Histological sections prepared from cortical parts of 25 bovine ovaries were used to study initiation of follicle growth in vivo. Small follicles were measured and characterized. Initiation of follicle growth consisted of two distinct consecutive phases. The first phase was characterized by transformation of granulosa cells from a flattened to a cuboidal shape and by their proliferation. In the second phase an increase in the number of granulosa cells was accompanied by a rapid increase in the size of the oocyte. Oocytes commenced growth when there were at least 40 granulosa cells in the largest cross-section (fourth generation of follicle cells). The oocyte diameter increased from 29.74 +/- 0.30 microns (mean +/- SEM) in primordial follicles to 92.90 +/- 4.50 microns in small antral follicles. The zona pellucida first appeared as an island of periodic acid-Schiff positive material in small preantral follicles, but formed a complete ring around the oocyte when the late preantral stage was reached. Organ culture of ovarian cortical explants was used to study initiation of follicle growth in vitro. Within 2 days of culture most of the primordial follicles entered the growth phase: granulosa cells changed from a flattened to a cuboidal shape and entered S-phase as demonstrated by autoradiography after [3H]thymidine incorporation. On day 2, 48.6% of follicles were labelled compared with 3% on day 0. Follicle growth started in the absence of gonadotrophins, in the serum-free medium, confirming the notion that gonadotrophins are not essential for this process. The culture system used here will be helpful in the study of the involvement of putative factor(s) in the initiation of follicle growth in large domestic animals.

In this study we have used acridine orange staining, as described by Evenson (1990), to follow changes in DNA packaging as they occur in hamster spermatozoa which have left the testis and are undergoing maturation in the epididymis. Measurement of the green and red fluorescent intensities of hamster sperm nuclei by flow cytometry demonstrated a decrease in acridine orange binding to DNA as sperm made their way from proximal corpus epididymis to the vas deferens. Using sperm from the cauda epididymis of the mature hamster as the standard, a method was developed for estimating the % of cells in a given sample that have matured with regard to DNA packaging. Staining with bromobimane was used to determine the extent of sulfhydryl oxidation in the nuclei. It was seen that sulfhydryl oxidation occurred mainly in the cauda epididymis whereas another process in chromatin condensation occurred earlier, during sperm passage through the caput epididymis. This earlier process could be mimicked by incubating sperm nuclei with alkaline phosphatase, suggesting that it consists of removal of phosphate in protamine.

During passage of hamster spermatozoa through the epididymis their maturation is shown to involve changes in the sperm head, midpiece (mitochondria) and tail. The sum of these changes results in a dramatic increase in the fertilizing potential of the spermatozoa. When comparable numbers of spermatozoa from the caput or corpus epididymis were injected into one uterine horn of mature females, following ovulation induction, and spermatozoa from the cauda epididymis were injected into the contralateral horn, no fertilization was observed with caput epididymal spermatozoa, 1.7% of oocytes were fertilized by corpus epididymal spermatozoa, whereas 79.5% fertilization was obtained with cauda epididymal spermatozoa. Total sperm numbers increased from caput to corpus to cauda [28.3 +/- 12.2, 40.6 +/- 20.8, 144 [corrected] +/- 62 million, respectively]. The percentage of progressively motile spermatozoa increased from 27.9 +/- 6.4 to 33.8 +/- 4.8 to 70 +/- 10.7 during this passage. Viability, measured by exclusion of the dye, propidium iodide, was significantly less in spermatozoa from the cauda than from the proximal or mid-caput epididymis. The percentage of the live cells that were stained intensely by rhodamine-123 (a measure of mitochondrial membrane potential) increased during epididymal passage from 22.8 +/- 7.8% in the proximal caput epididymis to 57.2 +/- 16.5% in the cauda epididymis. Staining with acridine orange (a measure of DNA packaging in the sperm head) indicated an increase in chromatin condensation in cauda epididymal spermatozoa, when compared to those obtained from the caput or corpus.

1. The effect of age on ovarian function was studied in 245-, 350-, 500-, 700- and 800-d-old Lohmann hens. The effect of three different methods for moult induction on ovarian function and corticosterone concentration was studied in 500-d-old hens. 2. No significant reductions in ovarian weight or in number of follicles before the age of 700 d were found. The ability to produce progesterone and oestradiol-17beta was unchanged up to the age of 700 d and the circadian secretion of these two steroids was identical in young (225 d) and old hens (600 d). 3. The effects of induced moulting by feed withdrawal (FW) and a high Zn (HZn) diet on body weight and ovarian function were very similar; those of a moderate Zn with low Ca (MZn/LCa) diet were smaller. 4. The first significant effect of moulting was a decrease in oestradiol-17beta plasma concentration (d 2). Plasma progesterone decreased more gradually than oestradiol-17beta, and reached a nadir on d 6 in FW- and HZn-treated hens and on d 9 in MZn/LCa-treated ones. 5. Hens treated with either FW or the MZn/LCa, but not those with the HZn diet, showed a very sharp rise in corticosterone concentration on d 2 of treatment. Thus the MZn/LCa diet was less efficient than the other treatments in induction of ovarian involution, but had a similar effect on stress induction, as indicated by increases in plasma corticosterone.

The aim of the present study was to characterize gene and protein expression of follistatin, inhibin alpha (alpha) and inhibin betaA (betaA) subunits in the ovaries of postnatal (3-week-old) and prepubertal (14 and 20-25-week-old) lambs. Northern blot analysis revealed the presence of two alpha and two betaA mRNAs. In postnatal ovary the a 1.2-kb transcript was abundant, its amount gradually falling, while the 2.0-kb mRNA increased and became a major band at 20-25 weeks. Both betaA mRNAs, 4.5 kb and 6.0-7.5 kb, were weakly expressed in postnatal ovary, but whereas the 4.5-kb mRNA expression remained at a low level, that of the 6.0-7.5 kb mRNA increased about five fold in prepubertal ovary. The ratio of total alpha mRNAs to the dominant betaA form (6.0-7.5 kb) varied from 1.27 at 3 weeks to 0.33 at 20-25 weeks of age. One major follistatin mRNA of 2.5-3.6 kb was recognized and was constitutively expressed during ovarian growth. Several molecular-mass forms of alpha and betaA subunits with different compositions were seen in prepubertal compared with postnatal ovaries, the latter exhibiting more active follicular growth. In summary, ovine ovaries undergo distinct changes early in life, both morphological and functional, and show a changing pattern of inhibin subunit expression.