Trim66's paternal deficiency causes intrauterine overgrowth.

The tripartite motif-containing protein 66 (TRIM66, also known as TIF1-delta) is a PHD-Bromo-containing protein primarily expressed in post-meiotic male germ cells known as spermatids. Biophysical assays showed that the TRIM66 PHD-Bromodomain binds to H3 N-terminus only when lysine 4 is unmethylated. We addressed TRIM66's role in reproduction by loss-of-function genetics in the mouse. Males homozygous for Trim66-null mutations produced functional spermatozoa. Round spermatids lacking TRIM66 up-regulated a network of genes involved in histone acetylation and H3K4 methylation. Profiling of H3K4me3 patterns in the sperm produced by the Trim66-null mutant showed minor alterations below statistical significance. Unexpectedly, Trim66-null males, but not females, sired pups overweight at birth, hence revealing that Trim66 mutations cause a paternal effect phenotype.

Spatially revealed roles for lncRNAs in Drosophila spermatogenesis, Y chromosome function and evolution.

Unlike coding genes, the number of lncRNA genes in organism genomes is relatively proportional to organism complexity. From plants to humans, the tissues with highest numbers and levels of lncRNA gene expression are the male reproductive organs. To learn why, we initiated a genome-wide analysis of Drosophila lncRNA spatial expression patterns in these tissues. The numbers of genes and levels of expression observed greatly exceed those previously reported, due largely to a preponderance of non-polyadenylated transcripts. In stark contrast to coding genes, the highest numbers of lncRNAs expressed are in post-meiotic spermatids. Correlations between expression levels, localization and previously performed genetic analyses indicate high levels of function and requirement. More focused analyses indicate that lncRNAs play major roles in evolution by controlling transposable element activities, Y chromosome gene expression and sperm construction. A new type of lncRNA-based particle found in seminal fluid may also contribute to reproductive outcomes.

The Planar Cell Polarity Protein Fat1 in Sertoli Cell Function.

Fat (FAT atypical cadherin) and Dchs (Dachsous cadherin-related protein) in adjacent Sertoli:Sertoli, Sertoli:spermatid, and spermatid:spermatid interfaces create an important intercellular bridge whose adhesive function is in turn supported by Fjx1, a nonreceptor Ser/Thr protein kinase. This concept is derived from earlier studies of Drosophila, which has been confirmed in this and earlier reports as well. Herein, we use the approach of knockdown of Fat1 by RNAi using primary cultures of Sertoli cells that mimicked the blood-testis barrier (BTB) in vivo, and a series of coherent experiments including functional assays to monitor the Sertoli cell tight junction (TJ) permeability barrier and a functional in vitro TJ integrity assay to assess the role of Fat1 in the testis. It was shown that planar cell polarity (PCP) protein Fat1 affected Sertoli cell function through its modulation of actin and microtubule cytoskeletal function, altering their polymerization activity through the Fat1/Fjx1 complex. Furthermore, Fat1 is intimately associated with ß-catenin and α-N-catenin, as well as with Prickle 1 of the Vangl1/Prickle 1 complex, another PCP core protein to support intercellular interactions to confer PCP. In summary, these findings support the notion that the Fat:Dchs and the Vangl2:Fzd PCP intercellular bridges are tightly associated with basal ES/TJ structural proteins to stabilize PCP function at the Sertoli:Sertoli, Sertoli:spermatid, and spermatid:spermatid interface to sustain spermatogenesis.

RpS25 is required for sperm elongation and individualization during Drosophila spermatogenesis.

Ribosomal protein 25 (RPS25) has been related to male fertility diseases in humans. However, the role of RPS25 in spermatogenesis has yet to be well understood. RpS25 is evolutionarily highly conserved from flies to humans through sequence alignment and phylogenetic tree construction. In this study, we found that RpS25 plays a critical role in Drosophila spermatogenesis and its knockdown leads to male sterility. Examination of each stage of spermatogenesis from RpS25-knockdown flies showed that RpS25 was not required for initial germline cell divisions, but was required for spermatid elongation and individualization. In RpS25-knockdown testes, the average length of cyst elongation was shortened, the spermatid nuclei bundling was disrupted, and the assembly of individualization complex from actin cones failed, resulting in the failure of mature sperm production. Our data revealed an essential role of RpS25 during Drosophila spermatogenesis through regulating spermatid elongation and individualization.

