Gene expression programs in mammalian spermatogenesis.

Mammalian spermatogenesis, probably the most complex of all cellular developmental processes, is an ideal model both for studying the specific mechanism of gametogenesis and for understanding the basic rules governing all developmental processes, as it entails both cell type-specific and housekeeping molecular processes. Spermatogenesis can be viewed as a mission with many tasks to accomplish, and its success is genetically programmed and ensured by the collaboration of a large number of genes. Here, I present an overview of mammalian spermatogenesis and the mechanisms underlying each step in the process, covering the cellular and molecular activities that occur at each developmental stage and emphasizing their gene regulation in light of recent studies.

VCP promotes tTAF-target gene expression and spermatocyte differentiation by downregulating mono-ubiquitylated H2A.

Valosin-containing protein (VCP) binds and extracts ubiquitylated cargo to regulate protein homeostasis. VCP has been studied primarily in aging and disease contexts, but it also affects germline development. However, the precise molecular functions of VCP in the germline, particularly in males, are poorly understood. Using the Drosophila male germline as a model system, we find that VCP translocates from the cytosol to the nucleus as germ cells transition into the meiotic spermatocyte stage. Importantly, nuclear translocation of VCP appears to be one crucial event stimulated by testis-specific TBP-associated factors (tTAFs) to drive spermatocyte differentiation. VCP promotes the expression of several tTAF-target genes, and VCP knockdown, like tTAF loss of function, causes cells to arrest in early meiotic stages. At a molecular level, VCP activity supports spermatocyte gene expression by downregulating a repressive histone modification, mono-ubiquitylated H2A (H2Aub), during meiosis. Remarkably, experimentally blocking H2Aub in VCP-RNAi testes is sufficient to overcome the meiotic-arrest phenotype and to promote development through the spermatocyte stage. Collectively, our data highlight VCP as a downstream effector of tTAFs that downregulates H2Aub to facilitate meiotic progression.

Kinesin-7 CENP-E mediates chromosome alignment and spindle assembly checkpoint in meiosis I.

In eukaryotes, meiosis is the genetic basis for sexual reproduction, which is important for chromosome stability and species evolution. The defects in meiosis usually lead to chromosome aneuploidy, reduced gamete number, and genetic diseases, but the pathogenic mechanisms are not well clarified. Kinesin-7 CENP-E is a key regulator in chromosome alignment and spindle assembly checkpoint in cell division. However, the functions and mechanisms of CENP-E in male meiosis remain largely unknown. In this study, we have revealed that the CENP-E gene was highly expressed in the rat testis. CENP-E inhibition influences chromosome alignment and spindle organization in metaphase I spermatocytes. We have found that a portion of misaligned homologous chromosomes is located at the spindle poles after CENP-E inhibition, which further activates the spindle assembly checkpoint during the metaphase-to-anaphase transition in rat spermatocytes. Furthermore, CENP-E depletion leads to abnormal spermatogenesis, reduced sperm count, and abnormal sperm head structure. Our findings have elucidated that CENP-E is essential for homologous chromosome alignment and spindle assembly checkpoint in spermatocytes, which further contribute to chromosome stability and sperm cell quality during spermatogenesis.

Dynein-dynactin segregate meiotic chromosomes in C. elegans spermatocytes.

The microtubule motor cytoplasmic dynein 1 (dynein) and its essential activator dynactin have conserved roles in spindle assembly and positioning during female meiosis and mitosis, but their contribution to male meiosis remains poorly understood. Here, we characterize the G33S mutation in the C. elegans dynactin subunit DNC-1, which corresponds to G59S in human p150Glued that causes motor neuron disease. In spermatocytes, dnc-1(G33S) delays spindle assembly and penetrantly inhibits anaphase spindle elongation in meiosis I, which prevents the segregation of homologous chromosomes. By contrast, chromosomes segregate without errors in the early dnc-1(G33S) embryo. Deletion of the DNC-1 N-terminus shows that defective meiosis in dnc-1(G33S) spermatocytes is not due to the inability of DNC-1 to interact with microtubules. Instead, our results suggest that the DNC-1(G33S) protein, which is aggregation prone in vitro, is less stable in spermatocytes than the early embryo, resulting in different phenotypic severity in the two dividing tissues. Thus, the dnc-1(G33S) mutant reveals that dynein-dynactin drive meiotic chromosome segregation in spermatocytes and illustrates that the extent to which protein misfolding leads to loss of function can vary significantly between cell types.

