General and Comparative Endocrinology 112, 356–363 (1998) Article No. GC987134 The Effects of Estradiol on Gonadotropin-Releasing Hormone Neurons in the Developing Mouse Brain Matthew S. Grober,1 Greg M. Winterstein, Asif A. Ghazanfar,2 and Victor P.Eroschenko* Department of Biological Sciences, *WAMI Medical Program, University of Idaho, Moscow, Idaho 83844-3051 Accepted March 30, 1998 The hypothalamic–pituitary–gonadal (HPG) axis plays a The perinatal organization of the vertebrate brain critical role in the control of reproduction. Two key plays a major role in determining adult reproductive hormonal components of the HPG axis are gonadal function. Two brain regions that are involved in the steroids and gonadotropin-releasing hormone (GnRH). control of reproductive behavior and physiology are Gonadal steroids are known to organize the development the hypothalamus and the neurohypophysis, which, in of neural substrates which control adult reproductive conjunction with the gonads, constitute the hypotha- behavior; GnRH is required for normal reproductive lamic–pituitary–gonadal axis (HPG axis). Two impor- structure and function. The possibility that gonadal tant hormonal components of the HPG axis are go- steroids may produce organizational changes in the nadal steroids and gonadotropin-releasing hormone pattern of GnRH staining observed in the brain is (GnRH). investigated through the use of injections of estradiol to Gonadal steroids mediate developmental organiza- neonatal mice and subsequent GnRH immunocytochem- tion of the neural and nonneural substrates that are istry at 2 months of age. Our results indicate that the critical for adult reproductive function (Phoenix et al., number of GnRH-immunoreactive (GnRH-ir) cells is 1959). Experimental manipulation of testosterone re- normally lower in females than males. Estradiol did not sults in changes in the expression of male-like external affect the number of GnRH-ir cells in females, but genitalia and frequency of mounting behavior by significantly increased the number of GnRH-ir cells in female offspring (Phoenix et al., 1959), the number of males, suggesting that early exposure to estradiol results neurons in the spinal nucleus of the bulbocavernosus in masculinization of the GnRH axis of males. ௠ 1998 in both males and females (Breedlove and Arnold, Academic Press 1983), and anogenital distance (AGD) (Clemens et al., Key Words: GnRH; sexual differentiation; hypothalam- 1978). The sexually dimorphic nucleus of the preoptic ic–preoptic area; neonates; estradiol; immunocytochem- area (SDN-POA) is one of several regions of the rodent istry; mouse; organization. brain that is influenced during development by circu- lating gonadal steroids (Gorski et al., 1978). The SDN- POA is larger in males than females (Gorski et al., 1980), and injection with testosterone in early life 1To whom correspondence should be addressed at: Department of causes this region to increase in size in both gonadecto- Life Sciences, Arizona State University West, P.O. Box 37100, 4701 mized males and normal females (Rhees et al., 1990). West Thunderbird Avenue, Phoenix, AZ 85069–7100. Fax: (602) 543–6073. E-mail: [email protected]. The estrogen antagonist tamoxifen and the androgen 2Present address: Department of Neurobiology, Box 3209, Duke antagonist cyproterone acetate have been used to show University Medical Center, Durham, NC 27710. the estrogenic mediation of sexual dimorphism in the 0016-6480/98 $25.00 Copyright ௠ 1998 by Academic Press 356 All rights of reproduction in any form reserved. Estradiol and GnRH Neuron Development 357 SDN-POA (Do¨hler et al., 1984, 1986). More recently, ever, this analysis was qualitative and thus did not antisense oligonucleotides which block the production examine quantitative differences in the effect of estra- of the estrogen receptor have been used to show that in diol on the number of GnRH cells. Data from radioim- female rats SDN-POA size and parastrial nuclear size munoassays on brain tissue from neonatally estro- are mediated by estrogen and that estrogen influences genized rats suggest that early estrogen treatment the development of both lordosis and open field modifies the hypothalamic mechanism involved in the behavior (McCarthy et al., 1993). These data suggest release of LHRH (Hayashi et al., 1991). Two previous that aromatization of testosterone to estradiol is respon- studies have quantitatively examined GnRH cells in sible for several key components of the masculiniza- mice (Hoffman and Finch, 1986; Wray et al., 1989). tion process. Thus, gonadal steroids have dramatic Wray et al. (1989) examined the progenitor cells that organizational effects on areas of the hypothalamus give rise to forebrain GnRH cells and provided counts and preoptic area that play a key role in the control of of GnRH cells from embryonic day 10.5 through reproduction. It is possible that the organizational adulthood. Hoffman and Finch (1986) looked at GnRH effects of gonadal steroids may also influence the cells during aging in a different strain of mice and development of the hypothalamic