Efecto de la Vitamina E en Células Peritubulares de Testículos de Ratones Mus musculus Tratados con Ácido Valproico Durante el Estado Fetal y Pubertad / Effect of Vitamin E on Peritubular Cells from Testis of Mus musculus Mice Treated with Valproic Acid During the Fetal Stage and Puberty

El ácido valproico (VPA) es un fármaco antiepiléptico teratógenico que, al ser administrado durante etapas tempranas del embarazo, puede producir alteraciones en el desarrollo embriofetal, las que se manifiestan tanto a nivel del sistema nervioso como del testículo. No obstante, se ha reportado que la administración de vitamina E (VE) podría revertir dichas alteraciones. El objetivo del presente estudio fue determinar el efecto protector de la VE a nivel testicular en fetos y ratones púberes expuestos a VPA durante la fase embrionaria de su desarrollo. Se utilizó un total de 30 ratones hembra adultas gestantes (Mus musculus) cepa BALB/c, las cuales se dividieron en 6 grupos. El estudio contempló el análisis de fetos machos a los 17,5 días post-coital (dpc) y machos juveniles a las 6 semanas post-natal. A los grupos 1 y 4 se les administró 0,3 mL de solución fisiológica (grupos control para 17,5 dpc y 6 semanas postnatal, respectivamente). A los grupos 2 y 5 se les suministró la cantidad de 600 mg/kg de VPA (grupos VPA), en tanto que a los grupos 3 y 6 se les aplicó la misma dosis de VPA complementada con 200 UI de VE (grupos VPA+VE). Se describió la histología normal y patológica del compartimento peritubular del testículo. En los grupos VPA se evidenció una degeneración de la pared peritubular, y atrofia de túbulos seminíferos, así como exfoliación de las células germinales. Por el contrario, en los grupos VPA+VE tales signos no fueron observados y la morfología presentó aspecto normal solo con algunas alteraciones focales. Estos resultados corroboran el hecho que la administración de VE contrarresta en parte, los efectos deletéreos que ocasiona el VPA.

SUMMARY: Valproic acid (VPA) is a teratogenic antiepileptic drug that, when administered during the early stages of pregnancy, can produce alterations in embryo-fetal development, which manifest both at the level of the nervous system and the testicle. However, it has been reported that the administration of vitamin E (VE) could reverse these alterations. The study aimed to determine the protective effect of VE at the testicular level in fetuses and pubertal mice exposed to VPA during the embryonic phase of their development. 30 pregnant adult female mice (Mus musculus) BALB/c strain were used, which were divided into 6 groups. The study included the analysis of male fetuses at 17.5 days post-coital (dpc) and juvenile males at 6 weeks post-natal. Groups 1 and 4 were administered 0.3 mL of physiological solution. Groups 2 and 5 were given 600 mg/kg of VPA (VPA groups), while groups 3 and 6 were given the same dose of VPA supplemented with 200 IU of VE (VPA+VE). The normal and pathological histology of the peritubular compartment of the testis was described. In the VPA groups, degeneration of the peritubular wall, and atrophy of the seminiferous tubules, as well as exfoliation of the germ cells, were evident. On the contrary, in the VPA+VE groups such signs were not observed and the morphology presented a normal appearance with only some focal alterations. These results corroborate the fact that the administration of VE partially counteracts the deleterious effects caused by VPA.

Xiaokang Liuwei Dihuang decoction ameliorates the immune infertility of male rats induced by lipopolysaccharide through regulating the levels of sex hormones, reactive oxygen species, pro-apoptotic and immune factors.

