Introduction ER a Directory UMM :Data Elmu:jurnal:A:Animal Reproduction Science:Vol60-61.Issue1-4.Jul2000:

1. Introduction

Oestrogens, androgens and progestogens, produced by the gonads, exert feedback Ž . Ž actions to regulate the secretion of luteinizing hormone LH in domestic animals e.g. sheep, Clarke, 1995; Tilbrook and Clarke, 1995; cattle, Burke et al., 1996; pig, Elsaesser . et al., 1992 . The major site of this action is within the brain to regulate the secretion of Ž . Ž gonadotrophin-releasing hormone GnRH sheep, Clarke, 1995; Tilbrook and Clarke, . 1995; Skinner et al., 1998; cattle, Gazal et al., 1998 , although oestrogen has a direct Ž action on the gonadotrophs of the anterior pituitary gland also Clarke and Cummins, . 1984 . The actions of these hormones are most likely through the classical steroid Ž . receptors Skinner et al., 1998 , which are ligand-activated transcription factors, al- Ž though evidence is accumulating for signalling via membrane-bound receptors Moss et . Ž . al., 1997 . Two forms of the oestrogen receptor ER have now been identified, the Ž . original ER, now designated ERa and a newly identified ERb Kuiper et al., 1996 . Despite reports that a small number of GnRH neurones in the rat brain may contain ERa Ž . Ž . Ž . Butler et al., 1999 and progesterone receptors PRs King et al., 1995 , studies in a variety of species consistently indicate that GnRH neurones do not contain ERa , ERb, Ž . Ž PR or androgen receptors ARs Shivers et al., 1983; Fox et al., 1990; Leranth et al., . 1992; Herbison et al., 1993; Herbison et al., 1996; Laflamme et al., 1998 . It is generally considered, therefore, that the actions of the gonadal steroid hormones must be via neurones that contain these receptors and that projectrrelay to the GnRH neurones. The neural pathways involved in mediating the actions of the gonadal steroids on GnRH secretion in domestic species remain largely unknown. In recent years, work in our laboratory has focused on determining these pathways in female and male sheep. In this paper, we review some of the work undertaken by our group, and others, to determine the sites of action and pathways involved in the steroid regulation of GnRH secretion in domestic animals. It is worth noting that GnRH neurones from female sheep Ž . receive twice as many synaptic contacts as those from male sheep Kim et al., 1999 and, therefore, there are likely to be sex differences in the pathways mediating steroid feedback on GnRH secretion. Accordingly, this review will compare data obtained from both sexes.

