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Given the central role of the decapeptide gonadotropin-releasing hormone (GnRH) in reproductive function, our long-term objective is to delineate the underlying mechanism regulating these reproductive processes. The outcome of GnRH secretion is in part dependent on the proteolytic metabolism of this decapeptide. In contrast to the belief that the metabolism of GnRH serves only as a degradative process that removes excess GnRH, we have shown that a metabolite of the decapeptide, GnRH-(1-5), can directly regulate GnRH gene expression and reproductive behavior. To further characterize the effect of GnRH-(1-5) on GnRH neuronal function, we determined whether GnRH-(1-5) can directly regulate GnRH secretion and pulsatility using an in vitro perifusion system. We compared the effect of GnRH-(1-5) on GnRH secretion in the immortalized GnRH neuron (GT1-7 cell line), whole rat hypothalamic explant, and enzymatically dispersed rat hypothalamic cells. Tissue preparations were perifused continuously for 9 h during which a 3-h challenge with GnRH-(1-5) was administered (4-6 h). The results show that treatment with GnRH-(1-5) increased (p < 0.05) the mean GnRH secretion and the amplitude of the pulses but not the pulse frequency. The present study supports the notion that GnRH-(1-5) is functionally capable of regulating the reproductive neuroendocrine system.

The role of the hypothalamus in female reproductive senescence is unclear. Here we identified novel molecular neuroendocrine changes during the natural progression from regular reproductive cycles to acyclicity in middle-aged female rats, comparable with the perimenopausal progression in women. Expression of 48 neuroendocrine genes was quantified within three hypothalamic regions: the anteroventral periventricular nucleus, the site of steroid positive feedback onto GnRH neurons; the arcuate nucleus (ARC), the site of negative feedback and pulsatile GnRH release; and the median eminence (ME), the site of GnRH secretion. Surprisingly, the majority of changes occurred in the ARC and ME, with few effects in anteroventral periventricular nucleus. The overall pattern was increased mRNA levels with chronological age and decreases with reproductive cycle status in middle-aged rats. Affected genes included transcription factors (Stat5b, Arnt, Ahr), sex steroid hormone receptors (Esr1, Esr2, Pgr, Ar), steroidogenic enzymes (Sts, Hsd17b8), growth factors (Igf1, Tgfa), and neuropeptides (Kiss1, Tac2, Gnrh1). Bionetwork analysis revealed region-specific correlations between genes and hormones. Immunohistochemical analyses of kisspeptin and estrogen receptor-α in the ARC demonstrated age-related decreases in kisspeptin cell numbers as well as kisspeptin-estrogen receptor-α dual-labeled cells. Taken together, these results identify unexpectedly strong roles for the ME and ARC during reproductive decline and highlight fundamental differences between middle-aged rats with regular cycles and all other groups. Our data provide evidence of decreased excitatory stimulation and altered hormone feedback with aging and suggest novel neuroendocrine pathways that warrant future study. Furthermore, these changes may impact other neuroendocrine systems that undergo functional declines with age.

Polychlorinated biphenyls (PCBs) are industrial contaminants and known endocrine-disrupting chemicals. Previous work has shown that gestational exposure to PCBs cause changes in reproductive neuroendocrine processes. Here we extended work farther down the life spectrum and tested the hypothesis that early life exposure to Aroclor 1221 (A1221), a mixture of primarily estrogenic PCBs, results in sexually dimorphic aging-associated alterations to reproductive parameters in rats, and gene expression changes in hypothalamic nuclei that regulate reproductive function. Pregnant Sprague Dawley rats were injected on gestational days 16 and 18 with vehicle (dimethylsulfoxide), A1221 (1 mg/kg), or estradiol benzoate (50 μg/kg). Developmental parameters, estrous cyclicity (females), and timing of reproductive senescence were monitored in the offspring through 9 months of age. Expression of 48 genes was measured in 3 hypothalamic nuclei: the anteroventral periventricular nucleus (AVPV), arcuate nucleus (ARC), and median eminence (females only) by real-time RT-PCR. Serum LH, testosterone, and estradiol were assayed in the same animals. In males, A1221 had no effects; however, prenatal estradiol benzoate increased serum estradiol, gene expression in the AVPV (1 gene), and ARC (2 genes) compared with controls. In females, estrous cycles were longer in the A1221-exposed females throughout the life cycle. Gene expression was not affected in the AVPV, but significant changes were caused by A1221 in the ARC and median eminence as a function of cycling status. Bionetwork analysis demonstrated fundamental differences in physiology and gene expression between cycling and acyclic females independent of treatment. Thus, gestational exposure to biologically relevant levels of estrogenic endocrine-disrupting chemicals has sexually dimorphic effects, with an altered transition to reproductive aging in female rats but relatively little effect in males.

Pregnancy and lactation produce a plethora of hormonal changes in females that promote maternal care of offspring. Males in the biparental marmoset species (Callithrix jacchus) demonstrate high levels of parenting behaviour and express enhanced circulating reproductive hormones. Furthermore, these hormonal changes are influenced by paternal experience. To determine whether the paternally experienced male marmoset has altered neurocrine hypothalamic release, as the maternal females does, we examined the release of several reproductive neurocrines, dopamine (DA), oxytocin (OT), vasopressin (AVP) and prolactin (PRL), in cultured explants of the hypothalamus of paternally experienced male marmosets compared to naïve, paternally inexperienced males. DA levels secreted from the isolated hypothalamus were significantly lower in the experienced males, whereas OT and PRL levels were significantly higher than levels found in inexperienced males. PRL levels decreased rapidly in the hypothalamic media, suggesting that PRL production occurs elsewhere. AVP levels did not change. Stimulation of the cultured explants with oestradiol significantly decreased DA levels in the inexperienced males but did not alter the other neurocrines, suggesting a direct effect of oestradiol on DA suppression in the hypothalamus. Although other factors such as age and rearing experience with siblings may play a role in hypothalamic neurocrine levels, these results demonstrate that paternal experience may impact upon the secretion of neurocrines in a male biparental primate., (© 2011 The Authors. Journal of Neuroendocrinology © 2011 Blackwell Publishing Ltd.)

