Role of Sertoli Cell Proteins in Immunomodulation.

BACKGROUND: Sertoli cell, over the past 30 years, have been elevated from simple mechanical elements to the rank of a "sentinel" in spermatogenesis. By delivering potent immunomodulatory and trophic proteins, Sertoli cells are unique cell type with a pivotal role in maintaining testis immune privilege and the immune-protection of the antigenic germ cells. CONCLUSIONS: The findings from SC transplantation studies utilizing experimental animal models of disease, demonstrate the presence of the same immuno-modulation properties and mechanisms at tissue and organ sites far from testis. The complex pathways that generate and maintain the immune tolerance involve the production of several immunomodulatory or immune-related proteins such as cytokines, chemokines, growth factors, mediators of the inflammation, complement inhibitors or adhesion molecules. A better definition and understanding of these Sertoli cell proteins and the mechanisms of immunoprotection should help to elucidate their role in the spermatogenic process. The demonstration of their capabilities in transplantation experiments suggests that Sertoli cells may be good candidates in cell therapy for a number of cell-mediated chronic diseases.

Sertoli cells for cell transplantation: pre-clinical studies and future perspectives.

Sertoli cells are located in the testes where they control several key functions in spermatogenesis. Over the past 30 years, Sertoli cells have been upgraded from a simple scaffold-like structural system to a dynamic functional system of intercellular support that delivers potent immunomodulatory and trophic factors. Since the discovery of new Sertoli cell secretory products, these cells have been utilized in experimental cell transplantation and co-transplantation protocols aimed at treating both chronic inflammatory and degenerative disorders. For these reasons, this work reviews the application of both naked and microencapsulated Sertoli cells used in cell transplantation studies of several chronic or autoimmune diseases such as diabetes mellitus, Laron dwarfism, and Duchenne muscular dystrophy and in studies aimed at the prevention of skin allograft rejection.

Microparticle-loaded neonatal porcine Sertoli cells for cell-based therapeutic and drug delivery system.

Neonatal porcine Sertoli cells (NPSC) are immune privileged cells showing innate phagocytic and antibacterial activities. NPSC have been shown capable of immunoaltering the body's response and possess lung homing capacity. These properties encourage investigation of NPSC as functional components of cell-based therapeutic protocols to treat lung infections and related complications. In this work, for the first time, NPSC were tailored to carry an antibiotic drug loaded into poly(d,l lactic) acid microparticles (MP). A loading protocol was developed, which afforded 30% drug uptake and high stability over time, with little or no effects on NPSC viability, morphology, reactive oxygen species production and DNA integrity. FSH receptor integrity, and TGFß (transforming growth factor ß) and AMH (anti-Müllerian hormone) expressions were unchanged after 1month of cryopreservation. Protein tyrosine kinase activation due to phagocytosis may have had resulted in changes in inhibin B expression. The activity of MP-loaded or NPSC alone against Pseudomonas aeruginosa was maintained throughout 1month of storage. NPSC couple an innate antibacterial activity with the capacity to embody drug loaded MP. We showed for the first time that engineered NPSC can be cryopreserved with no loss of their basic properties, thereby possibly representing a novel approach for cell-based therapeutic and drug delivery system.

Xenograft of microencapsulated Sertoli cells for the cell therapy of type 2 diabetes mellitus in spontaneously diabetic nonhuman primates: preliminary data.

