RESUMEN
Exposure to antiandrogens during the critical developmental window (i.e. sexual differentiation) can permanently demasculinize the male phenotype. Here we have investigated the effects of developmental exposure to di-isononylphthalate (DINP) (250 and 750 mg/kg) and 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (p,p'-DDE) (50 and 100mg/kg) on 19.5-day-old fetal Sprague-Dawley rat testicular and adrenal steroidogenesis. Maternal exposure to DINP or p,p'-DDE on embryonic days (EDs) 13.5-17.5 did not down-regulate the activity of steroidogenesis in ED 19.5 male rat fetus. Protein expression levels of testicular and adrenal StAR, P450scc, 3beta-HSD and androgen receptor (AR) did not show any changes. However, p,p'-DDE caused clear abnormalities in the ultrastructure of steroidogenic cells in ED 19.5 rat testis and adrenal. These structural alterations can disturb the development and function of fetal testis and adrenal that may become evident later in life.
Asunto(s)
Corticoesteroides/biosíntesis , Glándulas Suprarrenales/efectos de los fármacos , Antagonistas de Andrógenos/toxicidad , Diclorodifenil Dicloroetileno/análogos & derivados , Hormonas Esteroides Gonadales/biosíntesis , Exposición Materna , Ácidos Ftálicos/toxicidad , Testículo/efectos de los fármacos , 3-Hidroxiesteroide Deshidrogenasas/genética , Glándulas Suprarrenales/embriología , Glándulas Suprarrenales/metabolismo , Glándulas Suprarrenales/ultraestructura , Antagonistas de Andrógenos/administración & dosificación , Animales , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/genética , Diclorodifenil Dicloroetileno/administración & dosificación , Diclorodifenil Dicloroetileno/toxicidad , Relación Dosis-Respuesta a Droga , Femenino , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Edad Gestacional , Masculino , Fosfoproteínas/genética , Embarazo , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores Androgénicos/genética , Testículo/embriología , Testículo/metabolismo , Testículo/ultraestructuraRESUMEN
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is the most toxic and widely investigated dioxin congener. In utero and lactational exposure to TCDD results in developmental and reproductive defects that are the most sensitive endpoints for TCDD toxicity. TCDD has a potential to interfere with steroid metabolism, but the mechanisms by which this occurs are not well understood. In this study, we investigated the effects of TCDD on prenatal rat steroidogenesis. Pregnant Sprague-Dawley female rats were treated once with TCDD (0, 0.3 or 1 microg/kg) by gavage on embryonic day (ED) 11 and the expression levels of androgen (AR) and estrogen receptors (ER), steroidogenic enzymes (P450scc and 3beta-HSD) and four regulatory factors (StAR, SF-1, GATA-4 and Insl-3) involved in foetal Leydig cell and adrenal function were analysed on ED 19.5. Hormonal status of male foetuses was determined by measuring testicular testosterone (T) levels, plasma luteinizing hormone (LH) and corticosterone concentrations. In utero exposure to TCDD reduced intratesticular T of foetal males (significant at 0.3 microg/kg TCDD) and tended to reduce the protein expression of ERalpha and AR of foetal male rat testis. Foetal male rat plasma LH levels were significantly reduced at the dose of 1 microg/kg TCDD, while corticosterone levels tended to be increased possibly because of the TCDD-induced stress. Only minor alterations in steroidogenesis were observed in rat adrenal. mRNA expression of developmental regulatory factors was not influenced by foetal TCDD exposure, except for significantly reduced adrenal SF-1. The results demonstrate that maternal exposure to TCDD suppressed testicular steroidogenesis of 19.5-day-old foetal male Sprague-Dawley rat. The highest dose of TCDD (1 microg/kg) had also an effect on pituitary LH secretion. Our data implicate that TCDD has direct testicular and pituitary effects on foetal male rat but with different dose-responses. These changes can lead to impaired steroidogenesis and it may result in the maldevelopment of the testis and weaken masculinization.
Asunto(s)
Dibenzodioxinas Policloradas/toxicidad , Esteroides/biosíntesis , Animales , Femenino , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
Exposure of adult male animals to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) decreases serum androgen concentrations. Reduction in androgen levels after maternal exposure has also been reported, but these results have not been reproduced. We have earlier shown that TCDD stimulates rather than inhibits testosterone synthesis in the prenatal rat testis. The aim of the present study was to elucidate in utero-induced effects of TCDD on testicular steroidogenesis in the 14-day-old infant rats. At that time the foetal Leydig cell population is still the prevailing source of androgens. Pregnant Sprague-Dawley dams were given a single oral dose of TCDD (0, 0.04, 0.2, or 1.0 microg/kg) on day 13 of pregnancy. On postnatal day 14, the body weight of male offspring was reduced after exposure to 1.0 microg/kg TCDD (from 33.9 +/- 1.66 g to 31.6 +/- 2.67 g). Relative testis weight, plasma testosterone, luteinizing hormone and follicle-stimulating hormone levels remained unaltered in all exposure groups. Moreover, in ex vivo incubations, testosterone and cAMP production was not affected. StAR protein level in the freshly isolated testes was increased in the 0.2 microg/kg group, and seminiferous cord diameter in the 0.04 microg/kg group. The present study confirms our earlier findings in in utero TCDD-exposed foetal testis indicating that maternal TCDD exposure does not negatively influence the developmental testosterone production of foetal type Leydig cells in rats.
