TSPYL5 Depletion Induces Specific Death of ALT Cells through USP7-Dependent Proteasomal Degradation of POT1.

A significant fraction (∼10%) of cancer cells maintain their telomere length via a telomerase-independent mechanism known as alternative lengthening of telomeres (ALT). There are no known molecular, ALT-specific, therapeutic targets. We have identified TSPYL5 (testis-specific Y-encoded-like protein 5) as a PML body component, co-localizing with ALT telomeres and critical for ALT+ cell viability. TSPYL5 was described as an inhibitor of the USP7 deubiquitinase. We report that TSPYL5 prevents the poly-ubiquitination of POT1-a shelterin component-and protects POT1 from proteasomal degradation exclusively in ALT+ cells. USP7 depletion rescued POT1 poly-ubiquitination and loss, suggesting that the deubiquitinase activates POT1 E3 ubiquitin ligase(s). Similarly, PML depletion suppressed POT1 poly-ubiquitination, suggesting an interplay between USP7 and PML to trigger POT1 degradation in TSPYL5-depleted ALT+ cells. We demonstrate that ALT telomeres need to be protected from POT1 degradation in ALT-associated PML bodies and identify TSPYL5 as an ALT+ cancer-specific therapeutic target.

Germ Cell-Specific Proteins AKAP4 and ASPX Facilitate Identification of Rare Spermatozoa in Non-Obstructive Azoospermia.

Non-obstructive azoospermia (NOA), the most severe form of male infertility, could be treated with intracytoplasmic sperm injection, providing spermatozoa were retrieved with the microdissection testicular sperm extraction (mTESE). We hypothesized that testis-specific and germ cell-specific proteins would facilitate flow cytometry-assisted identification of rare spermatozoa in semen cell pellets of NOA patients, thus enabling non-invasive diagnostics prior to mTESE. Data mining, targeted proteomics, and immunofluorescent microscopy identified and verified a panel of highly testis-specific proteins expressed at the continuum of germ cell differentiation. Late germ cell-specific proteins AKAP4_HUMAN and ASPX_HUMAN (ACRV1 gene) revealed exclusive localization in spermatozoa tails and acrosomes, respectively. A multiplex imaging flow cytometry assay facilitated fast and unambiguous identification of rare but morphologically intact AKAP4+/ASPX+/Hoechst+ spermatozoa within debris-laden semen pellets of NOA patients. While the previously suggested markers for spermatozoa retrieval suffered from low diagnostic specificity, the multistep gating strategy and visualization of AKAP4+/ASPX+/Hoechst+ cells with elongated tails and acrosome-capped nuclei facilitated fast and unambiguous identification of the mature intact spermatozoa. AKAP4+/ASPX+/Hoechst+ assay may emerge as a noninvasive test to predict retrieval of morphologically intact spermatozoa by mTESE, thus improving diagnostics and treatment of severe forms of male infertility.

Structural insights into instability of the nucleosome driven by histone variant H3T.

The testis-specific histone variant H3T plays a crucial role in chromatin reorganization during spermatogenesis by destabilizing nucleosomes. However, the structure basis for the nucleosome instability driven by H3T is not fully understand. In this study, we determinate the crystal structure of H3T-H4 in complex with histone chaperone ASF1a at 2.8 Å resolution. Our findings reveal that H3T-H4 binds ASF1a similarly to the conventional H3.1-H4 complex. However, significant structural differences are observed in the H3 α1 helix, the N- and C-terminal region of α2, and N-terminal region of L2. These differences are driven by H3T-specific residues, particularly Val111. Unlike the smaller Ala111 in H3.1, we find that bulkier residue Val111 fits well within the ASF1-H3T-H4 complex, but is difficult to arrange in nucleosome structure. Given that H3.1-Ala111/H3T-Val111 is located at the DNA binding and tetramerization interface of H3-H4, it is likely that Ala111Val substitution will lead to the instability of the corresponding area in nucleosome, contributing to instability of H3T-containing nucleosome. These structural findings may elucidate the role of H3T in chromatin reorganization during spermatogenesis.