Fance deficiency impaired DNA damage repair of prospermatogonia and altered the repair dynamics of spermatocytes.

BACKGROUND: Non-obstructive azoospermia (NOA) is the most severe form of male infertility and affects approximately 1% of men worldwide. Fanconi anemia (FA) genes were known for their essential role in DNA repair and growing evidence showed the crucial role of FA pathway in NOA. However, the underlying mechanisms for Fance deficiency lead to a serious deficit and delayed maturation of male germ cells remain unclear. METHODS: We used Fance deficiency mouse model for experiments, and collected testes or epididymides from mice at 8 weeks (8W), 17.5 days post coitum (dpc), and postnatal 11 (P11) to P23. The mice referred to three genotypes: wildtype (Fance +/+), heterozygous (Fance +/-), and homozygous (Fance -/-). Hematoxylin and eosin staining, immunofluorescence staining, and surface spread of spermatocytes were performed to explore the mechanisms for NOA of Fance -/- mice. Each experiment was conducted with a minimum of three biological replicates and Kruskal-Wallis with Dunn's correction was used for statistical analysis. RESULTS: In the present study, we found that the adult male Fance -/- mice exhibited massive germ cell loss in seminiferous tubules and dramatically decreased sperms in epididymides. During the embryonic period, the number of Fance -/- prospermatogonia decreased significantly, without impacts on the proliferation (Ki-67, PCNA) and apoptosis (cleaved PARP, cleaved Caspase 3) status. The DNA double-strand breaks (γH2AX) increased at the cellular level of Fance -/- prospermatogonia, potentially associated with the increased nonhomologous end joining (53BP1) and decreased homologous recombination (RAD51) activity. Besides, Fance deficiency impeded the progression of meiotic prophase I of spermatocytes. The mechanisms entailed the reduced recruitment of the DNA end resection protein RPA2 at leptotene and recombinases RAD51 and DMC1 at zygotene. It also involved impaired removal of RPA2 at zygotene and FANCD2 foci at pachytene. And the accelerated initial formation of crossover at early pachytene, which is indicated by MLH1. CONCLUSIONS: Fance deficiency caused massive male germ cell loss involved in the imbalance of DNA damage repair in prospermatogonia and altered dynamics of proteins in homologous recombination, DNA end resection, and crossover, providing new insights into the etiology and molecular basis of NOA.

WWC2 expression in the testis: Implications for spermatogenesis and male fertility.

The family of WWC proteins is known to regulate cell proliferation and organ growth control via the Hippo signaling pathway. As WWC proteins share a similar domain structure and a common set of interacting proteins, they are supposed to fulfill compensatory functions in cells and tissues. While all three WWC family members WWC1, WWC2, and WWC3 are found co-expressed in most human organs including lung, brain, kidney, and liver, in the testis only WWC2 displays a relatively high expression. In this study, we investigated the testicular WWC2 expression in spermatogenesis and male fertility. We show that the Wwc2 mRNA expression level in mouse testes is increased during development in parallel with germ cell proliferation and differentiation. The cellular expression of each individual WWC family member was evaluated in published single-cell mRNA datasets of murine and human testes demonstrating a high WWC2 expression predominantly in early spermatocytes. In line with this, immunohistochemistry revealed cytosolic WWC2 protein expression in primary spermatocytes from human testes displaying full spermatogenesis. In accordance with these findings, markedly lower WWC2 expression levels were detected in testicular tissues from mice and men lacking germ cells. Finally, analysis of whole-exome sequencing data of male patients affected by infertility and unexplained severe spermatogenic failure revealed several heterozygous, rare WWC2 gene variants with a proposed damaging function and putative impact on WWC2 protein structure. Taken together, our findings provide novel insights into the testicular expression of WWC2 and show its cell-specific expression in spermatocytes. As rare WWC2 variants were identified in the background of disturbed spermatogenesis, WWC2 may be a novel candidate gene for male infertility.

Birth of mice from meiotically arrested spermatocytes following biparental meiosis in halved oocytes.

