Gonadotropin-Inhibitory Hormone Inhibits Gonadal Development and Maintenance by Decreasing Gonadotropin Synthesis and Release in M
http://www.100md.com
《内分泌学杂志》
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
Department of Integrative Biology (G.E.B.), University of California, Berkeley, California 94720
Abstract
Until recently, any neuropeptide that directly inhibits gonadotropin secretion had not been identified. We recently identified a novel hypothalamic dodecapeptide that directly inhibits gonadotropin release in quail and termed it gonadotropin-inhibitory hormone (GnIH). The action of GnIH on the inhibition of gonadotropin release is mediated by a novel G protein-coupled receptor in the quail pituitary. This new gonadotropin inhibitory system is considered to be a widespread property of birds and provides us with an unprecedented opportunity to study the regulation of avian reproduction from an entirely novel standpoint. To understand the physiological role(s) of GnIH in avian reproduction, we investigated GnIH actions on gonadal development and maintenance in male quail. Continuous administration of GnIH to mature birds via osmotic pumps for 2 wk decreased the expressions of gonadotropin common and LH 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
THE DECAPEPTIDE GnRH is the primary factor responsible for hypothalamic control of gonadotropin secretion. GnRH was originally isolated from mammals (1, 2) and subsequently from birds (3, 4, 5) and other vertebrates. Gonadal sex steroids and inhibin can also modulate gonadotropin secretion via feedback from the gonads, but a neuropeptide inhibitor for gonadotropin secretion was, until recently, unknown in vertebrates. We recently identified a novel hypothalamic dodecapeptide (SIKPSAYLPLRF-NH2) that directly inhibits gonadotropin release from the cultured quail anterior pituitary and termed it gonadotropin-inhibitory hormone (GnIH) (6). This was the first demonstration of a hypothalamic neuropeptide inhibiting gonadotropin release in any vertebrate. GnIH is located in neurons of the paraventricular nucleus. These neurons project to the median eminence, thus providing the correct neuroanatomical infrastructure to control anterior pituitary function (6, 7, 8). We also cloned a cDNA encoding the GnIH precursor polypeptide in the quail brain (9). The GnIH precursor mRNA was expressed only in the paraventricular nucleus (7, 8, 9). Subsequently, we identified a cDNA encoding GnIH in the brain of the white-crowned sparrow (10). Both quail and sparrow GnIH inhibited gonadotropin release in vivo (10). Addition of GnIH to cultured chicken pituitary also depressed gonadotropin release and synthesis (11). Thus, the inhibitory action of GnIH on gonadotropin secretion occurs in at least two orders of birds and thus may be an evolutionarily conserved property.
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 , LH, and FSH 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.
Materials and Methods
Animals
Mature and immature male Japanese quail, Coturnix japonica, were used in this study. Newly hatched quail were housed in a temperature-controlled room (35 ± 2 C) under daily photoperiods of 16-h light and 8-h dark (lights on at 0700 h) during the first week of brooding. The temperature was gradually reduced at the rate of 3.5 C per week until 4 wk of age when quail were isolated in individual cages. All birds were given quail food and tap water freely, and the experimental protocol was approved in accordance with guidelines prepared by Hiroshima University (Higashi-Hiroshima, Japan).
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 , LH, and FSH 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 , LH, and FSH
To quantify mRNAs encoding the gonadotropin subunits (common , LH, and FSH) 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 , LH, and FSH 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 , LH, and FSH subunits (underlined in Table 1) were primed to native fragments of the -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 LH, 0.1–10 fmol/tube; FSH, 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 , LH, or FSH 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.
RIA of LH
A highly purified chicken LH (CANOMS-12442B) (22) was radioiodinated by our previous method (23). The RIA was performed as described previously (24, 25), using rabbit antichicken LH serum (HAC-CH27–01-RBP75) (26) supplied by the Institute for Molecular and Cellular Regulation (Gunma University, Maebashi, Japan). The antiserum cross-reacted with quail LH (22, 26). Chicken LH preparation (AGC112B) (27) was used as the standard (22), and LH concentrations were expressed in nanograms of chicken LH (AGC112B) per milliliter of plasma. The purity of AGC112B was approximately a third of the most purified chicken LH fraction (CANOMS-12442B) (22).
