Effects of endocrine disrupting chemicals on gonad development: Mechanistic insights from fish and mammals.

Over the past century, evidence has emerged that endocrine disrupting chemicals (EDCs) have an impact on reproductive health. An increased frequency of reproductive disorders has been observed worldwide in both wildlife and humans that is correlated with accidental exposures to EDCs and their increased production. Epidemiological and experimental studies have highlighted the consequences of early exposures and the existence of key windows of sensitivity during development. Such early in life exposures can have an immediate impact on gonadal and reproductive tract development, as well as on long-term reproductive health in both males and females. Traditionally, EDCs were thought to exert their effects by modifying the endocrine pathways controlling reproduction. Advances in knowledge of the mechanisms regulating sex determination, differentiation and gonadal development in fish and rodents have led to a better understanding of the molecular mechanisms underlying the effects of early exposure to EDCs on reproduction. In this manuscript, we review the key developmental stages sensitive to EDCs and the state of knowledge on the mechanisms by which model EDCs affect these processes, based on the roadmap of gonad development specific to fish and mammals.

Stability of the human sperm DNA methylome to folic acid fortification and short-term supplementation.

STUDY QUESTION: Do short-term and long-term exposures to low-dose folic acid supplementation alter DNA methylation in sperm? SUMMARY ANSWER: No alterations in sperm DNA methylation patterns were found following the administration of low-dose folic acid supplements of 400 µg/day for 90 days (short-term exposure) or when pre-fortification of food with folic acid and post-fortification sperm samples (long-term exposure) were compared. WHAT IS KNOWN ALREADY: Excess dietary folate may be detrimental to health and DNA methylation profiles due to folate's role in one-carbon metabolism and the formation of S-adenosyl methionine, the universal methyl donor. DNA methylation patterns are established in developing male germ cells and have been suggested to be affected by high-dose (5 mg/day) folic acid supplementation. STUDY DESIGN, SIZE, DURATION: This is a control versus treatment study where genome-wide sperm DNA methylation patterns were examined prior to fortification of food (1996-1997) in men with no history of infertility at baseline and following 90-day exposure to placebo (n = 9) or supplement containing 400 µg folic acid/day (n = 10). Additionally, pre-fortification sperm DNA methylation profiles (n = 19) were compared with those of a group of post-fortification (post-2004) men (n = 8) who had been exposed for several years to dietary folic acid fortification. PARTICIPANTS/MATERIALS, SETTING, METHODS: Blood and seminal plasma folate levels were measured in participants before and following the 90-day treatment with placebo or supplement. Sperm DNA methylation was assessed using the whole-genome and genome-wide techniques, MassArray epityper, restriction landmark genomic scanning, methyl-CpG immunoprecipitation and Illumina HumanMethylation450 Bead Array. MAIN RESULTS AND THE ROLE OF CHANCE: Following treatment, supplemented individuals had significantly higher levels of blood and seminal plasma folates compared to placebo. Initial first-generation genome-wide analyses of sperm DNA methylation showed little evidence of changes when comparing pre- and post-treatment samples. With Illumina HumanMethylation450 BeadChip arrays, no significant changes were observed in individual probes following low-level supplementation; when compared with those of the post-fortification cohort, there were also few differences in methylation despite exposure to years of fortified foods. LARGE SCALE DATA: Illumina HumanMethylation450 BeadChip data from this study have been submitted to the NCBI Gene Expression Omnibus under the accession number GSE89781. LIMITATIONS, REASONS FOR CAUTION: This study was limited to the number of participants available in each cohort, in particular those who were not exposed to early (pre-1998) fortification of food with folic acid. While genome-wide DNA methylation was assessed with several techniques that targeted genic and CpG-rich regions, intergenic regions were less well interrogated. WIDER IMPLICATIONS OF THE FINDINGS: Overall, our findings provide evidence that short-term exposure to low-dose folic acid supplements of 400 µg/day, over a period of 3 months, a duration of time that might occur during infertility treatments, has no major impact on the sperm DNA methylome. STUDY FUNDING/COMPETING INTERESTS: This work was supported by a grant to J.M.T. from the Canadian Institutes of Health Research (CIHR: MOP-89944). The authors have no conflicts of interest to declare.

Amifostine-doxorubicin association causes long-term prepubertal spermatogonia DNA damage and early developmental arrest.

