Defining the importance of phosphatidylserine synthase 2 in mice

Phosphatidylserine synthase 1 (Pss1) and phosphatidylserine synthase 2 (Pss2) produce phosphatidylserine by exchanging serine for the head groups of other phospholipids. Pss1 and Pss2 are structurally similar (approximately 32% amino acid identity) but differ in their substrate specificities, with Pss1 using phosphatidylcholine for the serine exchange reaction and Pss2 using phosphatidylethanolamine. Whether Pss1 and Pss2 are both required for mammalian growth and development is not known, and no data exist on the relative contributions of the two enzymes to serine exchange activities in different tissues. To address those issues and also to define the cell type-specific expression of Pss2, we generated Pss2-deficient mice in which a beta-galactosidase marker is expressed from Pss2 regulatory sequences. Histologic studies of Pss2-deficient mice revealed very high levels of beta-galactosidase expression in Sertoli cells of the testis and high levels of expression in brown fat, neurons, and myometrium. The ability of testis extracts from Pss2-deficient mice to catalyze serine exchange was reduced by more than 95%; reductions of approximately 90% were noted in the brain and liver. However, we found no perturbations in the phospholipid content of any of these tissues. As judged by Northern blots, the expression of Pss1 was not up-regulated in Pss2-deficient cells and tissues. Testis weight was reduced in Pss2-deficient mice, and some of the male mice were infertile. We conclude that Pss2 is responsible for the majority of serine exchange activity in in vitro assays, but a deficiency in this enzyme does not cause perturbations in phospholipid content or severe developmental abnormalities.     

The integrin binding site 2 (IBS2) in the talin rod domain is essential for linking integrin beta subunits to the cytoskeleton

Talin1 is a large cytoskeletal protein that links integrins to actin filaments through two distinct integrin binding sites, one present in the talin head domain (IBS1) necessary for integrin activation and a second (IBS2) that we have previously mapped to talin residues 1984-2113 (fragment J) of the talin rod domain (1 Tremuth, L., Kreis, S., Melchior, C., Hoebeke, J., Ronde, P., Plancon, S., Takeda, K., and Kieffer, N. (2004) J. Biol. Chem. 279, 22258-22266), but whose functional role is still elusive. Using a bioinformatics and cell biology approach, we have determined the minimal structure of IBS2 and show that this integrin binding site corresponds to 23 residues located in alpha helix 50 of the talin rod domain (residues 2077-2099). Alanine mutation of 2 highly conserved residues (L2094A/I2095A) within this alpha helix, which disrupted the alpha-helical structure of IBS2 as demonstrated by infrared spectroscopy and limited trypsin proteolysis, was sufficient to prevent in vivo talin fragment J targeting to alphaIIbbeta3 integrin in focal adhesions and to inhibit in vitro this association as shown by an alphaIIbbeta3 pulldown assay. Moreover, expression of a full-length mouse green fluorescent protein-talin LI/AA mutant in mouse talin1(-/-) cells was unable to rescue the inability of these cells to assemble focal adhesions (in contrast to green fluorescent protein-talin wild type) despite the presence of IBS1. Our data provide the first direct evidence that IBS2 in the talin rod is essential to link integrins to the cytoskeleton.     

Expression of the A-raf proto-oncogene in the normal adult and embryonic mouse.

We have determined the expression pattern of the A-raf proto-oncogene in the embryonic and adult mouse. Western blot analysis of protein lysates from tissues of adult mice show that p69A-raf is ubiquitously expressed, but that levels of expression vary among different tissues. To determine the cell-specific expression pattern of A-raf, we generated transgenic mice expressing the beta-galactosidase reporter gene from the A-raf promoter. We show that A-raf expression is highly specific within a given tissue, and we identify cell types expressing this gene in the adult testis, epididymis, vas deferens, seminal vesicle, ovary, oviduct, bladder, kidney, intestine, heart, spleen, thymus, and cerebellum. In the embryo, ubiquitous expression of the reporter gene is observed, but the highest levels of expression are specifically detected in the embryonic heart at stages 9.5-11.5 days post-coitum.     

Targeted disruption of the neurochondrin/norbin gene results in embryonic lethality

Neurochondrin/norbin is a cytoplasmic protein involved in dendrite outgrowth. The expression of the gene has been restricted to neural, bone, and chondral tissues. To identify the functions of the gene in vivo, we have generated mice with a disrupted mutation in the neurochondrin/norbin gene. Histological analysis of heterozygous mutant mice indicates the possibility of specific functions of neurochondrin/norbin in chondrocyte differentiation. We defined the expression patterns of neurochondrin/norbin-lacZ fusion protein in the central nervous system. In the developing olfactory bulb, beta-galactosidase activity was detected in the mantle layer at 12.5 dpc and the strongest activity was detected in the presumptive mitral or tufted cell layer at 15.5 dpc. beta-Galactosidase activity was also detected in the lateral choroid plexus. In homozygous (-/-) mutant mice, the disruption of the neurochondrin/norbin gene leads to early embryonic death between 3.5 and 6.5 dpc. This result indicates that neurochondrin/norbin gene function is essential for the early embryogenesis.     

