The distribution of progesterone receptors (PR) was mapped in the hypothalamus of the ewe using immunocytochemistry. These results were confirmed using in situ hybridization with a sheep-specific 35S-labelled riboprobe. In addition, the effect of oestrogen on the level of PR mRNA in the hypothalamus was examined in ovariectomized (OVX) ewes following treatment with an oestrogen implant or without treatment. PR immunoreactive (-ir) cells were readily detected in OVX animals. Labelled cells were observed in four main hypothalamic regions: the preoptic area (POA), including the organum vasculosum of the lamina terminalis, periventricular nucleus (PeVN), ventromedial nucleus (VMN) and the arcuate nucleus (ARC) (including the region ventral to the mamillary recess). In addition, lightly stained PR-ir cells were observed in the supraoptic nucleus and a few PR-ir cells were also found in the diagonal band of Broca. No PR-ir cells were found in the brainstem. PR mRNA-containing cells were found in the same hypothalamic regions as the PR-ir cells. Image analysis of emulsion-dipped slides following in situ hybridization indicated that oestrogen treatment increased (P<0.01) the mean number of silver grains/cell and the density of labelled cells in the VMN and ARC but had no effect on the level of PR mRNA expression in the POA or PeN. The distribution of PR-containing cells in the hypothalamus is similar to that described in other species and all cells were located in nuclei that contain large populations of oestrogen receptor-containing cells. These include regions implicated in the regulation of reproductive neuroendocrine function, and reproductive behaviour. Oestrogen and progesterone synergize to inhibit GnRH secretion and the present results suggest that these functions may involve cells of the VMN and ARC, with oestrogen acting to upregulate PR.

Using probes for rat neural nitric oxide synthase (NOS) mRNA and GnRH mRNA, we performed in situ hybridization to survey NOS mRNA distribution within the hypothalamus of the male and female rat and sought evidence for its expression in GnRH neurons. The NOS cRNA probe was radiolabelled with 35S, and a digoxigenin-labeled rat GnRH cRNA probe was used for double-label studies. NOS mRNA was localized in discrete hypothalamic areas, in grain clusters suggestive of individual neurons. NOS mRNA-positive cells were located mainly in the supraoptic and paraventricular nucleus, particularly overlying the magnocellular division. Rostrally, cells expressing NOS mRNA were especially prominent in the diagonal band of Broca, in a distribution very similar to GnRH neurons. Nevertheless, only one of 370 cells labeled for GnRH mRNA appeared to be positive for NOS mRNA. We conclude that NOS mRNA is located prominently in regions where CRH, AVP and oxytocin cells are located. NOS mRNA-positive cells are located in close proximity to GnRH neurons, but rarely do such neurons express NOS mRNA.

The basic premise of this study is that the GnRH-LH pulsatile activity, particularly its frequency characteristics, constitutes, in the absence of any considerable ovarian feedback, the intrinsic rhythm of the hypothalamic-pituitary unit at its maximal rate. Thus, LH pulse attributes determined in postpubertal hypogonadal subjects may be used as a reference in assessing the degree of influence exerted by endocrine factors that modulate GnRH-LH pulses. Accordingly, serum LH levels were determined in samples obtained at 15-min intervals for 24 h in 20 hypogonadal women: 13 postmenopausal women (PMW) and seven women with premature ovarian failure (POF). Similar measurements were performed in 60 normally cycling women: 25 in the early follicular phase (EFP), 13 in the late follicular phase (LFP), seven at midcycle surge (LH surge) and 15 in the midluteal phase (MLP). Significant pulses were identified by the cluster algorithm utilizing factors appropriate for 24 h data series of a sampling frequency of 15-min intervals. The results show a 24-h mean (+/- SE) LH pulse frequency of 78.2 +/- 2.8 and 85.5 +/- 2.4 min per pulse for young (POF) and older (PMW) hypogonadal women, respectively. During the follicular phase of the cycle, the LH pulse frequency is not significantly different from that of hypogonadal women, but there is a significant (P less than 0.05) increase from early to late follicular phases (95.4 +/- 3.3 vs 78.8 +/- 2.2 min per pulse). However, when the sleep periods are excluded from the 24-h data series because of the associated decrease of LH pulse frequency in EFP women, the resulting pulse frequencies are almost identical for EFP, LFP and PMW. An elevation beyond the basic pulse rhythm determined in PMW or POF is not observed in any phase of the menstrual cycle studied, including the midcycle surge. The decrease in LH pulse frequency during the luteal phase of the cycle (151.8 +/- 8.0 min per pulse, P less than 0.001 vs hypogonadal women) beyond the reference pulse frequency of hypogonadal women is unequivocal. By contrast, the pulse amplitude varies markedly among the groups with the largest found in POF (36.6 +/- 4.5 IU/l). It follows, in descending order, PMW (22.7 +/- 3.1 IU/l), midcycle surge (17.3 +/- 2.8 IU/l), MLP women (7.0 +/- 1.3 IU/l) and the EFP (4.9 +/- 0.3 IU/l) and LFP (4.0 +/- 0.4 IU/l).(ABSTRACT TRUNCATED AT 250 WORDS)

Using recently defined analytical tools that permit quantitative and integrated assessments of pulsatile activities of two or more hormones, we have examined the coincidence of pulses of estradiol (E2), progesterone (P4), and LH determined in blood withdrawn at 15-min sampling intervals for a duration of 24 h in each of 15 women during the midluteal phase of the human menstrual cycle. The occurrence of E2 and P4 pulses is simultaneous, as their peaks were maximally correlated at zero time lag (P less than 10(-4], and there were comparable periodicities for E2 (13.5 +/- 0.7 pulses/24 h) and P4 (11.2 +/- 0.7 pulses/24 h). This coupling of E2 and P4 pulses suggests cosecretion by the mature corpus luteum. These E2 and P4 pulses are significantly coupled with LH pulses, with a lag time of about 30 min and/or 45 min for P4 (P = 0.029) and 0 min and/or 15 min for E2 (P = 0.032). Further, when considered together, LH, E2, and P4 are found to be triply copulsatile (P = 0.0066). However, significant numbers of discrete pulses of P4 and E2 are observed without antecedent LH pulses, suggesting some degree of corpus luteum autonomy. In conclusion, orchestrated synchrony of pulsatile pituitary and ovarian (corpus luteum) signaling can be demonstrated by the coordinated temporal release of LH, E2, and P4 in normal cycling women.