Insulin resistance in type 2 diabetes mellitus (T2DM) may be due to a chronic inflammation of the visceral adipose tissue (VAT) leading to local and systemic increases in proinflammatory cytokines. Microencapsulated porcine Sertoli cells (MC-pSC), by provision of immunomodulatory and trophic factors, have been successfully used to reduce such inflammation in rodent animal models of type 1 diabetes with no complications or deleterious side effects. Herein, we have begun to investigate this novel and safe therapeutic approach in the spontaneously obese nonhuman primate with spontaneous, insulin-dependent T2DM. After MC-pSC intraperitoneal injection we have evaluated, throughout a 6-month follow-up period, daily ad libitum fed glucose levels, daily exogenous insulin supplementation, biweekly body weight measurements, periodic fasting blood glucose concentrations, glycated hemoglobin (HbA1c) levels, glucose tolerance tests (GTT), and fluorescence-activated cell sorting cytometry (FACS) assessment of peripheral blood mononuclear cells. Very preliminarily, we have observed a slight reduction in fasting (FPG) and mean nonfasting (NF) plasma glucose levels. We found minimal changes, only in 1 animal, in daily exogenous insulin requirements and HbA1c levels. Flow cytometric analysis was associated with decrease in CD8(+) cells only in 1 recipient with a reduction in mean regulatory T Cells (Treg), whereas interestingly, decrease of B lymphocytes was observed in both animals. These results may suggest that this novel MC-SC-based transplantation protocol might possibly impact the metabolic status of T2DM in higher mammals that are close to humans.

Effects of simulated microgravity on the morphology and function of neonatal porcine cell clusters cultured with and without Sertoli cells.

Human islet allografts are well known to induce full and sustained remission of hyperglycemia, with complete normalization of key metabolic parameters. Nevertheless, acquiring human islets, even from cadaveric human donor pancreases, remains a significant impediment to successful transplantation therapy for diabetes. To overcome this difficulty, neonatal porcine cell clusters (NPCCs) have been considered for human islet substitutes because they are easily obtained by collagenase digestion of the neonatal piglet pancreas. Currently, the major hurdle in using NPCCs for xenograft is the delay (time lag) in achieving the posttransplant normalization of blood glucose levels in animal diabetic recipients. The present work is the first attempt to evaluate whether incubation of NPCCs in simulated microgravity, in the presence or absence of Sertoli cells (SC), may reduce the maturation time lag of beta-cells by differentiation acceleration in vitro, thereby expediting production, viability, and acquisition of functional competence of pretransplantation beta-cell-enriched islets. Following a 3-day incubation period, NPCCs maintained in conventional culture, NPCCs incubated in simulated microgravity in the HARV biochamber, and NPCCs plus co-incubated SC in simulated microgravity were examined for viability, morphology, and insulin secretion. Results show that NPCCs grown alone in the HARV biochamber are superior in quality, both in terms of viability and functional competence, when compared to other culture pretreatment protocols. This finding strongly suggests that NPCC pretreatment in simulated microgravity may enhance the transplantation success of NPCCs in the diabetic recipient.

Sertoli cells induce systemic donor-specific tolerance in xenogenic transplantation model.

Cell therapy is a potentially powerful tool in the treatment of many grave disorders including leukemia, immune deficiencies, autoimmune diseases, and diabetes. However, finding matched donors is challenging and recipients may suffer from the severe complications of systemic immune suppression. Sertoli cells, when cotransplanted with both allo- and xenograft tissues, promote graft acceptance in the absence of systemic immunosuppression. How Sertoli cells do this is not, as yet, clearly defined. We have examined the ability of Sertoli cells to produce systemic immune tolerance. For this purpose, Sertoli cells were injected into an otherwise normal C57/BL6 mouse host via the lateral tail vein. No other immunosuppressive protocols were applied. Six to 8 weeks posttransplantation, blood was collected for analysis of cytokine levels. Tolerance to donor cells was determined by mixed lymphocytic culture, and production of T-cell-dependent antibody was determined by an in vitro anti-sheep red blood cell plaque-forming assay. Results showed a marked modulation of immune cytokines in the transplanted mouse host and donor-specific transplantation tolerance was achieved. Tolerant mouse lymphocytes maintained a competent humoral antibody response. Additionally, C57/BL6 mice transplanted with rat Sertoli cells tolerated rat skin grafts significantly longer than control non-Sertoli cell transplanted mice. We conclude that systemic administration of rat Sertoli cells across xenogenic barrier induces transplantation tolerance without altering systemic immune competence. These data suggest that Sertoli cells may be used as a novel and potentially powerful tool in cell transplantation therapy.

The role of connexins in the differentiation of NT2 cells in Sertoli-NT2 cell tissue constructs grown in the rotating wall bioreactor.

