Brinda Dass

Systematic variation in mRNA 3′-processing signals during mouse spermatogenesis

Gene expression and processing during mouse male germ cell maturation (spermatogenesis) is highly specialized. Previous reports have suggested that there is a high incidence of alternative 3′-processing in male germ cell mRNAs, including reduced usage of the canonical polyadenylation signal, AAUAAA. We used EST libraries generated from mouse testicular cells to identify 3′-processing sites used at various stages of spermatogenesis (spermatogonia, spermatocytes and round spermatids) and testicular somatic Sertoli cells. We assessed differences in 3′-processing characteristics in the testicular samples, compared to control sets of widely used 3′-processing sites. Using a new method for comparison of degenerate regulatory elements between sequence samples, we identified significant changes in the use of putative 3′-processing regulatory sequence elements in all spermatogenic cell types. In addition, we observed a trend towards truncated 3′-untranslated regions (3′-UTRs), with the most significant differences apparent in round spermatids. In contrast, Sertoli cells displayed a much smaller trend towards 3′-UTR truncation and no significant difference in 3′-processing regulatory sequences. Finally, we identified a number of genes encoding mRNAs that were specifically subject to alternative 3′-processing during meiosis and postmeiotic development. Our results highlight developmental differences in polyadenylation site choice and in the elements that likely control them during spermatogenesis.

Loss of polyadenylation protein τCstF-64 causes spermatogenic defects and male infertility

Polyadenylation, the process of eukaryotic mRNA 3′ end formation, is essential for gene expression and cell viability. Polyadenylation of male germ cell mRNAs is unusual, exhibiting increased alternative polyadenylation, decreased AAUAAA polyadenylation signal use, and reduced downstream sequence element dependence. CstF-64, the RNA-binding component of the cleavage stimulation factor (CstF), interacts with pre-mRNAs at sequences downstream of the cleavage site. In mammalian testes, meiotic XY-body formation causes suppression of X-linked CstF-64 expression during pachynema. Consequently, an autosomal paralog, τCstF-64 (gene name Cstf2t), is expressed during meiosis and subsequent haploid differentiation. Here we show that targeted disruption of Cstf2t in mice causes aberrant spermatogenesis, specifically disrupting meiotic and postmeiotic development, resulting in male infertility resembling oligoasthenoteratozoospermia. Furthermore, the Cstf2t mutant phenotype displays variable expressivity such that spermatozoa show a broad range of defects. The overall phenotype is consistent with a requirement for τCstF-64 in spermatogenesis as indicated by the significant changes in expression of thousands of genes in testes of Cstf2t−/− mice as measured by microarray. Our results indicate that, although the infertility in Cstf2t−/− males is due to low sperm count, multiple genes controlling many aspects of germ-cell development depend on τCstF-64 for their normal expression. Finally, these transgenic mice provide a model for the study of polyadenylation in an isolated in vivo system and highlight the role of a growing family of testis-expressed autosomal retroposed variants of X-linked genes.

The Gene for a Variant Form of the Polyadenylation Protein CstF-64 Is on Chromosome 19 and Is Expressed in Pachytene Spermatocytes in Mice

Many mRNAs in male germ cells lack the canonical AAUAAA but are normally polyadenylated (Wallace, A. M., Dass, B., Ravnik, S. E., Tonk, V., Jenkins, N. A., Gilbert, D. J., Copeland, N. G., and MacDonald, C. C. (1999)Proc. Natl. Acad Sci. U. S. A. 96, 6763–6768). Previously, we demonstrated the presence of two distinct forms of theM r 64,000 protein of the cleavage stimulation factor (CstF-64) in mouse male germ cells and in brain, a somaticM r 64,000 form and a variantM r 70,000 form. The variant form was specific to meiotic and postmeiotic germ cells. We localized the gene for the somatic CstF-64 to the X chromosome, which would be inactivated during male meiosis. This suggested that the variant CstF-64 was an autosomal homolog activated during that time. We have named the variant form “τ CstF-64,” and we describe here the cloning and characterization of the mouse τCstF-64 cDNA, which maps to chromosome 19. The mouse τCstF-64 protein fits the criteria of the variant CstF-64, including antibody reactivity, size, germ cell expression, and a common proteolytic digest pattern with τCstF-64 from testis. Features of mτCstF-64 that might allow it to promote the germ cell pattern of polyadenylation include a Pro → Ser substitution in the RNA-binding domain and significant changes in the region that interacts with CstF-77.