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 17-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.
Results
GnIH actions on gonadotropin synthesis and release in the adult
After GnIH administration to mature birds via osmotic pumps for 2 wk, gonadotropin common , LH, and FSH 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 LH 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 FSH mRNA expression, but the effect was not statistically significant because of the large variances among individual birds (Fig. 1C). In addition to reducing LH 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).
GnIH actions on plasma testosterone concentration, testicular apoptosis, and spermatogenic activity in the adult
To understand the physiological role of GnIH in testicular maintenance, we investigated circulating testosterone concentration, testicular apoptosis, and spermatogenic activity after GnIH administration to mature birds via osmotic pump for 2 wk. GnIH decreased plasma testosterone concentration in a dose-dependent manner (P < 0.05, control vs. 0.3 or 3 μg GnIH per hour; P < 0.05, 0.03 μg vs. 3 μg GnIH per hour; Fig. 2A). 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.
GnIH actions on testicular growth, spermatogenic activity, and plasma testosterone concentration during development
To analyze the physiological role of GnIH in testicular development, GnIH was injected sc to immature birds from 8–20 d of age. No significant change in body weight was detected after GnIH administration (Fig. 5A). 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).
GnIH action on the molt of juvenile plumage into adult plumage during development
An external indicator of sexual maturity in birds is their plumage. Therefore we analyzed the effect of GnIH on the molt of juvenile plumage. GnIH was injected sc to immature birds from 8–20 d of age. All immature birds at the beginning of GnIH administration had yellowish juvenile plumage (data not shown). At the end of GnIH administration, all control birds had completed molt of juvenile plumage into brownish and white adult plumage (Fig. 8A). 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).
Discussion
GnIH is the first and only identified hypothalamic neuropeptide that directly inhibits gonadotropin release in any vertebrate (6). The inhibitory action of GnIH on gonadotropin release is considered to be a widespread property in birds (6, 10, 11) and mammals (16). To understand the physiological role of GnIH in avian reproduction, we used male quail to characterize the actions of GnIH not only on gonadotropin synthesis and release but also on measures of gonadal activity, including circulating testosterone, spermatogenic activity, and testicular apoptosis. The present study provides evidence for GnIH’s inhibitory effects on testicular development and maintenance. Our data also indicate that these inhibitory actions of GnIH are mediated by the decrease in gonadotropin synthesis and release. Thus, GnIH appears to act as an important factor on avian reproduction. As already stated, other gonadotropin inhibitory properties of GnIH may be conserved in mammals. Thus, our findings are likely to have implications for vertebrates in general.
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 LH subunit mRNAs in the pituitary. The effect on FSH 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 FSH 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.
Acknowledgments
We thank the Institute for Molecular and Cellular Regulation for the supply of the antisera raised against avian LH and testosterone. We also thank Drs. M. Wada and A. Hattori for their technical assistance.
Footnotes
This work was supported in part by grants-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science, and Technology (Tokyo).
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.
Accepted for publication November 9, 2005.
References
Matsuo H, Baba Y, Nair RM, Arimura A, Schally AV 1971 Structure of the porcine LH- and FSH-releasing hormone. I. The proposed amino acid sequence. Biochem Biophys Res Commun 43:1334–1339
