Haploid Male Germ Cells Research Articles

Busulfan Chemotherapy Downregulates TAF7/TNF-α Signaling in Male Germ Cell Dysfunction.

Background: Busulfan is an FDA-approved alkylating drug used in the chemotherapy of advanced acute myeloid leukemia. The precise mechanisms by which Busulfan kills spermatogonia stem cells (SSCs) are not yet completely understood. Methods: Using a murine model, we evaluated Busulfan-induced apoptosis and DNA damage signaling between testis and ovary tissues. We executed RT-qPCR, analyzed single-nuclei RNA sequencing data and performed in situ hybridization for the localization of the gene expression in the tissues. Results: The results indicate that, in contrast to female germ cells, haploid male germ cells undergo significant apoptosis following Busulfan chemotherapy. Moreover, a gene enrichment analysis revealed that reactive oxygen species may activate the inflammatory response in part through the TNF-α/NF-κB signaling pathway. Interestingly, in the testis, the mRNA levels of TNF-α and TAF7 (TATA box-binding protein-associated factor 7) are downregulated, and testosterone levels suppressed. Mechanistically, the promoter of TNF-α has a conserved motif for binding TAF7, which is necessary for its transcriptional activation and may require further in-depth study. We next analyzed the tumorigenic function of TAF7 and revealed that it is highly overexpressed in several types of human cancers, particularly testicular germ cell tumors, and associated with poor patient survival. Therefore, we executed in situ hybridization and single-nuclei RNA sequencing, finding that less TAF7 mRNA is present in SSCs after chemotherapy. Conclusions: Thus, our data indicate a possible function of TAF7 in the regulation of SSCs and spermatogenesis following downregulation by Busulfan. These findings may account for the therapeutic effects of Busulfan and underlie its potential impact on cancer chemotherapy prognosis.

In vivo and in vitro sperm production: an overview of the challenges and advances in male fertility restoration.

Male infertility can be caused by genetic anomalies, endocrine disorders, inflammation, and exposure to toxic chemicals or gonadotoxic treatments. Therefore, several recent studies have concentrated on the preservation and restoration of fertility to enhance the quality of life for affected individuals. It is currently recommended to biobank the tissue extracted from testicular biopsies to provide a later source of spermatogonial stem cells (SSCs). Another successful approach has been the in vitro production of haploid male germ cells. The capacity of SSCs to transform into sperm, as in testicular tissue transplantation, SSC therapy, and in vitro or ex vivo spermatogenesis, makes them ideal candidates for in vivo fertility restoration. The transplantation of SSCs or testicular tissue to regenerate spermatogenesis and create embryos has been achieved in nonhuman mammal species. Although the outcomes of human trials have yet to be released, this method may soon be approved for clinical use in humans. Furthermore, regenerative medicine techniques that develop tissue or cells on organic or synthetic scaffolds enriched with bioactive molecules have also gained traction. All of these methods are now in different stages of experimentation and clinical trials. However, thanks to rigorous studies on the safety and effectiveness of SSC-based reproductive treatments, some of these techniques may be clinically available in upcoming decades.

Spem2, a novel testis-enriched gene, is required for spermiogenesis and fertilization in mice

Spermiogenesis is considered to be crucial for the production of haploid spermatozoa with normal morphology, structure and function, but the mechanisms underlying this process remain largely unclear. Here, we demonstrate that SPEM family member 2 (Spem2), as a novel testis-enriched gene, is essential for spermiogenesis and male fertility. Spem2 is predominantly expressed in the haploid male germ cells and is highly conserved across mammals. Mice deficient for Spem2 develop male infertility associated with spermiogenesis impairment. Specifically, the insufficient sperm individualization, failure of excess cytoplasm shedding, and defects in acrosome formation are evident in Spem2-null sperm. Sperm counts and motility are also significantly reduced compared to controls. In vivo fertilization assays have shown that Spem2-null sperm are unable to fertilize oocytes, possibly due to their impaired ability to migrate from the uterus into the oviduct. However, the infertility of Spem2−/− males cannot be rescued by in vitro fertilization, suggesting that defective sperm–egg interaction may also be a contributing factor. Furthermore, SPEM2 is detected to interact with ZPBP, PRSS21, PRSS54, PRSS55, ADAM2 and ADAM3 and is also required for their processing and maturation in epididymal sperm. Our findings establish SPEM2 as an essential regulator of spermiogenesis and fertilization in mice, possibly in mammals including humans. Understanding the molecular role of SPEM2 could provide new insights into future therapeutic treatment of human male infertility and development of non-hormonal male contraceptives.

