Developmentally regulated alternative splicing of densin modulates protein-protein interaction and subcellular localization

Densin is a member of the leucine-rich repeat (LRR) and PDZ domain (LAP) protein family that binds several signaling molecules via its C-terminal domains, including calcium/calmodulin-dependent protein kinase II (CaMKII). In this study, we identify several novel mRNA splice variants of densin that are differentially expressed during development. The novel variants share the LRR domain but are either prematurely truncated or contain internal deletions relative to mature variants of the protein (180 kDa), thus removing key protein–protein interaction domains. For example, CaMKIIα coimmunoprecipitates with densin splice variants containing an intact C-terminal domain from lysates of transfected HEK293 cells, but not with variants that only contain N-terminal domains. Immunoblot analyses using antibodies to peptide epitopes in the N- and C- terminal domains of densin are consistent with developmental regulation of splice variant expression in brain. Moreover, putative splice variants display different subcellular fractionation patterns in brain extracts. Expression of green fluorescent protein (GFP)-fused densin splice variants in HEK293 cells shows that the LRR domain can target densin to a plasma membrane-associated compartment, but that the splice variants are differentially localized and have potentially distinct effects on cell morphology. In combination, these data show that densin splice variants have distinct functional characteristics suggesting multiple roles during neuronal development.     

Nesprin2在小鼠睾丸各级生精细胞中的表达及其原核表达与纯化

目的:研究Nesprin2在小鼠睾丸各级生精细胞中的表达变化,并对Nesprin2进行原核表达和纯化,为进一步研究该蛋白在精子发生中的作用奠定基础。方法:制备小鼠睾丸细胞涂片,用Nesprin2特异性抗体进行细胞免疫荧光染色,观察Nesprin2在各级生精细胞中的表达;应用RT-PCR技术扩增Nesprin2 C端包含KASH domain的编码85个氨基酸的目的片段,将PCR产物插入到PUCm-T载体中测序,将测序验证后的目的片段亚克隆至原核表达载体PGEX-4T-1中,转化大肠杆菌BL21,IPTG诱导表达。对GST-Nesprin2 C85融合蛋白进行纯化,Western blot进行验证。结果:免疫荧光检测结果发现,Nesprin2在小鼠睾丸各级生精细胞中均有表达,主要分布在核膜及其周围,在减数分裂过程中Nesprin2可能参与核膜重塑。构建了PGEX-4T-1-Nesprin2 C85重组质粒,经IPTG诱导后成功表达了GST-Nesprin2 C85融合蛋白。对GST-Nesprin2 C85融合蛋白进行纯化后,Western blot进行检测,结果发现,原核诱导表达的融合蛋白为GST-Nesprin2 C85融合蛋白。结论:Nesprin2在小鼠各级生精细胞中均有表达,在减数分裂过程中可能参与核膜的重塑。     

Selective loss of Sertoli cell and germ cell function leads to a disruption in sertoli cell-germ cell communication during aging in the Brown Norway rat

We investigated the effects of aging on Sertoli cell-germ cell interactions from Brown Norway rats using the induction of four specific mRNAs as markers. The testes from aging (24 mo old) Brown Norway rats can be normal size or regressed. One marker, a von Ebner's-like protein, is expressed in coculture and "in vivo" in germ cells from normal testes of 6- and 24-mo-old rats but not in germ cells from regressed testes of 24-mo-old rats. A second germ cell marker, the Huntington disease protein, is expressed in all germ cells. Two Sertoli cell markers, a serotonin receptor and a novel gene, are induced in Sertoli cells by meiotic germ cells. The serotonin receptor mRNA is expressed in Sertoli cells from 20-day, 6-mo, and 24-mo normal testes but not in those from 24-mo regressed testes. The novel gene is induced in Sertoli cells from all testes. We conclude that Sertoli cells from aged regressed testes are unable to respond to selective signals from germ cells from young rats, and germ cells from regressed testes show a similar selective loss. Such disruptions in communication between Sertoli cells and germ cells likely contribute to germ cell loss during aging.     

