Spermatogenesis is the process by which a single spermatogonium develops into 256 spermatozoa, one of which will fertilize the ovum. Since the 1950s when the stages of the epithelial cycle were first described, reproductive biologists have been in pursuit of one question: How can a spermatogonium traverse the epithelium, while at the same time differentiating into elongate spermatids that remain attached to the Sertoli cell throughout their development? Although it was generally agreed upon that junction restructuring was involved, at that time the types of junctions present in the testis were not even discerned. Today, it is known that tight, anchoring, and gap junctions are found in the testis. The testis also has two unique anchoring junction types, the ectoplasmic specialization and tubulobulbar complex. However, attention has recently shifted on identifying the regulatory molecules that "open" and "close" junctions, because this information will be useful in elucidating the mechanism of germ cell movement. For instance, cytokines have been shown to induce Sertoli cell tight junction disassembly by shutting down the production of tight junction proteins. Other factors such as proteases, protease inhibitors, GTPases, kinases, and phosphatases also come into play. In this review, we focus on this cellular phenomenon, recapping recent developments in the field.

Sertoli-Sertoli and Sertoli-Germ Cell Interactions and Their Significance in Germ Cell Movement in the Seminiferous Epithelium during Spermatogenesis

The blood-testis barrier (BTB) is one of the tightest blood-tissue barriers in the mammalian body. It divides the seminiferous epithelium into the basal and the apical (adluminal) compartments. Meiosis I and II, spermiogenesis, and spermiation all take place in a specialized microenvironment behind the BTB in the apical compartment, but spermatogonial renewal and differentiation and cell cycle progression up to the preleptotene spermatocyte stage take place outside of the BTB in the basal compartment of the epithelium. However, the BTB is not a static ultrastructure. Instead, it undergoes extensive restructuring during the seminiferous epithelial cycle of spermatogenesis at stage VIII to allow the transit of preleptotene spermatocytes at the BTB. Yet the immunological barrier conferred by the BTB cannot be compromised, even transiently, during the epithelial cycle to avoid the production of antibodies against meiotic and postmeiotic germ cells. Studies have demonstrated that some unlikely partners, namely adhesion protein complexes (e.g., occludin-ZO-1, N-cadherin-β-catenin, claudin-5-ZO-1), steroids (e.g., testosterone, estradiol-17β), nonreceptor protein kinases (e.g., focal adhesion kinase, c-Src, c-Yes), polarity proteins (e.g., PAR6, Cdc42, 14-3-3), endocytic vesicle proteins (e.g., clathrin, caveolin, dynamin 2), and actin regulatory proteins (e.g., Eps8, Arp2/3 complex), are working together, apparently under the overall influence of cytokines (e.g., transforming growth factor-β3, tumor necrosis factor-α, interleukin-1α). In short, a “new” BTB is created behind spermatocytes in transit while the “old” BTB above transiting cells undergoes timely degeneration, so that the immunological barrier can be maintained while spermatocytes are traversing the BTB. We also discuss recent findings regarding the molecular mechanisms by which environmental toxicants (e.g., cadmium, bisphenol A) induce testicular injury via their initial actions at the BTB to elicit subsequent damage to germ-cell adhesion, thereby leading to germ-cell loss, reduced sperm count, and male infertility or subfertility. Moreover, we also critically evaluate findings in the field regarding studies on drug transporters in the testis and discuss how these influx and efflux pumps regulate the entry of potential nonhormonal male contraceptives to the apical compartment to exert their effects. Collectively, these findings illustrate multiple potential targets are present at the BTB for innovative contraceptive development and for better delivery of drugs to alleviate toxicant-induced reproductive dysfunction in men.

