Apoptotic spermatogenic cells can be energy sources for Sertoli cells.

Apoptotic spermatogenic cells and residual bodies are phagocytosed and degraded by Sertoli cells during mammalian spermatogenesis. The meaning of this event remains to be clarified. In this report, we demonstrate that apoptotic spermatogenic cells and residual bodies can be used to produce ATP by Sertoli cells after phagocytosis of them. Sertoli cells produced the highest level of ATP compared with other testicular cells. Phagocytosis assay in vitro showed that engulfment of apoptotic spermatogenic cells increases ATP production by Sertoli cells. The increased ATP production was detected in seminiferous tubules at the stages where phagocytosis occurs. Induced apoptosis of spermatogenic cells in vivo increased ATP production in seminiferous tubules. The augmentation of ATP production both in vitro and in vivo associated with the lipid formation in Sertoli cells after phagocytosis of apoptotic spermatogenic cells. The lipid beta-oxidation was a predominant pathway to produce ATP in Sertoli cells. We conclude that after phagocytosis by Sertoli cells, apoptotic spermatogenic cells are degraded to form lipids that are then used to produce ATP. The results suggest that apoptotic spermatogenic cells can be energy sources for Sertoli cells that may define a novel meaning of spermatogenic cell death.

Functions of TAM RTKs in regulating spermatogenesis and male fertility in mice.

Mice lacking TYRO3, AXL and MER (TAM) receptor tyrosine kinases (RTKs) are male sterile. The mechanism of TAM RTKs in regulating male fertility remains unknown. In this study, we analyzed in more detail the testicular phenotype of TAM triple mutant (TAM(-/-)) mice with an effort to understand the mechanism. We demonstrate that the three TAM RTKs cooperatively regulate male fertility, and MER appears to be more important than AXL and TYRO3. TAM(-/-) testes showed a progressive loss of germ cells from elongated spermatids to spermatogonia. Young adult TAM(-/-) mice exhibited oligo-astheno-teratozoospermia and various morphological malformations of sperm cells. As the mice aged, the germ cells were eventually depleted from the seminiferous tubules. Furthermore, we found that TAM(-/-) Sertoli cells have an impaired phagocytic activity and a large number of differentially expressed genes compared to wild-type controls. By contrast, the function of Leydig cells was not apparently affected by the mutation of TAM RTKs. Therefore, we conclude that the suboptimal function of Sertoli cells leads to the impaired spermatogenesis in TAM(-/-) mice. The results provide novel insight into the mechanism of TAM RTKs in regulating male fertility.

Expression patterns and functions of toll-like receptors in mouse sertoli cells.

Toll-like receptors (TLRs) play crucial roles in mediating innate and adaptive immunity. Sertoli cells create a microenvironment that protects seminiferous tubules from autoantigens and invading pathogens. Here we examined the expression and potential function of TLR family in mouse Sertoli cells. RT-PCR, Western blotting, and flow cytometry were used to analyze gene expression. Immunofluorescence staining was used to determine activation of nuclear factor-kappaB. ELISA was used to detect secreted cytokines in culture medium. The phagocytosis assay was performed by Oil Red O staining for lipid droplets. We demonstrated that TLR2, TLR3, TLR4, and TLR5 are highly expressed; TLR6, TLR7, and TLR13 are expressed at relatively low level; and TLR1, TLR8, TLR9, TLR11, and TLR12 are not detected in mouse Sertoli cells. We focused our study on the roles of TLR2-TLR5 in Sertoli cells. Our data indicated that TLR2-TLR5 can be activated by their ligands in mouse Sertoli cells and subsequently increase expression of the inflammatory cytokines IL-1alpha, IL-6, and interferon-alpha, and -beta. The augmented expression of the cytokines might be induced by activation of nuclear factor-kappaB. Notably, activation of TLR3 by its ligand, poly (I:C), specifically promoted phagocytosis of apoptotic spermatogenic cells by Sertoli cells. The TLR-induced Sertoli cell phagocytosis was found to be associated with the up-regulation of scavenger receptors. The results suggest that TLRs expressed in mouse Sertoli cells may play roles in defense against invasion of allo- and autoantigens in the seminiferous tubules.

The role of Tyro 3 subfamily receptors in the regulation of hemostasis and megakaryocytopoiesis.

