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Laboratory of Brain Science (T.U., K.U., K.T.), Faculty of Integrated Arts and Sciences, Hiroshima University, Higashi-Hiroshima 739-8521, Japan; Core Research for Evolutional Science and Technology (T.U., K.U., K.T.), Japan Science and Technology Corporation, Tokyo 150-0002, Japan; Division of Genetics and Genomics (P.J.S.), Roslin Institute, Midlothian, EH25 9PS, United Kingdom; and Department of Integrative Biology (G.E.B.), University of California, Berkeley, California 94720
Address all correspondence and requests for reprints to: Kazuyoshi Tsutsui, Laboratory of Brain Science, Faculty of Integrated Arts and Sciences, Hiroshima University, Higashi-Hiroshima 739-8521, Japan. E-mail: tsutsui{at}hiroshima-u.ac.jp
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Abstract |
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and LHß subunit mRNAs in a dose-dependent manner. Plasma LH and testosterone concentrations were also decreased dose dependently. Furthermore, administration of GnIH to mature birds induced testicular apoptosis and decreased spermatogenic activity in the testis. In immature birds, daily administration of GnIH for 2 wk suppressed normal testicular growth and rise in plasma testosterone concentrations. An inhibition of juvenile molt also occurred after GnIH administration. These results indicate that GnIH inhibits gonadal development and maintenance through the decrease in gonadotropin synthesis and release. GnIH may explain the phenomenon of photoperiod-induced gonadal regression before an observable decline in hypothalamic GnRH in quail. To our knowledge, GnIH is the first identified hypothalamic neuropeptide inhibiting reproductive function in any vertebrate class. |
Introduction |
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To elucidate the mode of action of GnIH, we further identified a novel G protein-coupled receptor for GnIH in quail (12). The identified GnIH receptor was expressed in the pituitary and specifically bound to GnIH in a concentration-dependent manner (12). Melatonin is a key factor for GnIH induction; it induces expression of GnIH mRNA and mature GnIH peptide in a dose-dependent manner (13). Furthermore, deprivation of melatonin by orbital enucleation and pinealectomy reduces expression of GnIH mRNA and peptide (13). Based on studies on birds (6, 7, 8, 9, 10, 11, 12, 13), GnIH is considered to be an important neuropeptide for the regulation of avian reproduction. In addition, we found that GnIH homologs were present in the brains of other vertebrates, such as mammals, amphibians, and fish (14, 15). These peptides possessed a LPXRF-amide (X represents L or Q) motif at their C termini in all investigated animals. The receptors (RFRP-R) for GnIH homologs were further characterized in vertebrates (14, 15). We recently cloned a homolog of GnIH from the brain of Siberian hamster, a photoperiodic mammal (Inoue, K., T. Ubuka, K. Ukena, L. Kriegsfeld, and K. Tsutsui, unpublished data). The expression of the GnIH homolog in hamster hypothalamus was also controlled by melatonin (Inoue, K., T. Ubuka, K. Ukena, L. Kriegsfeld, and K. Tsutsui, unpublished observation). Either central or peripheral administration of GnIH dose-dependently inhibited LH secretion in Syrian hamsters (16). Now that this entirely novel gonadotropin inhibitory system has been identified, it is critical to understand how it fits into the development and seasonal patterns of avian reproductive function.
To clarify the functional significance of GnIH and its potential role as a key neuropeptide involved in avian reproduction, in this study we investigated GnIH actions on gonadal development and maintenance in male quail. It is generally accepted that in avian species, LH stimulates testosterone formation in Leydig cells. FSH and testosterone stimulate growth, differentiation, and spermatogenic activity of the testis (17, 18, 19). In light of these stimulatory actions of gonadotropins on the gonad and considering GnIH’s inhibitory effects on gonadotropin secretion (6, 10, 11), we hypothesized that GnIH may decrease gonadal development and maintenance by inhibiting gonadotropin synthesis and release. GnIH was administered to reproductively mature birds by means of osmotic pumps, and the expression of gonadotropin common , LHß, and FSHß subunit mRNAs was quantified. Plasma LH and testosterone concentrations were also measured. Seasonal testicular regression is considered to be mediated by apoptosis in photoperiodic birds (20, 21). Therefore, testicular apoptosis and spermatogenic activity in response to GnIH administration were also analyzed in mature birds. To investigate whether GnIH inhibits testicular development as well as maintenance, GnIH was also administered to immature birds. Again, plasma testosterone concentrations and spermatogenic activity were analyzed. Our findings clearly show that GnIH inhibits testicular development and maintenance by decreasing gonadotropin synthesis and release.