Autonomous transposons tune their sequences to ensure somatic suppression.

Transposable elements (TEs) are a major constituent of human genes, occupying approximately half of the intronic space. During pre-messenger RNA synthesis, intronic TEs are transcribed along with their host genes but rarely contribute to the final mRNA product because they are spliced out together with the intron and rapidly degraded. Paradoxically, TEs are an abundant source of RNA-processing signals through which they can create new introns1, and also functional2 or non-functional chimeric transcripts3. The rarity of these events implies the existence of a resilient splicing code that is able to suppress TE exonization without compromising host pre-mRNA processing. Here we show that SAFB proteins protect genome integrity by preventing retrotransposition of L1 elements while maintaining splicing integrity, via prevention of the exonization of previously integrated TEs. This unique dual role is possible because of L1's conserved adenosine-rich coding sequences that are bound by SAFB proteins. The suppressive activity of SAFB extends to tissue-specific, giant protein-coding cassette exons, nested genes and Tigger DNA transposons. Moreover, SAFB also suppresses LTR/ERV elements in species in which they are still active, such as mice and flies. A significant subset of splicing events suppressed by SAFB in somatic cells are activated in the testis, coinciding with low SAFB expression in postmeiotic spermatids. Reminiscent of the division of labour between innate and adaptive immune systems that fight external pathogens, our results uncover SAFB proteins as an RNA-based, pattern-guided, non-adaptive defence system against TEs in the soma, complementing the RNA-based, adaptive Piwi-interacting RNA pathway of the germline.

Immunolocalization of apelin receptor (APJ) in mouse seminiferous epithelium.

The apelin receptor (APJ) belongs to the member of the G protein-coupled receptor family, and expression of APJ has been reported in the different cell types of testis. The seminiferous tubules in the testis can be identified as different stages (I-XII). It has been also suggested that different factors could be expressed in stage and cell-specific manner in the seminiferous tubules. Recently, we also shown that expression of APJ is developmentally regulated in the testis from PND1 to PND42. Therefore, we analyzed the expression of APJ in the testis of adult mice by immunohistochemistry. Immunohistochemistry showed that the APJ was highly specific for the round and elongated spermatids with stage-dependent changes. The seminiferous tubules at stages I-VII showed APJ immunostaining in the spermatid steps 1-8, not steps of 13-16. The seminiferous tubules at stages IX-XII showed APJ immunostaining in the spermatid steps 9-12. These results suggested the possible role of APJ in the spermiogenesis process. The intratesticular administration of APJ antagonist, ML221 showed a few round spermatids in the seminiferous tubules and some of the tubules with complete absence of round spermatid. Overall, we present evidence that APJ expression in spermatid is dependent on the stages of the seminiferous epithelium cycle and APJ could be involved in the differentiation of round spermatid to elongated spermatid.

Coiled-coil domain containing 159 is required for spermatid head and tail assembly in mice†.

The centrosome is critical for maintaining the sperm head-tail connection and the formation of flagellar microtubules. In this study, we found that in mouse testes, CCDC159 (coiled-coil domain-containing protein 159) is specifically localized to the head-tail coupling apparatus (HTCA) of spermatids, a structure that ensures sperm head-tail tight conjunction. CCDC159 contains a C-terminal coiled-coil domain that functions as the centrosomal localization signal. Gene knockout (KO) of Ccdc159 in mice resulted in acephalic spermatozoa, abnormal flagella, and male infertility. To explore the mechanism behind CCDC159 regulating spermatogenesis, we identified CCDC159-binding proteins using a yeast two-hybrid screen and speculated that CCDC159 participates in HTCA assembly by regulating protein phosphatase PP1 activity. Further RNA-sequencing analyses of Ccdc159 KO testes revealed numerous genes involved in male gamete generation that were downregulated. Together, our results show that CCDC159 in spermatids is a novel centrosomal protein anchoring the sperm head to the tail. Considering the limitation of KO mouse model in clarifying the biological function of CCDC159 in spermatogenesis, a gene-rescue experiment will be performed in the future.

Spermatid perinuclear RNA-binding protein promotes UBR5-mediated proteolysis of Dicer to accelerate triple-negative breast cancer progression.

