N-WASP Is Required for Structural Integrity of the Blood-Testis Barrier

During spermatogenesis, the blood-testis barrier (BTB) segregates the adluminal (apical) and basal compartments in the seminiferous epithelium, thereby creating a privileged adluminal environment that allows post-meiotic spermatid development to proceed without interference of the host immune system. A key feature of the BTB is its continuous remodeling within the Sertoli cells, the major somatic component of the seminiferous epithelium. This remodeling is necessary to allow the transport of germ cells towards the seminiferous tubule interior, while maintaining intact barrier properties. Here we demonstrate that the actin nucleation promoting factor Neuronal Wiskott-Aldrich Syndrome Protein (N-WASP) provides an essential function necessary for BTB restructuring, and for maintaining spermatogenesis. Our data suggests that the N-WASP-Arp2/3 actin polymerization machinery generates branched-actin arrays at an advanced stage of BTB remodeling. These arrays are proposed to mediate the restructuring process through endocytic recycling of BTB components. Disruption of N-WASP in Sertoli cells results in major structural abnormalities to the BTB, including mis-localization of critical junctional and cytoskeletal elements, and leads to disruption of barrier function. These impairments result in a complete arrest of spermatogenesis, underscoring the critical involvement of the somatic compartment of the seminiferous tubules in germ cell maturation.     

Extracellular matrix: recent advances on its role in junction dynamics in the seminiferous epithelium during spermatogenesis

Spermatogenesis takes place in the seminiferous epithelium of the mammalian testis in which one type A1 spermatogonium (diploid, 2n) gives rise to 256 spermatids (haploid, 1n). To accomplish this, developing germ cells, such as preleptotene and leptotene spermatocytes, residing in the basal compartment of the seminiferous epithelium must traverse the blood-testis barrier (BTB) entering into the adluminal compartment for further development into round, elongating, and elongate spermatids. Recent studies have shown that the basement membrane in the testis (a modified form of extracellular matrix, ECM) is important to the event of germ cell movement across the BTB because proteins in the ECM were shown to regulate BTB dynamics via the interactions between collagens, proteases, and protease inhibitors, possibly under the regulation of cytokines. While these findings are intriguing, they are not entirely unexpected. For one, the basement membrane in the testis is intimately associated with the BTB, which represents the basolateral region of Sertoli cells. Also, Sertoli cell tight junctions (TJs) that constitute the BTB are present side-by-side with cell-cell actin-based adherens junctions (AJ, such as basal ectoplasmic specialization [ES]) and intermediate filament-based desmosome-like junctions. As such, the relative morphological layout between TJs, AJs, and desmosome-like junctions in the seminiferous epithelium is in sharp contrast to other epithelia where TJs are located at the apical portion of an epithelium or endothelium, furthest away from ECM, to be followed by AJs and desmosomes, which in turn constitute the junctional complex. For another, anchoring junctions between a cell epithelium and ECM found in multiple tissues, also known as focal contacts (or focal adhesion complex, FAC, an actin-based cell-matrix anchoring junction type), are the most efficient junction type that permits rapid junction restructuring to accommodate cell movement. It is therefore physiologically plausible, and perhaps essential, that the testis is using some components of the focal contacts to regulate rapid restructuring of AJs between Sertoli and germ cells when germ cells traverse the seminiferous epithelium. Indeed, recent findings have shown that the apical ES, a testis-specific AJ type in the seminiferous epithelium, is equipped with proteins of FAC to regulate its restructuring. In this review, we provide a timely update on this exciting yet rapidly developing field regarding how the homeostasis of basement membrane in the tunica propria regulates BTB dynamics and spermatogenesis in the testis, as well as a critical review on the molecular architecture and the regulation of ES in the seminiferous epithelium.     