Uncoupling transcription and translation through miRNA-dependent poly(A) length control in haploid male germ cells.

As one of the post-transcriptional regulatory mechanisms, uncoupling of transcription and translation plays an essential role in development and adulthood physiology. However, it remains elusive how thousands of mRNAs get translationally silenced while stability is maintained for hours or even days before translation. In addition to oocytes and neurons, developing spermatids display significant uncoupling of transcription and translation for delayed translation. Therefore, spermiogenesis represents an excellent in vivo model for investigating the mechanism underlying uncoupled transcription and translation. Through full-length poly(A) deep sequencing, we discovered dynamic changes in poly(A) length through deadenylation and re-polyadenylation. Deadenylation appeared to be mediated by microRNAs (miRNAs), and transcripts with shorter poly(A) tails tend to be sequestered into ribonucleoprotein (RNP) granules for translational repression and stabilization. In contrast, re-polyadenylation might allow for translocation of the translationally repressed transcripts from RNP granules to polysomes. Overall, our data suggest that miRNA-dependent poly(A) length control represents a previously unreported mechanism underlying uncoupled translation and transcription in haploid male mouse germ cells.

Ubiquitin-Proteasome System-Regulated Protein Degradation in Spermatogenesis.

Spermatogenesis is a prolonged and highly ordered physiological process that produces haploid male germ cells through more than 40 steps and experiences dramatic morphological and cellular transformations. The ubiquitin proteasome system (UPS) plays central roles in the precise control of protein homeostasis to ensure the effectiveness of certain protein groups at a given stage and the inactivation of them after this stage. Many UPS components have been demonstrated to regulate the progression of spermatogenesis at different levels. Especially in recent years, novel testis-specific proteasome isoforms have been identified to be essential and unique for spermatogenesis. In this review, we set out to discuss our current knowledge in functions of diverse USP components in mammalian spermatogenesis through: (1) the composition of proteasome isoforms at each stage of spermatogenesis; (2) the specificity of each proteasome isoform and the associated degradation events; (3) the E3 ubiquitin ligases mediating protein ubiquitination in male germ cells; and (4) the deubiquitinases involved in spermatogenesis and male fertility. Exploring the functions of UPS machineries in spermatogenesis provides a global picture of the proteome dynamics during male germ cell production and shed light on the etiology and pathogenesis of human male infertility.

In vitro production of haploid germ cells from murine spermatogonial stem cells using a two-dimensional cell culture system

The in vitro propagation and differentiation of spermatogonial stem cells (SSCs) has many potential applications within reproductive science and medicine. We established a two-dimensional (2D) cell culture system to proliferate and differentiate prepubertal mouse SSCs as a model capable of maximizing on a small number of donor SSCs. We also investigated the effects of retinol on in vitro SSC differentiation. Testis cells were cultured for 10 days in a serum-free medium. This produced SSC colonies which were then dissociated and sub-cultured for an additional 20 days in a differentiation medium. Before inducing differentiation, colonies expressed genes specific for undifferentiated spermatogonia (Ngn3, Plzf). After 10 days in the differentiation medium, Stra8 expression was upregulated. After 20 days, Acr expression was upregulated, indicating the completion of meiosis. Immunofluorescence, RT-PCR and flow cytometry confirmed the presence of haploid male germ cells (4.4% of all cells). When retinol was added to the differentiation medium the proportion of haploid germ cells increased (8.1% of cells). We concluded that, under serum-free culture conditions, prepubertal SSCs will generate colonies that can differentiate into haploid germ cells in a 2D culture system. These cells demonstrate a relatively high efficiency of haploid-cell production, which can be further improved with retinol.

Cracd Marks the First Wave of Meiosis during Spermatogenesis and Is Mis-Expressed in Azoospermia Mice.

