Ž .
ERa mRNA-containing cells Hileman et al., 1999 . The highest levels of ERb
expression were found in the preoptic area, bed nucleus of the stria terminalis, paraventricular nucleus and supraoptic nucleus. In contrast to ERa mRNA, ERb
mRNA was observed in the retrochiasmatic area, but very few ERb mRNA-containing cells were observed in the ventromedial nucleus and arcuate nucleus.
Ž .
We have cloned a portion of ovine ERb Genebank accession number applied for and compared the distribution and abundance of ERb mRNA in the hypothalamus of
ewes in the luteal phase of the oestrous cycle and intact rams using in situ hybridisation ŽScott, C.J., Tilbrook, A.J., Simmons, D.M. Rawson, J.A., Chu, S., Fuller, P.J., Ing,
. N.H., Clarke, I.J., unpublished data . Preliminary results indicate that the distribution
and level of expression of ERb mRNA is similar in rams and ewes with the exception of the ventromedial and arcuate nuclei where moderate levels were detected in the ewe;
Ž .
there was almost no ERb mRNA in this region of the ram brain Fig. 1 . We compared Ž
the distribution of ERb mRNA with ERa mRNA in ewes and rams as outlined in .
Ž Section 2.1 and a number of differences were observed 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Simmons, D.M., Rawson, J.A., Chu, S., Fuller, P.J., Ing, N.H., Clarke, I.J., unpublished .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 . In both sexes, ERa-mRNA was widely distributed in the preoptic area, whereas in the rostral preoptic area, ERb mRNA was localised to a thin strip down the midline of
the brain. In addition, the preoptic area, lateral septum, ventromedial and arcuate nuclei all contained higher numbers of ERa than ERb mRNA-containing cells. By contrast,
the median preoptic nucleus, and the zona incerta contained higher numbers of ERb than ERa mRNA-containing cells, with the retrochiasmatic nucleus containing only
ERb mRNA.
3.2. A role for ER b — the control of LH secretion? Ž
. Male mice lacking a functional ERb appear to be fully fertile Krege et al., 1998 .
This, combined with a paucity of ERb-containing cells in hypothalamic nuclei known to Ž
. be important in the regulation of GnRH secretion vide supra , suggests that ERb may
not be involved in the feedback regulation of GnRH secretion. Nevertheless, the Ž
presence of ERb mRNA-containing cells in the retrochiasmatic nucleus A15 dopamin- .
ergic cell group of the sheep, raises an intriguing possibility of involvement in
Ž .
oestrogen negative feedback during anoestrus Thiery et al., 1995 . Implantation of oestrogen directly into the retrochiasmatic nucleus suppressed plasma LH levels in
Ž .
ovariectomised ewes during an inhibitory photoperiod Gallegos-Sanchez et al., 1997 , Ž
. Ž .
yet ERa has not been detected in this nucleus Lehman et al., 1993a vide supra . It
remains possible, therefore, that oestrogen acts via ERb within the retrochiasmatic nucleus to inhibit LH secretion during the non-breeding season in the sheep.
4. Progesterone receptor
4.1. Location of cells containing PR Recent evidence suggests that the negative feedback actions of progesterone are
Ž .
mediated via the classical nuclear PR Skinner et al., 1998 . We used immunocytochem-
Ž .
istry to map the distribution of PR in the hypothalamus of the ewe Scott et al., 2000 . The largest population of PR-containing cells was found in the preoptic area, especially
in the medial region, around the OVLT. The arcuate and ventromedial nuclei also contained large numbers of cells containing PR, with moderate numbers in the periven-
tricular and supraoptic nuclei, and scattered cells in the diagonal band of Broca. We did not detect any PR-containing cells in the brainstem.
