Phosphorylation of mouse sperm axoneme central apparatus protein SPAG16L by a testis-specific kinase, TSSK2.

The mammalian protein SPAG16L, the ortholog of Chlamydomonas Pf20, is an axoneme central apparatus protein necessary for flagellar motility. The SPAG16L protein sequence contains multiple potential phosphorylation sites, and the protein was confirmed to be phosphorylated in vivo. A yeast two-hybrid screen identified the testis-specific kinase, TSSK2, to be a potential SPAG16L binding partner. SPAG16L and TSSK2 interactions were confirmed by coimmunoprecipitation of both proteins from testis extracts and cell lysates expressing these proteins, and their colocalization was also noted by confocal microscopy in Chinese hamster ovary cells, where they were coexpressed. TSSK2 associates with SPAG16L via its C-terminal domain bearing WD repeats. The N-terminal domain containing a coiled coil motif does not associate with TSSK2. SPAG16L can be phosphorylated by TSSK2 in vitro. Finally, TSSK2 is absent or markedly reduced from the testes in most of the SPAG16L-null mice. These data support the conclusion that SPAG16L is a TSSK2 substrate.

Seneca Valley virus, a systemically deliverable oncolytic picornavirus, and the treatment of neuroendocrine cancers.

BACKGROUND: Numerous clinical trials have demonstrated that oncolytic viruses can elicit antitumor responses when they are administered directly into localized cancers. However, the treatment of metastatic disease with oncolytic viruses has been challenging due to the inactivation of viruses by components of human blood and/or to inadequate tumor selectivity. METHODS: We determined the cytolytic potential and selectivity of Seneca Valley Virus-001 (SVV-001), a newly discovered native picornavirus, in neuroendocrine and pediatric tumor cell lines and normal cells. Suitability of the virus for intravenous delivery in humans was assessed by blood inactivation assays. Safety was evaluated in vivo using an immune-competent mouse model, and efficacy was evaluated in vivo in athymic mice bearing tumors derived from human small-cell lung cancer and retinoblastoma cell lines. RESULTS: Cell lines derived from small-cell lung cancers and solid pediatric cancers were at least 10,000-fold more sensitive to the cytolytic activity of SVV-001 than were any of the adult normal human cells tested. Viral infectivity was not inhibited by human blood components. Intravenous doses up to 1 x 10(14) virus particles (vp) per kg were well tolerated, and no dose-limiting toxicity was observed in immune-competent mice. A single intravenous dose of 1 x 10(8) vp per kg into athymic mice bearing preestablished small-cell lung or retinoblastoma tumors resulted in complete, durable responses in ten of ten and five of eight mice, respectively. CONCLUSIONS: SVV-001 has potent cytolytic activity and high selectivity for tumor cell lines having neuroendocrine properties versus adult normal cells. Systemically administered SVV-001 has potential for the treatment of metastatic neuroendocrine cancers.

Protein domains govern the intracellular distribution of mouse sperm AKAP4.

A-kinase anchor proteins (AKAPs) spatially restrict cAMP-dependent protein kinase by tethering it to various cellular structures. In the polarized sperm cell, various compartmentalized functions, such as motility generated by the flagellum, are modulated by cAMP-dependent protein kinase. This important regulatory enzyme is associated with AKAP4, the principal component of the fibrous sheath; AKAP4 is synthesized as a precursor, pro-AKAP4, which is cleaved into mature AKAP4 during fibrous sheath assembly. To define the domains responsible for the intracellular distribution and assembly of AKAP4 into a macromolecular complex, various AKAP4-green fluorescent protein (GFP) constructs were introduced into somatic cell lines. The presence of the pro domain, either alone or as part of pro-AKAP4, resulted in a diffuse cytoplasmic localization of the GFP fusion protein, suggesting that, the pro domain keeps the AKAP4 precursor unassembled in vivo until it is transported to the developing tail structure and incorporated into the fibrous sheath. When the mature AKAP4-GFP fusion protein was expressed, it localized in a punctate cytoplasmic pattern. Two domains critical for this punctate localization, T2a and T2b, are homologous to the T2-tethering domain of rat AKAP5 that is important for binding to the actin cytoskeleton in transfected HEK293 cells. In contrast to AKAP5, the distribution of AKAP4 was dependent on the microtubular cytoskeleton. The interaction of AKAP4 with the microtubular network provides evidence that the longitudinal columns of the fibrous sheath, which contain AKAP4, may interact directly with the outer microtubular doublets of the sperm axoneme.

The "soluble" adenylyl cyclase in sperm mediates multiple signaling events required for fertilization.

Mammalian fertilization is dependent upon a series of bicarbonate-induced, cAMP-dependent processes sperm undergo as they "capacitate," i.e., acquire the ability to fertilize eggs. Male mice lacking the bicarbonate- and calcium-responsive soluble adenylyl cyclase (sAC), the predominant source of cAMP in male germ cells, are infertile, as the sperm are immotile. Membrane-permeable cAMP analogs are reported to rescue the motility defect, but we now show that these "rescued" null sperm were not hyperactive, displayed flagellar angulation, and remained unable to fertilize eggs in vitro. These deficits uncover a requirement for sAC during spermatogenesis and/or epididymal maturation and reveal limitations inherent in studying sAC function using knockout mice. To circumvent this restriction, we identified a specific sAC inhibitor that allowed temporal control over sAC activity. This inhibitor revealed that capacitation is defined by separable events: induction of protein tyrosine phosphorylation and motility are sAC dependent while acrosomal exocytosis is not dependent on sAC.

