Estrogenic control of preoptic area development in a carnivore,the ferret

Summary 1. Evidence is reviewed which shows that a sexually dimorphic nucleus located in the dorsomedial portion of the male ferret's preoptic area/anterior hypothalamus (POA/AH), called the male nucleus of the POA/AH (Mn-POA/AH), develops during fetal life in response to the action of estradiol, which is formed directly in the nervous system from circulating testosterone over the final quarter of a 41-day gestation.2. Results are summarized which establish that neurons which make up the Mn-POA/AH are born prior to the critical period of estradiol's action in the male brain. Other data show that some radial glial processes, visualized immunocytochemically using antibodies against GFAP, emanate from proliferative zones at the base of the lateral ventricles in a dorsal-ventral orientation, whereas other glial processes emanate laterally from proliferative zones lining the third ventricle.3. We suggest that at least some neurons which constitute the dorsomedial POA/AH are born in proliferative zones surrounding the lateral ventricles, raising the question of whether estradiol acts in developing males to influence the migration of these neurons along radial glial guides into the Mn-POA/AH.4. Finally, evidence is summarized showing that excitotoxic lesions of the dorsomedial POA/AH enhance males' preference to approach and interact with another sexually active male, as opposed to an estrous female, when adult subjects are castrated and treated with estradiol benzoate. These data suggest that the sexually dimorphic Mn-POA/AH is an essential part of a CNS circuit which determines heterosexual partner preference in the male ferret.     

Neurogenesis and cell migration into the sexually dimorphic preoptic area/anterior hypothalamus of the fetal ferret

A sexually dimorphic male nucleus (MN) of the preoptic area/anterior hypothalamus (POA/AH), comprising large, estradiol-receptor containing neurons, is formed in male ferrets due to the action of estradiol, derived from the neural aromatization of circulating testosterone, during the last quarter of a 41-day gestation. Two experiments were conducted to compare the birthdates and the migration pattern of cells into the sexually dimorphic portion of the dorsomedial POA/AH as well as the nondimorphic ventral nucleus (VN) of the POA/AH of males and females. In experiment 1 the thymidine analog, bromodeoxyuridine (BrdU), was injected into the amniotic sacs of fetuses of different mothers between embryonic (E) days 18 and 30. Kits from all mothers were sacrificed on E38, and brains were processed to localize BrdU immunoreactivity (IR) for determining the birthdates of neurons in the POA/AH. Cells in the MN-POA/AH of males and in a comparable region of females were born between E22 and E28; cells in the nondimorphic VN-POA/AH of both sexes were born between these same ages. These results suggest that cells in the sexually dimorphic as well as the nondimorphic subdivision of the ferret POA/AH are born during the same embryonic period. This is well before the ages (E30–E41) when administering testosterone to females can stimulate, and blocking androgen aromatization in males can inhibit, MN-POA/AH differentiation. In experiment 2 BrdU was injected on E24, and kits from different litters were perfused on E30, E34, or E38. Brains were processed for BrdU-IR as well as glial fibrillary acidic protein (GFAP), which served as a marker for radial glial processes. The orientation of radial glial processes in fetal brains of both sexes suggested that cells migrate into the dorsomedial POA/AH from proliferative zones lining the lateral as well as the third ventricles. Quantitative, computer-assisted image analysis of BrdU-IR in groups of male and female brains supported this hypothesis. There were no significant sex differences in the distribution of BrdU-IR over the three ages studied, suggesting that formation of the MN-POA/AH in males cannot be attributed to an effect of estradiol on the migration of those cells born on E24 into this sexually dimorphic structure. Finally, total BrdU-IR did not change significantly in the POA/AH of male and female kits killed at E30, E34, or E38 while the area of the POA/AH increased more than 2.5-fold over this period, suggesting that few of the POA/AH cells born on E24 die during this period in either sex. In the absence of evidence that formation of the male ferret's MN-POA/AH depends on steroid-induced changes in neurogenesis, cell migration, or death, we suggest that the specification of a particular neuronal phenotype (e.g., large somal size; capacity to produce some undetermined neurotransmitter or neuropeptide) may be responsible. © 1996 John Wiley & Sons, Inc.     

