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Molecular Endocrinology, doi:10.1210/me.2005-0337
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Molecular Endocrinology 21 (12): 3013-3027
Copyright © 2007 by The Endocrine Society

Pubertal Impairment in Nhlh2 Null Mice Is Associated with Hypothalamic and Pituitary Deficiencies

Tiziana Cogliati, Petra Delgado-Romero, Errol R. Norwitz, Jasenka Guduric-Fuchs, Ursula B. Kaiser, Susan Wray1 and Ilan R. Kirsch1

Genetics Branch, Center for Cancer Research, National Cancer Institute (T.C., P.D.-R., I.R.K.), and Cellular and Developmental Neurobiology Section, National Institute of Neurological Disorders and Stroke (S.W.), National Institutes of Health, Bethesda, Maryland 20889; Departments of Obstetrics, Gynecology, and Reproductive Biology (E.R.N.) and Medicine (U.B.K.), Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts 02115; and Centre for Vision Science (J.G.), Queen’s University Belfast, School of Biomedical Sciences, Belfast BT12 6BA, United Kingdom

Address all correspondence and requests for reprints to: Susan Wray, Cellular and Developmental Neurobiology Section, National Institute of Neurological Disorders and Stroke-National Institutes of Health, Building 35, Room 3A-1012 Bethesda, Maryland 20892-3714. E-mail: wrays{at}ninds.nih.gov; and Ilan R. Kirsch, Research Oncology, Amgen, 1201 Amgen Court West, AW1-J4144, Seattle, Washington 98119-3105. E-mail: lkirsch{at}amgen.com.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Pubertal development is impaired in mice lacking the basic helix-loop-helix transcription factor Nhlh2. The mechanisms underlying changes in reproduction in Nhlh2-deficient mice (Nhlh2–/–) are unclear. Here we show that hypothalamic GnRH-1 content is reduced in adult Nhlh2–/– mice as is the number of GnRH-1 neurons localized to mid- and caudal hypothalamic regions. This reduction was detected postnatally after normal migration of GnRH-1 neurons within nasal regions had occurred. Phenotype rescue experiments showed that female Nhlh2–/– mice were responsive to estrogen treatment. In contrast, puberty could not be primed in female Nhlh2–/– mice with a GnRH-1 regimen. The adenohypophysis of Nhlh2–/– mice was hypoplastic although it contained a full complement of the five anterior pituitary cell types. GnRH-1 receptors (GnRHRs) were reduced in Nhlh2–/– pituitary gonadotropes as compared with wild type. In vitro assays indicated that Nhlh2 expression is regulated in parallel with GnRHR expression. However, direct transcriptional activity of Nhlh2 on the GnRHR promoter was not found. These results indicate that Nhlh2 plays a role in the development and functional maintenance of the hypothalamic-pituitary-gonadal axis at least at two levels: 1) in the hypothalamus by regulating the number and distribution of GnRH-1 neurons and, 2) in the developing and mature adenohypophysis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
MAMMALIAN REPRODUCTIVE FUNCTION requires complex interactions within the hypothalamic-pituitary-gonadal (HPG) axis modulated by positive and negative feedback at both the hypothalamic and pituitary levels. Within the HPG, the GnRH-1 system forms the hypothalamic output pathway to the pituitary. In rodents, GnRH-1 cells are distributed in a bilateral continuum from the olfactory bulbs to the caudal hypothalamus (1). Independent of location, the majority of GnRH-1 neurons send their axons to the median eminence. Pulsatile release of GnRH-1 into the hypophysial portal circulation induces synthesis and secretion of pituitary gonadotropins, LH and FSH, and, as a consequence, activation of gonadal function. The GnRH-1 system integrates internal homeostatic and external environmental factors pertinent to reproduction. Perturbation of either the development or regulation of GnRH-1-producing neurons results in reproductive dysfunction (see Ref. 1 for review). Thus, loss of GnRH-1 neurons (2), disruption of their migration during development (3), or disruption of the GnRH-1 gene (4, 5) prevents sexual development and leads to reproductive failure.

Targeted deletion of the basic helix-loop-helix (bHLH) gene family member Nhlh2 showed that it is essential for pubertal development in mouse and established a role for Nhlh2 in acquisition of reproductive competence (6). Male Nhlh2–/– mice are hypogonadal and infertile, with alterations in circulating gonadotropins, a defect in spermatogenesis, and loss of instinctual male sexual behavior. By 8 wk of age, female Nhlh2–/– mice reared in a male-free environment are hypogonadal, whereas Nhlh2–/– females raised with male mice can be fertile (6).

Nhlh2 belongs to a subfamily of class B bHLH transcription factors specific to the nervous system that includes Nhlh1 as the only other member (7, 8). Genes encoding class B bHLH proteins (e.g. Nhlh, MyoD, and NeuroD/β2) exhibit a restricted pattern of expression, are tissue specific, and are often expressed transiently in differentiating or differentiated cell lineages (9). MyoD and NeuroD1/β2 are involved in the regulation of myogenesis and neurogenesis, respectively (10, 11). Both Nhlh1 and Nhlh2 are expressed in postmitotic neurons suggesting a function as late-acting differentiation genes and in the maintenance of differentiated cells (8, 12). However, their lineage specificity and their function have not yet been defined. To better understand these transcription factors, we undertook an investigation of the role of Nhlh2 in specific cell types involved in reproductive maturation.

Nhlh2 expression was recently shown in developing GnRH-1 neurons and in the postnatal anterior pituitary gland (13), suggesting a potential dual role for Nhlh2 in the development and function of the HPG axis. Nhlh2 is the first bHLH gene described in GnRH-1 neurons. In contrast, several candidate bHLH proteins have been identified in anterior pituitary extracts and have been shown to be differentially expressed in separate hormone-secreting cell lines derived from the anterior pituitary (14). This suggests that proteins of the bHLH family of transcription factors influence pituitary-specific gene expression and possibly play a role in the establishment or function of individual cell lineages in the anterior pituitary. One example is NeuroD1/β2, which is part of a transcriptional complex that triggers corticotroph-specific transcription and cell differentiation during pituitary ontogeny (15). To assess at which level(s) of the HPG axis and for which cell population(s) Nhlh2 function is essential, we studied development of the GnRH-1 system and pituitary gland in Nhlh2-null mice.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Sexual Maturity in Nhlh2-Deficient Mice
The first appearance of estrus in female Nhlh2–/– mice raised in the absence of males was delayed approximately 2 wk compared with wild-type (WT) littermates. Moreover, only 40% of the population reached first estrus by 12 wk, a time at which 100% of female WT mice were regularly cycling (Fig. 1AGo). At older ages, a similar percentage of Nhlh2–/– anestrous females were found. After attainment of puberty, estrous cycles in Nhlh2–/– females were irregular and prolonged (average time period between two consecutive estrus 9.6 ± 1.23 d in Nhlh2–/– vs. 5.9 ± 0.36 d in WT; P < 0.01). At 12 wk, ovaries and uteri of female Nhlh2–/– mice that had undergone first estrus were examined and found to be smaller than those of female WT but larger than those of female Nhlh2–/– that had not undergone first estrus (Fig. 1BGo). After extensive inbreeding of the colony (mating of littermates), 100% of females over 3 months of age (n = 10) showed absence of a fully developed vaginal opening (suggestive of missed puberty) even when maintained in non-male-free conditions. These data confirm impairment of pubertal development in Nhlh2–/– female mice.


Figure 1
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  		Fig. 1. Puberty Is Delayed in Nhlh2–/– Mice

Time of occurrence of first estrus (A) and size of uterus-ovaries (B) in untreated WT and Nhlh2–/– female mice. Animals were separated from the rest of the colony at 21 d (weaning) and housed in a male-free environment throughout their pubertal development. Under these conditions, Nhlh2–/– mice separated into two populations. One group (40%) underwent delayed puberty (sexually mature) whereas the second group remained anestrous into adulthood (sexually immature). The two groups of sexually mature (+) and sexually immature (–) Nhlh2–/– females are plotted independently in panel B.