Burgus R, Butcher M, Amoss M, Ling N, Monahan M, Rivier J, Fellows R, Blackwell R, Vale W, Guillemin R 1972 Primary structure of the ovine hypothalamic luteinizing hormone-releasing factor (LRF) (LH- hypothalamus-LRF-gas chromatography-mass spectrometry-decapeptide-Edman degradation). Proc Natl Acad Sci USA 69:278–282
King JA, Millar RP 1982 Structure of chicken hypothalamic luteinizing hormone-releasing hormone. I. Structural determination on partially purified material. J Biol Chem 257:10722–10728
Miyamoto K, Hasegawa Y, Minegishi T, Nomura M, Takahashi Y, Igarashi M, Kangawa K, Matsuo H 1982 Isolation and characterization of chicken hypothalamic luteinizing hormone-releasing hormone. Biochem Biophys Res Commun 107:820–827
Miyamoto K, Hasegawa Y, Nomura M, Igarashi M, Kangawa K, Matsuo H 1984 Identification of the second gonadotropin-releasing hormone in chicken hypothalamus: evidence that gonadotropin secretion is probably controlled by two distinct gonadotropin-releasing hormones in avian species. Proc Natl Acad Sci USA 81:3874–3878
Tsutsui K, Saigoh E, Ukena K, Teranishi H, Fujisawa Y, Kikuchi M, Ishii S, Sharp PJ 2000 A novel avian hypothalamic peptide inhibiting gonadotropin release. Biochem Biophys Res Commun 275:661–667
Ukena K, Ubuka T, Tsutsui K 2003 Distribution of a novel avian gonadotropin-inhibitory hormone in the quail brain. Cell Tissue Res 312:73–79
Ubuka T, Ueno M, Ukena K, Tsutsui K 2003 Developmental changes in gonadotropin-inhibitory hormone in the Japanese quail (Coturnix japonica) hypothalamo-hypophysial system. J Endocrinol 178:311–318
Satake H, Hisada M, Kawada T, Minakata H, Ukena K, Tsutsui K 2001 Characterization of a cDNA encoding a novel avian hypothalamic neuropeptide exerting an inhibitory effect on gonadotropin release. Biochem J 354:379–385
Osugi T, Ukena K, Bentley GE, O’Brien S, Moore IT, Wingfield JC, Tsutsui K 2004 Gonadotropin-inhibitory hormone in Gambel’s white-crowned sparrow (Zonotrichia leucophrys gambelii): cDNA identification, transcript localization and functional effects in laboratory and field experiments. J Endocrinol 182:33–42
Ciccone NA, Dunn IC, Boswell T, Tsutsui K, Ubuka T, Ukena K, Sharp PJ 2004 Gonadotrophin inhibitory hormone depresses gonadotrophin and follicle-stimulating hormone subunit expression in the pituitary of the domestic chicken. J Neuroendocrinol 16:999–1006
Yin H, Ukena K, Ubuka T, Tsutsui K 2005 A novel G protein-coupled receptor for gonadotropin-inhibitory hormone in the Japanese quail (Coturnix japonica): identification, expression and binding activity. J Endocrinol 184:257–266
Ubuka T, Bentley GE, Ukena K, Wingfield JC, Tsutsui K 2005 Melatonin induces the expression of gonadotropin-inhibitory hormone in the avian brain. Proc Natl Acad Sci USA 102:3052–3057
Ukena K, Tsutsui K 2005 A new member of the hypothalamic RF-amide peptide family, LPXRF-amide peptides: structure, localization, and function. Mass Spectrom Rev 24:469–486
Ikemoto T, Park MK 2005 Chicken RFamide-related peptide (GnIH) and two distinct receptor subtypes: identification, molecular characterization, and evolutionary considerations. J Reprod Dev 51:359–377
Bentley GE, Kriegsfeld LJ, Osugi T, Ukena K, O’Brien S, Perfito N, Moore I, Tsutsui K, Wingfield JC, Interactions of gonadotropin-releasing hormone (GnRH) and gonadotropin-inhibitory hormone (GnIH) in birds and mammals. J Exp Zool, in press
Johnson AL 1986 Reproduction in the male. In: Sturkie PD, ed. Avian physiology. 4th ed. New York: Springer-Verlag; 432–451
Follett BK 1984 Birds. In: Lamming GE, ed. Marshall’s physiology of reproduction. 4th ed. New York: Churchill-Livingstone; 283–350
Brown NL, Bayle JD, Scanes CG, Follett BK 1975 Chicken gonadotrophins: their effects on the testes of immature and hypophysectomized Japanese quail. Cell Tissue Res 156:499–520
Young KA, Nelson RJ 2001 Mediation of seasonal testicular regression by apoptosis. Reproduction 122:677–685
Young KA, Ball GF Nelson RJ 2001 Photoperiod-induced testicular apoptosis in European starlings (Sturnus vulgaris). Biol Reprod 64:706–713
Kikuchi M, Ishii S 1989 Radioiodination of chicken luteinizing hormone without affecting receptor binding potency. Biol Reprod 41:1047–1054
Tsutsui K, Ishii S 1980 Hormonal regulations of follicle-stimulating hormone receptors in the tests of Japanese quail. J Endocrinol 85:511–518