In mammals, sexual differentiation of the hypothalamus occurs during prenatal and early postnatal development due in large part to sex differences in hormones. These early organizational processes are critically important for the attainment and maintenance of adult reproductive functions. We tested the hypothesis that perinatal exposure to polychlorinated biphenyls (PCBs) that disrupt hormonal pathways would perturb reproductive maturation and the sexually dimorphic development of neuroendocrine systems in the preoptic area (POA). Pregnant Sprague-Dawley rats were injected on gestational d 16 and 18 with vehicle (dimethylsulfoxide), Aroclor 1221 (A1221, an estrogenic PCB mix), a reconstituted PCB mixture representing those highest in human body burden (PCBs 138, 153, 180), or estradiol benzoate, an estrogenic control. Male and female pups were monitored for somatic and reproductive development. In adulthood, some rats were perfused and used for immunohistochemistry of estrogen receptor α, kisspeptin, and coexpression of Fos in GnRH neurons. Other rats were used to obtain fresh-frozen POA dissections for use in a PCR-based 48-gene expression array. Pubertal onset was advanced and estrous cyclicity irregular in endocrine-disrupted females. Furthermore, sexual differentiation of female neuroendocrine systems was masculinized/defeminized. Specifically, in the adult female anteroventral periventricular nucleus, estrogen receptor α-cell numbers and kisspeptin fiber density were significantly decreased, as was GnRH-Fos coexpression. PCR analysis identified androgen receptor, IGF-I, N-methyl-d-aspartate receptor subunit NR2b, and TGFβ1 mRNAs as significantly down-regulated in endocrine-disrupted female POAs. These data suggest that developmental PCBs profoundly impair the sexual differentiation of the female hypothalamus.

The present study was conducted to quantify in vitro gonadotropin-releasing hormone (GnRH) release parameters in the male marmoset. We established primary cultures of marmoset hypothalamic tissues for approximately 2 days (marmosets) to assess GnRH release profiles in vitro in hypothalamic explants from testis-intact and gonadectomized males. Pulsatile GnRH release profiles were readily demonstrated from in vitro hypothalamic explants isolated from adult male marmoset monkeys. Gonadectomy of male marmosets resulted in elevated mean GnRH and pulse amplitude from hypothalamic explants on the 1st day of culture (day 0). GnRH pulse amplitude increased by day 2 in approximately 67% of hypothalamic explants from testis-intact marmosets, suggesting release from an endogenous regulator of GnRH. We also measured GnRH release profiles in vitro in hypothalamic explants from testis-intact and gonadectomized rats. Male rats showed no changes in any concentration or frequency release parameters for GnRH following gonadectomy or during successive days in culture. The present study represents a unique examination of GnRH release from male marmoset monkey hypothalamic tissue and compares release dynamics directly with those obtained from male rat, suggesting a species difference in feedback regulation of GnRH release.

Exposure to endocrine disrupting chemicals (EDCs) such as polychlorinated biphenyls (PCBs) causes functional deficits in neuroendocrine systems. We used an immortalized hypothalamic GT1-7 cell line, which synthesizes the neuroendocrine peptide gonadotropin-releasing hormone (GnRH), to examine the neurotoxic and endocrine disrupting effects of PCBs and their mechanisms of action. Cells were treated for 1, 4, 8, or 24 h with a range of doses of a representative PCB from each of three classes: coplanar (2,4,4',5-tetrachlorobiphenyl: PCB74), dioxin-like coplanar (2',3,4,4',5' pentachlorobiphenyl: PCB118), non-coplanar (2,2',4,4',5,5'-hexachlorobiphenyl: PCB153), or their combination. GnRH peptide concentrations, cell viability, apoptotic and necrotic cell death, and caspase activation were quantified. In general, GnRH peptide levels were suppressed by high doses and longer durations of PCBs, and elevated at low doses and shorter timepoints. The suppression of GnRH peptide levels was partially reversed in cultures co-treated with the estrogen receptor antagonist ICI 182,780. All PCBs reduced viability and increased both apoptotic and necrotic cell death. Although the effects for the three classes of PCBs were often similar, subtle differences in responses, together with evidence that the combination of PCBs acted slightly different from individual PCBs, suggest that the three tested PCB compounds may act via slightly different or more than one mechanism. These results provide evidence that PCB congeners have endocrine disrupting and/or neurotoxic effects on the hypothalamic GnRH cell line, a finding that has implications for environmental endocrine disruption in animals.

Perinatal exposures to endocrine-disrupting chemicals, such as polychlorinated biphenyls (PCBs), can cause latent effects on reproductive function. Here, we tested whether PCBs administered during late pregnancy would compromise reproductive physiology in both the fetally exposed female offspring (F1 generation), as well as in their female offspring (F2 generation). Pregnant Sprague-Dawley rats were treated with the PCB mixture, Aroclor 1221 (A1221; 0, 0.1, 1, or 10 mg/kg), on Embryonic Days 16 and 18. Somatic and reproductive development of F1 and their F2 female offspring were monitored, including ages of eye opening, pubertal landmarks, and serum reproductive hormones. The results showed that low doses of A1221 given during this critical period of neuroendocrine development caused differential effects of A1221 on F1 and F2 female rats. In both generations, litter sex ratio was skewed toward females. In the F1 generation, additional effects were found, including a significant alteration of serum LH in the 1 mg/kg A1221 group. The F2 generation showed more profound alterations, particularly with respect to fluctuations in hormones and reproductive tract tissues across the estrous cycle. On proestrus, the day of the preovulatory GnRH/gonadotropin surge, F2 females whose mothers had been exposed perinatally to A1221 exhibited substantially suppressed LH and progesterone concentrations, and correspondingly smaller uterine and ovarian weights on estrus, compared with F2 descendants of control rats. These latter changes suggest a dysregulation of reproductive physiology. Thus, low levels of exposure to PCBs during late fetal development cause significant effects on the maturation and physiology of two generations of female offspring. These findings have implications for reproductive health and fertility of wildlife and humans.

Background/aims: The loss of reproductive capacity during aging involves changes in the neural regulation of the hypothalamic gonadotropin-releasing hormone (GnRH) neurons controlling reproduction. This neuronal circuitry includes glutamate receptors on GnRH neurons. Previously, we reported an increase in the expression of the NR2b subunit protein of the NMDA receptor on GnRH neurons in middle-aged compared to young female rats. Here, we examined the functional implications of the NR2b subunit on the onset of reproductive aging, using an NR2b-specific antagonist ifenprodil., Methods: Young (3-5 months) and middle-aged (10-13 months) female rats were ovariectomized (OVX), 17beta-estradiol (E2) or vehicle (cholesterol) treated, and implanted with a jugular catheter. Serial blood sampling was undertaken every 10 min for 4 h, with ifenprodil (10 mg/kg) or vehicle injected (i.p.) after 1 h of baseline sampling. The pulsatile release of pituitary LH and levels of GnRH mRNA in hypothalamus were quantified as indices of the reproductive axis., Results: Our results showed effects of ifenprodil on both endpoints. In OVX rats given cholesterol, neither age nor ifenprodil had any effects on LH release. In E2-treated rats, aging was associated with significant decreases in pulsatile LH release. Additionally, ifenprodil stimulated parameters of pulsatile LH release in both young and middle-aged animals. Ifenprodil had few effects on GnRH mRNA; the only significant effect of ifenprodil was found in the middle-aged, cholesterol group., Conclusion: Together, these findings support a role for the NR2b subunit of the NMDAR in GnRH/LH regulation. Because most of these effects were exhibited on pituitary LH release in the absence of a concomitant change in GnRH gene expression, it is likely that NMDA receptors containing the NR2b subunit play a role in GnRH-induced LH release, independent of de novo GnRH gene expression., ((c) 2007 S. Karger AG, Basel)