Neural transplantation is developing as a successful treatment for neurodegenerative diseases such as Parkinson's disease. The human Ntera-2/D1 (NT2) cell line is an attractive alternative to the use of human fetal neurons as a cell source for transplantation. We have explored combining NT2 cells, as a neuronal source, and Sertoli cells, which may act as a graft facilitator to enhance neuronal survival and differentiation, and ameliorate the host immune response, into a tissue construct for use in cell replacement therapy for neurodegenerative disease. This Sertoli-NT2-aggregated cell (SNAC) tissue construct is formed in the high aspect ratio vessel (HARV) bioreactor. NT2 cells differentiate to dopaminergic NT2N neurons within the SNAC tissue construct without retinoic acid. We report here that the gap junction protein connexin 43 is decreased among differentiated NT2N neurons. Inhibition of connexin 43 with 18beta glycyrrhetinic acid and carbenoxolone, a glycyrrhetinic acid derivative, during formation of the SNAC tissue constructs disrupts the differentiation of NT2 cells. Therefore, connexin 43 is important in the differentiation of NT2 cells in the SNAC tissue construct.

Survival of rat or mouse ventral mesencephalon neurons after cotransplantation with rat sertoli cells in the mouse striatum.

Transplanting cells across species (xenotransplantation) for the treatment of Parkinson's disease has been considered an option to alleviate ethical concerns and shortage of tissues. However, using this approach leads to decreased cell survival; the xenografted cells are often rejected. Sertoli cells (SCs) are testis-derived cells that provide immunological protection to developing germ cells and can enhance survival of both allografted and xenografted cells. It is not clear whether these cells will maintain their immunosuppressive support of cografted cells if they are transplanted across species. In this study, we investigated the immune modulatory capacity of SCs and the feasibility of xenografting these cells alone or with allografted and xenografted neural tissue. Transplanting xenografts of rat SCs into the mouse striatum with either rat or mouse ventral mesencephalon prevented astrocytic infiltration of the graft site, although all transplants showed activated microglia within the core of the graft. Surviving tyrosine hydroxylase-positive neurons were observed in all conditions, but the size of the grafts was small at best. SCs were found at 1 and 2 weeks posttransplant. However, few SCs were found at 2 months posttransplant. Further investigation is under way to characterize the immune capabilities of SCs in a xenogeneic environment.

An artificial testis for production of rat haploid cells.

PURPOSE: We attempted to apply the microgravity cell culture system for rat testicular germ cell maturation in vitro. METHODS: Primary spermatocytes were isolated from immature male rat by sedimentation velocity. Sertoli cells were isolated from another immature male by enzyme digestions. Sertoli cell aggregates were plated into conventional tissue culture flasks and incubated at 37 degrees C for 48 hours. These pretreated Sertoli-enriched monocultures were used in preparing Sertoli cell-primary spermatocyte cocultures. And then, primary spermatocytes and Sertoli cells were cocultured in a microgravity cell culture device for 28 days. RESULTS: Cell viability rate is more than 50 % after a 28-day long period of incubation. Furthermore, about 23 % haploid germ cells are observed. CONCLUSIONS: These results using primary spermatocyte coculture with Sertoli cell aggregates under microgravity show that it is possible to mature these cells up to the round spermatid and even to elongating/elongated steps. It may be possible to overcome the male sterility due to maturation arrest at the primary spermatocyte stage.

A unique cytoplasmic marker for extratesticular Sertoli cells.

In the absence of a definitive cell marker for testis-derived Sertoli cells, their identification in cell culture or in Sertoli cell-facilitated cell transplantation protocols is difficult and limits the creditable evaluation of experimental results. However, the production by prepubertal Sertoli cells of Mullerian inhibiting substance (MIS) presents the possibility of specifically identifying extratesticular Sertoli cells as well as Sertoli cells in situ, by the immunodection of this unique glycoprotein. This study was designed to determine if isolated rat Sertoli cells could be identified by routine immunocytochemistry utilizing an antibody raised against MIS. Sertoli cells immunostained for MIS included Sertoli cells in situ and freshly isolated, cultured and cocultured Sertoli cells, and Sertoli cells structurally integrated with NT2 cells in simulated microgravity. Detection of MIS was also determined by Western blot analysis.