Tsutsui K 1991 Pituitary and gonadal hormone-dependent and -independent induction of follicle-stimulating hormone receptors in the developing testis. Endocrinology 128:477–487
Tsutsui K, Kawashima S, Saxena RN, Ishii S 1992 Annual changes in the binding of follicle-stimulating hormone to gonads and plasma gonadotropin concentrations in Indian weaver birds inhabiting the subtropical zone. Gen Comp Endocrinol 88:444–453
Hattori M, Wakabayashi K 1979 Isoelectric focusing and gel filtration studies on the heterogeneity of avian pituitary luteinizing hormone. Gen Comp Endocrinol 39:215–221
Sakai H, Ishii S 1980 Isolation and characterization of chicken follicle-stimulating hormone. Gen Comp Endocrinol 42:1–8
Tsutsui K, Kawashima S, Masuda A, Oishi T 1988 Effects of photoperiod and temperature on the binding of follicle-stimulating hormone (FSH) to testicular preparations and plasma FSH concentration in the Djungarian hamster, Phodopus sungorus. Endocrinology 122:1094–1102
Kano Y, Nakano T, Kumakura M, Wasa T, Suzuki M, Yamauchi K, Tanaka S 2005 Seasonal expression of LH and FSH in the male newt pituitary gonadotrophs. Gen Comp Endocrinol 141:248–258
Maung ZW, Follett BK 1977 Effects of chicken and ovine luteinizing hormone on androgen release and cyclic AMP production by isolated cells from the quail testis. Gen Comp Endocrinol 33:242–253
Maung SL, Follett BK 1978 The endocrine control by luteinizing hormone of testosterone secretion from the testis of the Japanese quail. Gen Comp Endocrinol 36:79–89
Tapanainen JS, Tilly JL, Vihko KK, Hsueh AJ 1993 Hormonal control of apoptotic cell death in the testis: gonadotropins and androgens as testicular cell survival factors. Mol Endocrinol 7:643–650
Woolveridge I, de Boer-Brouwer M, Taylor MF, Teerds KJ, Wu FC, Morris ID 1999 Apoptosis in the rat spermatogenic epithelium following androgen withdrawal: changes in apoptosis-related genes. Biol Reprod 60:461–470
Nandi S, Banerjee PP, Zirkin BR 1999 Germ cell apoptosis in the testes of Sprague Dawley rats following testosterone withdrawal by ethane 1,2-dimethanesulfonate administration: relationship to Fas Biol Reprod 61:70–75
Ottinger MA, Bakst MR 1981 Peripheral androgen concentrations and testicular morphology in embryonic and young male Japanese quail. Gen Comp Endocrinol 43:170–177
Brown NL, Follett BK 1977 Effects of androgens on the testes of intact and hypophysectomized Japanese quail. Gen Comp Endocrinol 33:267–277
Tsutsui K, Ishii S 1978 Effects of follicle-stimulating hormone and testosterone on receptors of follicle-stimulating hormone in the testis of the immature Japanese quail. Gen Comp Endocrinol 36:297–305
Payne RB 1972 Mechanism and control of molt. In: Farner DS, King JR, eds. Avian biology. New York: Academic Press; 118–131
Tanabe Y, Himeno K, Nozaki H 1957 Thyroid and ovarian function in relation to molting in the hen. Endocrinology 61:661–666
Sharp PJ, Talbot RT, Main GM, Dunn IC, Fraser HM, Huskisson NS 1990 Physiological roles of chicken LHRH-I and -II in the control of gonadotrophin release in the domestic chicken. J Endocrinol 124:291–299
Foster RG, Panzica GC, Parry DM, Viglietti-Panzica C 1988 Immunocytochemical studies on the LHRH system of the Japanese quail: influence by photoperiod and aspects of sexual differentiation. Cell Tissue Res 253:327–335
Dawson A, King VM, Bentley GE, Ball GF 2001 Photoperiodic control of seasonality in birds. J Biol Rhythms 16:365–380
Bentley GE, Perfito N, Ukena K, Tsutsui K, Wingfield JC 2003 Gonadotropin-inhibitory peptide in song sparrows (Melospiza melodia) in different reproductive conditions, and in house sparrows (Passer domesticus) relative to chicken-gonadotropin-releasing hormone. J Neuroendocrinol 15:794–802(Takayoshi Ubuka, Kazuyoshi Ukena, Peter )
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
Department of Integrative Biology (G.E.B.), University of California, Berkeley, California 94720
Abstract