Sex differences in brain developing in the presence or absence of gonads

Brain sexual differentiation results from the interaction of genetic and hormonal influences. This study used a unique agonadal mouse model to determine relative contributions of genetic and gonadal hormone influences in the differentiation of selected brain regions. SF-1 knockout (SF-1 KO) mice are born without gonads and adrenal glands and are not exposed to endogenous sex steroids during fetal/neonatal development. Consequently, male and female SF-1 KO mice are born with female external genitalia and if left on their own, die shortly after birth due to adrenal insufficiency. In this study, SF-1 KO mice were rescued by neonatal adrenal transplantation to examine their brain morphology in adult life. To determine potential brain loci that might mediate functional sex differences, we examined the area and distribution of immunoreactive calbindin and neuronal nitric oxide synthase in the preoptic area (POA) and ventromedial nucleus of the hypothalamus, two areas previously reported to be sexually dimorphic in the mammalian brain. A sex difference in the positioning of cells containing immunoreactive calbindin in a group within the POA was clearly gonad dependent based on the elimination of the sex difference in SF-1 KO mice. Several other differences in the area of ventromedial hypothalamus and in POA were maintained in male and female SF-1 KO mice, suggesting gonad-independent genetic influences on sexually dimorphic brain development.     

Ontogeny of Neurons Containing Calcium‐Binding Proteins in the Preoptic Area of the Guinea Pig: Sexually Dimorphic Development of Calbindin

This study characterizes for the first time the distribution and coexistence patterns of calbindin (CB), calretinin (CR), and parvalbumin (PV) in the female and male guinea pig preoptic area (POA) during brain development, using immunohistochemistry and quantitative real‐time PCR techniques. The results show that the prenatal development of the guinea pig POA takes place in elevated levels of CB and CR immunoreactivity with the peak at embryonic day 50 (E50) and generally in newborns both these proteins reach an adult‐like pattern of immunoreactivity, contrary to PV which appears later, peaks at postnatal day (PND) 10 (P10), and stabilizes at P20. CB and CR have also overlapping distributions which differed from that of PV, and much higher expressions at mRNA and protein levels. However, CB‐positive (+), CR+ and PV+ neurons create in the guinea pig POA separate populations as CB and CR coexisted only in a small number of neurons and CB+ cells never coexpressed PV. Moreover, the density of CB+ neurons, contrary to CR+ and PV+ cells, is sexually dimorphic favoring males at all the examined stages. In conclusion, elevated levels of CR and CB at the time of intense cell migration, differentiation, myelination, and synaptogenesis in the guinea pig brain suggest that these proteins may be engaged in similar processes in the POA, while late onset of PV may be rather linked with POA maturation. As the population of CB+ cells in the POA is very large, its dimorphic development may have huge impact on the sexual differentiation of this brain region.     

Regulation of the female rat estrous cycle by a neural cell-specific epidermal growth factor-like repeat domain containing protein,NELL2

NELL2, a protein containing epidermal growth factor-like repeat domains, is predominantly expressed in the nervous system. In the mammalian brain, NELL2 expression is mostly neuronal. Previously we found that NELL2 is involved in the onset of female puberty by regulating the release of gonadotropin-releasing hormone (GnRH), and in normal male sexual behavior by controlling the development of the sexually dimorphic nucleus of the preoptic area (POA). In this study we investigated the effect of NELL2 on the female rat estrous cycle. NELL2 expression in the POA was highest during the proestrous phase. NELL2 mRNA levels in the POA were increased by estrogen treatment in ovariectomized female rats. Blocking NELL2 synthesis in the female rat hypothalamus decreased the expression of kisspeptin 1, an important regulator of the GnRH neuronal apparatus, and resulted in disruption of the estrous cycle at the diestrous phase. These results indicate that NELL2 is involved in the maintenance of the normal female reproductive cycle in mammals.     

Ontogeny of the sexually dimorphic male nucleus in the preoptic/anterior hypothalamus of ferrets and its manipulation by gonadal steroids

