Yi-Nan Lin

Absence of tektin 4 causes asthenozoospermia and subfertility in male mice

Sperm flagellar motion is the outcome of a dynamic interplay between the axonemal cytoskel‐eton, the peri‐axonemal accessory structures, and multiple regulatory networks that coordinate to produce flagellar beat and waveform. Tektins are conserved components of the flagellar proteome in evolutionarily diverse species and are believed to play essential roles in the mechanics of sperm motility. Using database mining, we identified multiple new paralogs of previously annotated tektins, including tektin 4 (TEKT4), which shares 77.1% identity with its nearest human homologue. Mouse Tekt4 is a germ cell‐enriched gene, most abundantly expressed in haploid round sperma‐tids in the testis, and the protein is localized to the sperm flagella. Male mice lacking TEKT4 on a 129S5/ SvEvBrd inbred background are subfertile. Tekt4‐null sperm exhibit drastically reduced forward progressive velocity and uncoordinated waveform propagation along the flagellum. In Tekt4‐null sperm, flagellar ultra‐structure is grossly unaltered as revealed by transmission electron microscopy. However, the ineffective flagellar strokes lead to ~10‐fold higher consumption of intracellular ATP in Tekt4‐null sperm as compared to wild‐type, and null spermatozoa rapidly lose progressive motility when incubated for ≥1.5 h. Our studies demonstrate that TEKT4 is necessary for the proper coordinated beating of the sperm flagellum and male reproductive physiology.—Roy, A., Lin, Y.‐N., Agno, J. E., DeMayo, F. J., Matzuk, M. M. Absence of tektin 4 causes asthenozoospermia and subfertility in male mice. FASEB J. 21, 1013–1025 (2007)

Tektin 3 is Required for Progressive Sperm Motility in Mice

Tektins are evolutionarily conserved flagellar (and ciliary) filamentous proteins present in the axoneme and peri‐axonemal structures in diverse metazoan species. We have previously shown that tektin 3 (TEKT3) and tektin 4 (TEKT4) are male germ cell‐enriched proteins, and that TEKT4 is essential for coordinated and progressive sperm motility in mice. Here we report that male mice null for TEKT3 produce sperm with reduced motility (47.2% motility) and forward progression, and increased flagellar structural bending defects. Male TEKT3‐null mice however maintain normal fertility in two different genetic backgrounds tested, in contrast to TEKT4‐null mice. Furthermore, male mice null for both TEKT3 and TEKT4 show subfertility on a mixed B6;129 genetic background, significantly different from either single knockouts, suggesting partial nonredundant roles for these two proteins in sperm physiology. Our results suggest that tektins are potential candidate genes for nonsyndromic asthenozoospermia in humans. Mol. Reprod. Dev. 76: 453–459, 2009.

Regulation of Growth Differentiation Factor 9 Expression in Oocytes In Vivo: A Key Role of the E-Box

Abstract Growth differentiation factor 9 (GDF9) is preferentially expressed in oocytes and is essential for female fertility. To identify regulatory elements that confer high-level expression of GDF9 in the ovary but repression in other tissues, we generated transgenic mice in which regions of the Gdf9 locus were fused to reporter genes. Two transgenes (−10.7/+5.6mGdf9-GFP) and (−3.3/+5.6mGdf9-GFP) that contained sequences either 10.7 or 3.3 kb upstream and 5.6 kb downstream of the Gdf9 initiation codon demonstrated expression specifically in oocytes, thereby mimicking endogenous Gdf9 expression. In contrast, transgenes −10.7mGdf9-Luc and −3.3mGdf9-Luc, which lacked the downstream 5.6-kb region, demonstrated reporter expression not only in oocytes but also high expression in male germ cells. This suggests that the downstream 5.6-kb sequence contains a testis-specific repressor element and that 3.3 kb of 5′-flanking sequence contains all the cis-acting elements for directing high expression of Gdf9 to female (and male) germ cells. To define sequences responsible for oocyte expression of Gdf9, we analyzed sequences of Gdf9 genes from 16 mammalian species. The approximately 400 proximal base pairs upstream of these Gdf9 genes are highly conserved and contain a perfectly conserved E-box (CAGCTG) sequence. When this 400-bp region was placed upstream of a luciferase reporter (−0.4mGdf9-Luc), oocyte-specific expression was observed. However, a similar transgene construct (−0.4MUT-mGdf9-Luc) with a mutation in the E-box abolished oocyte expression. Likewise, the presence of an E-box mutation in a longer construct (−3.3MUT-mGdf9-Luc) abolished expression in the ovary but not in the testis. These observations indicate that the E-box is a key regulatory sequence for Gdf9 expression in the ovary.

Genetic manipulations to study reproduction

Fertility disorders affect approximately 15% of individuals worldwide. With the imminent completion of the human and mouse genome sequence, it will be more feasible to identify the relevant genes underlying many fertility disorders. Already, the mouse has been utilized extensively as a genetic tool for the dissection of gene function, often providing significant insights into the relationship between gene and disease. In fact, there are over 200 mouse models that display reproductive defects. However, the available mouse mutant resources provide functional information for a mere 10% of the total number of genes in the mouse or human genomes at best. The improvement of available genome annotations together with more powerful techniques to manipulate the mouse genome provide substantial improvements in our ability to identify genes involved in reproduction, and in the future will likely benefit patients with fertility problems.

