Maria E. Teves

Sperm-associated antigen 6 (SPAG6) deficiency and defects in ciliogenesis and cilia function: polarity, density, and beat.

SPAG6, an axoneme central apparatus protein, is essential for function of ependymal cell cilia and sperm flagella. A significant number of Spag6-deficient mice die with hydrocephalus, and surviving males are sterile because of sperm motility defects. In further exploring the ciliary dysfunction in Spag6-null mice, we discovered that cilia beat frequency was significantly reduced in tracheal epithelial cells, and that the beat was not synchronized. There was also a significant reduction in cilia density in both brain ependymal and trachea epithelial cells, and cilia arrays were disorganized. The orientation of basal feet, which determines the direction of axoneme orientation, was apparently random in Spag6-deficient mice, and there were reduced numbers of basal feet, consistent with reduced cilia density. The polarized epithelial cell morphology and distribution of intracellular mucin, α-tubulin, and the planar cell polarity protein, Vangl2, were lost in Spag6-deficient tracheal epithelial cells. Polarized epithelial cell morphology and polarized distribution of α-tubulin in tracheal epithelial cells was observed in one-week old wild-type mice, but not in the Spag6-deficient mice of the same age. Thus, the cilia and polarity defects appear prior to 7 days post-partum. These findings suggest that SPAG6 not only regulates cilia/flagellar motility, but that in its absence, ciliogenesis, axoneme orientation, and tracheal epithelial cell polarity are altered.

A novel ERAP2 haplotype structure in a Chilean population: implications for ERAP2 protein expression and preeclampsia risk

Single nucleotide polymorphisms (SNPs) in the endoplasmic reticulum aminopeptidase 2 (ERAP2) gene are associated with preeclampsia (PE) in different populations. rs2549782, a coding variant (N392K) that significantly affects substrate specificity, is in linkage disequilibrium (LD) with rs2248374, a marker SNP associated with ERAP2 protein expression in previously studied populations. As a result of nonsense‐mediated RNA decay, ERAP2 protein is not expressed from the rs2248374 G allele. We previously reported that the fetal rs2549782 minor G allele is associated with PE in African‐Americans, but not in Chileans. In this study, we found that rs2549782 was in LD with rs2248374 in African‐Americans, but not in Chileans. The unexpected lack of strong LD in Chileans raised the possibility that rs2248374 could be associated with PE in the absence of an association with rs2549782. However, we found no significant association for this allele with PE in Chileans. Chileans homozygous for the rs2248374 G allele did not express 110 kDa ERAP2 protein, consistent with nonsense‐mediated RNA decay, and carriers of the rs2248374 A allele did. We conclude that the Chilean ERAP2 haplotype structure allows for the expression of the major T allele of rs2549782 encoding 392N, which could impact peptide trimming and antigen presentation. Our discovery of racial differences in genetic structure and association with PE reveal heretofore unrecognized complexity of the ERAP2 locus.

Spag16, an Axonemal Central Apparatus Gene, Encodes a Male Germ Cell Nuclear Speckle Protein that Regulates SPAG16 mRNA Expression

Spag16 is the murine orthologue of Chlamydomonas reinhardtii PF20, a protein known to be essential to the structure and function of the “9+2” axoneme. In Chlamydomonas, the PF20 gene encodes a single protein present in the central pair of the axoneme. Loss of PF20 prevents central pair assembly/integrity and results in flagellar paralysis. Here we demonstrate that the murine Spag16 gene encodes two proteins: 71 kDa SPAG16L, which is found in all murine cells with motile cilia or flagella, and 35 kDa SPAG16S, representing the C terminus of SPAG16L, which is expressed only in male germ cells, and is predominantly found in specific regions within the nucleus that also contain SC35, a known marker of nuclear speckles enriched in pre-mRNA splicing factors. SPAG16S expression precedes expression of SPAG16L. Mice homozygous for a knockout of SPAG16L alone are infertile, but show no abnormalities in spermatogenesis. Mice chimeric for a mutation deleting the transcripts for both SPAG16L and SPAG16S have a profound defect in spermatogenesis. We show here that transduction of SPAG16S into cultured dispersed mouse male germ cells and BEAS-2B human bronchial epithelial cells increases SPAG16L expression, but has no effect on the expression of several other axoneme components. We also demonstrate that the Spag16L promoter shows increased activity in the presence of SPAG16S. The distinct nuclear localization of SPAG16S and its ability to modulate Spag16L mRNA expression suggest that SPAG16S plays an important role in the gene expression machinery of male germ cells. This is a unique example of a highly conserved axonemal protein gene that encodes two protein products with different functions.