Cytokines and junction restructuring events during spermatogenesis in the testis: An emerging concept of regulation

During spermatogenesis in mammalian testes, junction restructuring takes place at the Sertoli–Sertoli and Sertoli–germ cell interface, which is coupled with germ cell development, such as cell cycle progression, and translocation of the germ cell within the seminiferous epithelium. In the rat testis, restructuring of the blood–testis barrier (BTB) formed between Sertoli cells near the basement membrane and disruption of the apical ectoplasmic specialization (apical ES) between Sertoli cells and fully developed spermatids (spermatozoa) at the luminal edge of the seminiferous epithelium occur concurrently at stage VIII of the seminiferous epithelial cycle of spermatogenesis. These two processes are essential for the translocation of primary spermatocytes from the basal to the apical compartment to prepare for meiosis, and the release of spermatozoa into the lumen of the seminiferous epithelium at spermiation, respectively. Cytokines, such as TNFα and TGFβ3, are present at high levels in the microenvironment of the epithelium at this stage of the epithelial cycle. Since these cytokines were shown to disrupt the BTB integrity and germ cell adhesion, it was proposed that some cytokines released from germ cells, particularly primary spermatocytes, and Sertoli cells, would induce restructuring of the BTB and apical ES at stage VIII of the seminiferous epithelial cycle. In this review, the intricate role of cytokines and testosterone to regulate the transit of primary spermatocytes at the BTB and spermiation will be discussed. Possible regulators that mediate cytokine-induced junction restructuring, including gap junction and extracellular matrix, and the role of testosterone on junction dynamics in the testis will also be discussed.     

Drug transporter,P-glycoprotein (MDR1), is an integrated component of the mammalian blood–testis barrier

Throughout spermatogenesis, leptotene spermatocytes traverse the blood–testis barrier (BTB) to enter the adluminal compartment of the seminiferous epithelium for continued development. At the same time, the integrity of the BTB, which is constituted by co-existing tight junctions (TJ), basal ectoplasmic specializations (basal ES) and desmosome-like junctions, must be maintained since a breach in barrier function can result in spermatogenic arrest and even infertility. There is evidence to suggest that drug transporters may function at the BTB, but little is known about how they contribute to spermatogenesis. In this study, we investigate the role of P-glycoprotein (P-gp), a drug efflux pump, in BTB dynamics. A survey by RT-PCR revealed several transporter genes to be expressed by the testis, including Mdr1 (gene symbol for P-gp), Mrp1, Abcc5 and Slc15a1. It was also demonstrated that P-gp localizes to the BTB in all stages of the seminiferous epithelial cycle in the adult rat testis, as well as to the Sertoli cell–elongated spermatid interface in stages VII and VIII. We continued our study by examining the levels of several transporters in the testis following oral administration of Adjudin, a compound known to affect Sertoli–germ cell adhesion. In this experiment, the steady-state levels of P-gp, MRP1, ABCG1 and SLC15A1 were all found to increase by several-fold within hours of Adjudin treatment during junction restructuring. More importantly, an increase in P-gp association with TJ proteins (e.g., occludin, claudin-11 and JAM-A) was noted when testis lysates from Adjudin-treated rats were used for co-immunoprecipitation experiments, suggesting that P-gp may enhance BTB function during Sertoli–germ cell junction restructuring.     

Disruption of blood-testis barrier dynamics in ether-lipid-deficient mice

One of the major roles of Sertoli cells is to establish the blood-testis (Sertoli cell) barrier (BTB), which is permanently assembled and disassembled to accommodate the translocation of leptotene spermatocytes from the basal into the adluminal compartment of the seminiferous epithelium and to guarantee completion of meiosis and spermiogenesis. Recently, we have demonstrated spermatogenesis to be arrested before spermatid elongation in Gnpat-null mice with selective deficiency of ether lipids (ELs) whose functions are poorly understood. In this study, we have focused on the spatio-temporal expression of several BTB tight-junctional proteins in the first wave of spermatogenesis to obtain insights into the physiological role of ELs during BTB establishment and dynamics. Our data confirm the transient existence of Russell’s intermediate or translocation compartment delineated by two separate claudin-3-positive luminal and basal tight junctions and reveal that EL deficiency blocks BTB remodeling. This block is associated with (1) downregulation and mistargeting of claudin-3 and (2) impaired BTB disassembly resulting in deficient sealing of the intermediate compartment as shown by increased BTB permeability to biotin. These results suggest that ELs are essential for cyclic BTB dynamics ensuring the sluice mechanism for leptotene translocation into the adluminal compartment. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. This work was supported by the German Research Foundation (grants Go 432/2-1, Ju 166/3-1, and Sa 172/1-1).     