BACKGROUND AND OBJECTIVES: The molecular mechanisms regulating megakaryocytopoiesis and hemostasis remain largely unknown. The Tyro 3 subfamily of receptor tyrosine kinases (RTK), which is composed of three members (Tyro 3, Axl and Mer), plays important roles in various tissues, such as those in the nervous, immune and reproductive systems. Here, we investigate the roles of the Tyro 3 RTK subfamily in regulating megakaryocytopoiesis and hemostasis. DESIGN AND METHODS: Single, double, and triple knock-out mice for the three Tyro 3 RTK were used in the study. Bleeding time, platelet count, megakaryocyte count, megakaryocyte ploidy, rate of proplatelet formation, platelet aggregation and ATP release were used as criteria to evaluate hemostasis, megakaryocytopoiesis and platelet function. RESULTS: Mice lacking all three receptors had impaired hemostasis and mild thrombocytopenia, which may be due to platelet dysfunction and defective megakaryocytopoiesis. Mice lacking different combinations of two receptors of the Tyro 3 RTK subfamily had normal platelet counts in peripheral blood, but exhibited impaired hemostasis and platelet function. Although knock-out mice for any single receptor had normal hemostasis and megakaryocytopoiesis, they exhibited a mild platelet dysfunction. INTERPRETATION AND CONCLUSIONS: The Tyro 3 RTK subfamily plays important roles in regulating hemostasis, megakaryocytopoiesis and platelet function.

Structure-Based Screen Identification of a Mammalian Ste20-like Kinase 4 (MST4) Inhibitor with Therapeutic Potential for Pituitary Tumors.

Pituitary tumors of the gonadotrope lineage are often large and invasive, resulting in hypopituitarism. No medical treatments are currently available. Using a combined genetic and genomic screen of individual human gonadotrope pituitary tumor samples, we recently identified the mammalian sterile-20 like kinase 4 (MST4) as a protumorigenic effector, driving increased pituitary cell proliferation and survival in response to a hypoxic microenvironment. To identify novel inhibitors of the MST4 kinase for potential future clinical use, computational-based virtual library screening was used to dock the SelleckChem kinase inhibitor library into the ATP-binding site of the MST4 crystal structure. Several inhibitor candidates were identified with the potential to bind with high affinity. Using a TR-FRET in vitro recombinant kinase assay, hesperadin, initially described as an Aurora kinase inhibitor, exhibited potent inhibition of the MST4 kinase at nanomolar concentrations. The LßT2 gonadotrope pituitary cell hypoxic model was used to test the ability of this inhibitor to antagonize MST4 actions. Under short-term severe hypoxia (1% O2), MST4 protection from hypoxia-induced apoptosis was abrogated in the presence of hesperadin. Similarly, under chronic hypoxia (5%), hesperadin blocked the proliferative and colony-forming actions of MST4 as well as the ability to activate specific downstream signaling and hypoxia-inducible factor-1 effectors. Together, these data identify hesperadin as the first potent, selective inhibitor of the MST4 kinase with the capacity to block pituitary tumor cell growth in a hypoxic microenvironment.

Mammalian Ste20-like kinase 4 promotes pituitary cell proliferation and survival under hypoxia.

The genetic and molecular mechanisms that initiate and maintain pituitary tumorigenesis are poorly understood. Nonfunctioning tumors of the gonadotrope lineage represent 35% of all tumors; are usually macroadenomas, often resulting in hypopituitarism; and have no medical treatments. Using expression microarrays combined with whole-genome copy number screens on individual human tumors, we identified the mammalian sterile-20-like kinase (MST4) transcript, which was amplified within chromosome Xq26.2 in one tumor and up-regulated in all gonadotrope tumor samples. MST4 mRNA and protein were consistently overexpressed in human tumors compared with normal pituitaries. To mimic the pituitary tumor microenvironment, a hypoxia model using LßT2 murine gonadotrope cells was created to examine the functional role of the kinase. During long-term hypoxia, MST4 expression increased colony formation in a soft agar assay and rates of cell proliferation by activating p38 MAPK and AKT. Under short-term severe hypoxic stress, MST4 decreased the rates of apoptosis via p38 MAPK, AKT, hypoxia-inducible factor-1, and its cell-specific downstream targets. Analysis of MST4 mutants confirmed the importance of the kinase sequence but not the regulatory C terminus for its functional effects. Together these data identify the MST4 kinase as a novel candidate to mediate human pituitary tumorigenesis in a hypoxic environment and position it as a potential therapeutic target.

Induction of TGF-ß1 synthesis by macrophages in response to apoptotic cells requires activation of the scavenger receptor CD36.

BACKGROUND/OBJECTIVE: Phosphatidylserine (PS) exposed on apoptotic cells has been shown to stimulate production of transforming growth factor-ß (TGF-ß) and promote anti-inflammatory responses. However, the PS receptor(s) responsible for this induction has not been clearly determined. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, using RAWTßRII cells in which a truncated dominant negative TGF-ß receptor II was stably transfected in order to avoid auto-feedback induction of TGF-ß, we show that TGF-ß1 synthesis is initiated via activation of the scavenger receptor, CD36. The response requires exposure of PS on the apoptotic cell surface and was absent in macrophages lacking CD36. Direct activation of CD36 with an anti-CD36 antibody initiated TGF-ß1 production, and signaling pathways involving both Lyn kinase and ERK1/2 were shown to participate in CD36-driven TGF-ß1 expression. CONCLUSION/SIGNIFICANCE: Since CD36 has been previously implicated in activation of secreted latent TGF-ß, the present study indicates its role in the multiple steps to generation of this important biological mediator.