Differential functions of the Aurora-B and Aurora-C kinases in mammalian spermatogenesis

The Aurora kinases are cell cycle-regulatory serine-threonine kinases that have been implicated in the function of the centrosomes, kinetechores, chromosome dynamics, and cytokinesis. In comparison with other tissues, there are high levels of expression of Aurora-B and -C in testis. What their respective roles in mammalian spermatogenesis are is an open question. Here we describe the expression and distribution patterns of the three kinases in mouse testis using in situ hybridization and immunohistochemistry. Importantly, the localization of Aurora-B is tightly regulated during spermatogenesis, whereas Aurora-C expression appears to be testis specific. To address the function of Aurora-B in spermatogenesis, we have generated transgenic mice using a pachytene-stage-specific promoter driving the expression of either wild-type Aurora-B or an inactive form of the kinase. Expression of the inactive Aurora-B results in abnormal spermatocytes, increased apoptosis, spermatogenic arrest, and subfertility defects. The function of Aurora-C may also be targeted in the Aurora-B transgenic mutants. To address the function of Aurora-C in testis, we generated Aurora-C knockout mice by homologous recombination. Remarkably, Aurora-C null mice were viable, yet the males had compromised fertility. Aurora-C mutant sperm display abnormalities that included heterogenous chromatin condensation, loose acrosomes, and blunted heads. These findings indicate that Aurora-B and Aurora-C serve specialized functions in mammalian spermatogenesis.     

Muscle glycogen synthase isoform is responsible for testicular glycogen synthesis: Glycogen overproduction induces apoptosis in male germ cells

Glycogen is the main source of glucose for many biological events. However, this molecule may have other functions, including those that have deleterious effects on cells. The rate‐limiting enzyme in glycogen synthesis is glycogen synthase (GS). It is encoded by two genes, GYS1, expressed in muscle (muscle glycogen synthase, MGS) and other tissues, and GYS2, primarily expressed in liver (liver glycogen synthase, LGS). Expression of GS and its activity have been widely studied in many tissues. To date, it is not clear which GS isoform is responsible for glycogen synthesis and the role of glycogen in testis. Using RT‐PCR, Western blot and immunofluorescence, we have detected expression of MGS but not LGS in mice testis during development. We have also evaluated GS activity and glycogen storage at different days after birth and we show that both GS activity and levels of glycogen are higher during the first days of development. Using RT‐PCR, we have also shown that malin and laforin are expressed in testis, key enzymes for regulation of GS activity. These proteins form an active complex that regulates MGS by poly‐ubiquitination in both Sertoli cell and male germ cell lines. In addition, PTG overexpression in male germ cell line triggered apoptosis by caspase3 activation, proposing a proapoptotic role of glycogen in testis. These findings suggest that GS activity and glycogen synthesis in testis could be regulated and a disruption of this process may be responsible for the apoptosis and degeneration of seminiferous tubules and possible cause of infertility. J. Cell. Biochem. 114: 1653–1664, 2013. © 2013 Wiley Periodicals, Inc.     

Spermatogenesis and testis development are normal in mice lacking testicular orphan nuclear receptor 2

Early in vitro cell culture studies suggested that testicular orphan nuclear receptor 2 (TR2), a member of the nuclear receptor superfamily, may play important roles in the control of several pathways including retinoic acids, vitamin D, thyroid hormones, and ciliary neurotrophic factor. Here we report the surprising results showing that mice lacking TR2 are viable and have no serious developmental defects. Male mice lacking TR2 have functional testes, including normal sperm number and motility, and both male and female mice lacking TR2 are fertile. In heterozygous TR2(+/-) male mice we found that beta-galactosidase, the indicator of TR2 protein expression, was first detected at the age of 3 weeks and its expression pattern was restricted mainly in the spermatocytes and round spermatids. These protein expression patterns were further confirmed with Northern blot analysis of TR2 mRNA expression. Together, results from TR2-knockout mice suggest that TR2 may not play essential roles in spermatogenesis and normal testis development, function, and maintenance. Alternatively, the roles of TR2 may be redundant and could be played by other close members of the nuclear receptor superfamily such as testicular orphan receptor 4 (TR4) or unidentified orphan receptors that share many similar functions with TR2. Further studies with double knockouts of both orphan nuclear receptors, TR2 and TR4, may reveal their real physiological roles.     

Monophosphoryl lipid A alleviated radiation-induced testicular injury through TLR4-dependent exosomes

Radiation protection on male testis is an important task for ionizing radiation-related workers or people who receive radiotherapy for tumours near the testicle. In recent years, Toll-like receptors (TLRs), especially TLR4, have been widely studied as a radiation protection target. In this study, we detected that a low-toxicity TLR4 agonist monophosphoryl lipid A (MPLA) produced obvious radiation protection effects on mice testis. We found that MPLA effectively alleviated testis structure damage and cell apoptosis induced by ionizing radiation (IR). However, as the expression abundance differs a lot in distinct cells and tissues, MPLA seemed not to directly activate TLR4 singling pathway in mice testis. Here, we demonstrated a brand new mechanism for MPLA producing radiation protection effects on testis. We observed a significant activation of TLR4 pathway in macrophages after MPLA stimulation and identified significant changes in macrophage-derived exosomes protein expression. We proved that after MPLA treatment, macrophage-derived exosomes played an important role in testis radiation protection, and specially, G-CSF and MIP-2 in exosomes are the core molecules in this protection effect.     