A sexually dimorphic nucleus exists in the dorsal region of the ferret preoptic/anterior hypothalamic area (POA/AH), and is called the male nucleus of the POA/AH (MN-POA/AH) because it is found only in males. Development of the MN-POA/AH was studied in male ferrets, and for comparison a sexually nondimorphic ventral POA/AH nucleus was studied in both sexes. The MN-POA/AH was conspicuous in males as early as embryonic day 37 (E37) of a 41-day gestation, and its volume increased until postnatal day 56 (P56). No nucleus was present in the dorsal POA/AH of females at any age. The densities and average somal areas of cells in the dorsal POA/AH were similar in males and females at E33, before the MN-POA/AH could be visualized. However, at E37 and E41 dorsal cells were greater in density and/or somal area in males than in females, accounting for the appearance of a nucleus in males at these ages. To insure that the dorsal POA/AH nucleus seen in males at E37 and E41 was the presumptive MN-POA/AH present in adult males, pregnant ferrets were given progesterone and either implanted subcutaneously (s.c.) with testosterone (T) or ovariectomized and implanted s.c. with the aromatase inhibitor, 1,4,6-androstatriene-3,17-dione (ATD), on day 30 of gestation. As predicted from previous studies in which subjects were sacrificed in adulthood, formation of a dorsal POA/AH nucleus was promoted in female ferrets by T, and blocked in males by maternal ovariectomy and ATD treatment for animals sacrificed at E41. Much evidence suggests that behavioral sexual differentiation is accomplished in the male ferret between age E28 and P20. The MN-POA/AH is present and potentially functional in males during a considerable portion of this perinatal period.     

Neuronal nitric oxide synthase and calbindin delineate sex differences in the developing hypothalamus and preoptic area

Throughout the hypothalamus there are several regions known to contain sex differences in specific cellular, neurochemical, or cell grouping characteristics. The current study examined the potential origin of sex differences in calbindin expression in the preoptic area and hypothalamus as related to sources of nitric oxide. Specific cell populations were defined by immunoreactive (ir) calbindin and neuronal nitric oxide synthase (nNOS) in the preoptic area/anterior hypothalamus (POA/AH), anteroventral periventricular nucleus (AVPv), and ventromedial nucleus of the hypothalamus (VMN). The POA/AH of adult mice was characterized by a striking sex difference in the distribution of cells with ir-calbindin. Examination of the POA/AH of androgen receptor deficient Tfm mice suggests that this pattern was in part androgen receptor dependent, since Tfm males had reduced ir-calbindin compared with wild-type males and more similar to wild-type females. At P0 ir-calbindin was more prevalent than in adulthood, with males having significantly more ir-calbindin and nNOS than have females. Cells that contained either ir-calbindin or ir-nNOS in the POA/AH were in adjacent cell groups, suggesting that NO derived from the enzymatic activity of nNOS may influence the development of ir-calbindin cells. In the region of AVPv, at P0, there was a sex difference with males having more ir-nNOS fibers than have females while ir-calbindin was not detected. In the VMN, at P0, ir-nNOS was greater in females than in males, with no significant difference in ir-calbindin. We suggest that NO as an effector molecule and calbindin as a molecular biomarker illuminate key aspects of sexual differentiation in the developing mouse brain.     

Medial Preoptic/Anterior Hypothalamic Lesions Induce a Female-Typical Profile of Sexual Partner Preference in Male Ferrets

In T-maze tests given to gonadectomized ferrets treated daily with estradiol benzoate (EB), females consistently prefer to approach and interact sexually with a stud male whereas male subjects, on average, prefer an estrous female. In the present experiment this sexually allomorphic pattern of partner preference was changed in males given lesions of the medial preoptic area/anterior hypothalamus (mPOA/AH). Electrolytic lesions, which caused extensive bilateral damage to the mPOA/AH, including the sexually dimorphic male nucleus (MN) of the POA/AH, led males to shift their mean preference away from the estrous female to the stud male. Their postoperative profile of partner preference more closely resembled that of sham-operated females than that of sham-operated males or of males which sustained either partial or minimal bilateral damage to the mPOA/AH so as to spare the MN-POA/AH in one or both hemispheres. Males with extensive bilateral mPOA/AH lesions, like sham-operated females, showed an even stronger preference to approach the stud male during T-maze tests in which the subjects could smell, see, and hear the stimulus animals without physically interacting with them. After receiving testosterone propionate, male ferrets with either extensive or partial lesions of the mPOA/AH showed significant deficits in neck gripping and mounting performance in tests with either female or male stimulus animals which were sexually receptive after gonadectomy and EB treatment. The present results, coupled with those of a previous study using excitotoxic mPOA/AH lesions, suggest that the male-typical profile of preference for an estrous female depends on the functional integrity of sexually dimorphic mPOA/AH neurons and the reward engendered by coital interaction with such a female. When these neurons either are destroyed experimentally (as in male ferrets with extensive bilateral mPOA/AH lesions) or are absent (as in sham-operated females), subjects are attracted by distal (possibly chemosensory) incentive cues from a stud male.     

