Stages VII-VIII Research Articles

Decreasing sperm quality in mice subjected to chronic cannabidiol exposure: New insights of cannabidiol-mediated male reproductive toxicity

Cannabidiol (CBD) is a natural cannabinoid present in the Cannabis sativa plant, widely prescribed as an anticonvulsant drug, especially for pediatric use. However, its effects on male reproduction are still little investigated. Therefore, the present study assessed the effects of CBD on the spermatogenesis and sperm quality. For this, twenty-one-day-old Swiss mice received CBD for 34 consecutive days by gavage at doses of either 15 or 30 mg/kg. Chronic exposure to CBD decreased the frequency of stages VII-VIII and XII of spermatogenesis and an increase in the frequency of stage IX were noted. Furthermore, the seminiferous epithelium height reduced at stage IX and increased at stage XII in both CBD-treated groups. There was a significant rise of sperm DNA damage, while no genotoxic effects were observed in leukocytes. The activities of superoxide dismutase and catalase decreased, while malondialdehyde levels increased in the sperm of mice treated with a higher dose of CBD. Mice exposed to 30 mg/kg of CBD showed a reduction in the mobile spermatozoa percentage and in curvilinear velocity, while straight line and average path velocity decreased in both treated groups. The number of acrosome-intact spermatozoa declined in the CBD 30 group, and the number of abnormal acrosomes raised in both CBD groups. On the other hand, the weight of reproductive organs, sperm count, and hormone levels were not affected by CBD treatment. These findings show that dysregulation of the endocannabinoid system by CBD can reduce sperm quality. The mechanisms responsible may be associated with disorders during spermatogenesis, especially during the final stages of nuclear remodelling and assembly of acrosome. However, changes in mitochondrial function, as well as the reduction on the antioxidant enzyme activities during epididymal transit, at least partly, may also be involved.

Di-(2-ethylhexyl) phthalate induces testicular endoplasmic reticulum stress and germ cell apoptosis in adolescent mice.

Di-(2-ethylhexyl) phthalate (DEHP) is a male reproductive toxicant. This research is aimed at investigating the effect of pubertal DEHP exposure on testicular endoplasmic reticulum (ER) stress and germ cell apoptosis. Five-week-old male mice were orally administered with DEHP (0, 0.5, 50, or 500mg/kg/day) for 35 days. Testis weight and sperm count were reduced in mice exposed to 500mg/kg/day DEHP. The number of seminiferous tubules in stages VII-VIII, mature seminiferous tubules, was reduced and the number of seminiferous tubules in stages IX-XII, immature seminiferous tubules, was elevated in mice treated with 500mg/kg/day DEHP. Numerous apoptotic germ cells were observed in mouse seminiferous tubules exposed to 50 and 500mg/kg/day DEHP. Moreover, cleaved caspase-3 was elevated in mouse testes exposed to 500mg/kg/day DEHP. In addition, Bcl-2 was reduced and Bax/Bcl-2 was elevated in mouse testes exposed to 500mg/kg/day DEHP. Additional experiment showed that GRP78, an ER molecular chaperone, was downregulated in mouse testes exposed to 500mg/kg/day DEHP. Testicular p-IRE-1α, p-JNK, and CHOP, three markers of ER stress, were upregulated in mice exposed to 500mg/kg/day DEHP. These results suggest that pubertal exposure to high doses of DEHP induces germ cell apoptosis partially through initiating ER stress in testes.

