Role of microtubule +TIPs and -TIPs in spermatogenesis—Insights from studies of toxicant models

During spermatogenesis, preleptotene spermatocytes and haploid spermatids, lacking lamellipodia and filopodia to initiate cell movement per se, but rely on Sertoli cells for transport across the blood-testis barrier (BTB) and the adluminal compartment of the seminiferous epithelium, respectively. Tracks provided by microtubules (MTs) that lay across the epithelium are essential to support germ cell and other cargo transports, but the mechanism(s) remain elusive. Studies have provided insightful information through the use of toxicant models. Herein, we summarize findings based on studies of the microtubule plus (+)-end tracking proteins (+TIPs) and the microtubule minus (-)-end targeting proteins (-TIPs), at the corresponding plus (+)-end and minus (-)-end of the polarized MTs in rat testes. We also provide a model by which + TIPs and -TIPs that work in concert with microtubule-associated proteins (MAPs; e.g., MAP-1a), MARKs (microtubule affinity-regulating kinases), and microtubule-specific motor proteins (e.g., dynein 1) to support germ cell and cargo transports. This thus provides a framework to design experiments for future studies

Emerging role for SRC family kinases in junction dynamics during spermatogenesis

SRC family kinases (SFKs) are known regulators of multiple cellular events, including cell movement, differentiation, proliferation, survival, and apoptosis. SFKs are expressed virtually by all mammalian cells. They are non-receptor protein kinases that phosphorylate a variety of cellular proteins on tyrosine, leading to activation of protein targets in response to environmental stimuli. Among SFKs, SRC, YES, and FYN are the ubiquitously expressed and best studied members. In fact, SRC, the prototypical SFK, was the first tyrosine kinase identified in mammalian cells. Studies have shown that SFKs are regulators of cell junctions, and function in endocytosis and membrane trafficking to regulate junction restructuring events. Herein, we briefly summarize recent findings in the field regarding the role of SFKs in the testis in regulating spermatogenesis, particularly in Sertoli-Sertoli and Sertoli-germ cell adhesion. While it is almost 50 years since the identification of the oncogene v-Src encoded by Rous sarcoma transforming virus, the understanding of SFK involvement during spermatogenesis in the testis remains far behind that in other epithelia and tissues. The goal of this review aims to bridge this gap

Regulation of BTB dynamics in spermatogenesis—Insights from the adjudin model

During spermatogenesis, cell organelles, and germ cells, most notably haploid spermatids, are transported across the seminiferous epithelium so that fully developed spermatids line-up at the edge of the tubule lumen to undergo spermiation at stage VIII of the cycle. Studies have suggested that the microtubule (MT)-based cytoskeleton is necessary to support these cellular events. However, the regulatory molecule(s) and underlying mechanism(s) remain poorly understood. Herein, we sought to better understand this event by using an adjudin-based animal model. Adult rats were treated with adjudin at low-dose (10 mg/kg b.w.) which by itself had no notable effects on spermatogenesis. Rats were also treated with low-dose adjudin combined with overexpression of 2 endogenously produced blood-testis barrier (BTB) modifiers, namely rpS6 (ribosomal protein S6, the downstream signaling protein of mammalian target of rapamycin complex 1 [mTORC1]) and F5-peptide (a biological active peptide released from laminin-γ3 chain at the Sertoli-spermatid interface) versus the 2 BTB modifiers alone. Overexpression of these 2 BTB modifiers in the testis was shown to enhance delivery of adjudin to the testis, effectively inducing disruptive changes in MT cytoskeletons, causing truncation of MT conferred tracks that led to their collapse across the epithelium. The net result was massive germ cell exfoliation in the tubules, disrupting germ cell transport and cell adhesion across the seminiferous epithelium that led to aspermatogenesis. These changes were the result of disruptive spatial expression of several MT-based regulatory proteins. In summary, MT cytoskeleton supported by the network of MT regulatory proteins is crucial to maintain spermatogenesis

Endogenously produced LG3/4/5-peptide protects testes against toxicant-induced injury

Laminin-α2 chain is one of the major constituent proteins of the basement membrane in the mammalian testis. The laminin-type globular (LG) domains of LG3, 4 and 5 (LG3/4/5, an 80 kDa fragment) can be cleaved from laminin-α2 chain at the C-terminus via the action of matrix metalloproteinase 9 (MMP-9). This LG3/4/5 is a biologically active fragment, capable of modulating the Sertoli cell blood–testis barrier (BTB) function by tightening the barrier both in vitro and in vivo. Overexpression of LG3/4/5 cloned into a mammalian expression vector pCI-neo in Sertoli cells in a Sertoli cell in vitro model with a functional BTB also protected Sertoli cells from cadmium chloride (CdCl2, an environmental toxicant) mediated cell injury. Importantly, overexpression of LG3/4/5 in the testis in vivo was found to block or rescue cadmium-induced BTB disruption and testis injury. LG3/4/5 was found to exert its BTB and spermatogenesis promoting effects through corrective spatiotemporal expression of actin- and MT-based regulatory proteins by maintaining the cytoskeletons in the testis, illustrating the therapeutic implication of this novel bioactive fragment