Luteinizing hormone-releasing hormone-I (LHRH-I) was isolated from the mammalian hypothalamus and shown to be the primary regulator of reproduction through its initiation of pituitary gonadotropin release. Subsequently, it has also been shown to have non-pituitary actions. Although the regulation of LHRH-I synthesis and release has been extensively studied, there is additional evidence to suggest that processing of the peptide represents another layer of regulation. The focus of this review will be on evidence for the action of LHRH-(1-5), the pentapeptide metabolite of LHRH-I, in regulating LHRH-I synthesis, secretion and reproductive behavior. The involvement of LHRH-(1-5) in the control of aspects of reproduction might represent yet another level of regulatory complexity through neuropeptide processing.

2, 3, 7,8-Tetrachlorodibenzo-p-dioxin (TCDD) has adverse effects on reproduction, in part due to direct actions at the ovary. It is unclear whether effects are further mediated by glands that regulate ovarian function. We investigated whether effects of TCDD are mediated via the hypothalamic-pituitary axis. Hypothalamic and pituitary tissues were cultured in medium with and without TCDD. TCDD did not alter GnRH release from hypothalamic samples. It continued to be pulsatile with no differences in the average peak frequency, average peak amplitude, or baseline GnRH release. TCDD did not alter GnRH-induced release of gonadotropins from pituitary samples. There were no differences in average peak amplitude or baseline release. AhR, ARNT or ER alpha mRNA copy numbers in cultured pituitaries were not affected by TCDD. Our data suggest that TCDD effects on ovarian function are not mediated through the hypothalamic or pituitary release parameters tested in this study.

Unlike other mammals, including rodents, Old World primates and humans, common marmosets and probably all other New World primates synthesise and release chorionic gonadotrophin (CG), and not luteinising hormone (LH) from pituitary gonadotrophs. However, little is known about the physiological dynamics of gonadotrophin-releasing hormone (GnRH)-regulated CG release from gonadotrophs and whether such CG release has pulsatile release characteristics similar to those of LH in other mammalian species. Consequently, we performed a series of in vivo and in vitro studies in ovariectomised laboratory rats and female marmosets to compare GnRH-induced pituitary LH and CG release characteristics, respectively. Exogenous GnRH stimulated a slower onset of release of marmoset pituitary CG, both in vivo and in vitro, and induced an approximately 400% greater increase in the duration of marmoset pituitary CG release compared to that for rat LH. Not surprisingly, hypothalamic pulsatile release of GnRH in vivo was not obviously concordant with endogenous episodic changes in circulating levels of CG in marmosets, in contrast to the clear concordance observed between in vivo GnRH and LH release previously demonstrated in rats and other mammals. Pituitary CG release in marmosets thus demonstrates considerable divergence from the timely hypothalamic GnRH-regulated LH release in other female mammals, implying potentially different physiological dynamics in gonadotrophin regulation of marmoset ovarian function.

In vivo hypothalamic gonadotrophin-releasing hormone (GnRH) release was characterised for the first time in a New World primate. A nonterminal and repeatable push-pull perfusion (PPP) technique reliably measured GnRH in conscious common marmoset monkeys. Nineteen adult females (n = 8 ovary-intact in the mid-follicular phase; n = 11 ovariectomised) were fitted with long-term cranial pedestals, and a push-pull cannula was temporarily placed in unique locations within the pituitary stalk-median eminence (S-ME) 2 days prior to each PPP session. Marmosets underwent 1-3 PPPs (32 PPPs in total) lasting up to 12 h. Plasma cortisol levels were not elevated in these habituated marmosets during PPP, and PPP did not disrupt ovulatory cyclicity or subsequent fertility in ovary-intact females. GnRH displayed an organised pattern of release, with pulses occurring every 50.0 +/- 2.6 min and lasting 25.4 +/- 1.3 min. GnRH pulse frequency was consistent within individual marmosets across multiple PPPs. GnRH mean concentration, baseline concentration and pulse amplitude varied predictably with anatomical location of the cannula tip within the S-ME. GnRH release increased characteristically in response to a norepinephrine infusion and decreased abruptly during the evening transition to lights off. Ovary-intact (mid-follicular phase) and ovariectomised marmosets did not differ significantly on any parameter of GnRH release. Overall, these results indicate that PPP can be used to reliably assess in vivo GnRH release in marmosets and will be a useful tool for future studies of reproductive neuroendocrinology in this small primate.

Microscopic and in situ visual observations were used to relate circulating hormone levels to morphological changes in the oviduct of the snapping turtle Chelydra serpentina throughout the ovarian cycle. Increase in levels of progesterone (P), estradiol (E2) and testosterone (T) levels coincide with an increase in number and growth of endometrial glands, luminal epithelial cells and secretory droplets throughout the oviduct. Testosterone and estradiol levels rose significantly (P < 0.05) after the May-June period and remained high throughout the rest of the summer. Progesterone levels remained stable throughout the summer, with a brief decline in July due to luteolysis. Hormonal values declined significantly (P < 0.001) at the end of the ovarian cycle in the fall. In situ visual observation of fresh oviducts at different stages of gravidity in recently ovulated turtles revealed that proteinaceous like components from the endometrial glands were released into the lumen to form fibers. The morphological features of the oviduct remained active throughout the summer months even though the snapping turtle is a monoclutch species which deposits all the eggs in late-May to mid-June. The high steroid levels correlate with and may be responsible for the secretory activity present throughout the summer and their decline correlates with change to low secretory activity in the fall. Calcium deposition accompanied by morphological changes in luminal cells are suggestive of secretory activity. In the egg-bearing turtles, uterine Ca2+ concentrations measured by flame atomic absorption spectrophotometry revealed significantly higher Ca2+ concentrations (P < 0.001) in eggs with soft shell than eggs without shell. There was a significant increase in calcium granules and proteinaceous fibers in luminal surface of the uterus during the period of eggshelling. This supports the fact that in the snapping turtle like in other reptiles, eggshelling process occurs in the uterus.

