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Summary.The responsiveness of decapsulated testes and isolated Leydig cell preparations from rats (30–80 days of age) to a constant dose of 3 ng hCG/2 ml was assessed by comparison of the production of testosterone and 'total 17β-hydroxy androgen' (17β-HA). When testosterone secretion was used as the index of response, there was a marked increase in the production with age by decapsulated testes and also by equal numbers of Leydig cells. When 17β-HA was taken as the response parameter this increase was only marginal for the decapsulated testes and there was an age-dependent decrease when expressed per 106cells. These differences probably reflect changes in the metabolism of testosterone to 5α-reduced products with increasing age because 80% of androgen secreted at 30 days is 3α-androstanediol and 86% is secreted as testosterone at 80 days. We conclude that for studies on hCG responsiveness and the steroidogenic capacity of immature rat Leydig cells (a) testosterone is an inappropriate response parameter and (b) this response undergoes a decrease rather than an increase during prepubertal development.

Summary.The levels of testosterone, dihydrotestosterone (DHT), 5α-androstan-3α,17β-diol and androgen-binding protein (ABP) were measured in various segments of the epididymis from adult rats which had been unilaterally orchidectomized for 4 weeks. On the 'intact' side, ABP concentrations were highest in the caput region. The segmental distribution of DHT closely followed that of ABP with the highest concentration in the caput (40 ng/g tissue) and lowest in the cauda (10 ng/g tissue) epididymidis. There was a high degree of correlation (r= 0·98) between the concentration of DHT in the epididymis and ABP levels. 'Castration' completely abolished the DHT gradient. The levels of testosterone and androstanediol were lower than those of DHT; most was present in the corpus epididymidis. The relative differences were reduced after 'castration'. It is concluded that ABP in the rat epididymis is the primary factor for determining the concentration of DHT in the epididymal fluid.

Summary.Soluble Mn2+-dependent adenylate cyclase and protein carboxyl methylase are two enzymes that are primarily localized in haploid germ cells of rat testicular tissue, and both enzymes exhibit an increase in activity in association with sexual maturation. Experimental cryptorchidism (surgery at 17 days of age) in immature rats prevented the age-dependent increase in the activity of these two testicular enzymes. After orchidopexy at 34 days of age the activities of these two enzymes increased to normal control values in association with testicular growth. These observations show that biochemical markers such as soluble Mn2+-dependent adenylate cyclase and protein carboxyl methylase can be used to follow germ cell differentiation.

Summary.The seasonal changes in testicular weight in the blue fox were associated with considerable variations in plasma concentrations of LH, prolactin, androstenedione and testosterone and in FSH-binding capacity of the testis. An increase in LH secretion and a 5-fold increase in FSH-binding capacity were observed during December and January, as testis weight increased rapidly. LH levels fell during March when testicular weight was maximal. Plasma androgen concentrations reached their peak values in the second half of March (androstenedione: 0·9 ± 0·1 ng/ml; testosterone: 3·6 ± 0·6 ng/ml). A small temporary increase in LH was seen in May and June after the breeding season as testicular weight declined rapidly before levels returned to the basal state (0·5–7 ng/ml) that lasted until December. There were clear seasonal variations in the androgenic response of the testis to LH challenge. Plasma prolactin concentrations (2–3ng/ml) were basal from August until the end of March when levels rose steadily to reach peak values (up to 13 ng/ml) in May and June just before maximum daylength and temperature. The circannual variations in plasma prolactin after castration were indistinguishable from those in intact animals, but LH concentrations were higher than normal for at least 1 year after castration.

Summary.Isolated rat Sertoli cells were incubated in the presence of [1-14C]palmitate at a cell concentration of 1·54 ± 0·31 mg protein/flask (n= 7). The oxidation of palmitate was concentration dependent and maximal oxidation was obtained at 0·35 mm-palmitate. At a saturating concentration of palmitate the oxidation was linear for at least 6 h. About 65% of the total amount of palmitate oxidized during 5 h at 0·52 mm-palmitate (109 ± 44 nmol/flask, n= 5) was recovered as CO2and the rest as acid-soluble compounds. Almost all radioactive acid-soluble compounds which were secreted by the Sertoli cells were shown to be 3-hydroxybutyrate and acetoacetate. The palmitate recovery in cellular lipids and triacylglycerols was 9·4 ± 5·1 nmol/flask (n= 5) and 3·5 ± 2·8 nmol/flask (n= 5) respectively. Addition of glucose had no significant effect on palmitate oxidation but caused a 9-fold increase in esterification of palmitate into triacylglycerols. We conclude that cultured rat Sertoli cells can oxidize palmitate to CO2and ketone bodies and that fatty acids appear to be a major energy substrate for these cells.

Summary.The testes of the blue fox (Alopex lagopus) showed marked seasonal variations in size. Testicular weight and volume increased rapidly during January and February to reach maximal values by the beginning of the breeding season (~15 March). During May and June the weights and volumes of the testes declined gradually to the quiescent state which lasted from July until October. Quantitation by DNA flow cytometry of the seasonal changes in the relative numbers of haploid (1C), diploid (2C) and tetraploid (4C) cell numbers in the testis showed that the increase in testis size from December to February was associated with a rapid expansion of the haploid cell compartment as spermatogenesis resumed. In addition, an increase in number of more mature cell types within the haploid cell population was observed over a 2-month period before the breeding season. The decline in testicular size from the middle of April until October was associated with a reduction in both the absolute and relative sizes of the haploid and tetraploid cell populations and a concomitant increase in the relative numbers of diploid cells. Measurements of the activity of the soluble Mn2 +-dependent adenylate cyclase revealed seasonal variations that closely paralleled those of the haploid cell population, indicating that, as in other species, the enzyme may be associated with maturing germ cells.