Male immune infertility is a kind of disease that damages family life and happiness. The development of novel methods treating male immune infertility is of great significance. This study aimed to investigate the therapeutic effects of Chinese medicine Xiaokang Liuwei Dihuang decoction on immune infertility of male rats and explored the involved mechanisms. Model rats were established by lipopolysaccharide (LPS) injection. Anti-sperm antibody (AsAb) was detected by ELISA assay and testicular cell apoptosis was evaluated by TUNEL staining to verify the successful model establishment and screen suitable doses of Xiaokang Liuwei Dihuang decoction. Thirty rats were then divided into five groups (n = 6 per group): Control, LPS, Xiaokang Liuwei Dihuang decoction (15.12 g/kg, 30.24 g/kg and 45.36 g/kg). Results of HE staining showed that compared with LPS group, Xiaokang Liuwei Dihuang decoction treatments gradually improved the morphology of seminiferous tubules and elevated the number of spermatogenic cells as the doses increased. The sperm number and the levels of testosterone, luteinizing hormone (LH) and follicle stimulating hormone (FSH) in the serum of 15.12 g/kg, 30.24 g/kg and 45.36 g/kg Xiaokang Liuwei Dihuang decoction groups were much higher than those in LPS group. Results of TUNEL staining, ELISA assay and western blot showed that compared with LPS group, the testicular cell apoptosis and the levels of interleukin 1ß (IL-1ß), tumor necrosis factor α (TNF-α), AsAb, malondialdehyde (MDA) and toll-like receptor 2 (TLR2) in the testicular tissue significantly decreased in three Xiaokang Liuwei Dihuang decoction groups. Compared with LPS group, Bax expression in the 30.24 g/kg and 45.36 g/kg Xiaokang Liuwei Dihuang decoction groups was significantly down-regulated as well. In conclusion, Xiaokang Liuwei Dihuang decoction might ameliorate the immune infertility of male rats induced by LPS through regulating the levels of sex hormones, reactive oxygen species, pro-apoptotic and immune factors.

Conversion from spermatogonia to spermatocytes: Extracellular cues and downstream transcription network.

Spermatocyte (spc) formation from spermatogonia (spg) differentiation is the first step of spermatogenesis which produces prodigious spermatozoa for a lifetime. After decades of studies, several factors involved in the functioning of a mouse were discovered both inside and outside spg. Considering the peculiar expression and working pattern of each factor, this review divides the whole conversion of spg to spc into four consecutive development processes with a focus on extracellular cues and downstream transcription network in each one. Potential coordination among Dmrt1, Sohlh1/2 and BMP families mediates Ngn3 upregulation, which marks progenitor spg, with other changes. After that, retinoic acid (RA), as a master regulator, promotes A1 spg formation with its helpers and Sall4. A1-to-B spg transition is under the control of Kitl and impulsive RA signaling together with early and late transcription factors Stra8 and Dmrt6. Finally, RA and its responsive effectors conduct the entry into meiosis. The systematic transcription network from outside to inside still needs research to supplement or settle the controversials in each process. As a step further ahead, this review provides possible drug targets for infertility therapy by cross-linking humans and mouse model.

Histomorphometric evaluation of seminiferous tubules and stereological assessment of germ cells in testes following administration of aqueous leaf-extract of Lawsonia inermis on aluminium-induced oxidative stress in adult Wistar rats.

OBJECTIVES: This study aimed to investigate the 'Cytoprotective effect of Lawsonia inermis aqueous leaf-extract on aluminium-induced Oxidative stress in Histomorphometric of the Seminiferous tubule and Stereology of Germ Cells of adult male Wistar rats', assessing its effect on the Histomorphometry of the Seminiferous tubule and Stereology of Germ Cells. METHODS: Thirty-five adult male Wistar rats, weighing between 100-196g, and fifteen mice of the same weight range were used. Lawsonia inermis extracts and aluminum chloride (AlCl3) were administered for a period of three (3) weeks, with Five (5) rats per group. Group 1 (control), received rat pellets and distilled water. Group 2 received 60mg/kg/d aqueous extract. Group 3 received 0.5mg/kg/d of AlCl3. Group 4 received 0.5mg/kg/d of AlCl3 and 60mg/kg/d of aqueous extract orally. Group 5 received 0.5mg/kg/d of AlCl3 and 75mg/kg/d of aqueous extract orally. Group 6 received 0.5mg/kg/d of AlCl3 and 100mg/kg/d of aqueous extract orally. Group 7 received 0.5mg/k/d of AlCl3 and 5mg/Kg/d of ascorbic acid orally. Twenty-four hours after the last administration, the animals were weighed, sedated with chloroform and blood was collected. The testes were removed and weighed. RESULTS: There were statistically significant changes in the percentage of seminiferous tubular and seminiferous ductal diameter within the experimental animals in all the groups (p<0.05). Stereological findings revealed increase in spermatogonia, primary spermatocytes, round Spermatids and elongated spematids, spermatozoa, Sertoli cells population of the control rats while the rats given 0.5mg of aluminum chloride per kg of body weight had the lowest value (p<0.05). CONCLUSION: In this study, we demonstrated the affected histomorphometry of the seminiferous tubule and stereology of germ cells in testes, where stress impacts were most felt and subsequently translated into drastic reproductive dysfunction and distortion of spermatogenesis.

