Marga Balvers

Expression of the Insulin-Like Peptide 3 (INSL3) Hormone-Receptor (LGR8) System in the Testis

Abstract 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.

Oxytocin and Male Reproductive Function

In the male mammal, the small peptide hormone oxytocin is produced in similar quantities within the hypothalamo-pituitary magnocellular system as in the female, yet for the male little is known about the physiology associated with this hormone. The present review summarizes what is known about the function of oxytocin in the male mammal and tries to take account of both central and systemic effects, and those linked with a local production of oxytocin within the male reproductive organs. In several species a pulse of systemic oxytocin, presumably of hypothalamic origin, appears to be associated with ejaculation. The systemic hormone could act peripherally stimulating smooth muscle cells of the male reproductive tract, but could also reflect central effects in the brain modulating sexual behaviour. In addition to systemic oxytocin, the peptide is also made locally within the testis, and possibly also the epididymis and prostate. In the former tissue it appears to have an autocrine/paracrine role modulating steroid metabolism, but may in addition be involved in contractility of the seminiferous tubules. However, the latter function may involve the mediacy of Sertoli cells which under some circumstances can also exhibit the components of a local oxytocin system. In the prostate of the rat and the dog oxytocin is linked again to steroid metabolism and may also act as a growth regulator. Finally, oxytocin in seminal fluid is discussed and its possible role in respect to the fate of the semen following ejaculation.

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

BackgroundThe 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.MethodsThree 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.ResultsHuman 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.ConclusionsUsing 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.

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.

Structure and expression of the rat relaxin-like factor (RLF) gene

The relaxin‐like factor (RLF) is a novel member of the insulin‐IGF‐relaxin family of growth factors and hormones, and its mRNA is expressed very specifically in the Leydig cells of the testis and in the theca and luteal cells of the ovary. Here we report the cloning of the RLF gene and cDNA from the rat. The 0.8kb mRNA is produced from a small gene comprising two exons situated less than 1 kb downstream of the gene for the signalling factor JAK3. Northern hybridization confirms high RLF mRNA expression in the adult rat testis, and low expression in the ovary, but in no other tissues examined. Northern analysis of fetal and neonatal gonadal tissues showed that RLF mRNA is highly upregulated in the testes of day 19 embryos, but not in later neonatal stages, nor in any ovarian tissue from this period. This would indicate that RLF is a marker for the mature fetal as well as the adult‐type Leydig cell, but is not expressed in premature, precursor, or dedifferentiated Leydig cells of either cell type. Finally, RNA was analysed from the testes of rats which had been treated with ethylene dimethane sulfonate (EDS), an alkylating agent that specifically destroys rat Leydig cells. RLF mRNA was absent from the acutely treated testes, but became detectable between 15 and 20 days post‐treatment, concomitant with the repopulation of the testes by new Leydig cells. Continuous testosterone substitution of EDS‐treated rats suppressed the production of gonadotropins, and LH‐dependent Leydig cell differentiation, with the result that RLF mRNA remained undetectable throughout the study period. In conclusion, RLF is a very specific marker for the mature Leydig cell phenotype in both the adult‐type and fetal Leydig cell populations of the rat testis. Mol. Reprod. Dev. 54:319–325, 1999.

Relaxin-like factor (RLF) : A new specific marker for leydig cells in the ovary

Relaxin-like factor (RLF), also known as the Leydig cell insulin-like factor (Ley-I-L), is a novel member of the insulin-IGF-relaxin family of hormones and growth factors that has recently been shown to be strongly expressed in testicular Leydig cells. Expression of the RLF peptide in the human ovary and in ovarian tumors has not been studied. In the present study, the expression of the RLF peptide in the human ovary was investigated by immunohistochemistry using a specific antibody raised against human RLF. By this method, RLF was found to be expressed in hilus (Leydig) cells and theca interna cells but absent in granulosa cells, ovarian stromal cells, and surface epithelium. RLF expression was also observed in the corpus luteum, although at a lower level than in theca cells. Thirty-seven sex cord-stromal tumors and five cases of hilar Leydig cell hyperplasia were also investigated for RLF expression. RLF was found to be strongly expressed in hilar Leydig cell hyperplasia and sex cord-stromal tumors with a component of Leydig or luteinized cells. Of the analyzed theca cell tumors, two displayed a diffuse staining pattern. As expected, RLF was not expressed in granulosa cell tumors. In conclusion, RLF appears to be a useful marker for Leydig cells in the human ovary and may be a diagnostic supplement in hyperplasias and tumors derived therefrom.