Asunto(s)
Contaminantes Ambientales/toxicidad , Feto/efectos de los fármacos , Exposición Materna , Dibenzodioxinas Policloradas/toxicidad , Testículo/efectos de los fármacos , Testosterona/sangre , Animales , Animales Recién Nacidos , AMP Cíclico/biosíntesis , Relación Dosis-Respuesta a Droga , Femenino , Feto/metabolismo , Hormona Folículo Estimulante/sangre , Células Intersticiales del Testículo/efectos de los fármacos , Células Intersticiales del Testículo/metabolismo , Hormona Luteinizante/sangre , Masculino , Tamaño de los Órganos/efectos de los fármacos , Embarazo , Ratas , Ratas Sprague-Dawley , Testículo/anatomía & histologíaRESUMEN
TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) has a potency to induce decreased fertility and structural reproductive anomalies in male and female mammals. While the activity profile of sex steroid hormone production distinctly differs in developing males and females, we wanted to analyze sex-specific effects of TCDD introduced in utero and via lactation on gonadal steroidogenesis and gonadotropin levels in male and female rat infant pups. One oral dose of TCDD (0, 0.04, 0.2, or 1.0 microg/kg) was given to dams on gestational day (GD) 13. Plasma testosterone, estradiol, progesterone, follicle stimulating hormone (FSH), luteinizing hormone (LH), and gonadal mRNA levels for steroid acute regulatory protein (StAR), cytochrome P-450 cholesterol side-chain cleavage (P450scc), 3beta-hydroxy-steroid-dehydrogenase/Delta(5)-Delta(4) isomerase type I (3beta-HSD1), P-450 17alpha-hydroxylase/17,20-lyase (P450-17alpha), and cytochrome P-450 aromatase (P450arom) were determined on postnatal days (PND) 10-16. TCDD 1.0 mug/kg reduced body weights but did not affect relative testis weight or alter testicular and ovarian histology. Plasma estradiol levels in dams and female pups were reduced on PND 14 and 16. Progesterone levels remained unaltered, and FSH levels were increased in female pups. In males, testosterone levels were elevated on PND 10. Gonadal mRNA levels for StAR and steroidogenic enzymes increased during the postnatal growth. TCDD caused no changes in relatively low testicular mRNA levels. However, significant reductions in StAR and P450arom mRNA levels were seen in PND 14 ovaries, and P450arom activity was decreased in isolated ovarian follicles. We conclude that developing testis and male gonadotropin secretion are resistant to TCDD-induced toxicity. In female pups, reduced estradiol, ovarian P450arom expression and enzyme activity levels, and elevated FSH levels may have a role in the development of ovarian dysfunction reported in TCDD-exposed females.
Asunto(s)
Anomalías Inducidas por Medicamentos , Lactancia/efectos de los fármacos , Exposición Materna , Ovario/efectos de los fármacos , Dibenzodioxinas Policloradas/toxicidad , Teratógenos/toxicidad , Testículo/efectos de los fármacos , Animales , Animales Recién Nacidos , Relación Dosis-Respuesta a Droga , Enzimas/genética , Enzimas/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Hormonas/sangre , Masculino , Ovario/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Embarazo/sangre , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Testículo/metabolismoRESUMEN
Phenolic compounds, such as 4-tert-octylphenol (OP), have been shown to interfere with rat ovarian steroidogenesis. However, little is known about steroidogenic effects of infantile OP exposure on immature ovary. The aim of the present study was to investigate the effects of infantile OP exposure on plasma FSH, LH, estradiol, and progesterone levels in 14-day-old female rats. The effect on ovarian steroidogenic acute regulatory protein (StAR) and FSH receptor (FSHr) expression was analyzed by Western blotting. Ex vivo analysis was carried out for follicular estradiol, progesterone, testosterone, and cAMP production. Sprague-Dawley rats were given OP (0, 10, 50, or 100 mg/kg) subcutaneously on postnatal days 6, 8, 10, and 12. On postnatal day 14, plasma FSH was decreased and progesterone increased significantly at a dose of 100 mg OP/kg. In addition, the highest OP dose advanced the time of vaginal opening in puberty. OP had no effect on infantile LH and estradiol levels or ovarian FSHr content. Ovarian StAR protein content and ex vivo hormone and cAMP production were decreased at all OP doses compared to controls. However, hormone levels recovered independent on FSH and even increased above the control level during a prolonged culture. On postnatal day 35, no statistically significant differences were seen between control and OP-exposed animals in plasma FSH, LH, estradiol, and progesterone levels, or in ovarian StAR protein content. The results indicate that the effect of OP on the infantile ovary is reversible, while more permanent effects in the hypothalamus and pituitary, as described earlier, are involved in the reduction of circulating FSH levels and premature vaginal opening.
