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C Yan Cheng

Publications and source records attributed to C Yan Cheng.

53 records · Page 3Linked to original sources
PubMed2003

Sertoli-germ cell adherens junction dynamics in the testis are regulated by RhoB GTPase via the ROCK/LIMK signaling pathway.

During spermatogenesis, cell-cell actin-based adherens junctions (AJs), such as ectoplasmic specializations (ESs), between Sertoli and germ cells undergo extensive restructuring in the seminiferous epithelium to facilitate germ cell movement across the epithelium. Although the mechanism(s) that regulates AJ dynamics in the testis is virtually unknown, Rho GTPases have been implicated in the regulation of these events in other epithelia. Studies have shown that the in vitro assembly of the Sertoli-germ cell AJs but not of the Sertoli cell tight junctions (TJs) is associated with a transient but significant induction of RhoB. Immunohistochemistry has shown that the localization of RhoB in the seminiferous epithelium is stage specific, being lowest in stages VII-VIII prior to spermiation, and displays cell-specific association during the epithelial cycle. Throughout the cycle, RhoB was localized near the site of basal and apical ESs but was restricted to the periphery of the nuclei in elongating (but not elongated) spermatids, spermatocytes, and Sertoli cells. However, RhoB was not detected near the site of apical ESs at stages VII-VIII. Furthermore, disruption of AJs in Sertoli-germ cell cocultures either by hypotonic treatment or by treatment with 1-(2,4-dichlorobenzyl)-indazole-3-carbohydrazide (AF-2364) also induced RhoB expression. When adult rats were treated with AF-2364 to perturb Sertoli-germ cell AJs in vivo, a approximately 4-fold induction in RhoB in the testis, but not in kidney and brain, was detected within 1 h, at least approximately 1-4 days before germ cell loss from the epithelium could be detected by histological analysis. The signaling pathway(s) by which AF-2364 perturbed the Sertoli-germ cell AJs apparently began with an initial activation of integrin, which in turn activated RhoB, ROCK1, (Rho-associated protein kinase 1, also called ROKbeta), LIMK1 (LIM kinase 1, also called lin-11 isl-1 mec3 kinase 1), and cofilin but not p140mDia and profilin via phosphorylation. Immunoprecipitation and immunoblots revealed that the induction of LIMK1 was mediated via an increase in its phospho-Ser but not phospho-Tyr content. Furthermore, Y-27632 ([(R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexane-carboxamide, 2HCl]), a specific ROCK inhibitor, could effectively delay the AF-2364-induced germ cell loss from the seminiferous epithelium in vivo, illustrating that the integrin/RhoB/ROCK/LIMK pathway indeed plays a crucial role in the regulation of Sertoli-germ cell AJ dynamics. The fact that the RhoB pathway in the kidney and brain was not activated suggests that AF-2364 exerts its effects primarily at the testis-specific ES multiprotein complex structures between Sertoli cells and spermatids. In summary, this report illustrates that Sertoli germ cell AJ dynamics are regulated, at least in part, via the integrin/ROCK/LIMK/cofilin signaling pathway.

Adherens Junctions↗
PubMed2003

TGF-betas: their role in testicular function and Sertoli cell tight junction dynamics.

Transforming growth factor-betas (TGF-betas) are known to regulate multiple physiological functions in the testis, which include spermatogenesis, Leydig cell steroidogenesis, extracellular matrix synthesis and testis development. More recent studies have shown that TGF-beta3 also regulates Sertoli cell tight junction (TJ) dynamics in vitro via the p38 mitogen-activated protein (MAP) kinase pathway, suggesting that this cytokine plays a crucial role in regulating the opening and closing of the blood-testis barrier (BTB). This in turn regulates the passage of pre-leptotene and leptotene spermatocytes across the BTB at stages VIII-XI of the seminiferous epithelial cycle. This review summarizes recent advances of studies on TGF-betas in the testis, highlighting their regulatory role in TJ dynamics.

Animals↗
PubMed2003

Is the cadherin/catenin complex a functional unit of cell-cell actin-based adherens junctions in the rat testis?