The control of reproductive function is manifested centrally through the control of hypothalamic release of gonadotropin-releasing hormone (GnRH) in episodic events or pulses. For GnRH release to occur in pulses, GnRH neurons must coordinate release events periodically to elicit a bolus of GnRH. We used a perifusion culture system to examine the release of GnRH from both intact hypothalami and enzymatically dispersed hypothalamic cells after challenge with GnRH analogs to evaluate the role of anatomical neuronal connections on autocrine/paracrine signals by GnRH on GnRH neurons. The potent GnRH agonist des-Gly(10)-D-Ala(6)-GnRH N-ethylamide, potent GnRH antagonists D-Phe(2)-D-Ala(6)-GnRH and D-Phe(2,6)-Pro(3)-GnRH or vehicle were infused, whereas GnRH release from hypothalamic tissue and cells were measured. PULSAR analysis of GnRH release profiles was conducted to evaluate parameters of pulsatile GnRH release. Infusion of the GnRH agonist resulted in a decrease in mean GnRH (P < 0.001), pulse nadir (P < 0.01), and pulse frequency (P < 0.05) but no effect on pulse amplitude. Infusion of GnRH antagonists resulted in an increase in mean GnRH (P < 0.001), pulse nadir (P < 0.05), and pulse frequency (P < 0.05) and in GnRH pulse amplitude only in dispersed cells (P < 0.05). These results are consistent with the hypothesis that GnRH inhibits endogenous GnRH release by an ultrashort-loop feedback mechanism and that treatment of hypothalamic tissue or cells with GnRH agonist inhibits ultrashort-loop feedback, whereas treatment with antagonists disrupts normal feedback to GnRH neurons and elicits an increased GnRH signal.

A decline in somatic function with aging in women is associated with a decrease in GH release and a loss of estrogen after menopause. As an initial step to establish a monkey model for the neuroendocrine mechanisms underlying somatopause and menopause, we have conducted three experiments in unrestrained aged (approximately 25.7-yr-old) and young (approximately 5.4-yr-old) female rhesus monkeys. GH release was pulsatile, and mean GH release and pulse amplitude were significantly lower in aged monkeys than in young monkeys. Injection of GHRH alone, GH-releasing peptide-2 alone, or the combination of both induced an increase in GH release in both age groups. The mean LH level, pulse amplitude, and baseline LH levels were significantly higher in aged animals than in young animals. Both estrogen and IGF-I levels were lower in aged than young monkeys. These results suggest that in female rhesus monkeys 1) there is a clear decline in circulating GH and IGF-I levels with aging; 2) GHRH and GH-releasing peptide-2 stimulate GH release synergistically; and 3) circulating LH levels increase as estrogen decreases with aging. These results indicate that the rhesus monkey is an excellent model for studies of the neuroendocrine mechanisms of aging.

Polychlorinated biphenyls (PCBs) cause abnormal development and physiology of the reproductive system. We hypothesized that these effects may be mediated, at least in part, by neuroendocrine cells in the hypothalamus that integrate inputs to and outputs from the central nervous system and reproductive systems. The effects of two PCB mixtures, Aroclor 1221 and Aroclor 1254, were tested on the hypothalamic GT1-7 cells, which synthesize and secrete the key hypothalamic hormone, gonadotropin-releasing hormone (GnRH). GT1-7 cells were treated for 24 h in dose-response experiments and GnRH gene expression and release were quantified. Aroclor 1221 was stimulatory to GnRH gene expression, particularly at post-transcriptional levels (GnRH cytoplasmic mRNA), and increased GnRH peptide levels, suggesting a post-translational regulation of GnRH biosynthesis. It also caused a qualitative increase in GT1-7 neurite outgrowth and cell confluency. Aroclor 1254 had very different effects from Aroclor 1221. It inhibited GnRH nuclear mRNA levels at high dosages, and stimulated GnRH mRNA at low doses, suggesting a post-transcriptional mechanism of regulation. Aroclor 1254 did not alter GnRH peptide levels. Qualitatively, Aroclor 1254 caused a retraction of GT1-7 cell processes and neurotoxicity at high dosages. In order to gauge the involvement of the oestrogen receptor in these responses, the oestrogen receptor antagonist, ICI 182,780 (ICI) was coadministered in other studies with the PCBs. While effects of Aroclor 1221 on GnRH gene expression were not blocked by ICI, its effects on GnRH peptide levels were blocked by ICI, indicating that some but not all of the effects of Aroclor 1221 are mediated by the classical oestrogen receptor alpha and/or beta. The inhibitory effects of Aroclor 1254 on GnRH gene expression were not prevented by ICI, although ICI itself had stimulatory effects on GnRH gene expression that were blocked by cotreatment with Aroclor 1254. These results demonstrate a novel mechanism for effects of the two PCBs directly on GnRH gene expression, and indicate a hypothalamic level for endocrine disruption by these environmental toxicants.

During reproductive ageing, the oestrous cycles of female rats become irregular and eventually cease. The mechanisms for reproductive senescence in rodents are believed to involve changes in hypothalamic neurones, including gonadotropin-releasing hormone (GnRH) cells and their afferent inputs. In addition, effects of oestrogen on hypothalamic function may vary in animals of different ages. These issues were addressed using young (aged 4-5 months), middle-aged (12-14 months) and old (24-26 months) female Sprague-Dawley rats. Animals were ovariectomized and given oestrogen or vehicle replacement. They were killed and the preoptic area-anterior hypothalamus (POA-AH) and the medial basal hypothalamus-median eminence (MBH-ME) were dissected out, RNA extracted, and RNase protection assay used to quantify gene expression of several hypothalamic molecules. In the first experiment, GnRH RNA levels were measured in the POA-AH. No effects of ageing or oestrogen were observed on GnRH gene expression. This finding suggests that ageing and oestrogen may affect GnRH release from neuroterminals independently of de novo biosynthesis, and that this may involve other neurones that affect GnRH neurosecretory function. In the second experiment, we investigated changes in N-methyl-D-aspartate (NMDA) receptor subunit mRNA levels. These receptors play an important regulatory role in mediating effects of glutamate on GnRH function, and are themselves regulated by oestrogen and ageing. NMDA receptor subunit (NR) 1, 2a and 2b mRNA levels were quantified in the POA-AH and MBH-ME, the sites of GnRH perikarya and neuroterminals, respectively. In general, oestrogen had inhibitory effects on NR1 and NR2a, and differential effects on NR2b subunit mRNA levels. NMDA receptor subunit mRNA levels also changed during ageing: age-related decreases in NR1 mRNA occurred in the MBH-ME, and an age-related increase in NR2b mRNA occurred in the POA-AH. Taken together, these results demonstrate subunit- and region-specific changes in hypothalamic NMDA receptor subunit gene expression with oestrogen and ageing. These alterations could have implications for the physiological effects of glutamate on its NMDA receptor, and impact the regulation of reproductive and other neuroendocrine and autonomic functions by hypothalamic glutamatergic inputs.

