Scrotal heat stress causes sperm chromatin damage and cysteinyl aspartate-spicific proteinases 3 changes in fertile men.

PURPOSE: To observe changes in semen parameters, sperm DNA integrity, chromatin condensation and cysteinyl aspartate-spicific proteinases (Caspase-3) in adult healthy men after scrotal heat stress (SHS). METHODS: The scrotums of 19 healthy male volunteers were exposed to the condition of 40-43 °C SHS belt warming 40 min each day for successive 2 days per week. The course of SHS was continuously 3 months. Routine semen analysis, hypo-osmotic swelling (HOS) test, eosin Y (EY) staining sperm HOS and chromatin dispersion (HOS/SCD) test, HOS and aniline blue (HOS/AB) staining test were carried out before, during and after SHS. The activated Caspase 3 levels of spermatozoa were determined with a microtiter plate reader. RESULTS: The mean parameters of sperm concentration, motility and normal morphological sperm were significantly decreased in groups with sperm being collected during SHS 1, 2 and 3 months when compared with those in groups of pre-SHS (P < 0.01). Statistically significant differences of sperm DNA fragmentation, normal sperm membrane and vitality, and Caspase-3 activity were observed between the groups of before SHS and after SHS 3 months and the groups of during SHS 1, 2 and 3 months (P < 0.001). Three months the SHS stopped, various parameters recovered to the level before SHS. Abnormal sperm with HOS/AB and HOS/SCD showed a negatively significant correlation with normal sperm by HOS/EY test, and WBC in semen showed a positively significant correlation with Caspase-3 activity. The percentage of abnormal sperm by using the test of HOS/SCD showed a positively significant correlation with that of HOS/AB. CONCLUSIONS: The continuously constant SHS can impact the semen quality, sperm DNA integrity, chromatin condensation and Caspase-3, and the combination of HOS plus AB test may simultaneously determine the integrity of membrane and chromatin condensation at the same spermatozoon.

The KAT6B-related disorders genitopatellar syndrome and Ohdo/SBBYS syndrome have distinct clinical features reflecting distinct molecular mechanisms.

Genitopatellar syndrome (GPS) and Say-Barber-Biesecker-Young-Simpson syndrome (SBBYSS or Ohdo syndrome) have both recently been shown to be caused by distinct mutations in the histone acetyltransferase KAT6B (a.k.a. MYST4/MORF). All variants are de novo dominant mutations that lead to protein truncation. Mutations leading to GPS occur in the proximal portion of the last exon and lead to the expression of a protein without a C-terminal domain. Mutations leading to SBBYSS occur either throughout the gene, leading to nonsense-mediated decay, or more distally in the last exon. Features present only in GPS are contractures, anomalies of the spine, ribs and pelvis, renal cysts, hydronephrosis, and agenesis of the corpus callosum. Features present only in SBBYSS include long thumbs and long great toes and lacrimal duct abnormalities. Several features occur in both, such as intellectual disability, congenital heart defects, and genital and patellar anomalies. We propose that haploinsufficiency or loss of a function mediated by the C-terminal domain causes the common features, whereas gain-of-function activities would explain the features unique to GPS. Further molecular studies and the compilation of mutations in a database for genotype-phenotype correlations (www.LOVD.nl/KAT6B) might help tease out answers to these questions and understand the developmental programs dysregulated by the different truncations.

Activation of extracellular signal-related kinases 1 and 2 in Sertoli cells in experimentally cryptorchid rhesus monkeys.

AIM: To assess the spatiotemporal changes in the expression of extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Jun N-terminal kinases (JNK) and p38 mitogen-activated protein kinases (MAPK) in response to heat stress in the cryptorchid testis, and to investigate a possible relation to Sertoli cell dedifferentiation. METHODS: Immunohistochemistry and western blot were used to examine the expression and activation of ERK1/2, p38 and JNK in the cryptorchid testis at various stages after experimental cryptorchidism. RESULTS: The abdominal temperature did not obviously change the total ERK1/2 expression but significantly activated phospho-ERK1/2 in the Sertoli cells of the cryptorchid testis. Heat stress increased total JNK expression in the Sertoli cells of the cryptorchid testis but did not activate phospho-JNK. Neither total p38 nor phospho-p38 was induced by heat stress in the Sertoli cells of the cryptorchid testis. Changes in the spatiotemporal expression of cytokeratin 18 (CK18), a marker of immature or undifferentiated Sertoli cells, were induced in the cryptorchid testis in a pattern similar to the activation of ERK1/2. CONCLUSION: The activation of ERK1/2 in the testis may be related to dedifferentiation of Sertoli cells under heat stress induced by experimental cryptorchidism.

Pharmacology and thermosensitivity of the dartos muscle isolated from rat scrotum.