Formation of insulin-secreting, Sertoli-enriched tissue constructs by microgravity coculture of isolated pig islets and rat Sertoli cells.

Pancreatic islets, isolated from neonatal pigs, and Sertoli cells, isolated from prepubertal rats, were cocultured in simulated microgravity utilizing the NASA-developed highly accelerating, rotating vessel (HARV) biochamber. Following 5 d of incubation, three-dimensional Sertoli-islet cell aggregates (SICA) retained the ability to secrete insulin when exposed to elevated glucose. SICA contained FasL-positive Sertoli cells and insulin-positive beta-cells randomly organized within the spherical construct. The addition of 1% Matrigel induced the reorganization of aggregates (SICAs formed in the presence of Matrigel [SICAmgs]) showing the peripherialization and epithelialization of Sertoli cells and the centralization of islets in association with lumen-like spaces. The Sertoli cells, but not Matrigel, aided in preserving the structural integrity of HARV-incubated islets. Neither Matrigel nor Sertoli cells appeared to interfere with the ability of SICA or SICA mg to secrete insulin and express FasL.

Identification of causal relationships among traits related to drought resistance in Stylosanthes scabra using QTL analysis.

Previous studies have shown that a negative relationship exists between transpiration efficiency (TE) and carbon isotope discrimination (Delta) and between TE and specific leaf area (SLA) in Stylosanthes scabra. A glasshouse experiment was conducted to confirm these relationships in an F(2) population and to study the causal nature of these relationships through quantitative trait loci (QTL) analysis. One hundred and twenty F(2) genotypes from a cross between two genotypes within S. scabra were used. Three replications for each genotype were maintained through vegetative propagation. Water stress was imposed by maintaining plants at 40% of field capacity for about 45 d. To facilitate QTL analysis, a genetic linkage map consisting of 151 RAPD markers was developed. Results from this study show that Delta was significantly and negatively correlated with TE and biomass production. Similarly, SLA showed significant negative correlation with TE and biomass production. Most of the QTL for TE and Delta were present on linkage groups 5 and 11. Similarly, QTL for SLA, transpiration and biomass productivity traits were clustered on linkage groups 13 and 24. One unlinked marker was also associated with these traits. There were several markers coincident between different traits. At all the coincident QTL, the direction of QTL effects was consistent with phenotypic data. At the coincident markers between TE and Delta, high alleles of TE were associated with low alleles of Delta. Similarly, low alleles of SLA were associated with high alleles of biomass productivity traits and transpiration. At the coincident markers between trans-4-hydroxy-N:-methyl proline (MHP) and relative water content (RWC), low alleles of MHP were associated with high alleles of RWC. This study suggests the causal nature of the relationship between TE and Delta. Phenotypic data and QTL data show that SLA was more closely associated with biomass production than with TE. This study also shows that a cause-effect relationship may exist between SLA and biomass production.

Sites of lanthanum occlusion in the testis of the crayfish Procambarus paeninsulanus (Crustacea: Cambaridae).

The presence of stage-dependent occlusive junctions between adjacent Sertoli cells in the seminiferous epithelium of the crayfish testis was demonstrated by a lanthanum tracer study. The germinal epithelium did not appear to be compartmentalized, as evidenced by access of lanthanum to spermatogonia, spermatocytes, and spermatids. During late spermiogenesis, when encapsulated stage VI spermatids were concentrated in the center of an acinus, lanthanum was excluded apically, coincident with lumen formation. This is the first study examining occluding junctions using a barrier penetration method in the testis of a crustacean.

Formation of Sertoli cell-enriched tissue constructs utilizing simulated microgravity technology.