In this study we investigated the effects of treatment by luteinizing hormone-releasing hormone (LHRH) on the morphology and steroid release of ovarian tissues in the Western painted turtle, (Chrysemys picta). In Experiment I, four adult female turtles were injected with synthetic mammalian LHRH (i.p., 500 pg/g bodyweight) and four with saline 2-3 weeks prior to ovulation. Granulosa cells from LHRH-treated turtles vs controls contained both preovulatory follicles (16-20 mm in diameter) and small follicles (0.5-1.00mm in diameter) with increased RER, free ribosomes and mitochondria with swollen cristae. An increase in the amount of cytoskeletal material (microfilaments) was observed in granulosa cells of the experimental turtles compared to the controls. Cytoplasmic extensions of the oocyte and granulosa cells were longer in the small follicles of treated animals, accounting for the observed increase in the thickness of the zona pellucida (ZP) over the controls. In Experiment II, administration of LHRH (i.p.) to 10 turtles during the same period triggered a substantial increase in plasma progesterone and estradiol-17beta levels over the 10 saline-injected controls. This supports the idea that in this species, as in mammals, steroidogenic activity in the ovarian follicles are under the control of the hypothalamic-pituitary axis. The ultrastructure and hormonal levels of the experimental animals were typical of untreated turtles just prior to ovulation. In this species the development of follicles and steroidogenesis can be stimulated prematurely by a releasing hormone from a nonreptilian origin.

Reproductive aging in female rats is characterized by profound alterations in the neuroendocrine axis. The preovulatory luteinizing hormone (LH) surge is attenuated, and preovulatory expression of the immediate early gene fos in gonadotropin-releasing hormone (GnRH) neurons is substantially reduced in middle-aged compared with young rats. We tested the hypothesis that alterations in GnRH gene expression may be correlated with the attenuation of the LH surge and may be a possible mechanism involved in neuroendocrine senescent changes. Sprague-Dawley rats ages 4 to 5 mo (young), 12-14 mo (middle-aged), or 25 to 26 mo (old) were killed at 10:00 AM or 3:00 PM on proestrus, the day of the LH surge, or diestrus I in cycling rats, and on persistent estrus or persistent diestrus in acyclic rats. RNase protection assays of GnRH mRNA and GnRH primary transcript were performed. GnRH mRNA levels increased significantly with age, whereas GnRH primary transcript levels, an index of GnRH gene transcription, decreased in old compared to young and middle-aged rats. This latter result suggests that an age-related change in GnRH mRNA levels occurs independently of a change in gene transcription, indicating a potential posttranscriptional mechanism. On proestrus, GnRH mRNA levels increased significantly from 10:00 AM to 3:00 PM in young rats. This was in contrast to proestrous middle-aged rats, in which this afternoon increase in GnRH mRNA levels was not observed. Thus, the normal afternoon increase in GnRH mRNA levels on proestrus is disrupted by middle age and may represent a substrate for the attenuation of the preovulatory GnRH/LH surge that occurs in rats of this age, prior to reproductive failure.

The inhibitory effects of pituitary allografts on the prolactin (PRL)-secretory system are presumed to be consequences of the unabated release of PRL by the allografts. In the present studies we used pituitary allografts in the Golden Syrian hamster to address the following questions: (a) Do allografts of adult adenohypophysial tissue which elevate serum PRL levels decrease the concentration of PRL mRNA in the host's adenohypophysis? (b) Is this effect shared by allografts of neonatal hypophysial tissue or neonatal muscle tissue which do not elevate serum PRL levels? (c) Do any of these types of allograft alter growth hormone mRNA in the host's adenohypophysis? Prolactin mRNA concentration, but not growth hormone mRNA concentration, was decreased in the adenohypophyses in situ in the hosts bearing adult adenohypophysial allografts in which serum PRL levels were elevated. In contrast, serum PRL in hosts with neonatal hypophysial or muscle allografts were not elevated and PRL mRNA levels in the adenohypophysis in situ were not decreased when compared to the levels measured in hamsters with sham transplants. Prolactin mRNA levels in hosts with neonatal muscle allografts were not different from levels in hosts with neonatal hypophysial allografts but were increased when compared to the levels measured in hamsters with sham transplants. There were no differences in PRL concentration in the adenohypophyses in situ between any of the groups. Also, PRL concentrations in neonatal hypophysial allografts were similar to those in adult adenohypophysial allografts. To our knowledge these observations are the first demonstrating that short-loop feed-back of PRL includes a decrease in PRL mRNA concentration. The observations also support the working hypothesis that PRL and not another pituitary factor exerts the negative feedback.