BACKGROUND: In a previous study, we found that amifostine provides some protection to the seminiferous epithelium of prepubertal doxorubicin-treated male rats but does not improve their fertility status as adults. Based on these results, a long-term study was undertaken to evaluate the DNA damage caused to spermatogonia and the consequences for embryo development. METHODS: Twenty-four male prepubertal rats (30-day-old) were divided into four equal groups and treated with: doxorubicin (D--5 mg/kg), amifostine (A--400 mg/kg), amifostine/doxorubicin (AD--amifostine 15 min before doxorubicin) and control (C--0.9% saline solution). Sixty-four days after the treatment, animals were euthanized and the testes and epididymides were excised. The testes were fixed in Bouin's solution and historesin-embedded for histopathological analysis. Spermatozoa from the cauda epididymides were collected for chromatin structure analyses (Comet Assay and SCSA™). Adult rats (100-day-old) were mated with fertile females for embryo analyses on 2.5, 4.5 and 20 days post-coitum (d.p.c.). RESULTS: The seminiferous epithelium histopathology of AD group was better preserved compared with the D group. On the other hand, rats from the D and AD groups presented an increased percentage of sperm DNA strand breaks, as assessed by the comet assay, as well as an increased level of sperm chromatin denaturation, as assessed by the SCSA™ assay. In amifostine-treated groups (A and AD) there was a significant increase in the number of arrested embryos, as observed by the number of oocytes/zygotes on 2.5 d.p.c., when compared with control and doxorubicin groups; however, this number was increased when the AD group was compared with the A group. CONCLUSIONS: These results raise a concern about the effects of the association of these two drugs on the germ cell genome. Amifostine-doxorubicin-exposed rat spermatogonia produced long-term damage on sperm DNA, compromised conceptus development and reduced pregnancy outcome.

Characterization of sperm chromatin quality in testicular cancer and Hodgkin's lymphoma patients prior to chemotherapy.

BACKGROUND: Although the incidences of testicular cancer and Hodgkin's lymphoma have increased in young men over the past decade, combination chemotherapy has improved survival. As fertility is of importance to these patients, characterization of sperm chromatin structure is needed. We assessed sperm chromatin in testicular cancer and Hodgkin's lymphoma patients prior to chemotherapy, in comparison with control community and idiopathic infertile volunteers. METHODS: DNA damage was assessed with the sperm chromatin structure assay (SCSA), terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and comet assays; reactive thiols (SH) and DNA compaction were determined with the monobromobimane (mBBr) and chromomycin A3 (CMA3) assays, respectively. RESULTS: Both testicular cancer (37%) and Hodgkin's lymphoma (81%) patients had normospermic samples with increased DNA damage, compared with controls. Cancer patients also had higher reactive thiols and CMA3 staining, indicating low DNA compaction. CONCLUSIONS: Sperm DNA integrity and compaction were affected in testicular cancer and Hodgkin's lymphoma patients prior to chemotherapy. Although SCSA, TUNEL and comet assays all detected DNA damage, the latter was optimal for use in cancer patients. A combination of the comet assay with tests that evaluate sperm DNA compaction, such as flow cytometry-based CMA3 and mBBr assays, is a reliable strategy to characterize sperm chromatin quality in cancer patients at the time of sperm banking.

Sperm aneuploidy frequencies analysed before and after chemotherapy in testicular cancer and Hodgkin's lymphoma patients.

BACKGROUND Multicolour fluorescent in situ hybridization was utilized to detect sperm aneuploidy for chromosomes 13, 21, X and Y in testicular cancer and Hodgkin's lymphoma chemotherapy patients. METHODS Aneuploidy was assessed before, and 6, 12 and/or 18-24 months after, the initiation of chemotherapy, and compared with age matched controls. 635 396 sperm were scored blindly with 5000 sperm/patient/chromosome/ time point, where sperm was available. (First two phrases have been reversed). RESULTS Comparing testicular cancer and Hodgkin's lymphoma patients to each other and with controls, cancer-specific differences were identified. Hodgkin's lymphoma patients, particularly, exhibited significantly increased aneuploidy frequencies for all chromosomes throughout treatment. At 6 months, all cancer patients showed significantly increased frequencies of XY disomy and nullisomy for chromosomes 13 and 21. In general, aneuploidy frequencies declined to pretreatment levels 18 months after treatment initiation, but increased aneuploidy frequencies persisted in some chromosomes for up to 24 months. CONCLUSIONS Because of elevated aneuploidy frequencies prior to and up to 24 months from the start of chemotherapy, patients should receive genetic counselling about the potentially increased risk of an aneuploid conceptus from sperm cryopreserved prior to chemotherapy, and for conceptions up to 2 years after the initiation of treatment.

The effects of chemotherapy with bleomycin, etoposide, and cis-platinum on telomeres in rat male germ cells.

