Protein kinases and adherens junction dynamics in the seminiferous epithelium of the rat testis

Earlier studies in multiple epithelia have shown that cell-cell actin-based adherens junction (AJ) dynamics are regulated, at least in part, by the interplay of kinases and phosphatases that determines the intracellular phosphoprotein content. Yet it is virtually unknown regarding the role of protein kinases in Sertoli-germ cell AJ dynamics in the seminiferous epithelium of the testis. To address this issue, an in vitro coculture system utilizing Sertoli and germ cells was used to study the regulation of several protein kinases, including c-Src (the cellular form of the v-src transforming gene of Rous Sarcoma virus, RSV), carboxyl-terminal Src kinase (Csk), and casein kinase 2 (CK2), during AJ assembly. Both Sertoli and germ cells were shown to express c-Src, Csk, and CK2 with a relative Sertoli:germ cell ratio of approximately 1:1, suggesting both cell types contributed equally to the pool of these kinases in the epithelium. c-Src and Csk were shown to be stage-specific proteins during the epithelial cycle, being highest at stages VII-VIII. Studies using immunoprecipitation have illustrated that these kinases were structurally associated with the N-cadherin/beta-catenin, but not the nectin/afadin, protein complex, implicating that the cadherin/catenin protein complex is their likely putative substrate. An induction in c-Src, Csk, and CK2 were detected during Sertoli-germ cell AJ assembly in vitro but not when Sertoli cells were cultured alone. When adult rats were treated with 1-(2,4-dichlorobenzyl)-indazole-3-carbohydrazide (AF-2364), a compound known to induce germ cell loss from the seminiferous epithelium, in particular elongating/elongate and round spermatids, by disrupting Sertoli-germ cell AJs, an induction of c-Src and Csk, but not CK2, was detected. Furthermore, a transient increase in the intrinsic kinase activities of c-Src, but not CK2, was also detected. This event was also associated with a loss of protein-protein association of N-cadherin and beta-catenin from the cadherin/catenin/c-Src/Csk/CK2 protein complex. Administration of PP1, a c-Src inhibitor, into adult rats via the jugular vein could induce the loss of spermatocytes and round spermatids, but not elongating/elongate spermatids, from the seminiferous epithelium. This result thus implicates the importance of c-Src in maintaining the integrity of AJs and possibly desmosome-like junctions between Sertoli cells and spermatocytes/round spermatids. In short, the data reported herein have shown that c-Src, Csk, and CK2 are novel protein kinases in AJ dynamics in the testis.     

Loss and recovery of androgen receptor protein expression in the adult rat testis following androgen withdrawal by ethane dimethanesulfonate

Androgens are especially important for the maintenance of spermatogenesis in adulthood and the experimental withdrawal of testosterone (T) production by ethane dimenthanesulfonate (EDS) is a valuable tool for studying androgen-dependent events of spermatogenesis. The aim of the present study was to investigate the specific changes in immunoexpression of androgen receptor (AR) in the testis in relation to degeneration and regeneration of Leydig cell (LC) population and seminiferous epithelium. Immunohistochemistry for AR and 3beta-hydroxysteroid dehydrogenase (3beta-HSD) as well as TUNEL assay for apoptosis were performed on testicular sections of control and EDS-treated rats. Serum LH and T levels were measured by RIA. Our results revealed a total loss of AR immunoexpression from the nuclei of Sertoli (SCs), LCs and peritubular cells during the first week after EDS administration and that coincided with severe drop in T levels. Two weeks after EDS administration, the AR expression was recovered in these cells but normal stage-specificity in SCs was replaced by uniform intensity of AR immunostaining at all the stages of the spermatogenic cycle. The stage-specific pattern of androgen expression in SCs with a maximum at stages VII-VIII appeared 5 weeks after treatment. LC immunoreactivity for 3beta-HSD at different time points after EDS administration correlated with values of T concentration. The maximal germ cell apoptosis on day 7 was followed by total loss of elongated spermatids 2 weeks after EDS treatment. Regeneration of seminiferous epithelium 3 weeks after EDS administration and onwards occurred in tandem with the development of new LC population indicated by the appearance of 3beta-HSD-positive cells and gradual increase in T production. The specific changes in AR after EDS including their loss and recovery in Sertoli cells paralleled with degenerative and regenerative events in Leydig and germ cell populations, confirming close functional relationship between Sertoli, Leydig and germ cells.     