Sexual differentiation and the Kiss1 system: hormonal and developmental considerations

The nervous system (both central and peripheral) is anatomically and physiologically differentiated between the sexes, ranging from gender-based differences in the cerebral cortex to motoneuron number in the spinal cord. Although genetic factors may play a role in the development of some sexually differentiated traits, most identified sex differences in the brain and behavior are produced under the influence of perinatal sex steroid signaling. In many species, the ability to display an estrogen-induced luteinizing hormone (LH) surge is sexually differentiated, yet the specific neural population(s) that allows females but not males to display such estrogen-mediated "positive feedback" has remained elusive. Recently, the Kiss1/kisspeptin system has been implicated in generating the sexually dimorphic circuitry underlying the LH surge. Specifically, Kiss1 gene expression and kisspeptin protein levels in the anteroventral periventricular (AVPV) nucleus of the hypothalamus are sexually differentiated, with females displaying higher levels than males, even under identical hormonal conditions as adults. These findings, in conjunction with accumulating evidence implicating kisspeptins as potent secretagogues of gonadotropin-releasing hormone (GnRH), suggest that the sex-specific display of the LH surge (positive feedback) reflects sexual differentiation of AVPV Kiss1 neurons. In addition, developmental kisspeptin signaling via its receptor GPR54 appears to be critical in males for the proper sexual differentiation of a variety of sexually dimorphic traits, ranging from complex social behavior to specific forebrain and spinal cord neuronal populations. This review discusses the recent data, and their implications, regarding the bi-directional relationship between the Kiss1 system and the process of sexual differentiation.     

Brain aromatase in control versus castrated Norway Brown, Sprague-Dawley and Wistar adult rats.

Brain aromatase cytochrome P450 converts androgens to estrogens that play a critical role in the development of sexually dimorphic neural structures, the modulation of neuroendocrine function(s), and the regulation of sexual behavior. We characterized the influence of surgical castration on brain aromatase in Norway Brown and Wistar adult rats and compared their responses to Sprague-Dawley rats that were surgically or biochemically castrated (with flutamide, a known androgen receptor blocker). Aromata enzyme activity was measured by the tritiated water release assay in the medial basal hypothalmus/preoptic area (MBH/POA) and amygdala brain regions. The present results demonstrate that independent of the rat strain examined, MBH/POA aromatase is regulated by androgens (in Sprague-Dawley, Norway Brown and Wistar males). However, intact Wistar animals displayed significantly higher MBH/POA aromatase levels compared to Sprague-Dawley control values. Conversely, in the amygdala region, there was an apparent lack of androgen hormone action upon aromatase enzyme activity in some of the rat strains tested. The importance of brain aromatase regulating estrogen biosynthesis and influencing brain development and function is covered.     

Intracerebral Sex Differences in the Vasotocin System in Birds: Possible Implication in Behavioral and Autonomic Functions

The brain vasotocinergic system demonstrates clear sexual dimorphism in birds investigated so far. This paper examines the evidence obtained in studies on gallinaceous (domestic fowl, Japanese quail) and passerine (canary, junco, zebra finch) birds. Vasotocin (VT)-immunoreactive parvocellular neurons are present in the nucleus of stria terminalis of males, but they are less abundant or absent in the corresponding structure of females. A similar difference has been observed in the dorsal paraventricular area of domestic fowl. Sex-related differences in VT-gene expression have been confirmed byin situhybridization. Moreover, overall brain content of VT mRNA in cockerels is about twice that of hens, suggesting that VT synthesis may also be sexually dimorphic in other brain areas where morphological sex differences have not yet been revealed. The vasotocinergic system in birds is implicated in body fluid homeostasis, and during ontogeny it starts to respond to osmotic challenges in a sexually dimorphic way. Photoperiod, aging, or castration—all associated with changes in circulating testosterone levels—affect sexually dimorphic VT pathways and cell clusters. Sexually dimorphic vasotocinergic circuits are distributed in regions containing steroid-concentrating cells and are closely intermingled with aromatase-containing neurons that may mediate activational effects of gonadal steroids on this peptidergic system. However, it remains undetermined whether the observed neuroanatomical sex differences are related to sexually dimorphic autonomic and behavioral effects induced by VT. Most likely, VT in birds has a modulatory rather than a specific regulatory function in control of male sexual behavior and vocalization.     