 
GnRH-1 Content and Neurons in Nhlh2-Deficient Mice
Reproductive competence is dependent on hypothalamic GnRH-1 reaching the pituitary gland (16). Whole hypothalamic extracts from both male and female Nhlh2–/– mice contained significantly less GnRH-1 than those from WT littermates at postnatal d 21 (PN21) (prepubertal) as well as at 8–12 wk (Fig. 2AGo; P < 0.05). GnRH-1 neurons were quantified in 12-wk-old male and female Nhlh2–/– mice and their WT littermates to determine whether reduction in hypothalamic GnRH-1 content reflected decreased expression within the GnRH-1 neuronal population and/or a decrease in total number of GnRH-1-producing cells. A significant reduction (59% for female; 31% for male) in the number of GnRH-1 neurons was found in the brain of adult Nhlh2-deficient mice (Fig. 2BGo). We asked whether attainment of sexual maturity in Nhlh2–/– female mice correlated with the severity of disruption of GnRH-1 neurons at adult age. However, the number of GnRH-1 neurons at 12 wk was comparable in both groups of females (297 ± 34, sexually mature, n = 2; 361 ± 48, sexually immature, n = 3). Similarly, no differences were detected in GnRH-1 content between sexually immature (0.246 ±0.0421 pg GnRH-1/µg protein, n = 11) and sexually mature 12-wk-old Nhlh2–/– female mice (0.297 ± 0.0588 pg GnRH-1/µg protein; n = 8). In all Nhlh2-deficient adult mice, loss of GnRH-1 neurons did not appear homogeneously distributed along the rostro-caudal axis of the forebrain. Fewer GnRH-1 neurons were detected at the organum vasculosum lamina terminalis (OVLT) and in the caudal hypothalamus in Nhlh2–/– compared with WT mice (Fig. 3Go, WT: B and C, Nhlh2–/–: E and F). Chi square ({chi}2) analysis confirmed a significant difference in the distribution of GnRH-1 cells in Nhlh2–/– compared with WT hypothalami ({chi}2 = 211.56, P < 0.001; Nhlh2–/–, n = 5; WT, n = 4). Post hoc cell {chi}2 analysis was done to determine the relative contribution of each region to the final {chi}2 value (211.56). This analysis indicated that the greatest contributing regions were rostral to the OVLT (78%). Together the olfactory and preoptic areas of the Nhlh2–/– mice showed 16% more GnRH-1 cells compared with WT. This increase was compensated for with an overall 16% decrease in GnRH-1 cell number in OVLT/caudal hypothalamic regions. {chi}2 analysis indicated a significant shift in the location of GnRH-1 neurons, but interpretation can be complicated if both cell loss and a shift in cell location occur. Thus, subsequent analysis by area (ANOVA) showed a significant decrease in GnRH-1 cell number at the level of the OVLT as well as caudal hypothalamus in Nhlh2–/– compared with WT mice (P < 0.05, OVLT: Nhlh2–/– = 306 ± 31, WT = 591 ± 59; caudal hypothalamus: Nhlh2–/– = 65 ± 29, WT = 193 ± 22). In mutant mice, ectopic GnRH-1 neurons were not found elsewhere in the brain, i.e. outside areas already reported in the literature (16).


Figure 2
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  		Fig. 2. GnRH-1 Is Reduced in Nhlh2–/– Mice

GnRH-1 content (A) and total neuron counts (B) in prepubertal (PN21) and adult female and male Nhh2–/– mice compared with WT littermates.

 

Figure 3
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  		Fig. 3. Fewer GnRH-1-Positive Neurons Are Detected in Caudal Hypothalamic Regions in Adult Nhlh2–/– Mice

Photomicrographs of representative coronal sections of brains from adult WT (A–C) and Nhlh2–/– (D–F) littermates showing the distribution of GnRH-1 neurons in the rostral (A and D) and caudal (C and F) hypothalamus and at the OVLT (B and E). Sections were hybridized with [35S]-labeled GnRH-1 riboprobe. Reduced GnRH-1-positive neurons were observed at the OVLT and in the most caudal hypothalamus of Nhlh2–/– mice (arrows). ac, Anterior commissure. Scale bars, 200 µm for panels A, B, D, and E and 500 µm for panels C and F.

 
Nhlh2 Is Transiently Expressed in Migrating GnRH-1 Neurons and in the Neonatal Hypothalamus
GnRH-1 neurons are first found within the nasal placode at embryonic d 10.5 (E10.5), migrate through the nasal region (E12.5–E14.5), and enter the ventral forebrain (E14.5–E18.5) (1, 17). The GnRH-1 neuron phenotype and the known function of the bHLH transcription factor family pointed to a developmental role of Nhlh2 in these cells. Nhlh2 mRNA was robustly expressed in cells of the nasal pit and nasal epithelium at E11.5 with a pattern similar to that of GnRH-1 neurons (Fig. 4Go, A and B, dotted lines). At E12.5 the Nhlh2 expression pattern continued to parallel that of GnRH-1 neurons (Fig. 4Go, C and D, arrows). However, at this stage the Nhlh2 hybridization signal appeared lower than that observed at E11.5. By E13.5 Nhlh2 hybridization signal was clearly down-regulated in cells farther away from the nasal pit and closer to the nasal forebrain junction (Fig. 4EGo). From the expression pattern at E11.5–E12.5, analyzed in adjacent serial thin sections (Fig. 4Go), three populations of migratory cells could be distinguished: cells expressing both Nhlh2 and GnRH-1, cells expressing only GnRH-1, and cells expressing only Nhlh2. By E18.5 GnRH-1 neurons in the hypothalamus did not express detectable Nhlh2 transcript, yet Nhlh2 expression appeared strong in hypothalamic regions containing GnRH-1 cells (Fig. 4Go, G and H). In the postnatal hypothalamus (PN0–5) Nhlh2 expression was consistently observed in cell populations close to GnRH-1 neurons, but by adulthood (12 wk) no detectable signal for Nhlh2 mRNA coincident with or in proximity to GnRH-1 immunoreactive neurons was found (data not shown).


Figure 4
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  		Fig. 4. Nhlh2 Is Expressed in Nasal Regions Prenatally

Photomicrograph of representative adjacent parasagittal sections of Nhlh2 mRNA-expressing cells (left panels) and GnRH-1 immunopositive cells (right panels) through the nasal region of E11.5 (A and B), E12.5 (C and D), E13.5 (E and F), and at the OVLT of E18.5 (G and H) WT embryos. To optimize signals for both molecules, adjacent sections were used. One series was processed for Nhlh2 mRNA by ISH, and the adjacent series was processed for GnRH-1 using ICC. At E11.5, Nhlh2-positive neurons (A), like GnRH-1-immunoreactive neurons (B) are present in the nasal pit and migrating stream (arrows). However, Nhlh2-positive cells are also present in the olfactory epithelium (open arrow). Hatched lines in A and B outline the migratory path from the nasal pit. By E12.5, Nhlh2 expression becomes attenuated in cells in nasal regions (C). Arrowheads in C–F point to coincidental patterns of Nhlh2 and GnRH-1 localization still apparent in migrating cells and in the vomeronasal organ (VNO). Adjacent sections hybridized with Nhlh2 (G, dark-field image) and immunostained for GnRH-1 (H) are cut along midline at the OVLT and juxtaposed to show the degree of cell staining. nc, Nasal cavity; oe, olfactory epithelium; np, nasal pit; fb, forebrain.