Testis-Specific Protein Y-Encoded (TSPY) Is Required for Male Early Embryo Development in Bos taurus.

TSPY is a highly conserved multi-copy gene with copy number variation (CNV) among species, populations, individuals and within families. TSPY has been shown to be involved in male development and fertility. However, information on TSPY in embryonic preimplantation stages is lacking. This study aims to determine whether TSPY CNV plays a role in male early development. Using sex-sorted semen from three different bulls, male embryo groups referred to as 1Y, 2Y and 3Y, were produced by in vitro fertilization (IVF). Developmental competency was assessed by cleavage and blastocyst rates. Embryos at different developmental stages were analyzed for TSPY CN, mRNA and protein levels. Furthermore, TSPY RNA knockdown was performed and embryos were assessed as per above. Development competency was only significantly different at the blastocyst stage, with 3Y being the highest. TSPY CNV and transcripts were detected in the range of 20-75 CN for 1Y, 20-65 CN for 2Y and 20-150 CN for 3Y, with corresponding averages of 30.2 ± 2.5, 33.0 ± 2.4 and 82.3 ± 3.6 copies, respectively. TSPY transcripts exhibited an inverse logarithmic pattern, with 3Y showing significantly higher TSPY. TSPY proteins, detected only in blastocysts, were not significantly different among groups. TSPY knockdown resulted in a significant TSPY depletion (p < 0.05), with no development observed after the eight-cell stage in male embryos, suggesting that TSPY is required for male embryo development.

Structural and biochemical analyses of Bcl-xL in complex with the BH3 domain of peroxisomal testis-specific 1.

Antiapoptotic B-cell lymphoma-2 (Bcl-2) proteins suppress apoptosis by interacting with proapoptotic regulators. They commonly contain a hydrophobic groove where the Bcl-2 homology 3 (BH3) domain of Bcl-2 family members or BH3 domain-containing non-Bcl-2 family proteins can be accommodated. Peroxisomal testis-specific 1 (Pxt1) was previously identified as a male germ cell-specific protein whose overexpression causes germ cell apoptosis and infertility in male mice. Sequence and biochemical analyses also showed that human Pxt1, which is composed of 134 amino acids and is longer than mouse Pxt1 consisting of only 51 amino acids, has a BH3 domain that interacts with antiapoptotic Bcl-2 proteins, including Bcl-2 and Bcl-xL. In this study, we determined the crystal structure of Bcl-xL bound to the human Pxt1 BH3 domain. The five BH3 consensus residues are well conserved in the human Pxt1 BH3 domain and make a critical contribution to the complex formation in a canonical manner. Structural and biochemical analyses also demonstrated that Bcl-xL interacts with the BH3 domain of human Pxt1 but not with that of mouse Pxt1, and that residues 76-83 of human Pxt1, absent in mouse Pxt1, play a pivotal role in the intermolecular binding to Bcl-xL. While Bcl-xL consistently colocalized with human Pxt1 in mitochondria, it did not do so with mouse Pxt1, when expressed in HeLa cells. Collectively, these data verified that human and mouse Pxt1 differ in their binding ability to the antiapoptotic regulator Bcl-xL, which might affect their functionality in controlling apoptosis.

Testis-specific fascin component FSCN3 is dispensable for mouse spermatogenesis and fertility.