Evaluation of apoptotic- and autophagic-related protein expressions before and after IVM of fresh, slow-frozen and vitrified pre-pubertal mouse testicular tissue.

STUDY QUESTION: Do freezing and in vitro culture procedures enhance the expression of proteins involved in apoptotic or autophagic pathways in murine pre-pubertal testicular tissue? SUMMARY ANSWER: IVM strongly modified apoptosis- and autophagy-related relative protein levels in mice testicular tissue whereas the impact of cryopreservation procedures was minimal at the end of the culture. WHAT IS KNOWN ALREADY: In vitro spermatogenesis remains a challenging technical issue as it imposes to find a very close balance between survival and death of germ cell natural precursors (i.e. gonocytes and spermatogonia), which will eventually undergo a complete spermatogenesis close to in vivo conditions. The establishment of efficient culture conditions coupled with suitable cryopreservation procedures (e.g. controlled slow freezing [CSF] and solid surface vitrification [SSV]) of pre-pubertal testicular tissue is a crucial step in the fields of fertility preservation and restoration to improve the spermatic yield obtained in vitro. STUDY DESIGN, SIZE, DURATION: Here, we study cryopreservation procedures (i.e. CSF or SSV) and the impact of culture media compositions. A first set of 66 mouse pre-pubertal testes were directly cultured during 30, 36, 38 and 60 days (D) from 2.5 to 6.5-day-old CD-1 mice to evaluate the impact of time-aspect of culture and to endorse the reverse phase protein microarrays (RPPM) technique as an adapted experimental tool for the field of in vitro spermatogenesis. Ninety others fresh, slow-frozen and vitrified pre-pubertal testes were cultured during 30 days for the principal study to evaluate the impact of cryopreservation procedures before and after culture. Thirty-four testes dissected from 2.5, 6.5, 36.5, 40.5, 42.5 and 62.5 days postpartum (dpp) mice, corresponding to the time frames of spermatogenesis orchestrated in vitro, were used as in vivo controls. PARTICIPANTS/MATERIALS, SETTING, METHODS: After in vitro culture, testicular tissue samples originated from 2.5 or 6.5-day-old CD-1 male mice were analyzed using RPPM. This targeted proteomic technique allowed us to assess the expression level of 29 apoptosis- and autophagy-related factors by normalizing blank-corrected signal values. In addition, morphological analyses (e.g. HES, PAS, TRA98 and CREM) and DNA fragmentation in intra-tubular cells (i.e. terminal deoxynucleotidyl transferase dUTP nick end labeling; TUNEL) were assessed for the distinct experimental conditions tested as well as for in vivo control mouse testes. MAIN RESULTS AND THE ROLE OF CHANCE: A validation of the RPPM procedure in the field of in vitro spermatogenesis was completed with assay and array robustness before a principal study concerning the evaluation of the impact of in vitro culture and cryopreservation procedures. The proportion of elongated spermatids and the total cell number per seminiferous tubule tended to be very different between the in vivo and in vitro conditions (P < 0.05), suggesting the presence of a beneficial regulation on the first spermatogenesis wave by intrinsic apoptosis (Caspase_9) and autophagy (Atg5) factors (P < 0.0003 and r2 = 0.74). Concerning the impact of culture media compositions, a basic medium (BM) composed of αMEM plus 10% KnockOut™ serum replacement and gentamicin supplemented with retinol (Rol) and vitamin E (Vit. E) was selected as the best culture medium for fresh 6.5 dpp tissue cultured during 30D with 27.7 ± 8.10% of seminiferous tubules containing elongated spermatids. Concerning the impact of cryopreservation procedures, SSV did not have any impact on the morphological parameters evaluated after culture in comparison to fresh tissue (FT) controls. The proportion of tubules with elongated spermatids on testicular explants cultured with BMRol+Vit. E was not different between SSV (6.6 ± 1.6%) and CSF (5.3 ± 1.9%); however, round spermatids were observed more frequently for SSV (19 ± 6.2%) than CSF (3.3 ± 1.9%, P = 0.0317). Even if the proportion of TUNEL-positive cells for BMRol+Vit. E was higher at D30 after SSV (4.12 ± 0.26%) than CSF (1.86 ± 0.12%, P = 0.0022) and FT (2.69 ± 0.33%, P = 0.0108), the DNA damages observed at the end of the culture (i.e. D30) were similar to respective 6.5 dpp controls. In addition, the relative protein level expression ratio of an apoptotic factor, the phosphorylated FADD on Fas, was reduced by 64-fold in vitrified testes cultured with BMRol+Vit. E. Furthermore, we found in this study that the StemPro®-34 SFM culture medium supplemented with growth factors (e.g. EGF, bFGF, GDNF and LIF) prevented the differentiation of spermatogonial stem cells in favor of a significant proliferation with a better architectural pattern than in vivo 6.5 dpp controls with an increase of seminiferous tubules area for FT (P = 0.0357) and CSF (P = 0.0317). LIMITATIONS REASONS FOR CAUTION: Despite our promising results, the evaluation of apoptotic- and autophagic-related proteins was studied for a limited amount of proteins and on global testicular tissue. WIDER IMPLICATIONS OF THE FINDINGS: The data presented herein will help to improve apoptotic and autophagic understanding during the first spermatogenic wave. Moreover, our findings illustrate for the first time that, using finely-tuned experimental conditions, a testicular in vitro culture combined with proteomic technologies may significantly facilitate the study of cryopreservation procedures and in vitro culture evaluations. This study may also contribute to improve work on testicular tissues from pre-pubertal and adolescent cancer survivors. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by a Ph.D. grant from the Rouen Normandie Université and a financial support from 'la Ligue nationale contre le cancer' (both awarded to L.D.), funding from Institute for Research and Innovation in Biomedicine (IRIB), Agence de la Biomédecine, and co-supported by European Union and Région Normandie. Europe gets involved in Normandie with European Regional Development Fund (ERDF). The authors declare that there is no conflict of interest.

