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Pregnancy exposure to di(n-butyl) phthalate (DBP) in rats induces a testicular dysgenesislike syndrome (TDS) in male offspring. Earlier studies suggested altered Sertoli cell development/maturation may result, especially in testes that become cryptorchid. This study quantitatively assessed Sertoli cell numerical and functional development in DBP-exposed rats and compared (unilaterally) cryptorchid and scrotal testes. Pregnant rats were gavaged with 500 mg/kg/day DBP or corn oil from embryonic (E) Days 13.5 to 21.5. Male offspring were sampled on E21.5 or Postnatal Day 6, 10, 15, 25, or 90. Sertoli cell number in DBP-exposed males was reduced by approximately 50% at E21.5 but recovered to normal by Days 25-90, accompanied by significant changes in plasma inhibin B and testosterone levels. Sertoli cell maturational development in DBP-exposed males, assessed using five protein markers (anti-mullerian hormone, cytokeratin, androgen receptor, CDKN1B, and Nestin), was largely normal, with some evidence of delayed maturation. However, in adulthood, Sertoli cells (SC) in areas lacking germ cells (Sertoli cell-only [SCO] tubules) often exhibited immature features, especially in cryptorchid testes. Sertoli cells in DBP-exposed animals supported fewer germ cells during puberty, but this normalized in scrotal testes by adulthood. Scrotal and especially cryptorchid testes from DBP-exposed animals exhibited abnormalities (SCO tubules, focal dysgenetic areas) at all postnatal ages. Cryptorchid testes from DBP-exposed animals exhibited more Sertoli cell abnormalities at Day 25 compared with scrotal testes, perhaps indicating more severe underlying Sertoli cell malfunction in these testes. Our findings support the concept of altered Sertoli cell development in TDS, especially in cryptorchid testes, but show that maturational defects in Sertoli cells in adulthood most commonly reflect secondary dedifferentiation in absence of germ cells.

Regulation of spermatogenesis involves stage-dependent androgen action on Sertoli cells, but the pathways involved are unclear. We assessed if cyclin D2 could play a role. In rats, Sertoli cell nuclear, stage-dependent immunoexpression of cyclin D2 switched on after Day 10 and persisted through Day 35, but disappeared by adulthood. However, ethane dimethane sulfonate (EDS)-induced testosterone withdrawal in adult rats for 6 days induced stage-dependent cyclin D2 immunoexpression in Sertoli cells, with highest expression at stages IX-XII and nondetectable at stages VI–VIII (opposite that for androgen receptor [AR] immunoexpression). In EDS-treated rats, a single injection of testosterone but not of estrogen reversed this change in 4 h, and testosterone administration from the time of EDS treatment prevented expression of cyclin D2 in Sertoli cells. The EDS-induced changes in cyclin D2 immunoexpression were matched by changes in expression of Ccnd2 (cyclin D2) mRNA in isolated stage-dissected tubul...

Vacuolar type H 1 -ATPase is involved in lumenal acidification of the epididymis. This protein is highly expressed in narrow and clear cells where it is located in the apical pole, and it contributes to proton secretion into the lumen. We have previously shown that in rats, epididymal cells rich in H 1 ATPase appear during postnatal development and reach maximal numbers at 3‐4 wk of age. The factors that regulate the appearance of these cells have not been investigated, but androgens, estrogens, or both may be involved. This study examined whether neonatal administration of estrogens (diethylstilbestrol [DES] or ethinyl estradiol) or an antiandrogen (flutamide), or the suppression of androgen production via administration of a GnRH antagonist (GnRHa), was able to alter the appearance of cells rich in H 1 ATPase in the rat epididymis when assessed at age 25 days. Surprisingly, all of these treatments were able to significantly reduce the number of H 1 -ATPase positive cells; this was determined by immunofluorescence and confirmed by Western blotting. In contrast, neonatal coadministration of DES and testosterone maintained the expression of H 1 -ATPase in the epididymis at Day 25 despite the high level of concomitant estrogen exposure. These findings indicate that androgens, acting via the androgen receptor, are essential for the normal development of epididymal cells rich in H 1 -ATPase, and that treatments that interfere directly or indirectly with androgen production (GnRHa, DES) or action (flutamide, DES) will result in reduced expression of H 1 -ATPase. Our findings do not exclude the possibility that estrogens can directly suppress the postnatal development of cells in the epididymis that are rich in H 1 -ATPase, but if this is the case, this suppression can be prevented by testosterone administration. epididymis, estradiol, male reproductive tract, steroid hormones, testosterone

