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The assay for cyclic nucleotide phosphodiesterase has been applied, with certain modifications, to the measurement of the soluble forms of these enzymes in the rat testis. The homogenization and incubation conditions were adjusted to achieve linear product formation as a function of time and protein concentrations and the resulting products were isolated by ion exchange chromatography using 5 mM HCl as the eluting agent. Phosphodiesterase activities were present in the testicular cytosol (105,000 g supernatant) of adult rats which were capable of hydrolyzing cAMP with a high (Km2 microM) and low Km20 microM) affinity and GMP with a relatively high affinity (Km3 microM). The low affinity cAMP enzyme activity could be stimulated with divalent ions such as calcium, magnesium, and manganese. At 18 days of age, all three enzyme activities were present in the testis, although both the high and low affinity cAMP phosphodiesterase displayed maximal rates (Vmax) that were only one third of the adult testis (when expressed per mg protein).

In recent years a multiplicity in isoforms of cAMP-dependent protein kinases has been revealed. Gene products for four different regulatory subunits (RI alpha, RI beta, RII alpha, RII beta) and two different catalytic subunits (C alpha, C beta) have been identified. We hereby present the molecular cloning of rat cDNAs for RII beta, as well as full-length human cDNAs for RII beta and RI alpha. The amino acid sequences deduced from the cDNAs of the regulatory subunits, revealed dissimilarities which were primarily confined to the N-terminal part of the protein. Based on the vital role in testicular function played by gonadotropin-induced activation of cAMP-dependent protein kinases, mRNA levels for the various subunits of cAMP-dependent protein kinase have been studied in rat testis. A clear pattern of cellular localization of mRNAs for the various subunits of cAMP-dependent protein kinase has been demonstrated. Furthermore, stimulation of Sertoli cells by FSH and cAMP elicited a differential response in mRNA levels for various subunits. A dramatic increase (30-40 fold) in the mRNA for RII beta (3.2 kb) was seen with cAMP stimulation, whereas such treatment had minor effects on mRNAs for RI alpha, RII alpha and C alpha. A distinct pattern of expression for various subunits of cAMP-dependent protein kinase was observed during germ cell differentiation. RI alpha and RI beta were expressed at high levels at early stages of spermatogenesis, whereas unique mRNAs for RII alpha and RII beta appeared in post-meiotic germ cells. Altogether, the present results demonstrate specific expression of mRNAs for different subunits of cAMP-dependent protein kinase in different cell types, during hormonal stimulation and during cellular differentiation. This indicates that the individual subunits may confer specific functional properties to the cAMP-dependent protein kinase holoenzyme and to the cAMP signal pathway of the cell.

Testis nuclei of hypophysectomized rats selectively accumulate labeled testosterone and 5alpha-dihydrotestosterone following the injection of tritiated testosterone in vivo. Testosterone and 5alpha-dihydrotestosterone are bound to macromolecules in nuclei and can be extracted with 0.5 M KCl. Accumulation of protein bound radioactive androgens in nuclei of isolated seminiferous tubules is similar to that of whole testis. The relative amounts of testosterone and dihydrotestosterone in purified nuclei were similar to the relative amounts bound to cytoplasmic receptors, suggesting that cytoplasmic androgen-receptor complexes may be transported into the nuclei. Binding of labeled androgen is saturable and inhibited by prior injection of unlabeled testosterone or cyproterone acetate. Nuclear binding sites are destroyed by the proteolytic enzyme pronase, but not by DNase. Like the cytoplasmic androgen-receptor complexes in rat testis, nuclear androgen-protein complexes are heat labile and dissociate slowly at 0 degrees C. androgens fail to accumulate in testis nuclei of the Stanley-Gumbreck androgen insensitive rat, a species lacking cytoplasmic androgen receptors in testis and other androgen target tissues.