Short- and long-term exposure to methamidophos impairs spermatogenesis in mice

Methamidophos (MET) is a pesticide that has toxic properties, including effects on fertility. This study aimed to assess the joint action of treatment time and exposure to methamidophos on the male reproductive system. MET was orally administered to adult male Swiss mice at a dose of 0.004 mg.kg−1 for 15 and 50 consecutive days. The following parameters were evaluated: weight of reproductive organs, spermatogenesis, sperm and Sertoli cell count, daily sperm production and sperm transit time. Short-term exposure to methamidophos induced a decrease in epididymal weight. The frequency of stages V–VI of spermatogenesis increased and the frequency of stage IX decreased. In the epididymis, sperm transit time (caput/corpus) was reduced and the relative sperm number (cauda) increased. Long-term exposure induced an increase in the frequencies of stages I–IV and V-VI and decreased the stages VII-VIII and IX. The number of Sertoli cells with evident nucleoli was reduced in both exposures. These results confirm the reproductive toxicity of MET.

Effects of spermatogenic cycle on Stem Leydig cell proliferation and differentiation

We reported previously that stem Leydig cells (SLC) on the surfaces of rat testicular seminiferous tubules are able to differentiate into Leydig cells. The proliferation and differentiation of SLCs seem likely to be regulated by niche cells, including nearby germ and Sertoli cells. Due to the cyclical nature of spermatogenesis, we hypothesized that the changes in the germ cell composition of the seminiferous tubules as spermatogenesis proceeds may affect tubule-associated SLC functions. To test this hypothesis, we compared the ability of SLCs associated with tubules at different stages of the cycle to differentiate into Leydig cells in vitro. SLCs associated with stages IX-XI were more active in proliferation and differentiation than SLCs associated with stages VII-VIII. However, when the SLCs were isolated from each of the two groups of tubules and cultured in vitro, no differences were seen in their ability to proliferate or differentiate. These results suggested that the stage-dependent local factors, not the SLCs themselves, explain the stage-dependent differences in SLC function. TGFB, produced in stage-specific fashion by Sertoli cells, is among the factors shown in previous studies to affect SLC function in vitro. When TGFB inhibitors were included in the cultures of stages IX-XI and VII-VIII tubules, stage-dependent differences in SLC development were reduced, suggesting that TGFB may be among the paracrine factors involved in the stage-dependent differences in SLC function. Taken together, the findings suggest that there is dynamic interaction between SLCs and germ/Sertoli cells within the seminiferous tubules that may affect SLC proliferation and differentiation.

Chronic exposure to cannabidiol induces reproductive toxicity in male Swiss mice.

Children and adults with frequent and severe episodes of epilepsy that do not respond to standard treatments (such as carbamazepine, phenytoin and valproate) have long been prescribed cannabidiol (CBD) as an anticonvulsant drug. However, the safety of its chronic use in relation to reproduction has not been fully examined. This study aimed to assess the effects of chronic CBD exposure on the male reproductive system. CBD was orally administered to 21-day-old male Swiss mice at doses of 15 and 30mgkg-1 daily (CBD 15 and 30 groups, respectively), with a control group receiving sunflower oil, for 34 consecutive days. After a 35day recovery period, the following parameters were evaluated: weight of reproductive organs, testosterone concentration, spermatogenesis, histomorphometry, daily sperm production and its morphology. The CBD 30 group had a 76% decrease in total circulating testosterone, but it remained within the physiological normal range (240-1100ngdl-1 ). CBD treatment induced a significant increase in the frequency of stages I-IV and V-VI of spermatogenesis, and a decrease in the frequency of stages VII-VIII and XII. A significant decrease in the number of Sertoli cells was observed only in the CBD 30 group. In both CBD groups the number of spermatozoa in the epididymis tail was reduced by 38%, sperm had head abnormalities, and cytoplasmic droplets were observed in the medial region of flagellum. These results indicated that chronic CBD exposure was associated with changes in the male reproductive system, suggesting its reproductive toxicity.

Protective roles of Rutin against restraint stress on spermatogenesis in testes of adult mice.

