Wiley

ABSTRACT: Physical and psychosocial stress challenge homeostasis, increasing glucocorticoid secretion (in rodents, corticosterone [CORT]) while decreasing testosterone (T) levels. The dynamics of stress‐induced changes in T, CORT, and luteinizing hormone (LH) concentrations in mice have not been investigated previously. In particular, it remains to be established whether there is a rapid effect of CORT that is directly mediated by glucocorticoid receptors (GRs) in the testis. Therefore, serum and intratesticular T, serum CORT, and LH levels were measured during acute immobilization (IMO) stress, using the C57BL/6 strain of mice. The effects of testicular GR blockade were investigated by administration of the GR antagonist, RU486, via intratesticular (IT) or intraperitoneal (IP) injection. CORT levels increased in stressed males starting at 15 minutes, reaching a fivefold higher plateau by 1 hour compared with controls (P < .01). Conversely, starting from 30 minutes on, both serum and intratesticular T levels decreased in stressed males to 30% and 8% of control values, respectively, by 6 hours (P < .01). In contrast, LH was unchanged by IMO stress for up to 6 hours. Intratesticular treatment with RU486 partially prevented the IMO‐induced decline in T levels. CORT treatment reduced intracellular cyclic adenosine monophosphate (cAMP) content in Leydig cells by 15 minutes and T production by 30 minutes in vitro. We conclude that 1) the rapid changes in T suggest a suppression of T biosynthesis by glucocorticoid through a nongenomic mechanism, lowering the production of cytoplasmic cAMP; 2) changes in gonadotropic stimulation of Leydig cells are unlikely to explain the suppression of T levels during acute stress; and 3) the results are consistent with a direct inhibitory action of CORT on Leydig cells.

ABSTRACT: The current study was designed to examine the actions of a model endocrine disruptor on embryonic testis development and male fertility. Pregnant rats (F0) that received a transient embryonic exposure to an environmental endocrine disruptor, vinclozolin, had male offspring (F1) with reduced spermatogenic capacity. The reduced spermatogenetic capacity observed in the F1 male offspring was transmitted to the subsequent generations (F2‐F4). The administration of vinclozolin, an androgen receptor antagonist, at 100 mg/kg/day from embryonic day 8–14 (E8‐E14) of pregnancy to only the F0 dam resulted in a transgenerational phenotype in the subsequent male offspring in the F1‐F4 generations. The litter size and male/female sex ratios were similar in controls and the vinclozolin generations. The average testes/body weight index of the postnatal day 60 (P60) males was not significantly different in the vinclozolin‐treated generations compared to the controls. However, the testicular spermatid number, as well as the epididymal sperm number and motility, were significantly reduced in the vinclozolin generations compared to the control animals. Postnatal day 20 (P20) testis from the vinclozolin F2 generation had no morphological abnormalities, but did have an increase in spermatogenic cell apoptosis. Although the P60 testis morphology was predominantly normal, the germ cell apoptosis was significantly increased in the testes cross sections of animals from the vinclozolin generations. The increase in apoptosis was stage‐specific in the testis, with tubules at stages IX—XIV having the highest increase in apoptotic germ cells. The tubules at stages I—V also had an increase in apoptotic germ cells compared to the control samples, but tubules at stages VI—VIII had no increase in apoptotic germ cells. An outcross of a vinclozolin generation male with a wild‐type female demonstrated that the reduced spermatogenic cell phenotype was transmitted through the male germ line. An outcross with a vinclozolin generation female with a wild‐type male had no phenotype. A similar phenotype was observed in outbred Sprague Dawley and inbred Fisher rat strains. Observations demonstrate that a transient exposure at the time of male sex determination to the antiandrogenic endocrine disruptor vinclozolin can induce an apparent epigenetic transgenerational phenotype with reduced spermatogenic capacity.

Testicular germ cell apoptosis in the cryptorchid testis is induced by abdominal heat stress. p53‐dependent apoptosis appears responsible for the initial phase of germ cell loss in experimental cryptorchidism based on a 3‐day delay of apoptosis in p53⁻¹⁻ mice. However, the mechanisms underlying the subsequent p53‐independent apoptosis have not been identified. Although studies have suggested that Fas plays a role in testicular germ cell apoptosis, no direct evidence has been shown. To test the hypothesis that Fas is involved in testicular germ cell apoptosis and is responsible for the p53‐independent phase of apoptosis in the cryptorchid testis, p53⁻¹, Ipr/lpr (a spontaneous mutation in the Fas gene, which causes autoimmune disease) double‐mutant mice were generated and unilateral cryptorchidism was induced in these mice. It was found that testicular weight reduction and germ cell apoptosis were delayed by an additional 3 days, and the Fas production increased in the time frame of p53‐independent apoptosis in the experimental cryptorchid testis of wild‐type mice. These results suggest that Fas is involved in testicular germ cell apoptosis, and that Fas‐dependent apoptosis is responsible for the p53‐independent phase of germ cell apoptosis in the cryptorchid testis. The cascade of testicular germ cell apoptosis in response to heat stress implies the existence of sequential quality control mechanisms in spermatogenesis.