Much controversy exists regarding the presence of the cadherin/catenin complex and its intracellular attachment site in the testis, which is the functional unit for actin-based cell-cell adherens junctions (AJs) in multiple epithelia. Furthermore, whether germ and Sertoli cells are equipped with the necessary AJ-associated signaling molecules to regulate this cadherin/catenin complex during spermatogenesis is not known. In the present study, it was shown that both Sertoli and germ cells indeed express N-cadherin, E-cadherin, alpha-catenin, beta-catenin, and p120(ctn) by semiquantitative reverse transcription-polymerase chain reaction and immunoblotting. Furthermore, the assembly of AJs between Sertoli and germ cells was associated with a transient induction in the steady-state mRNA and protein levels of cadherins and catenins. These analyses reveal, to our knowledge for the first time, that the testis may indeed be using the cadherin/catenin complex as one of the functional units to regulate AJ dynamics between Sertoli and germ cells in addition to alpha(6)beta(1) integrin and the nectin/afadin complex. To further confirm the existence of such a complex between Sertoli and germ cells, immunoprecipitation experiments were performed using Sertoli-germ cell lysates during AJ assembly. An anti-N-cadherin antibody can pull out beta-catenin, whereas N-cadherin can also be pulled out using an anti-beta-catenin antibody. To further expand and validate these in vitro biochemical studies, immunofluorescent histochemistry was performed, which colocalized N-cadherin and beta-catenin to the same site of Sertoli-Sertoli and Sertoli-germ cell AJs, possibly ectoplasmic specializations near the basal compartment, at the lower third of the seminiferous epithelium in vivo as well as between Sertoli cells cultured in vitro. Furthermore, studies by cross-linking using dithiobis(succinimidylpropionate) confirmed that the cadherin/catenin complex between Sertoli cells as well as between Sertoli and germ cells indeed structurally linked to actin but not to vimentin (an intermediate filament protein) or to tubulin (a microtubule protein). These results thus unequivocally demonstrate that the cadherin/catenin complex, which can be up-regulated by testosterone, is indeed present between Sertoli and germ cells and is used for the assembly of functional AJs.

Actins↗
PubMed2003

Regulation of Sertoli cell tight junction dynamics in the rat testis via the nitric oxide synthase/soluble guanylate cyclase/3',5'-cyclic guanosine monophosphate/protein kinase G signaling pathway: an in vitro study.

Nitric oxide (NO) synthase (NOS) catalyzes the oxidation of L-arginine to NO. NO plays a crucial role in regulating various physiological functions, possibly including junction dynamics via its effects on cAMP and cGMP, which are known modulators of tight junction (TJ) dynamics. Although inducible NOS (iNOS) and endothelial NOS (eNOS) are found in the testis and have been implicated in the regulation of spermatogenesis, their role(s) in TJ dynamics, if any, is not known. When Sertoli cells were cultured at 0.5-1.2 x 10(6) cells/cm(2) on Matrigel-coated dishes or bicameral units, functional TJ barrier was formed when the barrier function was assessed by quantifying transepithelial electrical resistance across the cell epithelium. The assembly of the TJ barrier was shown to associate with a significant plummeting in the levels of iNOS and eNOS, seemingly suggesting that their presence by producing NO might perturb TJ assembly. To further confirm the role of NOS on the TJ barrier function in vitro, zinc (II) protoporphyrin-IX (ZnPP), an NOS inhibitor and a soluble guanylate cyclase inhibitor, was added to the Sertoli cell cultures during TJ assembly. Indeed, ZnPP was found to facilitate the assembly and maintenance of the Sertoli cell TJ barrier, possibly by inducing the production of TJ-associated proteins, such as occludin. Subsequent studies by immunoprecipitation and immunoblotting have shown that iNOS and eNOS are structurally linked to TJ-integral membrane proteins, such as occludin, and cytoskeletal proteins, such as actin, vimentin, and alpha-tubulin. When the cAMP and cGMP levels in these ZnPP-treated samples were quantified, a ZnPP-induced reduction of intracellular cGMP, but not cAMP, was indeed detected. Furthermore, 8-bromo-cGMP, a cell membrane-permeable analog of cGMP, could also perturb the TJ barrier dose dependently similar to the effects of 8-bromo-cAMP. KT-5823, a specific inhibitor of protein kinase G, was shown to facilitate the Sertoli cell TJ barrier assembly. Cytokines, such as TGF-beta and TNF-alpha, known to perturb the Sertoli cell TJ barrier, were also shown to stimulate Sertoli cell iNOS and eNOS expression dose dependently in vitro. Collectively, these results illustrate NOS is an important physiological regulator of TJ dynamics in the testis, exerting its effects via the NO/soluble guanylate cyclase/cGMP/protein kinase G signaling pathway.