 
To further evaluate Nhlh2 expression in late migratory GnRH-1 neurons, nasal explants were generated from E11.5 WT mouse embryos, cultured for 3, 7, and 14 d (18), and single cell RT-PCR analysis of mRNA isolated from GnRH-1 primary cells was performed (n ≥ 6 cells at each stage). GnRH-1 cells in this model system are known to recapitulate many aspects of GnRH-1 neuron development in vivo and correspond to E14.5, E18.5, and PN5, respectively (19). No amplicon for Nhlh2 was detected in 24 of 25 samples after two cycles of cDNA amplification (Fig. 5Go). Only one cell at 14 d in vitro (DIV) consistently showed a positive band. Furthermore, Nhlh2 expression was not detected by RT-PCR in RNA samples isolated from NLT (20) and GT1–7 (21) cells, two immortalized cell lines expressing GnRH-1, either as whole-cell culture lysates or single cells (Fig. 5Go). These data support and strengthen the in vivo data and together indicate that Nhlh2 is transiently expressed in GnRH-1 cells during development, with Nhlh2 transcript being down-regulated by the time GnRH-1 cells reach the hypothalamus in vivo.


Figure 5
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  		Fig. 5. Nhlh2 Is Not Expressed in Either GnRH-1 Single Cells from E11.5 Nasal Explants, NLT, or GT1–7 Cell Lines

PCR amplification with specific 3'-untranslated region Nhlh2 primers was performed on cDNAs from single cells (57 ). Ethidium bromide-stained agarose gels of representative amplifications show the expected amplicon of approximately 250 bp (upper lane) in the control brain (B) lane and in one of six single GnRH-1 cells after 14 DIV (no. 1). No amplicon is observed in water (W), NLT, and GT1–7 cells (n = 6, n = 3, respectively) and in all other single-cell preparations from 3 DIV (n ≥ 6), 7 DIV (n ≥ 6), 14 DIV (n ≥ 6) explants (only two cells per group are shown). All RT-PCR-generated cDNAs were previously checked for positive GnRH-1, β-tubulin, and L19 expression (as described in Materials and Methods). Lower lane shows the presence of the expected approximately 320 bp amplicon for GnRH-1, even in cells negative for Nhlh2.

 
Development of GnRH-1 Neurons Appears Normal until Birth in Nhlh2-Deficient Mice
To explain the GnRH-1 neuron phenotype in Nhlh2–/– mice postnatally (loss of cells), we initially focused on migration of the GnRH-1 neurons and their migratory pathway. Throughout prenatal development, the nerve fibers contributing to the GnRH-1 neuron migratory path as well as GnRH-1 neuron migration were normal in Nhlh2–/– mice. GnRH-1 cells were detected extending from the nasal pit to and across the cribriform plate into the hypothalamus (Fig. 6Go, compare WT: A and B with Nhlh2–/–: C and D). Furthermore, no differences were observed in olfactory axon outgrowth and/or GnRH-1 migratory behavior (18) in nasal explants from WT and Nhlh2–/– mice, maintained for 7 and 14 DIV (data not shown). Indeed, the number of prenatal GnRH-1 neurons was similar in Nhlh2–/– compared with WT mice up until birth (Table 1Go). These data indicate that Nhlh2 is not necessary for the initial differentiation of GnRH-1 cells and subsequent migration of GnRH-1 cells from nasal regions into the forebrain.


Figure 6
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  		Fig. 6. GnRH-1 Neuronal Migration Appears Normal in Nhlh2-Deficient Mice

Photomicrographs of parasagittal sections from WT (A and B) and Nhlh2–/– (C and D) littermate embryos at E12.5 (A and C) and E13.5 (B and D). A and C were immunocytochemically stained for GnRH-1 (black arrows), whereas B and D were double labeled for GnRH-1 (blue/purple) and peripherin (a marker for olfactory axons, brown). GnRH-1-immunoreactive neurons (black) are seen in the nasal region migrating in cords away from the olfactory pit (arrows in A and C), crossing the cribriform plate, and turning back toward the caudal hypothalamus (arrowheads and asterisks in B and D, respectively). Examination of E18.5-PN5 Nhlh2-deficient mice for GnRH-1 cells undergoing cell death, using caspase as a marker, showed no differences between Nhlh2-deficient and WT mice. However, caspase staining (E and F, brown staining, arrow), was more often found in the vicinity of GnRH-1 neurons (blue staining, arrowheads) in Nhlh2–/– (E and F) than in WT (data not shown) sections at the rostral forebrain (E) as well as at the level of the OVLT (F). op, Olfactory pit; cp, cribriform plate; h, hypothalamus. Scale bars, 100 µm for panels A–D and 200 µm for panels E and F.

 

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  		Table 1. GnRH-1 Neuron Counts in Neonatal Nhlh2–/– Mice

 
Although the number of GnRH-1 cells was similar in Nhlh2–/– and WT mice until PN0, the distribution of the GnRH-1 cells was significantly different at birth ({chi}2 = 157.5, P < 0.001; Nhlh2–/– n = 3; WT n = 2). By cell {chi}2 analysis, the olfactory and preoptic areas of the Nhlh2–/– mice taken together showed 20% more GnRH-1 cells compared with WT. This increase was compensated for with an overall 20% decrease in GnRH-1 cell number in OVLT/caudal hypothalamic regions. By PN5 the number of GnRH-1 neurons was reduced in Nhlh2–/– mice compared with WT (Table 1Go), in proportions similar to the changes found in adults (see above). No obvious necrosis was detected by light microscopy between PN0 and PN5. Thus, to begin to address the mechanism(s) underlying the loss of a subpopulation of GnRH-1 cells, terminal transferase dUTP nick end labeling (TUNEL) assay and caspase-3 immunocytochemistry (ICC) on hypothalamic sections from Nhlh2–/– and WT mice at different developmental ages (E16.5, E18.5, PN0, and PN5) was performed. At all stages only a few TUNEL-positive or caspase-3 immunoreactive cells were observed that were double labeled with GnRH-1, and these were detected in both WT and Nhlh2-deficient mice (Fig. 6Go, Nhlh2–/–: E and F and data not shown). Thus, if apoptotic cell death is preferentially occurring in the Nhlh2–/– mice, because of GnRH-1 peptide rapid loss, detection of double-labeled cells is prevented. In summary, early development and migration of GnRH-1 neurons in nasal areas appeared similar in Nhlh2-deficient and WT mice. However, a subpopulation of GnRH-1 cells that enter the central nervous system appear to pause/stop rostrally and then become undetectable in Nhlh2-deficient mice.

Peptidergic Involvement in Nhlh2–/– Phenotype
Because Nhlh2 message is not expressed by GnRH-1 neurons in the forebrain, we hypothesized that other Nhlh2-expressing cells in the hypothalamus influence GnRH-1 neuronal migration, survival, and/or peptide expression. Cell types in two areas, the arcuate nucleus (ARC) and anteroventral periventricular nucleus (AVPV), which expressed Nhlh2 around birth (E18.5–PN5, Fig. 7Go, C and F) and are known to influence reproductive function (22, 23, 24), were investigated as well as other hypothalamic cell types known to interact with GnRH-1 neurons postnatally (25). Neurotransmitter and neuropeptide cell types examined included {gamma}-aminobutyric acid (GABA), tyrosine hydroxylase, vasoactive intestinal peptide (VIP), somatostatin, vasopressin, and neuropeptide Y (NPY). Due to low levels of expression of many of these proteins before birth, PN4–PN5 brains of WT and Nhlh2–/– were examined. No detectable differences between Nhlh2–/– and WT hypothalami were observed for GABA, tyrosine hydroxylase, VIP, SP, somatostatin, or vasopressin. However, NPY staining in the ARC was reduced, indicative of fewer cells (Fig. 7Go, WT: A, Nhlh2–/–: B) and fibers in the AVPV appeared less dense (data not shown) in Nhlh2–/– mice as compared with WT. Notably, no differences were observed in NPY-immunoreactive cells in the paraventricular nucleus of the hypothalamus (PVN, Fig. 7Go, WT: D, Nhlh2–/–: E), another hypothalamic area expressing Nhlh2 (Fig. 7FGo). Postnatally GnRH-1 neurons express NPY receptors (25). Whether alterations in NPY in the ARC or AVPV contribute, either directly or indirectly, to the loss of GnRH-1 neurons found in the Nhlh2–/– mice requires further investigation. To our knowledge the number and distribution of GnRH-1 neurons have not been reported for NPY knockout mice.