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Materials and Methods |
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Experimental schedules
To determine the effects of GnIH on gonadotropin synthesis and release, GnIH was administered to mature birds at 3 months of age by means of mini-osmotic pumps (model 2002; Alzet, Cupertino, CA) at three different doses (0.03, 0.3, and 3 µg GnIH in 0.5 µl of 0.9% saline per hour; n = 7 in each group). Control birds (n = 7) received saline vehicle by means of identical osmotic pumps. Each osmotic pump was implanted ip under Nembutal anesthesia (40 mg/kg). After 2 wk of continuous administration of GnIH or vehicle, all birds were terminated by decapitation between 1400 and 1600 h. Trunk blood was collected into heparinized tubes and centrifuged at 1800 x g for 20 min at 4 C. Plasma was stored at –20 C. Immediately after blood collection, the pituitary was removed, snap-frozen, and stored at –80 C. The expression of gonadotropin common , LHß, and FSHß subunit mRNAs in the pituitary was quantified using competitive PCR. Plasma LH and testosterone concentrations were quantified using RIA. The sensitivity of the avian FSH assay is too low to measure FSH reliably in the plasma; thus, we do not report findings on FSH here.
To investigate the effects of GnIH on testicular apoptosis and spermatogenic activity, GnIH (0.3 µg per hour) or vehicle (n = 6 in each group) was administered to mature birds by means of osmotic pumps for 2 wk. Birds were anesthetized between 1400 and 1600 h and perfused transcardially with PBS (pH 7.3) followed by fixative solution (4% paraformaldehyde in PBS). Testes were removed and postfixed in fixative solution for 2 d. They were then dehydrated and embedded in paraffin wax. Sections were cut at 6 µm, and apoptotic cells were detected by terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling (TUNEL). The morphology of the testes was investigated by hematoxylin and eosin staining.
To investigate the effect of GnIH on testicular development, GnIH was injected sc at three different doses (0.01, 0.1, and 1 µg GnIH in 100 µl of 0.9% saline) to immature birds (n = 8 in each group) once daily between 1400 and 1600 h from 8–20 d of age. Injections were used instead of osmotic mini-pumps because of the small size of the immature quail. The body weight of quail on the average increased from 17.1 to 56.8 g as they grew over the course of the experiment. Thus, the administered middle dose of GnIH effectively decreased from 5.8 to 1.8 ng GnIH/body weight (g) per day. Control birds (n = 8) were given saline alone. All birds were killed at 21 d of age by decapitation between 1400 and 1600 h for the collection of blood and testes. Immediately after blood collection, testes were removed, weighed, and immersion fixed in Bouin’s solution for 2 d. Testes were then dehydrated and embedded in paraffin wax. They were sectioned at 6 µm and stained with hematoxylin and eosin for histological examination. Plasma testosterone concentration was quantified by RIA.
Finally, to understand the physiological role of GnIH in avian reproduction, we analyzed the effect of GnIH on the molt of juvenile plumage into adult plumage as birds matured. GnIH was injected sc at three different doses (0.01, 0.1, and 1 µg GnIH in 100 µl of 0.9% saline) to immature birds (n = 8 in each group) once daily between 1400 and 1600 h from 8–20 d of age. Control birds (n = 8) were given saline alone.