Testosterone micromilieu in staged rat seminiferous tubules

Endogenous testosterone concentrations in rat seminiferous tubules were measured in relation to different stages of the cycle of the seminiferous epithelium. For this purpose, the seminiferous tubules were mechanically separated from the interstitial tissue on a cooled (1 degree C) petri dish under a stereomicroscope without added medium. After recognition of the stages of the cycle by transillumination, the specimens were rapidly transferred by dry forceps into test tubes for testosterone radioimmunoassay. The results of the dry dissection method were compared with measurements on tubules that were kept after separation in phosphate buffered saline (PBS, pH 7.4), in order to reveal the possible leakage of testosterone from the tubules. The maximal concentration of testosterone per unit length of seminiferous tubule was found in stages VII and VIII of the cycle (288 +/- 60 fmol/cm, mean +/- SEM, n = 12), and the minimal in stages IX-XII (219 +/- 57 fmol/cm, P less than 0.01). If the levels were correlated with unit volumes of the seminiferous tubules, identical concentrations of testosterone (521-542 fmol/mm3, approx. 500 nmol/l) were found in the different stages of the cycle. Despite the similarity of testosterone concentrations in the different parts of the seminiferous tubules the local concentrations of biologically active (i.e. free) testosterone may be modulated by extracellular and intracellular androgen binding components.     

Response of the seminiferous epithelium of the rat testis to withdrawal of androgen: evidence for direct effect upon intercellular spaces associated with Sertoli cell junctional complexes

The morphological response of the Sertoli cells to partial or complete withdrawal of testosterone was studied in adult rats following hypophysectomy or administration of ethane dimethanesulphonate (EDS), a toxicant known to destroy selectively the Leydig cells of the testis. To assess the role of germ cells in effecting changes to Sertoli cells following withdrawal of testosterone, germ cell-deficient rats with Sertoli-cell-only testes (SCO) were treated with EDS to remove the source of testosterone. At 6 days after hypophysectomy or 4,6 and 8 days after EDS treatment, stage VII and VIII seminiferous tubules showed degenerating germ cells and numerous basally-located vacuoles approximately 1–15 m in diameter. Ultrastructural analysis indicated that most of the vacuoles were multiple focal dilations of the intercellular space associated with Sertoli cell junctional complexes. In SCO rats, treatment with EDS resulted in a significant (P<0.05) increase in the formation of many vacuoles particularly in the base but also in the trunk of the Sertoli cells and again electron microscopic analysis showed multiple, localized expansions of the intercellular space associated with Sertoli cell junctional complexes. The appearance of intercellular spaces in SCO testes following androgen withdrawal cannot be attributed to shrinkage of degenerating germ cells since the seminiferous tubules did not contain germ cells. It is concluded that withdrawal of androgen induces early morphological alterations of the Sertoli cell junctional complexes in which the sites of membrane fusions representing tight junctions remain intact whereas the intercellular spaces exhibit major focal dilations. The results are discussed in relation to the fluid secretion by the seminiferous tubules which is regulated by the Sertoli cells.     

Comparative testis morphometry and seminiferous epithelium cycle length in donkeys and mules

