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Male Wistar-MS (W/MS), Fisher-344 (F-344) and Sprague-Dawley (SD) rats were divided into four groups including a control group implanted with a cholesterol pellet. Rats in the three experimental groups were treated with gamma-ray irradiation (260 cGyt) alone, diethylstilbestrol (DES) pellet implantation alone or both irradiation and DES, and all rats were observed for detection of mammary tumors for 1 year. Morphologically, well-developed mammary glands were observed in the SD rats at ages corresponding to the time of irradiation. But, the mammary glands in the W/MS and F-344 rats showed a lower degree of differentiation than those in the SD rats. No mammary tumor developed spontaneously in the W/MS and F-344 strains of rats during the experimental period. The rats administered both DES and irradiation showed significantly increased incidence of mammary tumors compared with the control, the incidence being 80.9% in the SD rats, 35.0% in the W/MS rats, and 9.4% in the F-344 rats, respectively. The incidence of tumor in the SD rats treated with irradiation alone and with DES alone was 9.5% and 14.3%, respectively, but no tumor development was observed in the F-344 rats treated with either irradiation alone or DES alone or in the W/MS rats treated with DES alone. The magnitude of the decrease of testicular weight in the SD rats implanted with DES after irradiation (to 70% of the control weight) was slightly less marked than that in either the W/MS (35%) or F-344 (16%) rats. The testicular atrophy showed a correlation with the accessory sex organ weight at the end of the experiment, serum testosterone concentration, and incidence of mammary tumors. Following administration of DES pellets after the irradiation, the activity of delta 5-3 beta- and of 17 beta-hydroxysteroid dehydrogenase in the testes showed the order F-344 < W/MS = SD and F-344 = W/MS < SD, respectively. Compared with the control, the irradiated F-344 rats implanted with DES pellets showed hypertrophied pituitary glands (10.7-fold, P < 0.01) as well as increased serum prolactin concentration (21.4-fold, P < 0.01). Of the three strains treated with both irradiation and DES, the F-344 rats showed the highest concentration of serum prolactin but the lowest incidence of mammary tumors. Our results suggest that W/MS, F-344 and SD male rats have differing susceptibilities for the induction of mammary tumor following irradiation. We discuss the relationship between testicular and pituitary functions and male mammary tumorigenesis.

Because nerve growth factor beta (NGF beta) and its corresponding receptors have been implicated in the paracrine regulation of spermatogenesis, we examined the postnatal developmental expression of the low- and high-affinity NGF receptors in the rat testis, and localized their expression to specific testicular cell types. The neurotropin receptors consist of a low-affinity p75 nerve growth factor receptor (LNGFR) and a family of high-affinity tyrosine receptor kinases (trk). Both the p75 LNGFR gene product and the trk receptor gene product were detected in immature rat testes, with maximal expression in 10- and 20-day-old rats. Expression of the testicular p75 LNGFR and the trk receptor progressively declined in older animals so that they were barely detectable in 90-day-old adult rats. The 75-kDa LNGFR was detected in membrane fractions of Sertoli cells, whereas the p75 LNGFR was not detected by Western blot in membrane fractions of round spermatids and primary spermatocytes. Interestingly, microsomal fractions of peritubular myoid cells were immunoreactive for a 65-kDa band on Western blots with the p75 LNGFR monoclonal antibody. Immunoblot analysis of the trk receptor in cell lysates of isolated cell types was inconclusive. Excess NGF beta and round spermatid protein, which is known to contain a NGF-like protein, were both capable of displacing the binding of 125I-NGF beta from the surface of Sertoli cells.(ABSTRACT TRUNCATED AT 250 WORDS)

The expression of guanine nucleotide-binding proteins (G proteins) during the development of rat testes was investigated. Immunohistochemical studies on frozen sections and isolated testicular cells demonstrated that the expression of the GTP-binding proteins was developmentally regulated and specific for different cell types. The alpha subunit of the cholera toxin-sensitive stimulatory G protein (Gs alpha) was first detected in testes from 7-day-old rats; its value reached a maximum at 23 days and then decreased to very low or undetectable amounts in testes of 45-day-old and adult rats (60-90 days of age). The Gs alpha subunit appears to be expressed by Sertoli, peritubular myoid and interstitial cells. The common beta subunit (G beta) was present at all ages during development and was more prominent around the periphery of the tubules in younger animals but then became more evident in the cytoplasm of germ cells with increasing age. The pertussis toxin-sensitive inhibitory G proteins, Gi1/2 and Gi3, showed a similar pattern of expression. Sertoli cells and peritubular cells expressed Gi1/2 and Gi3 at very low levels at all ages, whereas pachytene spermatocytes and round spermatids expressed the inhibitory binding proteins only at later ages of development (45-day-old and adult testis). Northern blot analysis showed that with increasing age the Gs alpha mRNA in the testis decreased and this was confirmed by in situ hybridization. These latter studies showed localization of the transcripts to somatic cells but not to germ cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Kinetic and binding studies were performed with a purified microsomal cytochrome P-450 from neonatal pig testis, the C21 side chain cleavage system (17 alpha-hydroxylase/C17,20-lyase). Binding of substrates and inhibitors was measured by spectral methods and by equilibrium dialysis. Kinetic data revealed that pregnenolone inhibits lyase activity with 17 alpha-hydroxypregnenolone as substrate (Ki, 0.3 microM) and that progesterone inhibits lyase activity with 17 alpha-hydroxyprogesterone (Ki, 1.5 microM); inhibition is competitive in both cases. Binding and kinetic studies revealed that Km, Ks, and Kd (Michaelis constant and dissociation constants determined by spectral and dialysis methods, respectively) are all considerably lower for the delta 5 substrates than for the corresponding delta 4 compounds. Equilibrium dialysis shows that there is a single binding site for the substrates of both activities (hydroxylase and lyase). Spectral studies revealed a lag in the development of the spectral shift produced by the addition of steroids and gave results compatible with a single active site, although this spectral evidence is not conclusive by itself. It is concluded that (i) the powerful forward competitive inhibition by pregnenolone and progesterone may be important in regulating synthesis of androgens in vivo; (ii) the porcine enzyme uses delta 5 substrates in preference to delta 4 substrates, thereby accounting for extensive use of the delta 5 pathway by pig testis in vivo; (iii) the evidence presented suggests one active site for both hydroxylase and lyase activities.