Testicular development starts in utero and maturation continues postnatally, requiring a cascade of gene activation and differentiation into different cell types, with each cell type having its own specific function. As we had previously reported that the Capping protein inhibiting regulator of actin (Cracd) gene was expressed in the adult mouse testis, herein we examine when and where the β-catenin associated Cracd is initially expressed during postnatal testis development. Significantly, Cracd mRNA is present in both the immature postnatal and adult testis in round spermatid cells, with highest level of expression occurring during the first wave of meiosis and spermatogenesis. In the juvenile testes, Cracd is initially expressed within the innermost region but as maturation occurs, Cracd mRNA switches to a more peripheral location. Thereafter, Cracd is downregulated to maintenance levels in the haploid male germ cell lineage. As Cracd mRNA was expressed within developing round spermatids, we tested its effectiveness as a biomarker of non-obstructive azoospermia using transgenic knockout mice models. Meaningfully, Cracd expression was absent in Deleted in azoospermia like (Dazl) null testis, which exhibit a dramatic germ cell loss. Moreover, Cracd was abnormally regulated and ectopically mis-expressed in Polypyrimidine tract binding protein-2 (Ptbp2) conditional germ cell restricted knockout testis, which exhibit a block during spermatid differentiation and a reduction in the number of late stage spermatocytes coincident with reduced β-catenin expression. Combined, these data suggest that Cracd is a useful first wave of spermatogenesis biomarker of azoospermia phenotypes, even prior to an overt phenotype being evident.

Haploid male germ cells-the Grand Central Station of protein transport.

The precise movement of proteins and vesicles is an essential ability for all eukaryotic cells. Nowhere is this more evident than during the remarkable transformation that occurs in spermiogenesis-the transformation of haploid round spermatids into sperm. These transformations are critically dependent upon both the microtubule and the actin cytoskeleton, and defects in these processes are thought to underpin a significant percentage of human male infertility. This review is aimed at summarising and synthesising the current state of knowledge around protein/vesicle transport during haploid male germ cell development and identifying knowledge gaps and challenges for future research. To achieve this, we summarise the key discoveries related to protein transport using the mouse as a model system. Where relevant, we anchored these insights to knowledge in the field of human spermiogenesis and the causality of human male infertility. Relevant studies published in English were identified using PubMed using a range of search terms related to the core focus of the review-protein/vesicle transport, intra-flagellar transport, intra-manchette transport, Golgi, acrosome, manchette, axoneme, outer dense fibres and fibrous sheath. Searches were not restricted to a particular time frame or species although the emphasis within the review is on mammalian spermiogenesis. Spermiogenesis is the final phase of sperm development. It results in the transformation of a round cell into a highly polarised sperm with the capacity for fertility. It is critically dependent on the cytoskeleton and its ability to transport protein complexes and vesicles over long distances and often between distinct cytoplasmic compartments. The development of the acrosome covering the sperm head, the sperm tail within the ciliary lobe, the manchette and its role in sperm head shaping and protein transport into the tail, and the assembly of mitochondria into the mid-piece of sperm, may all be viewed as a series of overlapping and interconnected train tracks. Defects in this redistribution network lead to male infertility characterised by abnormal sperm morphology (teratozoospermia) and/or abnormal sperm motility (asthenozoospermia) and are likely to be causal of, or contribute to, a significant percentage of human male infertility. A greater understanding of the mechanisms of protein transport in spermiogenesis offers the potential to precisely diagnose cases of male infertility and to forecast implications for children conceived using gametes containing these mutations. The manipulation of these processes will offer opportunities for male-based contraceptive development. Further, as increasingly evidenced in the literature, we believe that the continuous and spatiotemporally restrained nature of spermiogenesis provides an outstanding model system to identify, and de-code, cytoskeletal elements and transport mechanisms of relevance to multiple tissues.

Spermatogonial Stem Cells for In Vitro Spermatogenesis and In Vivo Restoration of Fertility.

Spermatogonial stem cells (SSCs) are the only adult stem cells capable of passing genes onto the next generation. SSCs also have the potential to provide important knowledge about stem cells in general and to offer critical in vitro and in vivo applications in assisted reproductive technologies. After century-long research, proof-of-principle culture systems have been introduced to support the in vitro differentiation of SSCs from rodent models into haploid male germ cells. Despite recent progress in organotypic testicular tissue culture and two-dimensional or three-dimensional cell culture systems, to achieve complete in vitro spermatogenesis (IVS) using non-rodent species remains challenging. Successful in vitro production of human haploid male germ cells will foster hopes of preserving the fertility potential of prepubertal cancer patients who frequently face infertility due to the gonadotoxic side-effects of cancer treatment. Moreover, the development of optimal systems for IVS would allow designing experiments that are otherwise difficult or impossible to be performed directly in vivo, such as genetic manipulation of germ cells or correction of genetic disorders. This review outlines the recent progress in the use of SSCs for IVS and potential in vivo applications for the restoration of fertility.

