Mary M Wohlford Laboratory For Male Contraceptive Research

New York City, NY, United States

Mary M Wohlford Laboratory For Male Contraceptive Research

New York City, NY, United States
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Qian X.,Mary M Wohlford Laboratory For Male Contraceptive Research | Qian X.,Peking Union Medical College | Mruk D.D.,Mary M Wohlford Laboratory For Male Contraceptive Research | Wong E.W.P.,Mary M Wohlford Laboratory For Male Contraceptive Research | And 2 more authors.
Endocrinology | Year: 2013

In rat testes, the ectoplasmic specialization (ES) at the Sertoli-Sertoli and Sertoli-spermatid interface known as the basal ES at the blood-testis barrier and the apical ES in the adluminal compartment, respectively, is a testis-specific adherens junction. The remarkable ultrastructural feature of the ES is the actin filament bundles that sandwiched in between the cisternae of endoplasmic reticulum and apposing plasma membranes. Although these actin filament bundles undergo extensive reorganization to switch between their bundled and debundled state to facilitate bloodtestis barrier restructuring and spermatid adhesion/transport, the regulatory molecules underlying these events remain unknown. Herein we report findings of an actin filament cross-linking/bundling protein palladin, which displayed restrictive spatiotemporal expression at the apical and the basal ES during the epithelial cycle. Palladin structurally interacted and colocalized with Eps8 (epidermal growth factor receptor pathway substrate 8, an actin barbed end capping and bundling protein) and Arp3 (actin related protein 3, which together with Arp2 form the Arp2/3 complex to induce branched actin nucleation, converting bundled actin filaments to an unbundled/branched network), illustrating its role in regulating actin filament bundle dynamics at the ES. A knockdown of palladin in Sertoli cells in vitro with an established tight junction (TJ)-permeability barrier was found to disrupt the TJ function, which was associated with a disorganization of actin filaments that affected protein distribution at the TJ. Its knockdown in vivo also perturbed F-Actin organization that led to a loss of spermatid polarity and adhesion, causing defects in spermatid transport and spermiation. In summary, palladin is an actin filament regulator at the ES. Copyright © 2013 by The Endocrine Society.


Mok K.-W.,Mary M Wohlford Laboratory For Male Contraceptive Research | Mok K.-W.,University of Hong Kong | Mruk D.D.,Mary M Wohlford Laboratory For Male Contraceptive Research | Lee W.M.,University of Hong Kong | Cheng C.Y.,Mary M Wohlford Laboratory For Male Contraceptive Research
FASEB Journal | Year: 2013

In the mammalian testis, coexisting tight junctions (TJs), basal ectoplasmic specializations, and gap junctions (GJs), together with desmosomes near the basement membrane, constitute the blood-testis barrier (BTB). The most notable feature of the BTB, however, is the extensive network of actin filament bundles, which makes it one of the tightest blood-tissue barriers. The BTB undergoes restructuring to facilitate the transit of preleptotene spermatocytes at stage VIII-IX of the epithelial cycle. Thus, the F-actin network at the BTB undergoes cyclic reorganization via a yetto-be explored mechanism. Rictor, the key component of mTORC2 that is known to regulate actin cytoskeleton, was shown to express stage-specifically at the BTB in the seminiferous epithelium. Its expression was down-regulated at the BTB in stage VIII-IX tubules, coinciding with BTB restructuring at these stages. Using an in vivo model, a down-regulation of rictor at the BTB was also detected during adjudin-induced BTB disruption, illustrating rictor expression is positively correlated with the status of the BTB integrity. Indeed, the knockdown of rictor by RNAi was found to perturb the Sertoli cell TJ-barrier function in vitro and the BTB integrity in vivo. This loss of barrier function was accompanied by changes in F-actin organization at the Sertoli cell BTB in vitro and in vivo, associated with a loss of interaction between actin and α-catenin or ZO-1. Rictor knockdown by RNAi was also found to impede Sertoli cell-cell GJ communication, disrupting protein distribution (e.g., occludin, ZO-1) at the BTB, illustrating that rictor is a crucial BTB regulator. © FASEB.