Disruption of Sertoli-germ cell adhesion function in the seminiferous epithelium of the rat testis can be limited to adherens junctions without affecting the blood-testis barrier integrity: an in vivo study using an androgen suppression model

During spermatogenesis, both adherens junctions (AJ) (such as ectoplasmic specialization (ES), a testis-specific AJ type at the Sertoli cell-spermatid interface (apical ES) or Sertoli-Sertoli cell interface (basal ES) in the apical compartment and BTB, respectively) and tight junctions (TJ) undergo extensive restructuring to permit germ cells to move across the blood-testis barrier (BTB) as well as the seminiferous epithelium from the basal compartment to the luminal edge to permit fully developed spermatids (spermatozoa) to be sloughed at spermiation. However, the integrity of the BTB cannot be compromised throughout spermatogenesis so that postmeiotic germ cell-specific antigens can be sequestered from the systemic circulation at all times. We thus hypothesize that AJ disruption in the seminiferous epithelium unlike other epithelia, can occur without compromising the BTB-barrier, even though these junctions, namely TJ and basal ES, co-exist side-by-side in the BTB. Using an intratesticular androgen suppression-induced germ cell loss model, we have shown that the disruption of AJs indeed was limited to the Sertoli-germ cell interface without perturbing the BTB. The testis apparently is using a unique physiological mechanism to induce the production of both TJ- and AJ-integral membrane proteins and their associated adaptors to maintain BTB integrity yet permitting a transient loss of cell adhesion function by dissociating N-cadherin from beta-catenin at the apical and basal ES. The enhanced production of TJ proteins, such as occludin and ZO-1, at the BTB site can supersede the transient loss of cadherin-catenin function at the basal ES. This thus allows germ cell depletion from the epithelium without compromising BTB integrity. It is plausible that the testis is using this novel mechanism to facilitate the movement of preleptotene and leptotene spermatocytes across the BTB at late stage VIII through early stage IX of the epithelial cycle in the rat while maintaining the BTB immunological barrier function.     

Cytokines and junction restructuring during spermatogenesis--a lesson to learn from the testis

In the mammalian testis, preleptotene and leptotene spermatocytes residing in the basal compartment of the seminiferous epithelium must traverse the blood-testis barrier (BTB) at late stage VIII through early stage IX of the epithelial cycle during spermatogenesis, entering the adluminal compartment for further development. However, until recently the regulatory mechanisms that regulate BTB dynamics remained largely unknown. We provide a critical review regarding the significance of cytokines in regulating the 'opening' and 'closing' of the BTB. We also discuss how cytokines may be working in concert with adaptors that selectively govern the downstream signaling pathways. This process, in turn, regulates the dynamics of either Sertoli-Sertoli tight junction (TJ), Sertoli-germ cell adherens junction (AJ), or both junction types in the epithelium, thereby permitting TJ opening without compromising AJs, and vice versa. We also discuss how adaptors alter their protein-protein association with the integral membrane proteins at the cell-cell interface via changes in their phosphorylation status, thereby altering adhesion function at AJ. These findings illustrate that the testis is a novel in vivo model to study the biology of junction restructuring. Furthermore, a molecular model is presented regarding how cytokines selectively regulate TJ/AJ restructuring in the epithelium during spermatogenesis.     

