Analysis of Development Edited by Prof Benjamin H Willier, Prof Paul A Weiss and Prof Viktor Hamburger Pp xii + 735 (Philadelphia and London: W B Saunders Company, 1955) 105s

Mechanisms of Development

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

XBP1 Controls Diverse Cell Type- and Condition-Specific Transcriptional Regulatory Networks

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

Many exogenous sources of stress can lead to cell death. In recent years, endogenous cellular sources of stress have also been identified, including the stress that arises from the accumulation of unfolded proteins within a cell's endoplasmic reticulum (ER). To counterbalance this type of ER stress, higher eukaryotic cells possess a three-pronged signal-transduction pathway termed the unfolded-protein response (UPR). This Review focuses on the role of the UPR in the mammalian immune system and how manipulation of this complex signalling pathway may be of therapeutic benefit in human disease.

The endoplasmic reticulum stress response in immunity and autoimmunity

Several animal models have been developed to investigate the pathobiology of pancreatitis, but few studies have examined the effects that altered pancreatic gene expression have in these models. In this study, the sensitivity to secretagogue-induced pancreatitis was examined in a mouse line that has an altered acinar cell environment due to the targeted deletion of Mist1. Mist1 is an exocrine specific transcription factor important for the complete differentiation and function of pancreatic acinar cells. Mice lacking the Mist1 gene [Mist1 knockout (KO) mice] exhibit cellular disorganization and functional defects in the exocrine pancreas but no gross morphological defects. Following the induction of pancreatitis with caerulein, a CCK analog, we observed elevated serum amylase levels, necrosis, and tissue damage in Mist1 KO mice, indicating increased pancreatic damage. There was also a delay in the regeneration of acinar tissue in Mist1 KO animals. Molecular profiling revealed an altered activation of stress response genes in Mist1 KO pancreatic tissue compared with wild-type (WT) tissue following the induction of pancreatitis. In particular, Western blot analysis for activating transcription factor 3 and phosphorylated eukaryotic initiation factor 2alpha (eIF2alpha), mediators of endoplasmic reticulum (ER) stress, indicated limited activation of this pathway in Mist1 KO animals compared with WT controls. Conversely, Mist1 KO pancreatic tissue exhibits increased expression of growth arrest and DNA damage inducible 34 protein, an inhibitor of eIF2alpha phosphorylation, before and after the induction of pancreatitis. These finding suggest that activation of the ER stress pathway is a protective event in the progression of pancreatitis and highlight the Mist1 KO mouse line as an important new model for studying the molecular events that contribute to the sensitivity to pancreatic injury.

Mice lacking the transcription factor Mist1 exhibit an altered stress response and increased sensitivity to caerulein-induced pancreatitis.

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.

Mist1 is necessary for the establishment of granule organization in serous exocrine cells of the gastrointestinal tract.

Gap junctions are intercellular channels that provide direct passage of small molecules between adjacent cells. In pancreatic acini, the connexin26 (Cx26) and connexin32 (Cx32) proteins form functional channels that coordinate the secretion of digestive enzymes. Although the function of Cx26/Cx32 gap junctions are well characterized, the regulatory circuits that control the spatial and temporal expression patterns of these connexin genes are not known. In an effort to identify the molecular pathways that regulate connexin gene expression, we examined Cx26 and Cx32 gene activities in mice lacking the basic helix-loop-helix transcription factor Mist1 (Mist1KO). Mist1, Cx26 and Cx32 are co-expressed in most exocrine cell types, and acinar cells from Mist1KO mice exhibit a highly disorganized cellular architecture and an altered pattern of expression for several genes involved in regulated exocytosis. Analysis of Mist1KO mice revealed a dramatic decrease in both connexin proteins, albeit through different molecular mechanisms. Cx32 gene transcription was greatly reduced in all Mist1KO exocrine cells, while Cx26 gene expression remained unaffected. However, in the absence of Cx32 protein, Cx26 did not participate in gap junction formation, leading to a complete lack of intercellular communication among Mist1KO acinar cells. Additional studies testing Mist1 gene constructs in pancreatic exocrine cells confirmed that Mist1 transcriptionally regulates expression of the Cx32 gene. We conclude that Mist1 functions as a positive regulator of Cx32 gene expression and, in its absence, acinar cell gap junctions and intercellular communication pathways become disrupted.

Agnes S. Kowalik

Papers

Mice lacking the transcription factor Mist1 exhibit an altered stress response and increased sensitivity to caerulein-induced pancreatitis.

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

Mist1 is necessary for the establishment of granule organization in serous exocrine cells of the gastrointestinal tract.

Exocrine specific expression of Connexin32 is dependent on the basic helix-loop-helix transcription factor Mist1

MIST1 regulates the pancreatic acinar cell expression of Atp2c2, the gene encoding secretory pathway calcium ATPase 2.