Relaxin-like factor: A highly specific and constitutive new marker for Leydig cells in the human testis

The complete protein-coding region of the human relaxin-like factor (RLF; formerly Ley-l-L) was cloned by reverse transcription-polymerase chain reaction from human testis and subcloned into a bacterial expression plasmid for the production of recombinant human RLF in Escherichia coli. Polyclonal antibodies were raised against the recombinant RLF, as well as against a peptide epitope from the B-domain of the RLF polypeptide. Antibodies were used for immunohistochemistry of Bouin-fixed, paraffin-embedded samples of human testis tissues. Specific immunoreactivity was located exclusively in the Leydig cells with a consistent high intensity of staining, showing similar spatial distribution to other Leydig cell markers, such as the luteinizing hormone (LH) receptor and SP-hydroxysteroid dehydrogenase (SP-HSD), and to the pattern of RLF mRNA shown by insitu transcript hybridization. In biopsy samples from patients with severe disturbances of spermatogenesis, RLF staining intensity was consistently high in all cases, unlike staining for BP-HSD which varied considerably between patients. Immunostaining for HLF would thus appear to offer an interesting new marker for Leydig cells in human testis samples.