Cell death in the sexually dimorphic dorsal preoptic area/anterior hypothalamus of perinatal male and female ferrets

A sexually dimorphic male nucleus (MN) is present in Nissl-stained sections through the dorsal (d) preoptic area/anterior hypothalamus (POA/AH) of male ferrets. The MN-POA/AH is composed of a cluster of large cells which is organized in males by the action of estradiol, formed via the neural aromatization of circulating testosterone (T), during the last quarter of a 41-day gestation. Several recent studies using rodent species have raised the possibility that the hormone-induced masculinization of POA/AH morphology is mediated at least in part by a perinatal modulation of cell death. We asked whether a perinatal reduction in cell death contributes to the differentiation of the MN-POA/AH in the male ferret, which is a carnivore species. The appearance of internucleosomal DNA fragmentation, detected by in situ end labeling (ISEL) using the ApopTag™ kit (Oncor Corp.) and of pyknotic cell nuclei in Nissl-stained sections were used to estimate the occurrence of cell death. Male and female ferret kits were killed at four different ages spanning the perinatal period during which the MN-POA/AH is organized and assumes an adult phenotype. A peak density of dying cells was present in both sexes at postnatal day (P) 2, which is nearly 1 week after the age, embryonic day (E) 37, when the MN-POA/AH is first visible in male ferrets using Nissl stains. The density of cells in the sexually dimorphic dPOA/AH which were either ISEL-positive or pyknotic was similar in males and females on E34, as well as on P2, 10, and 20. In the nondimorphic ventral POA/AH, the highest density of dying cells was present in both sexes at E34, and there were significantly more ISEL-positive cells present in males than females at this particular age. In contrast to previous studies using rodents, our results suggest that in fetal male ferrets a modulation of the incidence of cell death contributes little to estradiol's organizational action in the dPOA/AH. © 1998 John Wiley & Sons, Inc. J Neurobiol 34: 242–252, 1998     

What Nature''s Knockout Teaches Us about GnRH Activity: Hypogonadal Mice and Neuronal Grafts

The hypogonadal mouse is one of “nature's knockouts,” bearing a specific deletion in the gene for gonadotropin-releasing hormone (GnRH), with the result that no GnRH peptide is detectable in the brain. The lack of reproductive development after birth provides an animal model that has proved fruitful in clarifying the role of GnRH in reproductive behavior and physiology. Behavioral studies with hypogonadal mice convincingly demonstrate that although GnRH may facilitate the appearance of sexual behavior, this peptide is not essential for either male or female sexual behavior in the mouse. Administration of GnRH to hypogonadal mice with regimens mimicking GnRH pulsatility initiates reproductive development. Surprisingly, continuous exposure to GnRH stimulates remarkable ovarian and uterine growth and increased FSH release, although pituitary content of LH and FSH remains unchanged. In contrast, when brain grafts of normal fetal preoptic area (POA), containing GnRH cells, are implanted in the third ventricle of adult hypogonadal mice, both pituitary and plasma gonadotropin levels increase. Grafted GnRH neurons innervate the median eminence of the host and support pulsatile LH secretion in the majority of animals with graft-associated gonadal development. Studies of hypogonadal mice with POA grafts demonstrate that distinct components of reproductive function are dissociable: hosts may demonstrate reflex but not spontaneous ovulation; others may show positive but not negative feedback. Activation of grafted GnRH cells in response to sensory input to the host, as revealed in Fos expression studies, is an example of the integration of the graft with the host brain that underlies such capabilities. A goal of these studies is to elucidate the specific connectivity underlying discrete aspects of reproductive function.     

A Working Module for the Neurovascular Unit in the Sexually Dimorphic Nucleus of the Preoptic Area

The neurovascular unit (NVU) can be conceptualized as a functional entity consisting of neurons, astrocytes, pericytes, and endothelial and smooth muscle cells that operate in concert to affect blood flow to a very circumscribed area. Although we are currently in a “golden era” of bioengineering, there are, as yet, no living NVUs-on-a-chip modules available and the development of a neural chip that would mimic NVUs is a seemingly lofty goal. The sexually dimorphic nucleus of the preoptic area (SDN-POA) is a tiny brain structure (between 0.001~0.007 mm³ in rats) with an assessable biological function (i.e., male sexual behavior). The present effort was undertaken to determine whether there are identifiable NVUs in the SDN-POA by assessing its vasculature relative to its known neural components. First, a thorough and systematic review of thousands of histologic and immunofluorescent images from 201 weanling and adult rats was undertaken to define the characteristics of the vessels supplying the SDN-POA: its primary supply artery/arteriole and capillaries are physically inseparable from their neural elements. A subsequent immunofluorescent study targeting α-smooth muscle actin confirmed the identity of an artery/arteriole supplying the SDN-POA. In reality, the predominant components of the SDN-POA are calbindin D28k-positive neurons that are comingled with tyrosine hydroxylase-positive projections. Finally, a schematic of an SDN-POA NVU is proposed as a working model of the basic building block of the CNS. Such modules could serve the study of neurovascular mechanisms and potentially inform the development of next generation bioengineered neural transplants, i.e., the construct of an NVU neural chip.     