Overexpression of the CstF-64 and CPSF-160 Polyadenylation Protein Messenger RNAs in Mouse Male Germ Cells

Abstract Messenger RNAs for several components of the transcriptional apparatus are greatly overexpressed in postmeiotic male germ cells in rodents (Schmidt and Schibler, Development 1995; 121:2373–2383). Because of the tight coupling of polyadenylation and transcription, we examined expression in germ cells of mRNAs for key polyadenylation factors. The mRNA for the 64 000 Mr subunit of the cleavage stimulation factor (CstF-64) was expressed at least 250-fold greater in mouse testicular RNA than in liver RNA. RNA blot analysis showed that the mRNA for the 160 000 Mr subunit of the cleavage and polyadenylation specificity factor was similarly overexpressed, as was the mRNA for the large subunit of RNA polymerase II. General transcription factors, such as the TATA-binding protein and transcription factor IIH, and splicing factors, such as components of the small nuclear ribonucleoproteins, were also expressed in meiotic and postmeiotic germ cells. The X-linked CstF-64 protein is expressed before and after but not during meiosis in the mouse (Wallace et al., Proc Natl Acad Sci U S A 1999; 96:6763–6768), which suggests that overexpression of mRNA transcription and processing factors plays an essential role in postmeiotic germ cell mRNA metabolism.

Sertoli cell line lacks the immunoprotective properties associated with primary Sertoli cells.

Sertoli cells are important for maintenance of the immune privileged environment of the testis and prolong survival of cotransplanted cells. The objective of the current study was to examine the immunoprotective properties of a mouse Sertoli cell line (MSC-1) in order to identify a Sertoli cell line that could be used to aid in investigation of the immunoprotective abilities of Sertoli cells. BALB/c islets were cotransplanted with 0–9 million primary BALB/c Sertoli cells or MSC-1 cells into diabetic C3H or BALB/c mice and protection of grafted islets was examined by monitoring blood glucose levels and immunohistochemical analysis. Additionally, expression of potential immunoprotective factors in MSC-1 cells was examined. Cotransplantation of islets with 3 million primary Sertoli cells significantly prolonged islet allograft survival (61.1 ± 6.9 days; p < 0.05) compared with control mice that received allogeneic islets alone (26.9 ±2.1 days). Grafts collected from normoglycemic C3H mice at 100 days posttransplant contained insulin-positive β-cells adjacent to allogeneic Sertoli cells arranged in tubule-like structures. In contrast, cotransplantation of islet allografts with MSC-1 cells did not prolong islet survival (average 29.8 ± 3.3 days) regardless of the number of MSC-1 cells transplanted and the rejected grafts contained very few β-cells and randomly arranged MSC-1 cells. The lack of islet cell survival was not due to detrimental effects of MSC-1 cells because syngneic islets cotransplanted with MSC-1 cells were functional throughout the study. MSC-1 cells were found to express known Sertoli cell-expressed, immunoprotective factors, clusterin, Fas ligand, and transforming growth factor-β1, suggesting additional factors may be involved in Sertoli cell immune privilege. These data indicate the MSC-1 cell line lacks the immunoprotective properties associated with primary Sertoli cells. Further study of this cell line could be useful in examining the mechanisms that enable Sertoli cells to provide immune privilege.