A local regulatory network in the testis mediated by laminin and collagen fragments that supports spermatogenesis

It is almost five decades since the discovery of the hypothalamic-pituitary-testicular axis. This refers to the hormonal axis that connects the hypothalamus, pituitary gland and testes, which in turn, regulates the production of spermatozoa through spermatogenesis in the seminiferous tubules, and testosterone through steroidogenesis by Leydig cells in the interstitium, of the testes. Emerging evidence has demonstrated the presence of a regulatory network across the seminiferous epithelium utilizing bioactive molecules produced locally at specific domains of the epithelium. Studies have shown that biologically active fragments are produced from structural laminin and collagen chains in the basement membrane. Additionally, bioactive peptides are also produced locally in non-basement membrane laminin chains at the Sertoli-spermatid interface known as apical ectoplasmic specialization (apical ES, a testis-specific actin-based anchoring junction type). These bioactive peptides are derived from structural laminins and/or collagens at the corresponding sites through proteolytic cleavage by matrix metalloproteinases (MMPs). They in turn serve as autocrine and/or paracrine factors to modulate and coordinate cellular events across the epithelium by linking the apical and basal compartments, the apical and basal ES, the blood-testis barrier (BTB), and the basement membrane of the tunica propria. The cellular events supported by these bioactive peptides/fragments include the release of spermatozoa at spermiation, remodeling of the immunological barrier to facilitate the transport of preleptotene spermatocytes across the BTB, and the transport of haploid spermatids across the epithelium to support spermiogenesis. In this review, we critically evaluate these findings. Our goal is to identify research areas that deserve attentions in future years. The proposed research also provides the much needed understanding on the biology of spermatogenesis supported by a local network of regulatory biomolecules

Planar cell polarity protein Dishevelled 3 (Dvl3) regulates ectoplasmic specialization (ES) dynamics in the testis through changes in cytoskeletal organization

In the mammalian testes, such as in rats, the directional alignment of polarized elongating/elongated spermatids, in particular step 17–19 spermatids, across the plane of seminiferous epithelium resembles planar cell polarity (PCP) found in hair cells of the cochlea. It is obvious that spermatid PCP is necessary to support the simultaneous development of maximal number of elongating/elongated spermatids to sustain the daily production of \u3e 50 million sperm per adult rat. Studies have shown that the testis indeed expresses multiple PCP proteins necessary to support spermatid PCP. Herein, using physiological and biochemical assays, and morphological analysis, and with the technique of RNA interference (RNAi) to knockdown PCP protein Dishevelled (Dvl) 1 (Dvl1), Dvl2, Dvl3, or Dvl1/2/3, Dvl proteins, in particular Dvl3, it was shown that Dvl3 played a crucial role of support Sertoli cell tight junction (TJ)-permeability barrier function through changes in the organization of actin- and microtubule (MT)-based cytoskeletons. More important, an in vivo knockdown of Dvl1/2/3 in the testis, defects of spermatid polarity were remarkably noted across the seminiferous epithelium, concomitant with defects of spermatid adhesion and spermatid transport, leading to considerably defects in spermatogenesis. More important, Dvl1/2/3 triple knockdown in the testis also impeded the organization of actin- and MT-based cytoskeletons owing to disruptive spatial expression of actin- and MT-regulatory proteins. In summary, PCP Dishevelled proteins, in particular, Dvl3 is a regulator of Sertoli cell blood–testis barrier (BTB) and also spermatid PCP function through its effects on the actin- and MT-based cytoskeletons in Sertoli cells