Asunto(s)
Ovario/efectos de los fármacos , Fenoles/toxicidad , Fosfoproteínas/análisis , Esteroides/biosíntesis , Animales , Peso Corporal/efectos de los fármacos , AMP Cíclico/biosíntesis , Femenino , Hormona Folículo Estimulante/sangre , Hormonas Esteroides Gonadales/sangre , Hormona Luteinizante/sangre , Ovario/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores de HFE/análisisRESUMEN
Changes in the perinatal testosterone surge have been related to demasculinization of the central nervous system and androgen-dependent growth of the reproductive organs in male mammals. Earlier reports suggest that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) interferes with androgen production, but the perinatal effects have remained elusive. In the present study we explored in utero-effects of TCDD (0.05, 0.1, 0.5, and 1.0 microg/kg), introduced on day 13.5 of pregnancy, on prenatal (day 19.5 post-conception [p.c.]) testosterone (T) surge and pituitary luteinizing hormone (LH) production in TCDD-resistant Han/Wistar (H/W) and TCDD-sensitive Long-Evans (L-E) rats. To elucidate estrogenic effects on T and LH production, Sprague-Dawley (S-D) fetuses with previously known DES-sensitivity were exposed in utero to diethylstilbestrol (DES, 100-300 microg/kg) on days 13.5, 15.5, and 17.5 p.c. For comparison, H/W fetuses that responded to TCDD treatments were exposed to DES at concentration of 100 microg/kg. It was found that TCDD has a stimulatory effect on testicular T synthesis in the H/W fetuses and that their circulating T concentrations increased significantly. The effect was not seen in the inbred L-E fetuses, which throughout the study showed considerably low testicular T levels. Pituitary LH concentrations also increased in the H/W fetuses exposed to TCDD. Effects of TCDD (1.0 microg/kg) in the H/W fetuses could be confirmed in vitro by human chorionic gonadotropin (hCG) stimulation assay showing the highest response rate in the TCDD exposed testes. Stimulation of cyclic AMP (adenosine-3', 5'-cyclic monophosphate[cAMP]) production was not considerably altered by in utero TCDD exposure. A significant depression in testicular and plasma T content was seen in the DES-exposed S-D and H/W fetuses, but pituitary LH levels did not alter considerably. In the presence of hCG, DES-exposed testes showed lower in vitro T and cAMP production rates compared to the untreated testes. TCDD (1.0 microg/kg) increased and DES decreased the male body weight gain, but the changes were not sex-dependent. It is concluded that TCDD may increase the amplitude of the prenatal testosterone surge in male rats by stimulating pituitary LH production and enhancing the sensitivity of the fetal testis to LH. DES, on the contrary, apparently impairs testicular steroidogenesis and pituitary function.
Asunto(s)
Dietilestilbestrol/toxicidad , Feto/efectos de los fármacos , Feto/metabolismo , Hormona Luteinizante/metabolismo , Dibenzodioxinas Policloradas/toxicidad , Testosterona/metabolismo , Animales , Peso Corporal/efectos de los fármacos , AMP Cíclico/biosíntesis , Femenino , Técnicas In Vitro , Masculino , Intercambio Materno-Fetal , Hipófisis/efectos de los fármacos , Hipófisis/metabolismo , Embarazo , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Diferenciación Sexual/efectos de los fármacos , Testículo/efectos de los fármacos , Testículo/metabolismoRESUMEN
The aim of the present study was to find a reliable functional criterion for the evaluation of the proliferation potential of bovine in vitro-produced embryos. We used immunocytochemical detection of proliferating cell nuclear antigen (PCNA) combined with propidium iodide (PI) staining and subsequent confocal laser scanning microscopy together with routine morphological evaluation under a stereomicroscope to study fresh Day 7, 8, and 9, and cryopreserved Day 7 and 8 embryos. The ratio of PCNA/PI-positive nuclei was equal in fresh Day 7 and Day 8 embryos and significantly lower in Day 9 embryos. In general, Day 7 embryos tolerated the cryopreservation treatments better than Day 8 embryos. Vitrification in normal straws was especially detrimental to Day 8 embryos. In fresh Day 7 and 8 embryos, the PCNA results were in agreement with stereomicroscopic evaluation. However, in Day 9 fresh and in Day 7 and 8 treated embryos, the missing PCNA revealed disorders that were not observed under morphological evaluation. PCNA immunocytochemistry is an effective method to obtain information about the functional state of nuclei. The ratio of PCNA-positive nuclei can provide more information and numerical data about the developmental potential of bovine embryos after cryopreservation.
