Cheng Cy

Spermatogonial stem cells alone are not sufficient to re-initiate spermatogenesis in the rat testis following adjudin-induced infertility.

The blood-testis barrier (BTB) is a unique ultrastructure in the testis, which creates a specialized microenvironment in the seminiferous epithelium known as the apical (or adluminal) compartment for post-meiotic germ-cell development and for maintenance of an immunological barrier. In this study, we have demonstrated unequivocally that a functional and intact BTB is crucial for the initiation of spermatogenesis, in particular, the differentiation of spermatogonial stem cells (SSCs). It was shown that adult rats (∼300 g body weight, b.w.) treated with adjudin at 50 (low-dose) or 250 (high-dose) mg/kg b.w. by gavage led to germ-cell depletion from the seminiferous tubules and that >98% of the tubules were devoid of germ cells by ∼2 week and rats became infertile in both groups after the sperm reserve in the epididymis was exhausted. While the population of SSC/spermatogonia in the seminiferous tubules from both groups was similar to that of normal rats, only rats from the low-dose group were capable of re-initiating spermatogenesis; and by 20 weeks, greater than 75% of the tubules displayed normal spermatogenesis and the fertility of these rats rebounded. Detailed analysis by dual-labelled immunofluorescence analysis and a functional BTB integrity assay revealed that in both treatment groups, the BTB was disrupted from week 6 to week 12. However, the disrupted BTB resealed in the low-dose group, but not in the high-dose group. Our findings illustrate that SSC/spermatogonia failed to differentiate into spermatocytes beyond A(aligned) spermatogonia in the high-dose group with a disrupted BTB. In short, these findings illustrate the critical significance of the BTB for re-initiation of spermatogenesis besides SSC and spermatogonia.

Adjudin, a potential male contraceptive, exerts its effects locally in the seminiferous epithelium of mammalian testes

Adjudin is a derivative of 1H-indazole-3-carboxylic acid that was shown to have potent anti-spermatogenic activity in rats, rabbits, and dogs. It exerts its effects most notably locally in the apical compartment of the seminiferous epithelium, behind the blood-testis barrier, by disrupting adhesion of germ cells, most notably spermatids to the Sertoli cells, thereby inducing release of immature spermatids from the epithelium that leads to infertility. After adjudin is metabolized, the remaining spermatogonial stem cells and spermatogonia repopulate the seminiferous epithelium gradually via spermatogonial self-renewal and differentiation, to be followed by meiosis and spermiogenesis, and thus fertility rebounds. Recent studies in rats have demonstrated unequivocally that the primary and initial cellular target of adjudin in the testis is the apical ectoplasmic specialization, a testis-specific anchoring junction type restricted to the interface between Sertoli cells and elongating spermatids (from step 8 to 19 spermatids). In this review, we highlight some of the recent advances and obstacles regarding the possible use of adjudin as a male contraceptive.

The β1-integrin-p-FAK-p130Cas-DOCK180-RhoA-vinculin is a novel regulatory protein complex at the apical ectoplasmic specialization in adult rat testes

During spermatogenesis, step 1 spermatids (round spermatids) derive from spermatocytes following meiosis I and II at stage XIV of the epithelial cycle begin a series of morphological transformation and differentiation via 19 steps in rats to form spermatozoa. This process is known as spermiogenesis, which is marked by condensation of the genetic material in the spermatid head, formation of the acrosome and elongation of the tail. Since developing spermatids are lacking the robust protein synthesis and transcriptional activity, the cellular, molecular and morphological changes associated with spermiogenesis rely on the Sertoli cell in the seminiferous epithelium via desmosome and gap junction between Sertoli cells and step 1-7 spermatids. Interestingly, a unique anchoring junction arises at the interface of step 8 spermatid and Sertoli cell known as apical ectoplasmic specialization (apical ES). Once it appears, apical ES is the only anchoring device restricted to the interface of step 8-19 spermatids and Sertoli cells to confer spermatid polarity, adhesion, signal communication, and structural support, and to provide nutritional support during spermiogenesis, replacing desmosome and gap junction. While the adhesion protein complexes that constitute the apical ES are known, the signaling protein complexes that regulate apical ES dynamics, however, remain largely unknown. Herein we report the presence of a FAK-p130Cas-DOCK180-RhoA-vinculin signaling protein complex at the apical ES, which is also an integrated component of the β1-integrin-based adhesion protein complex based on co-immunoprecipitation experiment. It was also shown that besides p-FAK-Tyr397 and p-FAK-Tyr576, β1-integrin, p130Cas, RhoA, and vinculin displayed stage-specific expression in the seminiferous epithelium during the epithelial cycle with predominant localization at the apical ES as demonstrated by immunohistochemistry. Based on these findings, functional studies can now be performed to assess the role of this b1-integrin-p-FAK-p130Cas-DOCK180-RhoA-vinculin protein complex in apical ES dynamics during spermiogenesis.