2. ER a

2.1. Location of cells containing ER a The distribution of ERa-containing cells in the hypothalamus of the ewe has been Ž well described using immunocytochemistry Herbison et al., 1993; Lehman et al., 1993a; . Ž Blache et al., 1994 . The greatest concentration is in the preoptic area the region . containing the majority of GnRH neurones in this species, Lehman et al., 1986 , the ventromedial nucleus and the arcuate nucleus. Moderate to high numbers of cells containing ERa are also found in the bed nucleus of the stria terminalis and the anterior hypothalamic area, with ERa-containing cells also found in the lateral septum, dorsome- dial hypothalamus and posterior hypothalamus. The distribution of ERa-containing cells in the hypothalamus of the female pig is similar, although numerous cells containing ERa were also found in the stigmoid nucleus and what the authors’ term the dorsome- Ž . dial extension of the supraoptic nucleus Van Leeuwen et al., 1995 . The distribution of ERa-containing cells in the hypothalamus of the male pigs appears to be the same as in Ž . females Van Leeuwen et al., 1995 . Notably, high numbers of ERa-containing cells are found in a region of the preoptic area of the female pig brain that corresponds to the sexually dimorphic nucleus of rodents. Female pigs also possess a greater number of Ž cells containing ERa in the arcuate and ventromedial nuclei than male pigs Van . Leeuwen et al., 1995 . The distribution of ERa-containing cells in the hypothalamus of Ž male sheep has not been delineated although Herbison unpublished observations cited . in Herbison, 1995 has described it as ‘‘approximately similar to that of AR-containing Ž . Ž . cells in the ram ’’ vide infra . Cells containing ERa cells have been described in a Ž . number of regions of the brainstem in the ewe Scott et al., 1998; Simonian et al., 1998 , including the ventrolateral medulla, nucleus of the solitary tract, and area postrema in the caudal brainstem, and parabrachial nucleus, subcoeruleus region and peri-aqueductal gray in the pons region. We examined the distribution of ERa mRNA-containing cells in the hypothalamus Ž and brainstem of ewes using in situ hybridisation Scott, C.J., Tilbrook, A.J., Simmons, . D.M., Rawson, J.A., Chu, S., Fuller, P.J., Ing, N.H., Clarke, I.J., unpublished data and found it to be essentially the same as reported for immunocytochemical studies. Two exceptions were the supraoptic and paraventricular nuclei, which contain only scattered ERa-containing cells as detected by immunocytochemistry, but a dense population of ERa mRNA-containing cells. It remains to be demonstrated if the difference in cell number reflects methodology or whether many ERa mRNA-containing cells in these regions do not produce the ERa protein. We compared the level of ERa mRNA expression in luteal phase ewes and intact rams and preliminary results indicate that the distribution is essentially the same in both sexes, although the amount of ERa mRNA Ž per cell is lower in the ventromedial nucleus in rams Scott, C.J., Tilbrook, A.J., Simmons, D.M., Rawson, J.A., Chu, S., Fuller, P.J., Ing, N.H., Clarke, I.J., unpublished . Ž . data Fig. 1 . 2.2. Projections of neurones containing ER a For steroid receptor-containing neurones to regulate GnRH secretion, they must project to GnRH neurones or regions important in the regulation of GnRH secretion. Little is known about the neuronal inputs to GnRH neurones in domestic animals. Retrograde neuronal tracing has been performed in the ewe to characterise the cells that Ž project to the region of the GnRH cells, in the medial preoptic area e.g. Tillet et al., . 1993 . ERa-containing cells that project to the medial preoptic area of the ewe originate in the preoptic area, lateral septum, arcuate nucleus and caudal ventromedial nucleus Ž . Ž Goubillon et al., 1999 , as well as the ventrolateral medulla of the brainstem Scott et . al., 1999 . While it remains to be verified if these neurones synapse directly onto GnRH Ž . Ž . Fig. 1. Photomicrographs of ER mRNA in the ventromedial nucleus: a ERa in a ewe; b ERa mRNA in a Ž . Ž . Ž ram; c ERb mRNA in a ewe; d ERb mRNA in a ram bright field at higher power to show lack of silver . grain accumulation over Nissl stained cells . 