Asunto(s)
Embrión de Mamíferos/citología , Desarrollo Embrionario y Fetal/fisiología , Antígeno Nuclear de Célula en Proliferación/metabolismo , Animales , Bovinos , Recuento de Células , División Celular , Núcleo Celular/ultraestructura , Colorantes , Criopreservación , Femenino , Inmunohistoquímica , Microscopía Confocal , Embarazo , PropidioRESUMEN
PURPOSE: Our previous studies indicate that neonatal estrogenization with diethylstilbestrol (neoDES) of male mice and rats causes partial outlet obstruction. In the present study, type XII and XIV collagens were localized in the bladder to study their role in the development of obstruction. MATERIALS AND METHODS: The bladder sections immunostained with smooth muscle specific a-actin antibody were double labeled either with collagen type XII or type XIV antibodies. The specimens were then analyzed with conventional and confocal fluorescence microscope. RESULTS: Type XII and XIV collagens were not evenly distributed in the bladder. Further, in neonatally estrogenized rats collagen XIV appeared inside smooth muscle fascicles. CONCLUSIONS: Non-overlapping distributions of collagen XII and XIV suggest their different roles in the urinary bladder. Penetration of collagen XIV inside smooth muscle fascicles may have a role in the development of DES-induced partial outlet obstruction.
Asunto(s)
Colágeno , Obstrucción del Cuello de la Vejiga Urinaria/patología , Vejiga Urinaria/patología , Animales , Colágeno/análisis , Masculino , Ratas , Vejiga Urinaria/químicaRESUMEN
In cell extracts all of the nonliganded steroid receptor molecules are found as an oligomeric complex with Hsp90 and other proteins. In previous studies we have shown that Wild-type Hsp90 and progesterone receptor (PR) are located in different cell compartments (Tuohimaa et al. [1993] Proc. Natl. Acad. Sci. USA 90:5848-5852). In the present work we studied whether PR and Hsp90 can efficiently associate provided they are present in the same cell compartment. The association of Hsp90 with PR in vivo was studied by nuclear cotranslocation and immunohistochemistry with an antibody (alphaD) which can distinguish between the oligomeric and dissociated form. Upon expression of a cytoplasmic mutant of PR with Wild-type (cytoplasmic) Hsp90 and Wild-type (nuclear) PR with NLS-Hsp90 (a Hsp90 with a nuclear localization signal), we noted that the epitope of alphaD in PR was exposed in both cases. Also, in vivo crosslinking and treatment of cells with substances which stabilize the oligomeric complex in vitro were inefficient in demonstrating or inducing a similar oligomeric receptor form detectable in vitro in cell homogenates. However, when the cytoplasmic PR mutant (DeltaPR) was coexpressed with a nuclear form of Hsp90 (NLS-Hsp90), a portion of PR was cotranslocated into the nucleus. This would indicate that steroid receptors are indeed associated with Hsp90 in intact cells, but the Hsp90-associated receptor pool represents only a small portion of the receptors. This suggests that the majority of oligomeric complexes seen in cell extracts are formed during cell fractionation.
Asunto(s)
Citoplasma/química , Proteínas HSP90 de Choque Térmico/metabolismo , Receptores de Progesterona/metabolismo , Animales , Células COS , Proteínas HSP90 de Choque Térmico/química , Molibdeno/farmacología , Estrés Oxidativo , Unión Proteica , Receptores de Progesterona/química , Receptores de Progesterona/inmunologíaRESUMEN
Peptide hormones and growth factors are involved in the regulation of prostatic cell proliferation, differentiation, and programmed cell death, which functions are primarily controlled by androgen. In carcinogenesis, prostatic cancer cells often lose androgen dependence and become largely dependent on local growth factors. The prostatic cancer cells able to respond to factors other than androgen by proliferation and inhibition of apoptosis are possibly able to survive. We demonstrate that prostatic epithelium expresses prolactin mRNA and protein in a characteristic manner. By using in situ hybridization, an overall distribution of prolactin mRNA was demonstrated in the epithelium of rat dorsal and lateral prostate, whereas a very specific localization of prolactin protein to single cells was observed by immunohistochemistry in the same tissues. In these cells, immunoelectron microscopy showed that prolactin was primarily localized to the secretory granules. These data demonstrate a selective regulation of prostatic prolactin at least at the level of transcript processing/translation and/or protein accumulation and secretion. In addition, the expression of prolactin protein in rat dorsal and lateral prostate was found to be androgen dependent in vivo in castrated and in castrated, testosterone-treated rats, as well as in vitro in organ cultures. Our results support the concept of an autocrine/paracrine loop of prolactin action in prostate where it could mediate some of androgen actions. Also, locally synthesized prolactin might belong to the factors that take over androgen regulation of prostatic cancer cells during the development of androgen-independent growth.