Coordination of Actin- and Microtubule-Based Cytoskeletons Supports Transport of Spermatids and Residual Bodies/Phagosomes During Spermatogenesis in the Rat Testis

Germ cell transport across the seminiferous epithelium during spermatogenesis requires the intricate coordination of cell junctions, signaling proteins, and both actin- and microtubule (MT)-based cytoskeletons. Although the involvement of cytoskeletons in germ cell transport has been suggested, the precise mechanism(s) remains elusive. Based on growing evidence that actin and MT interactions underlie fundamental cellular processes, such as cell motility, it is unlikely that actin- and MT-based cytoskeletons work independently to regulate germ cell transport in the testis. Using rats treated with adjudin, a potential male contraceptive that disrupts spermatid adhesion and transport in the testis, as a study model, we show herein that actin- and MT-based cytoskeletons are both necessary for transport of spermatids and residual bodies/phagosomes across the seminiferous epithelium in adult rat testes. Analysis of intratubular expression of F-actin and tubulin revealed disruption of both actin and MT networks, concomitant with misdirected spermatids and phagosomes in rats treated with adjudin. Actin regulatory proteins, epidermal growth factor receptor pathway substrate 8 and actin-related protein 3, were mislocalized and down-regulated at the actin-rich anchoring junction between germ and Sertoli cells (apical ectoplasmic specialization) after adjudin treatment. Nonreceptor tyrosine kinase p-FAK-Tyr(407), known to regulate F-actin nucleation via actin-related protein 3, was also mislocalized and down-regulated at the apical ectoplasmic specialization, corroborating the observation of actin cytoskeleton disruption. Additionally, spatiotemporal expression of MT regulatory protein end-binding protein 1, shown to be involved in MT-actin cross talk herein, was also disrupted after adjudin treatment. In summary, spermatid/phagosome transport across the epithelium during spermatogenesis requires the coordination between actin- and MT-based cytoskeletons.

Unraveling the molecular targets pertinent to junction restructuring events during spermatogenesis using the Adjudin-induced germ cell depletion model

During spermatogenesis, extensive restructuring takes place at the Sertoli-Sertoli and Sertoli-germ cell interface, which is regulated via intriguing interactions among cytokines, proteases, protease inhibitors, kinases, phosphatases, and transcription factors. This in turn determines the steady-state levels of integral membrane proteins at the cell junctions. We sought to further expand these observations using the Adjudin model. Adjudin is a potential male contraceptive that targets Sertoli-germ cell adhesion, causing exfoliation of spermatids and spermatocytes, but not spermatogonia, from the seminiferous epithelium. This model thus provides the means to identify crucial regulatory molecules and signaling pathways pertinent to junction restructuring events during spermatogenesis. In this study, genome-wide expression profiling of rat testes after treatment with Adjudin at the time of extensive junction restructuring was performed. Differentially regulated genes, such as cytokines, proteases, protease inhibitors, cell junction-associated proteins, and transcription factors pertinent to junction restructuring were identified. These data were consistent with earlier findings; however, much new information was obtained which has been deposited at the Gene Expression Omnibus data repository website: http://www.ncbi.nih.gov/geo/ with Accession number: GSE5131. The primary signaling events pertinent to junction restructuring in the testis induced by Adjudin were also delineated using bioinformatics. These findings were also consistent with recently published reports. The identified molecular signatures or targets pertinent to junction dynamics in the testis as reported herein, many of which have not been investigated, thus offer a framework upon which the regulation of junction restructuring events at the Sertoli-Sertoli and Sertoli-germ cell interface pertinent to spermatogenesis can be further studied.

Cellular localization of sphingomyelin synthase 2 in the seminiferous epithelium of adult rat testes.

