Phenotypic differences in the GnRH neuronal system of deer mice Peromyscus maniculatus under a natural short photoperiod

The neural mechanisms by which short photoperiod induces gonadal regression among seasonally breeding mammals are not well understood. One hypothesis suggests that the proximate cause of seasonal gonadal regression is a photoperiod-induced modification in GnRH secretion. This hypothesis is indirectly supported by our recent findings using immunocytochemistry which identified specific photoperiod-induced adjustments in the number and morphology of GnRH containing neurones between reproductively competent and reproductively regressed laboratory housed male deer mice. Herein, we report that the GnRH neuronal system is similarly affected in reproductively responsive and nonresponsive wild male deer mice Peromyscus maniculatus exposed to a natural short photoperiod. The distribution of immunoreactive (IR)-GnRH neurones was nearly identical in field caught animals and those housed under artificial photoperiod in the laboratory. Compared with reproductively nonresponsive males, reproductively responsive mice from the field population possessed a greater total number of IR-GnRH neurones, a greater number of IR-GnRH neurones within the lateral hypothalamus, and a greater proportion of bipolar IR-GnRH neurones. Each of these distributional and morphological characters was consistent with our findings in laboratory housed male deer mice exposed to an artificial short photoperiod. Taken together, these data underscore the validity of using an artificial photoperiod to evaluate seasonal adjustments in reproductive function in the laboratory.     

Photoperiod and temperature interact to affect the GnRH neuronal system of male prairie voles (Microtus ochrogaster)

Individuals of numerous species limit energy expenditure during winter by inhibiting reproduction and other nonessential functions. To time these adaptations appropriately with the annual cycle, animals rely on environmental cues that predict, well in advance, the onset of winter. The most commonly studied environmental factor that animals use to time reproduction is photoperiod. Rodents housed in short photoperiods in the laboratory or in naturally declining day lengths exhibit pronounced alterations in reproductive function concomitant with alterations in the hypothalamic gonadotropin-releasing hormone neuronal system. Because animals in their natural environment use factors in addition to photoperiod to time reproduction, the present study sought to determine the independent effects of photoperiod and temperature, as well as the interaction between these factors, on reproductive parameters and the GnRH neuronal system. Male prairie voles were housed in either long (LD 16:8) or short (LD 8:16) day lengths for 10 weeks. Animals in each photoperiod were further subdivided into groups housed in either mild (i.e., 20 degrees C) or low (i.e., 8 degrees C) temperatures. As shown with immunohistochemistry, voles that underwent gonadal regression in response to short photoperiods and long-day voles housed in low temperatures (and maintained large gonads) exhibit higher GnRH-immunoreactive (GnRH-ir) neuron numbers in the preoptic area/anterior hypothalamus (POA/AH) relative to all other groups. In addition, voles that underwent gonadal regression in response to both short days and low temperatures did not exhibit an increase in GnRH-ir neuron numbers compared to long-day, mild-temperature controls. These data suggest that photoperiod and temperature interact to influence reproductive function potentially by alterations of the GnRH neuronal system.     

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.     

Sex differences in cell migration in the preoptic area/anterior hypothalamus of mice.

The preoptic area/anterior hypothalamus (POA/AH) sits as a boundary region rostral to the classical diencephalic hypothalamus and ventral to the telencephalic septal region. Numerous studies have pointed to the region's importance for sex-dependent functions. Previous studies suggested that migratory guidance cues within this region might be particularly unique in their diversity. To better understand the early development and differentiation of the POA/AH, cytoarchitectural, birthdate, immunocytochemical, and cell migration studies were conducted in vivo and in vitro using embryonic C57BL/6J mice. A medial preoptic nucleus became discernible using Nissl stain in males and females between embryonic days (E) E15 and E17. Cells containing immunoreactive estrogen receptor-alpha were detected in the POA/AH by E13, and increased in number with age in both sexes. From E15 to E17, examination of the radial glial fiber pattern by immunocytochemistry confirmed the presence of dual orientations for migratory guidance ventral to the anterior commissure (medial-lateral and dorsal-ventral) and uniform orientation more caudally (medial-lateral). Video microscopy studies followed the migration of DiI-labeled cells in coronal 250-microm brain slices from E15 mice maintained in serum-free media for 1-3 days. Analyses showed significant migration along a dorsal-ventral orientation in addition to medial-lateral. The video analyses showed significantly more medial-lateral migration in males than females in the caudal POA/AH. In vivo, changes in the distribution of cells labeled by the mitotic indicator bromodeoxyuridine (BrdU) suggested their progressive migration through the POA/AH. BrdU analyses also indicated significant movement from dorsal to ventral regions ventral to the anterior commissure. The significant dorsal-ventral migration of cells in the POA/AH provides additional support for the notion that the region integrates developmental information from both telencephalic and diencephalic compartments. The sex difference in the orientation of migration of cells in the caudal POA/AH suggests one locus for the influence of gonadal steroids in the embryonic mouse forebrain.     