Cell transplantation therapy for diabetes and Parkinson's disease offers hope for long-term alleviation of symptoms. However, successful protocols remain elusive due to obstacles, including rejection and lack of tropic support for the graft. To enhance engraftment, testis-derived postmitotic Sertoli cells have been cotransplanted with islets in the diabetic rat (Db) and neurons in the Parkinsonian rat (PD). Sertoli cell tropic, regulatory, and nutritive factors that nourish and stimulate germ cells also support isolated neurons and islets in vitro. Likewise, immunosuppressive properties of Sertoli cells, extant in the testis, are expressed by extratesticular Sertoli cells evidenced by allo- and xenograft immunoprotection of grafts in both the CNS (in the PD model) and the periphery (in the Db model). On this basis, we have created Sertoli islet cell aggregates (SICA) and Sertoli neuron aggregated cells (SNAC) using simulated microgravity culture technology developed by NASA. Isolated rat and pig Sertoli cells were cocultured with neonatal pig islets (SICA) and with immortalized N-Terra-2 (NT2) neurons (SNAC) in the HARV biochamber. Formed aggregates were assayed for desirable functional and structural characteristics. Cell viability in SICA and SNAC exceeded 90% and FasL immunopositive Sertoli cells were present in both. Sertoli cells did not interfere with insulin secretion by SICA and promoted differentiation of NT2 cells to the dopaminergic hNT cell type in SNAC. Addition of Matrigel resulted in structural reorganization of the aggregates and enhanced insulin secretion. We conclude that SICA, SNAC, and Matrigel-induced islet- and neuron-filled "Sertoli cell biochambers" are suitable for long-term transplantation treatment of Db and PD.

Endurance training increases the expression of mitochondrial and nuclear encoded cytochrome c oxidase subunits and heat shock proteins in rat skeletal muscle.

Cytochrome c oxidase (CCO) is an enzyme complex found on the inner mitochondrial membrane and serves as the final electron acceptor in mitochondrial electron transport. Heat shock proteins (HSPs) are involved in the import of nuclear encoded protein subunits into the mitochondria and induce conformational changes to form active enzyme complexes. As both the nuclear and mitochondrial encoded subunits of CCO have been shown to increase in activity and expression in muscle subsequent to artificial loading, and as exercise has been shown to induce HSPs, we sought to determine whether 16-20 weeks of treadmill exercise would result in enhanced CCO subunit expression, and to determine if there was a relationship between this expression and HSP content in medial gastrocnemius muscle of Fischer 344 rats. Our results indicated that endurance training resulted in a 53%, 87% and 80% increase (P<0.05) in the levels of HSP 60, CCO subunit II and CCO subunit VI, respectively. Enzymatic activity of CCO was 84% greater (P<0.05) after endurance training. Mann Whitney U analyses showed that CCO subunit II and VI increased to the same extent as HSP 60 after endurance training. It appears that 16-20 weeks of endurance training leads to uniform increases in CCO subunits and parts of the transport and assembly mechanisms required for CCO enzyme assembly. The similarity among the increases in CCO subunits II and VI protein levels and the increase in CCO enzyme activity suggest that this increase in activity is due to an increase in the amount of CCO enzyme.

Temporal relation between leptin and various indices of sexual maturation in the male rat.

Recent studies in humans and rhesus monkeys have suggested the possibility that the adipose tissue hormone leptin has a stimulatory and/or permissive effect on the onset of puberty in the male. We evaluated this hypothesis by measuring leptin in groups of male rats between the ages of 26 days and 96 days. A statistically significant positive correlation was present between serum leptin and age, body weight, prostate, seminal vesicle, and testes weight (both absolute and as a function of body weight). A statistically significant negative correlation was present between leptin and serum FSH and alpha-inhibin. There was not a statistically significant correlation between leptin and testosterone or LH. There was a statistically significant increase in the serum leptin concentrations at day 47. This rise was coincident with the peripubertal growth spurt in the secondary sexual organs and the peripubertal testosterone rise but occurred after the prepubertal rise in testicular weight, the appearance of elongating spermatids in the testes, and the start of the decline in FSH. In animals in which the peripubertal testosterone rise was delayed by the administration of EDS, serum leptin showed statistically significant differences from control. These data do not support the hypothesis that leptin provides a trigger for the onset of puberty in the male rat. They do suggest that leptin may be involved in the secondary sexual organ growth spurt and are consistent with the hypothesis that testosterone stimulates leptin synthesis during puberty.