Until recently, any neuropeptide that directly inhibits gonadotropin secretion had not been identified. We recently identified a novel hypothalamic dodecapeptide that directly inhibits gonadotropin release in quail and termed it gonadotropin-inhibitory hormone (GnIH). The action of GnIH on the inhibition of gonadotropin release is mediated by a novel G protein-coupled receptor in the quail pituitary. This new gonadotropin inhibitory system is considered to be a widespread property of birds and provides us with an unprecedented opportunity to study the regulation of avian reproduction from an entirely novel standpoint. To understand the physiological role(s) of GnIH in avian reproduction, we investigated GnIH actions on gonadal development and maintenance in male quail. Continuous administration of GnIH to mature birds via osmotic pumps for 2 wk decreased the expressions of gonadotropin common and LH 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
THE DECAPEPTIDE GnRH is the primary factor responsible for hypothalamic control of gonadotropin secretion. GnRH was originally isolated from mammals (1, 2) and subsequently from birds (3, 4, 5) and other vertebrates. Gonadal sex steroids and inhibin can also modulate gonadotropin secretion via feedback from the gonads, but a neuropeptide inhibitor for gonadotropin secretion was, until recently, unknown in vertebrates. We recently identified a novel hypothalamic dodecapeptide (SIKPSAYLPLRF-NH2) that directly inhibits gonadotropin release from the cultured quail anterior pituitary and termed it gonadotropin-inhibitory hormone (GnIH) (6). This was the first demonstration of a hypothalamic neuropeptide inhibiting gonadotropin release in any vertebrate. GnIH is located in neurons of the paraventricular nucleus. These neurons project to the median eminence, thus providing the correct neuroanatomical infrastructure to control anterior pituitary function (6, 7, 8). We also cloned a cDNA encoding the GnIH precursor polypeptide in the quail brain (9). The GnIH precursor mRNA was expressed only in the paraventricular nucleus (7, 8, 9). Subsequently, we identified a cDNA encoding GnIH in the brain of the white-crowned sparrow (10). Both quail and sparrow GnIH inhibited gonadotropin release in vivo (10). Addition of GnIH to cultured chicken pituitary also depressed gonadotropin release and synthesis (11). Thus, the inhibitory action of GnIH on gonadotropin secretion occurs in at least two orders of birds and thus may be an evolutionarily conserved property.
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 , LH, and FSH 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.
Materials and Methods
Animals
Mature and immature male Japanese quail, Coturnix japonica, were used in this study. Newly hatched quail were housed in a temperature-controlled room (35 ± 2 C) under daily photoperiods of 16-h light and 8-h dark (lights on at 0700 h) during the first week of brooding. The temperature was gradually reduced at the rate of 3.5 C per week until 4 wk of age when quail were isolated in individual cages. All birds were given quail food and tap water freely, and the experimental protocol was approved in accordance with guidelines prepared by Hiroshima University (Higashi-Hiroshima, Japan).
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 , LH, and FSH 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 , LH, and FSH
To quantify mRNAs encoding the gonadotropin subunits (common , LH, and FSH) 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 , LH, and FSH 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 , LH, and FSH subunits (underlined in Table 1) were primed to native fragments of the -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 LH, 0.1–10 fmol/tube; FSH, 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 , LH, or FSH 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.
RIA of LH
A highly purified chicken LH (CANOMS-12442B) (22) was radioiodinated by our previous method (23). The RIA was performed as described previously (24, 25), using rabbit antichicken LH serum (HAC-CH27–01-RBP75) (26) supplied by the Institute for Molecular and Cellular Regulation (Gunma University, Maebashi, Japan). The antiserum cross-reacted with quail LH (22, 26). Chicken LH preparation (AGC112B) (27) was used as the standard (22), and LH concentrations were expressed in nanograms of chicken LH (AGC112B) per milliliter of plasma. The purity of AGC112B was approximately a third of the most purified chicken LH fraction (CANOMS-12442B) (22).
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 17-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.
