Heiko T. Jansen

The GnRH system of Seasonal breeders : Anatomy and plasticity

Seasonal breeders, such as sheep and hamsters, by virtue of their annual cycles of reproduction, represent valuable models for the study of plasticity in the adult mammalian neuroendocrine brain. A major factor responsible for the occurrence of seasonal reproductive transitions is a striking change in the responsiveness of gonadotropin-releasing hormone (GnRH) neurons to the inhibitory effects of gonadal steroids. However, the neural circuitry mediating these seasonal changes is still relatively unexplored. In this article, we review recent findings that have begun to define that circuitry and its plasticity in a well-studied seasonal breeder, the ewe. Tract tracing studies and immunocytochemical analyses using Fos and FRAs as markers of activation point to a subset of neuroendocrine GnRH neurons in the MBH as potential mediators of pulsatile GnRH secretion. Because the vast majority of GnRH neurons lack estrogen receptors, seasonal changes in responsiveness to estradiol are most probably conveyed by afferents. Two possible mediators of this influence are dopaminergic cells in the A14/A15 cell groups of the hypothalamus, and estrogen receptor-containing cells in the arcuate nucleus that project to the median eminence. The importance of GnRH afferents in the regulation of season breeding is underscored by observations of seasonal changes in the density of synaptic inputs onto GnRH neurons. Thyroid hormones may participate in this remodeling, because they are important in seasonal reproduction, influence the morphology of other brain systems, and thyroid hormone receptors are expressed within GnRH neurons. Finally, in the hamster, neonatal hypothyroidism affects the number of caudally placed GnRH neurons in the adult brain, suggesting that thyroid hormones may influence development of the GnRH system as well as its reproductive functions in the adult brain.

Central connections of the ovine olfactory bulb formation identified using wheat germ agglutinin-conjugated horseradish peroxidase.

Pheromonal stimuli elicit rapid behavioral and reproductive endocrine changes in the ewe. The neural pathways responsible for these effects in sheep are unknown, in part, because the olfactory bulb projections have not been examined in this species. Using the anterograde and retrograde neuronal tracer, wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP), we describe the afferent and efferent olfactory bulb connections of the Suffolk ewe. Injections of WGA-HRP limited to the main olfactory bulb resulted in retrograde labeling of cells in numerous telencephalic, diencephalic, and metencephalic regions. Terminal labeling was limited to layer la of ipsilateral cortical structures extending rostrally from the anterior olfactory nucleus (AON), piriform cortex, anterior-, and posterolateral-cortical amygdaloid nuclei to lateral entorhinal cortex caudally. Injections involving the accessory olfactory bulb and AON produced additional labeling of cells within the bed nucleus of the stria terminalis (BNST), medial nucleus of the amygdala, and a few cells in the posteromedial cortical nucleus of the amygdala. Terminal labeling included a small dorsomedial quadrant of BNST and also extended to the far lateral portions of the supraoptic nucleus. A clearly defined accessory olfactory tract and nucleus was not evident, perhaps due to limitations in the sensitivity of the method. With this possible exception, the afferent and efferent olfactory connections in the sheep appear similar to those reported for other species.

Thyroid Hormone Receptor (α) Distribution in Hamster and Sheep Brain: Colocalization in Gonadotropin-Releasing Hormone and Other Identified Neurons1

Thyroid hormones appear to play an important role in the seasonal reproductive transitions of a number of mammalian and avian species. These seasonal transitions as well as the effects of thyroid hormones on the reproductive neuroendocrine axis are mediated by the GnRH system. How thyroid hormones affect the GnRH system is unclear. Double label immunocytochemistry was used to examine GnRH- and other neurotransmitter/neuropeptide-containing neurons for thyroid hormone receptor (alphaTHR) colocalization in two seasonal breeders, the golden hamster and the sheep. AlphaTHR was identified in hamster and sheep brain by Western blot analysis. Furthermore, alphaTHR immunoreactivity was widely distributed in brain and was colocalized in identified populations: GnRH neurons (hamster, 28%; sheep, 46%); dopaminergic neurons of the A14 (hypothalamic) and A16 (olfactory bulb) cell groups, but not in the hypothalamic A13 cell group; and neurophysin-immunoreactive neurons of the supraoptic and paraventricular nuclei. The finding of alphaTHR in GnRH and A14 dopamine neurons provides an anatomical substrate for direct thyroid hormone action on the reproductive neuroendocrine system of these two seasonally breeding species. It remains to be determined whether the GnRH gene itself or the gene of another constituent within the same GnRH neuron is responsive to thyroid hormones.