In order to investigate the effects of Rutin against restraint stress, 50 adult male mice were divided into five groups: control, restraint stress (RS), and RS with 2 doses of Rutin treatments. Mice were restrained in a conical tube for 4 h daily and Rutin was injected intraperitoneally for 15 consecutive days. Restraint stress significantly decreases body weights, testis and epididymis weights, thymus weights, visceral fats, serum concentrations of testosterone, sperm counts, sperm motility and sperm viability, while it increases serum epinephrine levels, adrenal gland weights and abnormal sperms. In addition, restraint stress severely damages the testicular histoarchitecture and spermatogenesis. Stressed groups also showed broken seminiferous tubules, few spermatozoa in lumen, less population of Leydig cells between the interstitial spaces, spermiation arrest in stage I-III and degenerated population of round spermatids in the lumen; as well as missing cells in stages IV-VI. Furthermore, lumen sizes increased in stages VII, VIII, IX and X. Residual bodies increased in stages IV-VI, VII-VIII and vacuoles found in stages XI-XII after restraint stress. PARP1 signaling is involved in apoptosis. In this study, expressional levels of cleaved PARP1 and cleaved Caspase-3 are significantly increased in testes after restraint stress. We demonstrate that Rutin significantly ameliorates the side effects induced by restraint stress.

A High Fat Diet during Adolescence in Male Rats Negatively Programs Reproductive and Metabolic Function Which Is Partially Ameliorated by Exercise.

An interaction between obesity, impaired glucose metabolism and sperm function in adults has been observed but it is not known whether exposure to a diet high in fat during the peri-pubertal period can have longstanding programmed effects on reproductive function and gonadal structure. This study examined metabolic and reproductive function in obese rats programmed by exposure to a high fat (HF) diet during adolescence. The effect of physical training (Ex) in ameliorating this phenotype was also assessed. Thirty-day-old male Wistar rats were fed a HF diet (35% lard w/w) for 30 days then subsequently fed a normal fat diet (NF) for a 40-day recovery period. Control animals were fed a NF diet throughout life. At 70 days of life, animals started a low frequency moderate exercise training that lasted 30 days. Control animals remained sedentary (Se). At 100 days of life, biometric, metabolic and reproductive parameters were evaluated. Animals exposed to HF diet showed greater body weight, glucose intolerance, increased fat tissue deposition, reduced VO2max and reduced energy expenditure. Consumption of the HF diet led to an increase in the number of abnormal seminiferous tubule and a reduction in seminiferous epithelium height and seminiferous tubular diameter, which was reversed by moderate exercise. Compared with the NF-Se group, a high fat diet decreased the number of seminiferous tubules in stages VII-VIII and the NF-Ex group showed an increase in stages XI-XIII. HF-Se and NF-Ex animals showed a decreased number of spermatozoa in the cauda epididymis compared with animals from the NF-Se group. Animals exposed to both treatments (HF and Ex) were similar to all the other groups, thus these alterations induced by HF or Ex alone were partially prevented. Physical training reduced fat pad deposition and restored altered reproductive parameters. HF diet consumption during the peri-pubertal period induces long-term changes on metabolism and the reproductive system, but moderate and low frequency physical training is able to recover adipose tissue deposition and reproductive system alterations induced by high fat diet. This study highlights the importance of a balanced diet and continued physical activity during adolescence, with regard to metabolic and reproductive health.

MKP-1 attenuates LPS-induced blood-testis barrier dysfunction and inflammatory response through p38 and IκBα pathways.

Sertoli cells create a local tolerogenic microenvironment to maintain testicular immune privilege especially through the formation of a blood-testis barrier (BTB). However, the molecular mechanisms underlying the immune modulation function and BTB dynamics of Sertoli cells remained elusive. MAP phosphatase (MKP)-1 acts as a crucial negative regulator of the inflammatory response. Nevertheless, the role of MKP-1 in regulating Sertoli cells has not been elucidated. In this study, we have for the first time uncovered distinct cellular localization of MKP-1 in the cells at different stages of mouse testis, and the level of MKP-1 expression was significantly up-regulated by LPS-induced acute testis inflammation. In addition, MKP-1 staining was strongly detected in nuclei and peri-nuclear regions of cytoplasm in the Sertoli cells, and it was presented at Sertoli cell tight junctions (TJs) at stages VII-VIII after LPS treatment. Moreover, we demonstrated that MKP-1 was capable of attenuating LPS-induced decrease of occludin by interaction with p38 MAP kinase and IκBα molecules. Taken together, our data highlight that MKP-1 was an important endogenous suppressor of innate immune responses involved in the regulation of BTB barrier dynamic. This study thus might offer novel targets for treating inflammatory diseases in the testis.

