Biomedical subjects

Vanesa Y Rawe

Publications and source records attributed to Vanesa Y Rawe.

7 recordsLinked to original sources

PubMed2006

Profilin and actin-related proteins regulate microfilament dynamics during early mammalian embryogenesis.

BACKGROUND: Profilins are ubiquitous proteins widely distributed in animals, including humans. They regulate actin polymerization by sequestering actin monomers in association with other actin-related proteins (Arps). Actin remodelling is essential for oocyte maturation, fertilization and embryo development; yet the role of profilins in these events is not well understood. Here we investigate profilin distribution and function during bovine fertilization and early embryogenesis, and we examine profilin localization with respect to the co-distribution of other Arps. METHODS AND RESULTS: Western blotting, confocal microscopy with immunofluorescence and protein inhibition studies with antibodies were implemented. Profilin distributes inside interphase nuclei, throughout the cytoplasm and near the cell cortex at different stages of bovine oocyte maturation, fertilization and embryo development. Expression is detected through the blastocyst stage, where profilin localizes to the inner cell mass as well as trophectoderm. Profilin co-distributes with actin monomers and Arps vasodilator-stimulated phospho protein, p140mDia, Arp 3 and p80 coilin in pronucleate-stage zygotes. Antiprofilin antibodies inhibit normal embryo development by disrupting microfilaments, but not microtubules, and result in a higher concentration of profilin and p140mDia mislocalized to the cortex. CONCLUSIONS: These findings demonstrate that profilin regulates actin dynamics both within the cytoplasm and inside the nuclei of developing mammalian embryos and that its function is essential during fertilization to ensure successful development.

Actin Cytoskeleton↗

PubMed2005

GDNF family receptor alpha1 phenotype of spermatogonial stem cells in immature mouse testes.

Spermatogonial stem cells (SSCs) are essential for spermatogenesis, and these adult tissue stem cells balance self-renewal and differentiation to meet the biological demand of the testis. The developmental dynamics of SSCs are controlled, in part, by factors in the stem cell niche, which is located on the basement membrane of seminiferous tubules situated among Sertoli cells. Sertoli cells produce glial cell line-derived neurotrophic factor (GDNF), and disruption of GDNF expression results in spermatogenic defects and infertility. The GDNF signals through a receptor complex that includes GDNF family receptor alpha1 (GFRA1), which is thought to be expressed by SSCs. However, expression of GFRA1 on SSCs has not been confirmed by in vivo functional assay, which is the only method that allows definitive identification of SSCs. Therefore, we fractionated mouse pup testis cells based on GFRA1 expression using magnetic activated cell sorting. The sorted and depleted fractions of GFRA1 were characterized for germ cell markers by immunocytochemistry and for stem cell activity by germ cell transplantation. The GFRA1-positive cell fraction coeluted with other markers of SSCs, including ITGA6 and CD9, and was significantly depleted of KIT-positive cells. The transplantation results confirmed that a subpopulation of SSCs expresses GFRA1, but also that the stem cell pool is heterogeneous with respect to the level of GFRA1 expression. Interestingly, POU5F1-positive cells were enriched nearly 15-fold in the GFRA1-selected fraction, possibly suggesting heterogeneity of developmental potential within the stem cell pool.

Animals↗

PubMed2005

A trial to restore defective human sperm centrosomal function.

BACKGROUND: In human fertilization, sperm centrosome function is essential for male and female pronuclear movement and fusion. In this study, we investigated the possibility of restoring human sperm centrosomal function in sperm exhibiting abnormalities in microtubule organization. METHODS: Semen was obtained from both a fertile donor and a patient with dysplasia of the fibrous sheath (DFS). Following heterologous ICSI using human sperm, we examined microtubules and chromatin configuration in bovine oocytes. Sperm were treated with dithiothreitol (DTT) prior to ICSI, while the oocytes were treated with the cytoskeletal stabilizer paclitaxel after ICSI. RESULTS: The combination of DTT and paclitaxel treatment induced microtubule organization in dead sperm from the fertile donor following heterologous ICSI. This treatment, however, was not effective for DFS sperm. In addition, expression of centrin, a protein functioning within the sperm centrosome, was reduced in DFS sperm from that of the normal levels observed in fertile donor sperm. CONCLUSION: These results indicate that sperm centrosomal function could be induced by the treatment of sperm with DTT before ICSI and of oocytes with paclitaxel after ICSI. DFS sperm are likely to exhibit such severe dysfunction of sperm centrosome that cannot be compensated for by this treatment; therefore, this method may be a practical way to discern the degree of sperm centrosomal dysfunction.

Adult↗

PubMed2005

The expression of mitochondrial DNA transcription factors during early cardiomyocyte in vitro differentiation from human embryonic stem cells.

Mitochondrial biogenesis and activation of both oxidative phosphorylation, as well as transcription and replication of the mitochondrial genome, are key regulatory events in cell differentiation. Mitochondrial DNA transcription and replication are highly dependent on the interaction with nuclear-encoded transcription factors translocated from the nucleus. Using a human embryonic stem cell line, HSF 6, we analyzed the proliferation of mitochondria and the expression of mtDNA-specific transcription factors in undifferentiated, migratory embryonic stem cells and spontaneously derived cardiomyocytes. Mitochondrial proliferation and mtDNA transcription are initiated in human embryonic stem cells as they undergo spontaneous differentiation in culture into beating cardiomyocytes. Undifferentiated, pluripotent human embryonic stem cells have few mitochondria, and, as they differentiate, they polarize to one extremity of the cell and then bipolarize the differentiating cell. The differentiated cell then adopts the cytoplasmic configuration of a somatic cell as evidenced in differentiating cardiomyocytes. Transcription and replication of the extranuclear mitochondrial genome is dependent on nuclear encoded factors exported to the mitochondrion. However, the differentiating cardiomyocytes have reduced or absent levels of these transcription and replication factors, namely mitochondrial transcription factors A, B1, B2, and nuclear respiratory factor 1 and polymerase gamma. Therefore, final embryonic stem cell commitment may be influenced by mitochondrial proliferation and mtDNA transcription. However, it is likely that differentiating cardiomyocytes are in mitochondrial arrest, awaiting commitment to a final cell fate.

