Kumagai J, et al 2002 reported that INSL3/Leydig insulin-like peptide activates the LGR8 receptor important in testis descent.
Several orphan G protein-coupled receptors homologous to gonadotropin and thyrotropin receptors have recently been identified and named as LGR4-8. INSL3, also known as Leydig insulin-like peptide or relaxin-like factor, is a relaxin family member expressed in testis Leydig cells and ovarian theca and luteal cells. Male mice mutant for INSL3 exhibit cryptorchidism or defects in testis descent due to abnormal gubernaculum development whereas overexpression of INSL3 induces ovary descent in transgenic females. Because transgenic mice missing the LGR8 gene are also cryptorchid, INSL3 was tested as the ligand for LGR8. The authors show that treatment with INSL3 stimulated cAMP production in cells expressing recombinant LGR8, but not LGR7. In addition, interactions between INSL3 and LGR8 were demonstrated following ligand receptor cross-linking. Northern blot analysis indicated that the LGR8 transcripts are expressed in gubernaculum whereas treatment of cultured gubernacular cells with INSL3 stimulated cAMP production and thymidine incorporation. The present study identified the ligand for an orphan GPCR based on common phenotypes of ligand and receptor null mice. Demonstration of INSL3 as the ligand for LGR8 facilitates understanding of the mechanism of testis descent and allows studies on the role of INSL3 in gonadal and other physiological processes.
NCBI Summary:
The receptors for glycoprotein hormones such as follicle-stimulating hormone (FSH; see MIM 136530) and thyroid-stimulating hormone (TSH; see MIM 188530) are G protein-coupled, 7-transmembrane receptors (GPCRs) with large N-terminal extracellular domains. Leucine-rich repeat (LRR)-containing GPCRs (LGRs) form a subgroup of the GPCR superfamily.[supplied by OMIM]
General function
Receptor
Comment
Cellular localization
Plasma membrane
Comment
Ovarian function
Luteinization, Oocyte maturation
Comment
Kawamura K, et al reported paracrine regulation of mammalian oocyte maturation and male germ cell survival.
Mammalian oocytes are arrested at the prophase of meiosis before induction of maturation by the preovulatory luteinizing hormone (LH) surge. LH also promotes the survival of meiotic male germ cells in the testis. Because LH binds somatic cells, the mechanism underlying its regulation of germ cell function is unclear. We found that LH stimulates Leydig insulin-like 3 (INSL3) transcripts in ovarian theca and testicular Leydig cells. INSL3, in turn, binds a G protein-coupled receptor, LGR8 (leucine-rich repeat-containing G protein-coupled receptor 8), expressed in germ cells to activate the inhibitory G protein, thus leading to decreases in cAMP production. Treatment with INSL3 initiates meiotic progression of arrested oocytes in preovulatory follicles in vitro and in vivo and suppresses male germ cell apoptosis in vivo, thus demonstrating the importance of the INSL3-LGR8 paracrine system in mediating gonadotropin actions.
Expression regulated by
Comment
Ovarian localization
Oocyte, Granulosa, Theca
Comment
Ovarian Expression of Insulin-Like Peptide 3 (INSL3) and Its Receptor (RXFP2) During Development of Bovine Antral Follicles and Corpora Lutea and Measurement of Circulating INSL3 Levels During Synchronized Estrous Cycles. Satchell L et al. Insulin-like peptide 3 (INSL3), a major product of testicular Leydig cells, is also expressed by the ovary, but its functional role remains poorly understood. Here, we quantified expression of INSL3 and its receptor RXFP2 in theca interna cell (TIC) and granulosa cell compartments of developing bovine antral follicles and in corpora lutea (CL). INSL3 and RXFP2 mRNA levels were much higher in TIC than granulosa cell and increased progressively during follicle maturation with INSL3 peaking in large (11-18 mm) estrogen-active follicles and RXFP2 peaking in 9- to 10-mm follicles before declining in larger (11-18 mm) follicles. Expression of both INSL3 and RXFP2 in CL was much lower than in TIC. In situ hybridization and immunohistochemistry confirmed abundant expression of INSL3 mRNA and protein in TIC. These observations indicate follicular TIC rather than CL as the primary site of both INSL3 production and action, implying a predominantly autocrine/paracrine role in TIC. To corroborate the above findings, we showed that in vitro exposure of TIC to a luteinizing concentration of LH greatly attenuated expression of both INSL3 and its receptor while increasing progesterone secretion and expression of STAR and CYP11A1. Moreover, in vivo, a significant cyclic variation in plasma INSL3 was observed during synchronized estrous cycles. INSL3 and estradiol-17 followed a similar pattern, both increasing after luteolysis, before falling sharply after the LH surge. Thus, theca-derived INSL3, likely from the dominant preovulatory follicle, is detectable in peripheral blood of cattle, and expression is down-regulated during luteinization induced by the preovulatory LH surge. Collectively, these findings underscore the likely role of INSL3 as an important intrafollicular modulator of TIC function/steroidogenesis, while raising doubts about its potential contribution to CL function.
Expression of insulin-like 3 (INSL3) and differential splicing of its receptor in the ovary of rhesus macaques. Hanna CB et al. ABSTRACT: BACKGROUND: Although insulin-like 3 (INSL3) has been identified in the gonad of both sexes in many species, there are only limited reports on the distribution of INSL3 and its receptor, relaxin/insulin-like family peptide receptor 2 (RXFP2), in the primate ovary. Since the hormone-receptor pair is believed to play a role in female reproduction, investigating the transcription of INSL3/RXFP2 genes and the spatiotemporal expression of INSL3 in the nonhuman primate may shed light on the functional aspects of the system in humans. METHODS: Database mining, molecular and immunological methods were applied. RESULTS: One single INSL3 transcript and three novel splice variant transcripts of RXFP2 were identified in the ovary of rhesus macaques. While the full-length RXFP2 transcript is barely detectable in granulosa cells during the periovulatory period, INSL3 transcript and protein are highly abundant in theca cells surrounding antral follicles. Moreover, the INSL3 level in follicular fluid is 3-4 times higher than that in female serum which remains low throughout the menstrual cycle. CONCLUSIONS: The presence of INSL3 and its receptor in the ovary implies a potential role of the ligand-receptor pair in female reproduction in nonhuman primates. However, the existence of multiple splice variants of RXFP2 indicates a very complex nature of the hormone-receptor system.