Influence of histological degree of seminiferous tubular degeneration and stage of seminiferous cycle on the proliferation of spermatogonia in aged Syrian hamster (Mesocricetus auratus)

The ageing testis is associated with germ loss in the seminiferous epithelium and a decrease in spermatogonia proliferation. In this work, we study whether the stages of the seminiferous epithelium cycle and/or the degree of histological tubular degeneration resulting from ageing is related with this decrease in spermatogonia proliferation. Eleven hamsters were used, five aged 6 months and six aged 24 months. In both groups, the proliferative activity was studied by BrdU immunostaining. The number of BrdU-positive and BrdU-negative cells was measured, providing the overall proliferation index in adult and aged testes. The mean number of BrdU-positive cells was also determined for each degree of histological degeneration of seminiferous epithelium, and a spermatogonia proliferation index was obtained for each stage of the seminiferous cycle. Ageing caused an overall decrease in the BrdU-positive cell percentage and a decrease in the number of BrdU-positive cells in the tubular sections with hypospermatogenesis, the sloughing of germ cells and maturation arrest, these changes being similar in both young and old animals. The spermatogonia proliferation index was only seen to be significantly lower in ageing hamster in stages VII-VIII of the seminiferous epithelium cycle. In conclusion, the overall decrease in proliferation observed in aged seminiferous epithelium is correlated with an increase in the number of degenerated sections of the seminiferous tubules, and this decrease is a phenomenon which occurs in specific stages of the seminiferous cycle.

The Scribble/Lgl/Dlg Polarity Protein Complex Is a Regulator of Blood-Testis Barrier Dynamics and Spermatid Polarity during Spermatogenesis

During spermatogenesis, spermiogenesis that releases sperm into the tubule lumen and restructuring of the blood-testis barrier (BTB) that accommodates the transit of preleptotene spermatocytes take place simultaneously, but at the opposite ends of the seminiferous epithelium. These events are tightly regulated and coordinated; however, neither the underlying mechanism(s) nor the involving molecules are known. Herein, the Scribble/Lgl (Lethal giant larvae)/Dlg (Discs large) polarity complex was shown to regulate spermatid polarity during spermiogenesis and tight junction (TJ)-permeability barrier via changes in protein distribution at the apical ectoplasmic specialization and the BTB during the epithelial cycle, respectively. Scribble, Lgl2, and Dlg1 were found to be expressed by Sertoli and germ cells. Scribble also displayed stage-specific expression at the BTB, being highest at stages VII-VIII, colocalizing with TJ proteins occludin and ZO-1. Unlike components of other polarity complex modules, such as partitioning-defective 6, the knockdown of which by RNA interference was found to impede Sertoli cell TJ barrier, a knockdown of the Scribble complex (i.e. simultaneous knockdown of Scribble, Lgl and Dlg or Lgl alone; but not Scribble or Dlg alone) both in vitro and in vivo promoted the TJ integrity. This was mediated by reorganizing actin filament network at the Sertoli cell-cell interface, which, in turn, affected changes in the localization and/or distribution of occludin and/or β-catenin at the BTB. These knockdowns also perturbed F-actin organization at the Sertoli cell-spermatid interface, thereby modulating spermatid adhesion and polarity at the apical ectoplasmic specialization. In summary, the Scribble/Lgl/Dlg complex participates in the regulation of BTB dynamics and spermatid adhesion/polarity in the testis.