The Blood-Testis Barrier and Its Implications for Male Contraception

Spermatogenesis is an intriguing but complicated biological process. However, many studies since the 1960s have focused either on the hormonal events of the hypothalamus-pituitary-testicular axis or morphological events that take place in the seminiferous epithelium. Recent advances in biochemistry, cell biology, and molecular biology have shifted attention to understanding some of the key events that regulate spermatogenesis, such as germ cell apoptosis, cell cycle regulation, Sertoli-germ cell communication, and junction dynamics. In this review, we discuss the physiology and biology of junction dynamics in the testis, in particular how these events affect interactions of Sertoli and germ cells in the seminiferous epithelium behind the blood-testis barrier. We also discuss how these events regulate the opening and closing of the blood-testis barrier to permit the timely passage of preleptotene and leptotene spermatocytes across the blood-testis barrier. This is physiologically important since developing germ cells must translocate across the blood-testis barrier as well as traverse the seminiferous epithelium during their development. We also discuss several available in vitro and in vivo models that can be used to study Sertoli-germ cell anchoring junctions and Sertoli-Sertoli tight junctions. An in-depth survey in this subject has also identified several potential targets to be tackled to perturb spermatogenesis, which will likely lead to the development of novel male contraceptives.

https://www.physiology.org/doi/pdf/10.1152/physrev.00009.2002

Cell Junction Dynamics in the Testis: Sertoli-Germ Cell Interactions and Male Contraceptive Development

A nuclear framework structure termed the nuclear matrix has been isolated and characterized. This matrix forms the major residual structure of isolated nuclei and consists largely of protein with smaller amounts of RNA, DNA, carbohydrate, and phospholipid. The nuclear matrix can be further resolved by combined treatment with DNase and RNase. The remaining nuclear protein structure, after extraction of 90 percent of the nuclear protein, 99.9 percent of the DNA, and 98 percent of the RNA and phospholipid, is termed the nuclear protein matrix. Electron microscopy of this final nuclear protein matrix reveals an interior framework structure composed of residual nucleolar structures associated with a granular and fibrous internal matrix structure. The internal matrix framework is derived from the interchromatinic structures of the nucleus, and is connected to a surrounding residual nuclear envelope layer containing residual nuclear pore complex structures. Sodium dodecyl sulfate-acrylamide gel electrophoresis of the nuclear matrix proteins demonstrates three major polypeptide fractions, P-1, P-2, and P-3, with average molecular weights of approximately 69,000, 66,000 and 62,000, as well as several minor polypeptides which migrate at approximately 50,000 and at higher molecular weights (>100,000). Polypeptides with molecular weights identical to those of P-1, P-2 and P-3 are also components of isolated nuclear envelopes and nucleoli, whereas isolated chromatin contains no detectable matrix polypeptides. This suggests that the major matrix polypeptides are localized in specific structural regions of the nucleus, i.e., nuclear envelope, nucleoli, and interchromatinic structures. The presence of cytochrome oxidase activity in the isolated nuclear matrix indicates that at least some integral proteins of the nuclear membrane are associated with the matrix.

Nuclear matrix: Isolation and characterization of a framework structure from rat liver nuclei