nuclei responsible found that onset of reproductive dysfunction did not for GnRH release. correlate with a loss of GnRH forebrain cells. Presently, The gonadotropin-releasing hormones are a family no data are available on the effects of estradiol on the of decapeptide hormones found in all vertebrates development of the GnRH axis in mice. This study examined thus far and which represents an important tests the hypothesis that early postnatal estradiol link between the brain and reproduction. In rodents, treatment affects the number of GnRH-immunoreac- the majority of the GnRH-producing cells are in the tive (GnRH-ir) neurons in the preoptic area of mice of preoptic region of the hypothalamus (reviewed in both sexes during the first 2 months of development. Silverman et al., 1994). Most of the GnRH cells are located amidst the diagonal band of Broca, the bed nucleus of the stria terminalis, the preoptic region (periventricular, medial, and lateral), and the anterior MATERIALS AND METHODS hypothalamus (Silverman et al., 1994). The majority of GnRH cells located in the preoptic region project to the Animals median eminence and are associated with the hypophy- seal portal system (Silverman et al., 1994). Gonadotro- Adult mice of ND4 Swiss Webster strain were pin-releasing hormone is primarily responsible for purchased from Simonsen Labs (Gilroy, CA), given causing the release of luteinizing hormone and follicle- food and water ad libitum, kept on a 12-h light–12-h stimulating hormone from the anterior pituitary. It is dark regimen, and allowed to breed. Within 24 h after also involved in the activation of lordosis in the female birth, the dams with their litters (housed together) (Moser and Mathiesen, 1996; Pfaff, 1973; Sakuma and were randomly assigned to different experimental Pfaff, 1980). Moreover, in both mice and humans groups. Immediately after group assignment, neonatal (reviewed in Silverman et al., 1994), improper develop- pups received the first of 14 daily intraperitoneal ment and or molecular regulation of the GnRH fore- injections (0.05 ml) of either sesame oil (control) or 10.0 brain nuclei results in hypogonadism and subsequent mg of 17b-estradiol in sesame oil. All injections were infertility. Thus, early and proper development of the administered with a 27-gauge needle using filtered forebrain GnRH axis is a necessary requirement for sesame oil as a vehicle. The choice of estradiol dosage normal adult reproductive function in several mamma- was based on previous experiments where this dose lian species. induced significant morphological and biochemical Neonatal treatment of rats with estradiol decreases alterations in the reproductive organs of immature the number of cells stained for GnRH in the male and female mice (Eroschenko et al., 1995). We were notified increases the number of cells in the female within the as to the availability of brain tissue from these animals first 10 days of life (Elkind-Hirsch et al., 1981). How- after the study was completed, and the established Copyright ௠ 1998 by Academic Press All rights of reproduction in any form reserved. 358 Grober et al. experimental design places some limitations on our cific neuroanatomical landmarks including the merger methods and interpretations. of the right and left radiations of the frontal aspect of At 2 months of age (56–60 days), all treated mice the corpus callosum, anteriorly, to the appearance of were anesthetized, injected with 100 ml heparin (1000 the fornix and the disappearance of the anterior com- units/ml), and perfused with 0.9% sodium chloride missure, posteriorly. This forebrain area includes the and then with 4% paraformaldehyde in 0.1 M phos- medial and lateral preoptic areas, the medial and phate-buffered saline. Anogenital distance and body lateral septal nuclei, and the diagonal band of Broca. weight were recorded before perfusion, whereas ovar- Total cell counts in the forebrain for each animal were ian weight was recorded after perfusion (testes weight tabulated. The GnRH-ir cells in some brain sections was not recorded). The brains were removed, blocked, were not visible because of poor staining. It is not clear sunk in 30% sucrose overnight at 4° C for cryoprotec- why some sections did not stain well, but poorly tion, sectioned at 50 µm in the coronal plane on a stained sections were distributed haphazardly among cryostat, and then stored in 0.1 M phosphate buffer brains and no brains were composed entirely of poorly (PB) at 4°C until the immunostaining was performed. stained sections. Thus, GnRH-ir cell number in brains with poorly stained sections would not be a reliable Immunocytochemistry indicator of the total number of cells in those brains. In order to exclude brains with poorly stained sections, Immunocytochemistry was carried out on free-floating group averages were calculated from the four highest sections using culture plates. Briefly, the sections were values in each group. The top four brains were chosen rinsed twice in PB with 0.4% Triton X-100 (PBX) for 5 for two reasons: (1) one treatment group only had four min.