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Preantral follicles (140-160 microm) were isolated mechanically from the ovaries of 10-day-old rats and cultured in groups of four to six for 6 days in medium containing 0, 1, 10, or 100 ng/ml of activin A. Activin stimulated (P < 0.05) the growth of preantral follicles in a dose-dependent fashion and enhanced the proliferation of preantral, oocyte-free follicular cells. Furthermore, treatment with activin induced the majority of follicles to form an antrum-like structure and helped to maintain the ultrastructure of these follicles during culture. Activin A also induced further changes characteristic of follicle and oocyte maturation, such as the elongation of granulosa cells contacting the oocyte and migration of the cortical granules to the oocyte cortex. In addition, gene expression for activin and activin receptor type II (ActR II) was demonstrated in both the oocytes and the somatic cells using the reverse transcription-polymerase chain reaction, and immunohistochemical studies demonstrated the presence of activin and ActR II proteins in the somatic tissue and, especially, the oocytes of these follicles. It is concluded that, in vitro, activin A stimulates the growth of rat preantral follicles and promotes antrum formation. Furthermore, because activin A and ActR II are synthesized within preantral follicles, intrafollicular activin likely plays an important role in early follicular development.

The primary aim of this study was to establish a flow cytometric technique for determining the capacitation status of stallion spermatozoa. To this end, a flow cytometric technique that demonstrates changes in plasma membrane fluidity; namely, merocyanine 540 staining, was compared with the more conventional Ca(2+)-dependent fluorescence microscopic technique, chlortetracycline (CTC) staining, for assessing capacitation status. In addition, the effect of bicarbonate/CO(2) on the progress of capacitation and the acrosome reaction (AR) and on temporal changes in sperm motility, with particular regard to hyperactivation, was analyzed. For the study, fresh semen was washed and then incubated for 5 h in bicarbonate-containing or bicarbonate-free medium, with or without Ca(2+) ionophore to induce the AR, and at intervals during incubation aliquots were taken and analyzed for capacitation and acrosome status. The AR was assessed using both the CTC and fluorescein isothiocyanate-peanut agglutinin (FITC-PNA) staining techniques with similar results. In brief, it was found that merocyanine 540 detects capacitation-related changes much earlier than CTC does (0.5 h versus approximately 3 h), and that flow cytometry for evaluation of capacitation and AR was a quicker (10 sec per sample) and more accurate (10,000 cells counted) technique than fluorescence microscopy. Furthermore, it was observed that Ca(2+) ionophore could not induce the AR in the absence of bicarbonate, but that the ionophore synergized the bicarbonate-mediated induction of the AR as detected by CTC (although it was not significant when evaluated using FITC-PNA). The percentage of hyperactive sperm in each sample was not affected by time of incubation under the experimental conditions studied. In conclusion, merocyanine 540 staining is a better method than CTC staining for evaluating the early events of capacitation for stallion spermatozoa incubated in vitro. Furthermore, bicarbonate sperm activation clearly plays a vital role in the induction of the AR in stallion spermatozoa.

We describe isolation and characterization of the bovine ortholog of POU5F1 (bPOU5F1) encoding octamer-binding transcription factor-4 (Oct-4). The organization of bPOU5F1 is similar to its human and murine orthologs, and it shares 90.6% and 81.7% overall identity at the protein level, respectively. Transient transfection of luciferase reporter constructs in murine P19 embryonal carcinoma cells demonstrated that bPOU5F1 has a functional promoter and contains two enhancer elements, of which one is repressed by retinoic acid. bPOU5F1 was mapped to the major histocompatibility complex on chromosome 23. bPOU5F1 mRNA was detected by nested reverse transcription-polymerase chain reaction in immature oocytes and in in vitro-produced preattachment-stage embryos. Oct-4 in oocytes and in vitro-produced preattachment-stage embryos was demonstrated by indirect immunofluorescence. Confocal laser scanning microscopy revealed Oct-4 in both the inner cell mass and trophoblast cells of the blastocyst until Day 10 of development. Immunofluorescence performed on the outgrowths formed at Day 13 postfertilization from in vitro-produced Day 8 blastocysts showed Oct-4 staining in all cells. This expression pattern suggests that bPOU5F1 acts early in bovine embryonic development but that its expression is not restricted to pluripotent cells of the blastocyst.