In normal hamsters, we investigated whether the sex-specific, selective increase in serum FSH concentration in the juvenile female was associated with sex-specific changes in the percentages of adenohypophysial gonadotrophs. Serum LH concentrations did not rise between Day 4 and Day 19 in either sex and did not differ significantly between the sexes on Days 4, 7, 12, 14, and 19 after birth. Serum FSH concentrations were about 2-fold higher on Days 7, 12, and 14 than on Days 4 or 19 in males. In females, serum FSH rose markedly between Days 4 and 7, declined slightly by Day 12, rose to peak levels by Day 14, and declined slightly by Day 19 to levels not different from those seen on Day 7. Body weights rose between Days 4 and 19 and were similar in both sexes. There were no sex differences in pituitary gland weights, which rose between Days 4 and 12 and did not increase significantly further by Day 19. On Day 0, the percentages of immunoreactive LH and FSH cells were about 6 and 1%, respectively, in both sexes. These percentages increased progressively between Days 0 and 7 and between Days 7 and 14. On Day 7, but not on Day 14, the percentages of LH and FSH cells were greater in females than in males. There were more LH than FSH cells in males on Days 0, 7 and 14, and in females on Day 0 but not on Day 7 or 14. Matching of 10 FSH cells per gland with LH cells in serial sections of each of 30 glands showed FSH immunoreactivity to occur only in cells staining for LH. In hypophysectomized-gonadectomized adult hamster hosts with allografts of neonatal pituitary glands beneath the renal capsule, we investigated whether these sex-specific changes in the percentage of cells might be predetermined by the time of birth or dependent on sex differences in the internal environment existing in the postnatal hamster. Groups consisted of male donors-male hosts, male donors-female hosts, female donors-female hosts, and female donors-male hosts. The percentages of LH cells in allografts in all four groups increased from Days 0 to 7 and from Days 7 to 14. Percentages of LH cells on Day 14 in all four groups were not different from those in age-matched male or female adenohypophyses in situ. In contrast, the mean percentages of FSH cells were low (about 1-3%) on Days 0, 7, and 14 in all four groups. In other males hosts, administration of a low dose of LHRH for 7 days did not alter the percentage of LH cells in male allografts but increased the percentage of FSH cells to approach that observed in age-matched male adenohypophyses in situ. Administration of a larger dose of LHRH for 7 days to other male hosts with male allografts increased the percentages of LH and FSH cells to percentages not different from those in age-matched female adenohypophyses in situ. Matching of 10 FSH cells/allograft with LH cells in serial sections of each of 58 allografts showed FSH immunoreactivity to occur only in cells staining for LH. The results of experiments conducted on normal hamsters demonstrate that more marked increases in the percentages of adenohypophysial LH cells and FSH cells occur in females than in males in association with the onset of the selective increase in serum FSH levels in females. The results of experiments employing allografts suggest that the greater increase in LH and FSH cells in females is due to sex differences in the internal environment existing in the postnatal hamster, which can be accounted for by differences in LHRH secretion, rather than to inherent differences between female and male adenohypophyses at the time of birth. We conclude that the greater increases in gonadotrophs observed in female hamster pups on Day 7 after birth and the accompanying sex-specific, selective elevation in serum FSH concentration are probably due to sex differences in LHRH secretion during the juvenile period.

Luteinizing hormone releasing hormone (LHRH) stimulates the development of cellular FSH immunoreactivity in the perinatal hamster adenohypophysis. Because neuropeptide Y (NPY) can act directly on rat adenohypophysial cells to stimulate FSH and LH release and potentiate the stimulatory effect of LHRH on FSH and LH release, we investigated the effects of NPY alone and in combination with a low, ineffective dose of LHRH on inducing cellular FSH immunoreactivity in the neonatal hamster adenohypophysis. Neonatal female pituitary glands were grafted beneath the right renal capsules of hypophysectomized-ovariectomized adult hamster hosts with a catheter implanted in the external jugular vein. After treatment, hosts were decapitated and graft tissue was stained for FSH and LH immunoreactivity. The mean percentage of adenohypophysial cells that stained for FSH was low (2.8%) in grafts in hosts infused continuously with heparinized saline vehicle for 7 days. In other hosts, peptides were pulsed through the catheter every 12 h for 7 days. The mean percentage of FSH cells also was low after pulsing 6 ng LHRH or 2 micrograms NPY but increased substantially when the two peptides were pulsed simultaneously. No differences in the mean percentage of LH cells existed between any of the groups. The results demonstrate that NPY and LHRH can synergize to induce cellular FSH immunoreactivity in the neonatal female hamster.

We investigated the effectiveness of continuous vs intermittent LHRH stimulation of the neonatal female anterior pituitary gland on inducing cellular FSH immunoreactivity in the Golden Syrian hamster. Neonatal female pituitary glands were grafted beneath the right renal capsules of hypophysectomized-ovariectomized adult hosts with a catheter implanted in the external jugular vein. In experiment 1, vehicle or LHRH (6 ng/h) was infused continuously or LHRH was pulsed at 1 h (6 ng) or 12 h (72 ng) intervals through the catheters for 8 days. Hamsters were decapitated for collection of trunk blood shortly after the end of treatment, and grafts were prepared for immunocytochemical staining for LH and FSH. Anterior pituitary glands removed from neonatal (day 1) and day 9 female pups also were stained for LH and FSH. The mean percentage of adenohypophysial cells staining for LH increased from 11% in neonatal pups to mean percentages (24-28%) that were similar in day 9 pups and in all groups with grafts. The mean percentage of adenohypophysial cells staining for FSH increased from 1% in neonatal pups to percentages (16-21%) that were similar in day 9 pups and in grafts in hosts administered 6 or 72 ng LHRH pulses. By contrast, the mean percentage of FSH cells did not increase in grafts in hosts administered vehicle or LHRH by continuous infusion. Serum LH concentration was low in hosts given vehicle or LHRH by continuous infusion but elevated in hosts given 72 ng LHRH pulses and in all but one host given 6 ng LHRH pulses.(ABSTRACT TRUNCATED AT 250 WORDS)

In previous studies we have shown that the pulsatility of LH-releasing hormone (LHRH) release in gonadectomized monkeys is modulated by input from neuropeptide-Y (NPY) neurons: 1) the endogenous release of NPY in the stalk-median eminence (S-ME) was pulsatile; 2) NPY pulses were temporally correlated with LHRH pulses, with NPY pulses preceding LHRH pulses by approximately 5 min; and 3) infusion of NPY into the S-ME stimulated LHRH release, whereas 4) infusion of antiserum to NPY suppressed endogenous LHRH pulses. It is not known, however, whether ovarian steroid hormones alter the pulsatility of NPY and LHRH release or whether the temporal correlation of NPY and LHRH pulses is maintained during the LH surge. In the present study we examined the changes in pulsatile release of NPY and LHRH in ovariectomized monkeys treated with estradiol benzoate (EB) followed by progesterone or oil. Using push-pull perfusion, perfusate samples from S-ME were collected at 10-min intervals for 15 h. NPY and LHRH concentrations in the perfusates were measured by RIA. Circulating LH levels were also monitored by periodic blood sampling and RIA. Injection of progesterone (sc) after EB induced an LH surge with a peak latency of 7.3 +/- 1.3 h (mean +/- SE) in seven of seven monkeys, whereas oil injection after EB elicited an LH surge in none of seven monkeys. The progesterone-induced LH surge was associated with an increase in LHRH release; the mean, pulse amplitude, and pulse frequency increased significantly (for all, P < 0.05) 4-8 h after progesterone. NPY pulse frequency also increased significantly (P < 0.05) 4-8 h after progesterone treatment, whereas mean release and pulse amplitude did not change in response to progesterone. Oil treatment after EB administration did not alter any parameter of LHRH and NPY pulses. Interestingly, the NPY and LHRH pulses were highly correlated (P < 0.001) in monkeys treated with either EB-progesterone or EB-oil, and NPY pulses preceded LHRH pulses by 4.8 +/- 0.7 and 5.1 +/- 0.6 min, respectively. In summary, 1) an episode of increased LHRH release occurs before and during the progesterone-induced LH surge; 2) acceleration of LHRH pulse frequency and the increase in LHRH pulse amplitude after progesterone are accompanied by acceleration of NPY pulse frequency; and 3) ovarian steroids do not affect the temporal correlation between NPY and LHRH pulses.(ABSTRACT TRUNCATED AT 400 WORDS)