1 The dartos is a thin sheet of smooth muscle closely associated with the skin of the scrotum. Although known to play an important role in scrotal thermoregulation, there has been no detailed study into the pharmacology, or thermosensitivity, of the dartos from any species. Here, we investigate these two parameters in the isolated dartos muscle from rat. 2 Field stimulation of the rat dartos caused contractions that were abolished by tetrodotoxin, phentolamine and guanethidine, but unaffected by atropine or L-N(G)-nitroarginine. Exogenous noradrenaline also produced contractions blocked by both phentolamine and prazosin. In muscles with raised tone and negated sympathetic function, field stimulation failed to elicit relaxation. The dartos muscle did not contract in response to carbachol, nicotine, histamine, 5-hydroxytryptamine (all up to 100 micro M) or substance P (up to 1 micro M). 3 Contractile responses to field stimulation and noradrenaline were much greater at 30 degrees C compared with 40 degrees C; indeed, contractions to 1 micro M noradrenaline at 30 degrees C were relaxed by around 80% on heating to 40 degrees C. Similar heat-induced relaxations were observed during contractions to both U46619 (100 nM) and high K (70 mM). 4 In contrast, contractile responses to the myosin phosphatase inhibitor calyculin-A (1 micro M), either in the presence or absence of external calcium, were resistant to relaxation by heating. In calcium-free medium at 30 degrees C, U46619 continued to produce contractions that were again relaxed by 80% on heating to 40 degrees C. However, in the presence of calyculin-A, this heat-induced relaxation was greatly reduced. 5 Thus, the rat dartos muscle receives a functional sympathetic innervation and contracts to noradrenaline via alpha-adrenoceptors. There is no functional inhibitory innervation. Experiments with calyculin-A suggest that myosin phosphatase is a major contributor to the marked thermosensitivity of the dartos muscle.

Activin and inhibin have opposite effects on steroid 5 alpha-reductase activity in genital skin fibroblasts.

The transforming growth factor beta (TGF-beta) superfamily includes several closely related peptides including the activins and inhibins. Since we recently reported that TGF-beta 1 and beta 2 are potent inducers of steroid 5 alpha-reductase (5 alpha R), we have now studied the effects of these other peptides using primary cultures of human scrotal skin fibroblasts. Recombinant human activin A or inhibin A were added to cultured cells (2 x 10(5) cells) for 2 days in a serum free media and 5 alpha R activity was measured by the %-conversion of tracer [3H]-testosterone to dihydrotestosterone (DHT) over a 4-h period. Activin significantly stimulated 5 alpha R activity in a dose related manner (control 3.0 +/- 0.4%, activin (1.2 x 10(-9) M) 6 +/- 0.7%, P < 0.01, (2.4 x 10(-9) M) 8.5 +/- 0.6%, P < 0.001). In comparison, androgen (DHT 10(-7) M) induction of 5 alpha R was 4.7 +/- 0.2%, P < 0.05. Combined exposure of fibroblasts to activin (1.2 x 10(-9) M) and androgen (10(-7) M) did not result in additive or synergistic effect on 5 alpha R activity. In contrast, exposure of cells to an androgen (10(-7) M) and TGF-beta (2 x 10(-10) M) led to synergistic effects on 5 alpha R activity (control 1.5 +/- 0.1%, DHT 2.6 +/- 0.2% TGF-beta 1 4.8 +/- 0.5, TGF-beta 1 + DHT 9.2 +/- 1.2%).(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of transforming growth factor beta and epidermal growth factor on steroid 5 alpha-reductase activity in genital skin fibroblasts.

Regulation of steroid 5 alpha-reductase (5 alpha R) activity and dihydrotestosterone (DHT) formation is central to prostate and sexual skin (hair) growth and cell function. Transforming growth factor-beta 1 (TGF-beta 1) is a ubiquitous peptide present in skin and scrotal tissue and its receptor is universally expressed. We have explored the role of TGF-beta 1 and -beta 2 on androgen formation in skin. Rat or human sexual skin fibroblasts were grown in primary cultures (passage 3-7). 5 alpha-Reductase activity was measured by the %-conversion of tracer 3H-testosterone to dihydrotestosterone over a 4 h period. Incubation of scrotal fibroblasts (2 x 10(5) cells) in serum and growth factor free media with androgen, such as DHT for two days significantly stimulates 5 alpha R in these cells (1.6-fold, p < 0.05 vs control). TGF-beta 1 alone at picomolar concentrations (2 x 10(-11) M to 2 x 10(-10) M) was a potent inducer of 5 alpha R activity in both rat (1.8-fold and 2.8-fold, respectively, p < 0.001 vs control at both doses) and human cells (TGF-beta 1 2 x 10(-10) M 3.3-fold, p < 0.001 vs control). Combined exposure of these fibroblasts to TGF-beta 1 (2 x 10(-10) M) and androgen (10(-7) M) further potentiated 5 alpha R activity (rat cells 6.5-fold, human cells 6.4-fold, p < 0.001 vs DHT or TGF-beta 1 alone).(ABSTRACT TRUNCATED AT 250 WORDS)

5 alpha-Reductase activity in the genital skin of hirsute women.