In vitro differentiation of rat spermatogonia into round spermatids in tissue culture.

STUDY QUESTION: Do the organ culture conditions, previously defined for in vitro murine male germ cell differentiation, also result in differentiation of rat spermatogonia into post-meiotic germ cells exhibiting specific markers for haploid germ cells? SUMMARY ANSWER: We demonstrated the differentiation of rat spermatogonia into post-meiotic cells in vitro, with emphasis on exhibiting, protein markers described for round spermatids. WHAT IS KNOWN ALREADY: Full spermatogenesis in vitro from immature germ cells using an organ culture technique in mice was first reported 5 years ago. However, no studies reporting the differentiation of rat spermatogonia into post-meiotic germ cells exhibiting the characteristic protein expression profile or into functional sperm have been reported. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: Organ culture of testicular fragments of 5 days postpartum (dpp) neonatal rats was performed for up to 52 days. Evaluation of microscopic morphology, testosterone levels, mRNA and protein expression as measured by RT-qPCR and immunostaining were conducted to monitor germ cell differentiation in vitro. Potential effects of melatonin, Glutamax® medium, retinoic acid and the presence of epidydimal fat tissue on the spermatogenic process were evaluated. A minimum of three biological replicates were performed for all experiments presented in this study. One-way ANOVA, ANOVA on ranks and student's t-test were applied to perform the statistical analysis. MAIN RESULTS AND THE ROLE OF CHANCE: Male germ cells, present in testicular tissue pieces grown from 5 dpp rats, exhibited positive protein expression for Acrosin and Crem (cAMP (cyclic adenosine mono phosphate) response element modulator) after 52 days of culture in vitro. Intra-testicular testosterone production could be observed after 3 days of culture, while when epididymal fat tissue was added, spontaneous contractility of cultured seminiferous tubules could be observed after 21 days. However, no supportive effect of the supplementation with any factor or the co-culturing with epididymal fat tissue on germ cell differentiation in vitro or testosterone production was observed. LIMITATIONS, REASONS FOR CAUTION: The human testis is very different in physiology from the rat testis, further investigations are still needed to optimize the organ culture system for future use in humans. WIDER IMPLICATIONS OF THE FINDINGS: The successful differentiation of undifferentiated spermatogonia using the testis explant culture system might be employed in future to produce sperm from human spermatogonia as a clinical tool for fertility preservation in boys and men suffering infertility. LARGE SCALE DATA: None. STUDY FUNDING AND COMPETING INTERESTS: This work was supported financially by the Frimurare Barnhuset in Stockholm, the Paediatric Research Foundation, Jeanssons Foundation, Sällskåpet Barnåvard in Stockholm, Swedish Research Council/Academy of Finland, Emil and Wera Cornells Foundation, Samariten Foundation, the Swedish Childhood Cancer Foundation as well as through the regional agreement on medical training and clinical research (ALF) between Stockholm County Council and Karolinska Institutet. All authors declare no conflicts of interests.

