C. Yan Cheng

Disruption of Mtmr2 produces CMT4B1-like neuropathy with myelin outfolding and impaired spermatogenesis

Mutations in MTMR2, the myotubularin-related 2 gene, cause autosomal recessive Charcot-Marie-Tooth (CMT) type 4B1, a demyelinating neuropathy with myelin outfolding and azoospermia. MTMR2 encodes a ubiquitously expressed phosphatase whose preferred substrate is phosphatidylinositol (3,5)-biphosphate, a regulator of membrane homeostasis and vesicle transport. We generated Mtmr2-null mice, which develop progressive neuropathy characterized by myelin outfolding and recurrent loops, predominantly at paranodal myelin, and depletion of spermatids and spermatocytes from the seminiferous epithelium, which leads to azoospermia. Disruption of Mtmr2 in Schwann cells reproduces the myelin abnormalities. We also identified a novel physical interaction in Schwann cells, between Mtmr2 and discs large 1 (Dlg1)/synapse-associated protein 97, a scaffolding molecule that is enriched at the node/paranode region. Dlg1 homologues have been located in several types of cellular junctions and play roles in cell polarity and membrane addition. We propose that Schwann cell–autonomous loss of Mtmr2–Dlg1 interaction dysregulates membrane homeostasis in the paranodal region, thereby producing outfolding and recurrent loops of myelin.

Two New Male Contraceptives Exert Their Effects by Depleting Germ Cells Prematurely from the Testis

Abstract The three currently available male contraceptive approaches are 1) the barrier method such as the condom, 2) hormonal methods by disrupting the pituitary-testicular axis so as to impair spermatogenesis, and 3) immunological methods by preparing vaccines against male-specific antigens. We hereby describe an alternative approach in which attachments of developing germ cells onto the seminiferous epithelium are disrupted, thereby inducing their premature release into the tubular lumen. This in turn leads to infertility. A panel of analogues based on the core structure of 1-(2,4-dichlorobenzyl)-indazole-3-carboxylic acid was synthesized. These compounds were subjected to an in vivo screening assay assessing their effects in inducing the expression of testin, a testicular marker whose expression correlates with the integrity of Sertoli-germ cell junctions. An induction of testin expression in the testis signifies a disruption of Sertoli-germ cell junctions that is followed by depletion of germ cells from the seminiferous epithelium. Two compounds, namely 1-(2,4-dichlorobenzyl)-indazole-3-carbohydrazide (AF-2364) and 1-(2,4-dichlorobenzyl)-indazole-3-acrylic acid (AF-2785), were identified that caused detachment of germ cells, in particular round and elongated spermatids, from the epithelium inducing their premature release into the tubular lumen as confirmed by histological analysis. Adult rats receiving several oral doses of either one of these compounds became infertile within 3–7 wk after the epididymal sperm reserve was exhausted. Depending on the dosing of the administered compound, rats became infertile for 4–14 wk before their fertility gradually bounced back, illustrating the reversibility and efficacy of these new compounds. Also, these compounds did not appear to impair the hypothalamus-pituitary-testicular axis because the serum levels of LH, FSH, and testosterone of the treated animals did not change significantly when compared to control rats. In addition, results of serum microchemistry illustrate that liver and kidney function was not affected in animals treated with both compounds.

Enhanced chemiluminescence (ECL) for routine immunoblotting

Immunoblotting is an analytical technique used by many laboratories to study protein expression. It involves electrophoretic separation of proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), immobilization of these proteins onto a membrane (i.e., nitrocellulose or polyvinylidene difluoride), incubation in a monoclonal or polyclonal antibody and detection by a standard method such as enhanced chemiluminescence (ECL). To achieve this, most laboratories opt to use commercially-available chemiluminescence kits which are acceptable but relatively expensive. In this technical report, we show that a self-prepared chemiluminescence reagent is superior to a commercially obtained kit in terms of sensitivity, duration of signal, ease-of-use and shelf-life but at a fraction of the cost of a kit.

Quantification of prostaglandin D synthetase in cerebrospinal fluid: a potential marker for brain tumor.