BACKGROUND: Fascins belong to a family of actin-bundling proteins that are involved in a wide range of biological functions. FSCN3, a newly identified testis-specific actin-bundling protein, is specifically expressed in elongated spermatids. However, its in vivo function in mouse spermiogenesis remains unknown. METHODS AND RESULTS: We generated Fscn3 knockout mice through CRISPR/Cas9 gene-editing technology. Fscn3-/- mice displayed normal testis morphology and testis to bodyweight ratio, and sperm concentrations did not differ significantly between Fscn3+/+ and Fscn3-/- mice. Fertility assays consistently revealed that Fscn3-/- mice are completely fertile and their reproductive status does not differ from that of wild-type. Moreover, hematoxylin and eosin staining of the testis sections of Fscn3-/- mice detected various germ cells, ranging from spermatogonia to mature spermatozoa. Furthermore, the swimming velocity of the sperm of Fscn3-/- mice was comparable to that of their wild-type littermates. Both Fscn3+/+ and Fscn3-/-mice had normal sperm morphology, indicating that the disruption of Fscn3 does not affect sperm morphology. The analysis of meiotic prophase I progression demonstrated normal prophase-I phases (leptonema to diplonema) in both Fscn3+/+ and Fscn3-/- mice, suggesting that Fscn3 is not essential for meiosis I. CONCLUSION: Our study provides the first evidence that FSCN3 is a testis-specific actin-bundling protein that is not required for mouse spermatogenesis. Our results will help reproductive biologists focus their efforts on genes that are crucial for fertility and avoid research duplication.

lnc94638 is a testis-specific long non-coding RNA involved in spermatozoa formation in Zeugodacus cucurbitae (Coquillett).

Long non-coding RNAs (lncRNAs) generally display tissue-specific distributions, and testis-specific lncRNAs form the highest proportion of lncRNAs in many species. Here, we presented a detailed analysis of testis-specific lncRNAs in the melon fly, Zeugodacus cucurbitae, a highly destructive insect pest of cucurbitaceous and other related crops. Most testis-specific lncRNAs were found to be long intergenic non-coding RNAs (lincRNA). The size distribution of these lncRNAs ranged between 600 and 1000 nucleotides. Testis-specific lncRNAs that harboured one isoform number and two exons were the most abundant. Compared to other male tissues, the testis had more highly expressed lncRNAs. The quantitative real-time polymerase chain reaction results of 10 randomly selected testis-specific lncRNAs showed expression patterns consistent with RNA-seq data. Further analysis of the most highly expressed testis-specific lncRNA, lnc94638, was undertaken. Fluorescent in situ hybridization assays localized lnc94638 to the apical region of the testis that contains mature spermatozoa. RNA interference-mediated knockdown of lnc94638 expression reduced spermatozoa numbers and impaired the fertility of Z. cucurbitae male. This study provides a catalogue of testis-specific lncRNAs, shows that the testis-specific lnc94638 is involved in spermatogenesis and has the potential to be used for treating male sterility.

TSSK3, a novel target for male contraception, is required for spermiogenesis.

We have previously shown that members of the family of testis-specific serine/threonine kinases (TSSKs) are post-meiotically expressed in testicular germ cells and in mature sperm in mammals. The restricted post-meiotic expression of TSSKs as well as the importance of phosphorylation in signaling processes strongly suggest that TSSKs have an important role in germ cell differentiation and/or sperm function. This prediction has been supported by the reported sterile phenotype of the TSSK6 knock-out (KO) mice and of the double TSSK1/TSSK2 KO. The aim of this study was to develop KO mouse models of TSSK3 and to validate this kinase as a target for the development of a male contraceptive. We used CRISPR/Cas9 technology to generate the TSSK3 KO allele on B6D2F1 background mice. Male heterozygous pups were used to establish three independent TSSK3 KO lines. After natural mating of TSSK3 KO males, females that presented a plug (indicative of mating) were monitored for the following 24 days and no pregnancies or pups were found. Sperm numbers were drastically reduced in all three KO lines and, remarkably, round spermatids were detected in the cauda epididymis of KO mice. From the small population of sperm recovered, severe morphology defects were detected. Our results indicate an essential role of TSSK3 in spermiogenesis and support this kinase as a suitable candidate for the development of novel nonhormonal male contraceptives.

Identification of TEX101-associated Proteins Through Proteomic Measurement of Human Spermatozoa Homozygous for the Missense Variant rs35033974.

