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Gonadotropin-releasing hormone-1 (GnRH-1) neurons play critical roles in the development and maintenance of reproductive function in all vertebrates. Due to a truncation in the GnRH-1 gene, hypogonadal (hpg) mice are unable to synthesize GnRH-1 and are infertile. These animals develop in the complete absence of exposure to gonadal steroid hormones, making them an interesting model for understanding brain sexual differentiation and dimorphism. We studied expression of the estrogen receptors (ERs) alpha and beta in the medial anteroventral periventricular nucleus (mAVPV), an important reproductive neuroendocrine brain region, in wild-type and hpg mice of both sexes. Adult wild-type and hpg mice of the same genetic background were used to quantify numbers of ERalpha and ERbeta immunoreactive cells in the mAVPV using a stereologic approach. Quantitative analyses showed that ERalpha cell numbers were significantly higher in hpg than wild-type mice, irrespective of sex. Qualitatively, ERalpha-immunoreactive cells were concentrated more densely along the ventricular zone of the AVPV of wild-type female mice compared with wild-type male mice or hpg male and female mice. No ERbeta-immunoreactive cells were detected in the mAVPV of any mice, a result that was surprising because ERbeta expression is abundant in the mAVPV of rats. These results on ERalpha provide additional evidence that the female brain is not the "default" organizational pattern, because neither ERalpha cell number nor its distribution in hpg mice, which develops with a deficiency of reproductive hormones, resembles that of the wild-type female mouse. Differences in ERalpha expression may be due in part to the absence of gonadal steroid hormones, although they more likely to also involve other factors, potentially GnRH itself.

The established function of thyroid stimulating hormone (TSH) is to promote thyroid follicle development and hormone secretion. The osteoporosis associated with hyperthyroidism is traditionally viewed as a secondary consequence of altered thyroid function. We provide evidence for direct effects of TSH on both components of skeletal remodeling, osteoblastic bone formation, and osteoclastic bone resorption, mediated via the TSH receptor (TSHR) found on osteoblast and osteoclast precursors. Even a 50% reduction in TSHR expression produces profound osteoporosis (bone loss) together with focal osteosclerosis (localized bone formation). TSH inhibits osteoclast formation and survival by attenuating JNK/c-jun and NFkappaB signaling triggered in response to RANK-L and TNFalpha. TSH also inhibits osteoblast differentiation and type 1 collagen expression in a Runx-2- and osterix-independent manner by downregulating Wnt (LRP-5) and VEGF (Flk) signaling. These studies define a role for TSH as a single molecular switch in the independent control of both bone formation and resorption.

We describe the effects of the overexpression of noggin, a bone morphogenetic protein (BMP) inhibitor, on osteoblast differentiation and bone formation. Cells of the osteoblast and chondrocyte lineages, as well as bone marrow macrophages, showed intense beta-gal histo- or cytostaining in adult noggin+/- mice that had a LacZ transgene inserted at the site of noggin deletion. Despite identical BMP levels, however, osteoblasts of 20-month-old C57BL/6J and 4-month-old senescence-accelerated mice (SAM-P6 mice) had noggin expression levels that were approximately fourfold higher than those of 4-month-old C57BL/6J and SAM-R1 (control) mice, respectively. U-33 preosteoblastic cells overexpressing the noggin gene showed defective maturation and, in parallel, a decreased expression of Runx-2, bone sialoprotein, osteocalcin, and RANK-L. Noggin did not inhibit the ligandless signaling and pro-differentiation action of the constitutively activated BMP receptor type 1A, ca-ALK-3. Transgenic mice overexpressing noggin in mature osteocalcin-positive osteoblasts showed dramatic decreases in bone mineral density and bone formation rates with histological evidence of decreased trabecular bone and CFU-osteoblast colonies at 4 and 8 months. Together, the results provide compelling evidence that noggin, expressed in mature osteoblasts, inhibits osteoblast differentiation and bone formation. Thus, the overproduction of noggin during biological aging may result in impaired osteoblast formation and function and hence, net bone loss.

Changes in connectivity of the gonadotropin-releasing hormone (GnRH) neuronal system are believed to occur during the transition from juvenile to adulthood in females. Experiments were designed to investigate whether there is any difference in the number of galanin inputs to GnRH cells located in the organum vasculosum of lamina terminalis-rostral preoptic area (OVLT-rPOA) between juvenile (2 weeks old) and adult (10 weeks old) female mice. Triple label immunofluorescence staining of brain sections for galanin, GnRH and the presynaptic vesicle marker synaptophysin coupled with confocal microscopy was employed to identify galanin synapses to GnRH perikarya. The number of galanin synapses to GnRH cells and the proportion of GnRH cells with galanin input were significantly higher in adults than in juvenile mice. In adult mice, the proportions of GnRH cells with 0, 1, 2, 3, 4, 5 or 6 galanin synapses/cell were comparable to each other whereas in the juveniles the vast majority of them received no galanin synaptic input. A greater number of galanin synapses in adult as compared with juvenile female mice suggests a functional role for galanin in the maturation of the GnRH system.