Prostaglandin D synthetase (PGD‐S; prostaglandin‐H2 D‐isomerase, EC 5,3,99,2), a 30 kDa glycoprotein also known as β‐trace protein that catalyzes the formation of prostaglandin D2 (PGD2) from PGH2, was purified to apparent homogeneity from human cerebrospinal fluid (CSF) using a two‐step procedure involving HPLC on a Vydac C8 reversed‐phase column and high performance electrophoresis chromatography (HPEC) using a 10% T SDS‐polyacrylamide gel. The purity of PGD‐S isolated from CSF was confirmed by silver stained SDS‐polyacrylamide gel and direct protein microsequencing (NH2‐APEAQVSVQPNFQ). A highly specific polyclonal antibody was prepared against this protein for immunoassay development. Using an ELISA, it was found that the concentration of PGD‐S in CSF did not alter significantly in different pathological conditions of the central nervous system (CNS). These include dementia (n=9), hydrocephalus (n=4), neuropathy (n=11), optic neuritis (n=4), multiple sclerosis (n=11), and demyelinating syndrome (n=11), when compared to normal individuals (n=12); however, the level of PGD‐S in the CSF obtained from patients with brain tumor (n=11), was reduced by as much as 2‐fold when compared to control samples (n=12) illustrating PGD‐S is a potentially useful marker for brain tumor.

Rat testicular myotubularin, a protein tyrosine phosphatase expressed by Sertoli and germ cells, is a potential marker for studying cell–cell interactions in the rat testis

The full‐length cDNA encoding the entire open reading frame (ORF) of rat myotubularin (rMTM) was isolated from a rat testis expression library by PCR. Among the three ∼2.9‐kb cDNAs that were sequenced, one clone was different from the other two clones. It contained seven extra amino acids of FVVLNLQ; this short stretch of extra sequence was found between Gln421 and Phe422 within the SET (Suvar3–9, Enhancer‐of‐zeste, Trithorax) interacting domain (SID) of rMTM. The rMTM ORF had 1,713 bp encoding for a 571 amino acid polypeptide and a calculated molecular weight of 65.8 kDa. A comparison between its deduced amino acid sequence and the GenBank database using BLAST revealed a 53.1% identity with human myotubularin protein (hMTM1), which is a member of the protein tyrosine phosphatase (PTP) family associated with X‐linked myotubular myopathy. A 22 amino acid peptide NH2‐TKVNERYELCDTYPALLAVPAN was synthesized based on the deduced amino acid sequence of rMTM and used for antibody production. By using immunoblot analysis, a 66‐kDa protein was indeed detected in both Sertoli and germ‐cell cytosols. rMTM mRNA was found in various tissues but was predominantly expressed in the testis, ovary, and skeletal muscle. Sertoli cell rMTM expression was stimulated by germ cells and enhanced when inter‐Sertoli junctions were being assembled in vitro. A drastic reduction in testicular rMTM steady‐state mRNA level correlated with the depletion of germ cells from the testis in vivo following either glycerol or lonidamine treatment. These results indicate that rMTM is a rat homologue of hMTM1 that may be a useful marker in monitoring the events of cell–cell interactions in the testis. J. Cell. Physiol. 185:366–385, 2000.

Desmosomes in the testis: Moving into an unchartered territory

Desmosomes are cell-cell junctions that link to cytoplasmic intermediate filaments, and they are known to mediate robust and stable adhesion in organs such as the skin and heart. Desmosomes are also present between apposing Sertoli cells at the blood-testis barrier, and between Sertoli cells and all germ cells up to, but not including, step 8 spermatids in the seminiferous epithelium. Unfortunately, they remain to be one of the least studied cell junction types in the seminiferous epithelium of the mammalian testis. In this article, we briefly discuss how kinases and the actin cytoskeleton relate to the study of desmosomes in the testis. It is hoped that this information is used to initiate more studies on the biology of the desmosome in the future.

Focal adhesion kinase is a regulator of F-actin dynamics: New insights from studies in the testis.

During spermatogenesis, spermatogonia (2n, diploid) undergo a series of mitotic divisions as well as differentiation to become spermatocytes, which enter meiosis I to be followed by meiosis II to form round spermatids (1n, haploid), and then differentiate into spermatozoa (1n, haploid) via spermiogenesis. These events take place in the epithelium of the seminiferous tubule, involving extensive junction restructuring at the Sertoli-Sertoli and Sertoli-germ cell interface to allow the transport of developing germ cells across the epithelium. Although structural aspects of these cell-cell junctions have been studied, the underlying mechanism(s) that governs these events has yet to be explored. Earlier studies have shown that a non-receptor protein tyrosine kinase known as focal adhesion kinase (FAK) is a likely regulator of these events due to the stage-specific and spatiotemporal expression of its various phosphorylated/activated forms at the testis-specific anchoring junctions in the testis, as well as its association with actin regulatory proteins. Recent studies have shown that FAK, in particular its two activated phosphorylated forms p-FAK-Tyr407 and p-FAK-Tyr397, are crucial regulators in modulating junction restructuring at the Sertoli cell-cell interface at the blood-testis barrier (BTB) known as the basal ectoplasmic specialization (basal ES), as well as at the Sertoli-spermatid interface called apical ES during spermiogenesis via its effects on the filamentous (F)-actin organization at the ES. We herein summarize and critically evaluate the current knowledge regarding the physiological significance of FAK in regulating BTB and apical ES dynamics by governing the conversion of actin filaments at the ES from a “bundled” to a “de-bundled/branched” configuration and vice versa. We also provide a molecular model on the role of FAK in regulating these events based on the latest findings in the field.