Potential for polysialylated form of neural cell adhesion molecule-mediated neuroplasticity within the gonadotropin-releasing hormone neurosecretory system of the ewe

The GnRH neurosecretory system undergoes marked structural and functional changes throughout life. The initial goal of this study was to examine the neuroanatomical relationship between GnRH neurons and a glycoprotein implicated in neuroplasticity, the polysialylated form of neural cell adhesion molecule (PSA-NCAM). Using dual label immunocytochemistry in conjunction with confocal microscopy, we determined that fibers, terminals, and perikarya of GnRH neurons in adult ovariectomized ewes are intimately associated with PSA-NCAM. In the preoptic area, intense PSA-NCAM immunoreactivity was evident around the periphery of GnRH cell bodies. The second goal of this study was to determine whether PSA-NCAM expression associated with GnRH neurons varies in conjunction with seasonal changes in the activity of the GnRH neurosecretory system in ovariectomized ewes treated with constant release implants of estradiol. During the breeding season when reproductive neuroendocrine activity was enhanced, the expression of P...

The Ability of Estradiol to Induce Fos Expression in a Subset of Estrogen Receptor-α-Containing Neurons in the Preoptic Area of the Ewe Depends on Reproductive Status1

In the ewe, seasonal anestrus results from a change in the hypothalamic responsiveness to estradiol (E2) negative feedback. Considerable evidence has implicated a specific group of dopaminergic neurons (the A15 group) in this seasonally dependent E2 effect, but these neurons do not appear to contain estrogen receptor-α (ERα). This apparent discrepancy raises the possibility that at least one other neural system is also involved in mediating E2 inhibition. The purpose of this study was to determine whether ERα-containing neurons are activated by the negative feedback action of E2 in anestrus. In Exp 1, we examined the effects of E2 on expression of the immediate early gene products, Fos and Fos-related antigens, in ERα-positive cells in anestrous ewes. ERα and Fos/ Fos-related antigens were colocalized using a dual immunofluorescence procedure in sections throughout the hypothalamus from ovariectomized and E2-treated ovariectomized anestrous ewes. A low dose E2 treatment that inhibited LH pulse frequency a...

A Subset of Estrogen Receptor‐Containing Neurons Project to the Median Eminence in the Ewe

The neural pathways responsible for conveying the steroid feedback signals that ultimately affect reproductive neuroendocrine function remain largely undefined. One possibility involves a direct projection from estrogen receptor (ER)‐containing neurons to the median eminence (ME), a site of neuroendocrine peptide release. To examine this possibility, 8 ewes received stereotaxic injections of the retrograde neuronal tract‐tracing compound cholera toxin‐β subunit (CTβ) into the ME. Neurons sending projections to the ME and containing ER were identified using a dual‐label immunoperoxidase method. Double‐labeled cells were found in distinct regions: (1) the ER‐rich arcuate nucleus (ARC) that contained the greatest number of double‐labeled cells, and (2) the organum vasculosum of the lamina terminalis (OVLT) which contained a very consistent, but low, number of double‐labeled cells. While a fairly large number of retrogradely‐labeled ARC neurons containing ER were identified, the majority of ER‐containing ARC neurons were unlabeled and thus send projections elsewhere. Other regions containing high concentrations of ER‐positive cells such as the medial preoptic area (MPOA), anterior hypothalamic area, and ventrolateral portion of the ventromedial hypothalamic nucleus, were devoid of double‐labeled cells. Similarly, regions rich in neuroendocrine neurons such as the periventricular hypothalamus and paraventricular and supraoptic hypothalamic nuclei contained no double‐labeled cells. These results suggest that modulation of neuroendocrine secretory activity may occur directly at the level of the ME by ER‐containing neurons located within restricted regions of the hypothalamus and forebrain. However, the relatively low proportion of ER‐containing neurons projecting to the ME suggests that the influence of estradiol upon neuroendocrine function also may include target sites other than the ME.

Disruption of Reproductive Rhythms and Patterns of Melatonin and Prolactin Secretion Following Bilateral Lesions of the Suprachiasmatic Nuclei in the Ewe

