Neural Versus Gonadal GnIH: Are they Independent Systems? A Mini-Review.

Based on research in protochordates and basal vertebrates, we know that communication across the first endocrine axes likely relied on diffusion. Because diffusion is relatively slow, rapid responses to some cues, including stress-related cues, may have required further local control of axis outputs (e.g., steroid hormone production by the gonads). Despite the evolution of much more efficient circulatory systems and complex nervous systems in vertebrates, production of many "neuro"transmitters has been identified outside of the hypothalamus across the vertebrate phylogeny and these neurotransmitters are known to locally regulate endocrine function. Our understanding of tissue-specific neuropeptide expression and their role coordinating physiological/behavioral responses of the whole organism remains limited, in part, due to nomenclature and historic dogma that ignores local regulation of axis output. Here, we review regulation of gonadotropin-inhibitory hormone (GnIH) across the reproductive axis in birds and mammals to bring further attention to context-dependent disparities and similarities in neuropeptide production by the brain and gonads. We find that GnIH responsiveness to cues of stress appears conserved across species, but that the response of specific tissues and the direction of GnIH regulation varies. The implications of differential regulation across tissues remain unclear in most studies, but further work that manipulates and contrasts function in different tissues has the potential to inform us about both organism-specific function and endocrine axis evolution.

The direct response of the gonads to cues of stress in a temperate songbird species is season-dependent.

The gonadotropin releasing hormone (GnRH) system in the hypothalamus is often considered the final point in integration of environmental cues as they pertain to the reproductive axis. However, cues such as stress and food availability are detectable in the plasma (as glucocorticoid and metabolic fuel fluctuations). Vertebrate gonads express glucocorticoid receptor, therefore we hypothesized that the gonads can detect and respond directly to cues of stress. We provide evidence here that, in addition to regulation by the brain, the gonads of European starlings (Sturnus vulgaris) respond directly to fluctuations in corticosterone and metabolic fuels by modulating sex steroid secretion. Using a 4-h gonad culture, we show that physiologically-relevant concentrations of corticosterone and metabolic stress (via use of the glucose utilization inhibitor 2-deoxy-D-glucose and the fatty acid oxidation inhibitor ethyl 2-mercaptoacetate (2DG/MA)) can directly decrease testosterone and estradiol secretion from luteinizing hormone and follicle-stimulating hormone (LH/FSH)-stimulated testes and ovaries. This effect is regulated seasonally. Prior to the breeding season, testes and ovaries respond to corticosterone and 2DG/MA by significantly decreasing gonadal steroid release. Within the breeding season, the testes do not respond to these cues of stress, while the ovaries respond only to corticosterone. This seasonal difference in response may be due in part to the influence of these cues of stress on gonadal neuropeptide expression: corticosterone upregulates GnIH expression in the testes while metabolic stress upregulates GnIH in the ovaries. Thus the gonads can directly respond to fluctuations in corticosterone and metabolic fuels during a time of critical importance to the onset of breeding.

Effects of melatonin on peripheral reproductive function: regulation of testicular GnIH and testosterone.

Study of seasonal reproduction has focused on the brain. Here, we show that the inhibition of sex steroid secretion can be seasonally mediated at the level of the gonad. We investigate the direct effects of melatonin on sex steroid secretion and gonadal neuropeptide expression in European starlings (Sturnus vulgaris). PCR reveals starling gonads express mRNA for gonadotropin inhibitory hormone (GnIH) and its receptor (GnIHR) and melatonin receptors 1B (Mel 1B) and 1C (Mel 1C). We demonstrate that the gonadal GnIH system is regulated seasonally, possibly via a mechanism involving melatonin. GnIH/ GnIHR expression in the testes is relatively low during breeding compared with outside the breeding season. The expression patterns of Mel 1B and Mel 1C are correlated with this expression, and melatonin up-regulates the expression of GnIH mRNA in starling gonads before breeding. In vitro, GnIH and melatonin significantly decrease testosterone secretion from LH/FSH-stimulated testes before, but not during, breeding. Thus local inhibition of sex steroid secretion appears to be regulated seasonally at the level of the gonad, by a mechanism involving melatonin and the gonadal GnIH system.

A functional neuropeptide system in vertebrate gonads: Gonadotropin-inhibitory hormone and its receptor in testes of field-caught house sparrow (Passer domesticus).

UNLABELLED: Previously, the expression and action of GnIH (Gonadotropin-inhibitory hormone) has been characterized in the hypothalamus and pituitary, respectively. The action of this neurohormone is to inhibit the synthesis and secretion of luteinizing hormone and follicle-stimulating hormone. Several hormone systems identified in the vertebrate brain and classified as neurohormones are synthesized in and have a localized action on the gonads as well. Here we present several lines of evidence for the expression and action of GnIH and its receptor (GnIHR) in the testis of house sparrow (Passer domesticus). EXPRESSION: Transcripts for GnIH and GnIHR isolated from house sparrow testis were initially identified by PCR, then sequenced and found to be homologous to transcripts from European starling (96% to GnIH, 98% to GnIHR), Gambel's white-crowned sparrow (94% to GnIH) and Japanese quail (90% to GnIHR) brains. Further investigation using in situ hybridization confirmed the presence of GnIH precursor mRNA in the interstitium of the testis and GnIHR mRNA in the interstitium and on spermatocytes. ACTION: The effect of this system on the secretion of testosterone by the testis was investigated using gonadal culture. Testosterone secretion was significantly decreased by 1 microM and 10 microM GnIH in gonadotropin-stimulated testis cultures. In summary, these results indicate the GnIH/GnIHR system is expressed in the testis of house sparrows and its function is to reduce gonadotropin-stimulated testosterone production.