Histological characterization of gonadotropin-releasing hormone (GnRH) in the hypothalamus of the South American plains vizcacha (Lagostomus maximus)

In contrast to most mammalian species, females of the South American plains vizcacha, Lagostomus maximus, show an extensive suppression of apoptosis-dependent follicular atresia, continuous folliculogenesis, and massive polyovulation. These unusual reproductive features pinpoint to an eventual peculiar modulation of the hypothalamo-hypophyseal-gonadal axis through its main regulator, the gonadotropin-releasing hormone (GnRH). We explored the hypothalamic histological landscape and cellular and subcellular localization of GnRH in adult non-pregnant L. maximus females. Comparison to brain atlases from mouse, rat, guinea pig and chinchilla enabled us to histologically define and locate the preoptic area (POA), the ventromedial nucleus, the median eminence (ME), and the arcuate nucleus (Arc) of the hypothalamus in vizcacha's brain. Specific immunolocalization of GnRH was detected in soma of neurons at medial POA (MPA), ventrolateral preoptic nucleus, septohypothalamic nucleus (SHy) and Arc, and in beaded fibers of MPA, SHy, ventromedial hypothalamic nucleus, anterior hypothalamic area and ME. Electron microscopy examination revealed GnRH associated to cytoplasmic vesicles of the ME and POA neurons, organized both in core and non-core vesicles within varicosities, and in neurosecretory vesicles within the myelinated axons of the MPA. Besides the peculiar and unusual features of folliculogenesis and ovulation in the vizcacha, these results show that hypothalamus histology and GnRH immune-detection and localization are comparable to those found in other mammals. This fact leads to the possibility that specific regulatory mechanisms should be in action to maintain continuous folliculogenesis and massive polyovulation.     

Sex differences in cell migration in the preoptic area/anterior hypothalamus of mice

The preoptic area/anterior hypothalamus (POA/AH) sits as a boundary region rostral to the classical diencephalic hypothalamus and ventral to the telencephalic septal region. Numerous studies have pointed to the region's importance for sex‐dependent functions. Previous studies suggested that migratory guidance cues within this region might be particularly unique in their diversity. To better understand the early development and differentiation of the POA/AH, cytoarchitectural, birthdate, immunocytochemical, and cell migration studies were conducted in vivo and in vitro using embryonic C57BL/6J mice. A medial preoptic nucleus became discernible using Nissl stain in males and females between embryonic days (E) E15 and E17. Cells containing immunoreactive estrogen receptor‐α were detected in the POA/AH by E13, and increased in number with age in both sexes. From E15 to E17, examination of the radial glial fiber pattern by immunocytochemistry confirmed the presence of dual orientations for migratory guidance ventral to the anterior commissure (medial‐lateral and dorsal‐ventral) and uniform orientation more caudally (medial‐lateral). Video microscopy studies followed the migration of DiI‐labeled cells in coronal 250‐μm brain slices from E15 mice maintained in serum‐free media for 1–3 days. Analyses showed significant migration along a dorsal‐ventral orientation in addition to medial‐lateral. The video analyses showed significantly more medial‐lateral migration in males than females in the caudal POA/AH. In vivo, changes in the distribution of cells labeled by the mitotic indicator bromodeoxyuridine (BrdU) suggested their progressive migration through the POA/AH. BrdU analyses also indicated significant movement from dorsal to ventral regions ventral to the anterior commissure. The significant dorsal‐ventral migration of cells in the POA/AH provides additional support for the notion that the region integrates developmental information from both telencephalic and diencephalic compartments. The sex difference in the orientation of migration of cells in the caudal POA/AH suggests one locus for the influence of gonadal steroids in the embryonic mouse forebrain. © 1999 John Wiley & Sons, Inc. J Neurobiol 41: 252–266, 1999     

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.     