The 6-h secretory profiles of LH and FSH and the possible concordance between the episodic release of LH and FSH were studied in 6 beagle bitches during early, mid-, and late anestrus and during the follicular and luteal phases of the estrous cycle. Plasma samples were obtained at 10-min intervals via jugular venipuncture. In all stages of anestrus and in the luteal phase, FSH and LH secretion was pulsatile. All FSH pulses coincided with LH pulses. However, the mean duration of the FSH pulse (115 min) was significantly longer than that of the LH pulse (72 min). The basal plasma LH concentration was low compared with the maximum peak levels, whereas FSH pulses were characterized by relatively low peaks compared with the basal levels. In contrast to the basal plasma LH levels and the area under the curve (AUC) for LH, the basal plasma FSH levels and the AUC for FSH increased significantly as anestrus progressed. During the follicular phase, the secretory pattern of LH was characterized by frequent increases of short duration. During this phase, the basal plasma FSH concentration was relatively low, whereas the basal plasma LH level was high in comparison with that in the other phases of the estrous cycle. The luteal phase was characterized by an increased frequency of LH pulses, a shorter duration of the LH peaks, and a tendency to a lower amplitude of both LH and FSH peaks compared with values observed during anestrus. It is concluded that in the bitch, FSH and LH pulses are released in concordance and that progression from early to late anestrus is associated with an increase in basal plasma FSH concentration without a concomitant rise in basal plasma LH concentrations. The latter suggests that in the bitch an increase in circulating FSH should be considered to be a critical event required for the initiation of ovarian folliculogenesis and consequently for the termination of anestrus.

The aim of the present study was to investigate whether the induction of stallion sperm acrosome reaction (AR) by progesterone is mediated by binding of progesterone to a receptor on the sperm plasma membrane or to an intracellular progesterone receptor. Progesterone-BSA conjugate labeled with fluorescein isothiocyanate (P-BSA-FITC) in combination with a vital stain, ethidium homodimer, was applied to visualize the presence of the progesterone receptor on living spermatozoa. Alternatively, an indirect immunofluorescence technique employing a monoclonal antibody (C-262) against human intracellular progesterone receptor was conducted to validate the presence of the progesterone receptor. Immunogold labeling techniques enabled ultrastructural localization of P-BSA-FITC or C-262 with transmission electron microscopy. The dynamic changes in labeling patterns were monitored for sperm cells, using fluorescence microscopy and flow cytometry during a 5-h capacitation period. An increasing number of viable cells showed affinity for P-BSA-FITC or C-262 at the acrosomal plasma membrane region of the sperm head, while a decreasing number of viable cells were not labeled. In contrast, almost all deteriorated cells were labeled in the cytosol of the postequatorial region of the sperm head. Incubation with P-BSA-FITC resulted in the induction of AR but to a lesser extent than that for sperm incubated with free progesterone. Therefore, coupling of progesterone to its receptor on the sperm plasma membrane appears to be an important step in the induction of the AR.

The aim of this study was to investigate whether the stimulatory effect of growth hormone (GH) on the in vitro maturation and cumulus expansion of bovine oocytes is exerted through the cAMP or the tyrosine kinase pathway. Therefore bovine cumulus-oocyte complexes (COCs) were cultured in Medium 199 without fetal calf serum and gonadotropins, but supplemented with 100 ng/ml bovine GH (bGH; NIH-GH-B18) with or without 10 microM methyl 2,5-dihydroxycinnamate (erbstatin analogue), a specific tyrosine kinase inhibitor; 100 microM 2',3'-dideoxyadenosine (DDA), a specific adenylate cyclase inhibitor; or 10 microM H-89, a specific inhibitor of cAMP-dependent protein kinase A. Epidermal growth factor (EGF; 20 ng/ml) was added as a positive control for tyrosine kinase activation, and FSH (0.05 IU/ml) was added as a positive control for cAMP mediation during in vitro maturation in the absence or presence of the inhibitors. Culture was performed at 39 degrees C in a humidified atmosphere with 5% CO2 in air. To assess the effect on nuclear maturation, the proportion of oocytes in metaphase II stage after 16 h of culture was determined using 4,6-diamino-2-phenylindole staining. To determine the effect on cumulus expansion, the diameter of COCs at the onset and after 24 h of culture was measured. The stimulatory effects of GH on oocyte maturation and cumulus expansion were blocked by DDA and H-89 (p < 0.01). Similarly, FSH-induced cumulus expansion was abolished by DDA and H-89 (p < 0.05), while DDA did not block either EGF-induced oocyte maturation or cumulus expansion. Erbstatin analogue significantly blocked the stimulation of oocyte maturation and cumulus expansion by EGF (p < 0.02) but did not inhibit GH action on the COCs. It is concluded that the stimulatory effect of GH on oocyte maturation and cumulus expansion is mediated by the cAMP signal transduction pathway and not by JAK2 phosphorylation.