The mule (Equus mulus mulus) is a sterile hybrid domestic animal that results from the breeding of a male donkey (Equus asinus) to a female horse (Equus caballus). Usually, spermatogenesis in mules does not advance beyond spermatocytes. In the present study, we performed a comparative and more accurate morphometric and functional investigation of the testis in donkeys and mules. Due to the smaller testis size, lower seminiferous tubule volume density, and fewer germ cells, the total length of seminiferous tubules in mules was significantly smaller than in donkeys. However, the percentage of seminiferous tubules containing germ cells (spermatogonia and spermatocytes) in mules was approximately 95%. The total number of Sertoli cells per testis observed in donkeys and mules was very similar. However, the total number of Leydig cells in mules was approximately 70% lower than in donkeys. At least in part, this difference was probably related to the lower number of germ cells present in mule seminiferous tubules. Although spermatogenesis in mules did not advance beyond secondary spermatocytes/newly formed round spermatids, germ cell associations in the seminiferous epithelium and pachytene spermatocytes nuclear volume in donkeys and mules were similar. The duration of spermatogenesis was estimated using intratesticular injections of tritiated thymidine. Each spermatogenic cycle in donkeys lasted 10.5 days. A similar value was found in mules ( approximately 10.1 days). Considering that the entire spermatogenic process takes approximately 4.5 cycles to be completed, its total duration in donkeys was estimated to last 47.2 days. The results found for mules suggest that the mechanisms involved in the determination of testis structure and function are probably originated from donkeys. Also, the data found for mules suggest that their seminiferous tubules are able to sustain complete spermatogenesis. In this regard, this species is a potential model for transplants of germ cells originated from donkeys and horses or other large animals.     

Short-time effects of taxol on the seminiferous epithelium of the rat

Summary The effect of taxol, an inhibitor of microtubule degradation, on the seminiferous epithelium was studied. Taxol arrested spermatogenesis at metaphase in both mitotic and meiotic germ cell division. Microtubules were seen to accumulate, especially in the cytoplasm of the spermatogonia, and also in the early spermatids and Sertoli cells. No microtubule accumulation was observed in germ cells during meiotic prophase. Formation of the flagellum was affected in developing spermatids. Peculiar lamellar structures, probably derived from degenerating mitochondria, were seen in the cytoplasm of late spermatids and Sertoli cells.The results are compared with the effects of other mitotic inhibitors such as colchicine and vinca alcaloids.     

Blockage of testicular connexins induced apoptosis in rat seminiferous epithelium

Spermatogenesis, a tightly regulated developmental process of male germ cells in testis, is associated with temporal and spatial expression of gap junction proteins, such as the connexin family members. Perturbation of their expressions may lead to spermatogenic arrest as manifested by disruption of cell-cell interaction. To explore the role(s) of connexins during spermatogenesis, we utilized the small peptide antagonistic approach to specifically deplete connexin 31, connexin 33, and pan-connexin. Three connexin peptides corresponding to the extracellular binding domain of connexin 31 and connexin 33 and to the extracellular conserved domain of connexins were designed and synthesized commercially. Peptides (at single dosage of 0.5, 1, or 2 mg per animal) were injected into rat testes and testes were collected on day 0, 1, 3, 5, 10, 15, and 30 after microinjection. In situ TUNEL assay demonstrated the induction of apoptosis in the testes after pan-connexin peptide treatment in a dose-dependent manner from day 3 and onward. Unlike the pan-connexin peptide, connexin 31 and connexin 33 peptides appeared to have little effect on inducing apoptosis and germ cell loss. CD45 staining also detected the occasional presence of infiltrating lymphocytes in the seminiferous tubules. Accompanied with the apoptotic events, two apoptotic markers, NF-κB and caspase 3, demonstrated a general up-regulation in their expressions. In adjacent testis sections, eliminations of connexin 31, 32, and 43 were observed. However, an induction of connexin 33 expression was detected. This suggests the versatility and functional diversity of connexins in the testis. The expression of ZO-1, the only known adaptor of connexins in the testis, was reduced and remained in a low level in the seminiferous epithelium. As such, the alterations of connexins in seminiferous epithelium may induce apoptotic signaling in the testis via the caspase 3 and the NF-κB pathway. This demonstrates the significant role of testicular connexins to maintain the survival of germ cells by regulating inter-cellular communications among germ cells and adjacent supporting cells during spermatogenesis. In addition, the inter-relationship between connexins and other junction proteins and associated signaling protein were investigated. After pan-connexin peptide treatment, a dys-localization of N-cadherin, an adherens junction protein, and diminution of occludin, a tight junction protein, level were detected. In addition, inductions of junction regulatory protein, cathepsin L, was observed during the course of peptide-mediated germ cell loss in the testes. In summary, pan-connexin peptide treatment triggered apoptosis and germ cell loss in the testes. This event influenced the localization and expression of different junction proteins and junction-associated protein in the testes. Financial support: The work was funded by a grant from the Research Grants Council of Hong Kong (HKU 7272/01M).     