ZFP628 Is a TAF4b-Interacting Transcription Factor Required for Mouse Spermiogenesis.

TAF4b is a subunit of the TFIID complex that is highly expressed in the ovary and testis and required for mouse fertility. TAF4b-deficient male mice undergo a complex series of developmental defects that result in the inability to maintain long-term spermatogenesis. To decipher the transcriptional mechanisms upon which TAF4b functions in spermatogenesis, we used two-hybrid screening to identify a novel TAF4b-interacting transcriptional cofactor, ZFP628. Deletion analysis of both proteins reveals discrete and novel domains of ZFP628 and TAF4b protein that function to bridge their direct interaction in vitro Moreover, coimmunoprecipitation of ZFP628 and TAF4b proteins in testis-derived protein extracts supports their endogenous association. Using CRISPR-Cas9, we disrupted the expression of ZFP628 in the mouse and uncovered a postmeiotic germ cell arrest at the round spermatid stage in the seminiferous tubules of the testis in ZFP628-deficient mice that results in male infertility. Coincident with round spermatid arrest, we find reduced mRNA expression of transition protein (Tnp1 and Tnp2) and protamine (Prm1 and Prm2) genes, which are critical for the specialized maturation of haploid male germ cells called spermiogenesis. These data delineate a novel association of two transcription factors, TAF4b and ZFP628, and identify ZFP628 as a novel transcriptional regulator of stage-specific spermiogenesis.

Three-dimensional decellularized amnion membrane scaffold promotes the efficiency of male germ cells generation from human induced pluripotent stem cells

Cells grow differently in conventional 2D cell culture than when they grow in the physiological microenvironment. In this study, we developed a 3D cell culture model for generating male germ cells from human iPSCs using a human decellularized amnion membrane (DAM) scaffold. To this end, human iPSCs were generated using retroviral vectors and characterized for pluripotency properties by immunofluorescence assay, flow cytometry, ALP staining, cytogenetic assay, and differentiation capacity. The iPSCs were used for investigating male germ cells differentiation efficiency in both conventional 2D culture and 3D-DAM scaffold. The expression of male germ cell markers was evaluated at day 21 of differentiation using immunofluorescence assay, flow-cytometry, and RT-qPCR. The results indicated a successful reprogramming of human foreskin fibroblast cells into pluripotent iPSCs. The reprogrammed cells were positive for pluripotency markers and differentiated into the three germ layers. During male germ cell differentiation, the cells tend to aggregate and form colony-like structures in both 2D and 3D conditions. However, significant expression of VASA, DAZL, PLZF, STELLA, and NANOS3 markers and more efficient haploid male germ cell production were observed in the 3D condition when compared to the 2D model. Considering the effect of the 3D-DAM scaffold in prompting male germ cell-specific markers and increased efficiency of germ cell differentiation in 3D culture, it appears that DAM scaffold is a useful tool for in vitro studies of human germ cell development and ultimately future clinical application.

Efficient GFP-labeling and analysis of spermatogenic cells using the IRG transgene and flow cytometry.

SummarySpermatogenesis is a highly ordered developmental program that produces haploid male germ cells. The study of male germ cell development in the mouse has provided unique perspectives into the molecular mechanisms that control cell development and differentiation in mammals, including tissue‐specific gene regulatory programs. An intrinsic challenge in spermatogenesis research is the heterogeneity of germ and somatic cell types present in the testis. Techniques to separate and isolate distinct mouse spermatogenic cell types have great potential to shed light on molecular mechanisms controlling mammalian cell development, while also providing new insights into cellular events important for human reproductive health. Here, we detail a versatile strategy that combines Cre‐lox technology to fluorescently label germ cells, with flow cytometry to discriminate and isolate germ cells in different stages of development for cellular and molecular analyses.

Sperm nuclear protamines: A checkpoint to control sperm chromatin quality.

Protamines are nuclear proteins which are specifically expressed in haploid male germ cells. Their replacement of histones and binding to DNA is followed by chromatin hypercondensation that protects DNA from negative influences by environmental factors. Mammalian sperm contain two types of protamines: PRM1 and PRM2. While the proportion of the two protamines is highly variable between different species, abnormal ratios within a species are known to be associated with male subfertility. Therefore, it is more than likely that correct protamine expression represents a kind of chromatin checkpoint during sperm development rendering protamines as suitable biomarkers for the estimation of sperm quality. This review presents an overview of our current knowledge on protamines comparing gene and protein structures between different mammalian species with particular consideration given to man, mouse and stallion. At last, recent insights into the possible role of inherited sperm histones for early embryo development are provided.