Triple-negative breast cancer (TNBC) is the most lethal subtype of breast cancer with no targeted therapy. Spermatid perinuclear RNA binding protein (STRBP), a poorly characterized RNA-binding protein (RBP), has an essential role in normal spermatogenesis and sperm function, but whether and how its dysregulation contributing to cancer progression has not yet been explored. Here, we report that STRBP functions as a novel oncogene to drive TNBC progression. STRBP expression was upregulated in TNBC tissues and correlated with poor disease prognosis. Functionally, STRBP promoted TNBC cell proliferation, migration, and invasion in vitro, and enhanced xenograft tumor growth and lung colonization in mice. Mechanistically, STRBP interacted with Dicer, a core component of the microRNA biogenesis machinery, and promoted its proteasomal degradation through enhancing its interaction with E3 ubiquitin ligase UBR5. MicroRNA-sequencing analysis identified miR-200a-3p as a downstream effector of STRBP, which was regulated by Dicer and affected epithelial-mesenchymal transition. Importantly, the impaired malignant phenotypes of TNBC cells caused by STRBP depletion were largely rescued by knockdown of Dicer, and these effects were compromised by transfection of miR-200a-3p mimics. Collectively, these findings revealed a previously unrecognized oncogenic role of STRBP in TNBC progression and identified STRBP as a promising target against TNBC.

Intronless Pabpc6 encodes a testis-specific, cytoplasmic poly(A)-binding protein but is dispensable for spermatogenesis in the mouse†.

Besides ubiquitous poly(A)-binding protein, cytoplasmic 1 (PABPC1), testis-specific PABPC2/PABPt (in humans, referred to as PABPC3), and female and male germline-specific PABPC1L/ePAB, have been reported in the mouse testis. Recent in silico analysis additionally identified testis-specific Pabpc6 in the mouse. In this study, we characterized PABPC6 and its mutant mice. PABPC6 was initially detectable in the cytoplasm of pachytene spermatocytes, increased in abundance in round spermatids, and decreased in elongating spermatids. PABPC6 was capable of binding to poly(A) tails of various mRNAs and interacting with translation-associated factors, including EIF4G, PAIP1, and PAIP2. Noteworthy was that PABPC6, unlike PABPC1, was barely associated with translationally active polysomes and enriched in chromatoid bodies of round spermatids. Despite these unique characteristics, neither synthesis of testicular proteins nor spermatogenesis was affected in the mutant mice lacking PABPC6, suggesting that PABPC6 is functionally redundant with other co-existing PABPC proteins during spermatogenesis.

Dopamine receptor D2 regulates genes involved in germ cell movement and sperm motility in rat testes†.

The function of dopamine receptor D2 (D2R) is well associated with sperm motility; however, the physiological role of D2R present on testicular cells remains elusive. The aim of the present study is to delineate the function of testicular D2R. Serum dopamine levels were found to decrease with age, whereas testicular D2R expression increased. In rat testicular sections, D2R immunolabeling was observed in interstitial cells, spermatogonia, spermatocytes and mature elongated spermatids, whereas tyrosine hydroxylase immunolabeling was selectively detected in Leydig cells. In vitro seminiferous tubule culture following bromocriptine (D2R agonist) treatment resulted in decreased cAMP levels. Microarray identified 1077 differentially expressed genes (511 up-regulated, 566 down-regulated). The majority of differentially expressed genes were present in post-meiotic cells including early and late spermatids, and sperm. Gene ontology elucidated processes related to extra-cellular matrix to be enriched and was supported by differential expression of various collagens and laminins, thereby indicating a role of dopamine in extra-cellular matrix integrity and transport of spermatids across the seminiferous epithelium. Gene ontology and enrichment map also highlighted cell/sperm motility to be significantly enriched. Therefore, genes involved in sperm motility functions were further validated by RT-qPCR. Seven genes (Akap4, Ccnyl1, Iqcf1, Klc3, Prss55, Tbc1d21, Tl18) were significantly up-regulated, whereas four genes (Dnah1, Dnah5, Clxn, Fsip2) were significantly down-regulated by bromocriptine treatment. The bromocriptine-stimulated reduction in seminiferous tubule cyclic AMP and associated changes in spermatid gene expression suggests that dopamine regulates both spermatogenesis and spermiogenesis within the seminiferous epithelium, and spermatozoa motility following spermiation, as essential processes for fertility.

Adropin, a novel hepatokine: localization and expression during postnatal development and its impact on testicular functions of pre-pubertal mice.