Molecular dynamics of the blood–testis barrier components during murine spermatogenesis

The blood–testis barrier (BTB) separates the seminiferous epithelium into the adluminal and basal compartments. During murine spermatogenesis, preleptotene/leptotene spermatocytes migrate from the basal to the adluminal compartment through the BTB during stages VIII–IX. In the present study, we focused on the tight junction (TJ) molecules and analyzed their spatiotemporal expression during the murine seminiferous epithelial cycle. Structural analysis revealed that the principal components of the BTB, for example, claudin‐3, claudin‐11, occludin, and zonula occludens‐1 (ZO‐1), were localized at the basal and luminal sides of the preleptotene/leptotene spermatocytes during the migration stages (VIII–IX). Although we detected claudin‐11, occludin, and ZO‐1 throughout spermatogenesis, claudin‐3 was only detected during stages VI–IX. Quantitative PCR using dissected seminiferous tubules from three stages (Early: II–VI, Middle: VII–VIII, Late: IX–I) clarified that the mRNA levels of TJ molecules were not correlated with the histoplanimetrical protein levels during spermatogenesis. Additionally, tubulobulbar complexes, considered to be involved in the internalization of TJ, were observed at the BTB site. Furthermore, a significant reduction in the mRNA levels of genes for the degradation of occludin (Itch) and endocytic recycling (Rab13) were observed during the Late and Middle stages, respectively. Therefore, we hypothesized that the lag between mRNA and protein expression of TJ molecules may be due to posttranslational modulation, for example, tubulobulbar complexes and endocytic recycling processes. In conclusion, these findings indicate that the integrity of the BTB is maintained throughout spermatogenesis, and the stage‐specific localization of claudin‐3 protein plays an important role in regulating BTB permeability. Mol. Reprod. Dev. 77: 630–639, 2010. © 2010 Wiley‐Liss, Inc.     

Nitric oxide/nitric oxide synthase, spermatogenesis, and tight junction dynamics

During spermatogenesis, preleptotene and leptotene spermatocytes, residing in the basal compartment of the seminiferous epithelium, must traverse the blood-testis barrier (BTB) to gain entry to the adluminal compartment for further development at late stage VIII and early stage IX of the epithelial cycle. As such, the timely opening and closing of the BTB is crucial to spermatogenesis. A compromise in this process can lead to infertility. Moreover, the BTB is unique in its relative localization in the seminiferous epithelium compared to the tight junctions (TJs) found in other epithelia. Sertoli cell TJs are situated near the basal lamina in the testis, closest to the basement membrane (a modified form of extracellular matrix [ECM]), unlike TJs found in other epithelia, which are found nearest the apical portion of an epithelium, farthest away from ECM. Needless to say, BTB function in the testis is maintained by intricate regulatory mechanisms. In addition to hormones and cytokines, nitric oxide (NO) was recently shown to be a putative TJ regulator in the testis. Perhaps equally important, TJ dynamics in the testis were shown to be regulated, at least in part, by occludin, a TJ-integral membrane protein, via the NO/soluble guanylate cyclase/cGMP/protein kinase G signaling pathway. This minireview summarizes recent advances in the field regarding the role of NO in testicular function, with special emphasis regarding its role in TJ dynamics and the likely implications of these studies for male contraceptive development.     

Differential interactions between transforming growth factor-beta3/TbetaR1, TAB1, and CD2AP disrupt blood-testis barrier and Sertoli-germ cell adhesion