Distribution of lamprey gonadotropin-releasing hormone-III (GnRH-III) in brains of larval lampreys (Petromyzon marinus)

Two immunoreactive forms of gonadotropinreleasing hormone (GnRH), lamprey GnRH-I and lamprey GnRH-III, were found in neurons in larval sea lampreys (Petromyzon marinus). Using antisera preferentially directed against either lamprey GnRH-I or-III, dense reaction product was seen in cell bodies in the rostral hypothalamus and preoptic area. Reaction product was also dense in fibers to and within the neurohypophysis, in addition to numerous fibers which projected caudally, beyond the neurohypophysis through the mesencephalon. The majority of immunoreactive GnRH was lamprey GnRH-III, and when lamprey GnRH-I was seen, it was in cells that appeared to contain both forms of GnRH. A small number of cells found in the caudal hypothalamus contained only immunoreactive lamprey GnRH-III, and these may constitute a functional subgroup within the population of GnRH neurons. In animals undergoing metamorphosis there was a large increase in reaction product in all GnRH-containing cells and fibers. A striking change within the distribution of GnRH cells was localized to a distinct group of GnRH-immunoreactive cells (GnRH-I and-III) in the ventral anterior hypothalamic area. These cells were minimally detectable in larvae, but during metamorphosis became densely filled with immunoreactive product in perikarya and distal processes. The results are consistent with the hypothesis that lamprey GnRH-III is an important form of GnRH during the maturation of GnRH cells and fibers, and further indicates that these cells have attained their normal positions in the preoptic area and hypothalamus before metamorphosis.     

Distribution of salmon gonadotrophin releasing-hormone in the brain and pituitary of the sea bass (Dicentrarchus labrax)

Summary The distribution of salmon gonadotrophin-releasing hormone (sGnRH) was studied in the brain and pituitary of two-year-old immature sea bass (Dicentrarchus labrax) by means of an enzymoimmunoassay (EIA) for sGnRH and immunocytochemistry. The EIA for sGnRH is a competitive assay using a tracer made of sGnRH coupled to acetylcholinesterase from an electric eel. The separation of free and bound tracer is achieved by coating the plates with mouse anti-rabbit IgG monoclonal antibodies. Displacement curves generated by sGnRH and extracts from pituitary and different brain regions showed a good parallelism allowing the assay to be used for sGnRH measurements in this species. Although all parts of the brain contained measurable levels of sGnRH, the highest concentrations were found in the pituitary, the olfactory bulbs and the telencephalon. These data were confirmed by immunocytochemistry. Cell bodies were found in the olfactory bulbs, ventral telencephalon, preoptic region and mediobasal hypothalamus. Immunoreactive fibers could be observed in all parts of the brain including the optic tectum, the cerebellum (corpus and valvula), the vagal lobe, the medulla oblongata and the rostral spinal cord. In most cases, these fibers do not form well defined bundles; however, there was clearly a continuum of immunoreactive fibers, extending from the olfactory bulbs to the pituitary, and along which all the cell bodies described above were located. In the ventral telencephalon and the preoptic region, clear pictures of varicose positive fibers contacting immunoreactive perikarya could be observed. These data indicate that sGnRH is most likely an endogenous peptide in the brain of the sea bass, although the presence of other forms of GnRH cannot be excluded at this point. This study also demonstrates that the general organization of the GnRH systems in the sea bass is highly similar to what has been described in most freshwater teleost species, and provides basis for further studies on the neuroendocrine control of gonadotrophin release in this commercially important species.     

Can Gonadal Steroids Influence Cell Position in the Developing Brain?

The preoptic area/anterior hypothalamus (POA/AH) is a site where hormones dramatically influence development. The POA/AH is comprised of multiple subgroups, but little is known about the derivation of these subgroups during development. Results from several laboratories suggest that some cells in the POA/AH originate from progenitor cells in other regions of the developing nervous system. We are exploring pathways for migration in the developing POA/AH in two ways. First, we are examining the distribution of radial glial processes as potential migratory guides using immunocytochemistry. We have identified a transient pattern of radial glial processes from the lateral ventricles to the pial surface at the base of the POA/AH. Additionally, the expression of a molecule in radial glial processes originating in the third ventricle was decreased by prenatal treatment with testosterone. Second, we are utilizing time-lapse video microscopy in vitro to assess the extent and direction of movements of fluorescent dye-labeled cells at different ages in brain slice preparations from the POA/AH of developing rats. Data from these studies indicate that cell migration in the POA/AH includes movements along dorsal-ventral routes and from lateral to medial positions, in addition to the predicted medial to lateral pathway away from the third ventricle. Several researchers have examined effects of gonadal steroids on neurite outgrowth, cell differentiation, cell death, and synaptogenesis. The determination of cell position, however, may be a key event influenced by gonadal steroids earlier in development. The characterization of migratory pathways that contribute to permanent changes in brain structure and ultimately function is essential for unraveling the process of sexual differentiation.     

