Jannette M. Dufour

The Blood-Testis and Blood-Epididymis Barriers Are More Than Just Their Tight Junctions

The terms blood-testis barrier (BTB) or blood-epididymis barrier (BEB), are often described as Sertoli cell-Sertoli cell tight junctions (TJs) or TJs between the epithelial cells in the epididymis, respectively. However, in reality, the BTB and BEB are much more complex than just the TJ. The focus of this minireview is to remind readers that the complete BTB and BEB are comprised of three components: anatomical, physiological, and immunological. The TJs form the anatomical (physical) barrier that restricts passage of molecules and cells from entering or exiting the lumen. The physiological barrier is comprised of transporters that regulate movement of substances in or out of the lumen, thus creating a microenvironment, which is critical for the proper development and maturation of germ cells. The immunological barrier limits access by the immune system and sequesters the majority of the autoantigenic germ cells. Combined with the overall immune-privilege of the testis, this suppresses detrimental immune responses against the autoantigenic germ cells. These three components on their own do not create a complete functional barrier; instead, it is the interaction between all three components that create a barrier of maximal competence.

Expansion of mesenchymal stem cells from human pancreatic ductal epithelium

Fibroblast-like cells emerging from cultured human pancreatic endocrine and exocrine tissue have been reported. Although a thorough phenotypic characterization of these cells has not yet been carried out, these cells have been hypothesized to be contaminating fibroblasts, mesenchyme and/or possibly beta-cell progenitors. In this study, we expanded fibroblast-like cells from adult human exocrine pancreas following islet isolation and characterized these cells as mesenchymal stem cells (MSCs) based on their cell surface antigen expression and ability to differentiate into mesoderm. Analysis by flow cytometry demonstrated that pancreatic MSCs express cell surface antigens used to define MSCs isolated from bone marrow such as CD13, CD29, CD44, CD49b, CD54, CD90 and CD105. In addition, utilizing protocols used to differentiate MSCs isolated from other somatic tissues, we successfully differentiated pancreatic MSCs into: (1) osteocytes that stained positive for alkaline phosphatase, collagen, mineralization (calcification) and expressed osteocalcin, (2) adipocytes that contained lipid inclusions and expressed fatty acid binding protein 4 and (3) chondrocytes that expressed aggrecan. We also demonstrated that pancreatic MSCs are multipotent and capable of deriving cells of endodermal origin. Pancreatic MSCs were differentiated into hepatocytes that stained positive for human serum albumin and expressed endoderm and liver-specific genes such as GATA 4 and tyrosine aminotransferase. In addition, preliminary protocols used to differentiate these cells into insulin-producing cells resulted in the expression of genes necessary for islet and beta-cell development such as Pax4 and neurogenin 3. Therefore, multipotent MSCs residing within the adult exocrine pancreas could represent a progenitor cell, which when further manipulated could result in the production of functional islet beta-cells.

Immunoprotective Sertoli cells: making allogeneic and xenogeneic transplantation feasible

The testis as an immune-privileged site allows long-term survival of allogeneic and xenogeneic transplants. Testicular Sertoli cells (SCs) play a major role in this immunoprotection and have been used to create an ectopic immune-privileged environment that prolongs survival of co-transplanted allogeneic and xenogeneic cells, including pancreatic islets and neurons. Extended survival of such grafts testifies to the immunoprotective properties of SCs. However, there is still variability in the survival rates of the co-grafted cells and rarely are 100% of the grafts protected. This emphasizes the need to learn more about what is involved in creating the optimal immunoprotective milieu. Several parameters including organization of the SCs into tubule-like structures and the production of immunomodulatory factors by SCs, specifically complement inhibitors, cytokines, and cytotoxic lymphocyte inhibitors, are likely important. In addition, an intricate interplay between several of these factors may be responsible for providing the most ideal environment for protection of the co-transplants by SCs. In this review, we will also briefly describe a novel use for the immune-privileged abilities of SCs; engineering them to deliver therapeutic proteins for the treatment of diseases like diabetes and Parkinsons disease. In conclusion, further studies and more detailed analysis of the mechanisms involved in creating the immune-protective environment by SCs may make their application in co-transplantation and as engineered cells clinically feasible.

