Donal C. Skinner

Distribution of Estrogen Receptor-Immunoreactive Cells in the Preoptic Area of the Ewe: Co-Localization with Glutamic Acid Decarboxylase but Not Luteinizing Hormone-Releasing Hormone

Using immunocytochemical techniques we have examined the distribution of cells containing estrogen receptors (ERs) in the preoptic and anterior hypothalamic regions of short-term (1 week) ovariectomized ewes. Subsequent double-labelling experiments examined the co-localization patterns of ER and luteinizing hormone-releasing hormone (LHRH) or glutamic acid decarboxylase (GAD) immunoreactivities. ER-immunoreactive (-IR) cells were identified throughout the central and medial aspects of the preoptic area in a continuum which begins at the level of the organum vasculosum of the lamina terminalis and terminates in the caudal anterior hypothalamic area. A conspicuous sub-population of densely clustered ER-IR cells was identified within this distribution extending from the central region of the preoptic area into the bed nucleus of the stria terminalis. ER-IR cells were also identified in the ventrolateral septum and supraoptic nuclei. Double-labelling experiments showed that although rostral LHRH neurons were surrounded by ER-IR cells, they did not themselves possess ER immunoreactivity. In marked contrast, we estimate that approximately 40% of GAD-IR cells in the central aspect of the medial preoptic area are immunoreactive for the ER and that these cells account for nearly 30% of all ER-IR cells in this region. These results indicate that, in common with other species, LHRH neurons in the ewe do not possess ERs and suggest therefore, that these neurons are unlikely to be modulated directly by circulating estrogens. However, large numbers of adjacent GABA neurons possess ERs and may comprise a major neuronal population mediating gonadal steroid input to LHRH neurons.

High Melatonin Concentrations in Third Ventricular Cerebrospinal Fluid Are Not due to Galen Vein Blood Recirculating through the Choroid Plexus

Melatonin has been implicated in several neurotropic effects, but few studies have investigated the bioavailability of melatonin in the brain. The discovery of periventricular sites of action adjacent to the third ventricle forced us to investigate the dynamics of cerebrospinal fluid (CSF) melatonin release and the source of this melatonin. Our first study demonstrated unequivocally that third ventricle CSF melatonin, like jugular plasma melatonin, accurately reflects the duration of the night and is rapidly suppressed by light. However, third ventricle CSF melatonin levels are 20-fold higher than nocturnal plasma concentrations. A further study showed that melatonin increased in plasma before third ventricle CSF, raising the possibility that melatonin is taken up from the blood after recirculation through the Galen vein. However, a final experiment suggested strongly that CSF melatonin is released directly into the third ventricle, as melatonin levels in the lateral ventricle were 7-fold lower than those...

Maternal Undernutrition from Early- to Mid-Gestation Leads to Growth Retardation, Cardiac Ventricular Hypertrophy, and Increased Liver Weight in the Fetal Sheep

Abstract Early gestation is critical for placentomal growth, differentiation, and vascularization, as well as fetal organogenesis. The fetal origins of adult disease hypothesis proposes that alterations in fetal nutrition and endocrine status result in developmental adaptations that permanently change structure, physiology, and metabolism, thereby predisposing individuals to cardiovascular, metabolic, and endocrine disease in adult life. Multiparous ewes were fed to 50% (nutrient restricted) or 100% (control fed) of total digestible nutrients from Days 28 to 78 of gestation. All ewes were weighed weekly and diets adjusted for individual weight loss or gain. Ewes were killed on Day 78 of gestation and gravid uteri recovered. Fetal body and organ weights were determined, and numbers, morphologies, diameters, and weights of all placentomes were obtained. From Day 28 to Day 78, restricted ewes lost 7.4% of body weight, while control ewes gained 7.5%. Maternal and fetal blood glucose concentrations were reduced in restricted versus control pregnancies. Fetuses were markedly smaller in the restricted group than in the control group. Further, restricted fetuses exhibited greater right- and left-ventricular and liver weights per unit fetal weight than control fetuses. No treatment differences were observed in any gross placentomal measurement. However, caruncular vascularity was enhanced in conceptuses from nutrient-restricted ewes but only in twin pregnancies. While these alterations in fetal/placental development may be beneficial to early fetal survival in the face of a nutrient restriction, their effects later in gestation as well as in postnatal life need further investigation.