ABSTRACT: Androgen‐dependent male urogenital development occurs via mesenchymal‐epithelial interactions in which mesenchyme induces epithelial morphogenesis, regulates epithelial proliferation, and evokes expression of tissue‐specific secretory proteins. Mesenchymal‐epithelial interactions continue to be important into adulthood. For example, mesenchyme of the urogenital sinus (UGM) and seminal vesicle (SVM) induce dramatic morphologic and functional changes in various adult epithelia. Since adult epithelial cells are unquestionably responsive to mesenchymes that can elicit expression of alternative morphologic and functional phenotypes, established carcinomas might also be influenced by their connective tissue environment. In this regard, Dunning prostatic tumor has been induced by UGM or SVM to differentiate into tall columnar secretory epithelial cells. This change in cytodifferentiation is associated with a reduction in growth rate and loss of tumorigenesis. The role of soluble growth factors in the mechanism of mesenchymal‐epithelial interactions is discussed.

The effects of synthetic salmon calcitonin on primary Sertoli cell‐enriched cultures and on an established cell line (TM₄ cells, derived from immature mouse Sertoli cells) were studied. Synthetic salmon calcitonin stimulated the conversion of [³H]adenine to [³H]cyclic AMP in both cell systems. In addition, this peptide stimulated the secretion of rABP in primary Sertoli cellenriched cultures prepared from rat testis. Calcitonin also increased the total concentration of both androgen and estrogen receptors in TM₄ cells. Because cAMP analogs decreased androgen and estrogen receptor concentrations, the effect of calcitonin on sex steroid receptors may not be mediated by its effect on cyclic AMP in these cells. The possibility that the action of synthetic salmon calcitonin on the receptors might be mediated by a change in cellular Ca²⁺ was investigated. Lowering extracellular Ca²⁺ concentrations from 1.5 mM to less than 0.01 mM markedly reduced the concentration of androgen and estrogen receptors; restoration of Ca²⁺ to 1.5 mM returned receptor levels to normal. When the receptor concentrations were decreased by lowering extracellular Ca²⁺ concentrations to 0.5 mM, treatment with the calcium ionophore, A23187, restored receptor levels to normal. Although the calcium channel blocker, verapamil, decreased receptor levels, calcitonin partially counteracted its effect. Trifluoperazine, an inhibitor of calmodulin, also diminished androgen and estrogen receptor levels in the cytosol of TM₄ cells. It was concluded that calcitonin stimulates the formation of cyclic AMP and the secretion of rABP by Sertoli cells. This peptide also increases the concentration of androgen and estrogen receptors, possibly by a mechanism that is, in part, Ca²⁺‐mediated. These results, along with those on Leydig cells, suggest that calcitonin could be a regulator of testicular function.

ABSTRACT: Although the process of glycolysis is highly conserved in eukaryotes, several glycolytic enzymes have unique structural or functional features in spermatogenic cells. We previously identified and characterized the mouse complementary DNA (cDNA) and a gene for 1 of these enzymes, glyceraldehyde 3‐phosphate dehydrogenase‐s (Gapds). This gene is expressed only in spermatids. The enzyme appears to have an essential role in energy production required for fertilization, and it is reported to be susceptible to inhibition by certain environmental chemicals. We have now cloned and sequenced the cDNA for the human homo‐logue of glyceraldehyde 3‐phosphate dehydrogenase (GAPD2) and determined the structure of the gene. The messenger RNA (mRNA) was detected in testis, but not in 15 other human tissues analyzed by Northern blot technique. The deduced GAPD2 protein contains 408 amino acids and is 68% identical with somatic cell GAPD. GAPD2 has a 72‐amino acid segment at the amino terminal end that is not present in somatic cell GAPD. This segment is prolinerich but contains smaller stretches of polyproline and is 30 amino acids shorter than the comparable segment of mouse GAPDS. The structure of the human GAPD2 gene was determined by polymerase chain reaction (PCR) to identify exonintron junctions in a genomic clone and in total genomic DNA. The locations of these junctions in the GAPD2 gene corresponded precisely to those of the 11 exonintron junctions in the mouse Gapds gene. Immunohistochemical studies found that GAPD2 is located in the principal piece of the flagellum of human spermatozoa, as are GAPDS in mouse and rat spermatozoa. GAPD2 extracted from human spermatozoa and analyzed by Western blot technique migrated with an apparent molecular weight of ∼56 000, although the calculated molecular weight is 44501. The conserved nature of the mouse, rat, and human enzymes suggests that they serve similar roles in these and other mammalian species.