Figure 7
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  		Fig. 7. NPY Neurons Are Reduced in Arcuate Nucleus (ARC) of Nhlh2–/– Hypothalamus

Photomicrographs of representative coronal sections of hypothalami from PN4 WT (A, D, C, and F) and Nhlh2–/– (B and E) littermates. NPY immunoreactivity in the ARC (A and B) but not in the PVN (D and E) was reduced in Nhlh2–/– hypothalami. ISH shows Nhlh2 expression in PN4 WT ARC (C) and PVN (F). PVN, Paraventricular nuclei; 3rd, third ventricle. Scale bars, 100 µm.

 
Pituitary Function in Nhlh2-Deficient Mice
In an attempt to rescue the Nhlh2–/– phenotype, GnRH-1 or 17β-estradiol (E2) was administered to prepubertal Nhlh2–/– and WT female mice (Fig. 8AGo). E2 primed 100% of Nhlh2–/– female mice to enter first estrus, with no significant delay compared with WT littermates (solid and open triangles, respectively). After treatment withdrawal, the estrous cycle in the Nhlh2–/– female mice continued but with an irregular pattern similar to that observed in untreated Nhlh2–/– females. Onset of puberty was primed in WT female mice by GnRH-1 (open squares), validating the administration protocol. However, no effect was observed in Nhlh2–/– mice (solid squares). Weight of ovaries and uteri at 12 wk of age confirmed the data obtained by observing the onset of estrus. No difference was detected between treated and untreated WT mice, reflecting attainment of full sexual maturity in all. However, as observed in the untreated group, ovaries and uteri of female Nhlh2–/– mice that had undergone first estrus were smaller than those of female WT but larger than those of female Nhlh2–/– that had not undergone first estrus (Fig. 8BGo), suggesting only partial attainment of sexual maturity. Because GnRH-1-mediated activation requires an intact pituitary gland and Nhlh2 is expressed in the anterior pituitary (6, 13) we next focused on the pituitary gland.


Figure 8
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  		Fig. 8. Nhlh2-Deficient Mice Respond to E2 But Not GnRH-1 Treatment

Daily single injections of 300 ng E2 (ip) or 10 µg GnRH-1 (sc) were administered starting at 28 d and were continued for 14 d. First detectable estrus was measured by vaginal smear examination (A). Size of uterus-ovaries at 12 wk of age in WT and Nhlh2–/– female mice untreated (white bars), treated with E2 (light gray bars), or treated with GnRH-1 (dark gray bars) (B). The two groups of sexually mature (+) and sexually immature (–) Nhlh2–/– females are plotted independently where distinguishable.

 
Pituitary dissection and histological analysis showed that the anterior lobe of 12-wk-old pituitaries were smaller in Nhlh2–/– than WT mice whereas the posterior and intermediate lobes were normal (Fig. 9Go, panels A and C, WT; panels B and D, Nhlh2–/–). The decrease in pituitary size was already noticeable in neonatal (PN2) Nhlh2–/– mice compared with WT littermates (Fig. 9GGo). Ultrastructural analysis of Nhlh2–/– mouse adult adenohypophysis performed by electron microscopy showed no obvious alterations (Fig. 9Go, panel E, WT; panel F, Nhlh2–/–). No sign of cell necrosis or apoptosis could be detected in the adult Nhlh2–/– pituitaries.


Figure 9
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  		Fig. 9. Pituitary Changes Occur in Nhlh2-Deficient Mice

WT (A, C, and E) and Nhlh2-deficient (B, D, and F) mouse pituitary glands at 12 wk of age. Based on gross morphology, Nhlh2–/– mice (B) had smaller pituitary glands compared with those of WT littermates (A), and the difference appeared confined to the anterior lobe (H+E stained sections, C and D). Enlarged ER is indicated by the arrowheads in the electron microscopic photographs (E and F). Decreased pituitary size was apparent by PN2 (G). Histogram was generated by comparison of average anterior pituitary area in H+E-stained serial coronal sections of PN2 Nhlh2–/– and WT littermate heads. The number of pituitary sections obtained was the same for both genotypes. AP, Anterior lobe; IP, intermediate lobe; PP, posterior lobe.

 
ICC with antisera to anterior pituitary hormones (ACTH, TSHβ, LHβ, F-SHβ, GH, prolactin, and proopiomelanocortin) showed that Nhlh2–/– pituitaries contained all pituitary cell types (data not shown). Gonadotrope cell density appeared reduced in PN2 Nhlh2–/– adenohypophysis compared with WT (Fig. 10Go, panel A, WT; panel C, Nhlh2–/–), but no differences in cell counts were measured in the adult pituitaries (Fig. 10Go, panel B, WT; panel D, Nhlh2–/–). Northern blot analysis of female Nhlh2–/– pituitary mRNA showed reduced LHβ expression compared with WT (Fig. 11AGo). These changes were confirmed by in situ hybridization (ISH) (Fig. 11Go, panel D, WT; panel F, Nhlh2–/–). Changes in hormone release were measured by ELISA for LH and RIA for FSH (Fig. 11Go, B and C). Consistent with Northern blot analysis and ISH data, LH serum content was reduced (~ 50%) in Nhlh2–/– female mice at 12 wk of age compared with WT littermates. Unlike LH, RIA data showed normal serum levels of FSH (Fig. 11CGo), in accordance with the mRNA ISH results (Fig. 11Go, E and G).


Figure 10
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  		Fig. 10. LH Is Altered in Nhlh2-Deficient Mice

The proportion of LH-positive cells in the adenohypophysis appeared reduced in Nhlh2–/– (C) compared with WT (A) mice at PN2 but not in the adult (D and B, respectively). ST, Sella turcica; H, hypothalamus; AP, anterior lobe; IP, intermediate lobe; PP, posterior lobe.

 

Figure 11
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  		Fig. 11. Gonadotropin Expression and Release Are Altered in Nhlh2-Deficient Mice

Northern blot analysis of pituitary RNA (A) shows decreased LHβ mRNA levels in Nhlh2–/– pituitary gland as shown also by ISH with LHβ (D and F) riboprobe. β-Actin was used as loading control in Northern blot. No difference was observed between Nhlh2–/–and WT ISH signal for FSHβ mRNA (E and G). LH (B) but not FSH (C) serum content was attenuated in Nhlh2–/– mice. Samples were collected on the first day of diestrus to allow for optimal comparison.

 
Gonadotropin expression and release are under hypothalamic regulation through the GnRH-1 receptor (GnRHR). ISH showed reduced expression of GnRHR mRNA (Fig. 12Go, panel A, WT; panel B, Nhlh2–/–) paralleled by a 20% reduction (Fig. 12EGo) in the number of GnRHR on the membrane of Nhlh2–/– pituitary cells (Fig. 12DGo) compared with WT (Fig. 12CGo), as detected by receptor ligand-binding assay.


Figure 12
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  		Fig. 12. Reduced Expression of GnRHR in Adenohypophysis of 12-wk-old Nhlh2–/– Mice

Dark-field photomicrographs of hybridization signal for GnRHR mRNA in WT (A) and Nhlh2–/– (B) pituitary sections. Autoradiographic film images of [125I]-labeled GnRH-1 ligand ([D-Ala6,N{alpha}MeLeu7,Pro9Net]-LHRH) bound to its receptor on pituitary sections from WT (C) and Nhlh2–/– (D) mice and corresponding densitometric analysis (E). Four series of sections were obtained cutting through each pituitary. ISH as well as GnRHR ligand-receptor binding assay were performed on all sections from one whole series. Densitometric analysis incorporates average measurements taken from all sections in the series. d.p.m., Disintegrations per minute.