Effects of Photoperiod on Estrogen Receptor, Tyrosine Hydroxylase, Neuropeptide Y, and β-Endorphin Immunoreactivity in the Ewe Hypothalamus

The neural components underlying the influence of photoperiod upon reproductive functioning are poorly understood. In this study, we have used immunocytochemistry to examine whether changes in photoperiod may influence specific neuronal cell populations implicated in mediating gonadal steroid feedback actions on GnRH neurons. Short day (SD) exposed ewes in the midluteal stage of the estrous cycle and long day (LD) anestrous ewes were perfused in pairs and hypothalamic brain sections immunostained for tyrosine hydroxylase (TH), neuropeptide Y (NPY), beta-endorphin (betaE), and the estrogen receptor (ER). The number of ER-immunoreactive cells detected within the preoptic area, but not the hypothalamus, was approximately 20% higher (P < 0.05) in LD ewes compared with SD animals. The numbers of TH-immunoreactive neurons comprising the A12, A14, and A15 cell groups were not different between LD and SD ewes, and the percentage of A12 (approximately 15%) and A14 (approximately 25%) neurons expressing ERs was similarly unaffected by photoperiod. The number of betaE neurons detected in the arcuate nucleus was 50% lower (P < 0.05) in SD vs. LD ewes, whereas NPY-immunoreactive cell numbers in the median eminence were 300% higher (P < 0.05). Approximately 3% of NPY neurons in the median eminence, and 10% in the arcuate nucleus, expressed ER immunoreactivity in a photoperiod-independent manner. These studies indicate that changes in photoperiod may regulate ER expression within the preoptic area and suggest that hypothalamic NPY and betaE neurons are involved in the seasonal regulation of reproductive activity in the ewe.

Control of the circannual rhythm of reproduction by melatonin in the ewe

Annual variations in day length are responsible for seasonal changes in reproductive activity in sheep. However, in constant photoperiodic conditions, ewes express an endogenous rhythm characterized by alternations of reproductive activity and quiescence that are not synchronized among animals. Thus, the main role of photoperiod in the natural environment appears to be the synchronization of this endogenous rhythm. Photoperiodic information is processed through a complex nervous and endocrine pathway to modulate reproductive activity. Light information perceived at the level of the retina is transformed through neural processing into an endocrine signal by the pineal gland: the nocturnal increase in melatonin release. Recent studies strongly suggest that melatonin has a hypothalamic target to modulate the reproductive neuroendocrine axis. Most LHRH perikarya are located in the preoptic area, but this region is devoid of melatonin receptors, and microimplants of melatonin placed in the preoptic area do not effect LHRH release. Thus, melatonin influences LHRH neurones indirectly and must involve interneurons. Good evidence now exists to demonstrate that a population of dopaminergic neurons with axons projecting to the median eminence is one of these interneurons.

Androgen Receptor-lmmunoreactive Cells in Ram Hypothalamus: Distribution and Co-Localization Patterns with Gonadotropin-Releasing Hormone, Somatostatin and Tyrosine Hydroxylase

Testosterone exerts important feedback effects on the hypothalamus of the ram to influence reproductive functioning. To provide a neuroanatomical basis for understanding this androgen action, the present study has examined androgen receptor (AR) immunoreactivity within the hypothalamus and adjacent brain areas of the intact non-breeding season ram. The largest populations of AR-immunoreactive cells were detected in the medial preoptic area, infundibular and premammillary nuclei in addition to the ventromedial nucleus (VMN) where cells were found distributed throughout its medial and lateral divisions. Smaller numbers of AR-expressing cells were identified in the bed nucleus of the stria terminalis and anterior hypothalamic area (AHA) including the paraventricular, but not the supraoptic, nucleus. Double-labelling immunocytochemistry revealed the presence of AR immunoreactivity in only 2 of 460 gonadotropin-releasing hormone (GnRH) neurons. A very small population of TH-immunoreactive cells located in the lateral aspect of the AHA was found to contain ARs. Dopaminergic cells elsewhere in the hypothalamus, including the infundibular nucleus, did not display AR immunoreactivity. Nearly 50% of AR-expressing cells in the lateral VMN were immunoreactive for somatostatin while less than 5% of periventricular somatostatin neurons displayed AR immunoreactivity. These results show where ARs are expressed in the ram hypothalamus and indicate the neuroanatomical sites at which androgen may act to influence reproductive function. The absence of ARs in the neuroendocrine GnRH and tuberoinfundibular dopaminergic cells suggests that androgens do not influence the genome of these cells in any direct manner. In contrast, the somatostatin neurons of the VMN appear to be an important target for circulating androgens in the non-breeding season ram.

Effects of gonadotrophin-releasing hormone outside the hypothalamic-pituitary-reproductive axis.

Gonadotrophin‐releasing hormone (GnRH) is a hypothalamic decapeptide with an undisputed role as a primary regulator of gonadal function. It exerts this regulation by controlling the release of gonadotrophins. However, it is becoming apparent that GnRH may have a variety of other vital roles in normal physiology. A reconsideration of the potential widespread action that this traditional reproductive hormone exerts may lead to the generation of novel therapies and provide insight into seemingly incongruent outcomes from current treatments using GnRH analogues to combat diseases such as prostate cancer.