TEX101 is a germ-cell-specific protein and a validated biomarker of male infertility. Mouse TEX101 was found essential for male fertility and was suggested to function as a cell surface chaperone involved in maturation of proteins required for sperm migration and sperm-oocyte interaction. However, the precise functional role of human TEX101 is not known and cannot be studied in vitro due to the lack of human germ cell lines. Here, we genotyped 386 men for a common missense variant rs35033974 of TEX101 and identified 52 heterozygous and 4 homozygous men. We then discovered by targeted proteomics that the variant allele rs35033974 was associated with the near-complete degradation (>97%) of the corresponding G99V TEX101 form and suggested that spermatozoa of homozygous men could serve as a knockdown model to study TEX101 function in humans. Differential proteomic profiling with label-free quantification measured 8,046 proteins in spermatozoa of eight men and identified eight cell-surface and nine secreted testis-specific proteins significantly down-regulated in four patients homozygous for rs35033974. Substantially reduced levels of testis-specific cell-surface proteins potentially involved in sperm migration and sperm-oocyte interaction (including LY6K and ADAM29) were confirmed by targeted proteomics and Western blotting assays. Because recent population-scale genomic data revealed homozygous fathers with biological children, rs35033974 is not a monogenic factor of male infertility in humans. However, median TEX101 levels in seminal plasma were found fivefold lower (p = 0.0005) in heterozygous than in wild-type men of European ancestry. We conclude that spermatozoa of rs35033974 homozygous men have substantially reduced levels of TEX101 and could be used as a model to elucidate the precise TEX101 function, which will advance biology of human reproduction.

Identification and characterization of male reproduction-related genes in pig (Sus scrofa) using transcriptome analysis.

BACKGROUND: The systematic interrogation of reproduction-related genes was key to gain a comprehensive understanding of the molecular mechanisms underlying male reproductive traits in mammals. Here, based on the data collected from the NCBI SRA database, this study first revealed the genes involved in porcine male reproduction as well their uncharacterized transcriptional characteristics. RESULTS: Results showed that the transcription of porcine genome was more widespread in testis than in other organs (the same for other mammals) and that testis had more tissue-specific genes (1210) than other organs. GO and GSEA analyses suggested that the identified test is-specific genes (TSGs) were associated with male reproduction. Subsequently, the transcriptional characteristics of porcine TSGs, which were conserved across different mammals, were uncovered. Data showed that 195 porcine TSGs shared similar expression patterns with other mammals (cattle, sheep, human and mouse), and had relatively higher transcription abundances and tissue specificity than low-conserved TSGs. Additionally, further analysis of the results suggested that alternative splicing, transcription factors binding, and the presence of other functionally similar genes were all involved in the regulation of porcine TSGs transcription. CONCLUSIONS: Overall, this analysis revealed an extensive gene set involved in the regulation of porcine male reproduction and their dynamic transcription patterns. Data reported here provide valuable insights for a further improvement of the economic benefits of pigs as well as future treatments for male infertility.

Linker histone variant H1T functions as a chromatin de-condenser on genic regions.

Linker histone H1 is mainly localized in the linker DNA region, between two nucleosome cores, and regulates chromatin structures linking gene expression. There are 11 variants in histone H1, and each variant has unique functions. Our previous study demonstrates that one of the H1 variants, H1T is mainly localized in the nucleolus and targets the rDNA repeat region. Moreover, H1T condenses the chromatin structures on rDNA to repress pre-rRNA expression. Although H1T is partially localized in the nucleoplasm area, the functions of H1T in the non-repeat genic region are unclear. In this study, we aimed to identify the target loci and the role of H1T in the genic region. Chromatin immunoprecipitation sequencing analysis showed that H1T is localized around the transcriptional start site and the chromatin structures of the region were relaxed. H1T knockdown and overexpression experiments revealed that H1T induced chromatin de-condensation and was associated with the increased expression of target genes. Moreover, we observed H1T co-localization with transcriptional factor SPZ1 on the genic region. Collectively, H1T has opposing roles in the genic region and in rDNA repeats; H1T functions to facilitate chromatin relaxation linked gene activation.

