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

Abstract

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.

The Primary Structure and the Disulfide Links of the Bovine Relaxin-like Factor (RLF)

“…An unknown convertase ignores an arginine in position P2 and cleaves between Glu(B40) and Leu to remove the connecting peptide from the C terminus of the B chain. In fact, all pro-RLF cDNAs (1,2,11,12,(22)(23)(24)(25)(26)(27)(28)(29), except for canine pro-RLF (30), show a Glu-Leu-Leusequence in positions P1, P1′, and P2′, which suggests that this enzyme is guided by a -Glu-Leu-Leu-sequence to affect C-peptide removal. This is probably also true for human and marmoset RLF even though these molecules have a pair of basic amino acids eight residues earlier in the C-terminal region of the B chain (EARRP) (22,24).…”

The Primary Structure and the Disulfide Links of the Bovine Relaxin-like Factor (RLF)

“…An unknown convertase ignores an arginine in position P2 and cleaves between Glu(B40) and Leu to remove the connecting peptide from the C terminus of the B chain. In fact, all pro-RLF cDNAs (1,2,11,12,(22)(23)(24)(25)(26)(27)(28)(29), except for canine pro-RLF (30), show a Glu-Leu-Leusequence in positions P1, P1′, and P2′, which suggests that this enzyme is guided by a -Glu-Leu-Leu-sequence to affect C-peptide removal. This is probably also true for human and marmoset RLF even though these molecules have a pair of basic amino acids eight residues earlier in the C-terminal region of the B chain (EARRP) (22,24).…”

Expression of the Insulin-Like Peptide 3 (INSL3) Hormone-Receptor (LGR8) System in the Testis1

“…Rat gubernacula were dissected from various pre-and postnatal stages of Wistar strain rats and immediately extracted for RNA preparation. Whole rat testes from control Wistar rats and from rats treated with a single bolus of ethane dimethylsulfonate (EDS) were as in Spiess et al [30]; primary rat Leydig cells and seminiferous tubules were prepared as in Ivell et al [31]. Various rat germ cell stages were prepared independently on two occasions by differential elutriation.…”

Novel Expression and Functional Role of Ghrelin in Rat Testis

“…Thus, this setting provides an optimal experimental background in which to test Leydig cell-specific expression of testis-derived factors (as an example, see Ref. 20).…”

The Primary Structure and the Disulfide Links of the Bovine Relaxin-like Factor (RLF)

“…An unknown convertase ignores an arginine in position P2 and cleaves between Glu(B40) and Leu to remove the connecting peptide from the C terminus of the B chain. In fact, all pro-RLF cDNAs (1,2,11,12,(22)(23)(24)(25)(26)(27)(28)(29), except for canine pro-RLF (30), show a Glu-Leu-Leusequence in positions P1, P1′, and P2′, which suggests that this enzyme is guided by a -Glu-Leu-Leu-sequence to affect C-peptide removal. This is probably also true for human and marmoset RLF even though these molecules have a pair of basic amino acids eight residues earlier in the C-terminal region of the B chain (EARRP) (22,24).…”

Expression of the Insulin-Like Peptide 3 (INSL3) Hormone-Receptor (LGR8) System in the Testis1

“…Rat gubernacula were dissected from various pre-and postnatal stages of Wistar strain rats and immediately extracted for RNA preparation. Whole rat testes from control Wistar rats and from rats treated with a single bolus of ethane dimethylsulfonate (EDS) were as in Spiess et al [30]; primary rat Leydig cells and seminiferous tubules were prepared as in Ivell et al [31]. Various rat germ cell stages were prepared independently on two occasions by differential elutriation.…”

Novel Expression and Functional Role of Ghrelin in Rat Testis

“…Thus, this setting provides an optimal experimental background in which to test Leydig cell-specific expression of testis-derived factors (as an example, see Ref. 20).…”

The Primary Structure and the Disulfide Links of the Bovine Relaxin-like Factor (RLF)

“…An unknown convertase ignores an arginine in position P2 and cleaves between Glu(B40) and Leu to remove the connecting peptide from the C terminus of the B chain. In fact, all pro-RLF cDNAs (1,2,11,12,(22)(23)(24)(25)(26)(27)(28)(29), except for canine pro-RLF (30), show a Glu-Leu-Leusequence in positions P1, P1′, and P2′, which suggests that this enzyme is guided by a -Glu-Leu-Leu-sequence to affect C-peptide removal. This is probably also true for human and marmoset RLF even though these molecules have a pair of basic amino acids eight residues earlier in the C-terminal region of the B chain (EARRP) (22,24).…”

Expression of the Insulin-Like Peptide 3 (INSL3) Hormone-Receptor (LGR8) System in the Testis1

“…Rat gubernacula were dissected from various pre-and postnatal stages of Wistar strain rats and immediately extracted for RNA preparation. Whole rat testes from control Wistar rats and from rats treated with a single bolus of ethane dimethylsulfonate (EDS) were as in Spiess et al [30]; primary rat Leydig cells and seminiferous tubules were prepared as in Ivell et al [31]. Various rat germ cell stages were prepared independently on two occasions by differential elutriation.…”

Novel Expression and Functional Role of Ghrelin in Rat Testis

“…Thus, this setting provides an optimal experimental background in which to test Leydig cell-specific expression of testis-derived factors (as an example, see Ref. 20).…”