Altered phosphorylation and distribution status of vimentin in rat seminiferous epithelium following 17β-estradiol treatment

Vimentin, type III intermediate filament, has stage-specific localization in the Sertoli cell. In the rat, during stages I-V and XI-XIV of the seminiferous epithelium, vimentin is localized in the perinuclear area with filaments projecting into the apical region toward the developing germ cells. These filaments decrease in length at stages VI-VII with perinuclear staining in stages VIII-IX, when spermiation occurs. Our earlier studies following 17β-estradiol treatment to adult male rats demonstrated an increase in germ cell apoptosis, spermiation failure and disruption of Sertoli cell microfilaments and microtubules. The present study was undertaken to determine the stage-specific distribution of vimentin and its involvement in spermiation failure and germ cell apoptosis. Immunofluorescence studies revealed that in contrast to the perinuclear localization with small extensions in control stages VII-IX, long extensions radiating apically to the spermatids in deep recess were observed in the treated group. Immunoprecipitation studies showed marked absence of phosphorylated vimentin in stages VII-VIII in the treated group. Further, localization of plectin, cytoskeletal linker protein, showed decrease in all the stages of spermatogenesis following estradiol treatment. Interestingly, for the first time the localization of plectin in the tubulobulbar complex was observed. In conclusion, the study suggests that estradiol treatment leads to an effect on vimentin phosphorylation, which could have inhibited the disassembly of vimentin leading to retention of apical projection in stages VII-VIII. These effects could be presumably due to a decrease in plectin, affecting the reorganization of vimentin and therefore the apical movement of spermatids, leading to spermiation failure.

Immunolocalization of galectin-3 in mouse testicular tissue.

Galectin-3 (Gal-3) is a member of the ß-galactoside-binding lectins which is expressed in a variety of tissues and plays a role in diverse biological events, such as embryogenesis, adhesion, cellular proliferation, and apoptosis. In this study, the presence and distribution of galectin-3 (Gal-3) in the mouse testicular tissue was investigated. Eight adult NMRI mice were used in this study. Animals were sacrificed by decapitation under ether anesthesia. The testes were excised, fixed in 10% buffered formalin and embedded in paraffin for immunohistochemical (IHC) studies. H-score, a semi-quantitative method, was used for scaling the immunostaining. Positive immunoreactivity to Gal-3 was detected in the connective tissues of the interstitium, Leydig cells and presumably peritubular myoid cells. Seminiferous tubules showed immuno-positive reaction in a stage dependent manner. Stages I-III and IX-XII of spermatogenic cycle showed mild immunostaining, while moderate immunostaining was observed at stages IV-VI. Highest immunoreactivity was observed at stages VII-VIII. H-score assessment showed a significant increase in stages of VII-VIII in comparison to other stages (P< 0.05). Expression of Gal-3 in interstitial tissue and seminiferous tubules indicated that this protein probably has multifunctional roles in the mouse testicular tissue.

Fenvalerate induces germ cell apoptosis in mouse testes through the Fas/FasL signaling pathway

Fenvalerate has a potentially adverse effect on male reproduction and spermatogenesis, whereas the precise mechanism remains obscure. The present study investigated the effects of fenvalerate on germ cell apoptosis in testes. Adult male mice were administered with fenvalerate (15 or 60mg/kg) by gavage for 28days. Germ cell apoptosis was determined by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL). The number of TUNEL+ germ cells per tubule and the percentage of tubules with TUNEL+ germ cells were significantly increased in testes of mice treated with fenvalerate in a dose-dependent manner. TUNEL+ germ cells were observed mainly in stages VII-VIII and also stages IX-XII seminiferous tubules in testes. Additional experiments showed that fenvalerate increased the level of active caspase-8 and caspase-3 in testes. In addition, fenvalerate upregulated the expression of Fas and FasL in testes. No significant difference on the expression of Bcl-2 and Bax in testes was observed between fenvalerate-treated mice and controls. Fenvalerate did not affect the leakage of cytochrome c from mitochondria into cytoplasm. In addition, fenvalerate did not cause the activation of caspase-9 in testes. Taken together, these results suggest that fenvalerate induces germ cell apoptosis in testes through the Fas/FasL signaling pathway.