In recent years, several studies have shown that neuropeptide-Y (NPY) is involved in the control of LH secretion. We determined the effects of estrogen on NPY-induced LH and FSH release in the absence or presence of LH-releasing hormone (LHRH) at the level of the anterior pituitary gland (APG). Adult female rats were ovariectomized. Fifteen to 20 days later, they were given a blank or estrogen-filled capsule subdermally and killed 17-19 h later. APG cells were isolated and cultured for 3 days in medium containing 12.5% rat serum collected at death from the same rats used to make the respective APG cell pools. The cells were then challenged for 3 h with vehicle, NPY (10(-12)-10(-6) M), LHRH (10(-9)-10(-6) M), or combinations of NPY (10(-9)-10(-7) M) and LHRH (10(-9) M). LHRH stimulated LH and FSH release from nonestrogen and estrogen-primed cells. NPY at 6.7 x 10(-8)-10(-6) M increased (P < 0.05) LH release and at 10(-6) M increased (P < 0.05) FSH release from estrogen-primed cells, but was without effect on nonestrogen-primed cells. In contrast, NPY at 10(-9)-10(-7) M potentiated the action of LHRH (10(-9) M) to increase the release of LH and FSH from nonestrogen-primed cells, but was without potentiating effects in cultures of estrogen-primed cells. The results demonstrate that 1) NPY can release LH and FSH by a direct action on estrogen-primed APG cells; and 2) NPY can potentiate the action of LHRH to increase the release of LH and FSH by a direct action on nonestrogen-primed APG cells.

The effect of estrogen on the responsiveness of the luteinizing hormone-releasing hormone (LHRH) neurosecretory system to neuropeptide Y (NPY) stimulation was examined using a push-pull perfusion method in conscious monkeys. NPY, at doses of 10(-6) to 10(-12) M, was infused into the stalk-median eminence (S-ME) of ovariectomized monkeys with or without estrogen, while perfusates were continuously collected. LHRH in perfusates was measured by RIA. The results indicate that in both estrogen-primed and unprimed monkeys, NPY infusion into the S-ME elicited LHRH release in a dose-dependent manner (p < 0.01). Moreover, estrogen priming enhanced the responsiveness of LHRH release to NPY infusion: (1) the minimum NPY dose necessary to elicit a significant LHRH response was reduced, (2) the peak LHRH response to NPY at a dose of 10(-6) to 10(-10) M was increased, and (3) the total release of LHRH in response to NPY at doses of 10(-6) to 10(-12) M was increased. These results suggest that NPY stimulates LHRH release in the S-ME in the presence or absence of estrogen and that estrogen enhances the responsiveness of the LHRH neurosecretory system to NPY stimulation in the rhesus monkey.

In a previous study, we have demonstrated that infusion of neuropeptide Y (NPY) into the stalk-median eminence (S-ME) of gonadectomized rhesus monkeys stimulated LHRH in a dose-dependent manner. This finding led us to address the following questions: 1) What are the characteristics of NPY release in vivo? 2) How does NPY release relate to LHRH release? 3) Is endogenous NPY essential to pulsatile LHRH release? To answer these questions, three experiments using push-pull perfusion were performed in adult gonadectomized rhesus monkeys. Perfusate samples from the S-ME were collected at 10-min intervals for 6 to 12-h periods, and the concentrations of LHRH and NPY in perfusates were determined by RIA. In Exp I, the release pattern of NPY and LHRH in the S-ME was independently determined in a group of 11 conscious monkeys: NPY release in the S-ME was pulsatile with an interpulse interval of 44.9 +/- 3.3 min (n = 11). This interpulse interval was similar to that seen for LHRH release (43.8 +/- 1.1 min, n = 7). Exp II was designed to determine whether NPY pulses and LHRH pulses occur synchronously and to examine whether NPY release in the S-ME is correlated with circulating LH pulses. NPY and LHRH concentrations in aliquots of the same perfusate sample from the S-ME and circulating LH levels were concurrently measured in 8 monkeys sedated with Saffan. It was found that NPY pulses were temporally correlated (P less than 0.001) with LHRH pulses, which were also temporally correlated (P less than 0.001) with LH pulses. Moreover, NPY pulses were correlated (P less than 0.05) with LH pulses. NPY peaks preceded LHRH peaks by 4.5 +/- 0.6 min, LHRH peaks preceded LH peaks by 5.5 +/- 0.6 min, and NPY peaks preceded LH peaks by 9.7 +/- 0.8 min. In Exp III, the role of endogenous NPY in LHRH release was evaluated by infusing a specific antiserum to NPY into the S-ME during push-pull perfusion in 8 conscious monkeys. Infusion of a specific antiserum to NPY into the S-ME at 1:100 and 1:1000 dilutions suppressed pulsatile LHRH release significantly (P less than 0.05). Infusion of nonimmune serum as a control was without effect. These results are summarized as follows: 1) NPY release in the S-ME is pulsatile, 2) NPY pulses occur synchronously with LHRH and LH pulses, and 3) immunoneutralization of endogenous NPY in the S-ME suppresses pulsatile LHRH release.(ABSTRACT TRUNCATED AT 400 WORDS)