Results
GnIH actions on gonadotropin synthesis and release in the adult
After GnIH administration to mature birds via osmotic pumps for 2 wk, gonadotropin common , LH, and FSH 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 LH 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 FSH mRNA expression, but the effect was not statistically significant because of the large variances among individual birds (Fig. 1C). In addition to reducing LH 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).
GnIH actions on plasma testosterone concentration, testicular apoptosis, and spermatogenic activity in the adult
To understand the physiological role of GnIH in testicular maintenance, we investigated circulating testosterone concentration, testicular apoptosis, and spermatogenic activity after GnIH administration to mature birds via osmotic pump for 2 wk. GnIH decreased plasma testosterone concentration in a dose-dependent manner (P < 0.05, control vs. 0.3 or 3 μg GnIH per hour; P < 0.05, 0.03 μg vs. 3 μg GnIH per hour; Fig. 2A). 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.
GnIH actions on testicular growth, spermatogenic activity, and plasma testosterone concentration during development
To analyze the physiological role of GnIH in testicular development, GnIH was injected sc to immature birds from 8–20 d of age. No significant change in body weight was detected after GnIH administration (Fig. 5A). 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).
GnIH action on the molt of juvenile plumage into adult plumage during development
An external indicator of sexual maturity in birds is their plumage. Therefore we analyzed the effect of GnIH on the molt of juvenile plumage. GnIH was injected sc to immature birds from 8–20 d of age. All immature birds at the beginning of GnIH administration had yellowish juvenile plumage (data not shown). At the end of GnIH administration, all control birds had completed molt of juvenile plumage into brownish and white adult plumage (Fig. 8A). 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).
Discussion
GnIH is the first and only identified hypothalamic neuropeptide that directly inhibits gonadotropin release in any vertebrate (6). The inhibitory action of GnIH on gonadotropin release is considered to be a widespread property in birds (6, 10, 11) and mammals (16). To understand the physiological role of GnIH in avian reproduction, we used male quail to characterize the actions of GnIH not only on gonadotropin synthesis and release but also on measures of gonadal activity, including circulating testosterone, spermatogenic activity, and testicular apoptosis. The present study provides evidence for GnIH’s inhibitory effects on testicular development and maintenance. Our data also indicate that these inhibitory actions of GnIH are mediated by the decrease in gonadotropin synthesis and release. Thus, GnIH appears to act as an important factor on avian reproduction. As already stated, other gonadotropin inhibitory properties of GnIH may be conserved in mammals. Thus, our findings are likely to have implications for vertebrates in general.
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 LH subunit mRNAs in the pituitary. The effect on FSH 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 FSH 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.
Acknowledgments
We thank the Institute for Molecular and Cellular Regulation for the supply of the antisera raised against avian LH and testosterone. We also thank Drs. M. Wada and A. Hattori for their technical assistance.
Footnotes
This work was supported in part by grants-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science, and Technology (Tokyo).
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.
Accepted for publication November 9, 2005.
References
Matsuo H, Baba Y, Nair RM, Arimura A, Schally AV 1971 Structure of the porcine LH- and FSH-releasing hormone. I. The proposed amino acid sequence. Biochem Biophys Res Commun 43:1334–1339
