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

Abstract

The complete protein-coding region of the human relaxin-like factor (RLF; formerly Ley-I-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 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), and to the pattern of RLF mRNA shown by in-situ transcript hybridization. In biopsy samples from patients with severe disturbances of spermatogenesis, RLF staining intensity was consistently high in all cases, unlike staining for 3 beta-HSD which varied considerably between patients. Immunostaining for RLF would thus appear to offer an interesting new marker for Leydig cells in human testis samples.

Steroidogenesis and steroidogenic gene expression in postnatal fetal rat Leydig cells

“…Our finding that PFLCs express Insl3, a factor of importance for the first phase of testicular descent (Ivell et al, 1997) is agreed well with previous studies demonstrating high levels of Insl 3 expression in FLCs (Balvers et al, 1998;McKinnell et al, 2005). We demonstrated that PFLCs lost their capacity to express Insl3 after long-term culturing and that (Bu) 2 cAMP prevented this down-regulation, indicating that Insl3 gene expression in PFLCs might be controlled by cAMP-dependent signaling pathway(s).…”

“…These cells secrete androgens that are critical for the normal development of male reproductive organs as well as insulin-like factor 3 (Insl3) required for the abdominal phase of the scrotal descent of the testis (Huhtaniemi and Pelliniemi, 1992;Ivell et al, 1997). In rats, FLCs start to differentiate and produce testosterone at gestational age of 14 days before they undergo functional regression from fetal day 18.5 onward (Habert and Picon, 1984;Kuopio et al, 1989).…”

Peripheral insulin-like factor 3 concentrations are reduced in men with type 2 diabetes mellitus: effect of glycemic control and visceral adiposity on Leydig cell function

“…Indeed, the present findings firstly suggest the existence of subtle alterations at gonadal level also in diabetic patients with normal testosterone values, regardless of the presence of symptoms of androgen deficiency. The global impairment of Leydig cell function in T2DM is confirmed by the finding of reduced circulating levels of INSL3, a novel peptide hormone mainly derived from Leydig cells (15,16,25,26), which have been indicated as an absolute measure of either quality or number of the Leydig cells, independently from gonadotropin stimulation (16,17,(27)(28)(29)(30). The higher LH levels in diabetic patients than in controls, though in the normal range, might suggest that when few or poor-quality Leydig cells are present, more LH is required to achieve normal circulating testosterone levels.…”

Testicular Descent

“…103 The Insl3 gene is maximally expressed in both fetal and mature adult-type Leydig cells, whereas it is only weakly expressed in prepubertal immature Leydig cells and the ovary (with an exception being the ruminant ovary that exhibits a very high degree of expression). 104,105 Insl3 transcripts are found in the developing testis of all mammalian species 106 but not in the gubernacular bulb or in other neighboring tissues. 101 They are first detected at 13.5 pcd in Leydig cells (exclusive source in the testis) 105 and remain constant through the 3 rd postnatal week when increases are observed coincident with the first wave of spermatogenesis/germ cell maturation reaching their highest level in the adult testis.…”