Even though the cells producing gonadotropin-releasing hormone (GnRH) are scattered in the basal forebrain, a large proportion of them is present in the organum vasculosum of the lamina terminalis (OVLT) and in the preoptic area. The present studies were undertaken to investigate whether there is any difference in the number of synaptic inputs between GnRH cells located in the OVLT and those located at more anterior levels of the brain. Immunohistochemical staining for the synaptic marker synaptophysin coupled with confocal microscopy was employed to analyze synaptic inputs to GnRH cells located at the two levels examined. The results indicate that GnRH cells in the OVLT region receive a greater number of synaptophysin-immunoreactive appositions as compared with those located in the anterior septum. This supports the existence of subsets among the GnRH cells located in the basal forebrain. The effect of estradiol on the number of synaptophysin-immunoreactive appositions onto GnRH cells was also studied. Treatment of ovariectomized mice with estradiol significantly enhanced the number of synaptophysin-immunoreactive appositions to GnRH cells located at both levels examined. Thus the effect of estrogen on GnRH cells may be mediated in part by changes in the number of synaptic contacts., (Copyright 2001 S. Karger AG, Basel.)

The distribution of galanin-immunoreactive (GAL-IR) cell bodies in the basal forebrain of mice was investigated. The overall pattern of staining for GAL in the area of brain analyzed was similar to that reported in other species with noticeable variations. Distinctive groups of GAL-IR cells were present in the bed nucleus of stria terminalis (BNST), supraoptic nucleus, retrochiasmatic supraoptic nucleus (SOR), magnocellular paraventricular nucleus, arcuate nucleus (ARC) and the nucleus circularis which is one of the cell groups belonging to the accessory magnocellular system. Comparison of the number of GAL-IR cells between the sexes indicated sexual dimorphism in the BNST, SOR and the ARC. As compared with female mice, the mean number of GAL-IR cells/section in the BNST and the SOR was higher and that in the ARC was lower in the males. Unlike in rats, the preoptic area contained mostly scattered GAL-IR cell bodies. Intraperitoneal injection of the retrograde tracer fluoro-gold in male mice resulted in uptake of fluoro-gold by selective GAL-IR cell groups in the basal forebrain suggesting that only some of these cell groups may project outside the blood-brain barrier whereas others may be involved in intracerebral neural transmission., (Copyright 2000 S. Karger AG, Basel.)

This study examined the distribution and regulation of androgen receptor immunoreactivity (IR) in the brain of the hypogonadal (hpg) male mouse, genetically deficient in GnRH. Five groups of animals were studied: intact, castrated, or castrated and testosterone propionate (TP)-treated normal adult male mice, and intact or TP-treated hpg adult male mice. All groups were studied 1 week after treatment. Five regions of the brain with high concentrations of androgen receptors in normal animals were examined, including the medial preoptic area, the lateral ventral septum, the ventromedial hypothalamus, the bed nucleus of the stria terminalis and the medial amygdala. The results showed that the congenital absence of GnRH results in minimal expression of androgen receptor-IR in mice in all regions examined. However, treatment with exogenous testosterone for 1 week was sufficient to induce the numbers of neurons containing androgen receptors, as detected by immunocytochemistry, into the range seen in normal male mice in all the areas studied except the VMH. Similar plasticity was also observed in normal males after 1 week of castration and TP replacement., (Copyright 1999 Elsevier Science B.V.)

A deletion in the gene encoding gonadotropin-releasing hormone (GnRH) induces hypogonadism in mice caused by the deficiency of GnRH. Activation of the reproductive axis can be achieved in these hypogonadal (hpg) mice by third cerebro-ventricular transplantation of preoptic area (POA) containing GnRH neurons, obtained from normal fetal mice. The present study was carried out in female hpg mice with POA grafts (hpg/POA) to investigate anatomical integration of the GnRH cells required for the functional activation of the reproductive system. Ovarian development was present only in mice in which the graft tissue was located close to the median eminence (ME). The total lack of ovarian development in individuals with grafts containing GnRH cells located elsewhere in the brain suggests that the mere presence of GnRH cells does not guarantee ovarian development, but that the location of the graft may be important. Activation of the grafted GnRH cells following mating, as evidenced by the induction of Fos immunoreactivity, was observed in hpg/POA mice in which there was no ovarian development or detectable GnRH immunoreactive fiber innervation of the ME. Although ovarian development was evident in individuals with grafts located close to the ME, release of luteinizing hormone (LH) in response to mating was apparent in only some of these mice. The occurrence of mating and pregnancy only in hpg/POA mice with ovarian development and the reflex release of LH in response to mating suggests that both the efferent and afferent connections of the GnRH system are important for the full functioning of the system.