Reg I protein in healthy and seminoma human testis

Regenerating gene (Reg), encodes a secretory protein with growth and differentiation stimulating effects mostly in digestive tissues. Overexpression of Reg proteins and specifically of Reg I, one member of the Reg family, is associated with several human diseases and cancers. In the present study we analyzed the expression of Reg I in normal rodent and human testes where germ cells normally proliferate and differentiate into spermatozoa, and in seminoma testis, the most common cancer of young men. Western blot analyses demonstrated the presence of a specific band at 19 kDa in human and rodent testis extracts. Immunofluorescence and deconvolution microscopy demonstrated that Reg I was present within the seminiferous tubules in both Sertoli and germ cells. By using a Sertoli cell line we demonstrated that Reg I was localized at the plasma membrane even in the absence of contact between neighboring cells and appeared before the tight junction associated protein ZO-1 was revealed at this location. Reg I was strongly expressed in human seminoma testis tissue and in a human tumor germ cell line where the immunoreactive signal was mainly detected at the plasma membrane level. These data showing for the first time the weak presence of Reg I in the normal testis and its strong expression in the testis cancer suggest a potential role of Reg I in normal and neoplastic germ cell proliferation.     

Immunohistochemical study of a membrane skeletal molecule, protein 4.1G, in mouse seminiferous tubules

Protein 4.1 families have recently been established as potential organizers of an adherens system. In the adult mouse testis, protein 4.1G (4.1G) localized as a line pattern in both basal and adluminal compartments of the seminiferous tubules, attaching regions of germ cells and Sertoli cells. By double staining for 4.1G and F-actin, their localizations were shown to be different, indicating that 4.1G was localized in a region other than the basal and apical ectoplasmic specializations, which formed the Sertoli–Sertoli cell junction and Sertoli–spermatid junction, respectively. By electron microscopy, immunoreactive products were seen exclusively on the cell membranes of Sertoli cells, attaching to the various differentiating germ cells. The immunolocalization of cadherin was identical to that of 4.1G, supporting the idea that 4.1G may be functionally interconnected with adhesion molecules. In an experimental mouse model of cadmium treatment, in which tight and adherens junctions of seminiferous tubules were disrupted, the 4.1G immunostaining in the seminiferous tubules was dramatically decreased. These results indicate that 4.1G may have a basic adhesive function between Sertoli cells and germ cells from the side of Sertoli cells.     

Sterol carrier protein 2 (non-specific lipid transfer protein) is localized in membranous fractions of Leydig cells and Sertoli cells but not in germ cells.

The cellular and subcellular distribution of sterol carrier protein 2 (SCP2; nsL-TP) was reinvestigated in rat testicular cells by Western blotting and immunocytochemistry, using the affinity purified antibody against rat liver SCP2. Western blot analysis revealed high levels of the protein in the somatic cells of the testis, e.g., Leydig and Sertoli cells whereas it could not be detected in germ cells. This cellular localization of SCP2 was confirmed by Northern blotting. Immunocytochemical techniques revealed that in Leydig cells, immunoreactive proteins were concentrated in peroxisomes. Although SCP2 was also detected in Sertoli cells, a specific subcellular localization could not be shown. SCP2 was absent from germ cells. Analysis of subcellular fractions of Leydig cells showed that SCP2 is membrane bound without detectable amounts in the cytosolic fraction. These results are at variance with data published previously which suggested that in Leydig cells a substantial amount of SCP2 was present in the cytosol and that the distribution between membranes and cytosol was regulated by luteinizing hormone. The present data raise the question in what way SCP2 is involved in cholesterol transport between membranes in steroidogenic cells but also in non-steroidogenic cells.     

Establishment of human embryonic stem cell line stably expressing Epstein-Barr virus-encoded nuclear antigen 1

Human embryonic stem (hES) cells have the capability of unlimited undifferentiated proliferation, yet maintain the potential to form perhaps any cell type in the body. Based on the high efficiency of the Epstein-Barr virus-based episomal vector in introducing exogenous genes of interest into mammalian cells, we applied this system to hES cells, expecting that this would resolve the problem of poor transfection efficiency existing in current hES cell research. Therefore, the first step was to establish EBNA1-positive hES cells. Using the Fugene 6 transfection reagent, we transfected hES cells with the EBNA1 expression vector and subsequently generated hES cell clones that stably expressed EBNA1 under drug selection. These clones were confirmed to express EBNA1 mRNA by RT-PCR and to express EBNA1 protein by Western blotting. Furthermore, luciferase reporter gene analysis was performed on the EBNA1 clones and revealed that the expressed EBNA1 protein was functional. When the EBNA1-positive cells were injected into severe combined immunodeficient (SCID) mice, they formed teratoma tissues containing all three embryonic germ layers and EBNA1 protein was detected in these teratoma tissues by Western blotting. All the results show that we have successfully created stable EBNAI-hES cells, thus laying a good foundation for further research.     