Burgus R, Butcher M, Amoss M, Ling N, Monahan M, Rivier J, Fellows R, Blackwell R, Vale W, Guillemin R 1972 Primary structure of the ovine hypothalamic luteinizing hormone-releasing factor (LRF) (LH- hypothalamus-LRF-gas chromatography-mass spectrometry-decapeptide-Edman degradation). Proc Natl Acad Sci USA 69:278–282
King JA, Millar RP 1982 Structure of chicken hypothalamic luteinizing hormone-releasing hormone. I. Structural determination on partially purified material. J Biol Chem 257:10722–10728
Miyamoto K, Hasegawa Y, Minegishi T, Nomura M, Takahashi Y, Igarashi M, Kangawa K, Matsuo H 1982 Isolation and characterization of chicken hypothalamic luteinizing hormone-releasing hormone. Biochem Biophys Res Commun 107:820–827
Miyamoto K, Hasegawa Y, Nomura M, Igarashi M, Kangawa K, Matsuo H 1984 Identification of the second gonadotropin-releasing hormone in chicken hypothalamus: evidence that gonadotropin secretion is probably controlled by two distinct gonadotropin-releasing hormones in avian species. Proc Natl Acad Sci USA 81:3874–3878
Tsutsui K, Saigoh E, Ukena K, Teranishi H, Fujisawa Y, Kikuchi M, Ishii S, Sharp PJ 2000 A novel avian hypothalamic peptide inhibiting gonadotropin release. Biochem Biophys Res Commun 275:661–667
Ukena K, Ubuka T, Tsutsui K 2003 Distribution of a novel avian gonadotropin-inhibitory hormone in the quail brain. Cell Tissue Res 312:73–79
Ubuka T, Ueno M, Ukena K, Tsutsui K 2003 Developmental changes in gonadotropin-inhibitory hormone in the Japanese quail (Coturnix japonica) hypothalamo-hypophysial system. J Endocrinol 178:311–318
Satake H, Hisada M, Kawada T, Minakata H, Ukena K, Tsutsui K 2001 Characterization of a cDNA encoding a novel avian hypothalamic neuropeptide exerting an inhibitory effect on gonadotropin release. Biochem J 354:379–385
Osugi T, Ukena K, Bentley GE, O’Brien S, Moore IT, Wingfield JC, Tsutsui K 2004 Gonadotropin-inhibitory hormone in Gambel’s white-crowned sparrow (Zonotrichia leucophrys gambelii): cDNA identification, transcript localization and functional effects in laboratory and field experiments. J Endocrinol 182:33–42
Ciccone NA, Dunn IC, Boswell T, Tsutsui K, Ubuka T, Ukena K, Sharp PJ 2004 Gonadotrophin inhibitory hormone depresses gonadotrophin and follicle-stimulating hormone subunit expression in the pituitary of the domestic chicken. J Neuroendocrinol 16:999–1006
Yin H, Ukena K, Ubuka T, Tsutsui K 2005 A novel G protein-coupled receptor for gonadotropin-inhibitory hormone in the Japanese quail (Coturnix japonica): identification, expression and binding activity. J Endocrinol 184:257–266
Ubuka T, Bentley GE, Ukena K, Wingfield JC, Tsutsui K 2005 Melatonin induces the expression of gonadotropin-inhibitory hormone in the avian brain. Proc Natl Acad Sci USA 102:3052–3057
Ukena K, Tsutsui K 2005 A new member of the hypothalamic RF-amide peptide family, LPXRF-amide peptides: structure, localization, and function. Mass Spectrom Rev 24:469–486
Ikemoto T, Park MK 2005 Chicken RFamide-related peptide (GnIH) and two distinct receptor subtypes: identification, molecular characterization, and evolutionary considerations. J Reprod Dev 51:359–377
Bentley GE, Kriegsfeld LJ, Osugi T, Ukena K, O’Brien S, Perfito N, Moore I, Tsutsui K, Wingfield JC, Interactions of gonadotropin-releasing hormone (GnRH) and gonadotropin-inhibitory hormone (GnIH) in birds and mammals. J Exp Zool, in press
Johnson AL 1986 Reproduction in the male. In: Sturkie PD, ed. Avian physiology. 4th ed. New York: Springer-Verlag; 432–451
Follett BK 1984 Birds. In: Lamming GE, ed. Marshall’s physiology of reproduction. 4th ed. New York: Churchill-Livingstone; 283–350
Brown NL, Bayle JD, Scanes CG, Follett BK 1975 Chicken gonadotrophins: their effects on the testes of immature and hypophysectomized Japanese quail. Cell Tissue Res 156:499–520
Young KA, Nelson RJ 2001 Mediation of seasonal testicular regression by apoptosis. Reproduction 122:677–685
Young KA, Ball GF Nelson RJ 2001 Photoperiod-induced testicular apoptosis in European starlings (Sturnus vulgaris). Biol Reprod 64:706–713