Prostaglandin-E2: a point of divergence in estradiol-mediated sexual differentiation

The preoptic area of the mammalian forebrain is a critical substrate for the development and maintenance of many sexually dimorphic behaviors relevant to reproduction. Normal development of the male rodent brain requires completion of two processes: (1) masculinization-induction of the male phenotype, and (2) defeminization-removal of the female phenotype. Both processes, although distinct, are largely directed by the same steroid, estradiol. Whether estradiol achieves both ends via the same or separate mechanisms has been unknown. Here, we report that prostaglandin-E(2) (PGE(2)) acting downstream of estradiol, is necessary and sufficient to masculinize sexual behavior but does not affect defeminization of sexual behavior or maternal behavior. Moreover, the volume of the sexually dimorphic nucleus of the preoptic area predicts defeminization of sexual behavior, but not masculinization of sexual behavior. Another sexually dimorphic cellular endpoint regulated by estradiol, spinophilin protein expression in the mediobasal hypothalamus, was not affected by PGE(2). Thus, PGE(2) is a key divergence point in the downstream actions of estradiol to simultaneously masculinize and defeminize sexual behavior.     

Serotonin promotes feminization of the sexually dimorphic nucleus of the preoptic area,but not the calbindin cell group

Testosterone and its metabolites masculinize the brain during a critical perinatal window, including the relative volume of sexually dimorphic brain areas such as the sexually dimorphic nucleus of the preoptic area (SDN), which is larger in males than females. Serotonin (5HT) may mediate this hormone action, since 5HT given during the second week of life decreases (i.e., feminizes) SDN volume in males and testosterone‐treated females. Although previous work indicates that the 5HT_2A/2C receptor is sufficient to induce feminization, it is unclear whether other serotonin receptors are required and which subpopulation(s) of SDN cells are specifically organized by 5HT. Therefore, we injected male and female Sprague‐Dawley rat pups with saline, a nonselective 5HTR agonist, a 5HT_2A/2C agonist, or a 5HT_2A/2C antagonist over several timecourses in early life, and measured the Nissl‐SDN as well as a calbindin+ subdivision of the SDN, the CALB‐SDN. When examined on postnatal day 18 or early adulthood, the size of the Nissl‐SDN was feminized in males treated with any of the serotonergic drugs, eliminating the typical sex difference. In contrast, the sex difference in CALB‐SDN size was maintained regardless of serotoninergic drug treatment. This pattern suggests that although gonadal hormones shape the whole SDN, individual cellular phenotypes respond to different intermediary signals to become sexually dimorphic. Specifically, 5HT mediates sexual differentiation of non‐calbindin population(s) within the SDN. The results also caution against using measurement of the CALB‐SDN in isolation, as the absence of an effect on the CALB‐SDN does not preclude an effect on the overall nucleus. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 76: 1241–1253, 2016     

Number of preoptic GnRH-immunoreactive cells correlates with sexual phase in a protandrously hermaphroditic fish, the dusky anemonefish (Amphiprionmelanopus)

The populations of gonadotropin-releasing hormone (GnRH)-producing cells within the preoptic area (POA) and terminal nerve (TN) of the brain have been suggested as the neuronal systems mediating social control of sex and gonadogenesis in sequentially hermaphroditic teleosts. In the present study, the number and soma size of GnRH-immunoreactive (GnRH-ir) cells in the POA and TN were studied in male, female and juvenile individuals of the dusky anemonefish (Amphiprionmelanopus), a species which displays both male to female sex change and socially controlled sexual maturation. The results showed that the number of POA (but not TN) GnRH-ir cells differ significantly between sexual phases, with males displaying higher cell numbers than both females and juveniles. Soma sizes of POA and TN GnRH-ir cells were larger in females than in males and juveniles. However, this relationship was fully explained by differences in body size. The results indicate that high POA GnRH cell numbers are part of a masculinizing mechanism and support the hypothesis that the POA GnRH cell population plays a central role in initiating or mediating the process of socially induced gonadal and/or behavioural transformations in sequential hermaphrodites. Accepted: 9 June 1997