Endogenous opioid peptides (EOP) mediate progesterone-negative feedback in many species, but the specific EOP systems involved remain unresolved. We first addressed this question in sheep by determining the role of different EOP receptor subtypes in the medial basal hypothalamus (MBH) and preoptic area (POA). Local administration of EOP receptor antagonists to luteal phase ewes indicated that kappa-, but not micro- or delta-, receptors mediate the inhibition of LH secretion in the MBH. In contrast, both kappa- and micro-, but not delta-receptor, antagonists increased LH pulse frequency when placed in the POA. We next examined close appositions between dynorphin (kappa ligand) and beta-endorphin (micro ligand) containing varicosities and GnRH perikarya in luteal phase ewes using dual immunocytochemistry and light microscopy. Approximately 90% of MBH GnRH neurons had close associations by dynorphin-containing varicosities, but only 40-50% of GnRH perikarya elsewhere had such close associations. In contrast, the percentage of beta-endorphinergic varicosities close to GnRH neurons was similar among all regions. Electron microscopic analysis demonstrated both dynorphinergic synapses and beta-endorphinergic synapses onto GnRH perikarya. These and other data lead to the hypothesis that dynorphin neurons play a major role in progesterone-negative feedback in the ewe and that this inhibition may be exerted directly on GnRH perikarya within the MBH, whereas dynorphin and beta-endorphin input to GnRH neurons in the POA provide redundancy to this system or are involved in other actions of progesterone or estradiol in the control of the GnRH surge.

Puberty onset in female sheep is marked by a decrease in estradiol-negative feedback, allowing for the increase in GnRH and LH pulses that heralds the first ovulation. Based on recent genetic studies in humans, two possible neuropeptides that could promote puberty onset are kisspeptin and neurokinin B (NKB). Our first experiment determined whether the NKB agonist, senktide, could stimulate LH secretion in prepubertal ewes. A second study used prepubertal and postpubertal ewes that were intact or ovariectomized (OVX) to test the hypothesis that expression of kisspeptin and NKB in the arcuate nucleus increased postpubertally. For comparison, kisspeptin and NKB expression in age-matched intact, and castrated males were also examined. In experiment 1, the percentage of ewes showing an LH pulse immediately after injection of senktide (100 μg, 60%; 500 μg, 100%) was greater than that for water-injected controls (experiment 1a, 25%; experiment 1b, 20%). In experiment 2, kisspeptin-positive cell numbers in the arcuate nucleus increased after puberty in intact females and were increased by OVX in prepubertal but not postpubertal ewes. Changes in kisspeptin cell numbers were paralleled by changes in kisspeptin-close contacts onto GnRH neurons in the medial preoptic area. NKB cell numbers did not differ significantly between intact prepubertal and postpubertal ewes but increased with OVX in both age groups. NKB fiber immunoreactivity was greater in postpubertal than in prepubertal intact ewes. In age-matched males, kisspeptin and NKB cell numbers increased with castration, but decreased with age. These results support the hypothesis that kisspeptin is a gatekeeper to female ovine puberty and raise the possibility that NKB may also play a role, albeit through different means.

Recent studies suggest that the endogenous opioid peptide, dynorphin, is an important mediator of progesterone negative feedback on GnRH pulse frequency in the ewe. These experiments tested this hypothesis by examining the effects of progesterone on dynorphin A concentrations in cerebrospinal fluid (CSF) collected from the third ventricle and expression of preprodynorphin (PPD) mRNA in hypothalamic nuclei. CSF was collected every 10 min for 5 h in three groups of ewes: 1) ovary-intact ewes during the luteal phase (d 6-7 of estrous cycle); 2) ewes 6-7 d after ovariectomy (OVX); and 3) OVX ewes treated for 6-7 d with implants that produced luteal-phase progesterone levels. Diencephalic tissue from these ewes was then collected and processed for in situ hybridization using an ovine cDNA probe against PPD. Progesterone treatment increased dynorphin A concentrations in CSF over that observed in untreated OVX ewes; CSF dynorphin A concentrations in ovary-intact ewes were midway between the other groups. OVX significantly decreased the number of PPD mRNA-expressing cells in the preoptic area (POA), anterior hypothalamic area (AHA), and arcuate nucleus (ARC), with no change seen in any other PPD-expressing nuclei. Progesterone treatment of OVX ewes restored PPD expression in the POA and AHA to levels seen in luteal-phase animals but had no effect on PPD expression in the ARC. These results are consistent with the hypothesis that progesterone acts via dynorphin neurons to inhibit pulsatile GnRH secretion and point to dynorphin neurons in the POA, AHA, and ARC as potential mediators of this action during the luteal phase.