The aim of the present study was to identify specific, novel germ cell markers that could be used to monitor normal and abnormal spermatogenesis. Of several cloned cDNAs isolated from an adult rat testis cDNA library using an expression screening strategy, clone 813B4 (700 base pairs) hybridized exclusively to three mRNA transcripts in samples isolated from rat testes on and after Day 21 of life and to epididymides from some, but not all, adult rats. After further screening, two identical clones encoding a 2.2-kilobase cDNA (KTT4) were isolated and found to contain an open reading frame of 578 amino acids including two leucine zipper motifs. On Northern blots, KTT4 mRNA was abundant in samples from round spermatids, and homologous mRNAs were present in testes from mice and marmosets. A zoo blot revealed that the KTT4 gene is conserved in humans, monkeys, mice, dogs, and cattle. On sections of rat testes, KTT4 mRNA was first detectable in pachytene spermatocytes at stage VII and thereafter was abundant in round and elongating spermatids until step 15. Expression of KTT4 was not altered by ethane dimethane sulphonate-induced androgen withdrawal, but in rats treated 14 days previously with methoxyacetic acid, a marked reduction in KTT4 was noted associated with the depletion of round spermatids. In conclusion, the present study identified a conserved gene expressed in meiotic and post-meiotic germ cells; database searches have shown it to be homologous to recently published sequences for an outer dense fiber protein of the sperm tail (Odf2/Odf84).

Pregnancy exposure to di(n-butyl) phthalate (DBP) in rats induces a testicular dysgenesislike syndrome (TDS) in male offspring. Earlier studies suggested altered Sertoli cell development/maturation may result, especially in testes that become cryptorchid. This study quantitatively assessed Sertoli cell numerical and functional development in DBP-exposed rats and compared (unilaterally) cryptorchid and scrotal testes. Pregnant rats were gavaged with 500 mg/kg/day DBP or corn oil from embryonic (E) Days 13.5 to 21.5. Male offspring were sampled on E21.5 or Postnatal Day 6, 10, 15, 25, or 90. Sertoli cell number in DBP-exposed males was reduced by approximately 50% at E21.5 but recovered to normal by Days 25-90, accompanied by significant changes in plasma inhibin B and testosterone levels. Sertoli cell maturational development in DBP-exposed males, assessed using five protein markers (anti-müllerian hormone, cytokeratin, androgen receptor, CDKN1B, and Nestin), was largely normal, with some evidence of delayed maturation. However, in adulthood, Sertoli cells (SC) in areas lacking germ cells (Sertoli cell-only [SCO] tubules) often exhibited immature features, especially in cryptorchid testes. Sertoli cells in DBP-exposed animals supported fewer germ cells during puberty, but this normalized in scrotal testes by adulthood. Scrotal and especially cryptorchid testes from DBP-exposed animals exhibited abnormalities (SCO tubules, focal dysgenetic areas) at all postnatal ages. Cryptorchid testes from DBP-exposed animals exhibited more Sertoli cell abnormalities at Day 25 compared with scrotal testes, perhaps indicating more severe underlying Sertoli cell malfunction in these testes. Our findings support the concept of altered Sertoli cell development in TDS, especially in cryptorchid testes, but show that maturational defects in Sertoli cells in adulthood most commonly reflect secondary dedifferentiation in absence of germ cells.