Microinjection of spermatozoa or spermatids into oocytes is a major choice for infertility treatment. However, the use of premeiotic spermatocytes has never been considered because of its technical problems. Here, we show that the efficiency of spermatocyte injection in mice can be improved greatly by reducing the size of the recipient oocytes. Live imaging showed that the underlying mechanism involves reduced premature separation of the spermatocyte's meiotic chromosomes, which produced much greater (19% vs. 1%) birth rates in smaller oocytes. Application of this technique to spermatocyte arrest caused by STX2 deficiency, an azoospermia factor also found in humans, resulted in the production of live offspring. Thus, the microinjection of primary spermatocytes into oocytes may be a potential treatment for overcoming a form of nonobstructive azoospermia caused by meiotic failure.

Cortical recruitment of centralspindlin and RhoA effectors during meiosis I of Caenorhabditiselegans primary spermatocytes.

Haploid male gametes are produced through meiosis during gametogenesis. Whereas the cell biology of mitosis and meiosis is well studied in the nematode Caenorhabditis elegans, comparatively little is known regarding the physical division of primary spermatocytes during meiosis I. Here, we investigated this process using high-resolution time-lapse confocal microscopy and examined the spatiotemporal regulation of contractile ring assembly in C. elegans primary spermatocytes. We found that centralspindlin and RhoA effectors were recruited to the equatorial cortex of dividing primary spermatocytes for contractile ring assembly before segregation of homologous chromosomes. We also observed that perturbations shown to promote centralspindlin oligomerization regulated the cortical recruitment of NMY-2 and impacted the order in which primary spermatocytes along the proximal-distal axis of the gonad enter meiosis I. These results expand our understanding of the cellular division of primary spermatocytes into secondary spermatocytes during meiosis I.This article has an associated First Person interview with the first author of the paper.

Orbit/CLASP determines centriole length by antagonising Klp10A in Drosophila spermatocytes.

After centrosome duplication, centrioles elongate before M phase. To identify genes required for this process and to understand the regulatory mechanism, we investigated the centrioles in Drosophila premeiotic spermatocytes expressing fluorescently tagged centriolar proteins. We demonstrated that an essential microtubule polymerisation factor, Orbit (the Drosophila CLASP orthologue, encoded by chb), accumulated at the distal end of centrioles and was required for the elongation. Conversely, a microtubule-severing factor, Klp10A, shortened the centrioles. Genetic analyses revealed that these two proteins functioned antagonistically to determine centriole length. Furthermore, Cp110 in the distal tip complex was closely associated with the factors involved in centriolar dynamics at the distal end. We observed loss of centriole integrity, including fragmentation of centrioles and earlier separation of the centriole pairs, in Cp110-null mutant cells either overexpressing Orbit or depleted of Klp10A Excess centriole elongation in the absence of the distal tip complex resulted in the loss of centriole integrity, leading to the formation of multipolar spindle microtubules emanating from centriole fragments, even when they were unpaired. Our findings contribute to understanding the mechanism of centriole integrity, disruption of which leads to chromosome instability in cancer cells.

Differential responsiveness of spermatogonia to retinoic acid dictates precocious differentiation but not meiotic entry during steady-state spermatogenesis†.

The foundation of mammalian spermatogenesis is provided by undifferentiated spermatogonia, which comprise of spermatogonial stem cells (SSCs) and transit-amplifying progenitors that differentiate in response to retinoic acid (RA) and are committed to enter meiosis. Our laboratory recently reported that the foundational populations of SSCs, undifferentiated progenitors, and differentiating spermatogonia are formed in the neonatal testis in part based on their differential responsiveness to RA. Here, we expand on those findings to define the extent to which RA responsiveness during steady-state spermatogenesis in the adult testis regulates the spermatogonial fate. Our results reveal that both progenitor and differentiating spermatogonia throughout the testis are capable of responding to exogenous RA, but their resulting fates were quite distinct-undifferentiated progenitors precociously differentiated and proceeded into meiosis on a normal timeline, while differentiating spermatogonia were unable to hasten their entry into meiosis. This reveals that the spermatogonia responding to RA must still complete the 8.6 day differentiation program prior to their entry into meiosis. Addition of exogenous RA enriched testes with preleptotene and pachytene spermatocytes one and two seminiferous cycles later, respectively, supporting recent clinical studies reporting increased sperm production and enhanced fertility in subfertile men on long-term RA analog treatment. Collectively, our results reveal that a well-buffered system exists within mammalian testes to regulate spermatogonial RA exposure, that exposed undifferentiated progenitors can precociously differentiate, but must complete a normal-length differentiation program prior to entering meiosis, and that daily RA treatments increased the numbers of advanced germ cells by directing undifferentiated progenitors to continuously differentiate.