Dissecting the axoneme interactome: the mammalian orthologue of Chlamydomonas PF6 interacts with sperm-associated antigen 6, the mammalian orthologue of Chlamydomonas PF16.

The axoneme central apparatus is thought to control flagellar/ciliary waveform and maintain the structural integrity of the axoneme, but proteins involved in these processes have not been fully elucidated. Moreover the network of interactions among them that allows these events to take place in a compact space has not been defined. PF6, a component of the Chlamydomonas central apparatus, is localized to the 1a projection of the C1 microtubule. Mutations in the Chlamydomonas PF6 gene result in flagellar paralysis. We characterized human and murine orthologues of PF6. The murine Pf6 gene is expressed in a pattern consistent with a role in flagella and cilia, and the PF6 protein is indeed localized to the central apparatus of the sperm flagellar axoneme. We discovered that a portion of PF6 associates with the mammalian orthologue of Chlamydomonas PF16 (sperm-associated antigen 6 (SPAG6)), another central apparatus protein that is localized to the C1 microtubule in algae. A fragment of PF6 corresponding to the PF6 domain that interacts with SPAG6 in yeast two-hybrid assays and colocalizes with SPAG6 in transfected cells was missing from epididymal sperm of SPAG6-deficient mice. SPAG6 binds to the mammalian orthologue of PF20, which in Chlamydomonas is located in bridges connecting the C2 and C1 microtubules. Thus, PF6, SPAG6, and PF20 form a newly identified network that links together components of the axoneme central apparatus and presumably participates in its dynamic regulation of ciliary and flagellar beat.

Patterns of expression of sperm flagellar genes: early expression of genes encoding axonemal proteins during the spermatogenic cycle and shared features of promoters of genes encoding central apparatus proteins.

Sperm are motile cells. Thus, a significant component of the spermatogenic cycle is devoted to the formation of flagellum, a process that must be coordinated to insure proper construction. To document the temporal pattern of flagellar gene expression, we employed real-time PCR to assess changes in accumulation of a cohort of genes encoding axoneme, outer dense fibre (ODF) and fibrous sheath (FS) proteins during the first wave of spermatogenesis in the mouse. Axoneme genes were expressed first at the pachytene spermatocyte stage, followed by expression of transcripts encoding ODF and FS components. However, there were differences among these families with respect to the time of initial expression and the rate of mRNA accumulation. To gain understanding of factors that determine these patterns of expression, we cloned the promoters of three axoneme central apparatus genes (Pf6, Spag6 and Pf20). These promoters shared common features including the absence of a TATA box, and putative binding sites for several factors implicated in spermatogenesis (CREB/CREM, SOX17 and SPZ1) as well as ciliogenesis (FOXJ1). Collectively, our findings demonstrate a sequential pattern of expression of flagellar component genes, differential times of expression or rates of transcript accumulation within each class and shared promoter features within a class.

Haploinsufficiency for the murine orthologue of Chlamydomonas PF20 disrupts spermatogenesis.

PF20 was first identified in Chlamydomonas rheinhardtii as an essential component of the axoneme central apparatus. We discovered that the mouse Pf20 gene encodes two major transcripts (2.5 and 1.4 kb), which are expressed in different patterns during spermatogenesis, yielding proteins of 71 and 35 kDa, respectively. Both proteins contain contiguous WD repeats in their C termini. The meiotically expressed 71-kDa protein is incorporated into the central apparatus, whereas the 35-kDa protein, which accumulates in postmeiotic male germ cells, is abundant in the nucleus. We disrupted the Pf20 gene domains that encode the C-terminal WD repeats in embryonic stem cells. Highly chimeric mice carrying the mutant Pf20 allele had impaired spermatogenesis with a significant loss of germ cells at the round spermatid stage, in association with disorganization of sperm axoneme structure. The mutated Pf20 allele was never transmitted, indicating that Pf20 haploinsufficiency caused the defects in spermatogenesis. The 35-kDa PF20 protein was shown to bind to meiosis-expressed gene 1 (MEIG1), a chromosome/chromatin-binding protein initially expressed during meiosis but retained in the germ cell nucleus throughout later stages of spermatogenesis. Our findings reveal an essential role for Pf20 in mouse spermatogenesis, sustaining postmeiotic germ cell viability. The different patterns of expression of the two PF20 proteins suggest the possibility that the Pf20 gene has multiple functions during spermatogenesis.

Requirements for glucose beyond sperm capacitation during in vitro fertilization in the mouse.

In both the mouse and the human, it is a point of controversy whether glucose is necessary for in vitro fertilization. Some of this controversy has resulted from a failure to distinguish between requirements for glucose during sperm capacitation versus requirements during the multistage process of fertilization. Using the mouse as a model, we performed a series of experiments designed to identify specific processes that might require glucose. We observed a positive correlation between increasing glucose concentrations during capacitation and fertilization, and increasing fertilization of zona pellucida (ZP)-intact eggs. These data supported a requirement for glucose in the fertilization medium even when sperm were first capacitated in the presence of 5.5 mM glucose. This glucose requirement was observed for both ZP-intact and ZP-free eggs. During ZP-free in vitro fertilization, some binding and fusion between the plasma membrane of the sperm and egg occurred in the absence of glucose and at concentrations less than 1 mM, suggesting that this substrate is not absolutely required. However, glucose concentrations of 1 mM or higher greatly facilitated both binding and fusion under these conditions. These subtle distinctions suggest that during ZP-free in vitro fertilization, 1 mM glucose represents a threshold level that facilitates binding and fusion. Taken as a whole, the data suggest requirements for glucose during both capacitation and fertilization under normal physiologic conditions.