Animals↗
PubMed2003

TGF-beta3 regulates the blood-testis barrier dynamics via the p38 mitogen activated protein (MAP) kinase pathway: an in vivo study.

Recent studies using Sertoli cells cultured in vitro to permit tight junction (TJ) assembly have shown that TJ dynamics are regulated, at least in part, by TGF-beta3 via the p38 mitogen activated protein (MAP) kinase pathway. This in turn regulates the production of occludin, a TJ-integral membrane protein, by Sertoli cells. Yet it is not known if this pathways is used by Sertoli cells to regulate the blood-testis barrier (BTB) function in vivo. Using an in vivo model for studying BTB dynamics, we report herein the CdCl(2)-induced BTB damage in rats was associated with a significant reduction in testicular occludin along with a loss of immunoreactive occludin in the seminiferous epithelium at the site of the BTB. Also, this CdCl(2)-induced occludin loss from the BTB coincided with a surge in testicular TGF-beta3, as well as p-p38 MAP kinase (the phosphorylated/activated form of p38), but not p38 MAP kinase and neither extracellular signal-regulated kinase nor its phosphorylated form (ERK/p-ERK), consistent with results of in vitro studies. More important, intratesticular administration of SB202190, a specific p38 MAP kinase inhibitor, could block the CdCl(2)-induced occludin loss from the BTB. These results illustrate that BTB dynamics in vivo are regulated by the TGF-beta3/p38 MAP kinase pathway, which in turn determines the level of occludin at the site of Sertoli cells TJs.

Animals↗
PubMed2003

The interplay of collagen IV, tumor necrosis factor-alpha, gelatinase B (matrix metalloprotease-9), and tissue inhibitor of metalloproteases-1 in the basal lamina regulates Sertoli cell-tight junction dynamics in the rat testis.

During spermatogenesis, preleptotene and leptotene spermatocytes must translocate across the blood-testis barrier formed by inter-Sertoli cell-tight junctions (TJs) from the basal compartment of the seminiferous epithelium adjacent to the basement membrane to the adluminal compartment at stages VIII-IX for further development. Because of the close proximity between extracellular matrix (ECM) that constitutes the basement membrane and the blood-testis barrier, we sought to investigate the role of ECM in Sertoli cell TJ dynamics. When Sertoli cells were cultured in vitro to initiate the assembly of the Sertoli cell TJ-permeability barrier, the presence of an anticollagen IV antibody indeed perturbed the barrier. Because ECM is known to maintain a pool of cytokines and TNFalpha has been shown to regulate TJ dynamics in other epithelia, we investigated whether TNFalpha can regulate Sertoli cell TJ function via its effects on collagen alpha3(IV) and other proteins that maintain the homeostasis of ECM. As expected, recombinant TNFalpha perturbed the Sertoli cell TJ-barrier assembly in vitro dose dependently. TNFalpha also inhibited the timely induction of occludin, which is known to associate with the Sertoli cell TJ-barrier assembly. Furthermore, TNFalpha induced the expression of Sertoli cell collagen alpha3(IV), gelatinase B (matrix metalloprotease-9, MMP-9) and tissue inhibitor of metalloproteases-1 but not gelatinase A (matrix metalloprotease-2), and promoted the activation of pro-MMP-9. These results thus suggest that the activated MMP-9 induced by TNFalpha is used to cleave the existing collagen network in the ECM, thereby perturbing the TJ-barrier. This in turn creates a negative feedback that causes TNFalpha to induce collagen alpha3(IV) and tissue inhibitor of metalloproteases-1 expression so as to replenish the collagen network in the disrupted TJ-barrier and limit the activity of MMP-9. Taken collectively, these observations strengthen the notion that ECM is involved in the regulation of junction dynamics in addition to its structural role in the testis.

Animals↗
PubMed2003

Adhering junction dynamics in the testis are regulated by an interplay of beta 1-integrin and focal adhesion complex-associated proteins.