Competitive PCR analyses of gonadotropin subunits, common , LHß, and FSHß
To quantify mRNAs encoding the gonadotropin subunits (common , LHß, and FSHß) in the pituitary, competitive PCR analyses were performed as described previously (8, 13). Briefly, total RNA (including rRNA and mRNA) was isolated by the Sepasol extraction method (Sepasol-RNA I Super; Nacalai tesque, Kyoto, Japan) from each pituitary and reverse transcribed using oligo(deoxythymidine)12–18 primer (Amersham Pharmacia Biotech, Piscataway, NJ) and reverse transcriptase (Moloney murine leukemia virus reverse transcriptase; Promega, Madison, WI). Competitor DNAs for competitive PCR analyses of common , LHß, and FSHß were produced by PCR using cDNA generated from the cerebrum and primers indicated in Table 1
. Using these primers, 20-bp fragments of gonadotropin common , LHß, and FSHß subunits (underlined in Table 1) were primed to native fragments of the ß-actin gene at both 5' and 3' ends. The PCR was conducted at 94 C for 3 min and then 30 cycles at 94 C for 1 min, 60 C for 1 min, and 72 C for 1 min, with an additional incubation at 72 C for 3 min. PCR products were purified by spin column (MicroSpin S-400HR column; Amersham Pharmacia Biotech) and spectrophotometrically quantified, and aliquots were used as standards for competitive PCR analyses. For competitive PCR, an aliquot of the cDNA solution corresponding to 1 µg of the initial total RNA of each sample and competitor standard (common and LHß, 0.1–10 fmol/tube; FSHß, 1–100 amol/tube) were used as templates. Oligonucleotides used as competitive PCR primers are indicated in Table 1. The PCR was conducted at 94 C for 3 min and then 35 cycles at 94 C for 1 min, 54 C for 1 min, and 72 C for 1 min, with an additional incubation at 72 C for 10 min. PCR products were quantified by fluorescence of ethidium bromide on a 3UV Transilluminator (UVP, Inc., Upland, CA) and subsequent two-dimensional analysis of the gel image with an NIH Image software package. Intensity data so derived were subjected to quantitative analyses to calculate gonadotropin common , LHß, or FSHß mRNA concentrations as described previously (8). Each gonadotropin subunit mRNA level was expressed as the mRNA concentration on a unit weight basis (µg) of total RNA derived from the same pituitary sample.
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RIA of testosterone
Testosterone concentrations were measured by RIA after extraction of plasma samples by ether as described previously (24, 28), using rabbit antitestosterone serum (HAC-AA61-02-RBP81) (29) supplied by the Institute for Molecular and Cellular Regulation. The antiserum was specific for testosterone and 5-dihydrotestosterone; cross-reactivity was less than 0.001% with progesterone, 0.003% with 17ß-estradiol and dehydroepiandrosterone, and 0.04% with androstenedione.
In situ TUNEL
Apoptotic cell death in the testis of GnIH-administered mature birds was assessed by in situ TUNEL according to the manufacturer’s instruction (in situ cell death detection, POD; Roche Diagnostics, Mannheim, Germany). Negative control sections, processed without terminal deoxynucleotidyltransferase, did not show positive labeling. Apoptotic activity was quantified by counting the number of cells positive for TUNEL staining and expressed as the number of apoptotic germ or Sertoli cells per seminiferous tubule within each cross-section.
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Results |
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, LHß, and FSHß subunit mRNA levels were measured in the pituitary. Plasma LH concentrations were also measured. As shown in Fig. 1A, the expression of common mRNA on a unit per weight basis (micrograms) of total RNA decreased significantly (P < 0.01) in birds that received 3 µg GnIH per hour compared with control birds that received saline alone. GnIH also induced a significant decrease in the expression of LHß mRNA in a dose-dependent manner (P < 0.01, control vs. 3 µg GnIH per hour; P < 0.05, 0.03 µg vs. 3 µg GnIH per hour; Fig. 1B). GnIH appeared to decrease the FSHß mRNA expression, but the effect was not statistically significant because of the large variances among individual birds (Fig. 1C). In addition to reducing LHß mRNA expression, GnIH also decreased plasma LH concentration dose dependently (Fig. 1D; P < 0.01, control vs. 3 µg GnIH per hour; P < 0.05, control vs. 0.3 µg GnIH per hour).