Co-administration of bleomycin, etoposide, and cis-platinum (BEP) has increased the 5-year survival rate of testis cancer patients to over 90%; however, this treatment induces chemotoxic effects on male germ cells. Treatment of male rats with BEP, using a similar schedule to that used in man, affects reproductive organ weights and sperm count, motility, and DNA integrity, as well as pup survival rates. Telomeres, specialized structures at the termini of chromosomes, play an important role in the maintenance of genetic stability. In previous studies, we demonstrated, using a spermatogonial cell line, that cis-platinum and bleomycin damage telomeres and that cis-platinum also inhibits telomerase activity. Our objective here was to test the hypothesis that in vivo exposure to the BEP regimen used to treat testis cancer targets telomeres in the male germ line. Adult male Brown Norway rats received chronic treatment with a BEP regimen. DNA double strand breaks were increased significantly in zygotene germ cells, as assessed by γ-H2AX immunofluorescence. Interestingly, treatment with this BEP regimen increased γ-H2AX foci in the telomere region of zygotene spermatocytes, but not in other germ cell types, such as pachytene cells, round spermatids, or elongating spermatids. Mean telomere lengths were reduced in zygotene, pachytene, round spermatid, elongating spermatid and cauda epididymal spermatozoa compared with the saline control group. Thus, telomere lengths did not recover during germ cell development. These studies demonstrate that BEP treatment is associated with an effect on telomeres.

Steroid delta 4-5 alpha-reductase and 3 alpha-hydroxysteroid dehydrogenase in the rat epididymis during development.

delta 4-5 alpha-Reductase activity is apparently regulated by a testicular factor(s) secreted directly into the epididymis, whereas 3 alpha-hydroxysteroid dehydrogenase activity, in this tissue, appears to reflect circulating androgen levels. To test whether the factor(s) regulating delta 4-5 alpha-reductase activity is directly associated with spermatozoa, a developmental study was undertaken to temporally correlate various parameters of the male reproductive tract with enzymatic activities. delta 4-5 alpha-Reductase activity is first detectable at 21 days of age. Activity increases until day 77, after which time enzymatic activity decreases by more than 60%, reaching steady adult values at 105 days. 3 alpha-Hydroxysteroid dehydrogenase activity is detectable as early as 7 days. Levels of this enzyme increase until day 63, after which time constant adult values are maintained until at least 1 yr. Spermatids and/or spermatozoa are first seen in the testes at 42 days, and plateau levels are reached by day 77. Spermatozoa are first seen in the epididymis at 49 days and reach maximal values by 91 days; no significant change occurs thereafter (until 365 days). Increases in seminal vesicle and ventral prostate weights are of a sigmoidal type, paralleling increases in plasma androgens, with the greatest rate of rise between days 35--63. This sigmoidal type of increase in tissue weights and plasma androgens is similar to that seen for epididymal 3 alpha-hydroxysteroid dehydrogenase but markedly different from that found for delta 4-5 alpha-reductase. The importance of delta 4-5 alpha-reductase and 3 alpha-hydroxysteroid dehydrogenase activities in the epididymis before the entry of spermatozoa and the decline in delta 4-5 alpha-reductase activity with age is discussed.

Orchidectomy induces a wave of apoptotic cell death in the epididymis.

The epididymis is the site where spermatozoa are matured and stored. After orchidectomy, this tissue loses up to 80% of its weight. In the prostate, androgen withdrawal by orchidectomy is associated with apoptotic cell death. The objective of the present study was to investigate whether apoptotic cell death is involved in the androgen-dependent weight loss found in the rat epididymis after orchidectomy. Adult male Sprague-Dawley rats were orchidectomized, and apoptotic cells were identified by in situ TUNEL (TdT-mediated dUTP-digoxigenin nick end-labeling) apoptosis detection. Apoptosis first appeared in the epithelium of the initial segment of the epididymis 18 h after orchidectomy, reached a maximum on day 2, and disappeared by day 5 postorchidectomy. In the caput epididymidis, apoptosis was first found after 24 h, reached a maximum by day 3, and was detectable until day 5. In the corpus epididymidis, apoptosis was first seen on day 4, peaked on day 5, and was undetectable by day 6 postorchidectomy. In the cauda epididymidis, apoptosis was first seen on day 5, peaked on day 6, and was occasionally detected on day 7. Throughout the rat epididymis, apoptotic cell death was localized specifically to principal cells. The presence of apoptosis was confirmed with the observation of a ladder of nucleosomal sized DNA fragmentation by using agarose gel electrophoresis. Androgen replacement therapy after orchidectomy demonstrated that apoptosis in the caput, corpus, and cauda epididymidis was androgen dependent. However, androgens alone could not completely prevent apoptosis in the initial segment of the epididymis. Efferent duct ligation induced a similar pattern of apoptosis in the initial segment of the epididymis as that seen after orchidectomy, but there were fewer apoptotic cells in the caput epididymidis, and no apoptotic cell death in the corpus and cauda epididymidis. We conclude that withdrawal of androgen by orchidectomy induces a wave of apoptotic cell death in the epididymis; we hypothesize that apoptosis in the initial segment is caused primarily by withdrawal of androgen as well as by luminal components coming from the testis.

Episodic prolactin secretion in the adult male rat is affected by orchidectomy and testosterone replacement.

