The acrosomal protein SP-10 (Acrv1) is an ideal marker for staging of the cycle of seminiferous epithelium in the mouse.

The study of spermatogenesis requires accurate identification of the stages of the cycle of the seminiferous epithelium. A stage refers to the unique association of germ cell types at a particular phase of development, as seen in a cross-sectioned seminiferous tubule. Stage-identification, however, is a daunting task. There are 12 stages represented in the mouse seminiferous epithelium. Stages are typically identified on the basis of the morphology of the developing acrosome of spermatids. Although the characteristic features of the acrosome are well-documented in ultrastructure images, a reagent that can highlight the subtle differences in acrosome shape under the light microscope is lacking. Here we demonstrate that a polyclonal antibody raised against the mouse acrosomal protein SP-10 is extremely useful for stage identification. Immunohistochemistry showed that the anti-SP-10 antibody is highly specific for the acrosome of spermatids, as no other cell type in the epithelium showed immunoreactivity. At lower magnification, the gross shape of the acrosome and the increasing intensity of immunostaining served as a guide for the identification of stages I-XII. At higher magnification, characteristic morphological features-such as whether the part of the acrosome that contacts the nuclear surface is round (stage III) or flat (stage IV) or curved (stage VI)-could be identified unambiguously. Overall, we present evidence that SP-10 is a useful marker for staging the cycle of the seminiferous epithelium. The anti-SP-10 antibody works well in different fixatives, on paraffin-embedded as well as cryosections, and has been shown to be useful for characterizing spermatogenic defects in mutant mice.

JunB mediates basal- and TGFß1-induced smooth muscle cell contractility.

Smooth muscle contraction is a dynamic process driven by acto-myosin interactions that are controlled by multiple regulatory proteins. Our studies have shown that members of the AP-1 transcription factor family control discrete behaviors of smooth muscle cells (SMC) such as growth, migration and fibrosis. However, the role of AP-1 in regulation of smooth muscle contractility is incompletely understood. In this study we show that the AP-1 family member JunB regulates contractility in visceral SMC by altering actin polymerization and myosin light chain phosphorylation. JunB levels are robustly upregulated downstream of transforming growth factor beta-1 (TGFß1), a known inducer of SMC contractility. RNAi-mediated silencing of JunB in primary human bladder SMC (pBSMC) inhibited cell contractility under both basal and TGFß1-stimulated conditions, as determined using gel contraction and traction force microscopy assays. JunB knockdown did not alter expression of the contractile proteins α-SMA, calponin or SM22α. However, JunB silencing decreased levels of Rho kinase (ROCK) and myosin light chain (MLC20). Moreover, JunB silencing attenuated phosphorylation of the MLC20 regulatory phosphatase subunit MYPT1 and the actin severing protein cofilin. Consistent with these changes, cells in which JunB was knocked down showed a reduction in the F:G actin ratio in response to TGFß1. Together these findings demonstrate a novel function for JunB in regulating visceral smooth muscle cell contractility through effects on both myosin and the actin cytoskeleton.

FosB regulates stretch-induced expression of extracellular matrix proteins in smooth muscle.

Fibroproliferative remodeling in smooth muscle-rich hollow organs is associated with aberrant extracellular matrix (ECM) production. Although mechanical stimuli regulate ECM protein expression, the transcriptional mediators of this process remain poorly defined. Previously, we implicated AP-1 as a mediator of smooth muscle cell (SMC) mechanotransduction; however, its role in stretch-induced ECM regulation has not been explored. Herein, we identify a novel role for the AP-1 subunit FosB in stretch-induced ECM expression in SMCs. The DNA-binding activity of AP-1 increased after stretch stimulation of SMCs in vitro. In contrast to c-Jun and c-fos, which are also activated by the SMC mitogen platelet-derived growth factor, FosB was only activated by stretch. FosB silencing attenuated the expression of the profibrotic factors tenascin C (TNC) and connective tissue growth factor (CTGF), whereas forced expression of Jun~FosB stimulated TNC and CTGF promoter activity. Chromatin immunoprecipitation revealed enrichment of AP-1 at the TNC and CTGF promoters. Bladder distension in vivo enhanced nuclear localization of c-jun and FosB. Finally, the distension-induced expression of TNC and CTGF in the detrusor smooth muscle of bladders from wild-type mice was significantly attenuated in FosB-null mice. Together, these findings identify FosB as a mechanosensitive regulator of ECM production in smooth muscle.

An Akt- and Fra-1-dependent pathway mediates platelet-derived growth factor-induced expression of thrombomodulin, a novel regulator of smooth muscle cell migration.

Overdistension of hollow organs evokes pathological changes characterized by smooth muscle remodeling. Mechanical stimuli induce smooth muscle cell (SMC) growth through acute activation of signaling cascades and by increased expression of soluble mitogens. Physical forces have also been implicated in ligand-independent activation of receptor tyrosine kinases, including the platelet-derived growth factor (PDGF) receptor, although the extent to which this occurs in intact tissue is unknown. Previously, we implicated Akt and activator protein-1 (AP-1) as mediators of growth and gene expression in SMC exposed to cyclic stretch or PDGF. Here we show that bladder wall distension leads to PDGFR activation and identify thrombomodulin (TM) as an Akt and AP-1 target in SMC. We demonstrate that TM, also induced by bladder stretch injury, is regulated at the transcriptional level by the AP-1 components c-jun and Fra1. Mutation of an AP-1 motif at -2010/-2004 abolished both AP-1 binding and PDGF responsiveness of the TM promoter. Fra1 silencing diminished PDGF-induced TM expression and SMC cell cycle transit. In contrast, TM knockdown did not affect cell growth but attenuated PDGF-stimulated SMC migration. Taken together, these results reveal new facets of TM regulation in SMC and provide the first demonstration of a role for endogenous TM in PDGF-induced cell migration. Moreover, TM induction on bladder injury suggests that it may be a biomarker for pathological smooth muscle remodeling.

