Search

Estrogen receptors (ER) in the neuroendocrine tissues of female sheep have been studied with respect to breed, season, and stage of the estrous cycle. In the first study, cytoplasmic ER measured by Scatchard analysis in neuroendocrine tissues from seasonally breeding Merino × Border Leicester (Crossbred) ewes demonstrated no seasonal variation in the pineal, pituitary, and cerebral cortex; in contrast, total cytoplasmic ER levels in the hypothalamus were almost twofold higher (31.5 ± 2.7 fmoles/mg protein; mean ± SEM) in anestrous ewes than in cycling ewes (18.5 ± 2.7 fmoles/mg protein).In a second study, cytoplasmic and nuclear ER levels were measured in pituitary glands from Crossbred ewes in anestrus and Merino and Crossbred ewes during the luteal and follicular phases of the estrous cycle. Cytoplasmic ER levels during the luteal phase were significantly (P<0.005) higher in Crossbred ewes (9717 ± 952 fmoles/g wet wt; mean ± SEM) than in Merino ewes (5477 ± 858 fmoles/g wet wt). In both breeds, nuclear ER levels were significantly (P<0.001) higher during the follicular phase (Crossbred, 1052 ± 147; Merino, 841 ± 156) than during the luteal phase (Crossbred, 292 ± 31; Merino, 394 ± 47). In Crossbred ewes nuclear ER levels during anestrus (347± 55) were similar to luteal phase levels.These data indicate that hypothalamic control of the circannual breeding pattern in the sheep may be related to ER levels. In the short term, during the estrous cycle, pituitary nuclear ER levels reflect estrogen secretion. A breed difference in ER levels may have a fundamental genotypic basis that explains breed disparities in responsiveness to estrogen.

Measurement of estradiol-17β, testosterone, and androstenedione in follicular fluid obtained from groups of antral follicles of known diameter and stage of morphological atresia indicates two phases of steroidogenesis during nonatretic growth. In pools of fluid obtained from follicles of 1 to 3 mm diameter, a transient fourfold increase in testosterone concentrations from 99 to 420 nM was observed soon after antrum formation, whereas concentrations of androstenedione and estradiol-17βranged from 4 to 16 and 10 to 47 nM, respectively. In the pooled fluid of nonatretic follicles of greater than 3 mm diameter, concentrations of estradiol-17βexceeded that of either testosterone or androstenedione, and increased from 9 to 341 nM as follicular diameter increased. Testosterone concentrations progressively decreased from 247 to 56 nM, and the concentration of androstenedione decreased from 22 to 7 nM as follicular diameter increased beyond 3 mm. Concentrations of all three steroids in pools of fluid from early atretic follicles were not significantly different from those of nonatretic follicles, but in the later stages of atresia concentrations of testosterone remained elevated and those of estradiol stayed low at all stages of growth. Data obtained from individual follicles confirmed the initial finding that a reduction in the ratio of estrogen to total follicular androgen in pools of follicular fluid was associated with atresia of all sizes of follicles. We propose that the changes in patterns of steroidogenesis which accompany atresia reflect some of the earliest changes in the functional capacity of follicles undergoing atresia, but are not necessarily the primary cause of atresia.

Ovariectomized (OVX), hypothalamo/pituitary-disconnected (HPD) ewes were used to ascertain the short-term effects of estradiol on the number of gonadotropin-releasing hormone (GnRH) receptors in the pituitary gland. The time course of the study was such that measurements were made during the period of short-term negative feedback and positive feedback. Groups of 4 OVX-HPD ewes were given 250-ng pulses of GnRH each hour and an i.m. injection of oil (Group 1) or 50 micrograms estradiol benzoate in oil (Groups 2−4). Blood samples were collected from each ewe prior to treatment with estradiol or oil and again immediately before slaughter. Groups 2, 3, and 4 were killed 6, 16, and 20 h, respectively, after administration of estradiol.Amplitudes of luteinizing hormone (LH) pulses and average plasma concentrations of LH were reduced 6 h after estradiol treatment. Sixteen and 20 h after injection, the average plasma LH levels were elevated, but pulse amplitudes were similar to preinjection values. The number of GnRH receptors was significantly (p < 0.01) increased within 6 h of estrogen treatment and further increased 16 and 20 h after treatment. Pituitary content of LH was similar in all groups.These data indicate that the number of GnRH receptors in the pituitary gland of ewes can be acutely influenced by a direct effect of estradiol. However, the magnitude and direction of the change in receptors number does not account for the changes in pituitary responsiveness to GnRH, suggesting estradiol also modifies post-receptor mechanisms that influence secretion of LH.