To characterize the plasma PRL pattern in adult male rats and elucidate the modulatory effects of testosterone on this circulating PRL pattern, serial blood samples were obtained from both intact rats and rats orchidectomized and given various doses of testosterone via sustained release polydimethylsiloxane implants. Adult male rats were equipped with chronic indwelling jugular catheters. One week later the animals were orchidectomized and given either empty (12-mm) or testosterone-filled polydimethylsiloxane implants measuring 3, 5, or 12 mm that maintain plasma testosterone at 0%, 20%, 30%, or 60%, respectively, of those concentrations found in normal animals. Plasma samples for PRL RIA were obtained every 5-10 min for 3 h, before (intact control) and 3, 6, 9, 15, 21, and 28 days after orchidectomy and testosterone replacement. The plasma PRL pattern in intact animals was pulsatile; on the average, three or four pulses per 3 h, with amplitudes of 3.6 ng/ml on a 2.7 ng/ml nadir, were seen. After orchidectomy PRL pulse nadir, peak, and amplitude were rapidly attenuated. These parameters stabilized between days 6 and 15 at levels approximately 40% of those recorded in intact rats. In contrast, PRL pulse frequency remained in the control range for the first 9 days after orchidectomy. Thereafter, pulse frequency accelerated and reached stable plateau levels by day 15 at 145% of the values seen before orchidectomy. The administration of 3-mm testosterone implants completely prevented the effects of orchidectomy on PRL pulse nadir, peak, and amplitude, but only partially prevented the postorchidectomy rise in pulse frequency. Although the two larger implants (5 and 12 mm) had no further effect on pulse nadir, peak, and amplitude over that seen with the 3-mm implant, only the 12-mm implant completely prevented the acceleration in PRL pulse frequency accompanying orchidectomy. These results indicate that testosterone is intimately involved in regulation of the circulating PRL pattern and that this steroid has effects on the neuroendocrine system controlling PRL pulse frequency independent of those regulating pulse nadir, peak, and amplitude.

Regulation of sulfated glycoprotein-2 (clusterin) messenger ribonucleic acid in the rat epididymis.

Sulfated glycoprotein-2 (SGP-2) is secreted by the principal cells of the caput epididymidis and binds to spermatozoa as they transit through this segment. The regulation of SGP-2 in the epididymis is poorly understood. The objectives of these studies were to determine if SGP-2 messenger RNA (mRNA) concentrations in the epididymis are regulated by testosterone or during postnatal development. Northern blot analysis was done using denatured plasmid containing a complementary DNA insert for rat SGP-2. A single 2.1-kilobase transcript was present throughout the epididymis. SGP-2 mRNA concentrations were highest in the caput followed by the initial segment, the cauda, and the corpus epididymidis. To determine if androgens regulate SGP-2 mRNA concentrations, adult rats were bilaterally orchidectomized and testosterone was replaced for 7 days using steroid-filled capsules measuring 2.5 or 18.6 cm. In the initial segment and the caput epididymidis, neither orchidectomy nor testosterone replacement, at either dose, had any effect on SGP-2 mRNA concentrations. In the corpus and cauda epididymidis, bilateral orchidectomy resulted in a 3.5- and 9.4-fold increase, respectively, in SGP-2 mRNA concentrations, whereas testosterone replacement caused a dose-dependent decrease in SGP-2 mRNA concentrations. Unilateral orchidectomy was done to determine if SGP-2 mRNA concentrations are dependent on testicular factors released in the lumen of the epididymis. In the corpus and the cauda epididymidis, unilateral orchidectomy resulted in elevated SGP-2 mRNA concentrations in the ipsilateral epididymis. There were no changes in SGP-2 mRNA concentrations in the initial segment and caput epididymidis. These results provide complementary evidence that the message for SGP-2 is differentially regulated along the epididymis. During postnatal development SGP-2 mRNA concentrations in the caput-corpus epididymidis increased dramatically between 14 and 21 days as well as between 49 and 63 days. Interestingly, between 28 and 42 days, when serum testosterone concentrations are increasing, there was no change in the concentration of SGP-2 mRNA in the caput-corpus epididymidis. Similar results were observed in the cauda epididymidis with the exception that between 28 and 42 days, there was a dramatic decrease in SGP-2 mRNA in the cauda epididymidis. Together these experiments demonstrate that the regulation of SGP-2 mRNA concentrations is segment specific. In the initial segment and caput epididymidis there is no apparent regulation of SGP-2 by testicular factors,whereas in the corpus and cauda epididymidis testosterone can repress SGP-2 mRNA concentrations.

Expression of 4-ene steroid 5 alpha-reductase messenger ribonucleic acid in the rat epididymis during postnatal development.