The biochemical basis that regulates the timely and selective opening of the blood-testis barrier (BTB) to migrating preleptotene/leptotene spermatocytes at stage VIII of the epithelial cycle in adult rat testes is virtually unknown. Recent studies have shown that cytokines (e.g. transforming growth factor (TGF)-beta3) may play a crucial role in this event. However, much of this information relies on the use of toxicants (e.g. CdCl(2)), making it difficult to relay these findings to normal testicular physiology. Here we report that overexpression of TGF-beta3 in primary Sertoli cells cultured in vitro indeed perturbed the tight junction (TJ) barrier with a concomitant decline in the production of BTB constituent proteins as follows: occludin, N-cadherin, and ZO-1. Additionally, local administration of TGF-beta3 to testes in vivo was shown to reversibly perturb the BTB integrity and Sertoli-germ cell adhesion via the p38 MAPK and ERK signaling pathways. Most importantly, the simultaneous activation of p38 and ERK signaling pathways is dependent on the association of the TGF-beta3-TbetaR1 complex with adaptors TAB1 and CD2AP because if TbetaR1 was associated preferentially with CD2AP, only Sertoli-germ cell adhesion was perturbed without compromising the BTB. Collectively, these data illustrate that local production of TGF-beta3, and perhaps other TGF-betas and cytokines, by Sertoli and germ cells into the microenvironment at the BTB during spermatogenesis transiently perturbs the BTB and Sertoli-germ cell adhesion to facilitate germ cell migration when the activated TbetaRI interacts with adaptors TAB1 and CD2AP. However, TGF-beta3 selectively disrupts Sertoli-germ cell adhesion in the seminiferous epithelium to facilitate germ cell migration without compromising BTB when TbetaRI interacts only with adaptor CD2AP.     

c-Yes regulates cell adhesion at the blood-testis barrier and the apical ectoplasmic specialization in the seminiferous epithelium of rat testes

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.     

Regulation of spermiogenesis, spermiation and blood-testis barrier dynamics: novel insights from studies on Eps8 and Arp3

Spermiogenesis in the mammalian testis is the most critical post-meiotic developmental event occurring during spermatogenesis in which haploid spermatids undergo extensive cellular, molecular and morphological changes to form spermatozoa. Spermatozoa are then released from the seminiferous epithelium at spermiation. At the same time, the BTB (blood-testis barrier) undergoes restructuring to facilitate the transit of preleptotene spermatocytes from the basal to the apical compartment. Thus meiotic divisions take place behind the BTB in the apical compartment to form spermatids. These germ cells enter spermiogenesis to transform into elongating spermatids and then into spermatozoa to replace those that were released in the previous cycle. However, the mole-cular regulators that control spermiogenesis, in particular the dynamic changes that occur at the Sertoli cell-spermatid interface and at the BTB, are not entirely known. This is largely due to the lack of suitable animal models which can be used to study these events. During the course of our investigation to develop adjudin [1-(2,4-dichlorobenzyl)-1H-indazole-3-carbohydrazide] as a potential male contraceptive, this drug was shown to 'accelerate' spermiation by inducing the release of premature spermatids from the epithelium. Using this model, we have identified several molecules that are crucial in regulating the actin filament network and the unique adhesion protein complex at the Sertoli cell-spermatid interface known as the apical ES (ectoplasmic specialization). In the present review, we critically evaluate these and other findings in the literature as they relate to the restricted temporal and spatial expression of two actin regulatory proteins, namely Eps8 (epidermal growth factor receptor pathway substrate 8) and Arp3 (actin-related protein 3), which regulate these events.     

Improvement of spermatogenesis in adult cryptorchid rat testis by intratesticular infusion of lactate.

In order to test the hypothesis that a lack of energy could be a cause of germ cell death at high temperatures, cryptorchid rats testes were infused with lactate, delivered by osmotic pumps over 3-15 days. In cryptorchid testes, the spermatids and spermatocytes were lost between 3 and 8 days. In cryptorchid testes supplemented with lactate, elongated spermatids persisted in a few seminiferous tubules at Day 15. Elimination of round spermatids occurred progressively between 3 and 15 days, mostly at stage VIII. The loss of spermatocytes increased after 8 days, and 30% of seminiferous tubules still contained meiotic or meiotic plus spermiogenetic cells at Day 15. After 8 days, the chromatin of step 8 round spermatids was abnormal and nuclear elongation did not commence. The Sertoli cell cytoplasm that was retracted toward the basal compartment of the seminiferous epithelium could not hold the germ cells of the adluminal compartment. Therefore, attachment of germ cells to Sertoli cells and the supply of lactate seem necessary for the development of germ cells at high temperatures. The improvement in spermatogenesis in cryptorchid supplemented testes for several days is a new finding.     