Chemosensory cues detected by the vomeronasal (VN) organ modulate a variety of social interactions in many species. In particular, activation of the VN system by pheromones regulates sexual behavior in the rodent. Although the exact nature of stimulus access to the organ is not clearly defined, the neuroanatomical pathway connecting the VN organ to hypothalamic centers controlling reproductive function is well established and relatively straightforward. Electrophysiological techniques have provided insight into the signal transduction process throughout the VN system. Combining behavioral studies with immunocytochemical detection of immediate early genes and neuropeptides reveals that gonadotropin hormone releasing hormone (GnRH)-containing neurons are specifically activated by stimulation of the VN organ. Furthermore, some of the activated GnRH neurons project to the ventromedial hypothalamus where they are hypothesized to induce sexual responsiveness. Early anecdotal evidence of an influence of the VN organ on human reproductive events has been substantiated by more recent anatomical, behavioral, and electrophysiological studies. Thus, further deciphering of the signal transduction process within the VN system of the rodent may yield unique insights into behaviors associated with human reproduction.

Olfactory information processed by the vomeronasal system is reported to influence reproductive functions in a variety of mammals. The present studies were designed to determine if male-associated cues affect the luteinizing hormone-releasing hormone (LHRH) neuronal system, and, if so, to determine the extent to which these cues are processed by the vomeronasal organ (VNO). Ovariectomized rats underwent VNO removal (VNX) or sham surgery (VN-Sham). Forty-eight hours after estrogen priming (5 micrograms), they were subjected to one of the following treatments: repeated mating, repeated exposure to male-soiled bedding or repeated exposure to clean bedding. In animals treated for 180 min, coronal brain sections were double labelled for Fos protein and LHRH. An intense Fos immunoreactivity was induced following mating in the majority of LHRH neurons in the VN-Sham females, whereas removal of the VNO significantly suppressed the mating-induced Fos staining. Exposure of female rats to male-soiled bedding or clean bedding did not induce appreciable Fos immunoreactivity in LHRH neurons. Following 90 min of mating or exposure to bedding, blood samples were assayed for luteinizing hormone (LH). Mating stimulated the release of LH in VN-Sham females, while the removal of the VNO significantly suppressed the mating-induced LH release. Exposure of the females to male-soiled bedding or clean bedding did not induce an LH surge. The present results demonstrate that male-originating sensory cues (i.e. repeated mating) can influence the LHRH neuronal system, as evidenced by the presence of Fos immunoreactivity in LHRH cell bodies, and indicate that this effect is mediated through the VNO to a certain extent.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of the vomeronasal organ (VNO) in the male-induced enhancement of sexual receptivity in ovariectomized estrogen-primed rats was investigated. Removal of the VNO significantly reduced the enhancement of sexual receptivity following mating, as compared with the sham-operated controls. The sham-operated females exhibited a surge of luteinizing hormone (LH) following mating; however, LH release induced by pairing with males was less manifested in the VNO-removed females. This suggests that in female rats, VNO removal impairs the male-induced release of LH-releasing hormone (LHRH). Since LHRH enhances sexual receptivity in ovariectomized estrogen-primed rats, the present results suggest that the increase in sexual receptivity in female rats following mating probably results from a VNO-mediated LHRH release.

Even though castration abolished the ability of alien males to induce implantation failure (the Bruce effect) in newly inseminated females, treatment of intact alien males with the steroidal antiandrogen, cyproterone acetate, for 14 days (short term) did not significantly depress their ability to induce the Bruce effect. However, prolonged treatment (42 days) with cyproterone acetate suppressed the pregnancy-blocking ability of alien males to some extent, possibly due to the antigonadotrophic properties of the drug. Ovariectomized alien females treated with implants of testosterone (androgenized females) exhibited the ability to block implantation in newly inseminated females, but concurrent treatment of androgenized females with cyproterone acetate did not depress this ability. The results strongly suggest that the pheromone involved in the male-induced implantation failure is not the product of an androgen-dependent tissue, but is likely to be a product of androgen metabolism.

The ability of exogenous progesterone administration to prevent implantation failure in newly inseminated females exposed to alien males for 3 days (from 10.00 hr on day 1 to 10.00 hr on day 4 post coitum) was evaluated. Administration of progesterone, 1 mg/female/day, for 5 days beginning at 10.00 hr on day 1 or at 10.00 hr on day 2 prevented the return of oestrus and induced implantation of blastocysts in alien male-exposed females. Administration of progesterone beginning at 22.00 hr on day 2 was only partially effective in preventing return of oestrus in the male-exposed females; however, the majority of the females which exhibited vaginal oestrus showed implanted embryos. Progesterone injection beginning at 10.00 hr on day 3 or later was totally ineffective in preventing return of oestrus in the females. Administration of progesterone beginning at 10.00 hr or at 22.00 hr on day 3 failed to induce implantation in the male-exposed females which returned to oestrus on day 3; however, the treatment induced implantation in the majority of the females which returned to oestrus on day 4. Progesterone administration beginning at 10.00 hr on day 4 was totally ineffective in inducing implantation in the male-exposed females exhibiting the Bruce effect. The results indicate that administration of progesterone beginning at any time prior to the appearance of oestrus facilitates implantation of blastocysts in male-exposed females; however, progesterone administration beginning after the onset of oestrus is totally ineffective in inducing implantation in females. The results also strongly suggest that in females exhibiting the Bruce effect there is a total loss of the blastocysts.