The regulation of epididymal 5 alpha-reductase mRNA is multifactorial and segment-specific. To further investigate the regulation of the message for the enzyme, the expression of 5 alpha-reductase mRNA in the rat epididymis was studied as a function of postnatal development. Developmental changes in 5 alpha-reductase mRNA concentrations were assessed by probing Northern blots with the full-length cDNA for rat steroid 5 alpha-reductase. In the first experiment the effect of postnatal age on 5 alpha-reductase mRNA concentrations in the caput-corpus and cauda epididymides was studied. Male rats, taken at 1-week intervals between the ages of 7-91 days, were used. In both epididymal regions, the mRNA for 5 alpha-reductase was present at all ages examined; it appeared in the immature animal at least 2 weeks before detectable 5 alpha-reductase enzyme activity. In the caput-corpus epididymidis, mRNA levels for 5 alpha-reductase decreased by half between postnatal days 7 and 21, rose 5-fold by day 56, and then remained constant through day 91. No change with postnatal age, however, was observed in the cauda epididymidis. In the second experiment, the longitudinal distribution of 5 alpha-reductase mRNA on postnatal days 21, 42, 49, 56, 77, and 91 was studied. The mRNA levels for 5 alpha-reductase increased remarkably, by 6- to 7-fold, in the initial segment of the caput epididymidis between postnatal days 21 and 42 and stayed constant thereafter. However, no significant change comparable to that found in the initial segment was observed in the adjacent proximal caput region or in any of the other epididymal segments. Thus, the 5-fold rise in 5 alpha-reductase mRNA concentrations that occurred in the caput-corpus epididymidis in the first experiment can be attributed solely to changes in the initial segment. We conclude that steady state concentrations of epididymal 5 alpha-reductase mRNA vary dramatically at different postnatal ages and are highly specific with respect to epididymal segment.

Differential regulation of steady state 4-ene steroid 5 alpha-reductase messenger ribonucleic acid levels along the rat epididymis.

Epididymal nuclear 5 alpha-reductase enzyme activity is regulated by a testosterone-dependent factor from the testis. Regulation at the mRNA level, however, has not been investigated. Endocrine manipulation experiments were designed to determine whether 5 alpha-reductase is regulated at the steady state mRNA level. Steady state mRNA concentrations were assessed using the full-length cDNA for female rat liver 5 alpha-reductase. Longitudinal distribution showed that the highest mRNA concentrations were present in the initial segment of the caput epididymidis and were 3- to 7-fold higher than in the other tissue segments. The androgen dependence of the mRNA levels for 5 alpha-reductase was assessed by bilateral orchidectomy and simultaneous testosterone replacement therapy. One week after surgery, mRNA concentrations in orchidectomized rats were decreased to 15% of control levels in the initial segment of the caput epididymidis and to 40-50% of control levels in the remaining epididymal segments. Administration of testosterone at a dose that mimics normal serum concentrations (2.5-cm Silastic implant) restored 5 alpha-reductase mRNA concentrations to control levels in the corpus and cauda epididymidis, but these were not significantly different from orchidectomized levels (P greater than or equal to 0.05) in the initial segment and caput epididymidis. Administration of testosterone at a dose designed to approximate 5- to 8-fold normal serum concentrations (18.6-cm implant) maintained 5 alpha-reductase mRNA concentrations at only 50% of control levels in the initial segment, while complete maintenance was observed in the rest of the tissue. The effects of unilateral orchidectomy revealed that 5 alpha-reductase mRNA concentrations decrease selectively in the initial segment of the orchidectomized side. This is the first report that epididymal 5 alpha-reductase is regulated at the mRNA level and that the regulation is different with respect to the segment being studied.

Immunocytochemical localization of 4-ene steroid 5 alpha-reductase type 1 along the rat epididymis during postnatal development.

Dihydrotestosterone (DHT), the active androgen in many tissues, is synthesized from testosterone by the enzyme 4-ene steroid 5 alpha-reductase (5 alpha-reductase; EC 1.3.1.22). In the epididymis, the maturation and storage of spermatozoa are dependent on the presence of 5 alpha-reduced androgens. The regulation of epididymal 5 alpha-reductase is complex. To date, the regulation of this enzyme has been studied extensively at the level of enzymatic activity and more recently at the mRNA level. Regulation at the level of the protein, however, remains poorly understood. We have raised rabbit polyclonal antibodies to a 24-mer synthetic peptide whose sequence was determined from the predicted amino acid sequence for rat 5 alpha-reductase type 1 to immunolocalize the 5 alpha-reductase type 1 protein in the rat epididymis during postnatal development. Western blot analysis revealed a specific immunoreactive band of 26 kilodaltons in male rat liver, epididymis, and prostate; this apparent molecular size is identical to that obtained when the 5 alpha-reductase type 1 cDNA is expressed in mammalian cells. Furthermore, the relative protein levels, liver > epididymis > prostate, were consistent with the mRNA levels for type 1 rat 5 alpha-reductase. Perfusion-fixed paraffin-embedded epididymal tissue sections were used to immunolocalize type 1 5 alpha-reductase. In the adult rat epididymis, the most intense immunoperoxidase reaction was observed in a discrete lobule of the initial segment of the epididymis. A progressive decrease in staining intensity occurred distally along the tissue to the cauda epididymis. The staining reaction was specific to cytoplasmic elements of epithelial principal cells; no reaction was evident over nuclei. However, specifically in the initial segment, very intense staining was seen in the infranuclear region of the principal cells. In the proximal caput epididymidis, the staining was primarily confined to an oval region above the nuclei, whereas in the remaining epididymal regions, weak staining was seen throughout the cytoplasm. Thus, the intracellular localization of the 5 alpha-reductase type 1 protein changed as one moved down the epididymis. Finally, the pattern of immunolocalization of 5 alpha-reductase type 1 protein was different in the epididymis of rats of different postnatal ages. On day 7, no reactivity was noted; by day 28, a weak apical staining of principal cells was seen throughout the epididymis; by day 47, the adult pattern of staining had been established. Our results revealed that the expression and intracellular localization of the 5 alpha-reductase type 1 protein are both age dependent and epididymal segment specific.