Testis-specific serine kinase protein family in male fertility and as targets for non-hormonal male contraception†.

Male contraception is a very active area of research. Several hormonal agents have entered clinical trials, while potential non-hormonal targets have been brought to light more recently and are at earlier stages of development. The general strategy is to target genes along the molecular pathways of sperm production, maturation, or function, and it is predicted that these novel approaches will hopefully lead to more selective male contraceptive compounds with a decreased side effect burden. Protein kinases are known to play a major role in signaling events associated with sperm differentiation and function. In this review, we focus our analysis on the testis-specific serine kinase (TSSK) protein family. We have previously shown that members of the family of TSSKs are postmeiotically expressed in male germ cells and in mature mammalian sperm. The restricted postmeiotic expression of TSSKs as well as the importance of phosphorylation in signaling processes strongly suggests that TSSKs have an important role in germ cell differentiation and/or sperm function. This prediction has been supported by the reported sterile phenotype of the Tssk6 knockout (KO) mice and of the double Tssk1 and Tssk2 KO mice and by the male subfertile phenotype observed in a Tssk4 KO mouse model.

TSGA10 overexpression inhibits angiogenesis of HUVECs: A HIF-2α biased perspective.

Testis-specific gene antigen 10 (TSGA10) is a protein overexpressed in most cancers; except for some certain types where its expression is reduced. TSGA10 overexpression in HeLa cells has been shown to disrupt hypoxia inducible factor-1α (HIF-1α) axis and exert potent inhibitory effects. Since HIF-1α is structurally and biochemically similar to HIF-2α, TSGA10 is expected to bind HIF-2α and inhibit its function as well. This study elucidated that increased expression of TSGA10 in manipulated human umbilical vein endothelial cells (HUVECs) decreased the proliferation and migration of these cells as affirmed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and wound healing tests, respectively. It also inhibited in vitro angiogenesis of these cells in 3D collagen-cytodex model. Expression levels of genes controlled by HIF-2α including autocrine vascular endothelial growth factor (VEGF) were also assessed using real-time PCR. Our bioinformatic analysis also showed that TSGA10 could bind HIF-2α. Moreover, flow cytometry results indicated a cell cycle arrest in G2/M. Therefore, this study showed that overexpression of TSGA10, as a tumor suppressor gene, in endothelial cells resulted in decreased proliferation, migration and therefore, angiogenic activity of HUVECs. Since angiogenesis is vital for tumor development and metastasis, our findings could be of clinical significance in cancer therapy.

Evolutionarily conserved and testis-specific gene, 4930524B15Rik, is not essential for mouse spermatogenesis and fertility.

Thousands of genes are involved in spermatogenesis, however, the functional roles of most these genes for male fertility remain to be discovered. This research focused to explore the function of evolutionarily conserved and testis-specific expressed gene 4930524B15Rik, which is known as C5orf47 in human. We generated 4930524B15Rik knockout mice by CRISPR/Cas9 technology and found 4930524B15Rik-/- mice were fertile. Furthermore, no averted abnormalities were observed in testis morphology, epididymal sperm contents and sperm morphology in 4930524B15Rik knockout mice. Subsequently, histological analysis of testicular tissue revealed intact structure of seminiferous tubules along with the presence of all types of germ cells in 4930524B15Rik-/- mice similar to wild type. Additionally, cytological analysis of spermatocytes displayed no significant differences in the prophase I progression of meiosis, further indicating that 4930524B15Rik have no essential function in mammalian spermatogenesis. Altogether, these results indicated that 4930524B15Rik is dispensable for fertility of male mice and these findings will help researchers to avoid future research overlap and to focus on genes that are crucial for spermatogenesis and reproduction.