Asunto(s)
Andrógenos/metabolismo , Prolactina/metabolismo , Próstata/metabolismo , Animales , Gránulos Citoplasmáticos/metabolismo , Epitelio/efectos de los fármacos , Epitelio/metabolismo , Epitelio/ultraestructura , Regulación de la Expresión Génica/efectos de los fármacos , Hibridación in Situ , Masculino , Microscopía Inmunoelectrónica , Orquiectomía , Técnicas de Cultivo de Órganos , Prolactina/genética , Próstata/efectos de los fármacos , Próstata/ultraestructura , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Testículo/fisiología , Testosterona/farmacologíaRESUMEN
We have studied the localization of the expression of FGF-8 mRNA in adult and developing rat and mouse gonads by in situ hybridization. The expression of FGF-8 mRNA was high in oocytes of small and large antral follicles of adult mouse ovaries. No signal was observed in fetal ovaries, or in primordial and atretic follicles of adult ovary. In mouse testis, the FGF-8 mRNA signal could be demonstrated in prespermatogonia during a short period covering the fetal days 15 to 17, but not any more on day 19 of fetal life, or in adult testis. The time course of the expression of FGF-8 mRNA in mouse testis was confirmed by RT-PCR reaction. Corresponding in situ results were obtained by studying rat tissues. The observed germ cell-specific expression of FGF-8 mRNA in maturing oocytes and fetal prespermatogonia suggests that FGF-8, which is a secretory protein, has a paracrine function during the specific phases of the maturation of the follicle and fetal seminiferous epithelium.
Asunto(s)
Factores de Crecimiento de Fibroblastos , Regulación del Desarrollo de la Expresión Génica , Sustancias de Crecimiento/genética , Oocitos/metabolismo , Espermatogonias/metabolismo , Animales , Femenino , Factor 8 de Crecimiento de Fibroblastos , Masculino , Ratones , Ratones Endogámicos BALB C , Ovario/embriología , Ovario/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Testículo/embriología , Testículo/metabolismoRESUMEN
PURPOSE: Analysis of voiding pattern, urodynamic measurements and immunohistochemical methods were performed in order to evaluate the effects of neonatal estrogenization on voiding functions of adult male mice. MATERIALS AND METHODS: Metabolic cages were used for recording the voiding volumes and frequencies. Bladder pressure and mean flow during voiding were measured in transvesical cystometry. Location of estrogen receptors and organization of smooth muscles in lower urinary track were demonstrated using immunohistochemical staining. RESULTS: Neonatally estrogenized (neoDES) male mice had lower voided urine volumes (the average voided urine volume and average of the three largest volumes) and higher voiding frequencies than control mice. In transvesical cystometry, the maximum bladder pressure during the high-frequency oscillation phase of voiding was significantly elevated. The average urinary flow rate was decreased. CONCLUSIONS: Urodynamically, these findings are consistent with the concept that neonatally estrogenized mice have infravesical obstruction. The predominance of estrogen receptors in the periurethral region and changes in urethral smooth muscle cells immunocytochemically stained with alpha-actin-antibody support the concept of urethral wall musculature as a target of estrogen action.
Asunto(s)
Dietilestilbestrol/administración & dosificación , Estrógenos no Esteroides/administración & dosificación , Obstrucción Uretral/inducido químicamente , Factores de Edad , Animales , Animales Recién Nacidos , Femenino , Masculino , Ratones , Músculo Liso/patología , Receptores de Estrógenos/análisis , Uretra/química , Uretra/patología , Vejiga Urinaria/química , Vejiga Urinaria/patología , Vejiga Urinaria/fisiopatología , MicciónRESUMEN
Heat shock protein 60 (HSP60), a member of the chaperonin family, has an essential role in mediating correct folding of nuclear encoded proteins imported to mitochondria. We have investigated immunocytochemical expression of HSP60 in developing fetal, newborn, postnatal, and pubertal testis and ovary, and in the adult ovary of the rat. In the fetal gonads, HSP60 was expressed in the germ cells organized into sex cords and in the developing Leydig cells of the testis. In the pubertal testis, Leydig cells were strongly, spermatogonia and premeiotic spermatocytes moderately labeled, spermatids unlabeled. In the postnatal ovary, oocytes at all stages of folliculogenesis were positive for HSP60. In the pubertal ovary, glandular theca cells, and in the mature ovary, also the cells of the corpora lutea exhibited intense cytoplasmic labeling. At the electron microscopic level, immunogold particles were localized in the mitochondrial matrix, and in the Western blot analysis the antibody detected one single band of 60 kDa. Anti-HSP60 labeling in male and female sex steroid producing cells and their progenitors seems to be coordinated with the functional differentiation of these endocrine cells of the gonad. In the oocytes, a key element required for proper folding of imported mitochondrial proteins seems to be constitutively expressed throughout folliculogenesis. However, the data suggest that in the male germ cells mitochondrial chaperonin HSP60 is either not needed during the haploid phase of spermatogenesis or its level becomes extensively reduced and therefore undetectable by the methods used in the study.