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). Although GnIH administration for 2 wk failed to decrease testicular weight (Fig. 2B), GnIH induced testicular apoptosis (Figs. 3 and 4) and decreased the diameter of seminiferous tubules in birds that received 0.3 µg GnIH per hour (Figs. 3 and 4). Apoptotic activity in the testis was assessed by in situ TUNEL, and spermatogenic stages of germ cells were characterized based on their position within the seminiferous epithelium. Sertoli cells, which are somatic support cells within the seminiferous tubule, were identified from their morphology. GnIH administration increased apoptotic cell death, which was primarily observed in germ cells (spermatogonia or spermatocytes; arrows in Fig. 3B) and Sertoli cells (arrowhead in Fig. 3B). Significant increases in the numbers of TUNEL-positive germ cells (P < 0.05; Fig. 4A) and Sertoli cells (P < 0.05; Fig. 4B) per seminiferous tubule within a cross-section were detected. Spermatogenic activity in the testis was then analyzed by hematoxylin and eosin staining (Fig. 3, C and D). GnIH administration was also followed by a significant decrease in the diameter of seminiferous tubules within a cross-section (P < 0.01; Fig. 4C). Although the seminiferous epithelium of GnIH-administered birds contained all stages of germ cells including spermatozoa (Fig. 3D), the reduced seminiferous tubule diameter indicates the decrease in spermatogenic activity.
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). In contrast, GnIH significantly suppressed testicular growth compared with control birds that received saline alone (P < 0.01, control vs. 0.1 or 1 µg GnIH per day; P < 0.05, control vs. 0.01 µg GnIH per day; Fig. 5B). GnIH administration to immature birds also suppressed the rise in plasma testosterone concentration during development (P < 0.05, control vs. 0.1 or 1 µg GnIH per day; Fig. 5C). Subsequently, we analyzed spermatogenic activity in the testis of immature birds after GnIH administration (Fig. 6). Although spermatogonia and spermatocytes were observed in the seminiferous epithelium of immature birds after GnIH administration (Fig. 6B), GnIH significantly decreased the diameter of seminiferous tubules (P < 0.01, control vs. 0.01, 0.1, or 1 µg GnIH per day; Fig. 7A) and the number of germ cells per seminiferous tubule within a cross-section (P < 0.01, control vs. 0.1 or 1 µg GnIH per day; P < 0.05, control vs. 0.01 µg GnIH per day; Fig. 7B).
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). In contrast, administration of GnIH dose-dependently suppressed development of adult plumage (Fig. 8, B–D). In birds that received 1 µg GnIH per day, birds had mostly juvenile plumage, and adult plumage was very sparse (Fig. 8D).
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Discussion |
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The decrease in plasma LH concentrations after GnIH administration to mature male quail in vivo confirms and extends previous in vitro observations in quail (6) and chickens (11). In addition to the inhibition of LH release, administration of GnIH to mature male quail decreased the expression of gonadotropin common and LHß subunit mRNAs in the pituitary. The effect on FSHß subunit mRNA expression was not significant, but given the magnitude of the observed trend and the observed variance, sample size would need to have been increased only to 10 for the effect to be significant at the 0.05 level. In chickens, GnIH suppresses the expression of FSHß subunit mRNA in the pituitary in vitro (11). Thus, both in vivo and in vitro studies on birds taken together suggest that GnIH inhibits the synthesis and release of gonadotropins. We previously demonstrated the expression of a novel G protein-coupled receptor for GnIH in the quail pituitary (12). Therefore, GnIH, acting through GnIH receptor in the pituitary, exerts direct inhibitory effects on gonadotropin synthesis and release.
We hypothesized that GnIH may inhibit gonadal development and maintenance through the decrease in gonadotropin synthesis and release. Our hypothesis was confirmed by the results of the present in vivo study. GnIH administration to mature male quail for 2 wk decreased plasma testosterone concentration in a dose-dependent manner. Because LH stimulates the synthesis and release of testosterone in the Leydig cells of birds (19, 30, 31), the decrease in circulating testosterone after GnIH administration is likely to be a result of the decrease in LH synthesis and release. Furthermore, GnIH administration induced testicular apoptosis in mature male quail. Apoptotic cell death was detected in spermatocytes, spermatogonia, and Sertoli cells, the same cell types that undergo apoptosis in the testis of starlings during seasonal testicular regression (21). It is considered that in starlings the decrease in circulating testosterone is the main cause of testicular apoptosis (20, 21). Testosterone is known as a testicular cell-survival factor in rodents (32, 33, 34). It is likely that GnIH decreases testicular testosterone and consequently induces apoptotic cell death in the testis. The decrease in the survival of germ and Sertoli cells would account for the observed reduction in seminiferous tubule diameter. We recently demonstrated that the expression of GnIH in the hypothalamus increases at the onset of testicular regression in adult quail exposed to short-day photoperiod (13). Therefore, the increase in GnIH action may be one of the main causes of gonadal regression in birds.