Studies in the rat and rabbit indicate that facilitatory effects of neuropeptide Y (NPY) as well as norepinephrine (NE) on LH and LHRH release are dependent on the presence of the ovarian steroid estrogen. However, we have previously found the NE and an alpha-1-adrenergic agonist are both stimulatory to pulsatile LHRH release in ovariectomized rhesus monkeys. In the present experiment the effects of NPY on LHRH release were examined in conscious monkeys using a push-pull perfusion method. Twelve gonadectomized monkeys (8 females and 4 males) were used. Perfusate samples from the stalk-median eminence (S-ME) were obtained through a push-pull cannula at 10-min intervals for 12 h, and the amount of LHRH in samples were determined with RIA. NPY dissolved in a modified Krebs-Ringer phosphate buffer solution at concentrations of 10(-8), 10(-7), 10(-6), and 10(-5) M was directly infused into the S-ME through the push cannula for 10 min at 90-min intervals. Vehicle was infused as a control. Since sex differences in LHRH response to NPY were not present, data from males and females were combined for analysis. NPY infusion into the S-ME stimulated LHRH release in a dose-dependent manner (P less than 0.001). The peak LHRH responses (mean +/- SEM) to NPY at different concentrations were: 10(-8) M = 2.1 +/- 0.4 pg/ml; 10(-7) M = 2.6 +/- 0.5 pg/ml; 10(-6) M = 6.5 +/- 1.1 pg/ml; 10(-5) M = 15.1 +/- 2.9 pg/ml, whereas to vehicle 0.37 +/- 0.17 pg/ml. All NPY doses tested were significantly effective as compared to vehicle (P less than 0.01). The LHRH response to 10(-6) M was greater (P less than 0.01) than that of 10(-8) M or 10(-7) M, and the response to 10(-5) M was greater (P less than 0.01) than that of all lower doses. The results indicate that NPY infusion into the S-ME elicits the release of LHRH in vivo in a dose-dependent manner in the monkey. The data further suggest that LHRH neurons and/or neuroterminals in the monkey are responsive to NPY stimulation in the absence of gonadal steroids. It is concluded that in addition to NE, NPY is an important regulator of pulsatile LHRH release in the nonhuman primate.

Ultrastructural changes in the testes of the common snapping turtle, Chelydra serpentina, were observed throughout the year. Plasma testosterone levels were measured and compared with the occurrence of delta 5-3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), cholesterol, and steroidogenic ultrastructural features (smooth endoplasmic reticulum (SER), mitochondria with tubular cristae) in Sertoli and Leydig cells. The testosterone level is highest in May and October (mating) and relatively low during the rest of the year. Fluctuations in 3 beta-HSD and cholesterol are consistent with the interpretation that the Leydig cells are potentially active throughout the year. They undergo very little ultrastructural change, (tubular SER to vesiculate and loss of golgi during spermatogenesis and in the winter). Sertoli cells are active only during spermatogenesis from May through October and become inactive until the next cycle; 3 beta-HSD, cholesterol and ultrastructural features change more drastically in the Sertoli cells than in the Leydig cells. These results are discussed with reference to the hypothesis that testosterone of Leydig origin is concerned mainly with mating behavior and that of Sertoli origin with spermatogenesis and maturation of sperm.

Adult mice, made susceptible to Clostridium botulinum by feedings of metronidazole, were immobilized with an anesthetic and held for 30 min in isolators in which a fine powder of lyophilized pathogen spores was made airborne. Exposed mice were surface decontaminated before being kept for 2 days in holding isolators. Mice were intestinally colonized by the pathogen. Colonization rates were related to spore numbers (10(4) to 10(7) type A or B) seeded into isolators.

The administration of arginine vasotocin (AVT) to gravid snapping turtles with steroidogenically active corpora lutea and high plasma progesterone concentration (1480 +/- 155 pg/ml) did not trigger oviposition, whereas 12 days after ovulation when luteolysis occurred and plasma progesterone concentration was low (570 +/- 78 pg/ml), treatment with AVT caused oviposition. Controls with high plasma progesterone concentration (1605 +/- 185 pg/ml) oviposited 15-23 days after ovulation when plasma progesterone concentration dropped to 201 +/- 35 pg/ml. Deluteinization-induced oviposition was initiated 15 hr after surgery and was completed by 30 hr. Oviposition of complete clutches occurred and was correlated with a significant drop in plasma progesterone. Sham-operated turtles did not exhibit oviposition and no significant change in progesterone concentration was observed. A single injection of prostaglandin F2 alpha (PGF) in recently ovulated turtles induced early luteolysis and a significant decrease in plasma progesterone concentration after 24-30 hr. A single administration of PGF caused the disappearance of steroidogenic features such as the smooth endoplasmic reticulum and mitochondria with tubular cristae 48 hr later. Also PGF triggered the invasion of a hyaline-like material from the luteal theca into the luteal cell mass which eventually induced luteolysis. The role of AVT, PGF, and progesterone in relation to egg retention and oviposition is discussed.

Follicular development in snapping turtle, Chelydra serpentina, was studied in relation to changes in plasma levels of estradiol-17 beta, total protein, calcium, inorganic phosphate and cholesterol. Histochemical reaction for 3 beta-HSD in granulosa and theca cells of the follicles is strong during the preovulatory and vitellogenic phases when the follicles attain their maximum size (18-22 mm) and estradiol is at a high level. The reaction for 3 beta-HSD is weal during the postovulatory phase when the developing follicles are at their minimum size (5-7 mm) and estradiol is low. Administration of estradiol-17 beta causes a rise in plasma calcium, total protein, inorganic phosphate and cholesterol.

A high affinity progesterone-binding component was detected in the cytosol of the uterus of the snapping turtle, Chelydra serpentina. Density gradient centrifugation indicated that binding of [3H]progesterone and [3H]promegestone (R5020) was to a fraction with a heavier sedimentation coefficient than bovine serum albumin (BSA) appearing as a broader peak in the 6-7 S region; it was not affected by excess cortisol. Another binding peak, lighter than BSA and appearing with [3H]R5020 and [3H]progesterone near the 4 S region, was affected by excess cortisol. Excess progesterone decreased both the heavier and lighter peaks. Analysis of steroid specificity revealed that, of the natural steroids, progesterone had the highest affinity for the uterine cytosol. This was followed by deoxycorticosterone, 5 alpha-pregnanedione, testosterone, oestradiol-17 beta, corticosterone, 5 alpha-dihydrotestosterone and cortisol. Non-linear regression analysis of saturation data indicated the presence of two classes of high affinity binding sites: progesterone-binding sites (R-sites) with equilibrium association constants (Ka) of 2.9 +/- 0.28 litres/nmol (mean +/- 95% confidence limit) for [3H]R5020 and 0.34 +/- 0.20 litres/nmol for [3H]progesterone, and corticosteroid-binding globulin-like sites (G-sites) with Ka of 4.5 +/- 1.6 litres/nmol for progesterone. The concentration of R-sites was between 0.66 +/- 0.10 and 2.6 +/- 0.55 pmol/mg protein while that of G-sites was between 0.73 +/- 0.05 and 5.0 +/- 0.27 pmol/mg protein. DEAE-cellulose filtration assay also confirmed the presence of R-sites and G-sites in the cytosol. R-sites were detectable without oestrogen priming during the preovulatory and vitellogenic phases (low progesterone, high oestrogen concentrations) when the ovarian follicles are mature (18-22 mm diameter).(ABSTRACT TRUNCATED AT 250 WORDS)