Modulation of intrafollicular oestradiol in explanted hamster follicles does not affect oocyte meiotic status.

Mature antral follicles were collected from PMSG-primed hamsters before the LH surge on Day 4 of the oestrous cycle and were incubated individually for 6 or 10 h under 95% O2 and 5% CO2. The relationship between follicle size and follicular fluid volume was established, and both the oestradiol output during incubation and the LH sensitivity of the follicle with respect to induction of meiotic resumption were determined. The data obtained from these initial studies were then applied to experiments in which the oestrogenic status of follicles was altered by pressure injection of solutions into the antral cavity to result in known concentrations in follicular fluid of oestradiol (0-500 microM), the anti-oestrogens, tamoxifen (0-200 microM) or enclomiphene citrate (0-500 microM), or an oestradiol-specific antiserum (1:200 dilution of serum). The proportion of immature oocytes (% GV) was determined cytogenetically after follicular incubation. Significant correlations were established between follicle size and both follicular fluid volume (r = 0.90) and follicular oestradiol accumulation (r = 0.96). The minimum concentration of LH tested which induced maximal meiotic resumption (0% GV) was 0.5 microgram/ml. The % GV was not significantly reduced by intrafollicular injection of either of the anti-oestrogens over the concentration ranges tested, or of the oestradiol-specific antiserum, which was injected in an amount adequate to bind all oestradiol accumulated during the incubation. Intrafollicular injection of oestradiol failed to increase the % GV after follicular incubation in the presence of 0.5 microgram LH/ml. From these results we conclude that it is unlikely that oestradiol plays a primary role in the maintenance of meiotic arrest in follicle-enclosed hamster oocytes in vitro.

In vitro and in vivo studies reveal that hamster oocyte meiotic arrest is maintained only transiently by follicular fluid, but persistently by membrana/cumulus granulosa cell contact.

Studies were carried out with the golden Syrian hamster to investigate the capacity of follicular fluid to maintain oocyte meiotic arrest and to determine the importance of cumulus-membrana granulosa cell contact in the regulation of meiotic status. The follicular fluid studies were conducted by cytological assessment of meiotic stage up to 6 hr after transferring cumulus-free oocytes into antra of explanted "host" follicles in vitro or into follicles of anesthetized animals prior to the gonadotropin surge at proestrus in vivo. The cumulus-membrana granulosa contact studies were undertaken with explanted follicles in which the oocyte-cumulus complex was dislodged from the underlying membrana granulosa, released into the antrum, and subsequently allowed to reestablish contact during 6 hr of incubation within the follicle. The extent of recontact of the dislodged complex with the underlying membrana granulosa was assessed visually at the end of incubation and was classified as close, moderate, or none. These various degrees of contact typically involved the following number of cumulus cells, as determined by serial sectioning of a representative sample of follicles after dislodgement and subsequent incubation: close, 32.7 +/- 1.78; moderate, 9.0 +/- 2.1; and no contact, 0. After 6 hr of incubation either in vitro or in vivo, few transferred oocytes remained at the germinal vesicle (GV) stage (18.8 +/- 8.7 and 17.3 +/- 4.0% GV, respectively). However, time course experiments revealed that meiotic resumption was significantly delayed in transferred oocytes compared with either liberated oocytes, spontaneously maturing oocytes, or follicle-enclosed oocytes induced to mature by luteinizing hormone in vitro (after 4 hr, transferred, 31.3 +/- 6.0% GV; liberated, 0% GV; follicle-enclosed, 0% GV; after 6 hr, 0% transferred oocytes exhibited a GV). In the dislodgement studies, after 6 hr of incubation, 26% of complexes reestablished close contact with the underlying membrana granulosa, 67% showed moderate contact, while 7% revealed no contact. There was a significant increase in the percentage GV stage oocytes as the extent of recontact increased (no contact, 21.9 +/- 3.6% GV; moderate contact, 56.6 +/- 6.8% GV; close contact, 87.5 +/- 14.4% GV). These data argue in favor of a stringent control of hamster oocyte meiotic status by the follicle cell/oocyte syncytium and against the possibility that follicular fluid is independently responsible for maintaining meiotic arrest.(ABSTRACT TRUNCATED AT 400 WORDS)

Down-regulation of membrana granulosa cell gap junctions is correlated with irreversible commitment to resume meiosis in golden Syrian hamster oocytes.

One of the currently popular hypotheses for the regulation of meiotic resumption in mammalian oocytes proposes that the preovulatory surge of luteinizing hormone causes down-regulation of follicular gap junctions, which in turn disrupts transfer of a meiotic arrester from the somatic cells into the oocyte. The present study has investigated this hypothesis by examining the integrity of membrana granulosa cell gap junctions during the period of irreversible commitment to maturation of golden Syrian hamster oocytes in vivo. Our results have revealed a significant progressive decrease in the fractional area of cell surface occupied by gap junction membrane with increasing percentage of oocytes irreversibly committed to mature (1.946% and 0.921% fractional gap junction area at 0% and 100% oocytes irreversibly committed to mature, respectively, P less than 0.05). This net loss of membrana granulosa cell gap junctions from the cell surface was accompanied by a significant decrease in density of gap junction particles, whether they were arranged in rectilinear or non-rectilinear packing patterns. Furthermore, the number of gap junction particles per unit area of surface membrane scanned also underwent a significant progressive decrease with increasing percentage of oocytes irreversibly committed to mature. These data with the hamster are consistent with the hypothesis that down-regulation of membrana granulosa cell gap junctions may be of central importance in the regulation of gonadotropic stimulation of meiotic resumption in mammalian oocytes.

Direct effects of nicotine on the meiotic maturation of hamster oocytes.

The effect of nicotine on the meiotic maturation of cultured hamster oocytes was investigated. Oocytes were cultured for up to 24 h and meiotic status was scored cytogenetically from chromatin spreads. The highest concentration of nicotine tested (5 mM) caused marked perturbations at both the first and second meiotic divisions which resulted in degenerating blobs of chromatin at 24 h. The most pronounced of these perturbations was either blockage at Metaphase I or disruption of homologue segregation to result in two groups of bivalents formed at Anaphase I. Concentrations of nicotine at or below 0.5 mM did not adversely affect the meiotic process. These data show, therefore, that nicotine significantly interferes with oocyte meiotic maturation only at concentrations far in excess of 0.1 to 1.0 microM, the concentration range of nicotine anticipated to be present in the blood of an "average" smoker. Thus, we conclude that the documented reduced fertility of women who smoke cigarettes is unlikely to be due to a direct effect of nicotine on oocyte meiotic maturation.

Nicotine and cotinine effects on development of two-cell mouse embryos in vitro.

The independent and interactive effects of nicotine and cotinine on the development of cultured two-cell embryos were investigated. Cultures were maintained for 120 h and developmental stages of embryos were scored after 72 h and at the termination of culture. Concentrations of nicotine at or below 0.5 mM, and concentrations of cotinine at or below 0.008 mM, did not adversely affect development. In addition, neither nicotine nor cotinine produced synergistic effects at higher concentrations at which both independently impaired development. These data show, therefore, that nicotine and its major metabolite, cotinine, significantly interfere with preimplantation development of mouse embryos only at concentrations far in excess of those anticipated to be present in the blood of an "average" smoker. Thus, we conclude that the well documented adverse effects of smoking during pregnancy are unlikely to be attributable to a direct effect of nicotine or cotinine on the preimplantation embryo.