The paralogs' enigma of germ-cell specific genes dispensable for fertility: the case of 19 oogenesin genes†.

Gene knockout experiments have shown that many genes are dispensable for a given biological function. In this review, we make an assessment of male and female germ cell-specific genes dispensable for the function of reproduction in mice, the inactivation of which does not affect fertility. In particular, we describe the deletion of a 1 Mb block containing nineteen paralogous genes of the oogenesin/Pramel family specifically expressed in female and/or male germ cells, which has no consequences in both sexes. We discuss this notion of dispensability and the experiments that need to be carried out to definitively conclude that a gene is dispensable for a function.

Systematic assessment of hexavalent chromium-induced damage to male fertility and the preventive role of melatonin: a longitudinal study from the translational point of view.

Chromium (Cr) and its compounds are closely associated with individuals' lives and extensively used in industry. Excessive exposure to hexavalent chromium (Cr(VI)) induces oxidative damage of various organs including the testes, posing a serious threat to male reproductive fitness. As an endogenous antioxidant, melatonin holds potent antioxidative and anti-inflammatory properties, becoming a potential candidate for treatment of a variety of diseases, including reproductive disorders. Here, by using a mouse model, we systematically assessed Cr(VI)-induced damage to male fertility as well as the preventive role of melatonin. We analyzed the histology and pathology of the testis and epididymis, the density, viability, and malformation of caudal epididymal sperm, the proliferative activity and apoptosis of various spermatogenic subtypes and Sertoli cells, as well as the fertility of mice at five timepoints within one cycle of spermatogenesis (Days 0, 14, 21, 28, and 35) post 14 days of Cr(VI) and/or melatonin intraperitoneal injection. We identified that the testicular damage caused by Cr(VI) persisted to Day 21 after administration and then started to be alleviated, with clear alleviation on Day 35. Pretreatment with melatonin evidently reduced Cr(VI)-induced testicular damage and accelerated spermatogenic restoration, generating an almost normal phenotype on Day 35. Melatonin pretreatment also retained the sperm quality at all time points investigated. Moreover, melatonin to some extent preserved the fertility of Cr(VI)-treated mice without apparent side effects. The findings shed light on the future clinical application of melatonin as a therapeutic agent for environmental heavy metal toxicant-induced male subfertility or infertility.

TERT transcription and translocation into mitochondria regulate benzo[a]pyrene/BPDE-induced senescence and mitochondrial damage in mouse spermatocytes.

Telomere and mitochondria may be the targets of Benzo[a]pyrene (BaP) -induced male reproductive damage, and further elucidation of the toxic molecular mechanisms is necessary. In this study, we used in vivo and in vitro exposure models to explore the molecular mechanisms of TERT regulation in BaP-induced telomere and mitochondrial damage in spermatocytes. The results showed that the treatment of benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), the active metabolite of BaP, caused telomere dysfunction in mouse spermatocyte-derived GC-2 cells, resulting in S-phase arrest and increased senescence-associated secretory phenotype (SASP). These effects were significantly alleviated by telomerase agonist (ABG) pretreatment in GC-2 cells. SIRT1, FOXO3a, or c-MYC overexpressing GC-2 cell models were established to demonstrate that BPDE inhibited TERT transcriptional expression through the SIRT1/FOXO3a/c-MYC pathway, leading to telomere dysfunction. We also observed that BPDE induced mitochondrial compromise, including complex I damage, accompanied by reduced mitochondrial TERT expression. Based on this, we constructed wild-type TERT-overexpressing (OE-TERTwt) and mitochondria targeting TERT-overexpressing (OE-TERTmst) GC-2 cell models and found that OE-TERTmst GC-2 cells improved mitochondrial function better than OE-TERTwt GC-2 cells. Finally, ICR mice were given BaP by intragastric administration for 35 days, which verified the results of the in vitro study. The results shown that BaP exposure can lead to spermatogenesis disturbance, which is related to the telomere and mitochondrial damage in spermatocytes. In conclusion, our results suggest that BPDE causes telomere and mitochondrial damage in spermatocytes by inhibiting TERT transcription and mitochondrial TERT expression. This study elucidates the molecular mechanism of male reproductive toxicity due to environmental pollutant BaP, and also provides a new perspective for the exploration of interventions and protective measures against male reproductive damage by BaP.