During spermatogenesis, the movement of developing germ cells across the seminiferous epithelium associates with extensive restructuring of cell-cell actin-based adherens junctions (AJs), such as ectoplasmic specialization (ES, a testis-specific AJ junction), between Sertoli and germ cells. Although this event of germ cell movement is essential to the completion of spermatogenesis, the mechanism(s) that regulates AJ restructuring is largely unknown. Using Sertoli-germ cells cocultured in vitro to study the regulation of AJ assembly, it was shown that this event associated with a transient induction of beta 1-integrin, vinculin, p-FAK-Tyr(397), and phosphatidylinositol 3-kinase (PI3K) but not the nonphosphorylated form of focal adhesion kinase (FAK), paxillin, and p130 Cas. Furthermore, p-FAK-Tyr(397) was shown to coimmunoprecipitate with beta 1-integrin, vinculin, and c-Src both in vitro and in vivo using Sertoli-germ cell cocultures and seminiferous tubules, respectively. These results seemingly suggest that the testis is using constituent proteins of the focal adhesion complex (FAC) found in other epithelia between cell and extracellular matrix to regulate AJ dynamics. To further confirm that p-FAK, a putative FAC protein in other epithelia, is indeed present at the site of ES, immunohistochemistry and immunofluorescent microscopy were used. The p-FAK-Tyr(397) and p-FAK-Tyr(576) were found to localize almost exclusively at the site of apical ES with weak staining at the basal ES in the seminiferous epithelium in a stage-specific manner, being highest at stages VI-VIII. In contrast, FAK was largely restricted to the basal compartment but with weak staining at the apical compartment. When rats were treated with 1-(2,4-dichlorobenzyl)-indazole-3-carbohydrazide (AF-2364) to perturb Sertoli-germ cell AJs, an induction of beta 1-integrin, vinculin, p-FAK-Tyr(397), PI3K, and p130 Cas but not the nonphosphorylated form of FAK and paxillin was also detected in the testis, coinciding with the time spermatids began to deplete from the epithelium, indicating their involvement in AJ disassembly. Thereafter, the levels of vinculin, p-FAK-Tyr(397), PI3K, and p130 Cas in the testis plunged, coinciding with the declining events of AJ disruption when virtually all spermatids were depleted from the epithelium. Taken collectively, these results suggest a bifunctional role of p-FAK, being involved in the events of Sertoli-germ cell AJ assembly and disassembly. In summary, the events of AJ dynamics in the testis, in particular at the site of ES, are regulated, at least in part, by proteins that are found in the FAC in other epithelia, such as beta1-integrin, vinculin, and FAK utilizing the integrin/pFAK/PI3K/p130 Cas signaling pathway.

Adherens Junctions↗
PubMed2002

Transforming growth factor beta3 regulates the dynamics of Sertoli cell tight junctions via the p38 mitogen-activated protein kinase pathway.

Earlier studies have implicated the significance of transforming growth factor-beta3 (TGFbeta3) in the regulation of Sertoli cell tight junction (TJ) dynamics, possibly via its inhibitory effects on the expression of occludin, claudin-11, and zonula occludens-1 (ZO-1). Yet the mechanism by which TGFbeta3 regulates the Sertoli cell TJ-permeability barrier is not known. Using techniques of semiquantitative reverse transcription-PCR (RT-PCR), immunoblotting, immunohistochemistry, and inhibitors against different kinases coupled with physiological techniques to assess the Sertoli cell TJ barrier function, it was shown that this TGFbeta3-induced effect on Sertoli cell TJ dynamics is mediated via the p38 mitogen-activated protein (MAP) kinase pathway. First, the assembly of the Sertoli cell-TJ barrier was shown to be associated with a transient but significant decline in both the TGFbeta3 production and expression by Sertoli cells. Furthermore, addition of TGFbeta3 to Sertoli cell cultures during TJ assembly indeed perturbed the TJ barrier with an IC50 at approximately 9 pM. Second, the TGFbeta3-induced disruption of the TJ barrier was associated with a transient induction in MEKK2 but not the other upstream signaling molecules that mediate TGFbeta3 action, such as Smad2, Cdc42, Rac2, and N-Ras, suggesting this effect might be mediated via the p38 MAP kinase pathway. This postulate was confirmed by the observation that TGFbeta3 also induced the protein level of the activated and phosphorylated form of p38 MAP kinase at the time the TJ barrier was perturbed. Third, and perhaps the most important of all, this TGFbeta3-mediated inhibitory effect on the TJ barrier and the TGFbeta3-induced p-p38 MAP kinase production could be blocked by SB202190, a specific p38 MAP kinase inhibitor, but not U0126, a specific MEK1/2 kinase inhibitor. These results thus unequivocally demonstrate that TGFbeta3 utilizes the p38 MAP kinase pathway to regulate Sertoli cell TJ dynamics.