Cell lines: Valuable tools or useless artifacts

Cell lines are often used in place of primary cells to study biological processes. However, care must be taken when interpreting the results as cell lines do not always accurately replicate the primary cells. In this article, we will briefly talk about advantages and disadvantages of cell lines and then discuss results using the mouse Sertoli cell line, MSC-1, compared with primary mouse Sertoli cells. MSC-1 cells resemble Sertoli cells morphologically and possess several biochemical markers associated with Sertoli cells. Studies have demonstrated that the function and regulation of retinoic acid receptor α (RARα) is similar between MSC-1 and rat Sertoli cells. However, MSC-1 cells lack some of the immune privilege properties associated with primary Sertoli cells, including survival in animals with a fully functional immune system. Therefore, it has to be kept in mind that cell lines do not behave identically with primary cells and should not be used to replace primary cells. In order to strengthen the findings, key control experiments using primary cells should always be performed.

Differential effects of phthalates on the testis and the liver.

Abstract Phthalates have been shown to elicit contrasting effects on the testis and the liver, causing testicular degeneration and promoting abnormal hepatocyte proliferation and carcinogenesis. In the present study, we compared the effects of phthalates on testicular and liver cells to better understand the mechanisms by which phthalates cause testicular degeneration. In vivo treatment of rats with di-(2-ethylhexyl) phthalate (DEHP) caused a threefold increase of germ cell apoptosis in the testis, whereas apoptosis was not changed significantly in livers from the same animals. Western blot analyses revealed that peroxisome proliferator-activated receptor (PPAR) α is equally abundant in the liver and the testis, whereas PPARγ and retinoic acid receptor (RAR) α are expressed more in the testis. To determine whether the principal metabolite of DEHP, mono-(2-ethylhexyl) phthalate (MEHP), or a strong peroxisome proliferator, 4-chloro-6(2,3-xylindino)-2-pyrimidinylthioacetic acid (Wy-14,643), have a differential effect in Sertoli and liver cells by altering the function of RARα and PPARs, their nuclear trafficking patterns were compared in Sertoli and liver cells after treatment. Both MEHP and Wy-14,643 increased the nuclear localization of PPARα and PPARγ in Sertoli cells, but they decreased the nuclear localization of RARα, as previously shown. Both PPARα and PPARγ were in the nucleus and cytoplasm of liver cells, but RARα was predominant in the cytoplasm, regardless of the treatment. At the molecular level, MEHP and Wy-14,643 reduced the amount of phosphorylated mitogen-activated protein kinase (activated MAPK) in Sertoli cells. In comparison, both MEHP and Wy-14,643 increased phosphorylated MAPK in liver cells. These results suggest that phthalates may cause contrasting effects on the testis and the liver by differential activation of the MAPK pathway, RARα, PPARα, and PPARγ in these organs.

The Sertoli cell: one hundred fifty years of beauty and plasticity.

It has been one and a half centuries since Enrico Sertoli published the seminal discovery of the testicular ‘nurse cell’, not only a key cell in the testis, but indeed one of the most amazing cells in the vertebrate body. In this review, we begin by examining the three phases of morphological research that have occurred in the study of Sertoli cells, because microscopic anatomy was essentially the only scientific discipline available for about the first 75 years after the discovery. Biochemistry and molecular biology then changed all of biological sciences, including our understanding of the functions of Sertoli cells. Immunology and stem cell biology were not even topics of science in 1865, but they have now become major issues in our appreciation of Sertoli cells role in spermatogenesis. We end with the universal importance and plasticity of function by comparing Sertoli cells in fish, amphibians, and mammals. In these various classes of vertebrates, Sertoli cells have quite different modes of proliferation and epithelial maintenance, cystic vs. tubular formation, yet accomplish essentially the same function but in strikingly different ways.

Identification of a Novel Human Granzyme B Inhibitor Secreted by Cultured Sertoli Cells

Sertoli cells have long since been recognized for their ability to suppress the immune system and protect themselves as well as other cell types from harmful immune reaction. However, the exact mechanism or product produced by Sertoli cells that affords this immunoprotection has never been fully elucidated. We examined the effect of mouse Sertoli cell-conditioned medium on human granzyme B-mediated killing and found that there was an inhibitory effect. We subsequently found that a factor secreted by Sertoli cells inhibited killing through the inhibition of granzyme B enzymatic activity. SDS-PAGE analysis revealed that this factor formed an SDS-insoluble complex with granzyme B. Immunoprecipitation and mass spectroscopic analysis of the complex identified a proteinase inhibitor, serpina3n, as a novel inhibitor of human granzyme B. We cloned serpina3n cDNA, expressed it in Jurkat cells, and confirmed its inhibitory action on granzyme B activity. Our studies have led to the discovery of a new inhibitor of granzyme B and have uncovered a new mechanism used by Sertoli cells for immunoprotection.