Coxsackie and adenovirus receptor (CAR) is a product of Sertoli and germ cells in rat testes which is localized at the Sertoli-Sertoli and Sertoli-germ cell interface

The coxsackie and adenovirus receptor (CAR), a putative cell-cell adhesion molecule, has attracted wide interest due to its importance in viral pathogenesis and in mediating adenoviral gene delivery. However, the distribution pattern and physiological function of CAR in the testis is still not clear. Here, we identified CAR in Sertoli cells and germ cells of rats. In vivo studies have shown that CAR resides at the blood-testis barrier as well as at the ectoplasmic specialization. The persistent expression of CAR in rat testes from neonatal period throughout adulthood implicates its role in spermatogenesis. Using primary Sertoli cell cultures, we observed a significant induction of CAR during the formation of Sertoli cell epithelium. Furthermore, CAR was seen to be concentrated at inter-Sertoli cell junctions, co-localizing with tight junction protein marker ZO-1 and adherens junction protein N-cadherin. CAR was also found to be associated with proteins of Src kinase family and its protein level declined after TNFα treatment in Sertoli cell cultures. Immunofluorescent staining of isolated germ cells has revealed the presence of CAR on spermatogonia, spermatocytes, round spermatids and elongate spermatids. Taken together, we propose that CAR functions as an adhesion molecule in maintaining the inter-Sertoli cell junctions at the basal compartment of the seminiferous epithelium. In addition, CAR may confer adhesion between Sertoli and germ cells at the Sertoli-germ cell interface. It is possible that the receptor utilized by viral pathogens to breakthrough the epithelial barrier was also employed by developing germ cells to migrate through the inter-Sertoli cell junctions.     

Testin regulates the blood-testis barrier via disturbing occludin/ZO-1 association and actin organization

The blood-testis barrier (BTB) separates the seminiferous epithelium into the apical and basal compartments. The BTB has to operate timely and accurately to ensure the correct migration of germ cells, meanwhile maintaining the immunological barrier. Testin was first characterized from primary Sertoli cells, it is a secretory protein and a sensitive biomarker to monitor junctions between Sertoli and germ cells. Till now, the functions of testin on BTB dynamics and the involving mechanisms are unknown. Herein, testin acts as a regulatory protein on BTB integrity. In vitro testin knockdown by RNAi caused significant damage to the Sertoli cell barrier with no apparent changes in the protein levels of several major tight junction (TJ), adhesion junction, and gap junction proteins. Also, testin RNAi caused the diffusion of two TJ structural proteins, occludin and ZO-1, diffusing away from the Sertoli cell surface into the cytoplasm. Association and colocalization between ZO-1 and occludin were decreased after testin RNAi, examined by Co-IP and coimmunofluorescent staining, respectively. Furthermore, testin RNAi induced a dramatic disruption on the arrangement of actin filament bundles and a reduced F-actin/G-actin ratio. The actin regulatory protein ARP3 appeared at the Sertoli cell interface after testin RNAi without its protein level change, whereas overexpressing testin in Sertoli cells showed no effect on TJ barrier integrity. The above findings suggest that besides as a monitor for Sertoli-germ cell junction integrity, testin is also an essential molecule to maintain Sertoli–Sertoli junctions.     