An immunohistochemical study of the GnRH neuron morphology and topography in the adult female rabbit hypothalamus

The morphology and distribution of immunoreactive (GnRH) neural elements in the hypothalamus of the adult nulliparous female rabbit were examined. Approximately 1,000 GnRH cells (range 890-1136) were counted in the right half of the hypothalamus. Two distinct GnRH cell types were observed: GnRH cells with rough or spiny contours accounted for 64% of the total immunoreactive cells, and smooth-contoured cells represented 34% of the total. The majority of immunoreactive neural elements were found in the anterior hypothalamus. GnRH cells and processes were located primarily in the ventral and medial anterior hypothalamus forming an inverted V pattern. Processes were followed from the medial preoptic area and suprachiasmatic nucleus to the infundibular stem. Extrahypothalamic projections of GnRH cells were observed. Immunoreactive fibers were also found to contact the ependymal lining of the third ventricle. It is concluded that two morphologically distinct GnRH cell types exist and have a broad distribution in the rabbit hypothalamus. The functional significance of these cell types requires further study.     

Effect of hypophysectomy on immunocytochemically demonstrated growth hormone releasing factor (GHRF) in the rat brain

The effect of removal of CH by long-term hypophysectomy (HPX) on the GHRF-immunoreactivity in the rat hypothalamus was studied using immunocytochemical methods. HPX resulted in a dramatic reduction in immunostaining in the median eminence (ME), indicating a significant reduction in storage of GHRF. Other regions of the hypothalamus, including the dorsomedial nucleus, the medial preoptic and suprachiasmatic regions, and the lateral hypothalamus showed the distribution and intensity of GHRF-immunoreactive structures as seen in intact animals; i.e., scattered fibers only. A few weakly stained cell bodies were seen in experimental and control animals. This could be due to a lack of storage, and/or this peptide may possibly not be present in its matured and fully immunoreactive form in the cell bodies.     

Telencephalic and diencephalic origin of radial glial processes in the developing preoptic area/anterior hypothalamus

Neuronal birth-dating sudies using [³H]thymidine have indicated that neurons in the preoptic area/anterior hypothalamus (POA/AH) are derived primarily from progenitors in proliferative zones surrounding the third ventricle. Radial glial processes are potential guides for neuronal migration, and their presence and orientation during development may provide further information about the origin of cells in the POA/AH. In addition to determining the orientation of radial glial fibers, we examined the relationship of neurons with identified birth dates to radial glial processes in the developing POA/AH of ferrets. Neuronal birth dates were determined by injecting ferret fetuses with bromodeoxyuridine (BrdU) at several different gestational ages; brains were taken from ferret kits at subsequent prenatal ages. Sections were processed for immunocytochemistry to reveal vimentin or glial fibrillary acidic protein in radial glia, or BrdU-labeled cell nuclei. Numerous radial glial processes extended from the lateral ventricles through ventral portions of the septal region to the pial surface of the POA/AH. These fibers both encapsulated and coursed ventrally through and around the anterior commissure of ferret, rat, and mouse fetuses. These ventrally directed fibers were less evident at older ages. In double-labeled sections from ferrets, BrdU-labeled cells in the dorsal POA/AH were often aligned in the same dorsal-ventral orientation as adjacent radial glial fibers. We suggest that a subset of neurons, originating in telencephalic proliferative zones, migrates ventrally along radial glial guides into the dorsal POA/AH. © 1995 John Wiley & Sons, Inc.     

Response to selection for photoperiod responsiveness on the density and location of mature GnRH-releasing neurons

Natural variation in neuroendocrine traits is poorly understood, despite the importance of variation in brain function and evolution. Most rodents in the temperate zones inhibit reproduction and other nonessential functions in short winter photoperiods, but some have little or no reproductive response. We tested whether genetic variability in reproductive seasonality is related to individual differences in the neuronal function of the gonadotropin-releasing hormone network, as assessed by the number and location of mature gonadotropin-releasing hormone-secreting neurons under inhibitory and excitatory photoperiods. The experiments used lines of Peromyscus leucopus previously developed by selection from a wild population. One line contained individuals reproductively inhibited by short photoperiod, and the other line contained individuals nonresponsive to short photoperiod. Expression of mature gonadotropin-releasing hormone (GnRH) immunoreactivity in the brain was detected using SMI-41 antibody in the single-labeled avidin-biotin-peroxidase-complex method. Nonresponsive mice had 50% more immunoreactive GnRH neurons than reproductively inhibited mice in both short- and long-day photoperiods. The greatest differences were in the anterior hypothalamus and preoptic areas. In contrast, we detected no significant within-lines differences in the number or location of immunoreactive GnRH neurons between photoperiod treatments. Our data indicate that high levels of genetic variation in a single wild population for a specific neuronal trait are related to phenotypic variation in a life history trait, i.e., winter reproduction. Variation in GnRH neuronal activity may underlie some of the natural reproductive and life history variation observed in wild populations of P. leucopus. Similar genetic variation in neuronal traits may be present in humans and other species.     