The acrosome reaction is a prerequisite for zona pellucida penetration by mammalian spermatozoa. In some species, the sperm undergo the acrosome reaction before binding to the zona pellucida, and in other species only acrosome-intact sperm can initiate binding to the zona. In the present investigation, we addressed the question whether acrosome-intact or acrosome-reacted boar sperm initiate binding to the pig zona pellucida by studying the acrosomal status of sperm bound to zonae pellucidae. Our approach was to vary the percentage of acrosome-intact sperm in suspension by long preincubation before incubation with hemizonae for 1 min. We hypothesized that if only acrosome-intact sperm are able to initiate binding to the zona pellucida, the majority of the sperm on the zona surface would be acrosome-intact regardless of the percentage of acrosome-reacted sperm in suspension. Fluorescein isothiocyanate-conjugated peanut agglutinin (Arachis hypogaea; FITC-PNA) in combination with optical sectioning by confocal laser-scanning microscopy was used to study the acrosomal status of sperm bound to the hemizona. Electron microscope studies showed that the FITC-PNA binding site is mainly limited to the outer acrosomal membrane of boar sperm, thus validating the use of FITC-PNA as an accurate probe for studying boar sperm acrosome reaction. Over 90% of the sperm bound to a hemizona were acrosome-intact irrespective of whether the majority of sperm in the suspension were acrosome-intact, acrosome-reacting, or acrosome-reacted. There was a significant difference (Kruskal-Wallis test, p < 0.05) in the mean +/- SEM number of sperm bound to the outer side, inner side, and edge of a hemizona (48 +/- 8, 14 +/- 3, and 7 +/- 2; n = 58; respectively). The acrosomal status of sperm bound to the various surfaces of hemizonae was similar. Taking the respective zona pellucida surface area into consideration, it was calculated that an average of 1.9 +/- 0.3, 1.0 +/- 0.2, and 1.5 +/- 0.3 spermatozoa were bound per 1000 microm2 of outer side, inner side, and edge of a hemizona, respectively (mean +/- SEM, n = 38). These observations indicate that acrosome-intact boar spermatozoa initiate binding to the pig zona pellucida. A gradient of sperm binding sites also exists, decreasing from the outside to the inside of the zona pellucida.

The distribution of the neuropeptides vasoactive intestinal peptide (VIP) and neuropeptide Y (NPY) was studied immunocytochemically in bovine ovaries from 3 mo of gestation up to and including puberty, and from adult cows at three stages of the estrous cycle. The appearance of VIP and NPY immunoreactivity of 4.5-6 mo of gestation coincided with the onset of follicular development. In contrast to NPY, VIP was first found in the cortex. Both VIP and NPY immunoreactivity increased with age. From 9 mo of gestation onwards, VIP and NPY were found around blood vessels and non-vascular smooth muscle cells, in the stroma near preantral follicles, and in the theca externa of antral follicles. In addition, VIP-positive cells were observed exclusively in the granulosa layer of the preovulatory follicle at the time of the LH surge. The distribution of VIP- and NPY-immunoreactive fibers in the ovary may point to an effect of these neuropeptides on various physiological processes, including follicle development and ovarian blood flow. In addition, the presence of VIP-positive cells in the granulosa layer of the preovulatory follicle is indicative of a role for VIP in ovulation.

The temporal relationship among changes of the concentrations of the 13,14-dihydro-15-keto metabolite of prostaglandin F2 alpha (PGFM), estrone (E1) and estrone sulphate (E1S) in maternal arterial plasma (MP) and amniotic fluid (AF), the prepartum progesterone (P4) decline in MP, and the evolution of uterine electromyographic (EMG) activity was investigated in 6 cows. Calving was induced by a single i.m. injection of 5 mg flumethason on Day 270 of gestation. The period under investigation was subdivided into four consecutive periods: Period 1 covered the last 2 days before flumethason treatment; Period 2 (mean +/- SEM duration: 16.1 +/- 2.5 h), Period 3 (8.8 +/- 1.1 h), and Period 4 (13.0 +/- 1.5 h) together included the interval between injection and the onset of the expulsive stage of induced parturition. Each was defined by its pattern of uterine EMG activity. During Periods 1 and 2, this activity occurred in long episodes (2-20 min; contractures) at a similar mean (+/- SEM) frequency (0.51 +/- 0.14/h and 0.42 +/- 0.07/h, respectively). No significant differences in hormonal concentrations in MP and AF between these two periods were detected. During Period 3, contractures nearly disappeared (freq: 0.09 +/- 0.05/h), and in MP mean P4 levels were significantly lower and PGFM levels were significantly higher than before. Mean PGFM concentrations in AF were not significantly changed during Period 3. Finally, during Period 4, EMG activity reappeared and a parturient EMG pattern gradually evolved in the presence of a further significant decline of P4 levels and significant increase of PGFM concentrations in MP.(ABSTRACT TRUNCATED AT 250 WORDS)