The inductive effects of Centella asiatica on rat spermatogenic cell apoptosis in vivo.

Centella asiatica (L.) Urban has been traditionally used for the treatment of various disease and as a food for thousands of years in various parts of the world including eastern Asia, China and India. The goal of this study was to investigate the effects of Centella asiatica aqueous leaf extract on the induction of spermatogenic cell apoptosis in male rats. After lethal dose (LD(50)) assessment of plant extract, rats were divided in five groups. The experimental groups received orally 10, 50, 80 and 100 mg/kg aqueous leaf extract daily for 60 days and the control group received just water. After 60 days, body and testis weight were measured and blood samples were taken from the heart. To evaluate apoptosis and histological changes, tissue samples obtained from rat testes were stained by TUNEL assay and hematoxylin and eosin stain. Results showed that the sperm count, motility, and viability and the number of spermatogenic cells in the seminiferous tubules were significantly decreased compared with the control group. The number of apoptotic germ cells per seminiferous tubule cross-section was significantly increased in the experimental group (18.11 ± 3.5) compared with the control group (8.7 ± 0.81) (P < 0.05). Serum testosterone, follicle-stimulating hormone, and luteinizing hormone levels also showed significant decreases in the experimental groups (P < 0.05). There was also a significant decrease in testis weight in experimental groups compared with the control group (P < 0.05). It is concluded that Centella asiatica has toxicological effects on the reproductive system in male rats and, therefore, it is suggested that leaf extracts of Centella asiatica possess antifertility effects in the male rat.

Proteinaceous diet inhibits gossypol-induced spermatotoxicity.

The present study was designed to investigate the effect of a proteinaceous dietary supplement, fishmeal, on gossypol-induced spermatotoxicity. Twenty-five adult male Wistar rats, averaging 205 g b.w., were randomly sorted into four experimental groups (I-IV) of 5 animals each, and a control group. Crude cottonseed oil was administered orally to each animal in groups I-IV at a rate that provided 14 mg/kg/d free gossypol; in addition, 3 g/d, 7 g/d, and 10 g/d of fishmeal was provided as meal supplement to each animal in groups I, II and III respectively. The control group received rat pellets and water freely. At the end of the 53-day treatment period, all animals were placed under chloroform anaesthesia; the caudal epididymides were removed, minced and placed in Ham's F10 solution for the evaluation of sperm count and motility. The testes were also processed for histological studies using the eosin and haematoxylin (H & E) method. Our findings revealed a dose-dependent inhibition of gossypol-induced spermatotoxicity by the supplemented fishmeal; this suggests that proteinaceous diets are protective against gossypol-induced male infertility.

Effects of acitretin on spermatogenesis of rats.