The present paper describes some physiocochemical properties of the soluble 3 alpha-oxidoreductases in the rat testis and prostate, and comparison with rat epididymal 3 alpha-oxidoreductase, published previously (Hastings & Hansson 1979). The testicular enzyme shows properties very similar to that in the epididymis (size, stability, pH optium) except for minor differences in charge (iso-electric point). The prostatic enzyme revealed a slightly higher molecular weight, and was more sensitive to heating than those in the testis and epididymis, whereas the iso-electric point was the same as that in the testis (pI-5.25). The enzymes in all tissues exhibit very similar shapes (f/fo 1.14-1.17). The similar properties of the testicular and prostate 3 alpha-oxidoreductases to those previously reported for that in the epididymis may indicate that these enzymes represent identical peptide chains. The small differences observed in size, temperature stability and change may be due to their presence in different environments.

The case of a 74-year-old woman with the rare popliteal pterygium syndrome is presented. This syndrome is inherited as an autosomal dominant trait with incomplete penetrance and varying expression and consists of cleft lip and palate, lip pits, genital anomalies, popliteal pterygium, and malformations of the extremities. The various treatments our patient underwent over the years are reported. Treatment of popliteal pterygium involves special problems whem removing the skin fold because the nerve and vascular cords lie immediately anterior to the posterior fibrous cord. In the present case there are widespread arthrotic changes, both in the extremity joints and in the spine. These patients are short in stature. This, together with the general arthropathy, suggests a hereditary metabolic disturbance in the cartilaginous tissue.

In the present study we have examined the effects of FSH, forskolin, and (Bu)2cAMP on messenger RNA (mRNA) levels for all known subunits of cAMP-dependent protein kinase in rat Sertoli cells, using newly developed complementary DNA (cDNA) probes. mRNAs for the three regulatory subunits [RI alpha, RII51, (RII beta), and RII54 (RII alpha)] and the catalytic subunit C alpha were shown to be present in cultured rat Sertoli cells, whereas mRNAs for the subunits designated RI beta and C beta were below the level of detection. A high-levelled, concentration-dependent increase in a 3.2 kilobase mRNA for RII51 was observed when cultured immature Sertoli cells were incubated with increasing concentrations of (Bu)2cAMP (10(-6) to 5 X 10(-3) M) for 16 h. Densitometric scanning indicated a maximal stimulation by (Bu)2cAMP of 30- to 40-fold. Incubation with forskolin (100 microM) and FSH (200 ng/ml) gave rise to a smaller but significant increase in mRNA for RII51. When cultured Sertoli cells were incubated in the presence of 10(-4) M (Bu)2cAMP for varying time periods, there was a biphasic regulation of mRNA for RII51. (Bu)2cAMP caused an initial increase in mRNA for RII51 with maximal levels obtained after 10-16 h, after which a time-dependent decrease was observed. For the other three subunits present in Sertoli cells (RI alpha, RII54, and C alpha) a smaller but significant stimulation by (Bu)2cAMP and forskolin (2-4 fold) was seen. The functional implications of these changes in mRNA levels for the different subunits of cAMP-dependent protein kinase have not yet been revealed. However, our data clearly demonstrate differential regulation of the various subunits of cAMP-dependent protein kinase in Sertoli cells. Furthermore, these results document the presence of distinct adaptational changes taking place at the level of cAMP-dependent protein kinase in response to long term elevation of cAMP.

Production of testicular androgen binding protein (ABP), ceases following hypophysectomy and can be stimulated by FSH. Within 24 h after the administration of FSH, ABP can be measured in caput epididymis supernatant and by 4 days after FSH treatment, the concentration of ABP reaches a plateau. In a 3-day assay, ABP production in immature hypophysectomized rats was stimulated by 31 mug NIH-FSH-P1 per day (0.08 U NIH-FSH-P1 per 3 days) which is comparable to the sensitivity of the ovarian weight augmentation test in hypophysectomized rats. The relative ovarian weight augmenting and ABP stimulating activities of various FSH preparations were in agreement, suggesting that the biological stimulus of the ABP response is, in fact, FSH. The ABP response to FSH could become a useful testicular bioassay for FSH. Such an assay would be more practicle if ABP could be measured by a radioimmunoassay.