We were readily able to detect PR cells in ovariectomised ewes, which was surprising considering that, in other species, significant numbers have been detected following
Ž .
oestrogen treatment e.g. Blaustein and Turcotte, 1989; Bethea et al., 1992 . To confirm Ž
. this, we used in situ hybridisation with a riboprobe for ovine PR Ing et al., 1996 . PR
mRNA-containing cells were clearly observed in the hypothalamus of ovariectomised Ž
. ewes Scott et al., 2000 , with a similar distribution to the PR protein as detected with
immunocytochemistry. Treatment of ovariectomised ewes with a subcutaneous oestro- gen implant for 14 days increased the number of PR-containing cells in the ventromedial
Ž .
Ž .
and arcuate nuclei Fig. 2 , but not in other hypothalamic regions Scott et al., 2000 . This observation highlights the importance of these nuclei in the regulation of steroid
feedback in the sheep. In the ewe, LH secretion is altered by implantation of proges- Ž
. Ž
. terone Blache et al., 1996 or oestrogen Blache et al., 1991 into the ventromedial
nucleus. These steroids are thought to act in synergy to regulate LH secretion in the Ž
. sheep Goodman and Karsch, 1980 , with oestrogen enhancing the efficacy of proges-
Fig. 2. Low power photomicrographs showing the effect of oestrogen treatment on PR-immunoreactive cells in Ž .
Ž . the mediobasal hypothalamus: a ventromedial nucleus in an oestrogen treated ewe; b ventromedial nucleus
Ž . Ž .
in an OVX ewe; c arcuate nucleus in an oestrogen treated ewe; d arcuate nucleus in an OVX ewe. 3V: third ventricle. Scale bar s100 mm.
Fig. 3. Film autoradiograms showing the distribution of PR mRNA-containing cells in the hypothalamus of a Ž .
Ž . Ž .
Ž . ram and ewe: a preoptic area of a ram; b preoptic area of a ewe; c mediobasal hypothalamus of a ram; d
mediobasal hypothalamus of a ewe. 3V: third ventricle; ARC: arcuate nucleus; OC: optic chiasm; POA: preoptic area; VMN: ventromedial nucleus. Scale bar s 2 mm.
terone negative feedback. The results above suggest that this effect of oestrogen may be achieved via an increase in PR-containing cells. This reinforces the notion that, in the
ewe, the ventromedial and arcuate nuclei are major sites for these steroids to exert dual regulation of LH secretion.
Progesterone, although a precursor for testosterone synthesis, is not normally associ- ated with feedback regulation of gonadotropin secretion in males. Nonetheless, synthetic
Ž progestogens are showing great promise as a reversible contraceptive for men WHO,
. 1999 by virtue of their ability to suppress gonadotropin secretion. Accordingly, we used
in situ hybridisation to determine if PR mRNA is produced in the brain of male sheep Ž
. Scott, C.J., Tilbrook, A.J., Clarke, I.J., unpublished observations and found expression
Ž .
similar to that in females Fig. 3 . Whether these receptors have a functional role in the feedback regulation of GnRH secretion in male sheep is currently being determined.
4.2. Neurochemical identity of neurones containing PR The neurochemical identity of PR-containing cells in the brain of domestic animals is
unknown, but clues as to which cell types might be involved can be gained from pharmacological studies. In the sheep, progesterone alters g-aminobutyric acid levels in
Ž .
the preoptic area Robinson and Kendrick, 1992 , dopamine levels in the mediobasal Ž
. Ž
hypothalamus Fabre-Nys et al., 1994 , and b-endorphin in the arcuate nucleus Yang et .
al., 1988; Whisnant et al., 1991 . Progesterone treatment altered levels of pre-proen- Ž
kephalin mRNA in the ventromedial nucleus of ovariectomised ewes Broad et al., .
Ž 1993 , although we did not detect any changes across the estrous cycle Walsh, J.P.,
. Clarke, I.J., unpublished . There is also evidence that endogenous opioids mediate
Ž .
Ž .
progesterone feedback in cattle Stumpf et al., 1993 and pigs Okrasa et al., 1992 . Thus, in these species, the cells producing these neurotransmitters are good candidates to
also co-express PR.
5. AR
