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.
Relaxin-like factor (RLF) is a new member of the insulin-relaxin gene family known to be expressed in the ovarian follicular thecal cells of ruminants. To investigate the pattern of RLF expression in development and atresia of bovine follicles, antisera were raised in rats and rabbits to recombinantly expressed bovine pro-RLF and to chemically synthesized ovine RLF B chain, respectively. On dot blotting analysis, the rat antiserum bound to pro-RLF and less strongly to a synthetic mature ovine RLF lacking the C-domain, whereas the rabbit antiserum bound the mature form of ovine RLF. These antisera were used to immunostain bovine ovarian follicles of differing sizes and stages of health and atresia. 3beta-Hydroxysteroid dehydrogenase was colocalized with pro-RLF (n = 86 follicles), and cholesterol side-chain cleavage cytochrome P450 was localized in another section of many of the same follicles (n = 66). Not all follicles expressed pro-RLF in the theca interna, so the results are presented as the proportion of follicles expressing pro-RLF. Both mature and pro-RLF were immunolocalized to steroidogenic thecal cells of healthy follicles. As follicles enlarged to >5 mm, the proportion expressing pro-RLF declined (19/19 for <5 mm and 18/26 for >6 mm). Atresia was divided into antral (antral granulosa cells dying first) or basal (basal cells dying first) and further divided into early, middle, and late. For antral atresia of small follicles (2-5 mm), no decline in the proportion expressing pro-RLF was observed (early 6/6, middle 2/2) until the late stages (1/4). For basal atresia, which only occurs in small follicles (2-5 mm), the proportion expressing pro-RLF declined in the middle (2/5) and late (0/8) stages. In larger follicles (>6 to <10 mm), the proportion expressing pro-RLF also declined with atresia (1/13). These declines in RLF expression with atresia or increasing size were not accompanied by a decline in the expression of steroidogenic enzymes in the theca interna. A significant (P < 0.001) inverse relationship in the expression of pro-RLF and 3beta-hydroxysteroid dehydrogenase in the membrana granulosa was observed. We conclude that the expression of pro-RLF in the theca interna is switched off as follicles enlarge or enter atresia, whereas the expression of steroidogenic enzymes is maintained in the theca interna.
Expression of the relaxin-like factor (RLF) was studied at the messenger RNA (mRNA) and protein levels in the testes and ovaries of the mouse, as well as through testicular development and differentiation in the mouse testis. In situ hybridization or RT-PCR, and immunohistochemistry using a polyclonal antibody raised against a recombinant protein, provided mutually confirmatory results for a high expression of RLF in the Leydig cells of the adult testis and at a much lower level of expression in the luteal cells of the ovary through the cycle, pregnancy, and in lactation. Analysis of protein and mRNA expression, through postnatal testicular development, indicated moderate RLF expression also in the fetal population of Leydig cells, even in the hpg mutant mouse, lacking an active pituitary-gonadal axis. Prepubertal Leydig cells, however, exhibit only very low-level RLF gene expression, this phenotype persisting in the adult hpg mouse. In summary, fetal Leydig cells express RLF in an LH/human CG-independent fashion, whereas LH/human CG is essential to induce RLF expression in the adult-type Leydig cell. In cultured adult Leydig cells or in the mouse tumor MA-10 cell line, RLF mRNA is expressed in a constitutive fashion. RLF thus seems to be a useful marker of Leydig cell differentiation status.
Hypothalamic GnRH (gonadotropin-releasing hormone) neurons play a critical role in the initiation and maintenance of reproduction competence. Using the mouse GnRH neuronal cell line, GT1-7, we have characterized the expression of the gene mPer1, a recognized key element of the mammalian circadian clockwork. Both mPer1 transcripts and the 136 kDa mPER1 gene product could be detected in these cells. Immunocytochemical analysis also confirmed expression of mPER1 both in vitro and in vivo in GnRH neurons. Activation of cyclic AMP signalling pathways in vitro elevated GnRH secretion as well as mPer1 expression and nuclear mPER1 immunoreactivity. As mPER1 is known to feedback on transcriptional activities in many cell models, the data presented here point to a role for mPER1 in the regulation of gene expression in GnRH neurons, and thus in the control of neuroendocrine activities.
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