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Gonadotropin activation of cyclic adenosine 3′,5′-monophosphate (cAMP)-dependent protein kinases plays an important role in the regulation of testicular function. This study was undertaken to establish the expression of various subunits of cAMP-dependent protein kinases in different testicular cell types as well as during sexual maturation. RNA was extracted from cultured Sertoli cells, cultured peritubular cells, germ cells (pachytene spermatocytes, round spermatids), tumor Leydig cells, as well as whole testis from rats of various ages. Messenger RNA levels were studied by Northern analysis using available cDNA probes.The regulatory subunit (R) designated RII51was found to be predominantly expressed in cAMP-stimulated Sertoli cells and tumor Leydig cells. Much lower levels were found in cultured peritubular cells and germ cells. A 2.9- and 3.2-kb mRNA for the RI subunit were found at about similar levels in all cell types, whereas the smaller 1.7-kb mRNA was expressed in high levels in germ cells. Also, the catalytic subunit (C) of cAMP-dependent protein kinase, designated Cα, was expressed in all cell types; the highest mRNA levels for this subunit were found in germ cells and in tumor Leydig cells. The 1.7-kb mRNA for androgen-binding protein (ABP) was abundant in cAMP-stimulated Sertoli cells and was not present in other cell types of the testis. Furthermore, the cellular localization of the cAMP-dependent protein kinase subunits was also supported by developmental studies. The mRNA level of the RII51 3.2-kb species was relatively constant until Day 30, after which there was a tendency to decrease. A 1.6-kb message first appeared at greater ages. The mRNA for the smaller 1.7-kb species of RI, as well as the Cα, showed a significant increase during development, supporting an enrichment of these mRNAs in germ cells. Messenger RNA levels for ABP were not detected in testis from 5- to 10-day-old rats but increased up to Day 30. After this age, mRNA for ABP revealed an age-dependent decrease, which parallels the relative increase of germ cells in the testis.In summary, these results demonstrate a clear pattern of cellular localization of the various mRNA species for subunits of the cAMP-dependent protein kinase in the rat testis.

When Sertoli cells were cultured in the presence of follicle-stimulating hormone (FSH), a time-and concentration-dependent desensitization of FSH-responsive adenylyl cyclase (AC) was observed. Maximal desensitization (80%) was attained after 6-9 h of incubation with FSH (10 micrograms/ml; NIH-FSH-S12). During 24 h of incubation the concentration of FSH causing a half-maximal desensitization was about 100 ng/ml. Removal of the hormone from the culture medium was associated with a gradual reappearance of the FSH response. Follicle-stimulating hormone-induced desensitization of Sertoli cell AC was specific for homologous hormone, since AC activation by isoproterenol was unaffected. Furthermore, AC activity of control and FSH-desensitized cells was equally activated by GTP and fluoride, showing that the interaction of the guanyl nucleotide regulatory (N) component with the catalytic subunit is not affected during FSH-induced desensitization. A loss in specific FSH binding was detected after 9 and 24 h of exposure to FSH, but not at shorter times of incubation. Desensitization of Sertoli cell AC to both FSH and isoproterenol stimulation could also be achieved by dibutyryl cyclic AMP (dbcAMP); however, a 30-40% desensitization required a high nucleotide concentration (1 mM) and a long incubation time (24 h). These results show that desensitization of Sertoli cell AC by FSH is associated with normal function of the N component, and precedes any significant loss in specific FSH binding sites. Furthermore, exogenous addition of dbcAMP (1 mM) did not cause the same effects on Sertoli cell AC as did FSH.

Androgen binding protein (ABP) has been localized in the testis and epididymis using a doublebridge immunoperoxidase technique. In the testis, the intensity of ABP staining and the intracellular localization is dependent on the stage of the spermatogenic cycle. In stages VII and VIII, the immunoreactive ABP was primarily localized in the apical portion of the seminiferous epithelium, with much less staining in the basal area. The most intensely stained material next to the lumen could not be definitely located in Sertoli cells. In stages II–IV, ABP was primarily localized in the basal part of the tubular epithelium with heavy staining around groups of elongated spermatids. In those stages, the ABP staining corresponded to Sertoli cell cytoplasm. The localization of ABP in the base, as well as the apex of the seminiferous tubular epithelium, is consistent with the hypothesis that Sertoli cells secrete into both the basal and adluminal compartments.In the epididymis, the heaviest staining was seen in the proximal caput where immunoreactive material was found in the lumen coating spermatozoa and the brush border of the principal cells. In addition, considerable staining was seen in the epididymal epithelium, with the heaviest staining in the apical portion of the cell. In some cells, however, the entire cytoplasm was immunoreactive. The heavy staining of ABP in the epithelial cells of the first part of the caput epididymis is consistent with a considerable degree of endocytosis of luminal ABP into the epithelial cells, where it is probably degraded. In distal caput, corpus, and cauda epididymides, much less immunoreactive ABP was seen in the lumen and in epithelial cells. The uniform stains of a few cells suggest that transepithelial transport of ABP is possible in this organ.

The activities of two testicular enzymes, protein carboxylmethylase (PCM) and Mn2+-dependent adenylyl cyclase (AC), both of which have been shown to be primarily localized in haploid germ cells, were assessed at various stages of the spermatogenic cycle in the rat. The two enzymes displayed the lowest specific activity at stages VII and VIII of the cycle. A gradual increase in the activity of both enzymes was observed from stages XII to IV–V with maximal levels at stages II–III. During the study a highly positive correlation (r = 0.91, P<0.001) was seen between PCM and AC activities. These results show a close parallelism and a stage-dependent variation of PCM and AC activities during the seminiferous epithelial cycle in the rat.

Treatment of hypophysectomized, 30-day-old male rats with 5 mg dihydrotestosterone propionate (DHTP) or testosterone propionate (TP) daily for 5–6 days resulted in a marked reduction in the magnitude of the maximum steroidogenic response of Leydig cells to hCG. This effect was not mediated through alterations in the LH receptor binding to testicular particles and did not represent a direct competitive inhibition of the steroidogenic enzymes.The possible involvement of androgens in a ultra-short loop negative feedback on steroidogenesis is discussed in relation to the phenomenon of Leydig cell desensitization.

Suspensions of testicular cells from 15 to 150 day old rats were stained with a fluorochrome (ethidium bromide) and subjected to micro-flow fluorometry. The resulting DNA distribution patterns consisted of two to three peaks of fluorescence whose relative size altered with increasing age. Up to Day 20, two peaks of fluorescence were detected. One represented diploid cells (2C) and the other tetraploid cells (4C). Following a relative accumulation of tetraploid cells, beginning at Day 19, a third peak of fluorescence (1C) representing a haploid cell population became apparent in cell suspensions from animals 20 days of age. The size of the peak gradually increased in relative magnitude until Day 60 when the haploid cells constituted approximately 75 percent of the total testis cell population.It is concluded that micro-flow fluorometry offers a practical and sensitive technique for monitoring meiosis in the rat.