In this experimental study the possible effects of the acitretin on the spermatogenesis of the rats were investigated histopathologically. Thirty-nine male adult Wistar albino rats were divided into 3 groups as two experimental groups and one control group. The first group consisting 14 rats were applied orally standard dose (0.75 mg/kg/day) acitretin and the second group consisting 16 rats were applied high dose (1.5 mg/kg/day) acitretin. Acitretin was given within dimetil sulphoxide (DMSO), which was diluted with saline solution as a ratio of 1/10, in order to increase its solubility. The control group consisting 9 rats were given only saline solution including DMSO for 8 weeks. After 8 weeks of the administration, half of the rats in the first and second groups and the entire control group were sacrificed under deep ether anaesthesia and bilateral orchiectomy was made. The remaining rats were compared with the control group using a similar method at the end of 8 weeks of wash-off period. The orchiectomy materials were histopathologically evaluated under the light microscope for spermatogenesis according to parameters including spermatogenetic activity, spermatogenetic organization, seminiferous tubular diameter, interstitial Leydig cells and fibroblasts. The groups, which were evaluated at the end of the 8(th) and 16(th) weeks, were compared with the control group regarding the mentioned parameters and no statistical significance was observed among the groups. In our study it was concluded that the standard and high doses of acitretin do not have any effect on the spermatogenesis of the rats.

Effect of Stephania hernandifolia leaf extract on testicular activity in rats.

AIM: The testicular inhibitory effect of the aqueous fraction of methanol extract of Stephania hernandifolia leaf was studied in male Wistar rats. METHODS: The supernatent and the precipitate part of aqueous fractions of the methanol extract of the leaf were gavaged separately to rat at a similar dose of 200 mg/mL per 100 g body weight per day for 28 days. After cessation of treatment, various observations were conducted. RESULTS: In both treated groups, there were significant decreases in the relative weights of the sex organs, the testicular key androgenic enzymes activities, the plasma level of testosterone, the number of different germ cells at stage VII of seminiferous epithelial cell cycle and the seminiferous tubular diameter in comparison to the controls. Neither of the parts had somatic, renal and hepatic toxicity. This study suggested that the active molecules present in the aqueous fraction of methanol extract of Stephania hernandifolia leaves might be steroids as indicated by thin layer chromatography using specific staining substance for steroid molecules. CONCLUSION: In rats, the aqueous fraction of methanol extract of the S. hernandifolia leaves possesses certain testis-inhibitory substances, which may be steroid-like agents.

Hypogonadotropic hypogonadism and peripheral neuropathy in Ebf2-null mice.

Olf/Ebf transcription factors have been implicated in numerous developmental processes, ranging from B-cell development to neuronal differentiation. We describe mice that carry a targeted deletion within the Ebf2 (O/E3) gene. In Ebf2-null mutants, because of defective migration of gonadotropin releasing hormone-synthesizing neurons, formation of the neuroendocrine axis (which is essential for pubertal development) is impaired, leading to secondary hypogonadism. In addition, Ebf2(-/-) peripheral nerves feature defective axon sorting, hypomyelination, segmental dysmyelination and axonal damage, accompanied by a sharp decrease in motor nerve conduction velocity. Ebf2-null mice reveal a novel genetic cause of hypogonadotropic hypogonadism and peripheral neuropathy in the mouse, disclosing an important role for Ebf2 in neuronal migration and nerve development.

Male reproductive defects caused by puromycin-sensitive aminopeptidase deficiency in mice.

Male reproductive performance is composed of two principal elements, copulation and spermatogenesis. A wealth of literature has described the intricate web of endocrine events underlying these biological processes. In the present study we show that puromycin-sensitive aminopeptidase (Psa)-deficient mice are infertile, lack copulatory behavior, and have impaired spermatogenesis. The reproductive deficits of the mutants are not restored by androgen administration, although no aberrant localization of the sex steroid receptors was detectable in their brains and testes. Considering the strong expression of the Psa gene in the brain and Sertoli cells and the degenerative morphology of Sertoli cells in Psa-deficient mice, Psa may participate in testosterone-mediated reproductive signal pathways in the brain and testis.

Developmental expression, intracellular localization, and selenium content of the cysteine-rich protein associated with the mitochondrial capsules of mouse sperm.