Oestradiol exerts a season-specific negative feedback effect on the GnRH/LH neurosecretory system of the Suffolk ewe. This neuroendocrine suppression is mediated in part by dopamine A15 neurones, but these neurones do not possess the oestrogen receptor. Based on indirect evidence, we hypothesized that oestrogen receptor-containing neurones in the ventromedial preoptic area (vmPOA) may be the initial step in a neuronal system whereby oestradiol suppresses GnRH secretion during the non-breeding season. To test this, three experiments were conducted using ovariectomized ewes receiving either empty or oestradiol-containing bilateral microimplants directed at the vmPOA or s.c. subcutaneous oestradiol-containing implants. In the first experiment, LH pulse frequency was measured on days 0, 1, 7 and 14 of treatment during seasonal anoestrus. In vmPOA oestradiol and s.c. oestradiol groups only, LH pulse frequency was suppressed on days 7 and 14, with maximal suppression evident by day 7. In the second experiment, this protocol was repeated during the breeding season, with LH pulses examined on days 0 and 7; LH pulse frequency did not change in any group. The third experiment tested if the effect of vmPOA oestradiol during anoestrus could be overcome by an injection of the dopamine-D2 receptor antagonist (-)-sulpiride. The vmPOA microimplants and s.c. oestradiol implants again suppressed LH pulse frequency and this was reversed by sulpiride in vmPOA oestradiol ewes. We conclude that oestradiol acts on cells in the vmPOA to stimulate a system involving dopamine neurones that inhibits GnRH/LH pulsatility in the anoestrous ewe.

Thyroid hormones are permissive for various species to enter seasonal anestrus. In the ewe they act centrally to permit the onset of potent estradiol-negative feedback responsible for anestrus, but the specific sites of action are unknown. Therefore, we tested whether T(4) replacement via chronic microimplants in any of five brain areas could reverse the reproductive effects of thyroidectomy. Diffusion of (125)I-T(4) from the microimplant was largely (>98%) limited to a 1.2-mm radius. A marked decline in LH concentration in ovariectomized, estradiol-treated ewes was used as an index for anestrus. In experiment 1, all thyroidectomized (THX) ewes with microimplants in the medial preoptic area, A15 area, and medial basal hypothalamus failed to enter anestrus; instead, LH levels remained elevated, similar to those in untreated THX controls. In ventromedial preoptic area (vmPOA)-microimplanted ewes, only the two animals with the most caudal microimplants entered anestrus, as did thyroid-intact controls and THX ewes receiving icv or sc T(4) replacement. In experiment 2, all vmPOA-treated ewes with similar placements to those effective in experiment 1 along with all ewes microimplanted in the premammillary region entered neuroendocrine anestrus. Thus, the premammillary region and vmPOA are brain sites in which thyroid hormones act to permit the onset of seasonal anestrus.