Discovery of a Human Testis-specific Protein Complex TEX101-DPEP3 and Selection of Its Disrupting Antibodies.

TEX101 is a testis-specific protein expressed exclusively in male germ cells and is a validated biomarker of male infertility. Studies in mice suggest that TEX101 is a cell-surface chaperone which regulates, through protein-protein interactions, the maturation of proteins involved in spermatozoa transit and oocyte binding. Male TEX101-null mice are sterile. Here, we identified by co-immunoprecipitation-mass spectrometry the interactome of human TEX101 in testicular tissues and spermatozoa. The testis-specific cell-surface dipeptidase 3 (DPEP3) emerged as the top hit. We further validated the TEX101-DPEP3 complex by using hybrid immunoassays. Combinations of antibodies recognizing different epitopes of TEX101 and DPEP3 facilitated development of a simple immunoassay to screen for disruptors of TEX101-DPEP3 complex. As a proof-of-a-concept, we demonstrated that anti-TEX101 antibody T4 disrupted the native TEX101-DPEP3 complex. Disrupting antibodies may be used to study the human TEX101-DPEP3 complex, and to develop modulators for male fertility.

TCTE1 is a conserved component of the dynein regulatory complex and is required for motility and metabolism in mouse spermatozoa.

Flagella and cilia are critical cellular organelles that provide a means for cells to sense and progress through their environment. The central component of flagella and cilia is the axoneme, which comprises the "9+2" microtubule arrangement, dynein arms, radial spokes, and the nexin-dynein regulatory complex (N-DRC). Failure to properly assemble components of the axoneme leads to defective flagella and in humans leads to a collection of diseases referred to as ciliopathies. Ciliopathies can manifest as severe syndromic diseases that affect lung and kidney function, central nervous system development, bone formation, visceral organ organization, and reproduction. T-Complex-Associated-Testis-Expressed 1 (TCTE1) is an evolutionarily conserved axonemal protein present from Chlamydomonas (DRC5) to mammals that localizes to the N-DRC. Here, we show that mouse TCTE1 is testis-enriched in its expression, with its mRNA appearing in early round spermatids and protein localized to the flagellum. TCTE1 is 498 aa in length with a leucine rich repeat domain at the C terminus and is present in eukaryotes containing a flagellum. Knockout of Tcte1 results in male sterility because Tcte1-null spermatozoa show aberrant motility. Although the axoneme is structurally normal in Tcte1 mutant spermatozoa, Tcte1-null sperm demonstrate a significant decrease of ATP, which is used by dynein motors to generate the bending force of the flagellum. These data provide a link to defining the molecular intricacies required for axoneme function, sperm motility, and male fertility.

Testis-specific calcium-binding protein CBP86-IV (CABYR) binds with phosphoglycerate kinase 2 in vitro and in vivo experiment.

The investigation of the interacting proteins with testis-specific calcium-binding protein CBP86-IV (CABYR) was carried out in human spermatozoa. The total RNA from human spermatozoa was extracted, and the ORF sequence of TSCBP86-IV gene was amplified and cloned into expression vector pET-28a. The positive recombinant clones were transformed into Escherichia coli strain BL21 (DE3) to express fusion protein. Then, co-immunoprecipitation (Co-IP) of TSCBP86-IV was performed in BL21 cell lysate expressing CBP86-IV recombinant protein. The immune complex was captured and identified by mass spectrometry. Reverse Co-IP of potential interacting proteins was performed in human sperm cell lysate. The potential protein interactions were confirmed by yeast two-hybrid system. Thirteen proteins were successfully identified in immune complex from E. coli cell lysate. Phosphoglycerate kinase 2 (PGK2) further showed positive results both in reverse Co-IP and yeast two-hybrid experiments and was confirmed to be interacted with TSCBP86-IV in human sperm cells.

Mutant NR5A1/SF-1 in patients with disorders of sex development shows defective activation of the SOX9 TESCO enhancer.