The outer membranes of mitochondria of mammalian sperm are encased in a keratinous structure known as the mitochondrial capsule. The experiments in the present study were designed to resolve a controversy surrounding the intracellular localization, developmental expression, and selenium-content of a cysteine-rich 17-20 kD protein that has been reported to constitute the major structural protein in the mitochondrial capsule of mammals. An antibody to a synthetic oligopeptide based on the predicted sequence of mouse cysteinerich protein recognizes a 24 kD protein in epididymal sperm tails of mice. The 24 kD protein does not appear to be a selenoprotein because: (1) it is not labeled with 75Se-selenite in seminiferous tubule culture; (2) cleavage with cyanogen bromide and translation of T7 RNA polymerase transcripts in vitro indicate that the translation start site is located downstream of potential UGA selenocysteine codons in the mouse cysteine-rich mRNA; (3) the reading frame encoding the cysteine-rich protein in rat lacks inphase UGA selenocysteine codons. Light and electron microscopy immunocytochemistry detects the cysteine-rich protein first during step 11 of spermiogenesis in the mouse demonstrating that the cysteine-rich protein mRNA is under temporal translational control. Electron microscope immunocytochemistry reveals that the cysteine-rich protein is evenly distributed in the cytoplasm in spermatids in steps 11 through early step 16 in mouse, and that it is associated with the outer mitochondrial membranes of spermatids in late step 16 and epididymal spermatozoa.

Effects of selenium deficiency on testicular morphology and function in rats.

For four generations rats were fed a low selenium diet (2-7 micrograms Se kg-1) or the same diet with 250 or 300 micrograms Se kg-1 added as selenite. In male rats of the first generation that had been fed the diets from the age of 20 days onwards, selenium depletion led to slightly delayed testis growth during pubertal development that was compensated for in the later stages of maturation. In adult rats fed the low selenium diet for nearly a year no changes in testicular mass and morphology were observed. The serum concentration of testosterone of 6-month-old, selenium-depleted animals was, however, slightly lower than that of adequately supplied controls, and the stimulation of testosterone secretion by administration of GnRH or LH resulted in a significantly less marked rise in the serum concentration of testosterone. From the second generation onwards the testis mass, expressed as a percentage of the body mass, decreased and in the fourth generation was less than 50% of that of the controls. The male gonads of fourth generation animals showed a severe bilateral atrophy, in which the seminiferous tubules were considerably reduced in diameter and almost entirely lined by Sertoli cells and a few stem cells. Differentiated spermatozoa could not be detected. The alterations were reversible and spermatogenesis was restored by feeding the selenium-adequate diet. The findings indicate that testicular morphology and functions are affected by severe selenium deficiency and that the element is necessary for testosterone biosynthesis and the formation and normal development of spermatozoa.

Steroidogenic and morphogenic characteristics of human peritubular cells in culture.

We have explored the morphogenic and functional characteristics of human peritubular cells originating from seminiferous tubule (ST) fragments isolated from the testes of two prepubertal patients with the androgen insensitivity syndrome. These ST were cultured in Dulbecco's Modified Eagle's Medium-Ham's F-12 supplemented with antibiotics, transferrin, hydrocortisone, vitamin E, and 3% fetal bovine serum. A centrifugal growth of elongated fibroblast-like cells peripheral to the ST explants was observed. Muscle-specific actin and 3 beta-hydroxysteroid dehydrogenase were evident in the peritubular area and in the elongated cells growing from the tubules. Histochemistry was negative in the tubules themselves, revealing the mixed nature of these cultures. The ST fragments were lost after subculturing, leaving a homogeneous monolayer of fibroblast-like cells. The steroidogenic potential of these cells was demonstrated by the secretion of testosterone (T) to the culture medium. T secretion was stimulated by hCG in a time-dependent fashion (patient 1: Day 11, 84% and Day 15, 200%; patient 2: Day 8, 73% and Day 11, 32% over basal). FSH also stimulated T secretion (patient 1: Day 5, 136% and Day 8, 89%; patient 2: Day 8, 117% and Day 11, 129% over basal). Furthermore, T secretion by these cultures was 100% higher than that observed in mesenchymal cells obtained from the testicular intertubular space in the same patient. Spontaneous T secretion and hormone responses declined progressively to cease by 25 days in culture. These results suggested the involvement of Sertoli cell (SC)-secreted factor(s) in the regulation of T secretion by peritubular cells. In order to further explore possible paracrine interactions between peritubular and Sertoli cells, we carried out heterologous cocultures with rat SC. After 72 h a striking redistribution of both cell types was observed with the formation of cord-like structures. Ultrastructural examination of these cords showed the formation of a basement membrane between epithelial (Sertoli) and mesenchymal cells of peritubular origin. No resumption of T secretion was observed, but an increase in androgen-binding protein (ABP) production by rat SC under basal (37%) and FSH-stimulated (52%) conditions was evident. Our results show that in the human peritubular compartment, cells exist that can alternatively express steroidogenic functions, associate with SC in a specific mesenchymal-epithelial interaction, and exert regulatory influences on ABP secretion by SC. In addition they indicate that communicating events in the testis are preserved throughout evolution.