The mechanisms responsible for mediating the influence of sperm preparation protocols on human sperm function have been investigated. Techniques that involved the separation of motile spermatozoa prior to centrifugation were found to yield sperm suspensions of highest quality. If the spermatozoa were centrifuged prior to isolation of the motile cells, sperm function was impaired. The detrimental effects of centrifugation were associated with a sudden burst of reactive oxygen species production by a discrete subpopulation of cells (characterized by significantly diminished motility and fertilizing capacity) that could be separated from normal functional spermatozoa on Percoll gradients. If unfractionated sperm suspensions were subjected to centrifugation, the reactive oxygen species generated by this subpopulation impaired the functional competence of normal spermatozoa in the same suspension. Assessment of the ability of the antioxidants, butylated hydroxytoluene, and vitamin E, to curtail the peroxidative damage inflicted by such cells in response to centrifugation revealed a significant improvement of sperm function in the presence of vitamin E.

ABSTRACT: Varicocele is a condition of varicosity and tortuosity of the pampiniform plexus that is often associated with a reduction in the volume of the affected testicle. Today there is much debate about how much the varicocele actually damages the reproductive system and the mechanism through which this occurs. Furthermore, it has not yet clearly been established if treatment is truly useful to restore testicular function. The goal of this study was to evaluate changes in the volume of the affected testis after treatment and to examine any correlations between volume and seminal parameters. We evaluated 43 patients with left idiopathic varicocele with ultrasound scan of the testis before and after surgery; testicular volume was obtained using the ellipsoid formula. We also examined semen parameters before and at an average time of 1 year after the procedure, using the WHO indications. We performed 2 statistical analyses, comparing changes in testicular volume before and after surgery, and volume with seminal parameters. Statistical analysis shows a significant increase of testicular volume after varicocele treatment (P < .05). Furthermore, the total number of spermatozoa and fast progressive spermatozoa rates significantly increased after surgery (respectively P < .05 and P < .01) (Figure 1). The Spearman correlation coefficient shows a good relationship between testicular volume and total number of spermatozoa (r = .445; P = .01). Our data point to the possibility that the affected testicle could benefit in terms of trophism and function after varicocele treatment. Ultrasound scan at follow‐up permits assessment of not only the presence of recurrence, but it is also useful for evaluating trophism.

ABSTRACT: Hypoxia‐inducible factor‐1α (HIF‐1α) is a transcription factor that plays an essential role in oxygen homeostasis. HIF‐1α is constitutively made in cells; however, it is ubiquitinated and degraded under normoxic conditions. Hypoxia prevents the ubiquitination of HIF‐1α, resulting in stabilization of the protein and activation of target genes. Because of its vascular arrangement and the high metabolic demand of spermatogenesis, the testis has been described previously as functioning on the brink of hypoxia; thus, we have hypothesized that HIF‐1α is constitutively expressed and stabilized in the testis, where it could play a role in testicular homeostasis. Western blot analysis using nuclear proteins from liver, kidney, and testis revealed the presence of HIF‐1α only in the testis. Immunohistochemistry confirmed this result and revealed that HIF‐1α was specifically located in interstitial Leydig cells. Electromobility shift assays employing nuclear extracts from the TM3 Leydig cell line revealed that these cells express HIF‐1α that is capable of binding DNA under normoxic conditions. Furthermore, we found that protein levels can be increased further when the TM3 cells are cultured under hypoxic conditions. Finally, transient transfections of TM3 Leydig cells revealed that the promoter of the mouse 3β‐hydroxysteroid dehydrogenase type 1 (Hsd3b1) gene, which encodes a key enzyme in testosterone production, is a potential target of HIF‐1α. In conclusion, HIF‐1α is constitutively present in the Leydig cells of the murine testis, where it potentially regulates Hsd3b1 transcription, and thus male reproductive function.