3V: third ventricle. Scale bar s100 mm. neurones, the ERa-containing cells in these hypothalamic and brainstem nuclei are well placed to regulate GnRH neurones. It is also possible that oestrogen influences GnRH secretion through neurones that are activated by oestrogen and project to terminals within the neurosecretory zone of the Ž . median eminence. Jansen et al. 1996 injected retrograde tracer into the median eminence of ewes and showed that ERa-containing cells in the organum vasculosum of Ž . the lamina terminalis OVLT and arcuate nucleus project to the median eminence. It should be noted, however, that synaptic input is very difficult to find at this level and Ž . only one study Kuljis and Advis, 1989 has reported such interaction. There is much evidence that retrochiasmatic area is involved in enhancing the Ž . negative feedback actions of oestrogen during anoestrus in the ewe Thiery et al., 1995 , Ž although no ERa-containing cells have been detected in this nucleus Lehman et al., . 1993a . Nevertheless, the influence of estrogen could be via cells from other brain regions that project to the retrochiasmatic area. Using retrograde tracing coupled with immunocytochemistry, ERa-containing cells that input to the retrochiasmatic area of the ewe were identified as projecting from the rostral medial preoptic area, anterior Ž hypothalamic area, ventromedial nucleus and arcuate nucleus Jansen, Heiko T., per- . sonal communication . In a study comparing anoestrous and cycling ewes, Lehman et al. Ž . 1996 used immunocytochemistry for the Fos-related antigens as markers of long-term cellular activation by oestrogen. They found that only cells in the rostral preoptic area and the retrochiasmatic area showed activation specifically during anoestrus and not during the breeding season. Thus, there appears to be a population of cells in these nuclei that are oestrogen responsive only during anoestrus. Taken together, these two studies would suggest that the negative feedback action of oestrogen during anoestrus may be mediated in part via cells in the preoptic area that contain ERa and project to the retrochiasmatic area. An alternative hypothesis for the actions of oestrogen on this nucleus is outlined in Section 3.2. In an effort to determine which sites in the hypothalamus are relevant to the regulation of GnRH secretion by oestrogen, microimplants of oestrogen were placed into various brain sites of gonadectomised ewes and rams and the effect on LH was Ž . evaluated. Implants in the ventromedial nucleus but not the preoptic area induced an Ž . LH surge in ovariectomized ewes Blache et al., 1991 and implants into the arcuate nucleus–ventromedial region of castrated rams were effective in reducing LH secretion Ž . Blache et al., 1997; Scott et al., 1997 . These data, combined with those of retrograde tracing studies, suggest that one important pathway for oestrogen feedback in ewes and rams may involve ERa-containing cells in the ventromedial nucleus that project to the preoptic area. Notably, the nature of this feedback differs between sexes, with oestrogen having a positive feedback action at this site in ewes and a negative feedback action on GnRH secretion in males. The origin of ERa-containing cells that project to the preoptic area has not been described in males. 2.3. Neurochemical identity of neurones containing ER a A key to understanding the feedback actions of gonadal steroids is a determination of the neurochemical identity of the neurones containing their receptors. The neurochemi- cal identity of the majority of ERa-ir cells remain to be elucidated, even in the arcuate Ž . nucleus, which as been studied most intensively Table 1 . Studies so far have concentrated on identifying cells producing neurotransmitters that are thought to be involved in the regulation of GnRH secretion. However, ER-containing cells in the hypothalamus subserve a number of functions other than the regulation of GnRH secretion, including the regulation of reproductive behaviour, and these cells may well Table 1 Neuronal cells that contain ERa-ir in the sheep brain Neurotransmitter Nucleus Percentage of Reference or marker labelled cells that contain ERa-ir Ž . Ž Tyrosine hydroxylase Arcuate A12 -10 of Batailler et al. Ž . . Ž . Dopamine ER-ir cells 1992 3–5 Lehman and Ž . Karsch 1993b 15 Skinner and Ž . Herbison 1997 Ž . Preoptic area A14 Lehman and Ž . Karsch 1993b 13–18 Skinner and Ž . Herbison 1997 Anterior hypothalamus 3–5 Lehman and Ž . Ž . A14 Karsch 1993b 25 Skinner and Ž . Herbison 1997 Glutamic acid decarboxylase Preoptic area 44 Herbison et al. Ž . Ž . GABA 1993 b -Endorphin Arcuate 15–20 Lehman and Ž . Karsch 1993b Neuropeptide Y Arcuate 3 Skinner and Ž . Herbison 1997 Ž Ž . Somatostatin Ventromedial Nucleus ; 35 ; 70 Herbison 1995 . of ER-ir cells Ž . Dopamine b hydroxylase Ventrolateral medulla A1 20–70 Simonian et al. Ž . Ž . noradrenaline 1998 Ž . 4–20 Scott et al. 1999 Nucleus of the solitary 16–60 Simonian et al. Ž . Ž . tract A2 1998 Ž . 8–25 Scott et al. 1999 GnRH Preoptic area Herbison et al. Ž . 1993 Lehman and Karsch Ž . 1993b produce neurotransmitters not yet identified. The neurochemical identity of ERa-con- taining cells in the brain of male sheep has not been described.

3. ERb