Deletion of the novel oocyte-enriched gene, Gpr149, leads to increased fertility in mice.

Through in silico subtraction and microarray analysis, we identified mouse Gpr149, a novel, oocyte-enriched transcript that encodes a predicted orphan G-protein-coupled receptor (GPR). Phylogenetic analysis of GPR149 from fish to mammals suggests that it is widely conserved in vertebrates. By multitissue RT-PCR analysis, we found that Gpr149 is highly expressed in the ovary and also in the brain and the digestive tract at low levels. Gpr149 levels are low in newborn ovaries but increase throughout folliculogenesis. In the ovary, we found that granulosa cells did not express Gpr149, whereas germinal vesicle and meiosis II stage oocytes showed high levels of Gpr149 expression. After fertilization, Gpr149 expression declined, becoming undetectable by the two-cell stage. To study the function of GPR149 in oocyte growth and maturation, we generated Gpr149 null mice. Surprisingly, Gpr149 null mice are viable and have normal folliculogenesis, but demonstrate increased fertility, enhanced ovulation, increased oocyte Gdf9 mRNA levels, and increased levels of FSH receptor and cyclin D2 mRNA levels in granulosa cells. Thus, Gpr149 null mice are one of the few models with enhanced fertility, and GPR149 could be a target for small molecules to enhance fertility in the assisted reproductive technology clinic.

Acrosome-specific gene AEP1: Identification, characterization and roles in spermatogenesis

Spermatogenesis is a tightly regulated process leading to the development of spermatozoa. To elucidate the molecular spermatogenic mechanisms, we identified an acrosome‐specific gene AEP1 in spermatids, which is located in rat chromosome 17p14 with a transcript size of 3,091 bp encoding a signal peptide, zinc finger‐like motif, coiled‐coil region, several predicted glycosylation and phosphorylation sites. Northern blot and RT‐PCR analyses revealed the restricted expression of AEP1 to the testis only. In postnatal rat testes, AEP1 mRNA became detectable from postnatal 25 dpp (round spermatids) and onwards. By using in situ hybridization (ISH) and flow cytometry‐fluorescent ISH, only the haploid spermatids yielded the positive AEP1 signal. Immunohistochemistry showed that AEP1 was expressed in the acrosomal cap of late‐staged germ cells in rat testis, and co‐localized with the acrosomal marker, peanut agglutinin. The spatial expression of AEP1 immunoreactivity in testis was conserved among diverse mammalian species (rat, pig, monkey, human). To further study its roles in spermatogenesis, we showed AEP1 and β‐actin was associated together in complex by co‐immunoprecipitation in adult germ cells and by immunofluorescence assay in isolated spermatozoon. In human testes diagnosed with hypospermatogenesis, lower expression of AEP1 was observed, whereas there was no detectable signal in undescended testes. In short, AEP1 is an evolutionary‐conserved acrosome‐specific gene and likely functions in acrosome‐cap formation. J. Cell. Physiol. 209: 755–766, 2006.

Testicular cell adhesion molecule 1 (TCAM1) is not essential for fertility.

Testicular cell adhesion molecule 1 (Tcam1) is a testis-expressed gene that is evolutionarily conserved in most mammalian species. The putative location of TCAM1 on the cell surface makes it an attractive contraceptive target to study. We found that Tcam1 transcription is enriched in the adult testis, and in situ hybridization revealed that Tcam1 is expressed in pachytene to secondary spermatocytes. Immunofluorescence for TCAM1 protein showed strong expression along cell membranes of spermatocytes and weak localization to round spermatids. In light of this evidence, we hypothesized that TCAM1 interacts with an unknown receptor on the surface of Sertoli cells and that this interaction is important for germ cell-Sertoli cell interactions. However, Tcam1 knockout mice that we generated are fertile, and testis weights and sperm counts were not significantly altered. Therefore, we conclude that TCAM1 is not essential for male fertility or germ cell function in Mus musculus.

Genetics of Idiopathic Male Infertility

Nearly 7% of men suffer from male factor infertility. In one-fourth of infertile males, the etiology remains unexplained. Unlike other multifactorial disorders, gene-gene and gene-environment interactions in the regulation of male fertility have been poorly characterized. A candidate-gene approach that incorporates biological information from model organisms is likely to be critical in deciphering the genetic basis of idiopathic male fertility. Genes that fulfill essential roles in spermatogenesis often have orthologs in several species wherein they serve similar functions. By using a comparative cross-species approach, major susceptibility genes underlying male infertility can be identified in association studies. With a better understanding of the molecular regulation of spermatogenesis, proper diagnosis and treatment of male infertility should be realized in the foreseeable future.

Shaping the sperm head: an ER enzyme leaves its mark.

Lipid storage diseases are debilitating inherited metabolic disorders that stem from the absence of specific lysosomal enzymes that degrade selected lipids. Most characteristically, these disorders affect the nervous and the reticulo-endothelial systems, with massive organomegaly resulting from the presence of engorged, lipid-laden macrophages. In this issue of the JCI, Yildiz et al. describe the role of the ER-resident enzyme beta-glucosidase 2 (GBA2) in mice (see the related article beginning on page 2985). Surprisingly, GBA2 deficiency leaves bile acid and cholesterol metabolism intact, instead causing lipid accumulation in the ER of testicular Sertoli cells, round-headed sperm (globozoospermia), and impaired male fertility.