Localization of cellular retinol-binding protein mRNA in rat testis and epididymis and its stage-dependent expression during the cycle of the seminiferous epithelium

Anatomical localization of cellular retinol-binding protein (CRBP) mRNA was examined in normal rat testis and epididymis and also in retinoid-deficient rat testis. In situ hybridization was performed with 35S-labeled rat CRBP cRNA probes on frozen tissue sections. In normal testis, CRBP mRNA was mainly localized in the Sertoli cells and to some extent in peritubular cells. A distinct cyclic variation of the relative levels of hybridizable CRBP mRNA was observed during the spermatogenic cycle. The peak of CRBP mRNA content was seen in the stages of the cycle that preceded those in which peak CRBP protein content had been observed previously in our laboratory by immunohistochemistry. No appreciable amount of CRBP mRNA was observed in the interstitial space or in the lumen of the tubules. CRBP mRNA displayed the same anatomical localization in the retinoid-deficient testis, but the level of hybridizable CRBP mRNA was substantially reduced. A strong hybridization signal for CRBP mRNA was seen in proximal epididymis and was strikingly localized in the ductular epithelium. CRBP mRNA was not detectable in the distal portion of the epididymis. These studies provide information about the cell-specific expression of CRBP synthesis within the testis and epididymis and about its cyclic variation and regulation.     

Immunohistochemical localization of the transferrin receptor in the seminiferous epithelium of the rat

The transferrin receptor has been immunohistochemically localized in the seminiferous epithelium of the rat with a monoclonal antibody, MRC OX26, which recognizes the transferrin receptor glycoprotein. The receptor was detectable on mitotically and meiotically dividing germ cells and, less abundantly, on round spermatids. It was lost from germ cells during spermatid elongation and was undetectable on immature spermatozoa. The transferrin receptor was also present on Sertoli cells in the testes of immature animals and on Sertoli cells in the testes of aspermatogenic animals that had been irradiated in utero. It was not detectable on Sertoli cells in the testes of cryptorchid animals. These studies demonstrate that the transferrin receptor is abundant on dividing germ cells as well as dividing somatic cells.     

Testosterone response in arginine vasopressin desensitized immature rat testis

Direct injection of arginine vasopressin into immature rat testis inhibited basal testosterone synthesis. Simultaneous injection of arginine vasopressin with luteinizing hormone, norepinephrine or cholera toxin inhibited these agonists - induced testosterone response. In arginine vasopressin - desensitized testis, cAMP response to luteinizing hormone, norepinephrine and cholera toxin was not disturbed. However, testosterone response to luteinizing hormone, norepinephrine or cholera toxin was drastically reduced in arginine vasopressin-desensitized testis. This shows that the increased cAMP generated by luteinizing hormone, norepinephrine or cholera toxin in arginine vasopressin desensitized testis did not cause increase in steroidogenesis. This could be due to a lesion in steroidogenic pathway beyond cAMP generation caused by arginine vasopressin.     

Microtubule depolymerization in rat seminiferous epithelium is associated with diminished tyrosination of alpha-tubulin