Distribution of gonadotropin-releasing hormone-like peptides in the brain during development of juvenile male Rana esculenta

Summary The distribution and density of cell bodies and fibers immunoreactive to GnRH-like peptides were investigated in the brain of male juvenile frogs (Rana esculenta) during postmetamorphic development. An immunohistochemical technique was used, involving antisera raised against 4 variants of GnRH: mammalian GnRH, chicken GnRH-I, chicken GnRH-II and salmon GnRH. A comparison of the immunohistochemical distribution at 8 different developmental stages shows that the maximum density of immunoreactive-GnRH elements, and the full distributional complexity of this system, is attained at the completion of spermatogenesis. Immunoreactive-GnRH cell bodies first appear in the anterior preoptic area during the metamorphic climax, and then appear sequentially in the medial septal area, tegmentum and, lastly, in the retrochiasmatic area and olfactory bulb when immunoreactive-fibers also reach the cerebellum. The GnRH system reacts positively to antisera for all 4 GnRH variants, but immunoreactivity for chicken GnRH-I is the weakest.     

Direct innervation of GnRH neurons by encephalic photoreceptors in birds

In nonmammalian vertebrates, photic cues that regulate the timing of seasonal reproductive cyclicity are detected by nonretinal, nonpineal deep brain photoreceptors. It has long been assumed that the underlying mechanism involves the transmission of photic information from the photoreceptor to a circadian system, and thence to the reproductive axis. An alternative hypothesis is that there is direct communication between the brain photoreceptor and the reproductive axis. In the present study, light and confocal microscopy reveal that gonadotropin releasing hormone (GnRH) neurons and processes are scattered among photoreceptor cells (identified by their opsin-immunoreactivity) in the lateral septum (SL). In the median eminence (ME), opsin and GnRH immunoreactive fibers overlap extensively. Single and double label ultrastructural immunocytochemistry indicate that in the SL and preoptic area (POA), opsin positive terminals form axo-dendritic synapses onto GnRH dendrites. In the ME, opsin and GnRH terminals lie adjacent to each other, make contact with tanycytes, or terminate on the hypophyseal portal capillaries. These results reveal thatbrain photoreceptors communicate directly with GnRH-neurons; this represents a means by which photoperiodic information reaches the reproductive axis.     

Sex differences and hormone influences on tyrosine hydroxylase immunoreactive cells in the leopard frog

We examined sex differences in tyrosine hydroxylase immunoreactive (TH-ir) cell populations in the preoptic area (POA), suprachiasmatic nucleus (SCN), posterior tuberculum (TP), and caudal hypothalamus (Hy) in the leopard frog (Rana pipiens), in addition to the effects of natural variation in sex steroid hormones on these same populations in both sexes. All four of these populations have been shown to be dopaminergic. Gonadal sex, androgens, and estrogen all influenced TH-ir cell numbers, but in a complicated pattern of interactions. After factoring out the effects of sex steroids by multiple regression, TH-ir cell numbers in all four areas differed between the sexes, with males having a greater number of TH-ir cells. The influence of androgens and estrogen differed by region and sex of the animals. Androgens were the main influence on TH-ir cell numbers in the POA and SCN. Plasma androgen concentrations were positively correlated with TH-ir cell numbers in both areas in males. In females, androgen concentration was negatively correlated with TH-ir cell numbers in the POA; there was no significant relationship in the SCN in females. In the more caudal populations, estrogen (E2) levels were positively correlated with TH-ir cell numbers in the TP of both males and females. In the caudal hypothalamus, E2 levels were positively correlated with TH-ir cell numbers in females, but there was no significant correlation in males. The results indicate that gonadal sex imposes a baseline sex difference in the four TH-ir (dopamine) populations, resulting in a higher number of such cells in males. Individual and sex-linked differences in gonadal steroid hormones lead to variation around this baseline condition, with androgens having a greater influence on rostral populations and estrogen on caudal populations. Last, an individual's gonadal sex determines the effect that androgens and estrogen have on each population.