Asunto(s)
Chaperonina 60/biosíntesis , Ovario/metabolismo , Testículo/metabolismo , Animales , Animales Recién Nacidos , Western Blotting , Chaperonina 60/química , Chaperonina 60/genética , Cuerpo Lúteo/química , Cuerpo Lúteo/metabolismo , Femenino , Fase Folicular , Inmunohistoquímica , Células Intersticiales del Testículo/química , Células Intersticiales del Testículo/metabolismo , Masculino , Microscopía Inmunoelectrónica , Mitocondrias/ultraestructura , Oocitos/química , Oocitos/metabolismo , Ovario/embriología , Ovario/crecimiento & desarrollo , Ratas , Ratas Sprague-Dawley , Espermatogonias/química , Espermatogonias/metabolismo , Testículo/embriología , Testículo/crecimiento & desarrolloRESUMEN
Magnetization transfer (MT) imaging provides a novel opportunity to characterize interactions between tissue water and macromolecules. Although several in vitro investigations have shown that proteins and lipids are important determinants of MT, the contribution of DNA is still unknown. This study was designed to determine whether DNA and cell nuclear material exhibit MT. We measured the magnetization transfer effect of pure DNA strands and purified bovine sperm head nuclei. Although no transfer of magnetization could be detected in samples of pure DNA strands, the sperm head nuclei exhibited a strong MT effect that increased with increasing solid content of the samples. Since the purified bovine sperm head samples consist of large nuclei with only minor traces of perinuclear matrix, the measured MT effect arises from the chromatin of the nuclei. The DNA fills 90% of the nuclear volume and it is extremely tightly packed as chromatin fibers by nucleoproteins. We hypothesize that the numerous intra- and intermolecular disulfide bonds that stabilize the chromatin fibers restrict the movement of the surface water binding sites of both DNA and protamines and thus facilitate the transfer of magnetization. Therefore, the results indicate that the amount of nuclear material may positively contribute to MT in tissues.
Asunto(s)
ADN/química , Imagen por Resonancia Magnética/métodos , Espermatozoides/química , Animales , Bovinos , Núcleo Celular/química , ADN/aislamiento & purificación , Masculino , Espermatozoides/ultraestructuraRESUMEN
Concern about the reproductive potential of long-term survivors of acute lymphoblastic leukaemia (ALL) prompted an investigation into the impact of the disease on spermatogenic cells. Using rats as a model, histological, immunocytochemical and electron microscopic analysis was applied to investigate changes in the seminiferous epithelium. In rats transplanted with leukaemic cells at early puberty, degenerate primary spermatocytes and spermatids were prevalent within stage VIII tubules. Electron microscopically, step 8 spermatids showed acrosomal abnormalities and nuclear contour distortion. In the distorted step 9 spermatids, the microtubules of the manchette were abnormally oriented or deficient. Antitubulin antibody staining was reduced in elongating spermatids in the group transplanted with leukaemic cells at early puberty but was not observed in the older leukaemic group. Step 13 spermatids showed extracted chromatin and degenerate step 19 spermatids were occasionally found. Similar but less severe changes were seen in the group of rats transplanted with leukaemic cells at late puberty. We conclude that germinal cell damage induced by ALL is dependent on the developmental maturity of the seminiferous epithelium. The present findings are of particular importance when interpreting the impact of anticancer chemotherapeutics on germinal cells in patients with ALL.
Asunto(s)
Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Espermátides/patología , Animales , Modelos Animales de Enfermedad , Masculino , Microscopía Electrónica , Microscopía Fluorescente , Ratas , Espermátides/ultraestructura , Testículo/patología , Testículo/ultraestructuraRESUMEN
The cellular mechanisms controlling sexual differentiation of fetal gonads are poorly understood. By examining the protein and mRNA expression of tenascin-C in correlation with the immunocytochemical detection of alpha-smooth muscle actin (alpha-SMA) and basement membrane heparan sulfate proteoglycan (HSPG) we demonstrate a clear-cut sex-and development-dependent expression pattern of tenascin-C in the rat testis, ovary and mesonephros. Immunocytochemistry and in situ hybridization of tenascin-C in 15-day-pc fetal testis and ovary showed protein and mRNA accumulation within the mesenchyme of the mesogonadal connection. In addition to the male and female mesonephros, some labeling could also be seen within the testicular tunica albuginea and intraovarian mesenchymal septa. In the 17-day-pc testis abundant accumulation of tenascin mRNA and protein appeared in the tunica and mediastinum testis, but not at all in the intratesticular mesenchyme. A similar pattern was still seen in the newborns where, however, a decrease in the anti-tenascin immunoreactivity of the tunica and mediastinum could be demonstrated. In contrast to the testis, expression of tenascin in 17-day-pc ovaries was widespread within the hilus and the entire intragonadal mesenchyme where it continued to accumulate also in newborns. Northern blot analysis of tenascin-C mRNAs showed one message of 8.0 kb in the 15-day-pc male and female gonads. An additional weak signal of 6.5 kb was seen in the female mesonephros. In the 18-day-pc testis, the 6.5-kb signal appeared stronger than the 8.0-kb signal. In contrast to the testis, the 6.5-kb message was weak in the developing ovary where the 8.0-kb signal had an intense peak on the day 18 pc. Further, in the ovary, mesenchymal accumulation of HSPG coincided with the spatial distribution of tenascin. In the testicular tunica and in the mesenchyme of the male and female genital ducts expression of tenascin was parallel with the differentiation of smooth muscle tissue, detected by labeling for alpha-SMA, which also indicated the tenascin-negative myoid cells of the testis. Our results indicate that tenascin expression in the fetal rat internal genitalia is involved in the differentiation of smooth muscle cells but not intratesticular myoid cells. In the ovarian mesenchyme, tenascin-C may have a specific function in the dynamic remodeling of the ovarian cords.