Lie P.P.Y.,Mary M Wohlford Laboratory For Male Contraceptive Research | Mruk D.D.,Mary M Wohlford Laboratory For Male Contraceptive Research | Lee W.M.,University of Hong Kong | Cheng C.Y.,Mary M Wohlford Laboratory For Male Contraceptive Research
Philosophical Transactions of the Royal Society B: Biological Sciences | Year: 2010

Different cellular events occur during spermatogenesis, and these include (i) mitosis for self-renewa of spermatogonia, (ii) differentiation of type A spermatogonia into type B and commitment of type B spermatogonia to develop into preleptotene primary spermatocytes, (iii) transit of preleptotene, leptotene spermatocytes across the blood-testis barrier in coordination with germ cell cycle progression and meiosis, (iv) spermiogenesis and spermiation. These events also associate with extensive changes in cell shape and size, and germ cell movement. The cytoskeleton, which comprises actin, microtubules and intermediate filaments, is believed to function in these cellular events. However, few studies have been conducted by investigators in the past decades to unfold the role of the cytoskeleton during spermatogenesis. This review summarizes recent advances in the field relating to cytoskeletal dynamics in the testis, and highlights areas of research that require additional emphasis so that new approaches for male contraception, as well as therapeutic approaches to alleviate environmental toxicant-induced reproductive dysfunction in men, can possibly be developed. © 2010 The Royal Society.


Xiao X.,Mary M Wohlford Laboratory For Male Contraceptive Research | Mruk D.D.,Mary M Wohlford Laboratory For Male Contraceptive Research | Lee W.M.,University of Hong Kong | Cheng C.Y.,Mary M Wohlford Laboratory For Male Contraceptive Research
International Journal of Biochemistry and Cell Biology | Year: 2011

During spermatogenesis, extensive junction restructuring takes place at the blood-testis barrier (BTB) and the Sertoli cell-spermatid interface known as the apical ectoplasmic specialization (apical ES, a testis-specific adherens junction) in the seminiferous epithelium. However, the mechanism(s) that regulates these critical events in the testis remains unknown. Based on the current concept in the field, changes in the phosphorylation status of integral membrane proteins at these sites can induce alterations in protein endocytosis and recycling, causing junction restructuring. Herein, c-Yes, a non-receptor protein tyrosine kinase, was found to express abundantly at the BTB and apical ES stage-specifically, coinciding with junction restructuring events at these sites during the seminiferous epithelial cycle of spermatogenesis. c-Yes also structurally associated with adhesion proteins at the BTB (e.g., occludin and N-cadherin) and the apical ES (e.g., β1-integrin, laminins β3 and γ3), possibly to regulate phosphorylation status of proteins at these sites. SU6656, a selective c-Yes inhibitor, was shown to perturb the Sertoli cell tight junction-permeability barrier in vitro, which is mediated by changes in the distribution of occludin and N-cadherin at the cell-cell interface, moving from cell surface to cytosol, thereby destabilizing the tight junction-barrier. However, this disruptive effect of SU6656 on the barrier was blocked by testosterone. Furthermore, c-Yes is crucial to maintain the actin filament network in Sertoli cells since a blockade of c-Yes by SU6656 induced actin filament disorganization. In summary, c-Yes regulates BTB and apical ES integrity by maintaining proper distribution of integral membrane proteins and actin filament organization at these sites. © 2011 Elsevier Ltd. All rights reserved.


Su W.,Mary M Wohlford Laboratory For Male Contraceptive Research | Su W.,China Medical University at Heping | Mruk D.D.,Mary M Wohlford Laboratory For Male Contraceptive Research | Cheng C.Y.,Mary M Wohlford Laboratory For Male Contraceptive Research
Critical Reviews in Biochemistry and Molecular Biology | Year: 2013

In the mammalian testis, extensive restructuring takes place across the seminiferous epithelium at the Sertoli-Sertoli and Sertoli-germ cell interface during the epithelial cycle of spermatogenesis, which is important to facilitate changes in the cell shape and morphology of developing germ cells. However, precise communications also take place at the cell junctions to coordinate the discrete events pertinent to spermatogenesis, namely spermatogonial renewal via mitosis, cell cycle progression and meiosis, spermiogenesis and spermiation. It is obvious that these cellular events are intimately related to the underlying actin-based cytoskeleton which is being used by different cell junctions for their attachment. However, little is known on the biology and regulation of this cytoskeleton, in particular its possible involvement in endocytic vesicle-mediated trafficking during spermatogenesis, which in turn affects cell adhesive function and communication at the cell-cell interface. Studies in other epithelia in recent years have shed insightful information on the intimate involvement of actin dynamics and protein trafficking in regulating cell adhesion and communications. The goal of this critical review is to provide an updated assessment of the latest findings in the field on how these complex processes are being regulated during spermatogenesis. We also provide a working model based on the latest findings in the field including our laboratory to provide our thoughts on an apparent complicated subject, which also serves as the framework for investigators in the field. It is obvious that this model will be rapidly updated when more data are available in future years. © 2013 Informa UK Ltd All rights reserved: reproduction in whole or part not permitted.