Sphingomyelin synthase 2 (SMS2) is an enzyme that catalyzes the conversion of phosphatidylcholine and ceramide to sphingomyelin and diacylglycerol, and it is crucial to cellular lipid metabolism. Using the technique of subtraction hybridization, we have isolated a full-length cDNA encoding SMS2 from rat testes, which shared 93 and 87% identity at the nucleotide level with SMS2 in mice and humans respectively. A specific polyclonal antibody was prepared against a 20 amino acid peptide of NH(2)-FSWPLSWPPGCFKSSCKKYS-COOH near the C-terminus of SMS2. Studies by RT-PCR and immunoblotting have shown that the expression of SMS2 was limited to late round spermatids and elongating spermatids, but it was not detected in late elongate spermatids and Sertoli cells. Furthermore, SMS2 was shown to associate with the developing acrosome beginning in late round spermatid through elongating spermatids (but not late elongate spermatids) and the cell membrane in studies using fluorescent microscopy and immunohistochemistry. These data were further confirmed by studies using immunogold electron microscopy. The expression of SMS2 in the seminiferous epithelium is stage-specific with its highest expression detected in the acrosome region in late round spermatids from stages VIII-IX, and also in the acrosome in elongating spermatids with diminished intensity in stages X-V; however, it was not found in the acrosome in elongate spermatids in stages VI-VIII. Collectively, these results suggest that SMS2 may play a crucial role in the lipid metabolism in acrosome formation and the plasma membrane restructuring from late round spermatids to early elongating spermatids.

Connexin 43 reboots meiosis and reseals blood-testis barrier following toxicant-mediated aspermatogenesis and barrier disruption

Earlier studies have shown that rats treated with an acute dose of 1‐(2,4‐dichlorobenzyl)‐1H‐indazole‐3‐carbohydrazide (adjudin, a male contraceptive under development) causes permanent infertility due to irreversible blood‐testis barrier (BTB) disruption even though the population of undifferentiated spermatogonia remains similar to normal rat testes, because spermatogonia fail to differentiate into spermatocytes to enter meiosis. Since other studies have illustrated the significance of connexin 43 (Cx43)‐based gap junction in maintaining the homeostasis of BTB in the rat testis and the phenotypes of Sertoli cell‐conditional Cx43 knockout mice share many of the similarities of the adjudin‐treated rats, we sought to examine if overexpression of Cx43 in these adjudin‐treated rats would reseal the disrupted BTB and reinitiate spermatogenesis. A full‐length Cx43 cloned into mammalian expression vector pCI‐neo was used to transfect testes of adjudin‐treated rats versus empty vector. It was found that overexpression of Cx43 indeed resealed the Sertoli cell tight junction‐permeability barrier based on a functional in vivo assay in tubules displaying signs of meiosis as noted by the presence of round spermatids. Thus, these findings suggest that over‐expression of Cx43 reinitiated spermatogenesis at least through the steps of meiosis to generate round spermatids in testes of rats treated with an acute dose of adjudin that led to aspermatogenesis. It was also noted that the round spermatids underwent eventual degeneration with the formation of multinucleated cells following Cx43 overexpression due to the failure of spermiogenesis because no elongating/elongated spermatids were detected in any of the tubules examined. The mechanism by which overexpression of Cx43 reboots meiosis and rescues BTB function was also examined. In summary, overexpression of Cx43 in the testis with aspermatogenesis reboots meiosis and reseals toxicant‐induced BTB disruption, even though it fails to support round spermatids to enter spermiogenesis.—Li, N., Mruk, D. D., Mok, K.‐W., Li, M. W. M., Wong, C. K. C., Lee, W. M., Han, D., Silvestrini, B., Cheng, C. Y. Connexin 43 reboots meiosis and reseals blood‐testis barrier following toxicant‐mediated aspermatogenesis and barrier disruption. FASEB J. 30, 1436–1452 (2016). www.fasebj.org

Formin 1 Regulates Microtubule and F-Actin Organization to Support Spermatid Transport During Spermatogenesis in the Rat Testis

Formin 1 confers actin nucleation by generating long stretches of actin microfilaments to support cell movement, cell shape, and intracellular protein trafficking. Formin 1 is likely involved in microtubule (MT) dynamics due to the presence of a MT binding domain near its N terminus. Here, formin 1 was shown to structurally interact with α-tubulin, the building block of MT, and also end-binding protein 1 (a MT plus [+]-end-binding protein that stabilizes MT) in the testis. Knockdown of formin 1 in Sertoli cells with an established tight junction barrier was found to induce down-regulation of detyrosinated MT (a stabilized form of MT), and disorganization of MTs, in which MTs were retracted from the cell cortical zone, mediated through a loss of MT polymerization and down-regulation of Akt1/2 signaling kinase. An efficient knockdown of formin 1 in the testis reduced the number of track-like structures conferred by MTs and F-actin considerably, causing defects in spermatid and phagosome transport across the seminiferous epithelium. In summary, formin1 maintains MT and F-actin track-like structures to support spermatid and phagosome transport across the seminiferous epithelium during spermatogenesis.