Immunoprotection of rat islet xenografts by cotransplantation with sertoli cells and a single injection of antilymphocyte serum1

Background. Islet transplantation has become a clinical reality; however, before it can be extended to young juvenile diabetics an unlimited supply of tissue is needed and the use of chronic immunosuppression should be eliminated. This study was designed to determine whether Sertoli cells can immunoprotect islet xenografts. Methods. Lewis rat islets were cotransplanted with Balb/c Sertoli cells in diabetic Balb/c mice treated with one injection of anti-mouse lymphocyte serum (ALS). Results. When islets were transplanted alone, in combination with Sertoli cells, or in combination with ALS, mean graft survival times were 10.9±0.8, 14.0±1.2, or 12.2±0.7, respectively. When islets were combined with Sertoli cells and ALS, mean graft survival time increased to 64.9±8.1. Conclusions. Sertoli cells are able to prolong the survival of islet xenografts when combined with ALS, thereby supporting their use as a means to immunoprotect cellular grafts such as islets for the treatment of type 1 diabetes.

Long‐term survival of neonatal porcine Sertoli cells in non‐immunosuppressed rats

Abstract: Sertoli cells from the testis contain immunoprotective properties which allow them to survive as allografts and also to protect islets and adrenal chromafin cells from immune rejection without the use of immunosuppressive drugs. Experiments were designed to determine whether xenogeneic neonatal porcine Sertoli cells (NPSCs) survive transplantation in rats without the use of immunosuppression. NPSCs (92.2 ± 5.1%) were isolated, cultured and then transplanted under the kidney capsule of non‐immunosuppressed Lewis rats. To assess survival, grafts were removed after 4, 20, 30, 40, 60, and 90 days post‐transplant and immunostained for the Sertoli cell marker vimentin. Survival was confirmed by polymerase chain reaction (PCR) for the porcine mitochondrial cytochrome oxidase II (COII) subunit gene, a marker for porcine tissue. In both methods, NPSCs were detected in the grafts for at least 90 days. Histologically, NPSCs were clustered in small aggregates or organized in tubule‐like structures. When stained for the presence of proliferating cell nuclear antigen (PCNA), many Sertoli cells stained positive at 20 days post‐transplant, indicating not only cell survival but also Sertoli cell proliferation. The number of PCNA postive cells decreased somewhat by 40 days with almost no positive Sertoli cells at 60 and 90 days. These data demonstrate that NPSCs survive long‐term following xenotransplantation in rats, which to our knowledge is the first report of a discordant xenograft surviving without immunosuppression in a non‐immunoprivileged site. Further study of the mechanism of NPSC xenograft survival may provide clues for promoting a local tolerogenic environment.

Immunoprotective Properties of Primary Sertoli Cells in Mice: Potential Functional Pathways that Confer Immune Privilege

ABSTRACT Primary Sertoli cells isolated from mouse testes survive when transplanted across immunological barriers and protect cotransplanted allogeneic and xenogeneic cells from rejection in rodent models. In contrast, the mouse Sertoli cell line (MSC-1) lacks immunoprotective properties associated with primary Sertoli cells. In this study, enriched primary Sertoli cells or MSC-1 cells were transplanted as allografts into the renal subcapsular area of naive BALB/c mice, and their survival in graft sites was compared. While Sertoli cells were detected within the grafts with 100% graft survival throughout the 20-day study, MSC-1 cells were rejected between 11 and 14 days, with 0% graft survival at 20 days posttransplantation. Nonetheless, the mechanism for primary Sertoli cell survival and immunoprotection remains unresolved. To identify immune factors or functional pathways potentially responsible for immune privilege, gene expression profiles of enriched primary Sertoli cells were compared with those of MSC-1 cells. Microarray analysis identified 2369 genes in enriched primary Sertoli cells that were differentially expressed at ±4-fold or higher levels than in MSC-1 cells. Ontological analyses identified multiple immune pathways, which were used to generate a list of 340 immune-related genes. Three functions were identified in primary Sertoli cells as potentially important for establishing immune privilege: suppression of inflammation by specific cytokines and prostanoid molecules, slowing of leukocyte migration by controlled cell junctions and actin polymerization, and inhibition of complement activation and membrane-associated cell lysis. These results increase our understanding of testicular immune privilege and, in the long-term, could lead to improvements in transplantation success.