A family of splice variants of CstF-64 expressed in vertebrate nervous systems

BackgroundAlternative splicing and polyadenylation are important mechanisms for creating the proteomic diversity necessary for the nervous system to fulfill its specialized functions. The contribution of alternative splicing to proteomic diversity in the nervous system has been well documented, whereas the role of alternative polyadenylation in this process is less well understood. Since the CstF-64 polyadenylation protein is known to be an important regulator of tissue-specific polyadenylation, we examined its expression in brain and other organs.ResultsWe discovered several closely related splice variants of CstF-64 – collectively called βCstF-64 – that could potentially contribute to proteomic diversity in the nervous system. The βCstF-64 splice variants are found predominantly in the brains of several vertebrate species including mice and humans. The major βCstF-64 variant mRNA is generated by inclusion of two alternate exons (that we call exons 8.1 and 8.2) found between exons 8 and 9 of the CstF-64 gene, and contains an additional 147 nucleotides, encoding 49 additional amino acids. Some variants of βCstF-64 contain only the first alternate exon (exon 8.1) while other variants contain both alternate exons (8.1 and 8.2). In mice, the predominant form of βCstF-64 also contains a deletion of 78 nucleotides from exon 9, although that variant is not seen in any other species examined, including rats. Immunoblot and 2D-PAGE analyses of mouse nuclear extracts indicate that a protein corresponding to βCstF-64 is expressed in brain at approximately equal levels to CstF-64. Since βCstF-64 splice variant family members were found in the brains of all vertebrate species examined (including turtles and fish), this suggests that βCstF-64 has an evolutionarily conserved function in these animals. βCstF-64 was present in both pre- and post-natal mice and in different regions of the nervous system, suggesting an important role for βCstF-64 in neural gene expression throughout development. Finally, experiments in representative cell lines suggest that βCstF-64 is expressed in neurons but not glia.ConclusionThis is the first report of a family of splice variants encoding a key polyadenylation protein that is expressed in a nervous system-specific manner. We propose that βCstF-64 contributes to proteomic diversity by regulating alternative polyadenylation of neural mRNAs.

Polyadenylation proteins CstF-64 and τCstF-64 exhibit differential binding affinities for RNA polymers

CstF-64 (cleavage stimulation factor-64), a major regulatory protein of polyadenylation, is absent during male meiosis. Therefore a paralogous variant, tauCstF-64 is expressed in male germ cells to maintain normal spermatogenesis. Based on sequence differences between tauCstF-64 and CstF-64, and on the high incidence of alternative polyadenylation in testes, we hypothesized that the RBDs (RNA-binding domains) of tauCstF-64 and CstF-64 have different affinities for RNA elements. We quantified K(d) values of CstF-64 and tauCstF-64 RBDs for various ribopolymers using an RNA cross-linking assay. The two RBDs had similar affinities for poly(G)18, poly(A)18 or poly(C)18, with affinity for poly(C)18 being the lowest. However, CstF-64 had a higher affinity for poly(U)18 than tauCstF-64, whereas it had a lower affinity for poly(GU)9. Changing Pro-41 to a serine residue in the CstF-64 RBD did not affect its affinity for poly(U)18, but changes in amino acids downstream of the C-terminal alpha-helical region decreased affinity towards poly(U)18. Thus we show that the two CstF-64 paralogues differ in their affinities for specific RNA sequences, and that the region C-terminal to the RBD is mportant in RNA sequence recognition. This supports the hypothesis that tauCstF-64 promotes germ-cell-specific patterns of polyadenylation by binding to different downstream sequence elements.

Delivery of a therapeutic protein by immune-privileged Sertoli cells.