The affinity alkylating progesterone analogue 17-(bromoacetoxy)progesterone has been used to label the active site of a microsomal cytochrome P-450 enzyme from neonatal pig testis. The enzyme causes removal of the C20 and C21 side chains from the substrates progesterone and pregnenolone by catalyzing both 17-hydroxylase and C17,20-lyase reactions, which produce the corresponding C19 steroidal precursors of testosterone. The progesterone analogue causes simultaneous inactivation of the two catalytic activities of the enzyme by a first-order kinetic process that obeys saturation kinetics. Progesterone and 17-hydroxyprogesterone each protect the enzyme against inactivation. The progesterone and analogue is a competitive inhibitor of the enzyme with Ki values of 8.4 microM and 7.8 microM for progesterone and 17-hydroxyprogesterone, respectively. The enzyme inactivation and kinetic data are consistent with a theory proposing that the analogue and the two substrates compete for the same active site. The radioactive analogue 17-[( 14C]bromoacetoxy)progesterone causes inactivation of the enzyme with incorporation of 1.5-2.2 mol of the analogue per mole of inactivated enzyme. When this experiment is carried out in the presence of a substrate, then 0.9-1.2 mol of radioactive analogue is incorporated per mole of inactivated enzyme. The data suggest that the analogue can bind to two different sites, one of which is related to the catalytic site. Radiolabeled enzyme samples, from reactions of the 14C-labeled analogue with the enzyme alone or with enzyme in the presence of a substrate, were subjected to amino acid analysis and also to tryptic digestion and peptide mapping.(ABSTRACT TRUNCATED AT 250 WORDS)

Highly purified cytochrome P-450 from neonatal pig testicular microsomes is capable of catalyzing both 17 alpha-hydroxylation and C-17,20-lyase activity. Estradiol was found to inhibit both activities of the purified enzyme with delta 4 and with delta 5 substrates (progesterone, pregnenolone, and the corresponding 17 alpha-hydroxysteroids). For the delta 4 series, inhibition of lyase is competitive and that of 17 alpha-hydroxylase is noncompetitive; for the delta 5 series, inhibition was noncompetitive for both activities. Ki values for lyase activity were determined from enzyme kinetics (5.0 microM for the delta 4 substrate and 20 microM for the delta 5 substrate). Estradiol produces a typical type I spectral shift with the pure enzyme (Ks = 3.0 microM where Ks is the concentration of steroid required to give half maximal spectral shift), so that Ki values were also determined directly from binding studies by using substrate-induced difference spectroscopy. Fifty per cent inhibition of the maximal spectral shift induced by the 17 alpha-hydroxysubstrates (Ki) are 3.8 and 7.6 microM for the delta 4 and delta 5 substrates, respectively. Values for Ki are higher with the substrates of 17 alpha-hydroxylase (progesterone and pregnenolone), by either method, than the corresponding Ki values for the lyase substrates. The concentration of estradiol in Leydig cells of neonatal pig testis is approximately 1.5 nmol/g. It is proposed that estradiol may influence testicular steroidogensis under physiological conditions by competitive inhibition of lyase activity.

Epidermal growth factors receptor (EGFR) was localized immunocytochemically in the testes of mature and immature rats and immature monkeys. One polyclonal antibody, recognizing the intracellular domain (RK2) of the receptor, was used to carry out the EGFR immunodetection. The RK2 antibody revealed the presence of the EGFR predominantly in Sertoli cells of mature and immature rats and of immature monkeys, although limited interstitial localization of the EGFR was also discerned in the mature rat. In cultured Sertoli cells of immature rats, grown in the absence of epidermal growth factor (EGF), the EGFR was randomly distributed at the cell surface, whereas after the addition of EGF the receptor became aggregated into distinct focal regions. In addition, EGFR of cultured Sertoli cells exhibited autophosphorylation activity upon stimulation with EGF, but failed to transcytose iodinated EGF across a permeability barrier formed by the cultured cells. Instead, all of the added iodinated EGF was internalized and degraded.