Asunto(s)
Moléculas de Adhesión Celular Neuronal/biosíntesis , Proteínas de la Matriz Extracelular/biosíntesis , Proteínas Fetales/biosíntesis , Ovario/metabolismo , ARN Mensajero/biosíntesis , Caracteres Sexuales , Testículo/metabolismo , Actinas/análisis , Animales , Membrana Basal/química , Moléculas de Adhesión Celular Neuronal/genética , Diferenciación Celular/fisiología , Desarrollo Embrionario y Fetal/fisiología , Proteínas de la Matriz Extracelular/genética , Femenino , Proteoglicanos de Heparán Sulfato , Heparitina Sulfato/análisis , Inmunohistoquímica , Hibridación in Situ , Masculino , Ovario/citología , Ovario/embriología , Proteoglicanos/análisis , Ratas , Ratas Sprague-Dawley , Tenascina , Testículo/citología , Testículo/embriologíaRESUMEN
Calicin, a basic cytoskeletal protein has been proposed to be involved in the formation and maintenance of the highly regular organization of the postacrosomal perinuclear theca, the calyx of mammalian spermatozoa. The fate of human sperm calicin in the zona-free hamster egg fertilized in vitro has been analysed at the light and electron microscope level using polyclonal mouse anti-calicin antibodies. Calicin was localized in the postacrosomal dense lamina of the capacitated acrosome-reacted spermatozoa attached on the surface of the egg as well as in the spermatozoa at an early stage of ooplasmic incorporation. As determined by the aid of the DNA-specific fluorescent dye 4,6-diamidino-2-phenylindole (DAPI), the pattern of staining with anti-calicin changed from a funnel-shape into a ring soon after the onset of the nuclear decondensation. Later, no anti-calicin labelling could be detected around the more decondensed sperm nuclei. Because the residual, ring-like accumulation of calicin was associated with the least decondensed chromatin, it appears that the degradation of calicin-containing perinuclear theca is intimately involved with the posteriorly advancing nuclear disintegration. The ring formation also suggests that the calyx of human spermatozoa is not structurally homogeneous at least in terms of sensitivity to ooplasmic degradation. Calicin appears to be unaffected by lytic enzymes of the acrosome. The present study further shows that DAPI can be effectively used to analyse sperm nuclear decondensation in vitro.
Asunto(s)
Proteínas del Citoesqueleto/análisis , Fertilización In Vitro , Espermatozoides/química , Acrosoma/química , Animales , Anticuerpos Monoclonales , Cricetinae , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Masculino , Mesocricetus , Ratones , Microscopía Inmunoelectrónica , Óvulo/ultraestructuraRESUMEN
BACKGROUND: Characteristic membrane changes in spermatozoa culminating in acrosome reaction and sperm-egg fusion, and suspected involvement of actin-containing cytoskeleton in membrane changes in general, prompted us to investigate subcellular distribution of actin and actin-binding proteins in bovine spermatozoa subjected to various extractions which sequentially denude the sperm investments. METHODS: Spermatozoa were treated with either 1% SDS, 0.1% Triton X-100, 0.1% Hyamine, or 1 M MgCl2 or were sonicated. Immunostaining of actin, alpha-actinin, spectrin, and acrosin as well as electron microscopic analysis of extracted spermatozoa were carried out. RESULTS: Extractions caused evagination of the acrosomal lamina which retained focal contacts with the inner acrosomal membrane. Extractions further revealed lateral prongs at the anterior border of the postacrosomal sheath. Labeling for alpha-actinin and spectrin was localized in the acrosin-positive acrosomal lamina, neck, and principal piece, the latter containing also relatively extraction-resistant oligomeric or polymerized actin. In the postacrosomal area, actin was accumulated in the extraction-resistant posterior ring structure and anteriorly at the sites apparently related to the lateral prongs. Notably, spectrin reactivity was enhanced by MgCl2 in head, neck, and principal piece, and sonication abolished cytoskeletal immuno-reactivity in the head. CONCLUSIONS: Destabilization of membranes with selected extractions induces changes in the acrosomal lamina mimicking acrosomal vesicle formation. The lateral prongs and posterior ring structure, respectively, may serve as anterior and posterior anchors for the extraction-resistant post-acrosomal sheath. The lateral prongs may also be a merger zone for actin, alpha-actinin, and spectrin with important implication on sperm function. The latter two proteins may be involved in acrosomal vesicle formation. It is apparent that extractions have a significant effect on the detectability of sperm cytoskeletal elements.