Damaged spermatogenic cells induce inflammatory gene expression in mouse Sertoli cells through the activation of Toll-like receptors 2 and 4.

Testicular inflammation, including noninfectious inflammatory responses in the testis, may impair male fertility. Mechanisms underlying the initiation of noninfectious testicular inflammation are poorly understood. In the current study, we demonstrate that damaged spermatogenic cell products (DSCPs) induce expression of various inflammatory mediators, including TNF-α, IL-1ß, IL-6, and macrophage chemotactic protein 1 (MCP-1), in Sertoli cells. Notably, the DSCP-induced inflammatory gene expression was significantly reduced by knockout Toll-like receptor (TLR)2 or TLR4, and abolished by double knockout TLR2 and TLR4 (TLR2(-/-)TLR4(-/-)). MCP-1 secreted by Sertoli cells after stimulation with DSCPs promotes macrophage migration. We also provide evidence that busulfan-induced spermatogenic cell damages in vivo upregulate TNF-α and MCP-1 expression in Sertoli cells, and facilitate macrophage infiltration into the testis in wild-type mice. These phenomena were not observed in TLR2(-/-)TLR4(-/-) mice. Data indicate that DSCPs induce inflammatory gene expression in Sertoli cells via the activation of TLR2 and TLR4, which may initiate noninfectious inflammatory responses in the testis. The results provide novel insights into the mechanisms underlying damaged spermatogenic cell-induced testicular inflammation.

Gas6 and the Tyro 3 receptor tyrosine kinase subfamily regulate the phagocytic function of Sertoli cells.

The apoptotic spermatogenic cells and residual bodies are phagocytosed and degraded by Sertoli cells during spermatogenesis. The mechanisms of this process are largely unknown. Here, we demonstrate that Gas6 and its receptors, the Tyro 3 subfamily of receptor tyrosine kinases (RTKs; Tyro 3, Axl, and Mer), regulate the phagocytic function of Sertoli cells. The phagocytic ability of Sertoli cells increased by five times in the presence of Gas6 in serum-free medium when compared with controls. The Sertoli cells lacking Mer showed a 35% reduction in phagocytosis of apoptotic spermatogenic cells when compared with wild-type (WT) controls, whereas the Sertoli cells lacking Tyro 3 or Axl exhibited phagocytic activity comparable with the controls. Notably, the Sertoli cells lacking all three members of the Tyro 3 RTK subfamily showed a dramatic decrease in phagocytic ability of 7.6-fold when compared with WT Sertoli cells. The deficiency in phagocytosis by the triple-mutant Sertoli cells was due to the deficit in binding of the Sertoli cells to apoptotic germ cells. These findings suggest that Mer is responsible for triggering phagocytosis of apoptotic spermatogenic cells by Sertoli cells and that Tyro 3, Axl, and Mer participate in recognizing and binding apoptotic germ cells by Sertoli cells in a redundant manner. Gas6 is a functional ligand of the Tyro 3 RTK subfamily in mediating phagocytic ability of Sertoli cells.

Evaluation on the phagocytosis of apoptotic spermatogenic cells by Sertoli cells in vitro through detecting lipid droplet formation by Oil Red O staining.

During spermatogenesis, more than half of the differentiating spermatogenic cells undergo apoptosis before they mature into spermatozoa. Ultrastructure studies showed that the formation of lipid droplets in Sertoli cells was associated with phagocytosis of residual bodies and apoptotic germ cells by Sertoli cells. Here, a relationship between the phagocytosis of apoptotic spermatogenic cells and lipid droplet formation in Sertoli cells was studied in vitro by Oil Red O (ORO) staining. The results confirmed that the formation of lipid droplets was a result of phagocytosis of apoptotic spermatogenic cells in Sertoli cells. By comparing phagocytosis of apoptotic spermatogenic cells and thymocytes by Sertoli cells to that by macrophages, we demonstrated that the lipid droplets accumulation in phagocytes depended on phagocytosed apoptotic cell type, but not phagocyte type. However, the size of lipid droplets was related to the type of phagocytes. By this approach, we found that Sertoli cells at different postnatal stages of development had a similar phagocytic ability. These results suggested that the detection of lipid droplets by ORO staining was a practical method to evaluate the phagocytic functions of Sertoli cells in vitro. This approach could also be considered as an in vitro model to study the lipid formation, metabolism, and function in Sertoli cells.