Ultra-structure of the sperm head-to-tail linkage complex in the absence of the spermatid-specific LINC component SPAG4.

Tight connection between sperm head and tail is crucial for the transport of the male genome and fertilization. The linkage complex, the sperm head-to-tail coupling apparatus (HTCA), originates from the centrosome and anchors to the nuclear membrane. In contrast to its ultra-structural organization, which is already well known for decades, its protein composition largely still awaits future deciphering. SUN-domain proteins are essential components of a complex that links the cytoskeleton to the peripheral nucleoskeleton, which is the nuclear lamina. Here, we studied the impact of the SUN protein SPAG4/SUN4 on the formation of the HTCA. SPAG4/SUN4 is specifically expressed in haploid male germ cells showing a polarized distribution towards the posterior pole in late spermatids that corresponds to the tail attachment site. SPAG4-deficient male mice are infertile with compromised manchette formation and malformed sperm heads. Nonetheless, sperm tails are present demonstrating dispensability of a proper manchette for their formation. Ultra-structural analyses revealed that the development of the sperm head-to-tail linkage complex in the absence of SPAG4 resembles that in the wild type. However, in SPAG4-deficient sperm, the attachment site is diminished with obvious lateral detachment of the HTCA from the nucleus. Our results thus indicate that SPAG4, albeit not essential for the formation of the HTCA per se, is, nevertheless, required for tightening the sperm head-to-tail anchorage by provoking the correct attachment of the lateral parts of the basal plate to the implantation fossa.

Characterization of Post-Meiotic Male Germ Cell Genome Organizational States.

Dramatic and unique genome reorganizations accompany the differentiation of haploid male germ cells, characterized by a gradual loss of the vast majority of histones leading to a final tight compaction of the genome by protamines. Despite being essential for procreation and the life cycle, the mechanisms driving the transformation of nucleosomes into nucleoprotamines remain poorly understood. To address this issue, our laboratory has developed a number of specific approaches, ranging from the purification of spermatogenic cells at specific stages, the analysis of chromatin transitional states, the functional characterization of histone variants, histone-replacing proteins and their chaperones. This chapter will detail all related relevant techniques with a particular emphasis on methods allowing the functional studies of histone variants and the genome organizational states associated with the studied histones in spermatogenic cells undergoing histone-to-protamine exchange.

ALKBH5-dependent m6A demethylation controls splicing and stability of long 3′-UTR mRNAs in male germ cells

N6-methyladenosine (m6A) represents one of the most common RNA modifications in eukaryotes. Specific m6A writer, eraser, and reader proteins have been identified. As an m6A eraser, ALKBH5 specifically removes m6A from target mRNAs and inactivation of Alkbh5 leads to male infertility in mice. However, the underlying molecular mechanism remains unknown. Here, we report that ALKBH5-mediated m6A erasure in the nuclei of spermatocytes and round spermatids is essential for correct splicing and the production of longer 3'-UTR mRNAs, and failure to do so leads to aberrant splicing and production of shorter transcripts with elevated levels of m6A that are rapidly degraded. Our study identified reversible m6A modification as a critical mechanism of posttranscriptional control of mRNA fate in late meiotic and haploid spermatogenic cells.

Katanin-like 2 (KATNAL2) functions in multiple aspects of haploid male germ cell development in the mouse.

The katanin microtubule-severing proteins are essential regulators of microtubule dynamics in a diverse range of species. Here we have defined critical roles for the poorly characterised katanin protein KATNAL2 in multiple aspects of spermatogenesis: the initiation of sperm tail growth from the basal body, sperm head shaping via the manchette, acrosome attachment, and ultimately sperm release. We present data suggesting that depending on context, KATNAL2 can partner with the regulatory protein KATNB1 or act autonomously. Moreover, our data indicate KATNAL2 may regulate δ- and ε-tubulin rather than classical α-β-tubulin microtubule polymers, suggesting the katanin family has a greater diversity of function than previously realised. Together with our previous research, showing the essential requirement of katanin proteins KATNAL1 and KATNB1 during spermatogenesis, our data supports the concept that in higher order species the presence of multiple katanins has allowed for subspecialisation of function within complex cellular settings such as the seminiferous epithelium.