Animals↗
PubMed2002

Sertoli cell tight junction dynamics: their regulation during spermatogenesis.

During spermatogenesis, developing preleptotene and leptotene spermatocytes must translocate from the basal to the adluminal compartment of the seminiferous epithelium so that fully developed spermatids (spermatozoa) can be released to the tubular lumen at spermiation. It is conceivable that the opening and closing of the inter-Sertoli tight junctions (TJs) that constitute the blood-testis barrier are regulated by an array of intriguingly coordinated signaling pathways and molecules. Several molecules have been shown to regulate Sertoli cell TJ dynamics; they include, for example, transforming growth factor beta3 (TGFbeta3), occludin, protein kinase A, protein kinase C, and signaling pathways such as the TGFbeta3/p38 mitogen-activated protein kinase pathway. Yet the mechanisms that regulate these events are essentially not known. This minireview summarizes some of the recent advances in the study of TJ dynamics in the testis and reviews several models that can be used to study TJ dynamics. It also highlights specific areas for future research toward understanding the precise physiological relationship between junction dynamics and spermatogenesis.

Animals↗
PubMed2002

Indazole carboxylic acids in male contraception.

Two new chemical entities, 1-(2,4-dichlorobenzyl)-indazole-3-carbohydrazide and 1-(2,4-dichlorobenzyl)-indazole-3-acrylic acid, were synthesized based on the core structure of lonidamine (1-(2,4-dichlorobenzyl)-indazole-3-carboxylic acid). These compounds apparently exert their effects in the testis by perturbing the Sertoli-germ cell adherens junctions causing germ cell loss from the seminiferous epithelium. Recently completed studies in the rat have demonstrated the efficacy, reversibility, and potential use of these two compounds as oral contraceptives for men. Neither compound affected the hypothalamus-pituitary-testicular axis, and both compounds were neither hepatotoxic nor nephrotoxic. These results suggest that these two compounds are safe for further development.

Animals↗
PubMed2002

Antioxidant superoxide dismutase - a review: its function, regulation in the testis, and role in male fertility.

Extracellular superoxide dismutase (SOD(EX)), an antioxidant enzyme, was found to be present in the testis at a relatively high concentration versus other organs. In a more detailed survey of several rat tissues and cells by reverse transcriptase-polymerase chain reaction, it was shown that germ cells expressed approximately one-third that of Sertoli cells, suggesting both cell types are equipped with the machinery needed to defend themselves from radical-induced damage. When we used an in vitro model in which germ cells were co-cultured with Sertoli cells at a Sertoli:germ cell ratio of 1:1, we failed to detect any changes in the mRNA level of SOD(EX). However, the addition of increasing concentrations of germ cell secretory proteins into Sertoli cell cultures resulted in a decrease in Sertoli cell SOD(EX) expression, illustrating that germ cells can indeed regulate Sertoli cell SOD(EX). On the other hand, Sertoli cell SOD(EX) expression was stimulated when human recombinant interleukin-1alpha (IL-1alpha), a germ cell product, was included into Sertoli cells in vitro. These results, taken collectively, suggest SOD(EX) is an important antioxidant molecule in the testis that is under germ cell regulation.

Animals↗
PubMed2002

Cell junction dynamics in the testis: Sertoli-germ cell interactions and male contraceptive development.