Vacuolar type H(+)-ATPase is involved in lumenal acidification of the epididymis. This protein is highly expressed in narrow and clear cells where it is located in the apical pole, and it contributes to proton secretion into the lumen. We have previously shown that in rats, epididymal cells rich in H(+)ATPase appear during postnatal development and reach maximal numbers at 3-4 wk of age. The factors that regulate the appearance of these cells have not been investigated, but androgens, estrogens, or both may be involved. This study examined whether neonatal administration of estrogens (diethylstilbestrol [DES] or ethinyl estradiol) or an antiandrogen (flutamide), or the suppression of androgen production via administration of a GnRH antagonist (GnRHa), was able to alter the appearance of cells rich in H(+)-ATPase in the rat epididymis when assessed at age 25 days. Surprisingly, all of these treatments were able to significantly reduce the number of H(+)-ATPase positive cells; this was determined by immunofluorescence and confirmed by Western blotting. In contrast, neonatal coadministration of DES and testosterone maintained the expression of H(+)-ATPase in the epididymis at Day 25 despite the high level of concomitant estrogen exposure. These findings indicate that androgens, acting via the androgen receptor, are essential for the normal development of epididymal cells rich in H(+)-ATPase, and that treatments that interfere directly or indirectly with androgen production (GnRHa, DES) or action (flutamide, DES) will result in reduced expression of H(+)-ATPase. Our findings do not exclude the possibility that estrogens can directly suppress the postnatal development of cells in the epididymis that are rich in H(+)-ATPase, but if this is the case, this suppression can be prevented by testosterone administration.

In line TGR(mRen2)26 transgenic rats (TGR26) bearing a randomly inserted additional renin transgene, the males, but not the females, were found to be infertile. Tissue was obtained from TGR26 males and littermate controls after perfusion fixation, and the morphology of the testes and epididymides was examined. Testis size was normal as was gross morphology, but careful examination revealed that the release of many spermatozoa at stage IX of the spermatogenic cycle was impaired. In addition, the process of cytoplasmic elimination was abnormal, as cytoplasmic fragments of elongate spermatids were present in the epididymis. In TGR26 males, seminiferous tubule lumen size was significantly larger (p < 0.001) than in littermate controls, a difference that was most marked at stages IX-XIV--an effect that could be related to the retention of spermatozoa. In situ hybridization confirmed that expression of renin mRNA could be detected in testes of TGR26 rats but not in normal controls or in a fertile line (TGR27) of rats bearing the same transgene. Immunocytochemistry and in situ and Northern hybridization were used to elucidate the pattern of expression of genes that previous studies have implicated in the process of sperm maturation and/or release. Of the gene products examined (sulphated glycoproteins 1 and 2 [SGP-1, SGP-2], transition proteins 1 and 2 [TP-1, TP-2], urokinase, and cyclic protein 2 [CP-2]), none showed any major change in the pattern of expression compared with that in controls. We postulate that TGR26 transgenic male rats may be infertile because the expression of a gene (or genes) involved in the process of cytoplasmic elimination and/or sperm release has been disrupted by the presence of the transgene close to or within the gene(s). Future planned studies will involve determination of the insertion site(s) and ultrastructural analysis of the final phases of spermiogenesis.

Hormonal regulation of the expression of mRNA transcripts for cAMP response element-binding protein (CREB) and cAMP response element modulator (CREM) during spermatogenesis was studied in the adult rat testis. Northern analysis of CREB and CREM identified two mRNA transcripts for CREM (2.4 and 1.6 kb) and one transcript for CREB (2.0 kb). Analysis of mRNAs from isolated testicular cells by reverse transcriptase polymerase chain reaction (RT/PCR) showed that CREM mRNAs were expressed by the germ cells but not the Sertoli or interstitial cells, whereas CREB mRNA was located in germ cells, Sertoli cells, and interstitial cells. RNA was isolated and analyzed from the testes of 1) rats treated for 24 h with FSH, 2) rats in which androgen withdrawal had been induced by ethane dimethane sulphonate (EDS) treatment 6 days earlier (EDS-treated), 3) EDS-treated rats supplemented with testosterone (EDS + T), or 4) intratesticular administration or dibutyryl cAMP (dbcAMP) in the preceding 24 h. CREM mRNA transcript expression was found to be decreased after all of these treatments in samples from intact testis and from isolated cells. Expression of the CREB transcript was also decreased by EDS-induced androgen withdrawal, but not by FSH or EDS + T. In situ hybridization of paraffin-embedded testis sections probed with digoxigenin-labeled riboprobes confirmed the localization of CREB and CREM mRNA to the same cell types as found with RT/PCR. No stage-dependent expression of CREM mRNA transcripts could be observed. Hybridization of the CREB probe was highest around the base of stage VII-VIII tubules, and this was shown to be androgen-dependent. The data presented suggest that regulation of the expression of CRE-binding protein mRNAs in Sertoli and germ cells during spermatogenesis is dependent on both androgen and FSH. However, the effects of androgen or FSH on the regulation of CRE-binding protein mRNAs are different.