This paper reports the results of accumulated selected metals concentrations (Fe, Mn, Zn, Cu, Ni, Co, Cr, Pb, Cd, Hg and meHg) in the muscle of five common Slovak fish species (Chub-Leuciscus cephalus, Common carp-Cyprinus carpio, Prussian carp-Carassius gibelio, Roach-Rutilus rutilus, and Wels catfish-Silurus glanis). Furthermore, correlations among the selected metals and order of metal accumulation in the fish muscle were determined. The concentrations of metals (mg/kg wet weight basis) ranged as follows: Fe 3.70-21.10; Mn 0.27-1.50; Zn 3.72-42.82; Cu 0.26-1.82; Ni 0.02-0.29; Co 0.06-0.28; Cr 0.09-0.28; Pb 0.08-34.59; Cd 0.06-2.76, Hg 0.34-3.64 and meHg 0.08-1.20. The level of lead and mercury exceeded the maximum allowed concentration in Slovakia by the Codex Alimentarius for safe human consumption (0.2 and 0.5 mg/kg, respectively) in the majority of samples (94.6 and 82.1%, respectively). Content of Cd (0.88 +/- 0.76 mg/kg wet weight) in the fish muscle exceeded maximum allowed levels (0.05 mg/kg) in all samples. An average, the order of metal concentrations in the fish muscle was: Fe > Zn > Pb > Cd > Hg > Cu > Mn > meHg > Ni > Cr > Co.

There is now considerable evidence that dynorphin neurons mediate the negative feedback actions of progesterone to inhibit GnRH and LH pulse frequency, but the specific neurons have yet to be identified. In ewes, dynorphin neurons in the arcuate nucleus (ARC) and preoptic area (POA) are likely candidates based on colocalization with progesterone receptors. These studies tested the hypothesis that progesterone negative feedback occurs in either the ARC or POA by determining whether microimplants of progesterone into either site would inhibit LH pulse frequency (study 1) and whether microimplants of the progesterone receptor antagonist, RU486, would disrupt the inhibitory effects of peripheral progesterone (study 2). Both studies were done in ovariectomized (OVX) and estradiol-treated OVX ewes. In study 1, no inhibitory effects of progesterone were observed during treatment in either area. In study 2, microimplants of RU486 into the ARC disrupted the negative-feedback actions of peripheral progesterone treatments on LH pulse frequency in both OVX and OVX+estradiol ewes. In contrast, microimplants of RU486 into the POA had no effect on the ability of systemic progesterone to inhibit LH pulse frequency. We thus conclude that the ARC is one important site of progesterone-negative feedback in the ewe. These data, which are the first evidence on the neural sites in which progesterone inhibits GnRH pulse frequency in any species, are consistent with the hypothesis that ARC dynorphin neurons mediate this action of progesterone.

Thyroid hormones permit the increase in response to estradiol negative feedback in ewes at the transition to anestrus. In this study, we tested whether the thyroid hormones are also required for steroid-independent seasonal changes in pulsatile LH secretion. In experiment 1, Suffolk ewes were ovariectomized and thyroidectomized (THX) or ovariectomized only (controls) in late November. LH pulse frequency and amplitude were measured for 4 h in December, April, May, June, and August. Pulse frequency was also measured in the presence of estradiol-containing implants during the breeding (December) and early anestrus (March) seasons. As expected, in the presence of estradiol, pulse frequency declined between December and March in control but not THX ewes. In the absence of estradiol, a seasonal decline in frequency and an increase in amplitude occurred in control ewes, concurrent with lengthening photoperiod. A similar trend was seen in THX ewes, but the seasonal changes were lower in magnitude and not significant. In experiment 2, the same protocol was used (pulse measurements in December, May, and June) with a larger THX group size (n = 7). Results were similar to those of experiment 1 for controls. In THX ewes, pulse frequency did not change over time and was significantly elevated relative to that of controls during the summer. Pulse amplitude in THX ewes tended to increase during summer and did not differ from pulse amplitudes in control ewes. These results demonstrate that thyroid hormones are required for steroid-independent cycles in LH pulse frequency; however, some seasonal changes in amplitude still occur in the absence of thyroid hormones. This finding contrasts with the changes in estradiol negative feedback at the transition to anestrus, which are entirely thyroid hormone dependent.