Spermatogenesis is an intriguing but complicated biological process. However, many studies since the 1960s have focused either on the hormonal events of the hypothalamus-pituitary-testicular axis or morphological events that take place in the seminiferous epithelium. Recent advances in biochemistry, cell biology, and molecular biology have shifted attention to understanding some of the key events that regulate spermatogenesis, such as germ cell apoptosis, cell cycle regulation, Sertoli-germ cell communication, and junction dynamics. In this review, we discuss the physiology and biology of junction dynamics in the testis, in particular how these events affect interactions of Sertoli and germ cells in the seminiferous epithelium behind the blood-testis barrier. We also discuss how these events regulate the opening and closing of the blood-testis barrier to permit the timely passage of preleptotene and leptotene spermatocytes across the blood-testis barrier. This is physiologically important since developing germ cells must translocate across the blood-testis barrier as well as traverse the seminiferous epithelium during their development. We also discuss several available in vitro and in vivo models that can be used to study Sertoli-germ cell anchoring junctions and Sertoli-Sertoli tight junctions. An in-depth survey in this subject has also identified several potential targets to be tackled to perturb spermatogenesis, which will likely lead to the development of novel male contraceptives.

Animals↗
PubMed2002

Gonadotropins, via cAMP, negatively regulate GATA-1 gene expression in testicular cells.

We and others demonstrated that the mRNAs encoding GATA-binding proteins, GATA-1 and GATA-4, were detected in mouse and rat testis, and in isolated rat Sertoli cells and testicular tumor cell lines derived from Leydig and Sertoli cells. In this study, we investigated the possible effects of gonadotropins and cAMP on the expression of GATA-binding protein genes in testicular cells. Unexpectedly, FSH negatively regulated GATA-1 (but not GATA-4) mRNA in a dose-dependent manner in primary cultures of rat Sertoli cells isolated from 21-d-old animals. GATA-1 mRNA was also negatively regulated by cAMP in a dose- and time-dependent manner in MA-10, a mouse Leydig tumor cell line. When 0.3 mM cAMP was administered to MA-10 cell cultures for 4 h, more than 95% of the GATA-1 mRNA and protein was abolished. The reduction of GATA-1 mRNA by cAMP can be mimicked by treatment with forskolin, which elevates intracellular cAMP levels. The inhibitory effect of cAMP was specific to the GATA-1 gene, given that GATA-4 and alpha-tubulin mRNA levels were not changed by any of the cAMP treatments. Inhibin alpha-subunit mRNA, on the other hand, was evidently increased by cAMP treatment in both MA-10 and Sertoli cells. However, inhibin alpha-subunit mRNA levels were elevated at 60-90 min before the suppression of GATA-1 mRNA detected. The inhibitory effect of cAMP on GATA-1 mRNA and protein was shown to be specific to testicular cells. The GATA-1 mRNA expressed in MEL, a mouse erythroid leukemia cell line, was not affected by cAMP. The reduction of GATA-1 mRNA by cAMP can be prevented when a translational inhibitor, cycloheximide, is added. In summary, we demonstrated that gonadotropins via cAMP negatively regulate the mRNA and protein levels of GATA-1, but not GATA-4, in testicular cells. The inhibitory effect on GATA-1 gene expression was specific to testicular cells and was not observed in erythroid cells.

Animals↗
PubMedDate not supplied

Role of tissue inhibitor of metalloproteases-1 in junction dynamics in the testis.

Using multiple high-performance liquid chromatography steps, we have identified and purified a polypeptide to apparent homogeneity from primary Sertoli cell conditioned culture medium that consisted of 2 molecular variants of 31 and 29 kDa when electrophoresed on a sodium dodecyl sulfate-polyacrylamide gel run under reducing conditions. Partial N-terminal amino acid sequence analysis of these 2 proteins revealed a sequence of NH(2)-IKMAKMLKGFDAVGNATG, which is homologous to tissue inhibitor of metalloproteases-1 (TIMP-1). Studies by semiquantitative reverse transcription-polymerase chain reaction using a primer pair specific to rat TIMP-1 demonstrated that both Sertoli and germ cells express TIMP-1. During maturation, the steady-state TIMP-1 mRNA level in the testis increased significantly from 40 to 60 days of age, which suggests its role in the restructuring of the epithelium during spermiation. This increase in testicular TIMP-1 expression was apparently not due to the increase in germ cell number, because TIMP-1 expression decreased approximately fivefold in germ cells isolated from testes of aging rats. Using Sertoli cells cultured at low (0.05 x 10(6) cells/cm(2)) and high (0.5 x 10(6) cells/cm(2)) densities, it was found that TIMP-1 expression increased transiently but significantly during junction assembly. A similar induction of TIMP-1 mRNA was also detected in Sertoli-germ cell cocultures during germ cell adhesion onto Sertoli cells. More important, the inclusion of either alpha(2)-macroglobulin (a protease inhibitor produced by Sertoli cells) or aprotinin (a serine protease inhibitor) into an in vitro germ cell adhesion assay facilitated the attachment of fluorescently labeled germ cells onto the Sertoli cell epithelium when compared to control, which suggests that the assembly of adherens junctions may involve protease inhibitors.