Steady state steroid 5 alpha-reductase messenger ribonucleic acid levels and immunocytochemical localization of the type 1 protein in the rat testis during postnatal development.

Steroid 5 alpha-reductase is the rate-limiting enzyme in the production of 5 alpha-reduced steroids in many tissues. Developmental changes in 5 alpha-reductase activity play an important role in regulating the amount of testosterone that is secreted by the testis. To date, the regulation of testicular 5 alpha-reductase has been studied extensively at the level of enzyme activity. Regulation at the messenger RNA (mRNA) and protein levels, however, has not been investigated. The objectives of the present study were to determine the steady state mRNA levels for the 5 alpha-reductase isozymes, types 1 and 2, and to immunolocalize the 5 alpha-reductase type 1 protein in the developing rat testis (7-91 days postpartum). Consistent with previously reported enzyme activity studies, type 1 5 alpha-reductase mRNA levels were most abundant in the immature animal (days 21-28). Unlike 5 alpha-reductase activity, however, type 1 mRNA levels did not decline thereafter to reach nearly undetectable levels in the adult (day 91). In contrast, 5 alpha-reductase type 1 mRNA levels remained relatively constant between days 42-91. The 5 alpha-reductase type 1 transcript size did not remain constant during postnatal testicular development. The characteristic 2.5-kilobase type 1 transcript size was detected in immature rats (days 21-28), whereas in the adult (day 91), a slower migrating 2.7-kilobase type 1 mRNA species was observed. An antipeptide antiserum specific to rat 5 alpha-reductase type 1 was used to immunolocalize the 5 alpha-reductase type 1 protein. At all ages examined, the immunoperoxidase reaction was localized predominantly to the interstitial tissue of the testis. On postnatal day 7, clusters of interstitial cells resembling fetal Leydig cells were clearly immunoreactive. The staining intensity increased steadily from day 7 onwards, so that by days 21 and 28, interstitial cells with the appearance of immature Leydig cells were intensely immunoreactive (peak expression). This was followed by a progressive decrease in staining intensity between days 28-91, so that by day 91 (adult) Leydig cell immunoreactivity was barely detectable. Immunocytochemical staining revealed a predominantly cytoplasmic localization; significant nuclear staining was not evident. We conclude that the expression of the 5 alpha-reductase type 1 protein is primarily found in the cytoplasm of Leydig cells, is dependent on age, and that this expression closely parallels 5 alpha-reductase enzyme activity.

Developmental changes in epithelial cadherin messenger ribonucleic acid and immunocytochemical localization of epithelial cadherin during postnatal epididymal development in the rat.

The formation of junctional complexes between adjacent epithelial principal cells leads to formation of the blood-epididymal barrier; this barrier is complete by 21 days of postnatal age. Cadherins are cell surface proteins that mediate intercellular adhesion and are involved in the formation of adherence, gap, and tight junctions between epithelial cells. In the adult rat epididymis, epithelial cadherin (E-Cad) is localized in principal cells; E-Cad mRNA concentrations are androgen dependent in this tissue. The objectives of this study were to determine the regulation of E-Cad mRNA concentrations and the pattern of immunocytochemical localization of E-Cad during epididymal development. Using Northern blot analysis, we noted that in the caput-corpus epididymidis, there was a 3-fold increase in E-Cad mRNA concentrations between 7-14 days; an additional 3-fold increase between days 35-42, when E-Cad mRNA concentrations reached their peak, was noted. A dramatic decrease in E-Cad mRNA was observed between 42-49 days of age. This effect was transitory as E-Cad mRNA concentrations returned to almost 80% of peak concentrations on day 56 and remained constant thereafter. In the cauda epididymidis, E-Cad mRNA concentrations increased by only 1.6-fold between days 7-21. E-Cad mRNA concentrations then decreased by 70% to their lowest concentrations on day 56. There was a 2-fold increase in E-Cad mRNA concentrations between postnatal ages 56-91 days. These results suggest that the developmental regulation of E-Cad mRNA concentrations is segment specific. A subsequent study on the longitudinal distribution of E-Cad mRNA levels in six epididymal segments at 21, 42, and 56 days of age revealed that the relative proportion of E-Cad mRNA along the epididymis changes as a function of age. An immunocytochemical study with the light microscope, using an anti-E-Cad antibody, demonstrated that the localization and relative concentrations of E-Cad varied as a function of age. On day 15, the immunoperoxidase staining of the entire epididymal epithelium was apical, with the weakest staining in the cauda epididymidis. By day 21, the reaction spread to cover the supranuclear region of the principal cells in all segments, while on day 39, it covered the entire cytoplasm of these cells, suggesting a high rate of synthesis or storage of the protein. At later time intervals, the intensity of staining over the principal cells appeared to increase with age.(ABSTRACT TRUNCATED AT 400 WORDS)