Sperm Associated Antigen 6 (SPAG6) Regulates Fibroblast Cell Growth, Morphology, Migration and Ciliogenesis.

Mammalian Spag6 is the orthologue of Chlamydomonas PF16, which encodes a protein localized in the axoneme central apparatus, and regulates flagella/cilia motility. Most Spag6-deficient mice are smaller in size than their littermates. Because SPAG6 decorates microtubules, we hypothesized that SPAG6 has other roles related to microtubule function besides regulating flagellar/cilia motility. Mouse embryonic fibroblasts (MEFs) were isolated from Spag6-deficient and wild-type embryos for these studies. Both primary and immortalized Spag6-deficient MEFs proliferated at a much slower rate than the wild-type MEFs, and they had a larger surface area. Re-expression of SPAG6 in the Spag6-deficient MEFs rescued the abnormal cell morphology. Spag6-deficient MEFs were less motile than wild-type MEFs, as shown by both chemotactic analysis and wound-healing assays. Spag6-deficient MEFs also showed reduced adhesion associated with a non-polarized F-actin distribution. Multiple centrosomes were observed in the Spag6-deficient MEF cultures. The percentage of cells with primary cilia was significantly reduced compared to the wild-type MEFs, and some Spag6-deficient MEFs developed multiple cilia. Furthermore, SPAG6 selectively increased expression of acetylated tubulin, a microtubule stability marker. The Spag6-deficient MEFs were more sensitive to paclitaxel, a microtubule stabilizer. Our studies reveal new roles for SPAG6 in modulation of cell morphology, proliferation, migration, and ciliogenesis.

A MEIG1/PACRG complex in the manchette is essential for building the sperm flagella

A key event in the process of spermiogenesis is the formation of the flagella, which enables sperm to reach eggs for fertilization. Yeast two-hybrid studies revealed that meiosis-expressed gene 1 (MEIG1) and Parkin co-regulated gene (PACRG) interact, and that sperm-associated antigen 16, which encodes an axoneme central apparatus protein, is also a binding partner of MEIG1. In spermatocytes of wild-type mice, MEIG1 is expressed in the whole germ cell bodies, but the protein migrates to the manchette, a unique structure at the base of elongating spermatid that directs formation of the flagella. In the elongating spermatids of wild-type mice, PACRG colocalizes with α-tubulin, a marker for the manchette, whereas this localization was not changed in the few remaining elongating spermatids of Meig1-deficient mice. In addition, MEIG1 no longer localizes to the manchette in the remaining elongating spermatids of Pacrg-deficient mice, indicating that PACRG recruits MEIG1 to the manchette. PACRG is not stable in mammalian cells, but can be stabilized by MEIG1 or by inhibition of proteasome function. SPAG16L is present in the spermatocyte cytoplasm of wild-type mice, and in the manchette of elongating spermatids, but in the Meig1 or Pacrg-deficient mice, SPAG16L no longer localizes to the manchette. By contrast, MEIG1 and PACRG are still present in the manchette of Spag16L-deficient mice, indicating that SPAG16L is a downstream partner of these two proteins. Together, our studies demonstrate that MEIG1/PACRG forms a complex in the manchette and that this complex is necessary to transport cargos, such as SPAG16L, to build the sperm flagella. Summary: In the manchette, a structure at the base of the elongating spermatid, the proteins MEIG1 and PACRG act in a complex to control cargo transport and direct formation of the flagellum.

Characterization of membrane occupation and recognition nexus repeat containing 3, meiosis expressed gene 1 binding partner, in mouse male germ cells

Mammalian spermatogenesis is a well-organized process of cell development and differentiation. Meiosis expressed gene 1 (MEIG1) plays an essential role in the regulation of spermiogenesis. To explore potential mechanisms of MEIG1′s action, a yeast two-hybrid screen was conducted, and several potential binding partners were identified; one of them was membrane occupation and recognition nexus repeat containing 3 (MORN3). MORN3 mRNA is only abundant in mouse testis. In the testis, Morn3 mRNA is highly expressed in the spermiogenesis stage. Specific anti-MORN3 polyclonal antibody was generated against N-terminus of the full-length MORN3 protein, and MORN3 expression and localization was examined in vitro and in vivo. In transfected Chinese hamster ovary cells, the antibody specifically crossed-reacted the full-length MORN3 protein, and immunofluorescence staining revealed that MORN3 was localized throughout the cytoplasm. Among multiple mouse tissues, about 25 kDa protein, was identified only in the testis. The protein was highly expressed after day 20 of birth. Immunofluorescence staining on mixed testicular cells isolated from adult wild-type mice demonstrated that MORN3 was expressed in the acrosome in germ cells throughout spermiogenesis. The protein was also present in the manchette of elongating spermatids. The total MORN3 expression and acrosome localization were not changed in the Meig 1-deficient mice. However, its expression in manchette was dramatically reduced in the mutant mice. Our studies suggest that MORN3 is another regulator for spermatogenesis, probably together with MEIG1.