Zinc protects testicular injury induced by concurrent exposure to cadmium and lead in rats.

The effect of coexposure to lead and cadmium (each 50 ppm alone and 25 ppm in combination) on the testes of rats and the preventive role of zinc (50 ppm) was investigated by administering these metals through drinking water. Male weaned albino rats were exposed to these metals for 120 days. Testicular histology, sperm counts and sperm motility were studied in these rats. The animals coexposed to lead and cadmium exhibited much more pronounced pathological changes and reduced sperm counts compared to the animals exposed to either of the metals alone. Zinc supplementation to the lead + cadmium exposed rats revealed the protective effect of zinc on these parameters. The observed higher magnitude of changes in the testes of lead + cadmium exposed group seems to be due to the excessive cadmium accumulation.

Differentiation of pancreatic acinar carcinoma cells cultured on rat testicular seminiferous tubular basement membranes.

The use of rat testicular seminiferous tubular basement membrane (STBM) segments as a model substratum for the in vitro maintenance of tumor cells dissociated from a transplantable pancreatic acinar rat carcinoma (J. K. Reddy and M. S. Rao, Science (Wash. DC), 198: 78-80, 1977) is described. Ultrastructurally pure, hollow tubular segments of STBM were prepared by mechanical disaggregation, DNase digestion, and deoxycholate treatment. Dissociated pancreatic acinar carcinoma cells adhered readily to STBM segments within 1 to 6 hr, and these STBM-tumor cell aggregates were maintained for up to 7 days in serum-free chemically defined medium supplemented with hydrocortisone, insulin, vitamin C, and soybean trypsin inhibitor. The tumor cells formed acinar-like clusters and displayed intercellular junctions and polarization of secretory granules toward the center of these clusters. By 4 days, virtually all cells of this acinar carcinoma maintained on STBM in supplemented chemically defined medium contained numerous secretory granules. Cell replication, as determined by [3H]thymidine autoradiography, ceased within 18 hr of attachment of neoplastic cells to STBM; however, all cells incorporated [3H]leucine as evidenced by light and electron microscopic autoradiography. In addition, two-dimensional analysis and fluorography of newly synthesized secretory proteins discharged by these cells in response to carbamylcholine revealed the presence of Mr 24,000 protein and 19 other secretory proteins characteristic of this tumor (L. J. Hansen, M. K. Reddy, and J. K. Reddy, Proc. Natl. Acad. Sci. USA, 80: 4379-4383, 1983). The culture system utilizing STBM and supplemented chemically defined medium should allow investigation of the effects of a variety of factors on morphogenesis, cytodifferentiation, and gene expression in pancreatic acinar tumors.

Maturation of 17 beta-estradiol binding protein in the hypothalamus and anterior pituitary of male rats.

Levels of estrogen binding proteins in the cytosol of hypothalamic and pituitary tissue of male rats at different ages were estimated. Two classes of estrogen binding sites were detected in 13 and 20 days old animals. The number of high affinity estrogen binding sites (K = ca. 2 x 10(-10) mol) of the hypothalamus was slightly larger in 13 days old animals, then decreased to constant levels at the age of 20 days and no changes were observed thereafter. Levels of low affinity binding sites of the hypothalamus (K = ca. 5 x 10(-9) mol) decreased with age and were no longer detectable in 30 days old animals. Similarly, a decrease of low affinity estrogen binding sites with age was recorded for pituitary cytosol samples and this class of receptor sites was no longer observable in 30 days old male rat. At 13 days high levels of high affinity 17 beta-estradiol binding protein was registered and minimal numbers of binding sites were found at 20, 30 and 40 days. Thereafter, the levels remained constant until 100 days and were similar to values observed in the 13 days old male animals. Apparent affinity constants of two classes of binding sites in hypothalamus and pituitary were similar...