Identification of calcium-binding proteins associated with the human sperm plasma membrane.

BACKGROUND: The precise composition of the human sperm plasma membrane, the molecular interactions that define domain specific functions, and the regulation of membrane associated proteins during the capacitation process, still remain to be fully understood. Here, we investigated the repertoire of calcium-regulated proteins associated with the human sperm plasma membrane. METHODS: Surface specific radioiodination was combined with two-dimensional gel electrophoresis, a 45Ca-overlay assay, computer assisted image analysis and mass spectrometry to identify calcium-binding proteins exposed on the human sperm surface. RESULTS: Nine acidic 45Ca-binding sperm proteins were excised from stained preparative 2D gels and identified by mass spectrometry. Five of the calcium binding proteins; HSPA2 (HSP70-1), HSPA5 (Bip), HYOU1 (ORP150), serum amyloid P-component (SAP) and protein kinase C substrate 80K-H (80K-H) were found to be accessible to Iodo-Bead catalyzed 125I-labelling on the surface of intact human sperm. Agglutination and immunofluorescence analysis confirmed that SAP is situated on the plasma membrane of intact, motile sperm as well as permeabilized cells. Western blot analysis showed increased phosphorylation of human sperm 80K-H protein following in vitro capacitation. This is the first demonstration of the 80K-H protein in a mammalian sperm. CONCLUSION: The presence of SAP on the surface of mature sperm implies that SAP has a physiological role in reproduction, which is thought to be in the removal of spermatozoa from the female genital tract via phagocytosis. Since 80K-H is a Ca2+-sensor recently implicated in the regulation of both inositol 1,4,5-trisphosphate receptor and transient receptor potential (TRP) cation channel activities, its detection in sperm represents the first direct signaling link between PKC and store-operated calcium channels identified in human sperm.

Changing middle schoolers' attitudes about mental illness through education.

The field test of The Science of Mental Illness curriculum supplement for middle school (grades 6-8) children provided an opportunity to assess knowledge and attitudes about mental illness in more than 1,500 middle school students throughout the United States and to evaluate the impact of an educational intervention on stigma-related attitudes. Two primary questions were examined: (1) what are the baseline knowledge and attitudes about mental illness in this sample of middle school students, and (2) does participation in a curriculum about the science of mental illness increase knowledge and improve attitudes about mental illness? Consistent with findings from other studies, results indicate that students had some understanding of mental illness as a problem of the brain with biological and psychosocial causes; however, they lacked knowledge about treatment and overall were "not sure" about many aspects of mental illness. The students did not strongly endorse negative attitudes about mental illness at baseline. The curriculum produced significant improvements in both knowledge and attitudes at posttest and was most effective in improving attitudes among those with more negative baseline attitudes. These findings suggest that a brief educational program can be an effective intervention to increase knowledge and improve attitudes about mental illness.

Equatorial segment protein defines a discrete acrosomal subcompartment persisting throughout acrosomal biogenesis.

The equatorial segment of the acrosome underlies the domain of the sperm that fuses with the egg membrane during fertilization. Equatorial segment protein (ESP), a novel 349-amino acid concanavalin-A-binding protein encoded by a two-exon gene (SP-ESP) located on chromosome 15 at q22, has been localized to the equatorial segment of ejaculated human sperm. Light microscopic immunofluorescent observations revealed that during acrosome biogenesis ESP first appears in the nascent acrosomal vesicle in early round spermatids and subsequently segregates to the periphery of the expanding acrosomal vesicle, thereby defining a peripheral equatorial segment compartment within flattened acrosomal vesicles and in the acrosomes of early and late cap phase, elongating, and mature spermatids. Electron microscopic examination revealed that ESP segregates to an electron-lucent subdomain of the condensing acrosomal matrix in Golgi phase round spermatids and persists in a similar electron-lucent subdomain within cap phase spermatids. Subsequently, ESP was localized to electron-dense regions of the equatorial segment and the expanded equatorial bulb in elongating spermatids and mature sperm. ESP is the earliest known protein to be recognized as a marker for the specification of the equatorial segment, and it allows this region to be traced through all phases of acrosomal biogenesis. Based on these observations, we propose a new model of acrosome biogenesis in which the equatorial segment is defined as a discrete domain within the acrosomal vesicle as early as the Golgi phase of acrosome biogenesis.

TACC3 expression and localization in the murine egg and ovary.

A protein spot cored from a silver-stained two dimensional (2D) gel of germinal vesicle stage immature mouse oocytes was identified as Transforming Acidic Coiled Coil containing protein (TACC3) by tandem mass spectrometry. PCR amplification revealed two alternatively spliced forms, Tacc3a and Tacc3b, in mouse ovarian cDNA libraries. TACC3a encoded a 630 aa protein with a predicted mass of 70 kDa. It contained seven 24 aa repeats at the N-terminus and two coiled-coil domains at the C-terminus. TACC3b encoded a 426 aa protein with a predicted mass of 49 kDa also containing two coiled coil domains, but lacking the 168 aa repeat region. In addition to homology to the TACC family members, murine TACC3 also showed 35.7% identity to the Xenopus protein, Maskin, a cytoplasmic polyadenylation element binding protein (CPEB)-associated factor. Northern blot analysis demonstrated that TACC3a is abundantly expressed in adult testis and spleen and is moderately expressed in the ovary, heart, and lung, suggesting a wide tissue distribution. Both myc-tagged TACC3a and TACC3b targeted to the cytoplasm of transiently transfected CV-1 cells. In situ hybridization of mouse ovarian tissue sections displayed abundant expression of TACC3 specifically in the cytoplasm of growing oocytes, but not in primordial or atretic follicles. This pattern of expression suggests that TACC3 is expressed in ovarian cells undergoing active growth and development.