Adropin, a multifaceted peptide, was identified as a new metabolic hormone responsible for regulating gluco-lipid homeostasis. However, its role in the testicular function is not yet understood. We aimed to investigate the localization and expression of adropin and GPR19 during different phases of postnatal development. Immunohistochemical study revealed the intense reactivity of adropin in the Leydig cells during all phases of postnatal development, while GPR19 showed intense immunoreactivity in the pachytene spermatocytes and mild immunoreactivity in Leydig cells as well as primary and secondary spermatocytes. Western blot study revealed maximum expression of GPR19 in pre-pubertal mouse testis that clearly indicates maximum responsiveness of adropin during that period. So, we hypothesized that adropin may act as an autocrine/paracrine factor that regulates pubertal changes in mouse testis. To examine the effect of adropin on pubertal onset, we gave bilateral intra-testicular doses (0.5 and 1.5 µg/testis) to pre-pubertal mice. Adropin treatment promoted testicular testosterone synthesis by increasing the expression of StAR, 3ß-HSD, and 17ß-HSD. Adropin also promoted germ cell survival and proliferation by upregulating the expression of PCNA and downregulating the Bax/Bcl2 ratio and Caspase 3 expression resulting in fewer TUNEL-positive cells in adropin-treated groups. FACS analysis demonstrated that adropin treatment not only increases 1C to 4C ratio but also significantly increases the 1C (spermatid) and 1C to 2C ratio which demarcates accelerated germ cell differentiation and turnover of testicular cells. In conclusion, adropin promotes steroidogenesis, germ cell survival, as well as the proliferation in the pre-pubertal mouse testis that may hasten the pubertal transition in an autocrine/paracrine manner.

Transcriptional, biochemical, and immunohistochemical analyses of CaMKKß/2 splice variants that co-localize with CaMKIV in spermatids.

Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) phosphorylates and activates downstream protein kinases, including CaMKI, CaMKIV, PKB/Akt, and AMPK; thus, regulates various Ca2+-dependent physiological and pathophysiological pathways. Further, CaMKKß/2 in mammalian species comprises multiple alternatively spliced variants; however, their functional differences or redundancy remain unclear. In this study, we aimed to characterize mouse CaMKKß/2 splice variants (CaMKKß-3 and ß-3x). RT-PCR analyses revealed that mouse CaMKKß-1, consisting of 17 exons, was predominantly expressed in the brain; whereas, mouse CaMKKß-3 and ß-3x, lacking exon 16 and exons 14/16, respectively, were primarily expressed in peripheral tissues. At the protein level, the CaMKKß-3 or ß-3x variants showed high expression levels in mouse cerebrum and testes. This was consistent with the localization of CaMKKß-3/-3x in spermatids in seminiferous tubules, but not the localization of CaMKKß-1. We also observed the co-localization of CaMKKß-3/-3x with a target kinase, CaMKIV, in elongating spermatids. Biochemical characterization further revealed that CaMKKß-3 exhibited Ca2+/CaM-induced kinase activity similar to CaMKKß-1. Conversely, we noted that CaMKKß-3x impaired Ca2+/CaM-binding ability, but exhibited significantly weak autonomous activity (approximately 500-fold lower than CaMKKß-1 or ß-3) due to the absence of C-terminal of the catalytic domain and a putative residue (Ile478) responsible for the kinase autoinhibition. Nevertheless, CaMKKß-3x showed the ability to phosphorylate downstream kinases, including CaMKIα, CaMKIV, and AMPKα in transfected cells comparable to CaMKKß-1 and ß-3. Collectively, CaMKKß-3/-3x were identified as functionally active and could be bona fide CaMKIV-kinases in testes involved in the activation of the CaMKIV cascade in spermatids, resulting in the regulation of spermiogenesis.

Decabromodiphenyl ether induces the chromosome association disorders of spermatocytes and deformation failures of spermatids in mice.