Sertoli cells decrease microglial response and increase engraftment of human hNT neurons in the hemiparkinsonian rat striatum.

Sertoli cells (SCs) provide immune protection and nutritive support to the developing germ cells in the testis. Sertoli cells have also been shown to provide immune protection to islets transplanted outside the testes. In this study, the ability of these cells to diminish the infiltration/activation of microglia into a neural graft implanted in the lesioned striatum of a hemiparkinsonian rat was investigated. Human neuron-like cells (hNT neurons) were implanted either alone or in combination with rat SCs. Three months later, the animals were sacrificed and immunohistochemistry was performed to determine the survival of the xenografted neurons as well as microglial infiltration/activation. Cotransplantation of the SCs with the hNT neurons increased graft survival and was associated with an increase in graft size. Furthermore, there were fewer microglia present in the grafted tissue of the cotransplantation groups. These results show that SCs retain their immunosuppressive ability even within the brain. As immune responses to grafted neural tissue within the central nervous system become better understood, this ability of the SCs to provide localized immunosuppression to the transplanted tissue may become more important. This is particularly true as the search for alternative sources of neural tissue to treat neurodegenerative diseases expands to encompass other species.

Sertoli cells enhance the survival of co-transplanted dopamine neurons.

One of the major issues in neural transplantation is the low survival rate (<5%) of transplanted dopamine (DA) neurons [3]. Recently it has been shown that it is possible to enhance the survival of these neurons, which in turn may decrease the amount of tissue that is required for each transplantation patient. The present paper demonstrates a novel approach for enhancing neuronal survival by co-transplantation of neuronal tissue with Testis-derived Sertoli cells (SC). This strategy could improve neuronal survival through the provision of trophic support.

Sertoli cell transplants: their use in the treatment of neurodegenerative disease.

The efficacy of treating neurodegenerative diseases with the transplantation of fetal tissue has been demonstrated in animal models of Parkinson's disease, Huntington's disease and stroke. In the clinical setting, neural transplantation as a treatment for patients with Parkinson's disease has shown promising results. However, for this treatment method to be effective neuronal survival needs to be improved through either trophic support or localized immunoprotection. Co-transplanting Sertoli cells, which express many nutritive, regulatory, trophic and immunosuppressive factors, with fetal neural cells could provide both of these requirements. Such a strategy could enhance the recovery benefits associated with transplantation and decrease the need for, and the risks associated with, long-term systemic immunosuppression.

Development of Sertoli cell binding competency in the peripubertal rat.

The peripheral distribution of Sertoli cell F-actin, a cytoskeletal protein found in Sertoli cell ectoplasmic specializations, is associated with enhanced spermatid binding to the Sertoli cell and, as such, serves as a functional marker for its acquisition of binding competency. Previous studies suggest that the peripheral distribution of actin is dependent on follicle-stimulating hormone (FSH). To investigate the developmental pattern of Sertoli cell actin distribution in relation to peripubertal FSH and testosterone levels, we examined epithelial sheets from 2-8 week-old-rats. Tissues were processed for light microscopy and for the visualization of rhodamine-labeled F-actin. At 2 weeks, actin staining was diffuse throughout most of the Sertoli cells and was similar to that observed in binding-incompetent Sertoli cells. By 4 weeks, actin distribution was peripheral, acquiring the same staining pattern as observed in binding-competent Sertoli cells. Serum levels of FSH peaked at 4 weeks and declined to adult levels thereafter. Testosterone levels did not increase significantly until 6 weeks. Results show that Sertoli cell actin undergoes peripheral reorganization concurrent with the peripubertal peak of FSH but prior to the peripubertal rise of testosterone. The study demonstrates a temporal correlation between the peripubertal FSH rise and the actin redistribution in Sertoli cells that is consistent with an induction of this redistribution by FSH. These results suggest that FSH induces binding competency in Sertoli cells.