RAB-like 2 has an essential role in male fertility, sperm intra-flagellar transport, and tail assembly.

A significant percentage of young men are infertile and, for the majority, the underlying cause remains unknown. Male infertility is, however, frequently associated with defective sperm motility, wherein the sperm tail is a modified flagella/cilia. Conversely, a greater understanding of essential mechanisms involved in tail formation may offer contraceptive opportunities, or more broadly, therapeutic strategies for global cilia defects. Here we have identified Rab-like 2 (RABL2) as an essential requirement for sperm tail assembly and function. RABL2 is a member of a poorly characterized clade of the RAS GTPase superfamily. RABL2 is highly enriched within developing male germ cells, where it localizes to the mid-piece of the sperm tail. Lesser amounts of Rabl2 mRNA were observed in other tissues containing motile cilia. Using a co-immunoprecipitation approach and RABL2 affinity columns followed by immunochemistry, we demonstrated that within developing haploid germ cells RABL2 interacts with intra-flagella transport (IFT) proteins and delivers a specific set of effector (cargo) proteins, including key members of the glycolytic pathway, to the sperm tail. RABL2 binding to effector proteins is regulated by GTP. Perturbed RABL2 function, as exemplified by the Mot mouse line that contains a mutation in a critical protein-protein interaction domain, results in male sterility characterized by reduced sperm output, and sperm with aberrant motility and short tails. Our data demonstrate a novel function for the RABL protein family, an essential role for RABL2 in male fertility and a previously uncharacterised mechanism for protein delivery to the flagellum.

Cysteine-rich secretory protein 4 is an inhibitor of transient receptor potential M8 with a role in establishing sperm function.

The cysteine-rich secretory proteins (CRISPs) are a group of four proteins in the mouse that are expressed abundantly in the male reproductive tract, and to a lesser extent in other tissues. Analysis of reptile CRISPs and mouse CRISP2 has shown that CRISPs can regulate cellular homeostasis via ion channels. With the exception of the ability of CRISP2 to regulate ryanodine receptors, the in vivo targets of mammalian CRISPs function are unknown. In this study, we have characterized the ion channel regulatory activity of epididymal CRISP4 using electrophysiology, cell assays, and mouse models. Through patch-clamping of testicular sperm, the CRISP4 CRISP domain was shown to inhibit the transient receptor potential (TRP) ion channel TRPM8. These data were confirmed using a stably transfected CHO cell line. TRPM8 is a major cold receptor in the body, but is found in other tissues, including the testis and on the tail and head of mouse and human sperm. Functional assays using sperm from wild-type mice showed that TRPM8 activation significantly reduced the number of sperm undergoing the progesterone-induced acrosome reaction following capacitation, and that this response was reversed by the coaddition of CRISP4. In accordance, sperm from Crisp4 null mice had a compromised ability to undergo to the progesterone-induced acrosome reaction. Collectively, these data identify CRISP4 as an endogenous regulator of TRPM8 with a role in normal sperm function.

The dendritic cell subtype-restricted C-type lectin Clec9A is a target for vaccine enhancement.

A novel dendritic cell (DC)-restricted molecule, Clec9A, was identified by gene expression profiling of mouse DC subtypes. Based on sequence similarity, a human ortholog was identified. Clec9A encodes a type II membrane protein with a single extracellular C-type lectin domain. Both the mouse Clec9A and human CLEC9A were cloned and expressed, and monoclonal antibodies (mAbs) against each were generated. Surface staining revealed that Clec9A was selective for mouse DCs and was restricted to the CD8(+) conventional DC and plasmacytoid DC subtypes. A subset of human blood DCs also expressed CLEC9A. A single injection of mice with a mAb against Clec9A, which targets antigens (Ags) to the DCs, produced a striking enhancement of antibody responses in the absence of added adjuvants or danger signals, even in mice lacking Toll-like receptor signaling pathways. Such targeting also enhanced CD4 and CD8 T-cell responses. Thus, Clec9A serves as a new marker to distinguish subtypes of both mouse and human DCs. Furthermore, targeting Ags to DCs with antibodies to Clec9A is a promising strategy to enhance the efficiency of vaccines, even in the absence of adjuvants.

Perilipin 5 Deletion in Hepatocytes Remodels Lipid Metabolism and Causes Hepatic Insulin Resistance in Mice.