Vitamin B12 Supplement Exerts a Beneficial Effect on the Seminiferous Epithelium of Cimetidine-Treated Rats

Treatment of gastric ulcer with cimetidine reduces acid secretion and interferes in the vitamin B₁₂ absorption. Regarding the harmful effect of cimetidine on the seminiferous tubules, the aim of the present study was to verify if prolonged treatment with cimetidine causes vitamin B₁₂ deficiency and whether the testicular damages are attenuated by vitamin B₁₂ supplementation. Adult male rats received, for 50 days, cimetidine (CMTG), cimetidine and vitamin B₁₂ (CMT/B₁₂G), vitamin B₁₂ (B₁₂G) and saline solution (CG). Vitamin B₁₂ and homocysteine plasma levels were evaluated and the testes were embedded in glycol methacrylate for the morphometric analyses of total, epithelial and luminal areas of the seminiferous tubules, number of Sertoli cells and frequencies of tubules according to stages and containing Sertoli and germ cells in the lumen. Terminal deoxynucleotidyl-transferase mediated dUTP nick end labeling (TUNEL) method and proliferating cell nuclear antigen (PCNA) immunohistochemistry were carried out. CMTG showed TUNEL-positive Sertoli cells and significant reductions in the epithelial and total tubular areas, number of Sertoli cells and frequency of tubules VII-VIII. In the CMT/B₁₂G, the number of Sertoli cells and the epithelial and total tubular areas were similar to CG. The number of Sertoli cells (in B₁₂G) and the frequency of tubules at stages VII-VIII (in B₁₂G and CMT/B₁₂G) increased significantly; PCNA-positive Sertoli cells were found in these groups. Although cimetidine was not able to induce vitamin B₁₂ deficiency, this drug causes tubular atrophy due to Sertoli cell damage and loss of germ cells. However, vitamin B₁₂ supplement is able to stimulate spermatogenesis and restore the number of Sertoli cells, softening the harmful effect of cimetidine on spermatogenesis.

Expression Patterns ofSuperoxide DismutaseGenes in the Stage-specific Seminiferous Tubules of Mice Excised by a Laser Capture Microdissection

Spermatogenesis is a particularly difficult process to study the unique multiple cellular associations within the seminiferous epithelium. Laser capture microdissection (LCM) is a recently developed technique that enables the isolation of individual cell populations from complex tissues. The superoxide dismutase (SOD) is the first and most important enzyme of antioxidant defense systems against superoxide anion. The aim of this study was to investigate the quantitative changes of SOD gene expression according to the spermatogenic cycle in mouse testes using LCM and real-time polymerase chain reaction (PCR) techniques. Frozen sections (10 μm) were obtained from the testes of 8-weeks-old ICR mice. LCM was used to capture all cells in cross-sectioned seminiferous tubules which were grouped into stages Ⅰ-Ⅴ, ⅦⅧ, and Ⅸ-ⅩⅠ. The expression level of cytoplasmic Cu, Zn-SOD (SOD1) mRNA was remarkably higher than those of mitochondrial Mn-SOD (SOD2) and extracellular Cu, Zn-SOD (SOD3) mRNAs in mouse testes. During spermatogenesis, the expressions of SOD1 and SOD2 mRNAs were highest on stages I-V, began to decrease after stage Ⅶ, and showed a lowest level on stage Ⅸ-ⅩⅠ. However, the expression of SOD3 mRNA was highest on stages Ⅶ-Ⅷ. These findings suggest that the subtypes of SOD are expressed differentially in mouse testes during spermatogenesis.