Postnatal changes in the expression of p60c-Src in mouse testes

Src family non-receptor tyrosine kinases are involved in signaling pathways which mediate cell growth, differentiation, transformation and tissue remodeling in various organs. In an effort to elucidate functional involvement of p60c-Src (c-Src) in spermatogenesis, the postnatal changes in c-src mRNA and c-Src protein together with kinase activity and subcellular localization were examined in mouse testes. c-src mRNA levels in testes increased during the first 2 weeks of postnatal development (PND). Following a decrease at puberty (PND 28), the c-src mRNA levels re-increased at adulthood (PND 50). Src kinase activity of testes was low at PND 7 but sharply increased prepubertally (PND 15) and highest at adulthood. Upon Western blotting, the level of c-Src protein was the highest in prepubertal testes but rather decreased in adult testes at PND 50. In adult testes, ubiquitination of c-Src proteins was apparent compared with immature one at PND 7, suggesting active turnover of c-Src by ubiquitination. In immature testes, c-Src immunoreactivity was largely found in the cytoplasm of the Sertoli cells. By contrast, in pubertal and adult testes intense immunoreactivity was localized at the adluminal and basal cytoplasm of Sertoli cells bearing elongated spermatids and early germ cells, respectively. The immunoreactivity of c-Src in the Leydig cells was increased during pubertal development, suggesting the functional involvement of c-Src in differentiated adult Leydig cells. Throughout postnatal development, some spermatogonia and spermatocytes showed intensive c-Src immunoreactivity compared with other germ cells, suggesting a possible role of c-Src in germ cell death. Taken together, it is suggested that c-Src may participate in the remodeling of the seminiferous epithelia and functional differentiation of Leydig cells during the postnatal development of mouse testes.     

Molecular identification and functional characterization of the vitamin C transporters expressed by Sertoli cells

Vitamin C is an essential micronutrient for the development of male germ cells. In the gonad, the germ cells are isolated from the systemic circulation by the blood-testis barrier, which consists of a basal layer of Sertoli cells that communicate through an extensive array of tight junction complexes. To study the behavior of Sertoli cells as a first approach to the molecular and functional characterization of the vitamin C transporters in this barrier, we used the 42GPA9 cell line immortalized from mouse Sertoli cells. To date, there is no available information on the mechanism of vitamin C transport across the blood-testis barrier. This work describe the molecular identity of the transporters involved in vitamin C transport in these cells, which we hope will improve our understanding of how germ cells obtain vitamin C, transported from the plasma into the adluminal compartment of the seminiferous tubules. RT-PCR analyses revealed that 42GPA9 cells express both vitamin C transport systems, a finding that was confirmed by immunocytochemical and immunoblotting analysis. The kinetic assays using radioactive vitamin C revealed that both ascorbic acid (AA) transporters, SVCT1 and SVCT2, are functionally active. Moreover, the kinetic characteristics of dehydroascorbic acid (DHA) and 3-methylglucose (OMG) transport by 42GPA9 Sertoli cells correspond to facilitative hexose transporters GLUT1, GLUT2 and GLUT3 expressed in these cells. This data is consistent with the concept that Sertoli cells have the ability to take up vitamin C. It is an important finding and contributes to our knowledge of the physiology of male germ cells.     

Isolation of sertoli cells from adult rat testes: an approach to ex vivo studies of Sertoli cell function

Much of what is known about the molecular regulation and function of adult Sertoli cells has been inferred from in vitro studies of immature Sertoli cells. However, adult and immature cells differ in significant ways and, moreover, many Sertoli cell functions are regulated by conditions that are difficult to replicate in vitro. Our objective was to develop a procedure to isolate Sertoli cells rapidly and in sufficient number and purity to make it possible to assess Sertoli cell function immediately after the isolation of the cells. The isolation procedure described herein takes less than 4 h and does not require culturing the cells. From a single 4-mo-old adult rat, we routinely obtain 7.0 +/- 0.4 x 10(6) Sertoli cells per testis, and from a 21-mo-old rat, 7.2 +/- 0.4 x 10(6) Sertoli cells per testis. The purity, determined by morphologic analyses of plastic-embedded cells or after staining for tyrosine-tubulin or vimentin, averaged 80%. The contaminants typically included germ cells (10%) and myoid cells (10%). The germ cell-expressed genes protamine-2 and hemiferrin were not detected in the Sertoli cell preparations by Northern blot analyses, but the Sertoli cell-expressed genes clusterin, cathepsin L, and transferrin were highly expressed. Transferrin mRNA levels were greater in Sertoli cells isolated from aged than from young adult rats, consistent with previous analyses of whole testes; and cathepsin L mRNA levels were far more highly expressed in Sertoli cells isolated from stages VI-VII than from other stages of the cycle of the seminiferous epithelium, also consistent with previous analyses of whole testes and isolated tubules. These studies indicate that the freshly isolated cells retain differentiated function, and thus it should be possible to assess the in vivo function of adult Sertoli cells by isolating the Sertoli cells and immediately assessing their function.     

