Expression of the insulin-like peptide 3 (INSL3) hormone-receptor (LGR8) system in the testis.

The new peptide hormone insulin-like peptide 3 (INSL3) is a member of the insulin-relaxin family, yet, unlike insulin, it signals through a new G-protein coupled receptor, LGR8, distantly related to the receptors for LH and FSH. INSL3 is produced in large amounts by the Leydig cells of the testis in both fetal and adult mammals. Using a combination of mRNA analysis by RT-PCR, immunohistochemistry, ligand-binding, and/or bioactivity assays, the distribution of LGR8 expression was assessed in testicular tissues and cells and in the epididymis. There was consistent agreement that LGR8 was expressed in meiotic and particularly postmeiotic germ cells and in Leydig cells, though not in Sertoli or peritubular cells. Leydig cells appear to express only a low level of the LGR8 gene product; other transcripts may be present, representing nonfunctional products. Messenger RNA analysis suggested that LGR8 transcripts in germ cells represented mostly full-length forms. LGR8 mRNA was also expressed in the epididymis, though no function can yet be ascribed to this expression. Therefore, the INSL3/LGR8 system represents a further paracrine hormone-receptor system in the testis, which conveys information about Leydig cell status to germ cells, and possibly as part of an autocrine feedback loop.

Constitutive regulation of the Insl3 gene in rat Leydig cells.

Insulin-like factor 3 (Insl3) is a major new product of the Leydig cells in all mammalian species so far examined. The rat Insl3 gene is encoded by two exons in close juxtaposition to the Jak3 gene. Using RT-PCR analysis we now show that in the rat testis it is expressed as both major and minor splice variants, the former encoding the normal protein, the latter a truncated peptide comprising a C-terminally extended B-domain. Both transcripts are produced in constant relative amounts uniquely in the Leydig cells of the postnatal testis and in no other testicular cell type. Rat Insl3 protein is also expressed only in Leydig cells after postnatal day 30. Although specific mRNA is present at earlier times, corresponding protein is not detected. Semi-quantitative RT-PCR analysis of Insl3 transcripts in the mouse MA-10 tumour Leydig cell-line under a wide range of stimulation regimes shows that in an acute context, the Insl3 gene is expressed absolutely constitutively. This is confirmed by transfection and electrophoretic mobility shift (EMSA) analysis of the rat Insl3 gene promoter, wherein the importance of three putative SF-1 responsive elements is underscored, although these appear to differ in their relative importance from their counterparts in the mouse Insl3 gene.

Novel testis-expressed profilin IV associated with acrosome biogenesis and spermatid elongation.

A novel profilin, named profilin IV, was cloned and characterized as a testicular isoform, distinct from the previously described testis-specific profilin III. Profilin IV showed only 30% amino acid identity with the other mammalian profilins; nevertheless, database searches produced significant alignments with the conserved profilin domain. Northern blot analysis and in situ transcript hybridization suggested that profilin IV, like profilin III, is transcribed in the germ cells. However, the timing of their expression during post-natal development of rat testis and in the rat spermatogenetic cycle was distinct. In the human testis, profilin IV mRNA expression correlates with the presence of germ cells suggesting that it may be a suitable molecular diagnostic parameter to supplement conventional histopathological diagnostics in the assessment of testicular biopsies. The predicted profilin IV protein was verified employing an anti-oligopeptide antibody. Western blot analysis detected an immunorelated testicular protein of approximately 14 kDa. Immunohistochemistry revealed an intracellular protein of the rat, the mouse and the human testis accumulating asymmetrically in the cytoplasm of round and elongating spermatids with its perinuclear location coinciding with the position of the developing acrosome-acroplaxome and the manchette. Profilin IV thus may regulate testicular actin cytoskeleton dynamics and play a role in acrosome generation and spermatid nuclear shaping.

Immunoexpression of the relaxin receptor LGR7 in breast and uterine tissues of humans and primates.

BACKGROUND: The receptor for the peptide hormone relaxin has recently been identified as the heptahelical G-protein coupled receptor, LGR7. In order to generate molecular tools with which to characterize both in vivo and in vitro expression of this receptor in human and primate tissues, specific monotypic antibodies have been generated and applied to a preliminary analysis of human and primate female reproductive tissues. METHODS: Three peptide sequences were identified from the proposed open reading frame of the cloned LGR7 receptor gene, representing both extracellular and intracellular domains. Two to three rabbits were immunized for each epitope, and the resulting sera subjected to a systematic validation using cultured cells transiently transfected with a receptor-expressing gene construct, or appropriate control constructs. RESULTS: Human and monkey (marmoset, macaque) endometrium showed consistent and specific immunostaining in the stromal cells close to glands. Staining appeared to be more intense in the luteal phase of the cycle. Weak immunostaining was also evident in the endometrial epithelial cells of the marmoset. A myoma in one patient exhibited strong immunostaining in the circumscribing connective tissue. Uterine expression was supported by RT-PCR results from cultured primary endometrial and myometrial cells. Human breast tissue (healthy and tumors) consistently indicated specific immunostaining in the interstitial connective (stromal) tissue within the glands, but not in epithelial or myoepithelial cells, except in some tumors, where a few epithelial and tumor cells also showed weak epitope expression. CONCLUSIONS: Using validated monotypic antibodies recognizing different epitopes of the LGR7 receptor, and from different immunized animals, and in different primate species, a consistent pattern of LGR7 expression was observed in the stromal (connective tissue) cells of the endometrium and breast, consistent also with the known physiology of the relaxin hormone.