Mok K.-W.,Mary M Wohlford Laboratory For Male Contraceptive Research | Mruk D.D.,Mary M Wohlford Laboratory For Male Contraceptive Research | Silvestrini B.,University of Rome La Sapienza | Cheng C.Y.,Mary M Wohlford Laboratory For Male Contraceptive Research
Endocrinology | Year: 2012

During spermatogenesis, preleptotene spermatocytes residing near the basement membrane of the seminiferous tubule must traverse the blood-testis barrier (BTB) at stage VIII-IX of the epithelial cycle to continue their development in the adluminal compartment. Unlike other blood-tissue barriers (e.g. the blood-brain barrier) that are created by the endothelial tight junction (TJ) barrier of capillaries, the BTB is created by specialized junctions between Sertoli cells in which TJ coexists with basal ectoplasmic specialization (basal ES, a testis-specific adherens junction). The basal ES is typified by the presence of tightly packed actin filament bundles sandwiched between cisternae of endoplasmic reticulum and the apposing plasma membranes of Sertoli cells. These actin filament bundles also confer unusual adhesive strength to the BTB. Yet the mechanisms by which these filamentous actin (F-actin) networks are regulated from the bundled to the debundled state to facilitate the transit of spermatocytes remain elusive. Herein, we provide evidence that ribosomal protein S6 (rpS6), the downstream signaling molecule of the mammalian target of rapamycin complex 1 (mTORC1) pathway, is a major regulator of F-actin organization and adhesion protein recruitment at the BTB. rpS6 is restrictively and spatiotemporally activated at the BTB during the epithelial cycle. An activation of rpS6 led to a disruption of the Sertoli cell TJ barrier and BTB integrity. Its silencing in vitro or in vivo by using small interfering RNA duplexes or short hairpin RNA was found to promote the Sertoli cell TJ permeability barrier by the recruitment of adhesion proteins (e.g. claudin-11 and occludin) to the BTB. Thus, rpS6 in the mTORC1 pathway regulates BTB restructuring via its effects on the F-actin organization and protein recruitment at the BTB. Copyright © 2012 by The Endocrine Society.


Cheng C.Y.,Mary M Wohlford Laboratory For Male Contraceptive Research | Mruk D.D.,Mary M Wohlford Laboratory For Male Contraceptive Research
Nature Reviews Endocrinology | Year: 2010

Spermiationthe release of mature spermatozoa from Sertoli cells into the seminiferous tubule lumenoccurs by the disruption of an anchoring device known as the apical ectoplasmic specialization (apical ES). At the same time, the blood-testis barrier (BTB) undergoes extensive restructuring to facilitate the transit of preleptotene spermatocytes. While these two cellular events take place at opposite ends of the Sertoli cell epithelium, the events are in fact tightly coordinated, as any disruption in either process will lead to infertility. A local regulatory axis exists between the apical ES and the BTB in which biologically active laminin fragments produced at the apical ES by the action of matrix metalloproteinase 2 can regulate BTB restructuring directly or indirectly via the hemidesmosome. Equally important, polarity proteins play a crucial part in coordinating cellular events within this apical ES-BTB-hemidesmosome axis. Additionally, testosterone and cytokines work in concert to facilitate BTB restructuring, which enables the transit of spermatocytes while maintaining immunological barrier function. Herein, we will discuss this important autocrine-based cellular axis that parallels the hormonal-based hypothalamic-pituitary-testicular axis that regulates spermatogenesis. This local regulatory axis is the emerging target for male contraception. © 2010 Macmillan Publishers Limited. All rights reserved.


Mok K.W.,Mary M Wohlford Laboratory For Male Contraceptive Research | Mruk D.D.,Mary M Wohlford Laboratory For Male Contraceptive Research | Cheng C.Y.,Mary M Wohlford Laboratory For Male Contraceptive Research
International Review of Cell and Molecular Biology | Year: 2013