 
Regulation of GnRHR through Nhlh2
GnRHR expression is regulated in part through stimulation of the membrane-bound receptor by GnRH-1 itself (26). At concentrations of GnRH-1 agonist (GnRHAg) and exposure times that induce up-regulation of GnRHR in the pituitary gonadotrope-derived cell line {alpha}T3–1 (27, 28), Nhlh2 expression is up-regulated in parallel (Fig. 13AGo). bHLH transcription factors are known to bind gene promoters containing the loose consensus sequence CANNTG (E-box). The GnRHR promoter region was analyzed for the presence of E-boxes, and four putative binding sites were identified. To test whether Nhlh2 directly regulated the GnRHR promoter, an expression vector encoding Nhlh2 was transiently transfected into the {alpha}T3-1 cell line, along with a luciferase reporter driven by –1164/+62 of the mGnRHR gene (29), and response to GnRHAg stimulation was measured (30). No increase in luciferase activity was observed after transfection with increasing amounts of Nhlh2 (1, 2, and 4 µg/well) either in the absence or in the presence of GnRHAg stimulation (Fig. 13BGo), even when Nhlh2 was cotransfected with a vector containing a putative dimerization partner, the ubiquitous bHLH transcription factor E2A (data not shown). Given the lack of an effect of Nhlh2, a pFLAG-CMV expression vector encoding Nhlh2 with an N-terminal FLAG tag was generated and used in the transient transfection assays. This expression vector gave similar results in luciferase reporter gene assays, with no increase in mGnRHR promoter activity observed [–1164/+62mGnRHRLuc: 34,270 ± 6,065 relative luciferase units (RLU); –1164/+62mGnRHRLuc + GnRH: 56,281 ± 7,857 RLU; –1164/+62mGnRHRLuc + pFLAG-Nhlh2: 35,603 ± 1,959 RLU; –1164/+62mGnRHRLuc + pFLAG-Nhlh2 + GnRH: 49,181 ± 4,610 RLU]. Western blot analysis of cell lysates from transfected cells resulted in detection of a band of approximately 20 kDa, the expected size of the FLAG-Nhlh2 fusion protein, confirming expression of the transfected protein (Fig. 13CGo).


Figure 13
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  		Fig. 13. Regulation of Nhlh2 and GnRHR Expression in {alpha}T3 Cells

RT-PCR analysis of GnRHR and Nhlh2 expression regulation after 4 h stimulation with GnRHAg (A). β-Actin was used as a control. GnRHAg-stimulated luciferase activity of GnRHR gene promoter in the presence of overexpressed Nhlh2 in {alpha}T3–1 cells (B). {alpha}T3-1 cells were cotransfected with plasmids containing Nhlh2 (1 µg/well), the GnRHR promoter region –1164/+62 fused to a luciferase reporter, or control plasmids pXP2 and pcDNA3, and RSV-β-galactosidase in the indicated combinations, followed by treatment with GnRHAg (100 nM) or vehicle for 4 h. Measurements are expressed as luciferase/β-galactosidase. Results are mean ± SEM from four experiments performed in triplicate. Fold response to GnRHAg stimulation is shown on top. Western blot analysis of Nhlh2 overexpression in pFLAG-CMV-Nhlh2 transfected {alpha}T3-1 cells (C). A band of approximately 20 KDa (arrow) localizes the FLAG-tagged Nhlh2 protein in the lysates of cells transfected with pFLAG-CMV-Nhlh2 (pFLAG-Nhlh2) and not in the lysates of cells transfected with the vector pFLAG-CMV (pFLAG) alone.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The pubertal defect of the Nhlh2 null mouse suggested defects in the hypothalamus and specifically the GnRH-1 system and/or the adenohypophysis (gonadotropes). In this paper we report alterations in neuroendocrine GnRH-1 cell number and location, in GnRH-1 hypothalamic content, in NPY immunoreactive cell number in the ARC, and morphological and functional abnormalities of the adenohypophysis after Nhlh2 gene-targeted deletion in mouse.

Nhlh2 Deletion and GnRH-1 Neurons
The acquisition of reproductive competence (the onset of puberty) relies on a hierarchy of events, with a critical component being GnRH-1 (31). GnRH-1 neuron counts were reduced in adult caudal hypothalamic regions of Nhlh2–/– mice. Developmental analysis showed migration of the entire GnRH-1 population into the developing forebrain. Loss of GnRH-1 cells occurred within a relatively short period, between PN0–PN5. By PN0 however, GnRH-1 cells in the null mice were already limited in the caudal extent of their distribution. Accumulation of GnRH-1 cells in the olfactory and preoptic areas at PN0 suggests that GnRH-1 cells did not disappear from caudal regions, but rather did not make it caudally and may, in fact, disappear from more rostral regions. Death by apoptosis or necrosis could have explained the cellular mechanisms underlying loss of GnRH-1 neurons in Nhlh2–/– mice. However, examination of stages E18.5 to PN5 did not reveal an excess of GnRH-1-immunoreactive necrotic or apoptotic cells in null mice as compared with WT. It is possible that GnRH-1 stores are depleted before cell death is detectable, because one of the early events associated with both necrosis and apoptosis is an increase in intracellular Ca2+ that is known to trigger GnRH-1 secretion (32, 33). Availability of alternative markers for GnRH-1 neurons might help in definitively establishing the mechanism underlying partial GnRH-1 neuron loss in the Nhlh2–/– mice.

A number of reports suggest that the GnRH-1 neuron population is functionally heterogeneous (34, 35, 36, 37). Notably, the residual population of GnRH-1 neurons in Nhlh2–/– mice did not compensate for the loss of neurons by up-regulating GnRH-1 expression as indicated by the reported reduction of GnRH-1 total hypothalamic content. The partial loss of GnRH-1 neurons in Nhlh2–/– mice suggests that Nhlh2 contributes to the development and/or maintenance of a subpopulation of GnRH-1 neurons that normally reside at the level of the OVLT and in the caudal hypothalamus. Our expression data, in vivo by ISH and in GnRH-1 neurons in vitro by RT-PCR, indicate that Nhlh2 message is not detectable in GnRH-1 neurons in the hypothalamus or in cultured GnRH-1-expressing neurons of equivalent developmental ages. Nonetheless, Nhlh2 is expressed in hypothalamic areas through which GnRH-1 neurons migrate, or which correspond to their terminal location, or in areas containing cells with known projections to GnRH-1 neurons. This suggests that Nhlh2 transcriptional activity contributes to GnRH-1 neuron development by regulating expression of yet unknown environmental cues, which affect their migration, gene expression, and/or survival in the caudal hypothalamus. In accordance with a non-cell autonomous mechanism, a reduction in the NPY neuron subpopulation residing in the caudal hypothalamus of Nhlh2–/– mice was also found. These data are consistent with an Nhlh2-dependent factor influencing survival or gene expression of at least two cell phenotypes. Alternatively (though not exclusively) the partial loss of NPY cells may be causal to the decrease in GnRH-1 cells. It is known that NPY regulates pulsatile GnRH-1 release (38, 39) and that by PN2, NPY cells are present in ARC, and NPY-immunoreactive fibers are present throughout the hypothalamus, including the preoptic area (22). Thus, loss of cell circuitry within the hypothalamus at early ages may ultimately disrupt the GnRH-1 system.

A previous report invoked a cell autonomous mechanism to explain the GnRH-1 neuron loss phenotype of Nhlh2–/– mice (13). This mechanism is based on expression studies in the heterozygous Nhlh2 lacZ knock-in mice where β-galactosidase immunoreactivity was detected in GnRH-1 neurons throughout embryonic and neonatal development. On the contrary, we could not detect Nhlh2 expression in GnRH-1 neurons in the hypothalamus. Differences between Nhlh2 ISH and β-galactosidase immunoreactivity expression data could be explained by a relatively long half-life of β-galactosidase protein compared with Nhlh2 mRNA (40). Persistence of the reporter protein long after transcription of Nhlh2 subsided might have prevented the detection of expression modulation of the latter that was observed in the present study. Conclusive direct evidence of the regulation of Nhlh2 expression in GnRH-1 neurons in the postnatal mouse is key to the establishment of the functional relationship between Nhlh2 and GnRH-1 neuron development. However, lack of an antibody specific to Nhlh2 to use in ICC only allows consideration of indirect evidences such as those reported herein.