Nuclear receptor subfamily 5 group A member 1/Steroidogenic factor 1 (NR5A1; SF-1; Ad4BP) mutations cause 46,XY disorders of sex development (DSD), with phenotypes ranging from developmentally mild (e.g., hypospadias) to severe (e.g., complete gonadal dysgenesis). The molecular mechanism underlying this spectrum is unclear. During sex determination, SF-1 regulates SOX9 (SRY [sex determining region Y]-box 9) expression. We hypothesized that SF-1 mutations in 46,XY DSD patients affect SOX9 expression via the Testis-specific Enhancer of Sox9 core element, TESCO. Our objective was to assess the ability of 20 SF-1 mutants found in 46,XY DSD patients to activate TESCO. Patient DNA was sequenced for SF-1 mutations and mutant SF-1 proteins were examined for transcriptional activity, protein expression, sub-cellular localization and in silico structural defects. Fifteen of the 20 mutants showed reduced SF-1 activation on TESCO, 11 with atypical sub-cellular localization. Fourteen SF-1 mutants were predicted in silico to alter DNA, ligand or cofactor interactions. Our study may implicate aberrant SF-1-mediated transcriptional regulation of SOX9 in 46,XY DSDs.

Profiling of testis-specific long noncoding RNAs in mice.

BACKGROUND: Spermatogenesis, which is the complex and highly regulated process of producing haploid spermatozoa, involves testis-specific transcripts. Recent studies have discovered that long noncoding RNAs (lncRNAs) are novel regulatory molecules that play important roles in various biological processes. However, there has been no report on the comprehensive identification of testis-specific lncRNAs in mice. RESULTS: We performed microarray analysis of transcripts from mouse brain, heart, kidney, liver and testis. We found that testis harbored the highest proportion of tissue-specific lncRNAs (11%; 1607 of 14,256). Testis also harbored the largest number of tissue-specific mRNAs among the examined tissues, but the proportion was lower than that of lncRNAs (7%; 1090 of 16,587). We categorized the testis-specific lncRNAs and found that a large portion corresponded to long intergenic ncRNAs (lincRNAs). Genomic analysis identified 250 protein-coding genes located near (≤ 10 kb) 194 of the loci encoding testis-specific lincRNAs. Gene ontology (GO) analysis showed that these protein-coding genes were enriched for transcriptional regulation-related terms. Analysis of male germ cell-related cell lines (F9, GC-1 and GC-2) revealed that some of the testis-specific lncRNAs were expressed in each of these cell lines. Finally, we arbitrarily selected 26 testis-specific lncRNAs and performed in vitro expression analysis. Our results revealed that all of them were expressed exclusively in the testis, and 23 of the 26 showed germ cell-specific expression. CONCLUSION: This study provides a catalog of testis-specific lncRNAs and a basis for future investigation of the lncRNAs involved in spermatogenesis and testicular functions.

PAPOLB/TPAP regulates spermiogenesis independently of chromatoid body-associated factors.

Mutant mice lacking a testis-specific cytoplasmic poly(A) polymerase, PAPOLB/TPAP, exhibit spermiogenesis arrest and male infertility. However, the mechanism by which PAPOLB regulates spermiogenesis remains unclear. In this study, we examined the relationships between PAPOLB and other spermiogenesis regulators present in the chromatoid body (CB). The loss of PAPOLB had no impact either on the abundance of CB components such as PIWIL1, TDRD6, YBX2, and piRNAs, or on retrotransposon expression. In addition, localization of CB proteins and CB architecture were both normal in PAPOLB-null mice. No interactions were observed between PAPOLB and PIWIL1 or YBX2. While PIWIL1 and YBX2 were associated with translationally inactive messenger ribonucleoproteins and translating polyribosomes, PAPOLB was present almost exclusively in the mRNA-free fractions of sucrose gradients. These results suggest that PAPOLB may regulate spermiogenesis through a pathway distinct from that mediated by CB-associated factors.