Progress and future prospect of in vitro spermatogenesis.

Infertility has become a major health issue in the world. It affects the social life of couples and of all infertility cases; approximately 40–50% is due to “male factor” infertility. Male infertility could be due to genetic factors, environment or due to gonadotoxic treatment. Developments in reproductive biotechnology have made it possible to rescue fertility and uphold biological fatherhood. In vitro production of haploid male germ cell is a powerful tool, not only for the treatment of infertility including oligozoospermic or azoospermic patient, but also for the fertility preservation in pre-pubertal boys whose gonadal function is threatened by gonadotoxic therapies. Genomic editing of in-vitro cultured germ cells could also potentially cure flaws in spermatogenesis due to genomic mutation. Furthermore, this ex-vivo maturation technique with genomic editing may be used to prevent paternal transmission of genomic diseases. Here, we summarize the historical progress of in vitro spermatogenesis research by using organ and cell culture techniques and the future clinical application of in vitro spermatogenesis.

MicroRNAs control mRNA fate by compartmentalization based on 3\u2032 UTR length in male germ cells

BackgroundPost-transcriptional regulation of gene expression can be achieved through the control of mRNA stability, cytoplasmic compartmentalization, 3′ UTR length and translational efficacy. Spermiogenesis, a process through which haploid male germ cells differentiate into spermatozoa, represents an ideal model for studying post-transcriptional regulation in vivo because it involves a large number of transcripts that are physically sequestered in ribonucleoprotein particles (RNPs) and thus subjected to delayed translation. To explore how small RNAs regulate mRNA fate, we conducted RNA-Seq analyses to determine not only the levels of both mRNAs and small noncoding RNAs, but also their cytoplasmic compartmentalization during spermiogenesis.ResultAmong all small noncoding RNAs studied, miRNAs displayed the most dynamic changes in both abundance and subcytoplasmic localization. mRNAs with shorter 3′ UTRs became increasingly enriched in RNPs from pachytene spermatocytes to round spermatids, and the enrichment of shorter 3′ UTR mRNAs in RNPs coincided with newly synthesized miRNAs that target these mRNAs at sites closer to the stop codon. In contrast, the translocation of longer 3′ UTR mRNAs from RNPs to polysomes correlated with the production of new miRNAs that target these mRNAs at sites distal to the stop codon.ConclusionsmiRNAs appear to control cytoplasmic compartmentalization of mRNAs based on 3′ UTR length. Our data suggest that transcripts with longer 3′ UTRs tend to contain distal miRNA binding sites and are thus targeted to polysomes for translation followed by degradation. In contrast, those with shorter 3′ UTRs only possess proximal miRNA binding sites, which, therefore, are targeted into RNPs for enrichment and delayed translation.

Ccdc181 is a microtubule-binding protein that interacts with Hook1 in haploid male germ cells and localizes to the sperm tail and motile cilia

Disruption of murine Hook1 results in a disturbed spermatogenesis and consequently leads to male infertility in mice. Within these mice abnormal sperm development starts with a disorganization of the microtubular manchette in elongating spermatids that leads to an abnormal head shape as well as to distinctive structural changes in the flagella of the sperm. To elucidate Hook1 function in male germ cell differentiation a yeast two-hybrid screen was performed using a murine testicular library, which leads to the identification of several putative Hook1 interacting proteins. One of the isolated cDNA fragments encodes for the coiled-coil domain containing protein 181 (Ccdc181). The putative interaction of Ccdc181 with Hook1 was verified by FRET analysis and interacting regions were identified using yeast two-hybrid assays. Furthermore, Ccdc181 seems to interact directly with microtubules and localizes to the microtubular manchette of elongating spermatids, resembling the previously reported localization of Hook1. According to the observed immunostaining pattern the RNA expression of Ccdc181 is less prominent in pre-meiotic stages of sperm development but increases in the haploid phase of spermatogenesis and seems to be restricted to male germ cells. However, Ccdc181 expression is also observed to a lower extent in somatic tissues, particularly, in tissues containing ciliated epithelia. Additionally, Ccdc181 protein is found to localize to the sperm flagella and to the basal half of motile cilia, whereas Ccdc181 was not detected in primary non-motile cilia. Furthermore, we showed that Ccdc181 is a putative interacting partner of the different catalytic subunits of Pp1, raising the hypothesis that Ccdc181 plays a role in mediating ciliary motility.