Kinesin-14 KIFC1 modulates spindle assembly and chromosome segregation in mouse spermatocytes.

Kinesin-14 KIFC1 regulates spindle assembly and centrosome clustering in diverse organisms during cell division. KIFC1 proteins are essential for spindle assembly and chromosome alignment in mitosis. However, the roles and mechanisms of KIFC1 proteins in male spermatocytes remain largely unknown. In this study, we reveal that KIFC1 proteins mainly accumulate at the centrosomes and central spindle in mouse spermatocytes both in vitro and in vivo. We utilize two KIFC1 specific inhibitors, AZ82 and CW069, for the inhibition of KIFC1 in mouse spermatogenic cells and cultured GC-2 spd(ts) cells. We find that KIFC1 inhibition results in the increase of spermatocytes with micronuclei, the disorganization of the meiotic spindles, and the formation of multiple centrosomes. Furthermore, we demonstrate that KIFC1 inhibition leads to spindle defects, chromosome misalignment and the formation of aneuploidy in cultured GC-2 spd(ts) cells. In this study, we reveal that KIFC1 proteins are critical for centrosome maintenance and chromosome stability in mouse spermatocytes.

Global phosphoproteomic analysis identified key kinases regulating male meiosis in mouse.

Meiosis, a highly conserved process in organisms from fungi to mammals, is subjected to protein phosphorylation regulation. Due to the low abundance of phosphorylation, there is a lack of systemic characterization of phosphorylation regulation of meiosis in mammals. Using the phosphoproteomic approach, we profiled large-scale phosphoproteome of purified primary spermatocytes undergoing meiosis I, and identified 14,660 phosphorylation sites in 4419 phosphoproteins. Kinase-substrate phosphorylation network analysis followed by in vitro meiosis study showed that CDK9 was essential for meiosis progression to metaphase I and had enriched substrate phosphorylation sites in proteins involved in meiotic cell cycle. In addition, histones and epigenetic factors were found to be widely phosphorylated. Among those, HASPIN was found to be essential for male fertility. Haspin knockout led to misalignment of chromosomes, apoptosis of metaphase spermatocytes and a decreased number of sperm by deregulation of H3T3ph, chromosomal passenger complex (CPC) and spindle assembly checkpoint (SAC). The complicated protein phosphorylation and its important regulatory functions in meiosis indicated that in-depth studies of phosphorylation-mediated signaling could help us elucidate the mechanisms of meiosis.

Localization and expression of SLX4 in the testis of sterile male cattle-yak.

Cattle-yak, the hybrid offspring of yak (Bos grunniens) and cattle (Bos taurus), serves as a unique model to dissect the molecular mechanisms underlying reproductive isolation. While female cattle-yaks are fertile, the males are completely sterile due to spermatogenic arrest at the meiosis stage and massive germ cell apoptosis. Interestingly, meiotic defects are partially rescued in the testes of backcrossed offspring. The genetic basis of meiotic defects in male cattle-yak remains unclear. Structure-specific endonuclease subunit (SLX4) participates in meiotic double-strand break (DSB) formation in mice, and its deletion results in defects in spermatogenesis. In the present study, we examined the expression patterns of SLX4 in the testes of yak, cattle-yak, and backcrossed offspring to investigate its potential roles in hybrid sterility. The results showed that the relative abundances of SLX4 mRNA and protein were significantly reduced in the testis of cattle-yak. The results of immunohistochemistry revealed that SLX4 was predominately expressed in spermatogonia and spermatocytes. Chromosome spreading experiments showed that SLX4 was significantly decreased in the pachytene spermatocytes of cattle-yak compared with yak and backcrossed offspring. These findings suggest that SLX4 expression was dysregulated in the testis of cattle-yak, potentially resulting in the failure of crossover formation and collapses of meiosis in hybrid males.

[Long non-coding RNA in spermatogenesis regulation and male infertility: Progress in research].