Dexamethasone effects on Bax expression in the mouse testicular germ cells.

Exposure to glucocorticoids (GCs) leads to numerous changes in various biological systems including the reproductive system. The aim of the present study was to find out whether dexamethasone (Dex), a widely used GC, would influence the apoptosis and expression of Bax, an important proapoptotic protein, in the mouse testicular germ cells. Experimental groups of 8 male NMRI mice received one of the following treatments daily for 7 days: 4, 7 and 10 mg/kg Dex. Control groups were treated with equivalent volumes of saline. Experimental and control animals were sacrificed 24 h after the last injection. Immunohistochemical procedure was used to evaluation of Bax expression and the deoxyuridine nick-end labeling (TUNEL) was applied to assessment of the apoptotic germ cells. Bax expression was upregulated mainly at stages VII-VIII of spermatogenic cycle (p<0.05) in experimental groups. Apoptotic index was significantly increased in 7 and 10 mg/kg Dex treated mice (p<0.05). It appears that GCs such as Dex could induce apoptosis through the expression of proapoptotic proteins.

Opposing Roles of Insulin-Like Growth Factor Binding Protein 3 and Humanin in the Regulation of Testicular Germ Cell Apoptosis

Modulating germ cell death and survival have significant therapeutic potential for male infertility and contraception. We have shown previously that IGF binding protein 3 (IGFBP3) gene expression is up-regulated in human testis when germ cell apoptosis is induced by intratesticular hormonal deprivation created by testosterone administration. Humanin (HN) is a binding partner of IGFBP3, and both are expressed in rat testes. We therefore hypothesized that IGFBP3, a proapoptotic factor, and HN, an antiapoptotic factor, are important regulators of male germ cell apoptosis. Whereas baseline apoptosis in the testis was equivalent between Igfbp3 knockout and wild-type mice, treatment with GnRH antagonist (GnRH-A) for 2 wk induced germ cell apoptosis in wild type, which was dramatically reduced in Igfbp3 knockout mice. To investigate the direct effects of IGFBP3 and HN on germ cell apoptosis, intratesticular administration of IGFBP3 for 5 d in rats induced a 4.2- and 3.8-fold increase in apoptosis at stages VII-VIII and XIV-I of the seminiferous epithelium cycle, respectively. GnRH-A treatment for 5 d increased apoptosis, mainly at stages VII-VIII. Addition of IGFBP3 to GnRH-A treatment enhanced apoptosis to 39.3-fold at stages VII-VIII, which was higher than either treatment alone. Intratesticular injection of HN significantly decreased GnRH-A-induced apoptosis at stages XIV-I but not stages VII-VIII. We conclude that IGFBP3 and HN play key roles in the coordinated regulation of testicular germ cell homeostasis. Perturbation of this interaction is important in enhancing or preventing germ cell death, providing new targets for future therapies.

Opposite Effects of Interleukin-1α and Transforming Growth Factor-β2 Induce Stage-Specific Regulation of Junctional Adhesion Molecule-B Gene in Sertoli Cells