This review presents an overview of the highlights of major concepts involving the anatomical routes for the transport of macromolecules and the transmigration of cellular elements across the blood-brain barrier (BBB) during inflammation The particular focus will include inflammatory leukocytes, neoplastic cells and pathogenic microorganisms including specific types of viruses, bacteria and yeasts The experimental animal models presented here have been employed successfully by the authors in several independent experiments during the past twenty-five years for investigations of pathologic alterations of the BBB after a variety of experimentally induced injuries and inflammatory conditions in mammalian and non-mammalian animal species The initial descriptions of endothelial cell (EC) vesicles or caveolae serving as mini-transporters of fluid substances essentially served as a springboard for many subsequent discoveries during the past half century related to mechanisms of uptake of materials into ECs and whether or not pinocytosis is related to the transport of these materials across EC barriers under normal physiologic conditions and after tissue injury In the mid-1970's, the authors of this review independently applied morphologic techniques (transmission electron microscopy-TEM), in conjunction with the plant protein tracer horseradish peroxidase (HRP) to investigate macromolecular transport structures that increased after the brain and spinal cord had been subjected to a variety of injuries Based on morphologic evidence from these studies of BBB injury, the authors elaborated a unique EC system of modified caveolae that purportedly fused together forming transendothelial cell channels, and later similar EC profiles defined as vesiculo-canalicular or vesiculo-tubular structures (VTS, Lossinsky, et al, 1999) These EC structures were observed in association with increased BBB permeability of tracers including exogenously injected HRP, normally excluded from the intercellular milieu of the CNS Subsequent studies of non-BBB-type tumor ECs determined that the EC VTS and other vesicular structures were defined by others as vesiculo-vacuolar organelles (VVOs, Kohn et al, 1992; Dvorak et al, 1996) Collectively, these structures appear to represent a type of anatomical gateway to the CNS likely serving as conduits However, these CNS conduits become patent only in damaged ECs for the passage of macromolecules, and purportedly for inflammatory and neoplastic cells as well (Lossinsky et al, 1999) In this review, we focus attention on the similarities and differences between caveolae, fused racemic vesicular bundles, endothelial tubules and channels (VTS and the VVOs) that are manifest in normal, non-BBB-type blood vessels, and in the BBB after injury This review will present evidence that the previous studies by the authors and other researchers established a framework for subsequent transmission (TEM), scanning (SEM) and high-voltage electron microscopic (HVEM) investigations concerning ultrastructural, ultracytochemical and immunoultra-structural alterations of the cerebral ECs and the mechanisms of the BBB transport that occurs after CNS injury This review is not intended to include all of the many observations that might be included in a general historical overview of the development of the EC channel hypothesis, but it will discuss several of the major contributions We have attempted to present some of the structural evidence that supports our early contributions and those made by other investigators by highlighting major features of these EC structures that are manifest in the injured BBB We have focused on currently established concepts and principles related to mechanisms for the transendothelial transport of macromolecules after CNS injury and also offer a critical appraisal of some of this literature Finally, we describe more recent concepts of transBBB avenues for viruses, including HIV-1, bacterial and mycotic organisms, as well as inflammatory and neoplastic cell adhesion and migration across the injured mammalian BBB Data from studies of EC-related adhesion molecules, both from the literature and from the author's experimental results and observations made in other laboratories, as well as from personal communications underscore the importance of the adhesion molecules in facilitating the movement of leukocytic, neoplastic cell and human pathogens across the BBB during inflammatory and neoplastic events Exciting, ongoing clinical trials are addressing possible therapeutic intervention in neuroinflammatory diseases, including multiple sclerosis, by blocking certain glycoprotein adhesion molecules before cells have the ability to adhere to the ECs and migrate across the BBB Approaches whereby inflammation may be reduced or arrested using anti-adhesion molecules, by restructuring EC cytoskeletal, filamentous proteins, as well as remodeling cholesterol components of the modified VTS are discussed in the context of developing future therapies for BBB injury and inflammation Understanding new concepts about the mechanism(s) by which inflammatory cells and a variety of pathogenic microorganisms are transported across the BBB can be expected to advance our understanding of fundamental disease processes Taken together, the literature and the author's experiences during the past quarter of a century, will hopefully provide new clues related to the mechanisms of transendothelial cell adhesion and emigration across the injured BBB, issues that have been receiving considerable attention in the clinical arena Learning how to chemically modulate the opening and/or closure of EC VTS and VVO structural pathways, or junctional complexes prior to cellular or microorganism adhesion and breaching the BBB presents challenging new questions in modern medicine Future studies will be critically important for the development of therapeutic intervention in several human afflictions including traumatic brain and spinal cord injuries, stroke, cancer, multiple sclerosis and conditions where the immune system may be compromised including HIV infection, infantile and adult meningitis

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Structural pathways for macromolecular and cellular transport across the blood-brain barrier during inflammatory conditions. Review.