Cell Differentiation↗

PubMed2004

WAVE1 intranuclear trafficking is essential for genomic and cytoskeletal dynamics during fertilization: cell-cycle-dependent shuttling between M-phase and interphase nuclei.

A-kinase-anchoring proteins (AKAP) help regulate the intracellular organization of cyclic AMP-dependent kinase (PKA) and actin within somatic cells. Elevated levels of cAMP also help maintain meiotic arrest in immature oocytes, with AKAPs implicated as critical mediators but poorly understood during this process. Here we test the hypothesis that the AKAP WAVE1 is required during mammalian fertilization, and identify a nuclear localization of WAVE1 that is independent of actin and actin-related proteins (Arp). Immunofluorescence and immunoprecipitation experiments show a redistribution of WAVE1 from the cortex in germinal vesicle (GV) oocytes to cytoplasmic foci in oocytes arrested in second meiosis (Met II). Following sperm entry, WAVE1 relocalizes to the developing male and female pronuclei. Association of WAVE1 with a regulatory subunit of PKA is detected in both Met II oocytes and pronucleate zygotes, but interaction with Arp 2/3 is observed only in Met II oocytes. WAVE1 redistributes to the cytoplasm upon nuclear envelope breakdown at mitosis, and concentrates at the cleavage furrow during embryonic cell division. Blocking nuclear pore formation with microinjected wheat germ agglutinin does not inhibit the nuclear localization of WAVE1, suggesting that this event precedes nuclear envelope formation. Neither depolymerization nor stabilization of actin affects WAVE1 distribution. Microtubule stabilization with Taxol, however, redistributes WAVE1 to the centrosome, and anti-WAVE1 antibodies prevent both the nuclear distribution of WAVE1 and the migration and apposition of pronuclei. These findings show that WAVE1 sequestration to the nucleus is required during fertilization, and is an actin-independent event that relies on dynamic microtubules but not nuclear pores.

Active Transport, Cell Nucleus↗

PubMed2004

WAVE1, an A-kinase anchoring protein, during mammalian spermatogenesis.

BACKGROUND: Proper compartmentalization of signalling cascades is paramount to many intracellular activities during spermatogenesis and sperm function. In the present study we focus on the A-kinase-anchoring protein (AKAP) WAVE1, a member of the Wiskott-Aldrich syndrome (WASP) family of adaptor proteins, to study its localization throughout mammalian spermatogenesis. METHODS: Using transmission electron microscopy, immunocytochemistry and western blotting, we examined the distribution of WAVE1 and putative partners during mammalian spermatogenesis. The localization and association of PKA RII, the regulatory subunit II of protein kinase A, tyrosine kinase Abl, and small GTPase RAC1 were also explored. RESULTS: WAVE1 localization in spermatocytes and round spermatids coincided with Golgi apparatus distribution, whereas in elongated spermatids and testicular sperm WAVE1 localized to the mitochondrial sheath. Following epididymal passage, WAVE1 was found exclusively on the mitochondrial sheath, suggesting that the protein may function in this region. WAVE1 and PKA RII co-localized along the mitochondrial sheath, PKA RII concentrates in the mid-piece, and RAC1 associated with the post-acrosomal region and the connecting piece. The distribution of WAVE1, PKA RII and RAC1 is conserved in mature mouse, bull, baboon and human sperm. CONCLUSIONS: The data support the possibility of a functional signalling unit established by WAVE1 and its associated proteins in the mid-piece of maturing sperm.

Animals↗

PubMed2004

Spermatocyte/spermatid-specific thioredoxin-3, a novel Golgi apparatus-associated thioredoxin, is a specific marker of aberrant spermatogenesis.

Mammalian germ cells are endowed with a complete set of thioredoxins (Trx), a class of redox proteins located in specific structures of the spermatid and sperm tail. We report here the characterization, under normal and pathological conditions, of a novel thioredoxin with a germ line-restricted expression pattern, named spermatocyte/spermatid-specific thioredoxin-3 (SPTRX-3). The human SPTRX-3 gene maps at 9q32, only 50 kb downstream from the TRX-1 gene from which it probably originated as genomic duplication. Therefore, human SPTRX-3 protein comprises a unique thioredoxin domain displaying high homology with the ubiquitously expressed TRX-1. Among the tissues investigated, Sptrx-3 mRNA is found exclusively in the male germ cells at pachytene spermatocyte and round spermatid stages. Light and electron microscopy show SPTRX-3 protein to be predominately located in the Golgi apparatus of pachytene spermatocytes and round and elongated spermatids, with a transient localization in the developing acrosome of round spermatids. In addition, increased levels of SPTRX-3, possibly caused by overexpression, are observed in morphologically abnormal human spermatozoa from infertile men. In addition, SPTRX-3 is identified as a novel postobstruction autoantigen. In this report, we propose that SPTRX-3 can be used as a specific marker for diverse sperm and testis pathologies. SPTRX-3 is the first thioredoxin specific to the Golgi apparatus, and its function within this organelle might be related to the post-translational modification of proteins required for germ cell-specific functions, such as acrosomal biogenesis.

Acrosome Reaction↗