Identification of novel immunodominant epididymal sperm proteins using combinatorial approach.

Functionally immature spermatozoa leave the testis mature during epididymal transit. This process of maturation involves either addition of new proteins or modification of existing proteins onto the sperm domains that are responsible for domain-specific functions. Epididymal proteins are preferred targets for immunocontraception. In an attempt to identify epididymis-specific sperm proteins, we used a novel combinatorial approach comprising subtractive immunization (SI) followed by proteomics. Following SI, sera of mice were used for immunoproteomics, which led to the identification of 30 proteins, of which four proteins namely sperm head protein 1, sperm flagella protein 2 (SFP2), SFP3, and SFP4 are being reported for the first time on sperm. Another group of four proteins namely collagen alpha-2 (I) chain precursor, homeodomain-interacting protein kinase 1, GTP-binding protein Rab1, and ubiquinol cytochrome c reductase core protein II although reported earlier in testis are being reported for the first time in epididymal sperm. Furthermore, seven out of these eight novel proteins could be validated using peptide ELISA. These data are a useful repository, which could be exploited to develop targets for post-testicular immunocontraception or biomarkers for infertility diagnosis and management.

NF45 and NF90 in murine seminiferous epithelium: potential role in SP-10 gene transcription.

Identification of transcription factors involved in the progression of spermatogenic cell differentiation is important for understanding the molecular mechanisms controlling spermatogenesis. To this end, we utilized the mouse SP-10 gene encoding a conserved acrosomal protein as an experimental model. Promoter analysis in transgenic mice had previously shown that the -186/-91 region of the SP-10 promoter was critical for spermatid-specific expression. Here, we focus on a purine (Pu) box (-agaaaa) located at -154, which is conserved in the mouse, monkey, and human SP-10 gene promoters. NF45 and NF90, which belong to the family of nuclear factor of activated T cells (NFAT), are known as Pu-box-binding proteins. We tested the potential of NF45 and NF90 to activate the SP-10 promoter via the Pu-box element. Immunohistochemistry showed the presence of NF45 and NF90 in the nuclei of pachytene spermatocytes, round spermatids, and Sertoli cells. In gel shift assays, recombinant NF45 bound to the mouse SP-10 promoter in an AGAAAA site-specific manner. Cotransfection of NF45 and NF90 up-regulated SP-10 promoter-driven luciferase expression in transiently transfected spermatogenic GC2 cell line; this up-regulation required the -AGAAAA- site. Furthermore, stimulation of the endogenous NF45-NF90 complex in Jurkat cells by phorbol myristate acetate + ionomycin up-regulated the SP-10 promoter activity in plasmid-based assays. In the context of chromatin, however, stimulation of NF45-NF90 alone was not sufficient to activate an SP-10 promoter-driven green fluorescent protein transgene. Based on these results, we propose that NF45 and NF90 have the potential to activate SP-10 gene transcription, and that a chromatin modification event must occur first in order to provide access to these transcription factors.

Role of an insulator in testis-specific gene transcription.

Testis-specific promoters are unique in that relatively short proximal promoters of several genes have been shown to be capable of directing tissue- and cell-type-specific expression in transgenic mice. How such small promoter fragments perform the dual functions of maintaining a silenced state in somatic tissues and activating gene expression in the correct germ-cell type in testis remains poorly understood. Studies from our laboratory using the round spermatid-specific SP-10 gene as an experimental model have provided some insights into the mechanisms involved. It was found that the proximal promoter of the SP-10 gene acts as a chromatin insulator or boundary element in somatic tissues and prevents transcription of the SP-10 gene. In round spermatids, the insulator function is relieved, thus facilitating the SP-10 gene transcription. Insulators act as enhancer blockers and/or barriers to heterochromatin to protect the programmed expression of a gene. Typically, insulators are separable from promoters. In the case of the SP-10 gene, however, the insulator overlaps the promoter and operates in a facultative manner. We hypothesize that the proximal promoters of some testis-specific genes have adapted the insulator function to maintain transcriptional silence in the somatic tissues.

Monoclonal antibodies to epididymis-specific proteins using mice rendered immune tolerant to testicular proteins.

Monoclonal antibodies (mabs) have been used as a powerful tool for identification of newer sperm proteins. However, conventional hybridoma technology rarely provides chance to obtain mabs to epididymal proteins. To increase this chance, we have used an alternate method of neonatal tolerization. In this protocol, animals were tolerized at birth using testicular proteins followed by immunization with cauda epididymal sperm protein (which is a cocktail of proteins both from testicular and epididymal origin). This protocol induced a specific immune response to epididymal sperm proteins. Spleen from one of these animals was then used for preparation of mabs. This fusion resulted in a number of mabs reacting specifically to epididymal proteins. Although mabs identified a protein of approximately similar molecular weight on 1-dimensional Western blot analysis, there were differences in regional localization on rat sperm as seen by indirect immunofluorescence. Immunohistochemical localization of these proteins in rat epididymis showed region specific synthesis. The synthesis of proteins was seen in the distal caput epididymis, and maximum expression was seen in supranuclear region of corpus epithelium. The proteins were localized on sperm from corpus and cauda region. Epididymis specific synthesis of the proteins and agglutinating nature of the mabs to these underlines the functional importance of these proteins in sperm maturation in epididymis. These antibodies could therefore, be used as tools for understanding the physiology of maturation of sperm in epididymis and role of the epididymal protein in fertilization.