RESUMO
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.
Assuntos
DNA/biossíntese , Fator de Crescimento Epidérmico/farmacologia , Túbulos Seminíferos/efeitos dos fármacos , Animais , Betacelulina , Bromodesoxiuridina/metabolismo , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Receptores ErbB/genética , Receptores ErbB/metabolismo , Técnicas In Vitro , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Células Intersticiais do Testículo/efeitos dos fármacos , Células Intersticiais do Testículo/metabolismo , Masculino , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Receptor ErbB-3/genética , Receptor ErbB-3/metabolismo , Receptor ErbB-4 , Proteínas Recombinantes/farmacologia , Túbulos Seminíferos/metabolismo , Células de Sertoli/efeitos dos fármacos , Células de Sertoli/metabolismo , Espermatogônias/efeitos dos fármacos , Espermatogônias/metabolismo , Timidina/metabolismo , Fator de Crescimento Transformador alfa/farmacologiaRESUMO
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.