Studies were undertaken to determine if changes in the amplitude of luteinizing hormone (LH) pulses that occur in response to changes in the frequency of gonadotropin-releasing hormone (GnRH) pulses are due to an alteration in the number of GnRH receptors. Ewes were ovariectomized (OVX) and the hypothalamus was disconnected from the pituitary (HPD). Ewes were then given pulses of GnRH at a frequency of 1/h or 1/3 h. Two control groups were included: OVX ewes not subjected to HPD, and HPD ewes that were not OVX. At the end of one week of treatment, blood samples were collected to determine the amplitude of LH pulses. The treated ewes were killed just before the next scheduled pulse of GnRH, and the content of LH and number of GnRH receptors were measured in each pituitary. The amplitude of LH pulses was highly correlated with the amount of LH in the pituitary gland (0 = 0.71, p < 0.01), and both LH content and pulse amplitude (mean + SEM) were higher in ewes receiving GnRH once per 3 h (189.7 ± 39.3 μg/pituitary, 10.3 ± 1.1 ng/ml, respectively) than in ewes receiving GnRH once per h (77.8 ± 11.4 μg/pituitary, 5.2 ± 1.3 ng/ml). The pituitary content of LH was highest in the OVX ewes (260.2 ± 57.4 μg/pituitary) and lowest in the nonpulsed HPD ewes (61.7 ± 51.2 μg/pituitary). The number of GnRH receptors was similar in all groups, and was not correlated with any other variable. From these data, we conclude that changes in LH pulse amplitude in response to changes in GnRH pulse frequency are more likely to be associated with the content of LH in the pituitary gland than with the number of GnRH receptors.

The effects of changes in pulse frequency of exogenously infused gonadotropin-releasing hormone (GnRH) were investigated in 6 adult surgically hypothalamo/pituitary-disconnected (HPD) gonadal-intact rams. Ten-minute sampling in 16 normal animals prior to HPD showed endogenous luteinizing hormone (LH) pulses occurring every 2.3 h with a mean pulse amplitude of 1.11 ± 0.06 (SEM) ng/ml. Mean testosterone and follicle-stimulating hormone (FSH) concentrations were 3.0 ± 0.14 ng/ml and 0.85 ± 0.10 ng/ml, respectively. Before HPD, increasing single doses of GnRH (50–500 ng) elicited a dose-dependent rise of LH, 50 ng producing a response of similar amplitude to those of spontaneous LH pulses. The effects of varying the pulse frequency of a 100-ng GnRH dose weekly was investigated in 6 HPD animals; the pulse intervals explored were those at 1, 2, and 4 h. The pulsatile GnRH treatment was commenced 2–6 days after HPD when plasma testosterone concentrations were in the castrate range (< 0.5 ng/ml) in all animals. Pulsatile LH and testosterone secretion was reestablished in all animals in the first 7 days by 2-h GnRH pulses, but the maximal pulse amplitudes of both hormones were only 50 and 62%, respectively, of endogenous pulses in the pre-HPD state. The plasma FSH pattern was nonpulsatile and FSH concentrations gradually increased in the first 7 days, although not to the pre-HPD range. Increasing GnRH pulse frequency from 2- to 1-hour immediately increased the LH baseline and pulse amplitude. As testosterone concentrations increased, the LH responses declined in a reciprocal fashion between Days 2 and 7. FSH concentration decreased gradually over the 7 days at the 1-h pulse frequency. Slowing the GnRH pulse to a 4-h frequency produced a progressive fall in testosterone concentrations, even though LH baselines were unchanged and LH pulse amplitudes increased transiently. FSH concentrations were unaltered during the 4-h regime. These results show that 1) the pulsatile pattern of LH and testosterone secretion in HPD rams can be reestablished by exogenous GnRH, 2) the magnitude of LH, FSH, and testosterone secretion were not fully restored to pre-HPD levels by the GnRH dose of 100 ng per pulse, and 3) changes in GnRH pulse frequency alone can influence both gonadotropin and testosterone secretion in the HPD model.

To study the role of androgens in the control of gonadotropin and prolactin secretion in ther ewe, we have characterized androgen receptors in pituitary cytosol, and investigated the effect of androgens on pituitary hormone release in vivo and in vitro. High affinity, low capacity receptors, with an affinity for methyltrienolone (R1881) > 5α-dihydrotestosterone (5α-DHT) > testosterone (T) ≫ androstenedione (A4), estradiol-17β(E2) and progesterone (P), were identified in pituitary cytosol.Addition of 1 nM 5α-DHT, but not A4, inhibited luteinizing hormone (LH) release from pituitary cells in vitro, induced by 10−10to 10−7M luteinizing hormone releasing hormone (LHRH). The release of follicle-stimulating hormone (FSH) with 10−9M LHRH was inhibited when cells were incubated with 1 nM 5α-DHT. 5α-DHT had no effect when higher or lower doses of LHRH were used.In ovariectomized ewes, neither an i.v. injection of 1 mg, nor intracarotid injections of up to 1 mg, 5α-DHT affected plasma LH, FSH or prolactin levels, despite dose-related increases in plasma 5α-DHT levels. Daily or twice daily i.m. injections of 5 mg 5α-DHT in oil did not affect LH or FSH levels, but daily injections of 20 mg significantly reduced plasma LH levels within 4 days and plasma FSH levels within 6 days.Thus, despite the presence of androgen receptors in the ewe pituitary, we conclude that androgens per se are of minimal importance in the regulation of pituitary LH, FSH and prolactin secretion in the ewe. The low binding affinity of A4and the lack of its effect on hormone secretion in vitro suggests that A4may act as an estrogen precursor rather than an androgenic hormone. The function of the pituitary androgen receptor remains to be established.