Prolactin receptor expression in human testis and accessory tissues: localization and function

W.M.Hair 0 O.Gubbay 0 H.N.Jabbour 0 G.A.Lincoln 0 0 MRC Human Reproductive Sciences Unit, Centre for Reproductive Biology , 37 Chalmers Street, Edinburgh EH3 9EW, UK Experimental studies in animals have established prolactin (PRL) as a progonadal hormone that promotes the function of the testis and reproductive accessory glands. The present study investigated the localization of PRL receptor (PRL-R) expression in the human testis and accessory tissues. Expression of PRL-R was identified in human testis and vas deferens by RT-PCR, and further localized by immunohistochemistry to the Leydig cells and differentiating germ cells of the testis (developmental stages extending from pachytene spermatocytes to elongating spermatids). Positive staining for PRL-R was also clearly evident in the epithelium of vas deferens, epididymis, prostate and seminal vesicles. Functional activation of PRL-R was demonstrated in fresh samples of vas deferens collected at vasectomy by examination of the JAK/STAT (Janus kinase/signal transducer and activator of transcription) and MAP (mitogen-activated protein) kinase ERK (extracellular signal-regulated kinase) signalling pathways. Within the vas deferens, PRL induced rapid tyrosine phosphorylation of JAK 2 and STAT 5 (after 10 and 20 min respectively), and tyrosine and threonine phosphorylation of ERK 1 and 2 (after 5 min). The demonstration of function and localization of PRL-R presented here suggests multiple roles for PRL in the human male reproductive tract. - Experimental studies in animals have established that prolactin acts in concert with the classical gonadotrophins, LH and FSH, to stimulate full testicular function in the adult male rat, mouse and hamster (Bartke, 1971; Bartke et al., 1975; Zipf et al., 1978; Dombrowicz et al., 1992). In the ram, a functional role for prolactin (PRL) in the testis is indicated by the observation that hypothalamo-pituitary disconnected rams, that permanently lack gonadotrophin due to a blockade of GnRH secretion, continue to express cycles in testicular size in response to photoperiod-induced changes in PRL secretion. These gonadal changes are minor and occur with a long latency, but they indicate that PRL is a weak gonadotrophin in the absence of LH and FSH (Lincoln et al., 1996). Prolactin receptor (PRL-R) gene expression in the testis has been demonstrated in different species including rat, ram and red deer (Ouhtit et al., 1993; Hondo et al., 1995; Jabbour et al., 1998a; Jabbour and Lincoln, 1999). These studies show PRL-R to be localized to the Leydig cells in the interstitium and germ cells within the seminiferous tubules (Jabbour and Lincoln, 1999). The addition of PRL to testicular explants of ruminant species induces phosphorylation of JAK (Janus kinase) and STAT (signal transducer and activator of transcription) signalling proteins, consistent with a functional PRL-R in the testis (Jabbour et al., 1998b). PRL is believed to stimulate testicular steroidogenesis by regulating LH receptors (Bex and Bartke, 1977; Takase et al., 1990), or androgen/estrogen biosynthesis through the control of rate-limiting enzymes in the Leydig cells (Takeyama et al., 1986; Chandrashekar and Bartke, 1988). However, the mechanism of action of PRL on spermatogenesis remains to be clarified. Expression of PRL-R has also been demonstrated in the rat dorsal and lateral prostate, and seminal vesicles (Ouhtit et al., 1993; Nevalainen et al., 1996). In cultured prostatic cells, androgens and estrogens stimulate the expression of PRL-R (Nevalainen et al., 1996), and both gonadal steroids and PRL, induce the secretion of prostate-specific proteins (Costello and Franklin, 1994). In transgenic mice engineered to over-express the PRL gene, the prostate gland becomes grossly enlarged illustrating the importance of PRL in the control of accessory gland function (Wennbo et al., 1997). Chronic suppression of blood concentrations of PRL secretion in the ram produces a decrease in size and fructose content of the seminal vesicles, with no change in testosterone secretion (Ravault et al., 1977), and manipulations of PRL and androgens in the macaque monkey affect seminal vesicular enzymes (Arunakaran et al., 1988); thus, PRL may promote the function of various androgen-dependant male accessory structures. Clinical observations also support a role for PRL in the regulation of the testis and accessory glands in man. For example, the restoration of normal PRL levels in a cohort of subfertile, hypoprolactinaemic men caused an increase in sperm density and quality, and restored fertility (Ufearo et al., 1995). In another study, suppression of gonadotrophins and PRL secretion in eugonadal men treated for prostatic carcinoma caused a more marked reduction in testicular weight and spermatogenesis than suppression of gonadotrophin secretion alone (Huhtaniemi et al., 1991). Both observations are consistent with a progonadal role of PRL in the testis, although early studies using I125-iodo PRL failed to demonstrate the presence of PRL binding in the human testis, in contrast to the situation in the rat (Wahlstrom et al., 1983). PRL binding has been demonstrated in the human prostate (Leake et al., 1983), and other studies suggest that PRL may play a role in the aetiology of benign prostatic hyperplasia and cancer (Kadar et al., 1988; Nevalainen et al., 1997). The synchronous reduction in both PRL and androgen improves the efficacy of the treatment of prostatic carcinoma (Rana et al., 1995). The purpose of the present study was to provide direct evidence for a role of PRL in the regulation of the testis and reproductive tract in man. To this end, the expression of the PRL-R gene was investigated by RTPCR using RNA extracted from human testis and vas deferens. The localization of expression of the PRL-R protein was further studied using immunohistochemistry in sections prepared from the human testis, epididymis, vas deferens, prostate and seminal vesicles. Lastly, a functional PRL-R was identified in the vas deferens by investigating activation of the JAK/STAT and MAP (mitogen activated protein) kinase ERK (extracellular signal-regulated kinase) specific intracellular signalling pathways. Activation of JAK/STAT and ERK proteins, following binding of PRL to its receptor, mediates both proliferative and differentiating effects in target cells (Findiori and Kelly, 1995; Lewis et al., 1998). Materials and methods Testicular tissue (n 6), exhibiting normal morphology, was obtained by biopsy from men exhibiting unexplained infertility. Vas deferens tissue (n 10) was obtained from normal men undergoing vasectomy. Left and right vas deferens were used for comparison between treatment and control. Ethical approval was obtained from Lothian Paediatric and Reproductive Medicine Research Ethics Subcommittee, and written informed consent was obtained from each subject. Prostate and bladder tissues were obtained from archival surgical re-section sp (...truncated)