IGF1R activity and subsequent AKT activation.

Analysis over the first 48 h of development in vitro from the one-cell stage to the early four-cell stage indicated that (i) ethylenediaminetetraacetic acid (EDTA) exerts the major beneficial effect on culture to the blastocyst stage of F1 and MF1 embryos, (ii) glutamine assists development of MF1, but not F1, embryos to the blastocyst stage and probably functions as part of a metabolic response to oxidative damage to mitochondria and (iii) exposure to glucose at some time during early cleavage is essential for full development to blastocysts. None of the culture conditions examined affected significantly the increase in concentration of reactive oxygen species in late two-cell embryos in vitro, although F1 embryos in vitro often had lower peroxide concentrations than MF1 embryos. A decline in oxygen tension from 20 to 50% had no consistent effect on culture to the blastocyst stage or production of reactive oxygen species. Aminooxyacetate, an inhibitor of transaminase activity, prevented non-blocking embryos from developing beyond G2 of the second cell cycle. It is concluded that the chelation of transitional metals provides the most effective method of overcoming the block to development in vitro.

Progression through the mammalian cell cycle is regulated by the sequential activation and inactivation of the cyclin-dependent kinases. In adult cells, cyclin A2-dependent kinases are required for entry into S and M phases, completion of S phase, and centrosome duplication. However, mouse embryos lacking the cyclin A2 gene nonetheless complete preimplantation development, but die soon after implantation. In this report, we investigated whether a contribution of maternal cyclin A2 mRNA and protein to early embryonic cell cycles might explain these conflicting observations. Our data show that a maternal stock of cyclin A2 mRNA is present in the oocyte and persists after fertilization until the second mitotic cell cycle, when it is degraded to undetectable levels coincident with transcriptional activation of the zygotic genome. A portion of maternally derived cyclin A2 protein is stable during the first mitosis and persists in the cytoplasm, but is completely degraded at the second mitosis. The ability of cyclin A2-null mutants to develop normally from the four-cell to the postimplantation stage in the absence of detectable cyclin A2 gene product indicates therefore that cyclin A2 is dispensable for cellular progression during the preimplantation nongrowth period of mouse embryo development.

Human cleaving pre-embryos at 2 and 3 days and cavitated pre-embryos at 5 days post-insemination have been examined for cell number and the incidence of mononucleated cells. At least 60% of polynucleate or anucleate cells have been detected at all these stages and regardless of morphological grading at day 2. It is concluded that even by the time at which pre-embryo replacement would occur therapeutically, the majority of pre-embryos are unlikely to have full developmental potential. The possible origins of the abnormalities of nucleocytoplasmic ratios are discussed.

The activity of calmodulin-dependent protein kinase II (CaMKII) was measured in mouse oocytes arrested in metaphase II and following their activation parthenogenetically. In metaphase II-arrested oocytes CaMKII was inactive. However, following the exposure of oocytes to ethanol, the kinase was highly active, returning to baseline activity within 15 min of their removal from ethanol. The increase in kinase activity was similar in recently ovulated and older oocytes despite an age-dependent difference in their ability to progress to interphase. Moreover, the microtubule-depolymerizing drug nocodazole, which blocks the exit from M phase in mouse oocytes, had no effect on CaMKII activation. These results illustrate clearly that CaMKII is activated in mouse oocytes in response to a rise in intracellular calcium and is acting upstream of the microtubule-dependent cyclin destruction machinery.