Long non-coding RNA (lncRNA) is an RNA molecule transcribed by RNA polymerase II, longer than 200 nt, and not translated into proteins. During gonadal development and spermatogenesis, lncRNAs are involved in epigenetic mechanisms, including DNA methylation, chromatin remodeling, and histone tail modification, which play important regulatory roles at the transcriptional or post-transcriptional level. Epigenomics including lncRNA is considered to be the second dimension of DNA sequence that can be adapted to environmental factors to specifically regulate gene expressions in some cells. Based on the functional action mechanism of lncRNAs, we reviewed the advances in the studies of lncRNAs in the direction of spermatogenesis and male infertility and analyzed the potential of lncRNAs as a biomarker of male infertility. The potential application of lncRNA in the treatment of male infertility diseases can be further explored based on the lncRNA target, RNA interference, competitive binding closed target and structural disruption of lncRNAs.

Dual DNA staining enables isolation of multiple sub-types of post-replicative mouse male germ cells.

During spermatogenesis, mammalian male germ cells undergo multiple developmental processes, including meiosis and post-meiotic differentiation (spermiogenesis). To understand the transitions between different cellular states it is essential to isolate pure populations of cells at different stages of development. Previous approaches enabled the isolation of cells from different stages of meiotic prophase I, but techniques to sub-fractionate unfixed, post-meiotic spermatids have been lacking. Here we report the development of a protocol enabling simultaneous isolation of cells at different stages of meiotic prophase and post-meiotic differentiation from testes of adult mice. This approach builds on existing fluorescence activated cell sorting protocols designed to purify cells in different stages of meiotic prophase I. By utilizing the specific spectral properties that two different DNA dyes (Hoechst 33342 and SYTO 16) exhibit when bound to chromatin of different stage male germ cells, we obtain highly pure populations of cells in relatively large numbers. This FACS protocol will enable immunocytological and molecular characterization studies of fractionated meiotic and haploid germ cells from both wild type and genetically mutant animals.

A dual enhancer-silencer element, DES-K16, in mouse spermatocyte-derived GC-2spd(ts) cells.

The multifunctionality of genome is suggested at some loci in different species but not well understood. Here we identified a DES-K16 region in an intron of the Kctd16 gene as the chromatin highly marked with epigenetic modifications of both enhancers (H3K4me1 and H3K27ac) and silencers (H3K27me3) in mouse spermatocytes. In vitro reporter gene assay demonstrated that DES-K16 exhibited significant enhancer activity in spermatocyte-derived GC-2spd(ts) and hepatic tumor-derived Hepa1-6 cells, and a deletion of this sequence in GC-2spd(ts) cells resulted in a decrease and increase of Yipf5 and Kctd16 expression, respectively. This was consistent with increased and decreased expression of Yipf5 and Kctd16, respectively, in primary spermatocytes during testis development. While known dual enhancer-silencers exert each activity in different tissues, our data suggest that DES-K16 functions as both enhancer and silencer in a single cell type, GC-2spd(ts) cells. This is the first report on a dual enhancer-silencer element which activates and suppresses gene expression in a single cell type.

Cellular and subcellular localization of endogenous phospholipase D6 in seminiferous tubules of mouse testes.

Phospholipase D6 (PLD6) plays pivotal roles in mitochondrial dynamics and spermatogenesis, but the cellular and subcellular localization of endogenous PLD6 in testis germ cells is poorly defined. We examined the distribution and subcellular localization of PLD6 in mouse testes using validated specific anti-PLD6 antibodies. Ectopically expressed PLD6 protein was detected in the mitochondria of PLD6-transfected cells, but endogenous PLD6 expression in mouse testes was localized to the perinuclear region of pachytene spermatocytes, and more prominently, to the round (Golgi and cap phases) and elongating spermatids (acrosomal phase); these results suggest that PLD6 is localized to the Golgi apparatus. The distribution of PLD6 in the round spermatids partially overlapped with that of the cis-Golgi marker GM130, indicating that the PLD6 expression corresponded to the GM130-positive subdomains of the Golgi apparatus. Correlative light and electron microscopy revealed that PLD6 expression in developing spermatids was localized almost exclusively to several flattened cisternae, and these structures might correspond to the medial Golgi subcompartment; neither the trans-Golgi networks nor the developing acrosomal system expressed PLD6. Further, we observed that PLD6 interacted with tesmin, a testis-specific transcript necessary for successful spermatogenesis in mouse testes. To our knowledge, these results provide the first evidence of PLD6 as a Golgi-localized protein of pachytene spermatocytes and developing spermatids and suggest that its subcompartment-specific distribution within the Golgi apparatus may be related to the specific functions of this organelle during spermatogenesis.