Mechanistic insights into PFOS-mediated Sertoli cell injury

Rat and human primary Sertoli cells cultured in vitro are useful models to study toxicant-induced Sertoli cell injury. Using these two in vitro models, it was shown that environmental toxicants (e.g., PFOS) induced Sertoli cell injury by perturbing the Sertoli cell tight junction-permeability barrier function, localization of Sertoli adhesion proteins, organization of Sertoli cell cytoskeletons, and/or kinetics of polymerization or bundling activity of actin- and/or MT-based cytoskeletons. PFOS-induced Sertoli cell injury is mediated through p-FAK-Tyr407-based and mTORC1/rpS6/Akt1/2-based signaling complexes and/or pathways. PFOS-induced Sertoli cell injury can be blocked by manipulating either p-FAK-Tyr407 or mTORC1/rpS6/Akt1/2 signaling proteins, illustrating a potential therapeutic approach to manage toxicant-induced male reproductive dysfunction. Studies have proven that per- and polyfluoroalkyl substances are harmful to humans, most notably perfluorooctanesulfonate (PFOS). PFOS induces rapid disorganization of actin- and microtubule (MT)-based cytoskeletons in primary cultures of rodent and human Sertoli cells, perturbing Sertoli cell gap junction communication, thereby prohibiting Sertoli cells from maintaining cellular homeostasis in the seminiferous epithelium to support spermatogenesis. PFOS perturbs several signaling proteins/pathways, such as FAK and mTORC1/rpS6/Akt1/2. The use of either an activator of Akt1/2 or overexpression of a phosphomimetic (and constitutively active) mutant of FAK or connexin 43 has demonstrated that such treatment blocks PFOS-induced Sertoli cell injury by preventing actin- and MT-based cytoskeletal disorganization. These findings thus illustrate an approach to manage PFOS-induced reproductive dysfunction

Microtubule cytoskeleton and spermatogenesis—Lesson from studies of toxicant models

Studies have shown that mammalian testes, in particular the Sertoli cells, are highly susceptible to exposure of environmental toxicants, such as cadmium, PFOS (perfluorooctanesulfonate), phthalates, 2,5-hexanedione and bisphenol A. However, important studies conducted by reproductive toxicologists and/or biologists in the past have been treated as toxicology reports. Yet many of these studies provided important mechanistic insights on the toxicant-induced testis injury and reproductive dysfunction, relevant to the biology of the testis and spermatogenesis. Furthermore, recent studies have shown that findings obtained from toxicant models are exceedingly helpful tools to unravel the biology of testis function in particular spermatogenesis, including specific cellular events associated with spermatid transport to support spermiogenesis and spermiation. In this review, we critically evaluate some recent data, focusing primarily on the molecular structure and role of microtubules in cellular function, illustrating the importance of toxicant models to unravel the biology of microtubule cytoskeleton in supporting spermatogenesis, well beyond toxicity studies. These findings have opened up some potential areas of research which should be carefully addressed in the years to come

Investigation of Photoelastic Property and Stress Analysis for Optical Polyimide Membrane through Stress Birefringence Method

Optical polyimide (PI) membranes have been increasingly attractive in optoelectronic substrate and optical element material applications. Controlled stress distribution is very important to optical PI membrane-based optics. However, nondestructive absolute stress measurement inside optical PI membranes remains challenging. In this letter, we adopted the stress birefringence method to experimentally investigate the correlation between stress and retardation in uniaxially, biaxially, and circularly stretched PI membranes. The calculated value of the photoelastic coefficient was found to be around 400 nm/Mpa·cm. A theoretical model was established where the retardation angle is the negative arctan of the principal stress ratio in the biaxially stretched membrane. We also found that the average retardation angle is an important parameter for evaluating the uniformity of stretching force in the circularly stretched membrane. This work provides a better understanding of the stress birefringence measurement of membrane materials

CAMSAP2 is a microtubule minus-end targeting protein (-TIP) that regulates BTB dynamics through cytoskeletal organization

During spermatogenesis, microtubule (MT) cytoskeleton in Sertoli cells confers blood-testis barrier (BTB) function, but the regulator(s) and the mechanism(s) that modulate MT dynamics remains unexplored. Herein, we examined the role of CAMSAP2 (calmodulin-regulated spectrin-associated protein 2, a member of the CAMSAP/Patronin protein family), also a minis (-) end-targeting protein (-TIP) that binds to the minus-end (i.e., slow growing end) of polarized MTs involved in determining MT length, in Sertoli cell function. CAMSAP2 was found to localize at discrete sites across the Sertoli cell cytosol, different from EB1 (end binding protein 1, a microtubule plus (+)-end tracking protein, +TIP, that binds to the plus (+)-end of MTs), and co-localized with MTs. CAMSAP2 displayed stage-specific expression pattern, appearing as track-like structures across the seminiferous epithelium in adult rat testes that laid perpendicular to the basement membrane. CAMSAP2 knockdown by RNAi was found to promote Sertoli cell tight junction (TJ)-barrier function, illustrating its role in TJ remodeling under physiological conditions. To further examine the regulatory role of CAMSAP2 in BTB dynamics, we used a PFOS-induced Sertoli cell injury model for studies. CAMSAP2 knockdown blocked PFOS-induced Sertoli cell injury by promoting proper distribution of BTB-associated proteins at the cell-cell interface. This effect was mediated by the ability of CAMSAP2 knockdown to block PFOS-induced disruptive organization of MTs, but also F-actin, across cell cytosol through changes in cellular distribution/localization of MT and actin regulatory proteins. In summary, CAMSAP2 is a regulator of MT and actin dynamics in Sertoli cells to support BTB dynamics and spermatogenesis