Nhlh2 Deletion and Pituitary Gonadotropes
The importance of Nhlh2 in the development of GnRH-1 neurons is clear. However, the fact that no detectable differences were found in the GnRH-1 neuroendocrine system between sexually mature and immature Nhlh2–/– female mice suggests that, although GnRH-1 reduction and cell loss may explain in part the hypogonadal phenotype of Nhlh2-deficient mice, it is unlikely to be the only cause. The size of the adenohypophysis of male and female Nhlh2–/– mice was reduced at birth and in the adult, but contained all five pituitary cell types. Although no change in gonadotrope cell density was observed, a smaller Nhlh2–/– anterior pituitary contained fewer total cell counts than its WT counterpart. In other transgenic models (41) smaller anterior pituitary is associated with a defect in progenitor cell expansion during embryonic development (E10.5). Nhlh2 is not expressed in the pituitary gland at these early stages. However, its expression is strong in the ventral hypothalamus (Refs. 6 and 13 and our own observations), which has been suggested to play a role in pituitary development in the initial phase of progenitor cell expansion (42). Nhlh2 expression appears postnatally in the WT adenohypophysis and persists in the adult (6), suggesting a functional role in the adult pituitary as well. Using Northern blot and ISH for RNA detection and receptor-ligand binding assays and immunoassays for protein expression and release, we have shown that pituitary gonadotropins and GnRH-1 receptor are dysregulated in Nhlh2–/– mice. Nonetheless, the exact role of Nhlh2 in the developing and mature anterior pituitary gland remains to be determined.

Biosynthesis and secretion of LH and FSH by pituitary gonadotropes depend primarily on the amplitude and frequency of pulsatile GnRH-1 released from the hypothalamus (43) and correlate with the concentration of GnRHRs on the cell surface (44). Because GnRH-1 storage in the Nhlh2–/– hypothalamus is reduced, one could argue that the amplitude of GnRH-1 release is reduced. GnRHR density was decreased in Nhlh2–/– mice compared with WT littermates, with a pattern of suppression similar to that observed in rat pituitary during lactation (45). During lactation it is suggested that the suckling stimulus inhibits GnRH-1 secretion, which, in turn, has an effect on GnRHR expression. Similarly, chronic reduction in GnRH-1 stimulation could induce GnRHR down-regulation in Nhlh2–/– mice. Variable reduction in GnRH-1 stimulus to the pituitary gland might also explain the dimorphic response in gonadotropin release observed in these mice. The amount of GnRH-1 reaching Nhlh2–/– gonadotropes could be in the range of concentrations that elicits opposite responses on LH and FSH synthesis and release (43). Thus, LH serum content is reduced in Nhlh2–/– female mice, similar to the rat pituitary during lactation (45), whereas FSH serum content remains unchanged.

In our study, the Nhlh2–/– female hypogonadal phenotype was rescued by single daily injections of E2 but not of GnRH-1. E2 mediates feedback loop signaling from the ovaries to the hypothalamus and pituitary (31). Perhaps acting locally, E2 elicited estrous changes in vaginal cytology and stimulated an increase in uterine weight. In contrast, GnRH-1 administration at dosages that induced puberty in the WT failed to rescue the Nhlh2–/– female hypogonadal phenotype. It is possible that the dose of GnRH-1 used in our treatment protocol was inappropriate for Nhlh2–/– females, given the reduced number of receptors in Nhlh2–/– mice; hence it failed to elicit a response. More likely, an additional defect in pituitary gonadotropes affecting the regulation of the GnRHR gene by Nhlh2 was responsible for reduction in GnRHR and lack of response to GnRH-1 stimulation. Using an in vitro system, Nhlh2 and GnRHR expression in the {alpha}T3-1 cell line was examined (21). Nhlh2 expression was low in cultured {alpha}T3-1 cells. Coordinated up-regulation of Nhlh2 and GnRHR mRNA upon stimulation with GnRHAg suggested that Nhlh2 regulates expression of the receptor. However, in reporter gene assays using the GnRHR gene promoter, neither Nhlh2 cloned into two independent expression vectors and transfected alone at different concentrations nor in combination with its ubiquitous partner E2A showed trans-activating activity. Under our experimental conditions, the transient nature of Nhlh2 overexpression, the absence of other potential interacting factors, and the possible requirement for cis-regulatory element sequences absent from the reporter construct might have prevented the detection of Nhlh2 trans-activating activity. Thus, it remains to be established whether Nhlh2 regulates transcription of the GnRHR gene directly or indirectly through regulation of yet unknown genes involved in its autoregulatory loop.

Nhlh2 Deletion and Puberty
Pubertal development in Nhlh2-deficient mice is delayed or blocked. When delayed, it appears that beyond a certain threshold sexual maturity is accomplished in Nhlh2–/– female mice, although reproductive function is not completely normal. The pubertal defect of Nhlh2–/– mice is unique in that it is caused by a partial loss of GnRH-1 neurons combined with pituitary gonadotrope deficiencies. In SF1–/– mice, in which reproductive defects are the consequence of hypothalamic and pituitary dysfunctions, the absence of LH- and FSH-immunoreactive gonadotropes is associated with a normal number of GnRH-1 neurons (46). No hypothalamic GnRH-1 defect has been described for the Krox-24–/– mouse in which anterior pituitary size is reduced and gonadotropes fail to synthesize LH (47). The partial hypothalamic and pituitary phenotypes place Nhlh2–/– mice at the intersection of the GnRH-1-deficient hypogonadal (hpg) mouse (5) and of mice with targeted ablation of pituitary gonadotropes (48) and makes it an interesting model for system biology studies of the reciprocal interactions between GnRH-1 neurons and gonadotropes in the hypothalamic-pituitary axis.

In summary, partial loss of GnRH-1 neurons in association with pituitary deficiencies result in pubertal disruption and infertility of Nhlh2–/– mice. The contribution of both hypothalamic GnRH-1 neuron and pituitary defects to the pubertal phenotype of Nhlh2–/– mice point to combined/coordinated functions of Nhlh2 in the developing and mature hypothalamic-pituitary axis and to the importance of this gene in fine tuning reproductive functions in the mouse.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Mice
Nhlh2-deficient mice (Nhlh2–/–) on a C57Bl6-SV129 mixed background (6) were generated and maintained at the National Institutes of Health (NIH). Female Nhlh2–/– mice and WT littermates were separated from the rest of the colony at weaning and housed in a male-free, pathogen-free facility with a 12-h light, 12-h dark cycle (lights on at 0600 h and off at 1800 h) and fed ad libitum. Male mice were housed with the rest of the colony under the same conditions. Daily vaginal smears (20 µl in PBS) were collected from female mice after vaginal opening, and phase of estrous cycle was established after cytological analysis (49). Hormone treatments were started at 4 wk of age. Female mice were treated with single daily injections, between 0800 h-1000 h, of either E2 (300 ng/d ip; Sigma, St. Louis, MO) or GnRH-1 (GnRH-1, 10 µg/d sc, Sigma) for 2 wk. Attainment of sexual maturity was defined as the day of the first detectable estrus. Adult mice were euthanized at the indicated ages for tissue and blood collection. All female mice were euthanized between 1300 h and 1500 h on the first day of diestrus to allow comparison among regularly cycling WT, irregularly cycling Nhlh2–/–, and anestrous Nhlh2–/– mice. Embryos were obtained at the indicated time points from Nhlh2 heterozygous (Nhlh2+/–) or WT time-mating. Mice were genotyped by Southern blot analysis of their yolk sac (embryos) or tail (adults). In all experiments Nhlh2–/– mice were compared with WT littermates. Animals were maintained and all procedures were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals.