The environmental presence of decabromodiphenyl ether (BDE-209), which is toxic to the male reproductive system, is widespread. The current study investigated its mechanism of toxicity in mice. The results showed, that BDE-209 induced DNA damage, decreased the expression of the promoter of meiosis spermatogenesis- and oogenesis-specific basic helix-loop-helix 1 (Sohlh1), meiosis related-factors Lethal (3) malignant brain tumor like 2 (L3MBTL2), PIWI-like protein 2 (MILI), Cyclin-dependent kinase 2 (CDK2), Cyclin A, synaptonemal complex protein 1 (SYCP1) and synaptonemal complex protein 3 (SYCP3), and caused spermatogenic cell apoptosis, resulting in a decrease in sperm quantity and quality. Furthermore, BDE-209 downregulated the levels of anaphase-promoting complex/cyclosome (APC/C), increased the expression of PIWI-like protein 1 (MIWI) in the cytoplasm of elongating spermatids, and decreased the nuclear levels of RING finger protein 8 (RNF8), ubiquitinated (ub)-H2A/ub-H2B, and Protamine 1 (PRM1)/Protamine 2 (PRM2), while increasing H2A/H2B nuclear levels in spermatids. The reproductive toxicity was persistent for 50 days following the withdrawal of BDE-209 exposure. The results suggested that BDE-209 inhibits the initiation of meiosis by decreasing the expression of Sohlh1. Furthermore, the reduced expression of L3MBTL2 inhibited the formation of chromosomal synaptonemal complexes by depressing the expression of meiosis regulators affecting the meiotic progression and also inhibited histone ubiquitination preventing the replacement of histones by protamines, by preventing RNF8 from entering nuclei, which affected the evolution of spermatids into mature sperm.

Transcriptome analysis of meiotic and post-meiotic spermatogenic cells reveals the potential hub genes of aging on the decline of male fertility.

Genetic and epigenetic changes in sperm caused by male aging may be essential factors affecting semen parameters, but the effects and specific molecular mechanisms of aging on male reproduction have not been fully clarified. In this study, to explore the effect of aging on male fertility and seek the potential molecular etiology, we performed high-throughput RNA-sequencing in isolated spermatogenic cells, including pachytene spermatocytes (marked by the completion of chromosome synapsis) and round spermatids (produced by the separation of sister chromatids) from the elderly and the young men. Functional enrichment analysis of differentially expressed genes (DEGs) in round spermatids between the elderly and young showed that they were significantly enriched in gamete generation, spindle assembly, and cilium movement involved in cell motility. In addition, the expression levels of DEGs in round spermatids (post-meiotic cells) were found to be more susceptible to age. Furthermore, ten genes (AURKA, CCNB1, CDC20, CCNB2, KIF2C, KIAA0101, NR5A1, PLK1, PTTG1, RAD51AP1) were identified to be the hub genes involved in the regulation of sperm quality in the elderly through Protein-Protein Interaction (PPI) network construction and measuring semantic among GO terms and gene products. Our data provide aging-related molecular alterations in meiotic and post-meiotic spermatogenic cells, and the information gained from this study may explain the abnormal aging-related male fertility decline.

Regulatory Effects of the Kiss1 Gene in the Testis on Puberty and Reproduction in Hezuo and Landrance Boars.

Kisspeptin, a neuropeptide encoded by the Kiss1 gene, combines with its receptor Kiss1R to regulate the onset of puberty and male fertility by the hypothalamic-pituitary-gonadal axis. However, little is known regarding the expression signatures and molecular functions of Kiss1 in the testis. H&E staining revealed that well-arranged spermatogonia, spermatocytes, round and elongated spermatids, and spermatozoa, were observed in 4-, 6-, and 8-month-old testes compared to 1- and 3-month-old testes of Hezuo pigs; however, these were not observed in Landrance until 6 months. The diameter, perimeter, and cross-sectional area of seminiferous tubules and the perimeter and area of the tubular lumen increased gradually with age in both pigs. Still, Hezuo pigs grew faster than Landrance. The cloning results suggested that the Hezuo pigs' Kiss1 CDS region is 417 bp in length, encodes 138 amino acids, and is highly conserved in the kisspeptin-10 region. qRT-PCR and Western blot indicated that the expression trends of Kiss1 mRNA and protein were essentially identical, with higher expression levels at post-pubertal stages. Immunohistochemistry demonstrated that the Kiss1 protein was mainly located in Leydig cells and post-pubertal spermatogenic cells, ranging from round spermatids to spermatozoa. These studies suggest that Kiss1 is an essential regulator in the onset of puberty and spermatogenesis of boars.

A postmeiotically bifurcated roadmap of honeybee spermatogenesis marked by phylogenetically restricted genes.