Interrelationships between Sertoli cells and germ cells in the Syrian hamster

The interrelationships of the Sertoli cells and germ cells in the Syrian hamster were examined using the electron microscope. Demosome-like junctions were observed attaching Sertoli cells to spermatogonia and spermatocytes. In the region of the junctions dense plaques lay on the cytoplasmic surfaces of the plasmalemma of the opposing cells. Sertoli cell cytoplasmic filaments converged in the area of the junctions and inserted into the subsurface densities. Filaments were not observed associated with the subsurface densities of the germ cells. In the region of the junctions a 15...20 nm gap, filled with an attenuate amorphous substance, separated the plasmalemmata. Another attachment device termed "junctional specialization" occurred between Sertoli cells, and preleptotene spermatocytes and all successive developmental steps in the germ cell line in the hamster. The junctional specializations consisted of a mantel of Sertoli cell cytoplasmic filament lying subjacent to the Sertoli cell plasmalemma and an opposed cisterna of the endoplasmic reticulum. In stages VII-VIII preleptotene supermatocytes were observed in transit from the basal compartment to the adluminal compartment. While Sertoli-Sertoli junctions adluminal to the spermatocytes remained intact, typical Sertoli-Sertoli junctions formed between opposed Sertoli cell processes basal to the spermatocytes. It is proposed that, during the passage of spermatocytes in to the adluminal compartment, junctional specializations associated with preleptotene spermatocytes in the basal compartment migrate basal to the spermatocytes and contribute to formation of Sertoli-Sertoli junctions. Treatment of seminiferous tubules with hypertonic media was used to demonstrate that the junctional specializations function in cell-to-cell adhesion. Data indicated that these junctions function to retain the developing spermatids within the seminiferous epithelijm until the time of spermiation. At spermination the junctional specializations disappear and the spermatids drift off into the tubule lumen.     

An intracellular trafficking pathway in the seminiferous epithelium regulating spermatogenesis: a biochemical and molecular perspective

During spermatogenesis in adult rat testes, fully developed spermatids (i.e. spermatozoa) at the luminal edge of the seminiferous epithelium undergo “spermiation” at stage VIII of the seminiferous epithelial cycle. This is manifested by the disruption of the apical ectoplasmic specialization (apical ES) so that spermatozoa can enter the tubule lumen and to complete their maturation in the epididymis. At the same time, the blood–testis barrier (BTB) located near the basement membrane undergoes extensive restructuring to allow transit of preleptotene spermatocytes so that post-meiotic germ cells complete their development behind the BTB. While spermiation and BTB restructuring take place concurrently at opposite ends of the Sertoli cell epithelium, the biochemical mechanism(s) by which they are coordinated were not known until recently. Studies have shown that fragments of laminin chains are generated from the laminin/integrin protein complex at the apical ES via the action of MMP-2 (matrix metalloprotease-2) at spermiation. These peptides serve as the local autocrine factors to destabilize the BTB. These laminin peptides also exert their effects on hemidesmosome which, in turn, further potentiates BTB restructuring. Thus, a novel apical ES-BTB-hemidesmosome regulatory loop is operating in the seminiferous epithelium to coordinate these two crucial cellular events of spermatogenesis. This functional loop is further assisted by the Par3/Par6-based polarity protein complex in coordination with cytokines and testosterone at the BTB. Herein, we provide a critical review based on the latest findings in the field regarding the regulation of these cellular events. These recent findings also open up a new window for investigators studying blood–tissue barriers.     