Identification and developmental regulation of cadherin messenger ribonucleic acids in the rat testis.

Cellular interactions in the rat testis are suggested by the presence of gap junctions between developing germ cells and Sertoli cells as well as tight junctions between adjacent Sertoli cells. Cadherins are cell surface proteins that mediate calcium-dependent intercellular adhesion. In these experiments the presence and developmental regulation of three cadherins have been examined: epithelial cadherin (E-Cad), neural cadherin (N-Cad), and placental cadherin (P-Cad). Northern blot analysis of testicular RNA indicates the presence of N-Cad [4.3 and 3.5 kilobases (kb)] and P-Cad (3.5 kb) transcripts. No E-Cad message was detected. To determine whether mRNA concentrations for P-Cad and N-Cad are regulated during postnatal rat testicular development, testes from rats ranging in age from 7-91 days were subjected to Northern blot analysis. Relative P-Cad mRNA levels were highest at 7 days of age and decreased to almost half of these levels by day 14. P-Cad mRNA levels subsequently decreased to low levels and remained constant thereafter. This contrasted with the developmental pattern observed for the 4.3-kb N-Cad transcript, which was low early in testicular development but increased to peak levels on day 42, coincident with the shedding of the first sperm. N-Cad mRNA concentrations decreased from 42 to 56 days and then remained constant until 91 days. While mouse P-Cad antibody did not cross-react with rat P-Cad, immunoblots of testicular membrane protein preparations identified the presence of immunoreactive N-Cad protein in the testis. The presence of N-Cad protein confirms that N-Cad mRNA is translated in this tissue. The developmental patterns of P-Cad and N-Cad mRNA suggest a role for P-Cad early in testicular development, while N-Cad appears to play a role in later stages of spermatogenesis.

Distribution and regulation of epithelial cadherin messenger ribonucleic acid and immunocytochemical localization of epithelial cadherin in the rat epididymis.

The epithelium of the epididymis possesses an elaborate network of tight junctions between principal cells which is altered as a function of postnatal age. Cadherins are implicated in the formation of tight junctions. The objective of the present study was to determine whether RNA transcripts for cadherins were present in the epididymis, and if so, how they were hormonally regulated. Using specific cDNA probes for epithelial cadherin (E-Cad) and neural cadherin (N-Cad), Northern blot analysis was used to study steady state levels of cadherin mRNAs. A major E-Cad mRNA species of 4.7 kilobases and a weaker 4.3-kilobase species were observed in the epididymis. No signal for N-Cad was detected. Steady state mRNA levels for E-Cad were highest in the caput and corpus epididymidis and were almost 4 times higher than those in the initial segments and cauda epididymidis; no signal was detected in the vas deferens. Light microscopic immunocytochemical localization of E-Cad revealed a reaction over the principal cells of the entire epididymis. The relative intensities of the immunoreactivity suggested that the E-Cad protein concentration was highest in the corpus, followed by the caput, cauda, and initial segments of the epididymis. There was no reaction over the epithelial basal and clear cells or intraepithelial halo cells. Three days after bilateral orchidectomy, E-cad mRNA was decreased by 75% in the caput epididymidis. A dose-dependent maintenance of mRNA concentration for E-Cad was observed throughout the epididymis of orchidectomized rats after replacement with testosterone. Fourteen days after unilateral orchidectomy, no differences were observed in the concentrations of epididymal E-Cad mRNA between control and unilaterally orchidectomized rats. Together, these data demonstrate that mRNA for E-Cad is present and translated in the rat epididymis, is differentially distributed along this tissue, and can be regulated by circulating androgens.

Gene expression in Brown Norway rat Leydig cells: effects of age and of age-related germ cell loss.