Although the testis is under overall control by pituitary gonadotropins, intratesticular control mechanisms are important 1) because of the unique structural organization of the testis, and 2) because of the organization and local requirements of spermatogenesis. The avascular nature of the seminiferous tubules, which comprise 90% of testicular mass in most mammals, means that their exceptionally high energy and nutritional demand has to be met by local transport in testicular interstitial fluid (IF). The rate of formation of IF is determined by capillary permeability and local control of this process is thus a prerequisite for ensuring full nutritional and hormonal support for spermatogenesis. The latter is organized into specific stages arranged in a cycle, each stage having different requirements which only local control mechanisms can ensure. In the rat, Stage VII of the spermatogenic cycle has an absolute requirement for high levels of testosterone and maintenance of such levels in the testicular IF surrounding the tubules thus constitutes a crucially important function requiring local control. Because in this situation, testosterone works via the Sertoli cell and because the testosterone is produced locally by the Leydig cells in response to luteinizing hormone (LH), it is suggested that local control of intratesticular levels of testosterone is likely to be effected by a factor (or factors) produced by the Sertoli cell which acts on the Leydig cell, and which interacts with LH to modulate the levels of testosterone in testicular IF. The possibility that “testicular luteinizing hormone releasing hormone (LHRH)” may fulfill this role is examined in detail by investigating the local effects of an LHRH agonist (LHRH-A) on testicular capillary permeability and the IF levels of testosterone in normal intact adult rats. The results show that LHRH-A has dose-dependent effec-ts on capillary permeability and the IF levels of testosterone and that these effects are modulated according to the ambient level of LH. Alteration of the IF levels of testosterone by LHRH-A is shown to be biologically effective and to be mediated partly by direct effects on Leydig cell steroidogenesis and partly by the control of capillary permeability. The physiological implications and operation of this local mechanism are discussed.

lypophysectomized adult male rats with a pituitary transplant under the kidney capsule were used to study the effects of chronic treatment with LHRH, with or without CB154, on the secre- tion of LH, FSH and prolactin and the action of these hormones on testicular LH receptorsand testosterone secretion. The presence of a pituitary transplant resulted in the expected increase in circulating levels of prolactin. Treatment with LHRH (5 or 50 Mg) caused a significant increase in basal serum levels of FSH, but not LH, after 7 or 14 days of treatment. Treatment with CB1S4 reduced serum pro- lactin levels but did not affect the secretion of LH or FSH. The presence of a pituitary transplant alone increased testicular binding of hCG without in- creasing testis weight. Administration of LHRH was associated with an increase in testis weight and with the capacity of the whole testis to bind hCC. Administration of CB154 to rats having trans. plants and receiving 5 M5 LHRH reduced testicular binding of hCG without affecting testis weight. Basal testicular testosterone secretion in vitro was increased in all groups receiving pituitary transplants. However, hCG-stimulated testosterone secretion in vitro was increased significantly over that in hypophysectomized controls only when gonadotropin secretion was maintained by LHRH treatment with or without CB154. These results suggest that: 1) small amounts of LHRH stimulated a greater and more prolonged increase in the levels of FSH than of LH; 2) something from the pituitary transplant, presumably prolactin, induces or maintains LH receptors in the testis without affecting testis weight or hCG- stimulated testosterone secretion in vitro; and 3) LHRH treatment and the increased gonadotropin secretion caused by it, increase testis weight with an associated increase in LH binding and ability