In the testis, microtubule-disrupting agents cause breakdown of the Sertoli cell cytoskeleton and sloughing of germ cells with associated Sertoli cell fragments, although the mechanism underlying this event is not understood. In this study, we investigated the effects of carbendazim and colchicine on microtubule polymerization status and posttranslational modifications of tubulin in freshly isolated rat seminiferous tubules. Soluble and polymerized tubulin pools were separated and tubulin was quantified using a competitive ELISA. Carbendazim and colchicine caused extensive microtubule depolymerization, shifting the ratio of soluble to polymerized tubulin from 40%:60% to 78%:22%, and to 84%:16%, respectively. Total tubulin levels remained relatively constant after carbendazim treatment but decreased twofold after colchicine treatment. To determine if modifications to tubulin may be associated with polymerization status, tubulin pools were analyzed by immunoblotting. Acetylated alpha-tubulin and betaIII-tubulin distribution in tubulin pools was not affected by treatment. Tyrosinated alpha-tubulin (52 kDa) was localized in both tubulin pools and had decreased tyrosination in the microtubule pool after carbendazim treatment. A 47-kDa protein immunoreactive with both tyrosinated alpha-tubulin and general alpha-tubulin antibodies was found only in the microtubule pool. The 47-kDa protein (potentially an alpha-tubulin isoform) lost tyrosination, yet was still present in the microtubule pool based on detection with the general alpha-tubulin antibody, after carbendazim treatment. Similar effects were seen with colchicine, although loss of total tubulin protein was measured. Thus, decreased tyrosination of the microtubule pool of tubulin appears to be associated with depolymerization of microtubules.     

TGF-beta3 regulates anchoring junction dynamics in the seminiferous epithelium of the rat testis via the Ras/ERK signaling pathway: An in vivo study

Recent studies have shown that transforming growth factor (TGF)-beta3 regulates blood-testis barrier (BTB) dynamics in vivo, plausibly by determining the steady-state levels of occludin and zonula occludens-1 (ZO-1) at the BTB site via the p38 MAP kinase signaling pathway. Since BTB is composed of coexisting TJs and basal ectoplasmic specializations [ES, a testis-specific adherens junction (AJ) type] in the seminiferous epithelium of the rat testis, we sought to examine if TGF-beta3 would also regulate anchoring junction dynamics. Using an in vivo model in which rats were treated with AF-2364 [1-(2,4-dichlorobenzyl)-indazole-3-carbohydrazide] to perturb Sertoli-germ cell AJs without affecting the integrity of TJs at the BTB, it was noted that the event of germ cell loss from the epithelium was associated with a transient surge in TGF-beta3. Furthermore, it was also associated with a surge in the protein levels of Ras, p-ERK, and the intrinsic activity of ERK, illustrating TGF-beta3 apparently regulates Sertoli-germ cell ES function via the Ras/MEK/ERK signaling pathway. Indeed, pretreatment of rats with TbetaRII/Fc chimera, a TGF-beta antagonist, or U0126, a specific MEK inhibitor, could significantly delay and partially block the disruptive effects of AF-2364 in depleting germ cells from the epithelium. While the protein levels of the cadherin/catenin complex were significantly induced during AF-2364-mediated germ cell loss, perhaps being used to retain germ cells in the epithelium, this increase failed to reverse the loss of adhesion function between Sertoli and germ cells because of a loss of protein-protein interactions between cadherins and catenins. Collectively, these results illustrate that the testis has a novel mechanism in place in which an agent that primarily disrupts TJs can induce secondary loss of AJ function, leading to germ cell loss from the seminiferous epithelium. Yet an agent that selectively disrupts AJs (e.g., AF-2364) can limit its effects exclusively at the Sertoli-germ cell adhesive site without perturbing the Sertoli-Sertoli TJs.     

Effects of spaceflight on the spermatogonial population of rat seminiferous epithelium

Testes from rats flown on Cosmos 1887 were compared with vivarium control and synchronous control samples. The mean weights of flight testes, normalized for weight per 100 g, were 6.4% less when compared with the vivarium controls. Counts of spermatogonia from tissue sections (seminiferous tubules in maturation stage 6) from five animals in each group revealed 4% fewer spermatogonia in flight testes compared with synchronous controls and 11% fewer spermatogonia in flight samples compared with vivarium controls.     