Immune-privileged Sertoli cells survive long term after allogeneic or xenogeneic transplantation without the use of immunosuppressive drugs, suggesting they could be used as a vehicle to deliver therapeutic proteins. As a model to test this, we engineered Sertoli cells to transiently produce basal levels of insulin and then examined their ability to lower blood glucose levels after transplantation into diabetic SCID mice. Mouse and porcine Sertoli cells transduced with a recombinant adenoviral vector containing furin-modified human proinsulin cDNA expressed insulin mRNA and secreted insulin protein. Transplantation of 5–20 million insulin-expressing porcine Sertoli cells into diabetic SCID mice significantly decreased blood glucose levels in a dose-dependent manner, with 20 million Sertoli cells decreasing blood glucose levels to 9.8 ± 2.7 mM. Similar results were obtained when 20 million insulin-positive, BALB/c mouse Sertoli cells were transplanted; blood glucose levels dropped to 6.3 ± 2.4 mM and remained significantly lower for 5 days. To our knowledge, this is the first study to demonstrate Sertoli cells can be engineered to produce and secrete a clinically relevant factor that has a therapeutic effect, thus supporting the concept of using immune-privileged Sertoli cells as a potential vehicle for gene therapy.

Spermatogenetic but not immunological defects in mice lacking the τCstF-64 polyadenylation protein

Alternative polyadenylation controls expression of genes in many tissues including immune cells and male germ cells. The τCstF-64 polyadenylation protein is expressed in both cell types, and we previously showed that Cstf2t, the gene encoding τCstF-64 was necessary for spermatogenesis and fertilization. Here we examine consequences of τCstF-64 loss in both germ cells and immune cells. Spermatozoa from Cstf2t null mutant (Cstf2t(-/-)) mice of ages ranging from 60 to 108 days postpartum exhibited severe defects in motility and morphology that were correlated with a decrease in numbers of round spermatids. Spermatozoa in these mice also displayed severe morphological defects at every age, especially in the head and midpiece. In the testicular epithelium, we saw normal numbers of cells in earlier stages of spermatogenesis, but reduced numbers of round spermatids in Cstf2t(-/-) mice. Although Leydig cell numbers were normal, we did observe reduced levels of plasma testosterone in the knockout animals, suggesting that reduced androgen might also be contributing to the Cstf2t(-/-) phenotype. Finally, while τCstF-64 was expressed in a variety of immune cell types in wild type mice, we did not find differences in secreted IgG or IgM or changes in immune cell populations in Cstf2t(-/-) mice, suggesting that τCstF-64 function in immune cells is either redundant or vestigial. Together, these data show that τCstF-64 function is primarily to support spermatogenesis, but only incidentally to support immune cell function.

Infertility with Impaired Zona Pellucida Adhesion of Spermatozoa from Mice Lacking TauCstF-64

Fertilization is a multistep process requiring spermatozoa with unique cellular structures and numerous germ cell-specific molecules that function in the various steps. In the highly coordinated process of male germ cell development, RNA splicing and polyadenylation help regulate gene expression to assure formation of functional spermatozoa. Male germ cells express tauCstF-64 (Cstf2t gene product), a paralog of the X-linked CstF-64 protein that supports polyadenylation in most somatic cells. We previously showed that loss of tauCstF-64 causes male infertility because of major defects in mouse spermatogenesis. Surprisingly, although Cstf2t−/− males produce very few recognizable spermatozoa, some of the spermatozoa produced are motile. This led us to ask whether these Cstf2t−/− sperm were fertile. A motile cell-enriched population of spermatozoa from Cstf2t-null males dispersed cumulus cells of cumulus-oocyte complexes normally. However, motile spermatozoa from Cstf2t-null males failed to fertilize cumulus-intact mouse eggs in vitro. In addition, sperm adhesion to the zona pellucida (ZP) of cumulus-free eggs was significantly decreased, indicating tauCstF-64 is required for production of spermatozoa capable of ZP interaction. Acrosomal proteins involved in sperm-ZP recognition, including zonadhesin, proacrosin, SPAM1/PH-20, and ZP3R/sp56, were normally distributed in the apical head of Cstf2t−/− spermatozoa. We conclude that tauCstF-64 is required not only for expression of genes involved in morphological differentiation of spermatids but also for genes having products that function during interaction of motile spermatozoa with eggs. To our knowledge, this is the first demonstration that a gene involved in polyadenylation has a negative consequence on sperm-ZP adhesion.