Talin contains a C-terminal calpain2 cleavage site important in focal adhesion dynamics

Talin is a large (～2540 residues) dimeric adaptor protein that associates with the integrin family of cell adhesion molecules in cell-extracellular matrix junctions (focal adhesions; FAs), where it both activates integrins and couples them to the actin cytoskeleton. Calpain2-mediated cleavage of talin between the head and rod domains has previously been shown to be important in FA turnover. Here we identify an additional calpain2-cleavage site that removes the dimerisation domain from the C-terminus of the talin rod, and show that an E2492G mutation inhibits calpain cleavage at this site in vitro, and increases the steady state levels of talin1 in vivo. Expression of a GFP-tagged talin1 E2492G mutant in CHO.K1 cells inhibited FA turnover and the persistence of cell protrusion just as effectively as a L432G mutation that inhibits calpain cleavage between the talin head and rod domains. Moreover, incorporation of both mutations into a single talin molecule had an additive effect clearly demonstrating that calpain cleavage at both the N- and C-terminal regions of talin contribute to the regulation of FA dynamics. However, the N-terminal site was more sensitive to calpain cleavage suggesting that lower levels of calpain are required to liberate the talin head and rod fragments than are needed to clip off the C-terminal dimerisation domain. The talin head and rod liberated by calpain2 cleavage have recently been shown to play roles in an integrin activation cycle important in FA turnover and in FAK-dependent cell cycle progression respectively. The half-life of the talin head is tightly regulated by ubiquitination and we suggest that removal of the C-terminal dimerisation domain from the talin rod may provide a mechanism both for terminating the signalling function of the talin rod and indeed for inactivating full-length talin thereby promoting FA turnover at the rear of the cell.     

Impaired spermatogenesis and fertility in mice carrying a mutation in the Spink2 gene expressed predominantly in testes

Spermatogenesis is a complex process involving an intrinsic genetic program composed of germ cell-specific and -predominant genes. In this study, we investigated the mouse Spink2 (serine protease inhibitor Kazal-type 2) gene, which belongs to the SPINK family of proteins characterized by the presence of a Kazal-type serine protease inhibitor-pancreatic secretory trypsin inhibitor domain. We showed that recombinant mouse SPINK2 has trypsin-inhibitory activity. Distribution analyses revealed that Spink2 is transcribed strongly in the testis and weakly in the epididymis, but is not detected in other mouse tissues. Expression of Spink2 is specific to germ cells in the testis and is first evident at the pachytene spermatocyte stage. Immunoblot analyses demonstrated that SPINK2 protein is present in male germ cells at all developmental stages, including in testicular spermatogenic cells, testicular sperm, and mature sperm. To elucidate the functional role of SPINK2 in vivo, we generated mutant mice with diminished levels of SPINK2 using a gene trap mutagenesis approach. Mutant male mice exhibit significantly impaired fertility; further phenotypic analyses revealed that testicular integrity is disrupted, resulting in a reduction in sperm number. Moreover, we found that testes from mutant mice exhibit abnormal spermatogenesis and germ cell apoptosis accompanied by elevated serine protease activity. Our studies thus provide the first demonstration that SPINK2 is required for maintaining normal spermatogenesis and potentially regulates serine protease-mediated apoptosis in male germ cells.     

Impaired fertility and spermiogenetic disorders with loss of cell adhesion in male mice expressing an interfering Rap1 mutant

The guanosine trisphosphatase Rap1 serves as a critical player in signal transduction, somatic cell proliferation and differentiation, and cell-cell adhesion by acting through distinct mechanisms. During mouse spermiogenesis, Rap1 is activated and forms a signaling complex with its effector, the serine-threonine kinase B-Raf. To investigate the functional role of Rap1 in male germ cell differentiation, we generated transgenic mice expressing an inactive Rap1 mutant selectively in differentiating spermatids. This expression resulted in a derailment of spermiogenesis due to an anomalous release of immature round spermatids from the seminiferous epithelium within the tubule lumen and in low sperm counts. These spermiogenetic disorders correlated with impaired fertility, with the transgenic males being severely subfertile. Because mutant testis exhibited perturbations in ectoplasmic specializations (ESs), a Sertoli-germ cell-specific adherens junction, we searched for expression of vascular endothelial cadherin (VE-cadherin), an adhesion molecule regulated by Rap1, in spermatogenic cells of wild-type and mutant mice. We found that germ cells express VE-cadherin with a timing strictly related to apical ES formation and function; immature, VE-cadherin-positive spermatids were, however, prematurely released in the transgenic testis. In conclusion, interfering with Rap1 function during spermiogenesis leads to reduced fertility by impairment of germ-Sertoli cell contacts; our transgenic mouse provides an in vivo model to study the regulation of ES dynamics.