Kikuchi M, Ishii S 1989 Radioiodination of chicken luteinizing hormone without affecting receptor binding potency. Biol Reprod 41:1047–1054
Tsutsui K, Ishii S 1980 Hormonal regulations of follicle-stimulating hormone receptors in the tests of Japanese quail. J Endocrinol 85:511–518
Tsutsui K 1991 Pituitary and gonadal hormone-dependent and -independent induction of follicle-stimulating hormone receptors in the developing testis. Endocrinology 128:477–487
Tsutsui K, Kawashima S, Saxena RN, Ishii S 1992 Annual changes in the binding of follicle-stimulating hormone to gonads and plasma gonadotropin concentrations in Indian weaver birds inhabiting the subtropical zone. Gen Comp Endocrinol 88:444–453
Hattori M, Wakabayashi K 1979 Isoelectric focusing and gel filtration studies on the heterogeneity of avian pituitary luteinizing hormone. Gen Comp Endocrinol 39:215–221
Sakai H, Ishii S 1980 Isolation and characterization of chicken follicle-stimulating hormone. Gen Comp Endocrinol 42:1–8
Tsutsui K, Kawashima S, Masuda A, Oishi T 1988 Effects of photoperiod and temperature on the binding of follicle-stimulating hormone (FSH) to testicular preparations and plasma FSH concentration in the Djungarian hamster, Phodopus sungorus. Endocrinology 122:1094–1102
Kano Y, Nakano T, Kumakura M, Wasa T, Suzuki M, Yamauchi K, Tanaka S 2005 Seasonal expression of LH and FSH in the male newt pituitary gonadotrophs. Gen Comp Endocrinol 141:248–258
Maung ZW, Follett BK 1977 Effects of chicken and ovine luteinizing hormone on androgen release and cyclic AMP production by isolated cells from the quail testis. Gen Comp Endocrinol 33:242–253
Maung SL, Follett BK 1978 The endocrine control by luteinizing hormone of testosterone secretion from the testis of the Japanese quail. Gen Comp Endocrinol 36:79–89
Tapanainen JS, Tilly JL, Vihko KK, Hsueh AJ 1993 Hormonal control of apoptotic cell death in the testis: gonadotropins and androgens as testicular cell survival factors. Mol Endocrinol 7:643–650
Woolveridge I, de Boer-Brouwer M, Taylor MF, Teerds KJ, Wu FC, Morris ID 1999 Apoptosis in the rat spermatogenic epithelium following androgen withdrawal: changes in apoptosis-related genes. Biol Reprod 60:461–470
Nandi S, Banerjee PP, Zirkin BR 1999 Germ cell apoptosis in the testes of Sprague Dawley rats following testosterone withdrawal by ethane 1,2-dimethanesulfonate administration: relationship to Fas Biol Reprod 61:70–75
Ottinger MA, Bakst MR 1981 Peripheral androgen concentrations and testicular morphology in embryonic and young male Japanese quail. Gen Comp Endocrinol 43:170–177
Brown NL, Follett BK 1977 Effects of androgens on the testes of intact and hypophysectomized Japanese quail. Gen Comp Endocrinol 33:267–277
Tsutsui K, Ishii S 1978 Effects of follicle-stimulating hormone and testosterone on receptors of follicle-stimulating hormone in the testis of the immature Japanese quail. Gen Comp Endocrinol 36:297–305
Payne RB 1972 Mechanism and control of molt. In: Farner DS, King JR, eds. Avian biology. New York: Academic Press; 118–131
Tanabe Y, Himeno K, Nozaki H 1957 Thyroid and ovarian function in relation to molting in the hen. Endocrinology 61:661–666
Sharp PJ, Talbot RT, Main GM, Dunn IC, Fraser HM, Huskisson NS 1990 Physiological roles of chicken LHRH-I and -II in the control of gonadotrophin release in the domestic chicken. J Endocrinol 124:291–299
Foster RG, Panzica GC, Parry DM, Viglietti-Panzica C 1988 Immunocytochemical studies on the LHRH system of the Japanese quail: influence by photoperiod and aspects of sexual differentiation. Cell Tissue Res 253:327–335
Dawson A, King VM, Bentley GE, Ball GF 2001 Photoperiodic control of seasonality in birds. J Biol Rhythms 16:365–380
Bentley GE, Perfito N, Ukena K, Tsutsui K, Wingfield JC 2003 Gonadotropin-inhibitory peptide in song sparrows (Melospiza melodia) in different reproductive conditions, and in house sparrows (Passer domesticus) relative to chicken-gonadotropin-releasing hormone. J Neuroendocrinol 15:794–802(Takayoshi Ubuka, Kazuyoshi Ukena, Peter )