Summary. The inhibitory effects of follicular fluid on FSH secretion were similar in gonadectomized male and female sheep, and in the anoestrous and breeding seasons. Significant suppression of LH was variable and was observed only at the highest dose of follicular fluid when suppression rarely exceeded 50% of pretreatment values. Basal plasma FSH and LH concentrations were higher in castrated males than in ovariectomized females in both seasons. Plasma FSH concentrations in gonadectomized males and females and LH concentrations in the males were lower in the anoestrous than the breeding season. Therefore, in the absence of the gonads, sex and photoperiod can influence hypothalamic control of basal pituitary gonadotrophin secretion in males and females, whereas the feedback effect of non-steroidal factors in follicular fluid (inhibin) on FSH secretion is not influenced by photoperiod or sex.

Ewes actively immunized against αN, the N-terminal peptide of inhibin α₄₃ precursor, have lowered fertility associated with ovulation failure, restricted tissue remodelling and reduced matrix metalloproteinase-2 activity in the follicular fluid at the time of expected ovulation. This could be due to altered ratios of matrix metalloproteinase-2 and tissue inhibitor of matrix metalloproteinase (TIMP-1), or to the onset of atresia in antral follicles destined to ovulate. The objectives of the present study were to investigate the effects of immunization against αN on the localization of TIMP-1 in ovine follicles, and on follicular growth and atresia in the follicular phase. Ewes were either immunized against aN or remained as controls and the ovaries were removed before (0, n = 4) and at 12 h (n = 4) and 24 h (n = 4) after hCG administration in a synchronized follicular phase, 48 h after removal of intravaginal pessaries. Observations were made on a single section taken through the largest follicle present in the ovaries of each ewe. There were no healthy antral follicles > 1 mm in immunized ovaries (0/29) compared with controls (16/31) (P < 0.001), whereas the proportion of healthy antral follicles < 1 mm was the same in each group (9/19 versus 5/12). TIMP-1 immunoactivity was localized in large luteal cells, smooth muscle and endothelial cells, and in all antral follicles, including oocytes. At the time of hCG administration, no TIMP-1 immunoreactivity was detected in the apical region of the follicular wall of large follicles (> 6 mm) compared with the rest of the follicle wall, but staining appeared in the apical granulosa layer 24 h later. In newly formed corpora lutea, TIMP-1 expression was found along the invaginating vascular layer. There was no effect of immunization on the patterns of TIMP-1 immunoreactivity, suggesting that changes in TIMP-1 are not involved in the effects of aN. These data are consistent with a paracrine role for αN in the selection and atresia of antral follicles, and for TIMP-1 in tissue reorganization and steroidogenesis at the time of ovulation.

Immunization of ewes against the amino-terminal peptide (αN) of the pro-α-subunit of inhibin has been shown to reduce fertility, thought to be due to disruption of ovulation. The aims of this study were to examine the effects of active immunization of ewes against αN on circulating concentrations of FSH, LH and on ovarian inhibin and progesterone, and to relate these observations to number of corpora lutea and oocyte recovery rates. Ewes were immunized against one or both of two recombinant full length bovine-αN immunogens (FP₁ and FP₂). Three experiments were performed in which jugular venous plasma was sampled from control and immunized ewes: (1) hourly across the oestrous surge of gonadotrophins (Expt 1); (2) daily for one entire oestrous cycle, and in the subsequent cycle, oviducts were flushed to recover ovulated eggs (Expt 2); and (3) samples were taken at 10 min intervals during the follicular and luteal phases (Expt 3). Binding of ¹²⁵I-labelled αN_1–26 to serum was greater (P < 0.05) in immunized groups than in controls for all experiments. The number of eggs per corpus luteum recovered from the oviducts was lower (P < 0.05) in the αN-immunized groups (39%) than in controls (88%). There were more (P < 0.05) corpora lutea per ewe in FP₂ immunized groups 4 (1.8 ± 0.45) and 5 (1.75 ± 0.5) than in the control group (1.13 ± 0.13), but no increase in group 3 (FP₁; 1.4 ± 0.24). The oestrous surge of LH, basal values across cycle and the frequency of LH pulses were similar in treated and control groups. Circulating FSH concentrations were higher (P < 0.05) than those of controls in all immunized groups that had binding greater than 10% at all stages of the cycle, except during the oestrous surge. A corresponding decrease (P < 0.05) in circulating inhibin concentrations was observed in most immunized groups, with a significant negative correlation between inhibin values and αN-binding in the follicular phase of the cycle. The pattern of progesterone production during the cycle was similar, with a slight non-significant increase in immunized compared with control ewes. These data confirm the previous observation that ovulation is impaired in ewes immunized against the amino-terminal peptide of inhibin α₄₃ and also suggest that the mechanism of this effect does not involve disruption of pituitary function, implying a role for αN in the intraovarian events of ovulation.

Endothelin, which has potent vasoconstrictor and mitogenic actions, was measured by radioimmunoassay in tissue extracts of sheep endometrium and myometrium and was found to be present in similar amounts in both tissues during the oestrous cycle and in increasing amounts during the first 20 days of pregnancy 250–630 pg g⁻¹ wet weight). Immunoreactive endothelin extracted from endometrium eluted at the same position as standard endothelin-1 on reverse-phase HPLC. Immunohistochemical techniques demonstrated that during the oestrous cycle endothelin immunoreactivity was very low in caruncular and intercaruncular stroma, luminal epithelium, outer and inner glandular epithelium, myometrium and blood vessels until after day 12 (oestrus: day 0). Staining increased in all but the inner glands to day 16 and the most intense staining was found in intercaruncular luminal epithelium and outer glands and in myometrium, although endothelin in tissue extracts did not change over this period. During early pregnancy (days 4–20), staining in intercaruncular areas and in myometrium increased slightly from day 4 to day 12 to a maximum which was maintained from day 15 to day 20. Intensity of staining in caruncles increased only from day 15, particularly in the epithelium. Immunoreactive endothelin was also present in the trophoblast cells of the embryo on day 20 of pregnancy. Strong endothelin immunostaining was observed in uteri from ovariectomized ewes, particularly in epithelial cells and in blood vessels. The intensity of immunostaining in epithelium and stroma was increased slightly by oestradiol and decreased slightly by progesterone treatment, whereas treatment with oestradiol plus progesterone reduced staining intensity of endothelin in all types of tissue. Results of this study therefore demonstrate that immunoreactive endothelin-1 is present in the ovine uterus during the oestrous cycle and in increasing amounts during the first 20 days of pregnancy and is localized in most types of cell. Endothelin-1 is regulated by ovarian steroids and pregnancy-related factors and may play an important role in the regulation of uterine blood flow, in uterine development and the establishment of pregnancy.