Leydig cells express follicle-stimulating hormone receptors in African catfish

Abstract

This report aimed to establish, using African catfish, Clarias gariepinus, as model species, a basis for understanding a well-known, although not yet clarified, feature of male fish reproductive physiology: the strong steroidogenic activity of FSHs. Assays with gonadotropin receptor-expressing cell lines showed that FSH activated its cognate receptor (FSHR) with an at least 1000-fold lower EC50 than when challenging the LH receptor (LHR), whereas LH stimulated both receptors with similar EC50s. In androgen release bioassays, FSH elicited a significant response at lower concentrations than those required to cross-activate of the LHR, indicating that FSH stimulated steroid release via FSHR-dependent mechanisms. LHR/FSHR-mediated stimulation of androgen release was completely abolished by H-89, a specific protein kinase A inhibitor, pointing to the cAMP/protein kinase A pathway as the main route for both LH- and FSH-stimulated steroid release. Localization studies showed that intratubular Sertoli cells express FSHR mRNA, whereas, as reported for the first time in a vertebrate, catfish Leydig cells express both LHR and FSHR mRNA. Testicular FSHR and LHR mRNA expression increased gradually during pubertal development. FSHR, but not LHR, transcript levels continued to rise between completion of the first wave of spermatogenesis at about 7 months and full maturity at about 12 months of age, which was associated with a previously recorded approximately 3-fold increase in the steroid production capacity per unit testis weight. Taken together, our data strongly suggest that the steroidogenic potency of FSH can be explained by its direct trophic action on FSHR-expressing Leydig cells.

Figure 1

A, Effects of pituitary-purified AcfLH, rCcfFSH, and rCcfLH on HEK-T 293 cells transiently cotransfected with either the AcfFSHR or AcfLHR and a cAMP-responsive reporter gene construct. Data are expressed as arbitrary units (AU) corresponding to the level of reporter gene expression induced by 10 μm forskolin. Data shown correspond to a single representative experiment with triplicate observations per ligand concentration. B, EC₅₀s [nanograms per milliliter; mean (±sem) from three or four independent assays with triplicate observations each]. For comparison, the values between brackets show the EC₅₀s obtained previously using pituitary-purified or recombinant African catfish gonadotropins (rAcfFSH and rAcfLH) produced in the Dictyostelium discoideum expression system (2).

Figure 2

Effects of increasing concentrations of rCcfFSH or rCcfLH and 10 μm forskolin (Forsk) on OHA secretion by sexually mature (51 wk old) African catfish testis tissue in vitro. Data are expressed as fold induction over basal OHA release (nanograms per milligram testis tissue) and represent compiled data (mean ± sem) from three independent experiments with four to six replicates per ligand concentration each. The mean OHA level measured in the basal groups (0) was 1.38 ± 0.11 ng/mg testis tissue. *, Values are significantly different from basal OHA release (P < 0.05); #, significantly different from each other (P < 0.05).

Figure 3

Effects of increasing concentrations of the PKA inhibitor H-89 on 150 ng/ml AcfLH-stimulated OHA secretion by adolescent (32 wk old) African catfish testis tissue in vitro. Data are expressed as fold induction over basal OHA release (nanograms per milligram testis tissue) and represent compiled data (mean ± sem) from two independent experiments with four replicates per condition. The mean OHA levels measured in the basal group (0) was 0.67 ± 0.08 ng/mg testis tissue. Different letters denote significant differences among groups (P < 0.05).

Figure 4

Laser microdissection and real-time qPCR analysis of sexually mature (51 wk old) African catfish testis tissue fractions. A, Target areas for microdissection were selected and tagged as follows: interstitial (tagged with number 1) containing presumptive Leydig cells (arrow) and excluding blood vessels (BV) and intratubular (tagged with number 2) containing spermatogenic cysts (i.e. Sertoli and germ cells; asterisks). B, After selection, tissue was laser microdissected and pressure catapulted into separate tubes for interstitial and intratubular tissue. Stippled lines delineate the border between interstitial tissue and spermatogenic tubules. Scale bars, 30 μm. C, Relative African catfish gonadotropin receptor mRNA expression levels in microdissected fractions. Data correspond to values from two experiments with duplicate measurements each (mean ± sem) and normalized to Acf-glyceraldehyde-3-phosphate dehydrogenase mRNA levels.

Figure 5

In situ hybridization for AcfLHR and AcfFSHR on testis sections of sexually mature (51 wk old) African catfish. A, Antisense riboprobe for AcfFSHR; note the positive staining in Leydig cells (L) in the interstitial tissue (IT) and in the cytoplasmic extensions of Sertoli cells (S) lining the spermatogenic cysts. B, Antisense riboprobe for AcfLHR; note the positive staining in Leydig cells (L) grouped within the interstitial tissue (IT) between spermatogenic tubules. In all the cases, germ cells (G) were completely devoid of staining. C, Representative sense riboprobe signal for Acf gonadotropin receptors; note the absence of a clear staining. Scale bars, 25 μm.

Figure 6

Relative gonadotropin receptor mRNA expression levels in African catfish testis in juvenile fish (11 wk old), during the first wave of spermatogenesis (16 and 21 wk old), in adolescent fish (first wave of spermatogenesis completed; 32 wk old), and in sexually mature adults (51 wk old). Relative mRNA levels (mean ± sem) were normalized to African catfish 28S rRNA, corrected for the gonadosomatic index (100 × gonad weight × body weight⁻¹) and expressed as relative values of the expression levels measured in 51-wk-old fish testis. For each receptor, different letters denote significant differences among groups (P < 0.05). See text for sample sizes and description of the developmental stage of spermatogenesis in each age group.