支持细胞紧密连接与男性避孕

支持细胞的紧密连接是血睾屏障的主要组成成分,对支持细胞紧密连接结构与功能的深入研究有助于探讨男性避孕的新的研究方法。对紧密连接动力学的影响因素以及其与精子发生和男性避孕间的关系进行了分析。为进一步探讨男性避孕的研究方法提供新思路。     

Histological studies on male sterility of hybrids between laboratory and wild mouse strains

In this study the cellular mechanisms of male sterility in F1 hybrids (BNF1) between BALB/c and wild-derived M.MUS-NJL (NJL) was investigated. Cell proliferation and differentiation in the sterile testis were examined by bromodeoxyuridine-labeling and use of germ cell stage-specific antibodies. In BNF1 testes, spermatogonia actively proliferated with a seminiferous epithelial cycle, and were retained in the basal layer of the tubules. However, preleptotene, leptotene and zygotene spermatocytes moved to the adluminal region. Immunohistological data with germ cell stage-specific antibodies indicated the presence of few, if any, pachytene spermatocytes in BNF1 testes. Thus, spermatogenesis seemed to be blocked at the zygotene stage. For examination of germ cell-Sertoli cell interactions, testes of aggregation chimeras between BNF1 and C3H/HeN were analyzed immunohistologically with C3H-specific antibody. Results showed that spermatogenesis of C3H-germ cells was normal, even when these cells in contact with BNF1-Sertoli cells. Differentiation of BNF1-germ cells progressed from zygotene to pachytene stage spermatocytes when these cells were surrounded by C3H-Sertoli cells, but never proceeded beyond the pachytene stage. These observations suggest that at least two different cellular factors may be involved in spermatogenesis, one acting in the germ cells and the other mediated by Sertoli cells. Furthermore, mating experiments revealed that the degree of spermatogenesis varied in different F1 hybrids, and that the major sterility factor was closely linked to the T -locus on chromosome 17.     

Sertoli cell tight junction dynamics: their regulation during spermatogenesis

During spermatogenesis, developing preleptotene and leptotene spermatocytes must translocate from the basal to the adluminal compartment of the seminiferous epithelium so that fully developed spermatids (spermatozoa) can be released to the tubular lumen at spermiation. It is conceivable that the opening and closing of the inter-Sertoli tight junctions (TJs) that constitute the blood-testis barrier are regulated by an array of intriguingly coordinated signaling pathways and molecules. Several molecules have been shown to regulate Sertoli cell TJ dynamics; they include, for example, transforming growth factor beta3 (TGFbeta3), occludin, protein kinase A, protein kinase C, and signaling pathways such as the TGFbeta3/p38 mitogen-activated protein kinase pathway. Yet the mechanisms that regulate these events are essentially not known. This minireview summarizes some of the recent advances in the study of TJ dynamics in the testis and reviews several models that can be used to study TJ dynamics. It also highlights specific areas for future research toward understanding the precise physiological relationship between junction dynamics and spermatogenesis.     

Receptor-mediated endocytosis of testicular transferrin by germinal cells of the rat testis

Summary The present study examines events of the Sertoli cell iron delivery pathway following the secretion of diferric testicular transferrin (tTf) into the adluminal compartment of the rat seminiferous epithelium. The unidirectional secretion of tTf by Sertoli cells was verified, in vivo, and it was shown that this protein is internalized by adluminal germ cells. It was further determined by Scatchard analysis that this internalization was mediated by high affinity transferrin binding sites on the surface of round spermatids, numbering 1453/cell and displaying a K_d=0.6×10^-9 M. Northern blot analysis of RNA isolated from adluminal germ cells, namely spermatocytes, round spermatids and elongating spermatids, indicated that these cells expressed Tf receptor mRNA and ferritin mRNA in levels inversely related to their stage of maturation. Finally it was determined that following binding and internalization in round spermatids, Tf became associated with the endosomal compartment and was recycled back to the cell surface. This study illustrates the immediate fate of tTf once it is secreted by the Sertoli cell. Thus, diferric tTf binds of Tf receptor on the surface of adluminal germ cells, is internalized by receptor-mediated endocytosis and the apo Tf-Tf receptor complex is recycled back to the cell surface where apotTf is released into the adluminal fluid.