Organ specific underexpression renal of Na+-dependent B0AT1 in the SHR correlates positively with overexpression of NHE3 and salt intake

Densin is a member of LAP (leucine-rich repeat and PDZ domain) protein family that localizes in kidney to slit diaphragms, which are essential components of the glomerular filtration barrier. We have previously shown that densin interacts with a crucial slit diaphragm protein, nephrin. Here, we searched for novel binding partners of densin by yeast-two hybrid assay and identified beta-catenin. The interaction was confirmed by reciprocal co-immunoprecipitation assay and the binding site in densin was determined by GST-pull down assays. The GST-tagged densin was also able to pull down P-cadherin together with beta-catenin from human kidney glomerular lysates. Furthermore, densin co-localized with beta-catenin and F-actin in cell–cell contacts in cultured mouse podocytes. During cell–cell contact disruption and reformation densin and beta-catenin were dislocated from and relocated back to plasma membrane in a similar fashion. These and our previous findings suggest that densin may associate with the cadherin-catenin and nephrin complex(es), and may be involved in the formation of the cell–cell contacts including the slit diaphragm.     

Immunohistochemical study of protein 4.1B in the normal and W/W(v) mouse seminiferous epithelium.

Cell-cell adhesion is crucial not only for mechanical adhesion but also for tissue morphogenesis. Protein 4.1B, a member of the protein 4.1 family named from an erythrocyte membrane protein, is a potential organizer of an adherens system. In adult mouse seminiferous tubules, protein 4.1B localized in the basal compartment, especially in the attaching region of spermatogonia and Sertoli cells. Protein 4.1B localization and appearance were not different in each spermatogenic stage. Developmentally, protein 4.1B was not detected at postnatal day 3 (P3), was diffusely localized at P15, and was found in the basal compartment during the third week. By double staining for protein 4.1B and F-actin, their localizations were shown to be different, indicating that protein 4.1B was localized in a region lower than the basal ectoplasmic specialization that formed the Sertoli-Sertoli junction. By electron microscopy, immunoreactive products were seen mainly on the membranes of Sertoli cells. In the W/W(v) mutant mouse, the seminiferous epithelium had few germ cells. Protein 4.1B and beta-catenin were not detected, although the basal ectoplasmic specialization was retained. These results indicate that protein 4.1B may be related to the adhesion between Sertoli cells and germ cells, especially the spermatogonium.     

Extracellular domain of V-set and immunoglobulin domain containing 1 (VSIG1) interacts with sertoli cell membrane protein, while its PDZ-binding motif forms a complex with ZO-1

V-set and immunoglobulin domain containing 1 (VSIG1) is a newly discovered member of the junctional adhesion molecule (JAM) family; it is encoded by a gene located on human chromosome X and preferentially expressed in a variety of cancers in humans. Little is known about its physiological function. To determine the role(s) of VSIG1 in mammalian spermatogenesis, we first generated a specific antibody against mouse VSIG1 and examined the presence and localization of the protein in tissues. RTRCR and Western blot analysis of the mouse tissues indicated that VSIG1 was specifically expressed in the testis. Furthermore, the results of our trypsinization and biotinylation assays strongly support the assumption that VSIG1 is localized on the testicular germ cell surface. In order to determine whether VSIG1 is capable of participation in homotypic interactions, we performed a GST-pull down assay by using recombinant GST-fusion and Histagging proteins. The pull-down assay revealed that each GST-fusion Ig-like domain shows homotypic binding. We further show that mVSIG1 can adhere to the Sertoli cells through its first Ig-like domain. To identify the protein that interacted with cytoplasmic domain, we next performed co-immunoprecipitation analysis. This analysis showed that ZO-1, which is the central structural protein of the tight junction, is the binding partner of the cytoplasmic domain of mouse VSIG1. Our findings suggest that mouse VSIG1 interacts with Sertoli cells by heterophilic adhesion via its first Ig-like domain. In addition, its cytoplasmic domain is critical for binding to ZO-1.