Differentiation-dependent expression of 17beta-hydroxysteroid dehydrogenase, type 10, in the rodent testis: effect of aging in Leydig cells.

Expression of the new 17beta-hydroxysteroid dehydrogenase (HSD), type 10 (17beta-HSD-10), formerly known as endoplasmic reticulum-associated amyloid-binding protein, has been investigated in the testes of various mammals under normal and perturbed conditions. Results show that 17beta-HSD-10 is a major product of both fetal and adult-type Leydig cells. In the former, protein persists until late in postnatal development; and in the short-day hamster model, it does not disappear when Leydig cells involute. During puberty in the rat, immunohistochemical staining for 17beta-HSD-10 in adult-type Leydig cells first becomes evident on d 20, increasing to maximal staining intensity by d 35. In the rat, but not in the mouse or any other species examined, there is also staining in late spermatids. Examination of testes from rats subjected to perinatal treatment with either a GnRH antagonist or low and high doses of diethylstilbestrol revealed that expression of 17beta-HSD-10 follows closely Leydig cell differentiation status, correlating with 3beta-HSD expression in a previous study. In aging (23 months) rat testes, Leydig cell (but not germ cell) immunostaining for 17beta-HSD-10 is markedly reduced. 17beta-HSD-10 seems to preferentially convert 3alpha-androstanediol into dihydrotestosterone, and estradiol to estrone. Thus, perinatal expression of this enzyme in fetal Leydig cells may contribute to protecting these cells from estrogens and encourage androgen formation.

SPEER--a new family of testis-specific genes from the mouse.

Differential cloning revealed a partial mRNA sequence expressed in the mouse testis, which on further molecular characterization proved to be a member of a new family of 14 transcribed genes. Six of the genes appear to be expressed pseudogenes. The remainder indicate an open reading frame of approximately 200-220 amino acids encoding proteins with a very high proportion of alpha helical secondary structure, comprising approximately 15% glutamate residues. Because of this property, the family has been named SPErm-associated glutamate (E)-Rich protein (SPEER). Three members were chosen for more detailed characterization: SPEER-1 (pseudogene), SPEER-2, and SPEER-4D. All three are expressed tissue specifically in the testis of mice, with only very weak expression evident in the rat testis but in no other species tested. Using reverse transcription-polymerase chain reaction (RT-PCR), all three transcripts can be detected also in the epididymis, presumably due to the presence of spermatozoa. All three transcripts are expressed to high levels in haploid germ cells at the spermatocyte-spermatid transition. SPEER-1 mRNA is present in the cytoplasm as a sense transcript, SPEER-2 appears to be made mostly as an antisense transcript, whereas SPEER-4D appears to be localized within a subcellular compartment as a conventional sense transcript. Codon usage analysis suggests that all but the pseudogenes can be expressed as protein, confirmed for SPEER-2 and SPEER-4D by in vitro transcription/translation. An antibody raised against a peptide region of SPEER-4D, which probably cross-reacts with other SPEER members, immunohistochemically stains the nuclei of early round spermatids. While there are no true homologies to other proteins in the genome databases, some motifs are present that suggest a relationship to nuclear matrix proteins, implying that the SPEER family is a new group of haploid sperm-specific nuclear factors.

Testis-specific expression of rat mitochondrial glycerol-3-phosphate dehydrogenase in haploid male germ cells.

Mitochondrial glycerol-3-phosphate dehydrogenase (mGPDH) is regulated by multiple promoters in a tissue-specific manner. We characterized the testis-specific promoter C of the mGPDH gene and investigated the cellular localization of mGPDH within the testis. Electrophoretic mobility shift experiments identified a cAMP-response element (CRE) site at -57 that was active in the testis. An in vitro-translated CRE modulator (CREM) protein was able to bind this CRE site, and an anti-CREM antibody interfered with this complex. Ectopic expression of the testis-specific transcriptional activator CREMtau and protein kinase A in human hepatocarcinoma HepG2 cells activated a promoter C-driven luciferase construct in transient transfection experiments. Furthermore, mGPDH expression was undetectable in testis of CREM-deficient mice. The cellular localization of mGPDH expression and translation in adult rat testis was determined by in situ hybridization and immunohistochemistry techniques. The mGPDH transcripts were detected solely in postmeiotic germ cells. Expression of mGPDH was restricted from round spermatids to early elongating spermatids. The mGPDH protein was delayed in postmeiotic germ cells, restricted from late elongating spermatids to mature spermatids. Our results indicate that rat mGPDH is expressed by a testis-specific promoter from haploid male germ cells in a stage-specific manner.