Neuropeptides in the gonads: from evolution to pharmacology.

Vertebrate gonads are the sites of synthesis and binding of many peptides that were initially classified as neuropeptides. These gonadal neuropeptide systems are neither well understood in isolation, nor in their interactions with other neuropeptide systems. Further, our knowledge of the control of these gonadal neuropeptides by peripheral hormones that bind to the gonads, and which themselves are under regulation by true neuropeptide systems from the hypothalamus, is relatively meager. This review discusses the existence of a variety of neuropeptides and their receptors which have been discovered in vertebrate gonads, and the possible way in which such systems could have evolved. We then focus on two key neuropeptides for regulation of the hypothalamo-pituitary-gonadal axis: gonadotropin-releasing hormone (GnRH) and gonadotropin-inhibitory hormone (GnIH). Comparative studies have provided us with a degree of understanding as to how a gonadal GnRH system might have evolved, and they have been responsible for the discovery of GnIH and its gonadal counterpart. We attempt to highlight what is known about these two key gonadal neuropeptides, how their actions differ from their hypothalamic counterparts, and how we might learn from comparative studies of them and other gonadal neuropeptides in terms of pharmacology, reproductive physiology and evolutionary biology.

Gonadotropin-inhibitory hormone and its receptor in the avian reproductive system.

Many hormones that are classified as neuropeptides are synthesized in vertebrate gonads in addition to the brain. Receptors for these hormones are also expressed in gonadal tissue; thus there is potential for a highly localized autocrine or paracrine effect of these hormones on a variety of gonadal functions. In the present study we focused on gonadotropin-inhibitory hormone (GnIH), a neuropeptide that was first discovered in the hypothalamus of birds. We present different lines of evidence for the synthesis of GnIH and its receptor in the avian reproductive system including gonads and accessory reproductive organs by studies on two orders of birds: Passeriformes and Galliformes. Binding sites for GnIH were initially identified via in vivo and in vitro receptor fluorography, and were localized in ovarian granulosa cells along with the interstitial layer and seminiferous tubules of the testis. Furthermore, species-specific primers produced clear PCR products of GnIH and GnIH receptor (GnIH-R) in songbird and quail gonadal and other reproductive tissues, such as oviduct, epididymis and vas deferens. Sequencing of the PCR products confirmed their identities. Immunocytochemistry detected GnIH peptide in ovarian thecal and granulosa cells, testicular interstitial cells and germ cells and pseudostratified columnar epithelial cells in the epididymis. In situ hybridization of GnIH-R mRNA in testes produced a strong reaction product which was localized to the germ cells and interstitium. In the epididymis, the product was also localized in the pseudostratified columnar epithelial cells. In sum, these results indicate that the avian reproductive system has the capability to synthesize and bind GnIH in several tissues. The distribution of GnIH and its receptor suggest a potential for autocrine/paracrine regulation of gonadal steroid production and germ cell differentiation and maturation.

The control of reproductive physiology and behavior by gonadotropin-inhibitory hormone.

Gonadotropin-releasing hormone (GnRH) controls the reproductive physiology and behavior of vertebrates by stimulating synthesis and release of gonadotropin from the pituitary gland. In 2000, another hypothalamic neuropeptide, gonadotropin-inhibitory hormone (GnIH), was discovered in quail and found to be an inhibiting factor for gonadotropin release. GnIH homologs are present in the brains of vertebrates, including birds, mammals, amphibians, and fish. These peptides, categorized as RF amide-related peptides (RFRPs), possess a characteristic LPXRF-amide (X = L or Q) motif at their C-termini. GnIH/RFRP precursor mRNA encodes a polypeptide that is possibly cleaved into three mature peptides in birds and two in mammals. The names of these peptides are GnIH, GnIH-related peptide-1 (GnIH-RP-1) and GnIH-RP-2 in birds, and RFRP-1 and RFRP-3 in mammals. GnIH/RFRP is synthesized in neurons of the paraventricular nucleus of the hypothalamus in birds and the dorsomedial hypothalamic area in mammals. GnIH neurons project to the median eminence, thus providing a functional neuroanatomical infrastructure to regulate anterior pituitary function. In quail, GnIH inhibits gonadal activity by decreasing synthesis and release of gonadotropin. The widespread distribution of GnIH/RFRP immunoreactive fibers in all animals tested suggests various actions within the brain. In accordance, GnIH/RFRP receptor mRNA is also expressed widely in the brain and the pituitary. GnIH/RFRP immunoreactive axon terminals are in probable contact with GnRH neurons in birds and mammals, and we recently demonstrated expression of GnIH receptor mRNA in GnRH-I and GnRH-II neurons in European starlings. Thus, GnIH/RFRP may also inhibit gonadotropin synthesis and release by inhibiting GnRH neurons in addition to having direct actions on the pituitary gland. Intracerebroventricular administration of GnIH/RFRP further inhibits reproductive behaviors in songbirds and rodents, possibly via direct actions on the GnRH system. The expression of GnIH/RFRP is regulated by melatonin which is an internal indicator of day length in vertebrates. Stress stimuli also regulate the expression of GnIH/RFRP in songbirds and rodents. Accordingly, GnIH/RFRP may serve as a transducer of environmental information and social interactions into endogenous physiology and behavior of the animal. Recently, it was shown that GnIH/RFRP and its receptor are also expressed in the gonads of birds, rodents and primates. In sum, the existing data suggest that GnIH/RFRP is an important mediator of reproductive function acting at the level of the brain, pituitary, and the gonad in birds and mammals.