Hypothalamic GnRH-1 Content
Hypothalami were isolated, snap frozen on dry ice, and stored at –80 C until used. Subsequently, they were homogenized and briefly sonicated (0.1 M HCl, followed by adjustment of the pH to 7.5). Collected extracts were stored at –20 C before RIA using [125I]GnRH-1 (Amersham Pharmacia Biotech, Arlington Heights, IL), GnRH-1 standards (Peninsula Laboratories, Inc., Belmont, CA), and primary antibody against GnRH-1 [donated by Dr. V. D. Ramirez, University of Illinois, Urbana, IL (50)]. The intra- and interassay coefficients of variation at 80% binding in standard samples (15 pg/ml) were 12% and 14%, respectively. GnRH-1 content was normalized against total protein content measured by the Bio-Rad Protein Assay according to the manufacturer’s procedure (Bio-Rad Laboratories, Inc., Hercules, CA).

Single- and Double-Label ICC
Polyclonal antisera against pituitary hormones were obtained from the National Hormone and Pituitary Program (Torrance, CA) and were used at the following dilution: antirat LHβ (LHβ 1:2000), antirat FSHβ (FSHβ 1:2000), antirat prolactin (PRL 1:2000), antimonkey ACTH (1:1000), and antirat TSHβ (TSHβ, 1:1000). All other antisera were obtained or purchased as indicated and were used at the following dilution: anti-GnRH-1 [SW-1 (51), 1:3000], peripherin (Chemicon, Int., Inc., Temecula, CA; 1:2,000), GABA (Incstar Corp, Stillwater, MN, 1:5000), tyrosine hydroxylase (TH, Chemicon, 1:5000), VIP (Incstar Corp., 1:750), somatostatin (Incstar Corp., 1:3000), vasopressin [VA4 (52), 1:1500], and neuropeptide Y (NPY, Peninsula Laboratories, 1:4000). Biotinylated goat antirabbit secondary antibody was purchased from Vector Laboratories, Inc. (Burlingame, CA). Fresh frozen mouse head, brain, pituitary, and embryo sections were processed as described previously (18, 53). Single-label ICC (18) was performed using standard avidin-biotin-horseradish peroxidase (ABC Elite, Vector Laboratories) procedures and developed with diaminobenzidine (DAB) as the chromogen (alone or Ni-enhanced). Double-label ICC was performed as two consecutive immunoreactions using an avidin-biotin complex (ABC Elite, Vector Laboratories) and two distinct chromogens for horseradish peroxidase, (Ni-enhanced DAB, blue-black reaction, followed by DAB, brown reaction) (18).

In Situ Hybridization (ISH)
ISH on fresh frozen sections was performed using standard methods as described elsewhere (54). Mouse [35S]UTP-labeled riboprobes were prepared as follows: Nhlh2 (nucleotides 31–338), LHβ (nucleotides 142–326), and FSHβ (nucleotides 707–1212) were transcribed from cDNA fragments generated by PCR amplification using primers containing either T3 or T7 promoter sequences upstream to gene-specific nucleotide sequences in the sense and antisense orientation, respectively; GnRH-1 was transcribed from a linearized plasmid containing a 462-bp restriction fragment from rat pro-GnRH-1 cDNA encompassing the entire coding region (55); GnRHR was transcribed from a linearized plasmid containing a 416-bp PstI restriction fragment from mouse GnRHR cDNA, spanning the region from the first cytoplasmic loop to half of the second extracellular loop (45). Slides coated with autoradiographic emulsion (Kodak NTB3; Eastman Kodak, Rochester, NY) were developed after 7 d (GnRH-1, GnRHR, LHβ, and FSHβ) or 2 wk (Nhlh2) using Kodak Dektol at 18 C and counterstained with 0.5% methyl green. No signal was observed when sense probes were used for hybridization.

Nasal Explants, GnRH-1 Single-Cell Isolation, cDNA Library Construction, and Analysis
Nasal explants were prepared after bilateral olfactory pit dissection from E11.5 embryos as described previously (18). The explants were used for experiments after 3, 7, and 14 DIV for ICC or single GnRH-1 cell isolation.

For ICC analysis, explants were grown on glass coverslips, fixed in 4% buffered formaldehyde, blocked (1x PBS containing 10% normal goat serum, 0.3% Triton X-100, and 0.01% NaN3) and processed as described above for frozen sections. Single GnRH-1 neurons were identified in nasal explants based on their bipolar morphology and association with fibers extending from the main body of the explant (18). Single cells (n ≥ 6 per time point) were isolated from a minimum of two independent explants using microcapillary pipettes. NLT (20) and GT1–7 (21) cells were also plucked directly from the culture plates. cDNAs were produced, and PCR amplification was performed as described previously (56, 57, 58). Only cells that resulted positive for GnRH-1, β-tubulin, and L19 expression were further analyzed for Nhlh2 expression. Specific primers were designed in the 3'-untranslated region within 500 bp of the polyadenylation site but biased toward the 3'-end and screened using BLAST (basic local alignment search tool) to ensure specificity of the amplicon. GnRH-1 primers used for confirming identity of neurons as GnRH-1 cells were: 5'-GCT AGG CAG ACA GAA ACT TCG-3' and 5'-GGT GTT GTG GAT CCA CCT GG-3'. Primers used to detect Nhlh2 transcript in GnRH-1-positive cells were: 5'-CCC CTC AGT GTG TTA GGA CC-3' and 5'-GAC ACA GAA AGG TGT ACC AGC-3'. Brain cDNA served as a positive control. Specific bands were observed in all control brain lanes, and no bands were seen in water lanes. Each set of primers was run two times/single cell cDNA pool.

Cell Death Analysis
Cell death was examined using In Situ Cell Death Detection kit (Roche, Mannheim, Germany) following the manufacturer’s protocol. Signal was visualized with antifluorescein horseradish peroxidase-conjugated antibody and Ni-enhanced DAB (see above). Slides were then washed and incubated in GnRH-1 antiserum (SW1, 1:1000). Alexa(488) fluorescein-conjugated goat antirabbit (Molecular Probes, Eugene, OR) was used as the secondary antibody. Slides were analyzed under light (TUNEL) and fluorescence (GnRH-1 cells) microscopy. Double-label ICC was also performed using GnRH-1 and active caspase 3 (1:3000, BD Pharmingen, Franklin Lakes, NJ) antisera (see above). Reversal of primary antibodies and omission of primary antibodies served as controls.

Pituitary Histology and Electron Microscopy
Pituitaries were removed and fixed in Histochoice (Amresco, Solon, OH) overnight at room temperature, washed in 70% ethanol, and embedded in paraffin. Hematoxilin and eosin (H+E) staining was performed on 6-µm sections. For electron microscopy, pituitary gland anterior lobes were dissected, fixed overnight in 2% paraformaldehyde/1% glutaraldehyde in 1x PBS, postfixed in 1% osmium tetroxide, dehydrated through alcohol and propylene oxide, and embedded in Eponate 12 resin (Ted Pella, Inc., Redding, CA). Thin sections were prepared with a Leica Ultracut UCT ultramicrotome (Leica Corp., Deerfield, IL), stained with uranyl acetate and lead citrate, and examined under a Philips 201 electron microscope (Philips Technologies, Cheshire, CT).

Northern Blot Analysis and RT-PCR
Pituitary glands from Nhlh2–/– and WT female mice were collected and pooled according to genotype, and RNA was purified as described elsewhere (6). Samples (5–10 µg) were separated on agarose gel and transferred overnight onto 0.45-µm nitrocellulose. Filters were hybridized with [{alpha}-32P]deoxy-CTP-labeled mouse LHβ and β-actin at 42 C overnight, washed at 58 C, and exposed on film for up to 2 wk.