Summary.Cryptorchidism was associated with increased responsiveness of the isoproterenol-sensitive adenylate cyclase in membrane particles from rat testis. Abdominal testes from uni- and bilaterally cryptorchid rats showed the same activities. The change in isoproterenol-responsive adenylate cyclase was independent of the age at which the animals were made cryptorchid. The isoproterenol response was maximal 3–4 weeks after the rats were made cryptorchid. By 2–3 months after orchidopexy the isoproterenol response in the rat testis had decreased to normal control values.

Summary.The response of rat Leydig cells to hCG in vitrowas examined using decapsulated testes and enriched Leydig cell suspensions of a known purity. The secretion of testosterone and 5α-androstan-3α,17β-diol was used as the response and medium extracts were assayed before and after celite chromatography. At least 4 important phases in the development of steroidogenic function of the Leydig cells were detected. (1) A period after birth up to 10 days of age when the Leydig cell preferentially secretes testosterone. The cells may constitute the remnants of a fetal Leydig cell population. (2) A period which begins between 10 and 15 days of age and continues until 35–40 days of age, when the major products of the Leydig cell are 5α-reduced metabolites of testosterone, especially 5α-androstan-3α,17β-diol. (3) A phase at 20–30 days of age when responsiveness of the Leydig cell to hCG in vitroundergoes a distinct increase (the quantity of androgen produced with maximum hCG stimulation) and attains adult levels, although the sensitivity of the Leydig cells to hCG (the dose of hCG eliciting a half-maximal response, ED50) is gradually decreased after Day 20. (4) A period from 35–40 days of age into adulthood when testosterone increasingly becomes the major secretory product of the Leydig cell.Hypophysectomy for 8 days decreased the quantity of androgens produced per 106Leydig cells in response to hCG to an extent dependent on age. At 28 days of age steroidogenic function was impaired to a much greater extent (reduced by some 80–90%) than at 38 or 58 days (reduced by 40–50%). At all ages the sensitivity of the Leydig cells to hCG was increased after hypophysectomy.

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.

Soluble Mn2+ -dependent adenylate cyclase (MnAC) activity was found in testicular cytosol from blue foxes castrated during the breeding season. The rate of MnAC activity was approximately constant for 30 min at 35°C and for 2 hr after storage at 25°C. Activity was directly proportional to cytosol protein concentration and was optimal in the physiological pH range. Enzyme activity declined in the presence of an alkylating agent (N-ethyl maleimide, NEM) and was eliminated at a concentration of 1 mM NEM. Low concentrations (0.1—10 mM) of a reducing agent (β-mercapto ethanol, βME) did not increase MnAC activity, whereas a high concentration (100 mM) led to a significant reduction (p< 0.01) in activity. Substitution of Mn2- in the assay medium with Mg2- led to a total loss of enzyme activity, which could not be regained by adding hormones or by preincubation of cytosol for 60 min. The Km for Mn2+ was estimated to be 3.5 mM. The affinity of the enzyme for Mn2+ was not altered by varying the concentration of ATP. In contrast, increasing concentrations of Mn2+ appeared to increase the affinity of the enzyme for MnATP2-. The Km for MnATP2- thus varied from 6 to 18 mM.

Seminiferous tubules contain a cytoplasmic androgen receptor similar to the receptors in the epididymis and ventral prostate. The presence of a cytoplasmic receptor indicates that androgens maintain spermatogenesis by a direct action on certain types of cells within the seminiferous tubule. The Sertoli cell appears to be one of the cell types containing androgen receptors and the receptor might also be present in spermatogonia, primary spermatocytes, or peritubular cells. The Sertoli cell is stimulated by FSH to produce an androgen-binding protein which may serve to increase the accumulation of androgen in the seminiferous epithelium and make it available for binding by intracellular androgen receptors. This may be a way in which FSH enhances the action of androgen on spermatogenesis. Androgens act on the Sertoli cell to increase its response to FSH. This action of androgens on the Sertoli cell results in increased production of androgen-binding protein and may enhance the production of other substances which exert trophic effects on spermatogenesis.

Following incubation of slices from benign nodular prostatic hyperplasia (BNPH) with [3H] testosterone in vitro, a selective accumulation and retention of radioactivity was found in the prostatic tissue. The uptake and retention of androgen by the pyramidalis muscles was significantly lower.The 105 000 × g supernatant fraction of homogenates obtained from BNPH contained androphilic macromolecules excluded from Sephadex G-100 gel, both after incubation of the slices from [3H] testosterone and [3H] 5α-dihydrotestosterone, and after incubation of prostatic cytosol directly with [3H] 5α-dihydrotestosterone in vitro.The 600 × g crude nuclear fraction of homogenized BNPH contained salt extractable androgen-binding macromolecules. Similar results were also obtained when purified nuclear fractions were examined.