Adherens Junctions↗
PubMedDate not supplied

Zyxin, axin, and Wiskott-Aldrich syndrome protein are adaptors that link the cadherin/catenin protein complex to the cytoskeleton at adherens junctions in the seminiferous epithelium of the rat testis.

During spermatogenesis, the movement of germ cells across the seminiferous epithelium is associated with extensive junction restructuring. Yet the underlying mechanism (or mechanisms) that regulates these events is largely unknown. If the molecular architecture of the cell-cell actin-based adherens junction (AJ), such as ectoplasmic specialization (ES) and tubulobulbar complex- two testis-specific AJ types, is known, many functional mechanistic studies can be designed. We thus undertook an investigation to study 3 adaptors in the seminiferous epithelium: zyxin, axin, and Wiskott-Aldrich syndrome protein (WASP). All 3 adaptors were shown to be products of Sertoli and germ cells. Zyxin was shown to be a stage-specific protein that was most prominent during stages V-VII and restricted mostly to pachytene spermatocytes, but it could also be detected at the site of basal and apical ectoplasmic specialization (ES). Zyxin, axin, and WASP were shown to be structurally linked to the N-cadherin/beta-catenin/alpha-actinin/actin complex but not to the nectin-3/afadin or the beta 1-integrin-mediated protein complexes. Interestingly, zyxin, axin, and WASP are also structurally linked to vimentin (an intermediate filament protein) and alpha-tubulin (the subunit of a microtubule), which suggests that they have a role (or roles) in the regulation of the dynamics of the desmosome-like junction and microtubule. These results illustrate that zyxin, axin, and WASP are adaptors in both AJs and intermediate filament-based desmosome-like junctions. This raises the possibility that classic cadherins are also associated with vimentin-based intermediate filaments via these adaptors in the testis. While virtually no N-cadherin was found to associate with vimentin in the seminiferous tubules, it did associate with vimentin when testis lysates were used. Interestingly, about 5% of the E-cadherin associated with vimentin in isolated seminiferous tubules, and about 50% of the E-cadherin in the testis used vimentin as its attachment site. These data suggest that cadherins in the testis, unlike those in other epithelia, use different attachment sites to anchor the cadherin/catenin complex to the cytoskeleton. The levels of zyxin, axin, and WASP were also assessed during AF-2364-mediated AJ disruption of the testis, which illustrated a time-dependent protein reduction that was similar to the trends observed in nectin-3 and afadin but was the opposite of those observed for N-cadherin and beta-catenin, which were induced. Collectively, these results illustrate that while these adaptors are structurally associated with the cadherin/catenin complex in the testis, they are regulated differently.

Adherens Junctions↗
PubMedDate not supplied

Cytokines and junction restructuring during spermatogenesis--a lesson to learn from the testis.

In the mammalian testis, preleptotene and leptotene spermatocytes residing in the basal compartment of the seminiferous epithelium must traverse the blood-testis barrier (BTB) at late stage VIII through early stage IX of the epithelial cycle during spermatogenesis, entering the adluminal compartment for further development. However, until recently the regulatory mechanisms that regulate BTB dynamics remained largely unknown. We provide a critical review regarding the significance of cytokines in regulating the 'opening' and 'closing' of the BTB. We also discuss how cytokines may be working in concert with adaptors that selectively govern the downstream signaling pathways. This process, in turn, regulates the dynamics of either Sertoli-Sertoli tight junction (TJ), Sertoli-germ cell adherens junction (AJ), or both junction types in the epithelium, thereby permitting TJ opening without compromising AJs, and vice versa. We also discuss how adaptors alter their protein-protein association with the integral membrane proteins at the cell-cell interface via changes in their phosphorylation status, thereby altering adhesion function at AJ. These findings illustrate that the testis is a novel in vivo model to study the biology of junction restructuring. Furthermore, a molecular model is presented regarding how cytokines selectively regulate TJ/AJ restructuring in the epithelium during spermatogenesis.

Blood-Testis Barrier↗