Mutations in fetal genes involved in innate immunity and host defense against microbes increase risk of preterm premature rupture of membranes (PPROM)

Twin studies have revealed a significant contribution of the fetal genome to risk of preterm birth. Preterm premature rupture of membranes (PPROM) is the leading identifiable cause of preterm delivery. Infection and inflammation of the fetal membranes is commonly found associated with PPROM.

Intraflagellar transport protein IFT20 is essential for male fertility and spermiogenesis in mice

Study of intraflagellar transport protein IFT20 in male germ cell development shows that it is essential for male fertility and spermiogenesis in mice, playing a role in the building of sperm flagella and the disposal of cytoplasmic components.

Mammalian axoneme central pair complex proteins: Broader roles revealed by gene knockout phenotypes.

The axoneme genes, their encoded proteins, their functions and the structures they form are largely conserved across species. Much of our knowledge of the function and structure of axoneme proteins in cilia and flagella is derived from studies on model organisms like the green algae, Chlamydomonas reinhardtii. The core structure of cilia and flagella is the axoneme, which in most motile cilia and flagella contains a 9 + 2 configuration of microtubules. The two central microtubules are the scaffold of the central pair complex (CPC). Mutations that disrupt CPC genes in Chlamydomonas and other model organisms result in defects in assembly, stability and function of the axoneme, leading to flagellar motility defects. However, targeted mutations generated in mice in the orthologous CPC genes have revealed significant differences in phenotypes of mutants compared to Chlamydomonas. Here we review observations that support the concept of cell‐type specific roles for the CPC genes in mice, and an expanded repertoire of functions for the products of these genes in cilia, including non‐motile cilia, and other microtubule‐associated cellular functions.

Germ cell‐specific disruption of the Meig1 gene causes impaired spermiogenesis in mice

Meiosis expressed gene 1 (Meig1) was originally identified in a search for mammalian genes potentially involved in meiosis. Seven mouse Meig1 transcripts with the same coding region, but different 5′‐UTRs, have been identified. These transcripts have different tissue distributions, two are only present in the testis. In the testis, Meig1 is present in germ cells and Sertoli cells. A Meig1 conditional knockout model has been generated. When Meig1 was inactivated globally by crossing with Cmv‐Cre transgenic mice, the Meig1‐deficient males were sterile due to severe spermiogenic defects, and had no obvious defects in meiosis. To further study its role in individual cell types in the testis, the Meig1flox mice were crossed with Hsp2a‐Cre, Prm‐Cre, and Amh‐Cre mice, in which the Cre recombinase is driven by the heat shock protein 2 (Hsp2a) gene promoter (expressed in spermatocytes), the protamine 1 gene promoter (expressed in post‐meiotic spermatids) and the anti‐Mullerian hormone (Amh) gene promoter (expressed in Sertoli cells) respectively. Both Meig1 mRNA and protein were undetectable in testis of the Hsp2a‐Cre; Meig1flox/flox mice and all the mutant adult males tested were sterile. This phenotype mirrors that of the Cmv‐Cre; Meig1flox/flox mice. Even though the total testicular Meig1 mRNA and protein expression levels were dramatically reduced in testis of the Prm‐Cre; Meig1flox/flox males, all the mice tested were fertile, and there was no significant difference in sperm count and sperm motility compared with age‐matched Meig1flox/flox male mice. Disruption of Meig1 in the Sertoli cells did not affect the MEIG1 protein expression. Amh‐Cre; Meig1flox/flox males were fertile, and produced the same amount of spermatozoa as age‐matched Meig1flox/flox mice. The testicular histology was also normal. Our results indicate that MEIG1 regulates spermiogenesis through effects in germ cells alone, and that the Meig1 gene must be active during a discrete period in spermatogenesis after which it is dispensable.