{alpha}T3-1 cells [generously donated by Dr. P. Mellon, University of California, San Diego, CA (27)] were treated with 100 nM Des-Gly10[D-Ala6]-GnRH-ethylamide (GnRHAg; Sigma) for 4 h. RNA from treated and untreated cells was extracted with QIAGEN RNeasy extraction kit (QIAGEN, Inc., Valencia, CA) following manufacturer’s protocols and reverse transcribed with Superscript II RNase H-reverse transcriptase and random primers according to manufacturer’s instruction (Invitrogen, San Diego, CA). PCR primers were as follows: GnRHR, 5'-CAG TCT TCT CGC AAT GTG TGA CC-3' (sense) and 5'-GCA CGG GTT TAG GAA AGC AAA G-3' (antisense); Nhlh2, 5'-ACC AGA AGA GCC AAG AAG CCA C-3' (sense) and 5'-GCG GGT GTA TGG TTG TTC ACT TAG-3' (antisense).

Serum Gonadotropin RIA and Enzymatic Immunoassay
Mouse serum was collected by cardiac puncture after anesthesia with inhaled Metophane. LH was measured by the Accucyte LH enzyme immunoassay kit (Cytimmune, College Park, MD) according to the manufacturer’s protocol. FSH was measured by Biotrak rat FSH (rFSH) [125I] assay system (Amersham Pharmacia Biotech, Little Chalfont, UK) according to the manufacturer’s protocol.

GnRHR in Situ Receptor-Ligand Assay
In situ GnRHR receptor-ligand assay was performed as described elsewhere (59) on 14-µm cryostat pituitary sections using [125I][D-Ala6,N{alpha}MeLeu7,Pro9Net]-LHRH (0.16 x 106 dpm/ml, ~80 pM, Amersham Pharmacia Biotech). Nonspecific binding was determined in the presence of 1 µM unlabeled [D-Ala6,Pro9Net]-LHRH. Dried slides were exposed to autoradiography film (Hyper film 3H, Amersham Pharmacia Biotech) for 4 d. After manual film development, quantification of the autoradiographical data was performed as described elsewhere (60).

GnRHR Promoter Reporter Gene Assay
{alpha}T3-1 cells (27) were maintained in monolayer culture and transfected at 60–80% confluency by calcium phosphate coprecipitation as previously described (30) with a luciferase reporter plasmid containing 1.2 kb of 5'-flanking region of the mGnRHR gene [–1164/+62 (29)] or empty plasmid (pXP2). Cells were cotransfected with expression plasmids for Nhlh2 (pcDNA3-Nhlh2 or pFLAG-CMV-Nhlh2), E2A (pcDNA3-E2A), alone or in combination, or empty pcDNA3 or pFLAG-CMV vector. To optimize the transfection paradigm, increasing amounts of Nhlh2 expression plasmid (1, 2, and 4 µg/well) were used. An expression vector encoding the β-galactosidase gene driven by the Rous sarcoma virus (RSV) promoter (RSV-β-galactosidase, 1 µg/well) was cotransfected in all experiments and used as an internal standard to control for cell number and transfection efficiency. Cells were treated, 48 h after transfection, with 100 nM GnRHAg or vehicle for 4 h and harvested. This dose and time interval had previously been shown to give maximal response to GnRHAg stimulation in {alpha}T3-1 cells (29). Supernatants from cell lysates were assayed immediately for luciferase and β-galactosidase activity by standard protocols (30). Luciferase activity was normalized to β-galactosidase activity. Nhlh2 overexpression was verified in protein lysates of {alpha}T3–1 cells transfected with pFLAG-CMV-Nhlh2 construct by Western blot analysis using primary mouse anti-FLAG M5 monoclonal antibody (1:1500, Sigma-Aldrich, Poole, UK) followed by secondary goat antimouse IgG-horseradish peroxidase-conjugated polyclonal antibody (1:3000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and SuperSignal West Pico Chemiluminescent Substrate detection for horseradish peroxidase (Pierce Chemical Co., Rockford, IL) according to manufacturer’s instructions.

Statistical Analysis
GnRH-1 immunoreactive or ISH-positive neurons in embryonic and adult mice were counted, and the total number of neurons per animal was calculated (mean cell number from counted series multiplied by total number of series). Data are presented as the mean ± SEM. Location of the GnRH-1 cells was recorded to establish the overall pattern of GnRH-1 migration in the embryo and terminal location of the neurons in the adult. Topographical regions were identified at each stage: nose, nose-brain junction, brain at E12.5–E13.5; nose, olfactory bulb, rostral and caudal hypothalamus at E16.5–E18.5; olfactory bulb, rostral hypothalamus, OVLT, and caudal hypothalamus at PN0-adult. GnRH-1 cells were grouped by region and genotype, contingency tables were constructed, and the statistical test for independence, chi square ({chi}2), was performed. This nonparametric test was chosen because the number of cells per region in WT vs. Nhlh –/– mice was not the same. Using this analysis, the expected GnRH-1 cell numbers were calculated, based on the hypothesis of independence, and were compared with the counted cell numbers. Subsequent cell {chi}2 analysis determined the relative contribution of each region to the differences observed in the distribution of GnRH-1 neurons. A stringent P value of 0.001 was chosen for significance because this test is based on total GnRH-1 cell number (large N). If the cell {chi}2 analysis indicated significance, a one-way ANOVA was performed to compare GnRH-1 cell number within discrete regions between genotypes at each age. A P value of 0.05 was chosen for significance in these statistical tests because N here is the number of animals (small N). The distribution of GnRH-1 cells was analyzed using Statview Software (Abacus Concepts, Inc., Berkely CA). Statistical comparison of biochemical and morphological data was performed with GraphPad Prism version 3.0 (GraphPad Software, Inc., San Diego, CA). When not otherwise stated, unpaired t test was used and P < 0.05 was considered significant.


   ACKNOWLEDGMENTS
 
We thank L. Krsmanovic for GnRH-1 measurements; E. Mezey and M. Rusnak for assistance with the ISH; A. Reuss for embryonic nasal explants; J. Lee for single cell RT-PCR; M. Herkenham and S. Yu for assistance with the GnRHR in situ receptor-ligand assay; J. Owens for electron microscopic analysis; S. Xu for technical assistance with the promoter reporter gene assays; R. Molina for technical support in the mouse room; R. Dreyfuss for assistance with microphotography; and D. Good for useful discussion.


   FOOTNOTES
 
This research was supported in part by the Intramural Research Program of the National Institutes of Health (NIH), National Institute of Neurological Disorders and Stroke, and National Cancer Institute-CCR and by NIH Grant R01 HD19938 (to U.B.K.).

Present address for T.C.: Centre of Vision Science, Queen’s University School of Biomedical Sciences, Belfast BT12 6BA, United Kingdom.

Disclosure Statement: The authors have nothing to disclose.

First Published Online August 23, 2007

1 S.W. and I.R.K. contributed equally to this work. Back

Abbreviations: ARC, Arcuate nucleus; AVPV, anteroventral periventricular nucleus; bHLH, basic helix-loop-helix; DAB, diaminobenzidine; DIV, days in vitro; E2, estradiol; E10.5, embryonic d 10.5; GABA, {gamma}-aminobutyric acid; GnRHAg, GnRH-1 agonist; GnRHR, GnRH-1 receptor; H+E, hematoxylin and eosin; HPG, hypothalamic-pituitary-gonadal; ICC, immunocytochemistry; ISH, in situ hybridization; NPY, neuropeptide Y; OVLT, organum vasculosum lamina terminalis; PN21, postnatal d 21; RSV, Rous sarcoma virus; TUNEL, terminal transferase dUTP nick end labeling; VIP, vasoactive intestinal peptide; WT, wild type.

Received for publication August 22, 2005. Accepted for publication August 13, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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