Asunto(s)
Acrosoma/química , Proteínas de Microfilamentos/análisis , Espectrina/análisis , Acrosoma/ultraestructura , Actinina/análisis , Actinina/ultraestructura , Actinas/análisis , Actinas/ultraestructura , Animales , Bovinos , Masculino , Proteínas de Microfilamentos/ultraestructura , Microscopía Fluorescente , Espectrina/ultraestructura , Espermatozoides/química , Espermatozoides/ultraestructuraRESUMEN
BACKGROUND: Presence of immunocytochemically detectable actin in the rat and mouse sperm head has been enigmatic for years. In this study, we demonstrate actin in the perinuclear theca and show that the detection of actin epitopes in the rat and mouse epididymal spermatozoa can effectively be enhanced by pre-extraction of sperm cells with SDS. METHODS: The study with one monoclonal and one polyclonal anti-actin antibody was carried out at conventional and confocal fluorescence and electron microscope level, and by immunoblotting of proteins isolated from the head and tail fractions. RESULTS: In the head of the control methanol-acetone fixed rat spermatozoa, the polyclonal antibody gave a stronger immunostaining in the postacrosomal area and in the perforatorium than the monoclonal antibody. In the mouse sperm head, the monoclonal antibody labeled the ventral edge of the postacrosomal area and slightly the perforatorium, whereas the polyclonal antibody stained the entire perinuclear space. In the SDS-extracted spermatozoa, an intense postacrosomal and perforatorial labeling was obtained with both antibodies but, in particular in the rat spermatozoa, the middle lateral portion of the postacrosomal segment remained unlabeled. Sonication seemed to cause structural modifications which specifically impeded staining with the monoclonal antibody. Both antibodies detected actin in the basal plate and the monoclonal antibody in the neck. Amorphous matrix of the connecting piece showed immunogold labeling. In the tail, the monoclonal antibody recognized actin and a relatively basic 53 kDa polypeptide, whereas the polyclonal antibody reacted with several protein bands. SDS-soluble actin of the tail was addressed to the midpiece and the SDS-insoluble 53 kDa protein profoundly to the outer dense fibers of the principal piece. CONCLUSIONS: Intense labeling of actin in the SDS-extracted rat and mouse spermatozoa was presumably due to the generated demasking of actin epitopes embedded in the perinuclear cytoplasm. The results are important in confirming that actin in the rat and mouse sperm head is not lost during spermiogenesis but apparently contributes to the three-dimensional packing of the mature perinuclear cytoplasm. This study further demonstrates the importance of the methods used in sample preparation and advantages of confocal microscopy when attempting to detect cytoskeletal proteins which, as in spermatozoa, may occur in small quantities.
Asunto(s)
Actinas/análisis , Péptidos/análisis , Espermatozoides/química , Animales , Anticuerpos Monoclonales , Electroforesis en Gel de Poliacrilamida , Epidídimo , Técnica del Anticuerpo Fluorescente , Immunoblotting , Inmunohistoquímica , Masculino , Ratones , Microscopía Inmunoelectrónica , Ratas , Cabeza del Espermatozoide/química , Cola del Espermatozoide/química , Espermatozoides/ultraestructuraRESUMEN
BACKGROUND: Silver-stainability (argentophilia) of cytoplasmic structures occurring in spermatids have been localized into the organizing perinuclear theca, but the biochemical nature and structural associations of these proteins with the cytoskeletal and membranous elements are unresolved and, therefore, were the aim of the present study. METHODS: Light and electron microscopic analysis of the silver-stainability in the rat spermatids and spermatozoa was carried out in the intact testis tissue and epididymal spermatozoa and after their chemical and mechanical extraction. Correlation of argentophilia with specific proteins of rat and bovine spermatids and spermatozoa was investigated using a recently developed technique for silver nitrate staining of proteins on nitrocellulose. RESULTS: Sequential formation of the silver-stainable domains seemed to proceed from the argentophilic acrosomal ring. Various extractions indicated that argentophilia in the spermatids and spermatozoa was mainly associated with the perinuclear theca and to some extent to the plasma membrane. Hyamine-soluble extract from spermatozoa of rat and bull revealed only a single argentophilic protein of 130 kDa. Hyamine and SDS-soluble extracts of rat testis tissue contained an additional group of argentophilic polypeptides of lower molecular weight (115, 94, 36, 23, and 21 kDa). CONCLUSIONS: Reduction in the number of argentophilic proteins appears to be involved in a series of changes in the cyto-architecture of developing spermatids. Tentative cytoskeletal nature of argentophilic proteins remains to be identified. Nevertheless, they may have important physical relations with the higher-order organization of the sperm head cytoskeleton and overlying membranes.