The structure and regulation of the oxytocin receptor

The oxytocin receptor (OTR) is part of an ancient hormone system expressed in diverse phyla in relation to acute reproductive smooth muscle responses, such as egg‐laying, birth, or milk letdown. The regulation of the OTR gene, while correlating with steroid levels in vivo, remains elusive. There appear to be both inhibitory and stimulatory influences acting upon a constitutive pattern of basal expression. We have found no evidence, however, for an effect of the sex steroids either directly on gene transcription, or on the receptor itself at the protein level. In the prostatic carcinoma cell line Du145, we have shown that up‐regulation of the OTR gene transcription can be effected by cAMP. In an attempt to characterize the expression of the OTR protein in vivo, we have shown, using ligand‐blotting, that the OTR can be expressed at different sizes in transfected cells and in myometrium. Also, in the myometrium at term, immunohistochemistry suggests that there is both an increase in OTR protein per cell, as well as in the number of smooth muscle cells expressing OTR, emphasizing that perinatal changes are the results of both individual gene activation events and gross cellular differentiation. The OTR is a valuable model system reflecting molecular changes in the perinatal period. When we understand how this important molecule is regulated, we will also be a long way towards understanding the mechanisms controlling myometrial contractility at birth.

SPEER—A New Family of Testis-Specific Genes from the Mouse

Abstract 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.

The Rat Endozepine-Like Peptide Gene Is Highly Expressed in Late Haploid Stages of Male Germ Cell Development

Abstract The structure of the endozepine-like peptide (ELP) gene is closely related to the intracellular acyl-CoA binding protein (ACBP), but unlike the generalized distribution of the latter, it is restricted to the male germ cells of the testis. In the present study, a combination of nonradioactive in situ mRNA hybridization and immunohistochemistry was used to precisely determine the cellular expression patterns of ELP mRNA and protein in control and methoxyacetic acid (MAA)-treated rat testes. ELP transcripts are first detectable in late stages (step 6) of round spermatids, with transcription increasing through late-elongating steps. Translation of the ELP mRNA is delayed, with first immunohistochemical staining occurring in elongated spermatids at step 16, and protein accumulating through step 19. ELP immunoreactivity proves to be an excellent marker for late spermatid stages and highlights the presumably clonal recovery of spermatids following MAA treatment.

A Novel Endozepine-Like Peptide (ELP) is Exclusively Expressed in Male Germ Cells

A cDNA clone encoding a novel endozepine-like peptide (ELP) was isolated from mouse testes, sequenced, and its mRNA expression characterized by northern and in situ hybridization. ELP mRNA was found exclusively in the late spermatid stages of spermatogenesis in the testes of sexually mature mice and in no other tissue or cell type examined. It was also expressed in rat, bovine, porcine and sheep testes. Mouse ELP-encoding cDNA was used to construct expression vectors for the production of ELP in bacteria, and the purified bacterial protein used to raise polyclonal antibodies in rats. These antibodies identified the predicted endogenous ELP in extracts of mouse testis and epididymis and in no other tissue. Immunohistochemistry confirmed that the ELP antigen was present only in late spermatids and spermatozoa, particularly within the cytoplasmic droplet which is retained by the mature spermatozoa during their transit into the epididymis. We conclude that ELP is an intracytoplasmic peptide exclusively expressed in post-meiotic spermatozoa and which may be involved in the energy metabolism of the mature sperm.