Intratesticular injections of glycerol have been shown to result in a marked and prolonged reduction of spermatogenesis, accompanied by increased permeability of the blood-testis barrier. Because the permeability of the blood-testis barrier is regulated by Sertoli cell tight junctions, and tight junction organization is regulated by the cytoskeleton, we undertook to examine the effects of glycerol treatment on cytoskeletal actin microfilaments and microtubules, and on the tight junction protein, occludin, in Sertoli cells. Adult rats received a single intratesticular injection of either saline (controls) or a 10% glycerol solution. At 24 hours and 7, 15, and 21 days after injection, testes were collected and prepared for routine histology, cryosectioning, or whole seminiferous tubule immunohistochemical staining; and the preparations were viewed by light and confocal microscopy. In saline-injected testes, Sertoli cells had a cytoskeletal and junctional organization that resembled that of normal testes. F-actin microfilaments, located in the basal region, were arranged in regular bundles or chords that circumscribed the perimeter of each Sertoli cell at the level of the tight junction. Occludin colocalized with tight junction-associated actin filament distribution and microtubules formed a geometric array associated with spermatogenic cells. In contrast, in glycerol-treated Sertoli cells, microfilament and microtubule organization and occludin distribution were partially or completely disrupted. From these results we conclude that glycerol treatment either directly or indirectly disrupts tight junction-associated F-actin and occludin and tubulin organization in rat Sertoli cells. Perturbation of the tight junction-associated proteins could explain the increase in permeability of the blood-testis barrier observed after glycerol treatment. Impaired spermatogenesis following glycerol treatment is likely a consequence of a leaky blood testis barrier and disrupted Sertoli cell cytoskeleton. Glycerol injections may serve as a useful tool in studying the relationship between cytoskeletal organization and the stabilization of Sertoli-Sertoli cell junctions.

Glycerol disrupts tight junction-associated actin microfilaments, occludin, and microtubules in Sertoli cells.

Perinuclear actin shells have been reported in a variety of organisms. The shells have been identified by staining perinuclear material with fluorescently-labelled phalloidin, but have not been localized to a specific subcellular compartment at the ultrastructural level. We show here that the shells of 3T3 cells lie in the peripheral nuclear matrix. Nuclear shells and matrix actin in other parts of the nucleus are not usually detected by immunohistochemical staining because they are inaccessible to antibodies or to phalloidin. Immunohistochemical detection of nuclear actin is only possible during its deposition at the end of mitosis, or in interphase nuclei that have been extracted with detergent, digested with nucleases and washed with high salt buffers.

Peripheral nuclear matrix actin forms perinuclear shells

Intermediate filaments and microtubules are known to be involved in establishing and maintaining nuclear shape. F-actin may also be involved in determining nuclear shape, since we have found it associated with reforming nuclei very briefly after cell division. We stained cells from vertebrate tissue cultures (3T3 and NRK-49F) and epidermal cells from an insect with rhodamine-phalloidin and Hoechst #33342 to localize F-actin in relation to the nucleus. We found that F-actin forms shells only around nuclei during reorganization in late mitosis and early interphase. We suggest that perinuclear F-actin shells may be generally present in eukaryotes, but that they are easily missed because of their delicacy and transience.

F-actin forms transient perinuclear shells at the mitosis-interphase transition.

Since the extracellular matrix (ECM) has a role in regulating cell proliferation it has been hypothesized that unregulated growth of transformed cells results from an inability of cells to interpret the constraints on growth as dictated by the ECM. The most likely candidate to communicate the restraint on growth to the nucleus is the cytoskeleton because it is the only cytoplasmic structure to physically connect the nucleus and plasma membrane. The purpose of this study was to determine whether the cytoskeleton, specifically the microfilaments (MF), of the C6-glioma cell line prossesses the ability to respond to changes in the ECM. The C6-glioma cell line was chosen as a model because it exhibits the characteristics of a tumor cell, both in vivo and in vitro. In this study cells were grown on bare glass, Matrigel, laminin and type IV collagen prior to staining with phalloidin tetramethylrhodamine B isothiocyanate or antibodies specific to vinculin to visualize MF and adhesion plaques, respectively. On the basis of MF orientation, network complexity and location of adhesion plaques, this study reports that the cytoskeletal arrangements of C6 cells grown on various substrates are distinctly different. Each distinct organization pattern may reflect a change of cell behavior promoted by the different culture conditions. The significance of this study is that it demonstrates that the process of transformation in C6-glioma cells has not interfered with the cells ability to recognize, interpret and adapt to changes in the ECM.

Bryan H. Clubb

Papers

Glycerol disrupts tight junction-associated actin microfilaments, occludin, and microtubules in Sertoli cells.

Peripheral nuclear matrix actin forms perinuclear shells

F-actin forms transient perinuclear shells at the mitosis-interphase transition.

Extracellular matrix regulates microfilament and vinculin organization in C6-glioma cells