Environmental contaminants: Is male reproductive health at risk?

Contaminants such as cadmium, bisphenol A and lead which pollute our environment affect male reproductive function. There is evidence that toxicant exposure adversely affects fertility. Cadmium and bisphenol A exert their effects in the testis by perturbing blood-testis barrier function, which in turn affects germ cell adhesion in the seminiferous epithelium because of a disruption of the functional axis between these sites. In essence, cadmium mediated its adverse effects at the blood-testis barrier by disrupting cell adhesion protein complexes, illustrating toxicants dismantle cell junctions in the testis. Herein, we will discuss how environmental toxicants affect reproductive function. We will also examine how these adverse effects on fertility may be mediated in part by adipose tissue and bone. Lastly, we will briefly discuss how toxicant-induced damage may be effectively managed so that fertility can be maintaine-d. It is hoped that this information will offer a new paradigm for future studies.

Actin binding proteins and spermiogenesis: Some unexpected findings.

Drebrin E, an actin-binding protein lacking intrinsic activity in the regulation of actin dynamics (e.g., polymerization, capping, nucleation, branching, cross-linking, bundling, and severing), is known to recruit actin regulatory proteins to a specific cellular site. Herein, we critically evaluate recent findings in the field which illustrate that drebrin E works together with two other actin-binding proteins, namely Arp3 (actin-related protein 3, a component of the Arp2/3 complex that simultaneously controls actin nucleation for polymerization and branching of actin filaments) and Eps8 (epidermal growth factor receptor pathway substrate 8 that controls capping of the barbed ends of actin filaments, as well as actin filament bundling) to regulate the homeostasis of F-actin filament bundles at the ectoplasmic specialization (ES), a testis-specific adherens junction (AJ) in the seminiferous epithelium. This is mediated by the strict temporal and spatial expression of these three actin-binding proteins at the apical and basal ES at the Sertoli cell-spermatid (step 8-19) and Sertoli-Sertoli cell interface, respectively, during the seminiferous epithelial cycle of spermatogenesis. In this Commentary, we put forth a possible model by which drebrin E may be acting as a platform upon which proteins (e.g., Arp3) that are needed to alter the conformation of actin filament bundles at the ES can be recruited to the site, thus facilitating changes in cell shape and cell position in the epithelium during spermiogenesis and spermiation. In short, drebrin E may be acting as a “logistic” distribution center to manage different regulatory proteins at the apical ES, thereby regulating the dynamics of actin filament bundles and modulating the plasticity of the apical ES. This would allow adhesion to be altered continuously throughout the epithelial cycle to accommodate spermatid movement in the seminiferous epithelium during spermiogenesis and spermiation. We also describe a hypothetical model, upon which functional studies can be designed in the future.

NC1 domain of collagen α3(IV) derived from the basement membrane regulates Sertoli cell blood-testis barrier dynamics

The blood-testis barrier (BTB) is an important ultrastructure for spermatogenesis. Delay in BTB formation in neonatal rats or its irreversible damage in adult rats leads to meiotic arrest and failure of spermatogonial differentiation beyond type A. While hormones, such as testosterone and FSH, are crucial to BTB function, little is known if there is a local regulatory mechanism in the seminiferous epithelium that modulates BTB function. Herein, we report that collagen α3(IV) chain, a component of the basement membrane in the rat testis, could generate a noncollagenous (NC1) domain peptide [Colα3(IV) NC1] via limited proteolysis by matrix metalloproteinase-9 (MMP-9), and that the expression of MMP-9 was upregulated by TNFα. While recombinant Colα3(IV) NC1 protein produced in E. coli failed to perturb Sertoli cell tight junction (TJ)-permeability barrier function, possibly due to the lack of glycosylation, Colα3(IV) NC1 recombinant protein produced in mammalian cells and purified to apparent homogeneity by affinity chromatography was found to reversibly perturb the Sertoli cell TJ-barrier function. Interestingly, Colα3(IV) NC1 recombinant protein did not perturb the steady-state levels of several TJ- (e.g., occludin, CAR, JAM-A, ZO-1) and basal ectoplasmic specialization- (e.g., N-cadherin, α-catenin, β-catenin) proteins at the BTB but induced changes in protein localization and/or distribution at the Sertoli cell-cell interface in which these proteins moved from the cell surface into the cell cytosol, thereby destabilizing the TJ function. These findings illustrate the presence of a local regulatory axis known as the BTB-basement membrane axis that regulates BTB restructuring during spermatogenesis.