There is a marked reduction in circulating T and a commensurate decrease in Leydig cell function in males during aging. Aging is also accompanied by progressive loss of germ cells, leading to testicular atrophy. However, in aged animals, there is no difference in T production by Leydig cells from nonregressed testes and from regressed testes. We hypothesize that there are changes in Leydig cell gene expression that accompany aging, and that different changes in gene expression result from testicular regression. To test this hypothesis, the expression of stress response genes was compared in Leydig cells isolated from young rat testes, from aged testes that were not regressed, and from aged testes that were regressed, using an array approach. Similar numbers of transcripts (n = 56-63) were detected in Leydig cells isolated from all three groups of rats. Among these, 21 transcripts were increased in Leydig cells of testes from aged nonregressed animals compared with cells from young animals; 23 were increased with subsequent testicular regression. Only 3 of these transcripts were in common. Thus, age and testicular regression affected Leydig cell transcripts in dramatically different ways. Furthermore, none of the transcripts that decreased when comparing Leydig cells of young and aged nonregressed animals were the same as those that decreased when comparing aged nonregressed and aged regressed animals. In individual gene families, the steady state concentrations of transcripts in Leydig cells from aging and aging regressed testes often differed. Thus, there are major differences in the expression of a wide variety of stress response genes in Leydig cells associated with aging and testicular regression.

Effects of PNU157706, a dual 5alpha-reductase inhibitor, on gene expression in the rat epididymis.

The epididymis is the site of sperm maturation and storage. 5alpha-Reductases (types 1 and 2) are key enzymes in this tissue because they convert testosterone to dihydrotestosterone (DHT), the main androgen regulating epididymal functions. Examining the consequences of inhibiting DHT formation is likely to provide important information regarding the regulation of epididymal functions, yet few inhibitor studies have focused on this tissue. To understand better DHT-mediated regulation of epididymal gene expression, we employed a dual 5alpha-reductase inhibitor and cDNA microarrays to examine the effects of 5alpha-reductase inhibition on gene expression in the initial segment, caput, corpus, and cauda epididymidis. Inhibition of epididymal 5alpha-reductase activity by PNU157706 was confirmed by in vitro enzyme assays. Rats were treated with 0, 0.1, 1.0 or 10 mg/kg per day PNU157706 for 28 days. The weights of DHT-dependent tissues, including the epididymis, were decreased following treatment. The effect of treatment on gene expression was dose-dependent and highly segment-specific. The initial segment responded uniquely in that a similar number of genes increased and decreased in expression compared with the other segments where the majority of affected genes decreased in expression. Some of the more dramatically affected genes were involved in signal transduction as well as fatty acid and lipid metabolism, regulation of ion and fluid transport, luminal acidification, oxidative defense and protein processing and degradation. These are essential processes contributing to the formation of an optimal luminal microenvironment required for proper sperm maturation. These results provide a novel insight into the DHT-dependent mechanisms that control epididymal functions.

Reversal of long-term LH deprivation on testosterone secretion and Leydig cell volume, number and proliferation in adult rats.

The purpose of this study was to determine whether Leydig cell volume and function could recover fully from long-term LH deprivation upon restoration of endogenous LH secretion, and whether the restoration of LH would elicit a mitogenic response, i.e. stimulate Leydig cell proliferation or affect Leydig cell number per testis. LH secretion was inhibited by treating adult rats with testosterone and oestradiol-filled (TO) silicone elastomer implants (16 weeks), and was restored by removing the implants. Changes in serum concentrations of LH and FSH, LH-stimulated testosterone secretion by testes perfused in vitro, Leydig cell volume and number per testis, average Leydig cell volume and Leydig cell [3H]thymidine incorporation were measured at weekly intervals following implant removal. The TO implants inhibited (P less than 0.01) LH secretion, but serum concentrations of FSH were not significantly different (P greater than 0.10) from control values. After implant removal, serum LH returned to control values within 1 week, whereas serum FSH increased twofold (P less than 0.01) and returned to control values at 4 weeks. LH-stimulated in-vitro testosterone secretion was inhibited by more than 99% in TO-implanted rats, but increased (P less than 0.01) to 80% of control values by 8 weeks after implant removal. The total volume of Leydig cells per testis and the volume of an average Leydig cell were 14 and 19% of control values respectively, after 16 weeks of TO implantation (P less than 0.01), but returned to 83 and 86% of controls (P greater than 0.10) respectively, by 6 weeks after implant removal. Leydig cell proliferation ([3H]thymidine labelling index) was low (less than 0.1%) in both control and TO-implanted rats, increased (P less than 0.01) fivefold from 1 to 4 weeks after implant removal and then declined to control values at 6 weeks. The increase in Leydig cell [3H]thymidine incorporation was mimicked by treating TO-implanted rats with exogenous LH, but not FSH. Leydig cells were identified in both the interstitium and the lamina propria of the seminiferous epithelium. The proportion of Leydig cell nuclei in the lamina propria was 30-fold greater (P less than 0.01) at 1 and 3 weeks after implant removal (3%) compared with that for control and TO-implanted rats (0.1%). Total Leydig cell number per testis was marginally but not significantly (P = 0.06) decreased in rats treated with TO implants for 16 weeks when compared with controls (18.4 +/- 2.2 vs 25.4 +/- 1.2 x 10(6)).(ABSTRACT TRUNCATED AT 400 WORDS)