Follicle stages
Antral, Preovulatory, Corpus luteum
Comment
Possible Role of Insulin-Like Factor 3 in the Bovine Corpus Luteum. Abe M et al. Insulin-like factor 3 (INSL3) is a local regulator in mammalian gonads, but little is known of its function in bovine corpus luteum (CL). Here, we show that RXFP2 protein, the receptor of INSL3, was expressed throughout the estrous cycle and significantly high at the early luteal stage compared to the regressed luteal stage. INSL3 stimulated progesterone secretion, but not prostaglandin F2a and viability in cultured luteal cells. Together, these results suggest that INSL3 plays a luteotropic role as a local regulator in the bovine CL.
Phenotypes
Mutations
3 mutations
Species: mouse
Mutation name: None
type: null mutation fertility: fertile Comment:Overbeek PA, et al 2001 report a new mouse transgenic insertional mutation, cryptorchidism with white spotting (crsp). Males homozygous for crsp exhibit a high intraabdominal position of the testes, associated with complete sterility. Heterozygous males have a wild-type phenotype, and homozygous females are fertile. Surgically descended testes in crsp/crsp males show normal spermatogenesis. Using FISH and genetic analyses, the transgenic insert causing the crsp mutation has been mapped to the distal part of mouse chromosome 5. Transgene integration resulted in a 550-kb deletion located upstream of the Brca2 gene. A candidate gene encoding a novel G protein-coupled receptor (Great), the LGR8 ortholog, with an expression pattern suggesting involvement in testicular descent has been identified.
Species: human
Mutation name: None
type: naturally occurring fertility: fertile Comment: Role of INSL3 and LGR8 in cryptorchidism and testicular functions.
Foresta C, 2004 .
Cryptorchidism is the most frequent congenital anomaly of the urogenital tract in human males. INSL3 and LGR8/GREAT proteins seem to act as ligand and receptor respectively, and to have a role in gubernaculum development involved in testicular descent. Mutations in the INSL3 gene or LGR8/GREAT were found to be associated with cryptorchidism in humans. In a cohort of 135 ex-cryptorchid patients and 100 controls, mutations were sought in INSL3 and LGR8/GREAT genes by sequencing. Six patients were found with mutations in the INSL3 gene and four patients with LGR8/GREAT mutation (10/135, 7.4%). The 10 patients show different phenotypes, ranging from normozoospermia to complete azoospermia, and from bilateral cryptorchidism to retractile testes. Furthermore, the endocrine function of the testis appeared normal in all subjects. These findings demonstrate that INSL3-LGR8/GREAT mutations are frequently associated with human cryptorchidism, and that the only clinical consequence of alterations of the INSL3-LGR8/GREAT system seems to be failure of the testis to descend normally in the scrotum during embryonic development, without affecting the spermatogenic and endocrine components of the testis itself. The first analysis in humans of INSL3 was then performed using a novel radioimmunoassay kit to measure INSL3 concentrations in serum of adults. The results show that INSL3 circulates in adult men, it is a male-specific hormone, and it is of almost exclusively testicular origin. The role of this hormonal system in adulthood is, however, to date unknown.
Species: human
Mutation name: None
type: naturally occurring fertility: subfertile Comment: A missense mutation in LRR8 of RXFP2 is associated with cryptorchidism. Harris RM et al. Using genome-wide mutagenesis with N-ethyl-N-nitrosourea (ENU), a mouse mutant with cryptorchidism was identified. Genome mapping and exon sequencing identified a novel missense mutation (D294G) in Relaxin/insulin-like family peptide receptor 2 (Rxfp2). The mutation impaired testicular descent and resulted in decreased testis weight in Rxfp2 ( DG/DG ) mice compared to Rxfp2 (+/DG ) and Rxfp2 (+/+) mice. Testicular histology of the Rxfp2 ( DG/DG ) mice revealed spermatogenic defects ranging from germ cell loss to tubules with Sertoli-cell-only features. Genetic complementation analysis using a loss-of-function allele (Rxfp2 (-)) confirmed causality of the D294G mutation. Specifically, mice with one of each mutant allele (Rxfp2 ( DG/-)) exhibited decreased testis weight and failure of the testes to descend compared to their Rxfp2 (+/-) littermates. Total and cell-surface expression of mouse RXFP2 protein and intracellular cAMP accumulation were measured. Total expression of the D294G protein was minimally reduced compared to wild-type, but cell-surface expression was markedly decreased. When analyzed for cAMP accumulation, the EC50 was similar for cells transfected with wild-type and mutant RXFP2 receptor. However, the maximum cAMP response that the mutant receptor reached was greatly reduced compared to the wild-type receptor. In silico modeling of leucine rich repeats (LRRs) 7-9 indicated that aspartic acid 294 is located within the ?pleated sheet of LRR8. We thus postulate that mutation of D294 results in protein misfolding and aberrant trafficking. The ENU-induced D294G mutation underscores the role of the INSL3/RXFP2-mediated pathway in testicular descent and expands the repertoire of mutations known to affect receptor trafficking and function.