Estrogen receptor beta selective ligand 5alpha-Androstane-3beta, 17beta-diol stimulates spermatogonial deoxyribonucleic acid synthesis in rat seminiferous epithelium in vitro.

Gonadotropins and testosterone are important regulators of spermatogenesis, even though gonadotropin receptors and the androgen receptor are not expressed by germ cells. However, a functional role for estrogens in connection with male reproduction has been postulated on the basis of the phenotypes of mice lacking estrogen receptor (ER) and cytochrome P-450 aromatase. This has further support by findings of ER expression in the testis, including that of ERbeta in spermatogonia. 5alpha-Androstane-3beta, 17beta-diol (3betaAdiol), a metabolite of testosterone produced via the intermediate potent androgen 5alpha-dihydrotestosterone (DHT), has been reported to selectively bind ERbeta rather than EpsilonRalpha, but not androgen receptor. Here, we have characterized the influence of 17beta-estradiol (E), the major physiological estrogen, 3betaAdiol, and DHT on DNA synthesis in vitro by segments of the seminiferous epithelium at different stages of the seminiferous epithelial cycle in the rat. E and 3betaAdiol exerted similar stimulatory effects on premitotic DNA synthesis in stage I segments, whereas other stages tested (V, VIIa, and XIII-IX) remained unresponsive. In contrast, DHT had no effect on this process. 5-bromo-2'-deoxyuridine labeling of stage I segments revealed a 30-fold higher labeling index in the presence than in the absence of E, and the labeled cells were identified as spermatogonia. Moreover, high levels of 3betaAdiol were found in the testis of intact rats as well as in primary cultures of rat Leydig cells in response to human chorionic gonadotropin. We suggest that 3betaAdiol may serve as a growth factor for germ cells stimulating premitotic DNA synthesis in connection with spermatogenesis via an ERbeta-dependent pathway.

Anti-apoptotic factor humanin is expressed in the testis and prevents cell-death in leydig cells during the first wave of spermatogenesis.

Humanin (HN) is a 24 amino acids peptide with potent neuro-survival properties that protects against damage associated with Alzheimer's disease. In the present report, we have demonstrated by immunohistochemical analysis and Western blotting the pattern of expression of rat humanin (HNr) in the testis of 10- to 60-day-old rats. The Leydig cells of 10- and 40- day-old rats expressed this peptide at high levels; and in the testis of 60-day-old rats the expression of HNr expanded to include Leydig, endothelial, peritubular and germ cells. As monitored by Western blotting, HNr was released into the medium of cultures of Leydig cells isolated from 10-, 40-, and 60-days-old rats. HNr stimulated the incorporation of [(3)H]TdR into DNA of Leydig cells from 10-days-old rats, in a manner that indicated promotion of cell survival rather than an increase in the rate of cell multiplication. This peptide also enhanced steroidogenesis by cultured Leydig cells from 10- to 40-day-old rats both alone and synergistically with IGF-I. The expression of HNr in cultured Leydig cells increased in response to GH and IGF-I. In summary, we demonstrated here that HNr was expressed at all stages of maturation in the rat testis. This peptide promoted the survival of Leydig cells in culture and interacted with IGF-I to stimulate DNA synthesis and steroidogenesis. We propose that HNr is a novel testicular anti-apoptotic factor.

Interleukin-18 is expressed in rat testis and may promote germ cell growth.

Although host-defence mechanisms, designed to preserve the integrity of the developing germ cells are operative in the testis, the components of this protective system have yet to be characterised in detail. Here, we report that the cytokine interleukin-18 (IL-18) is expressed in the rat testis and may contribute to these defences. Thus, analysis by RT-PCR and Western blotting revealed pronounced testicular expression of pro-IL-18 from postnatal day 5 and onwards. Expression of both IL-18 mRNA and protein was found to be localised to meiotic and post-meiotic germ cells as evaluated by in situ hybridisation and immunohistochemistry, respectively. The mRNA species coding for the IL-18 receptor and IL-1beta converting enzyme, which activates pro-IL-18, were also shown to be expressed by the seminiferous tubules. Recombinant IL-18 was seen to stimulate spermatogonial DNA synthesis in cultures of staged segments of rat seminiferous tubules, without influencing germ cell apoptosis. These results suggest that IL-18 may have host-protective and growth-promoting functions in the testis, but further investigations need to be done to confirm this.

EGF stimulates rat spermatogonial DNA synthesis in seminiferous tubule segments in vitro.

Epidermal growth factor (EGF) superfamily of peptide growth factors (EGF-GFs) plays a role in male germ cell development, but the precise function is yet to be defined. The present study shows that EGF-GFs stimulate spermatogonial proliferation in vitro. The EGF-GF ligands, EGF, transforming growth factor-alpha and betacellulin all stimulated DNA synthesis in microdissected stage I segments of rat testis seminiferous tubules in vitro, as revealed by 3H-thymidine incorporation and 5-bromo-2'-deoxyuridine (BrdU) labeling. A fourfold increase over control of BrdU labeled cells, identified as spermatogonia, was seen after treatment with EGF. RT-PCR analysis revealed that the EGF receptors erbB1, erbB2, erbB3 and erbB4 were expressed at all stages of the spermatogenic wave, whereas differential expression was found in isolated Leydig, Sertoli and peritubular cells. The results show that EGF-GFs is spermatogonial growth factor(s) in vitro, although we have not discriminated between a direct action and an indirect effect via somatic cells. We suggest that EGF-GFs is involved in the paracrine control of spermatogenesis in vivo.

Expression and regulation of the prointerleukin-1alpha processing enzymes calpain I and II in the rat testis.

Interleukin-1alpha (IL-1alpha) is constitutively expressed in an age- and stage-dependent manner by rat Sertoli cells. However, the mechanism of regulation of IL-1alpha is unclear in testis. We studied this regulation at the level of the enzyme calpain, a potential regulator that cleaves 32 kDa proIL-1alpha to produce mature 17 kDa IL-1alpha. Both calpain I and II were found to cleave recombinant rat testis 32proIL-1alpha in vitro. A temporary age-related increase in messenger RNA (mRNA) levels of calpain I was found in testis of 20- and 25-day-old rats, coinciding with important events of spermatogenesis and a gradual increase in IL-1alpha, while calpain II expression was constant. In response to lipopolysaccharide (LPS), calpain I protein levels were down-regulated in the seminiferous tubules, while calpain II was less affected. By contrast, the liver after LPS treatment showed up-regulated calpain I and II immunoreactive protein and reverse transcriptase chain reaction (RT-PCR) signal. Depleting Leydig cells by ethane 1,2-dimethane sulphonate treatment resulted in down-regulated calpain I mRNA and protein expression, whereas calpain II remained unchanged. In summary, there is a differential expression of calpain I and II under pathological conditions induced either by endotoxin stimuli or Leydig cell depletion, which may produce a differential effect on IL-1alpha processing.