Frequency of the stages in the cycle of the seminiferous epithelium in the rat

This study determined the optimum number of tubules to be counted per testis cross section, and the number of animals per treatment group, when changes in stage frequencies in the cycle of the seminiferous epithelium are criteria for assessing effects of treatment on spermatogenesis. A data base of 9,672 observed and staged tubules was collected from testicular cross sections of 15 Sprague-Dawley rats. A significant variation between animals was found for the frequencies of Stages I, II, IV, VI, VIII, and XIII. Computer simulation was used to randomly select different combinations of animal and tubule numbers from the observed data. Stage frequency means from each simulation experiment were compared statistically to observed mean frequencies. A model that used data from all 14 stages was analyzed. The following conclusions were made: a) a minimum of 200 tubule cross sections/testis is recommended for estimating stage frequencies; b) for a fixed number of tubules scored, the number of animals sampled is more important than the number of tubules per animal in reducing variance; c) to detect a difference of 2 standard deviations from the mean with a 2% error rate and examining 200 tubules/testis, at least 12 animals must be used per group when assessing all 14 stages; d) when individual stages are examined using 10 animals per group, only Stage VII has 80% or greater power of test (alpha = 0.05) to detect a frequency difference; e) pooling stages into 3-4 groups is recommended to improve the power of detecting a treatment difference.     

Sertoli cell ectoplasmic specializations in the seminiferous epithelium of the testosterone-suppressed adult rat

The Sertoli cell ectoplasmic specialization is a unique junctional structure involved in the interaction between elongating spermatids and Sertoli cells. We have previously shown that suppression of testicular testosterone in adult rats by low-dose testosterone and estradiol (TE) treatment causes the premature detachment of step 8 round spermatids from the Sertoli cell. Because these detaching round spermatids would normally associate with the Sertoli cell via the ectoplasmic specialization, we hypothesized that ectoplasmic specializations would be absent in the seminiferous epithelium of TE-treated rats, and the lack of this junction would cause round spermatids to detach. In this study, we investigated Sertoli cell ectoplasmic specializations in normal and TE-treated rat testis using electron microscopy and localization of known ectoplasmic specialization-associated proteins (espin, actin, and vinculin) by immunocytochemistry and confocal microscopy. In TE-treated rats where round spermatid detachment was occurring, ectoplasmic specializations of normal morphology were observed opposite the remaining step 8 spermatids in the epithelium and, importantly, in the adluminal Sertoli cell cytoplasm during and after round spermatid detachment. When higher doses of testosterone were administered to promote the reattachment of all step 8 round spermatids, newly elongating spermatids associated with ectoplasmic specialization proteins within 2 days. We concluded that the Sertoli cell ectoplasmic specialization structure is qualitatively normal in TE-treated rats, and thus the absence of this structure is unlikely to be the cause of round spermatid detachment. We suggest that defects in adhesion molecules between round spermatids and Sertoli cells are likely to be involved in the testosterone-dependent detachment of round spermatids from the seminiferous epithelium.     

Newly synthesized proteins in seminiferous intertubular and intratubular compartments of the rat testis

Two-dimensional gel electrophoresis combined with autoradiography and Western blot procedures have been used to characterize newly synthesized proteins in testicular intertubular fluid (TIF) and seminiferous tubular fluid (SNF). Fluids were collected following in vivo and in vitro intratesticular injection of [35S]methionine into control and hypophysectomized adult rats. A discrete number of [35S]methionine-labeled proteins were detected within TIF and SNF. Their presence and relative abundance varied according to in vivo and in vitro labeling conditions. While two major blood plasma proteins, albumin and transferrin, were radioactively labeled after in vivo labeling, these two proteins were insignificantly labeled in samples collected after in vitro labeling. Three acidic proteins, possibly secreted by Sertoli cells (Mr = 72,000, 45,000 and 35,000), were more abundant in TIF samples collected after in vitro [35S]methionine labeling than after in vivo labeling. Incubated seminiferous tubules and TIF of hypophysectomized rats showed a decrease in [35S]methionine-labeling intensity of the Mr = 72,000 acidic protein, possibly reflecting changes in the seminiferous epithelium caused by pituitary hormonal deprivation. Autoradiographs of TIF and most remarkably, of SNF, showed many protein spots that suggested cell breakage and leakage during sample collection. Results of this study suggest that most albumin and transferrin found in TIF and SNF have an extratesticular origin and that proteins secreted by the Sertoli cell can gain access to both TIF and SNF.