A simplified, rapid, and highly reproducible technique is described for measuring 5 alpha-reductase activity (5 alpha RA) in small skin biopsies. Human genital skin was obtained from 23 nonhirsute and 20 hirsute premenopausal women (HW) and 5 normal men. Skin samples were minced at 4 C and incubated with RPMI-1640 in the presence of 95% O2-5% CO2 and 4.15 nmol [14C]testosterone ([14C]T) for 2 h at 37 C. Steroids were extracted with diethyl ether and separated by Celite and paper chromatography. Radioactivity in specific eluates was quantified, and the mass of each steroid was measured by RIA. The separate formation of 5 alpha-androstane-17 beta-ol-3-one (DHT), 5 alpha-androstane-3 alpha, 17B diol (3 alpha diol), androstenedione, and androsterone from [14C]T was measured. In separate experiments it was demonstrated that an incubation time of 2 h was optimum and that the addition of cofactors was unnecessary. Radiochemical purity was confirmed after chromatography. The mean +/- SE conversion ratio (CR) of T to DHT (in 2 h) in HW was higher than that in normal women (16.80 +/- 1.62% vs. 4.48 +/- 0.36%; P less than 0.01). In men, the CR of T to DHT averaged 31.60 +/- 3.96%. Individual values for the CR of T to DHT in HW and normal women did not overlap. The CR of T to 3 alpha diol was significantly higher in HW (9.66 +/- 0.86%) and men (15.98 +/- 2.0%) compared to that in normal women (2.96 +/- 0.32%; P less than 0.05). The CR of T to androstenedione was significantly greater in HW and men (6.18 +/- 0.42 and 7.28 +/- 1.92%) compared to that in normal women (2.64 +/- 0.64%; P less than 0.05). The CR of T to androsterone was very low and was similar in the three groups. The production of DHT in HW (4.50 +/- 1.0 pmol/mg X 2 h) was significantly greater than that in normal women (0.48 +/- 0.08; P less than 0.01) and was similar to the production in men (6.18 +/- 1.94 pmol/mg X 2 h). There was a significant correlation between the CR of T to DHT and DHT production, and the CR of T to 3 alpha diol and 3 alpha diol production as well as between the CRs of T to DHT and T to 3 alpha diol. These data suggest that measurements of DHT formation are best suited for the assessment of 5 alpha RA and that the measurement of 5 alpha RA in vitro from small skin biopsies is suitable for the clinical evaluation of hirsutism.

The melanogenic response to testosterone in scrotal epidermis: effects on tyrosinase activity and protein synthesis.

Pigmentation of the scrotum of the black-pelted rat, as expressed through melanocyte melanogenic activity, is controlled by androgens. Castration decreased in vitro incorporation of [14C]tyrosine into melanin. Testosterone pre-treatment for 4 days increased malanin radioactivity over castrate cont rols; the increment in vitro was prevented by an inhibitor of protein systhesis (cycloheximide) added to the incubation. However, cycloheximide only partially blocked melanin synthesis when added to tissue from animals hromone treated for 6 days in vivo, and was ineffective in tissue from intacts. Bulk protein snthesis in vitro (incorporation of [14C]tyrosine or -leucine) was not affected by castration or testosterone treatment but was uniformly inhibited by cycloheximide. The data suggest that new synthesis of specific protein in vitro was necessary for initial hormone-stimulation of melanogenesis, but with longer exposure to hormone sufficient protein was pre-synthetized in vivo to permit melanogenesis during incubation with the inhibitor.

Diminished 5alpha-reductase activity in extracts of fibroblasts cultured from patients with familial incomplete male pseudohermaphroditism, type 2.

The activity of 5alpha-reductase, the enzyme that converts testosterone to dihydrotestosterone, has been assessed in cell-free extracts of fibroblasts grown from foreskin, labia majora, scrotum, and nongenital skin from control subjects, from patients with developmental defects of the urogenital system, drom two subjects with the type 2 form of familial incomplete male pseudohermaphroditism and from individuals with other forms of hereditary male pseudohermaphoditism. Enzyme activity was shown to be maximal in the pH range of 5 to 6. Substrate specificity studies indicated that the enzyme so assayed is the 5alpha-reductase previously characterized in human foreskin. The activity of the enzyme was low in normal fibroblasts grown from nongenital skin and high in most fibroblasts grown from genital skin. 5alpha-Reductase activity in extracts of foreskin fibroblasts from two subjects with the type 2 disorder was undetectable at pH 5.5. Activity in comparable fibroblast extracts from most patients with other forms of hereditary male pseudohermaphroditism was easily measurable.