Seasonal breeding in sheep: Mechanism of action of melatonin

Abstract Melatonin, through its duration of nocturnal secretion, transduces the effects of daylength on the reproductive axis by modifying the pulsatile secretion of luteinising hormone-releasing hormone (LHRH). It is not yet known where or how this pineal indoleamine acts to exert this effect. Although melatonin binding sites are preferentially localised in the pars tuberalis (PT) of the adenohypophysis, which suggests that this may be the site of action, the direct delivery of melatonin into the PT does not influence LH secretion. In contrast, melatonin delivered in the mediobasal hypothalamus mimics the effects on LH secretion of peripherally administered melatonin. These results suggest that, despite the presence of high melatonin binding in the PT, the mediobasal hypothalamus contains the physiological target site of melatonin. The action of melatonin on LHRH secretion does not seem to be directly on LHRH neurones; rather, it appears to involve a complex circuit of interneurones. Both the A15 dopaminergic cell group and dopaminergic neuronal terminals of the median eminence are parts of that network but their anatomical and functional relationships are still unknown. Serotonin and excitatory amino acids also appear to be involved in the seasonal regulation of LHRH secretion. Characterisation of this neuronal circuit is a key to understanding photoperiodic regulation of reproduction.

The Ovine Pars Tuberalis does not Appear to be Targeted by Melatonin to Modulate Luteinizing Hormone Secretion, but may be Important for Prolactin Release

The pineal hormone, melatonin, transduces photoperiodic information to the neuroendocrine axis of seasonally breeding mammals to regulate reproduction. It is not known where or how melatonin achieves this effect, but the recent identification of the pars tuberalis (PT) as the area with the highest density of melatonin binding sites suggests that this pituitary subdivision may be an important target for the actions of this indoleamine on luteinizing hormone (LH) and prolactin release. The present study was designed to test this hypothesis. Ovariectomized oestradiol‐implanted ewes were exposed to inhibitory long days for 85 days and then received melatonin micro‐implants (Day 0) in the mediobasal hypothalamus (MBH; n = 7) or PT (Melatonin‐PT; n = 5). The effect of these micro‐implants was compared to ewes receiving empty micro‐implants in the PT (Sham‐PT; n = 5). For LH, bi‐weekly jugular blood samples were collected and for prolactin, samples were collected every 20 min for 5 h, with the first hour discarded, on Days ‐4, 26 and 69. Melatonin implanted in the MBH stimulated LH secretion in 3 ewes by Day 46±0 after implantation, and one ewe by Day 67 after implantation. In contrast, no Melatonin‐PT or Sham‐PT ewes exhibited an increase in LH secretion by the end of the study (Day 70). A subsequent experiment, in which the Sham‐PT ewes were implanted with melatonin both subcutaneously and in the PT showed that the micro‐implants did not impair the ability of the ovine reproductive neuroendocrine axis to respond to melatonin. For prolactin, both the Melatonin‐PT ewes and MBH ewes (which had displayed an increase in LH secretion) exhibited a significant decrease in prolactin secretion by Day 26 and this decrease persisted until the end of the study. This result suggests that melatonin may have two sites of action on prolactin secretion; the MBH and PT, or, if melatonin diffused from the MBH to the PT, or vice versa, that only one of these sites is important. In contrast, these data suggest that the PT is not an important target for the action of melatonin on LH secretion.

Progesterone Receptor, Estrogen Receptor α, and the Type II Glucocorticoid Receptor Are Coexpressed in the Same Neurons of the Ovine Preoptic Area and Arcuate Nucleus: A Triple Immunolabeling Study

Abstract The neuroendocrine reproductive and stress axes are known to be closely linked, but the mechanisms underlying these links remain poorly understood. In the ovine brain, GnRH neurons do not contain type II glucocorticoid (GR), progesterone (PR), or α estrogen (ERα) receptors. We sought to determine whether PR, ERα, and GR coexist within the same hypothalamic neurons. A triple immunocytochemical study, involving antisera raised in three different species, was performed on cryostat sections from ovariectomized ewes treated either with estradiol and progesterone or with progesterone alone. All PR-immunoreactive neurons contained ERα, and about 95% of ERα were PR immunoreactive in the preoptic area and arcuate nucleus. Although the PR with ERα colocalization ratio was not affected by the steroid treatments, immunolabeling for PR was weaker in animals that did not receive estradiol. Numerous PR- and ERα-immunoreactive cells contain GR. PR+ERα+GR-immunoreactive cells represent 70% of PR, 65% of ERα, and 72% of GR in the preoptic area and 70% of PR, 66% of ERα, and 63% of GR in the arcuate nucleus. These results suggest that estrogen, progesterone, and glucocorticoids may influence the activity of the same neurons to modulate both reproductive and stress axes.