To determine whether the photoperiodic responses of reproductive and prolactin (PRL) rhythms in the ewe requires an intact suprachiasmatic nucleus (SCN) driving the pineal rhythm of melatonin secretion, four groups of ovary‐intact ewes over a 6‐year period were subjected to bilateral (n = 40) or sham lesions (n = 15) of the SCN. Animals were exposed to an alternating 90–120 day photoregimen of 9L:15D and 16L: 8D photoperiods. Blood samples taken twice weekly were assayed for prolactin and for progesterone to monitor oestrous cycles. On several occasions blood samples also were taken at hourly intervals for 24 h and analyzed for melatonin. Melatonin concentrations in sham lesioned ewes were basal during the lights‐on period and rose robustly during darkness. Those sheep bearing unilateral lesions of the SCN (n = 13) or where the lesion spared the SCN entirely (n = 8) had patterns of melatonin secretion similar to sham ewes. The remaining ewes, having complete (n = 9) or incomplete bilateral (n = 8) destruction of the SCN, with one exception, had disrupted patterns of melatonin secretion. The nature of this disruption varied from complete suppression to continuously elevated levels. In lesioned ewes where melatonin secretion was not affected the onset and cessation of ovarian cycles were similar to sham ewes; stimulation of oestrous cycles under 9L:15D and cessation of oestrous cycles under 16L:8D. In contrast, 13 of 17 ewes with disrupted melatonin secretion also exhibited disrupted patterns of ovarian activity. In these animals oestrous cycles were no longer entrained by photoperiod but still occurred in distinct clusters, that is, groups of cycles began and ended spontaneously. Sheep with normal melatonin patterns showed low levels of PRL secretion during short days and elevated PRL levels during long days. However, 8 of 13 ewes with disrupted melatonin showed patterns of PRL secretion that were no longer entrained by photoperiod. A minority of ewes with disrupted melatonin patterns still showed reproductive (n = 4) and PRL (n = 5) responses similar to those of sham‐lesioned ewes. These results show that bilateral destruction of the SCN in the ewe disrupts the circadian pattern of melatonin secretion and that this disruption usually, but not always, is associated with altered photoperiodic responses. These results strongly suggest that the SCN are important neural elements within the photoperiod time–keeping system in this species. A role for the SCN in the generation of endogenous transitions in reproductive activity (refractoriness) and prolactin secretion is not supported.

Olfactory Bulb Removal Does Not Prevent Gonadotropin or Prolactin Responses to Changing Photoperiod in the Ewe

The purpose of this study was to determine if bilateral olfactory bulb removal (Bulbx) alters photoperiod-induced changes of gonadotropin and prolactin secretion in the ewe. Ovariectomized (group 1; n = 12) or ovariectomized estradiol-treated (group 2; n = 12) Suffolk ewes underwent Bulbx (7 per group) or sham operations (5 per group). All ewes subsequently were placed into photochambers and exposed to a photoregimen of alternating 16 h light/8 h dark and 10 h light/14 h dark photoperiods. Plasma concentrations of luteinizing hormone (LH), follicle-stimulating hormone (FSH) and prolactin were determined in blood samples taken twice weekly from group 2 ewes. At the end of each 90-day photoregimen blood samples from all ewes in both groups were taken at frequent intervals for 4 h to determine LH pulse parameters. During the initial 16 h light/8 h dark photoperiod, plasma melatonin concentrations were determined for group 2 ewes during a 24-hour period. The completeness of Bulbx was determined at time of necropsy for all Bulbx ewes. In addition, the functional completeness of Bulbx was determined in group 2 ewes by exposing them to rams wool and measuring changes in LH secretion. Bulbx did not affect either basal or photoperiod-induced changes in LH, FSH and prolactin in group 2 ewes. LH pulse parameters varied with photoperiod but were not significantly (p > 0.05) affected by Bulbx in either group 1 or group 2 ewes. The normal nocturnal elevation in plasma melatonin concentrations was unaffected by Bulbx.(ABSTRACT TRUNCATED AT 250 WORDS)

A re-evaluation of the effects of gonadal steroids on neuronal activity in the male rat

Single unit activity (SUA) was recorded from 77 cells located in the arcuate nucleus (ARC) and medial preoptic area (MPA) of anesthetized, intact male rats. Animals were administered vehicle, testosterone (T; 5 or 50 micrograms) or 17 beta-estradiol (E; 0.5 microgram) intravenously and SUA was monitored for 8-12 min. T (50 micrograms) reduced SUA in 50% of ARC units and 44% of MPA units within 2.1 +/- 0.46 and 3.3 +/- 0.92 min, respectively. Inhibition of ARC SUA was more pronounced than MPA SUA. A small percentage (9%) of ARC units were excited by T. E reduced SUA in 29% of ARC units and 27% of MPA units. Single doses of 5 micrograms T did not affect ARC activity. However, when followed within 10 min by an additional dose of 5 or 50 micrograms T, 30% and 43% of ARC units were inhibited, respectively. Doses (10 micrograms) of T produced plasma T concentrations within physiological limits, although 50 micrograms doses produced supraphysiological T levels. Neither dose affected circulating LH concentrations. We conclude that physiological and supraphysiological concentrations of T can rapidly affect SUA within the ARC.