In mammalian testes, haploid spermatozoa are formed from diploid spermatogonia during spermatogenesis, which is a complicated cellular process. While these cellular events were reported in the 1960s and 1970s, the underlying molecular mechanism(s) that regulates these events remained unexplored until the past ∼10 years. For instance, adhesion proteins were shown to be integrated components at the Sertoli cell-cell interface and/or the Sertoli-spermatid interface in the late 1980s. But only until recently, studies have demonstrated that some of the adhesion proteins serve as the platform for signal transduction that regulates cell adhesion. In this chapter, a brief summary and critical discussion are provided on the latest findings regarding these cell-adhesion proteins in the testis and their relationship to spermatogenesis. Moreover, antagonistic effects of two mammalian target of rapamycin (mTOR) complexes, known as mTORC1 and mTORC2, on cell-adhesion function in the testis are discussed. Finally, a hypothetic model is presented to depict how these two mTOR-signaling complexes having the " yin" and " yang" antagonistic effects on the Sertoli cell tight junction (TJ)-permeability barrier can maintain the blood-testis barrier (BTB) integrity during the epithelial cycle while preleptotene spermatocytes are crossing the BTB. © 2013 Elsevier Inc.


Cheng C.Y.,Mary M Wohlford Laboratory For Male Contraceptive Research | Mruk D.D.,Mary M Wohlford Laboratory For Male Contraceptive Research
Philosophical Transactions of the Royal Society B: Biological Sciences | Year: 2010

The physiological function of spermatogenesis in Caenorhabditis elegans, Drosophila melanogaster and mammals is to produce spermatozoa (In, haploid) that contain only half of the genetic material of spermatogonia (2n, diploid). This half number of chromosomes from a spermatozoon will then be reconstituted to become a diploid cell upon fertilization with an egg, which is also haploid. Thus, genetic information from two parental individuals can be passed onto their offspring. Spermatogenesis takes place in the seminiferous epithelium of the seminiferous tubule, the functional unit of the mammalian testis. In mammals, particularly in rodents, the fascinating morphological changes that occur during spermatogenesis involving cellular differentiation and transformation, mitosis, meiosis, germ cell movement, spermiogenesis and spermiation have been well documented from the 1950s through the 1980s. During this time, however, the regulation of, as well as the biochemical and molecular mechanisms underlying these diverse cellular events occurring throughout spermatogenesis, have remained largely unexplored. In the past two decades, important advancements have been made using new biochemical, cell and molecular biology techniques to understand how different genes, proteins and signalling pathways regulate various aspects of spermatogenesis. These include studies on the differentiation of spermatogonia from gonocytes; regulation of spermatogonial stem cells; regulation of spermatogonial mitosis; regulation of meiosis, spermiogenesis and spermiation; role of hormones (e.g. oestrogens, androgens) in spermatogenesis; transcriptional regulation of spermatogenesis; regulation of apoptosis; cell-cell interactions; and the biology of junction dynamics during spermatogenesis. The impact of environmental toxicants on spermatogenesis has also become an urgent issue in the field in light of declining fertility levels in males. Many of these studies have helped investigators to understand important similarities, differences and evolutionary relationships between C. elegans, D. melanogaster and mammals relating to spermatogenesis. In this Special Issue of the Philosophical Transactions of the Royal Society B: Biological Sciences, we have covered many of these areas, and in this Introduction, we highlight the topic of spermatogenesis by examining its past, present and future. © 2010 The Royal Society.


Su L.,Mary M Wohlford Laboratory For Male Contraceptive Research | Mruk D.D.,Mary M Wohlford Laboratory For Male Contraceptive Research | Lie P.P.Y.,Mary M Wohlford Laboratory For Male Contraceptive Research | Silvestrini B.,University of Rome La Sapienza | Yan Cheng C.,Mary M Wohlford Laboratory For Male Contraceptive Research
Nature Communications | Year: 2012

Cellular events that occur across the seminiferous epithelium in the mammalian testis during spermatogenesis are tightly coordinated by biologically active peptides released from laminin chains. Laminin-γ3 domain IV is released at the apical ectoplasmic specialization during spermiation and mediates restructuring of the blood-testis barrier, which facilitates the transit of preleptotene spermatocytes. Here we determine the biologically active domain in laminin-γ3 domain IV, which we designate F5 peptide, and show that the overexpression of this domain, or the use of a synthetic F5 peptide, in Sertoli cells with an established functional blood-testis barrier reversibly perturbs blood-testis barrier integrity in vitro and in the rat testis in vivo. This effect is mediated via changes in protein distribution at the Sertoli and Sertoli-germ-cell cell interface and by phosphorylation of focal adhesion kinase at Tyr407. The consequences are perturbed organization of actin filaments in Sertoli cells, disruption of the blood-testis barrier and spermatid loss. The impairment of spermatogenesis suggests that this laminin peptide fragment may serve as a contraceptive in male rats. © 2012 Macmillan Publishers Limited. All rights reserved.

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