Defects in hepatic lipid metabolism cause nonalcoholic fatty liver disease and insulin resistance, and these pathologies are closely linked. Regulation of lipid droplet metabolism is central to the control of intracellular fatty acid fluxes, and perilipin 5 (PLIN5) is important in this process. We examined the role of PLIN5 on hepatic lipid metabolism and systemic glycemic control using liver-specific Plin5-deficient mice (Plin5LKO ). Hepatocytes isolated from Plin5LKO mice exhibited marked changes in lipid metabolism characterized by decreased fatty acid uptake and storage, decreased fatty acid oxidation that was associated with reduced contact between lipid droplets and mitochondria, and reduced triglyceride secretion. With consumption of a high-fat diet, Plin5LKO mice accumulated intrahepatic triglyceride, without significant changes in inflammation, ceramide or diglyceride contents, endoplasmic reticulum stress, or autophagy. Instead, livers of Plin5LKO mice exhibited activation of c-Jun N-terminal kinase, impaired insulin signal transduction, and insulin resistance, which impaired systemic insulin action and glycemic control. Re-expression of Plin5 in the livers of Plin5LKO mice reversed these effects. Together, we show that Plin5 is an important modulator of intrahepatic lipid metabolism and suggest that the increased Plin5 expression that occurs with overnutrition may play an important role in preventing hepatic insulin resistance.

Obesity does not promote tumorigenesis of localized patient-derived prostate cancer xenografts.

There are established epidemiological links between obesity and the severity of prostate cancer. We directly tested this relationship by assessing tumorigenicity of patient-derived xenografts (PDXs) of moderate-grade localized prostate cancer in lean and obese severe combined immunodeficiency (SCID) mice. Mice were rendered obese and insulin resistant by high-fat feeding for 6 weeks prior to transplantation, and PDXs were assessed 10 weeks thereafter. Histological analysis of PDX grafts showed no differences in tumor pathology, prostate-specific antigen, androgen receptor and homeobox protein Nkx-3.1 expression, or proliferation index in lean versus obese mice. Whilst systemic obesity per se did not promote prostate tumorigenicity, we next asked whether the peri-prostatic adipose tissue (PPAT), which covers the prostate anteriorly, plays a role in prostate tumorigenesis. In vitro studies in a cellularized co-culture model of stromal and epithelial cells demonstrated that factors secreted from human PPAT are pro-tumorigenic. Accordingly, we recapitulated the prostate-PPAT spatial relationship by co-grafting human PPAT with prostate cancer in PDX grafts. PDX tissues were harvested 10 weeks after grafting, and histological analysis revealed no evidence of enhanced tumorigenesis with PPAT compared to prostate cancer grafts alone. Altogether, these data demonstrate that prostate cancer tumorigenicity is not accelerated in the setting of diet-induced obesity or in the presence of human PPAT, prompting the need for further work to define the at-risk populations of obesity-driven tumorigenesis and the biological factors linking obesity, adipose tissue and prostate cancer pathogenesis.

Fetuin B Is a Secreted Hepatocyte Factor Linking Steatosis to Impaired Glucose Metabolism.

Liver steatosis is associated with the development of insulin resistance and the pathogenesis of type 2 diabetes. We tested the hypothesis that protein signals originating from steatotic hepatocytes communicate with other cells to modulate metabolic phenotypes. We show that the secreted factors from steatotic hepatocytes induce pro-inflammatory signaling and insulin resistance in cultured cells. Next, we identified 168 hepatokines, of which 32 were differentially secreted in steatotic versus non-steatotic hepatocytes. Targeted analysis showed that fetuin B was increased in humans with liver steatosis and patients with type 2 diabetes. Fetuin B impaired insulin action in myotubes and hepatocytes and caused glucose intolerance in mice. Silencing of fetuin B in obese mice improved glucose tolerance. We conclude that the protein secretory profile of hepatocytes is altered with steatosis and is linked to inflammation and insulin resistance. Therefore, preventing steatosis may limit the development of dysregulated glucose metabolism in settings of overnutrition.

Glutamine or glucose starvation in hybridoma cultures induces death receptor and mitochondrial apoptotic pathways.

Glutamine and glucose are often controlled at low levels in fed-batch strategies to limit ammonia and lactate accumulation and improve productivity of mammalian cell cultures. However, this risks triggering apoptosis if cells are depleted of glutamine or glucose. To examine the apoptosis cascade during glutamine or glucose limitation, the transcriptional profile of FAS, FASL, FADD, FLIP, BAX, p53 and PEG3 in CRL 1606 hybridoma culture was investigated using quantitative real-time PCR. Activities of caspases 2, 3, 8 and 9 were also analyzed. Increase in the activities of the caspases was observed with up-regulation in the expression of FAS (6-8-fold) and PEG3 (2.5-fold), suggesting that the cells experienced apoptotic cell death via both the death receptor and mitochondrial pathways.