In the mammalian testis, junctional adhesion molecule-B (JAM-B) is found at the blood-testis barrier between Sertoli cells and the apical ectoplasmic specializations between Sertoli and germ cells. The expression of JAM-B is tightly regulated to allow the transit of developing germ cells across the blood-testis barrier and the timely release of mature spermatids at stage VIII. In this study, the basal transcription of JAM-B in the mouse Sertoli cell line, MSC-1 cells, was examined. We found that the constitutive expression of JAM-B is carried out by the binding of specificity proteins (Sps), ETS domain transcription factor Elk-1 (Elk1), neuron-restrictive silencer factor (NRSF), and E2F transcription factor 3 (E2F3) to various cis-acting elements including TG interacting factor (TGIF), Elk-1, NRSF, and proximal Sp1 (pSp1) + E2F binding motifs. We also investigated the effects of two cytokines IL-1alpha and TGF-beta2 on JAM-B expression. IL-1alpha promotes JAM-B expression by facilitating the binding of Elk-1 to TGIF and pSp1 + E2F motifs in a p38-dependent manner, which leads to an additive effect on Sp1- and NRSF-mediated JAM-B transactivation. TGF-beta2 inhibits JAM-B transcription via the activation of mothers against decapentaplegic (Smad) proteins and activated Smads compete with specificity proteins (Sp1 and Sp3) for the TGIF motif, resulting in JAM-B repression. IL-1alpha and Smad3 expression have been reported to be stage specific. IL-1alpha is absent in the seminferous epithelium at stages VII-VIII, whereas a high level of nuclear Smad3 level is found at the same stages. This study shows for the first time that IL-1alpha and TGF-beta2 regulate JAM-B expression in an opposite manner, and in vitro data obtained herein provide some clues on how junctions are regulated in the testis.

Intron Retention Generates a Novel Isoform of CEACAM6 That May Act as an Adhesion Molecule in the Ectoplasmic Specialization Structures Between Spermatids and Sertoli Cells in Rat Testis1

By differential display technique followed by RT-PCR and DNA sequence analyses, we isolated carcinoembryonic antigen-related cell adhesion molecule 6 (Ceacam6) and its novel spliced variant Ceacam6-Long (Ceacam6-L) from rat testis. Ceacam6-L mRNA was generated by retention of 67 nucleotide-length third intron in Ceacam6 gene. Ceacam6-L is a member of an immunoglobulin superfamily and encodes a protein of 50 kDa with a signal sequence at the N-terminus, one immunoglobulin (Ig)-like domain, three IgCAM domains, a transmembrane region, and a short intracellular region. Expression analyses by RT-PCR and Northern blot showed that Ceacam6-L was exclusively expressed in rat testis and first detectable at 5 wk during postnatal development of testis. We performed immunoblot analyses and immunohistochemistry using the anti-CEACAM6-L antibody. Confocal laser scanning microscopy revealed that CEACAM6-L was not present at blood-testis barrier junctions between Sertoli cells but localized at the interface between Sertoli cells and germ cells, possibly to work as an adhesion molecule in the apical compartment of the seminiferous epithelium. At stages VII-VIII, at which all of the elongated spermatids migrated to the luminal surface of the seminiferous tubules, CEACAM6-L was found to locate at the concave side of elongated spermatid heads, following the curvature of their sickle-shaped nuclei, suggesting that CEACAM6-L might be involved in the anchoring of spermatids to Sertoli cells and spermiation. We concluded that CEACAM6-L might be a novel adhesion molecule constructing the apical ectoplasmic specialization in testis.

Dexamethasone Effects on Fas Ligand Expression in Mouse Testicular Germ Cells

The aim of this study is to investigate the effect of Dex, a widely used GC, on apoptosis and expression of FasL protein in the mouse testicular germ cells. The effects of dexamethasone (Dex) on expression of Fas ligand (FasL), an important proapoptotic protein, in the mouse testicular germ cells were investigated. Six groups, each of 8 male NMRI mice were chosen for the experiment. Experimental groups received one of the following treatments daily for 7 days: 4, 7 and 10 mg kg(-1) Dex. Control groups were treated with equivalent volumes of saline. Experimental and control animals were sacrificed 24 h after the last injection. Immunohistochemical procedure was used to evaluation of FasL expression and the deoxyuridine nick-end labeling (TUNEL) was applied to assessment of the apoptotic germ cells. FasL expression of testicular germ cells were significantly increased in 10 mg kg(-1) Dex treated mice (p < 0.05), particularly at stages VII-VIII of spermatogenic cycle. Apoptotic indexes (AIs) of germ cells were significantly increased in 7 and 10 mg kg(-1) Dex treated mice.