In this study, we also investigated the inhibitory effect of GnIH on gonadal development using immature male quail. Previous findings indicated that circulating gonadotropin concentrations are negatively correlated with the GnIH content in the hypothalamus during quail development (8). In other words, GnIH decreases as gonadotropins increase during development. Based on this finding (8), we hypothesized that the decrease in hypothalamic GnIH content may be involved in the rapid increases in the plasma testosterone concentration and testicular growth observed during sexual development (8). In accordance with the hypothesis, administration of GnIH to immature male quail suppressed the normal rise in plasma testosterone concentrations. The growth of seminiferous tubules and proliferation of germ cells during development were also suppressed by GnIH administration. Because the rise in circulating testosterone is required for testicular development in quail (23, 35, 36, 37), the suppression of testosterone after GnIH administration may cause the decrease in spermatogenic activity. GnIH further inhibited the transition from juvenile plumage into adult plumage during development. This phenomenon might also be a result of suppressed plasma testosterone; gonadal steroids are known to maintain adult plumage in several birds (38, 39).
The neuropeptide control of gonadotropin secretion in birds, as in other vertebrates, is primarily through the stimulatory action of the hypothalamic decapeptide GnRH (3, 4, 5). Based on studies of different forms of GnRH and its receptors, the form of GnRH that controls reproductive function in birds is considered to be chicken GnRH-1 (40). However, gonadal regression caused by a decrease in photoperiod is not associated with a decrease in hypothalamic GnRH-1 in quail (41, 42). Thus, up until now we have had an unexplained paradox: gonads exhibiting regression in the presence of large amounts of chicken GnRH-1. The present study provides findings that GnIH, a newly discovered hypothalamic dodecapeptide, is involved in the regulation of testicular development and maintenance. In addition, GnIH inhibits the developmental transition from juvenile to adult plumage, indicating that GnIH not only has direct effects upon the reproductive axis but also has indirect effects upon secondary sexual characteristics. Thus, GnIH is an important factor for avian reproduction. Our data indicate that GnIH acts directly on the pituitary through GnIH receptor, to cause a decrease in gonadotropin synthesis and release. In consequence, testicular development and maintenance are inhibited. To our knowledge, GnIH is the first identified hypothalamic neuropeptide inhibiting reproductive function in any vertebrate class. Despite the compelling nature of our data, we cannot preclude a direct action of GnIH on the testis. However, the direct inhibition of LH in vitro and the hypothalamic localization of GnIH suggest its effects are through direct gonadotroph inhibition, but it is also possible that GnIH might act by inhibiting GnRH secretion. Histological evidence is suggestive of functional connectivity between GnIH-immunoreactive fibers and GnRH-I and -II cell bodies and fiber terminals (43). These possibilities will be the subjects of further study.
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Acknowledgments |
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Footnotes |
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Present address for T.U.: Department of Integrative Biology, University of California, Berkeley, California 94720.
T.U., K.U., P.J.S., G.E.B., and K.T. have nothing to declare.
First Published Online November 23, 2005
Abbreviations: GnIH, Gonadotropin-inhibitory hormone; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling.
Received September 14, 2005.
Accepted for publication November 9, 2005.
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and follicle-stimulating hormone ß subunit expression in the pituitary of the domestic chicken. J Neuroendocrinol 16:999–1006[CrossRef][Medline]This article has been cited by other articles:
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, P < 0.05, 0.03 µg vs. 3 µg GnIH per hour by one-way ANOVA, followed by Duncan’s multiple range test.