Haploid males of hymenopteran species produce gametes through an abortive meiosis I followed by meiosis II that can either be symmetric or asymmetric in different species. Thus, one spermatocyte could give rise to two spermatids with either equal or unequal amounts of cytoplasm. It is currently unknown what molecular features accompany these postmeiotic sperm cells especially in species with asymmetric meiosis II such as bees. Here we present testis single-cell RNA sequencing datasets from the honeybee (Apis mellifera) drones of 3 and 14 days after emergence (3d and 14d). We show that, while 3d testes exhibit active, ongoing spermatogenesis, 14d testes only have late-stage spermatids. We identify a postmeiotic bifurcation in the transcriptional roadmap during spermatogenesis, with cells progressing toward the annotated spermatids (SPT) and small spermatids (sSPT), respectively. Despite an overall similarity in their transcriptomic profiles, sSPTs express the fewest genes and the least RNA content among all the sperm cell types. Intriguingly, sSPTs exhibit a relatively high expression level for Hymenoptera-restricted genes and a high mutation load, suggesting that the special meiosis II during spermatogenesis in the honeybee is accompanied by phylogenetically young gene activities.

FAM71D is dispensable for spermatogenesis and male fertility in mice.

In mammals, the generation of sperm cells capable of fertilization is a highly complex process including spermatogenesis in the testis and maturation in the epididymis. In our previous study, we have demonstrated that FAM71D (Family with sequence similarity 71, member D), which could interact with calmodulin, was highly expressed in human and mouse testis. To investigate the physiological role of FAM71D in spermatogenesis, we next generate Fam71d loss-of-function mouse model using CRISPR/Cas9 technology. We performed immunofluorescence and RT-qPCR to examine the protein and mRNA expression in testicular cells. We found that FAM71D was predominantly localized in the round and elongated spermatids. And FAM71D KO mice displayed normal development of germ cell and fertility. Furthermore, testicular histology and sperm concentration showed no significant difference between WT and KO mice. These data demonstrate that FAM71D is dispensable for mouse spermatogenesis and male fertility.

Integrated single cell transcriptome sequencing analysis reveals species-specific genes and molecular pathways for pig spermiogenesis.

Mammalian spermatogenesis is a highly complicated and intricately organized process involving spermatogonia propagation (mitosis) and meiotic differentiation into mature sperm cells (spermiogenesis). In pigs, spermatogonia development and the role of somatic cells in spermatogenesis were previously investigated in detail. However, the characterization of key molecules fundamental to pig spermiogenesis remains less explored. Here we compared spermatogenesis between humans and pigs, focusing on spermiogenesis, by integrative testicular single-cell RNA sequencing (scRNA-seq) analysis. Human and pig testicular cells were clustered into 26 different groups, with cell-type-specific markers and signalling pathways. For spermiogenesis, pseudo-time analysis classified the lineage differentiation routes for round, elongated spermatids and spermatozoa. Moreover, markers and molecular pathways specific to each type of spermatids were examined for humans and pigs, respectively. Furthermore, high-dimensional weighted gene co-expression network analysis (hdWGCNA) identified gene modules specific for each type of human and pig spermatids. Hub genes (pig: SNRPD2.1 related to alternative splicing; human: CATSPERZ, Ca[2+] ion channel) potentially involved in spermiogenesis were also revealed. Taken together, our integrative analysis found that human and pig spermiogeneses involve specific genes and molecular pathways and provided resources and insights for further functional investigation on spermatid maturation and male reproductive ability.

Actl7b deficiency leads to mislocalization of LC8 type dynein light chains and disruption of murine spermatogenesis.

Actin-related proteins (Arps) are classified according to their similarity to actin and are involved in diverse cellular processes. ACTL7B is a testis-specific Arp, and is highly conserved in rodents and primates. ACTL7B is specifically expressed in round and elongating spermatids during spermiogenesis. Here, we have generated an Actl7b-null allele in mice to unravel the role of ACTL7B in sperm formation. Male mice homozygous for the Actl7b-null allele (Actl7b-/-) were infertile, whereas heterozygous males (Actl7b+/-) were fertile. Severe spermatid defects, such as detached acrosomes, disrupted membranes and flagella malformations start to appear after spermiogenesis step 9 in Actl7b-/- mice, finally resulting in spermatogenic arrest. Abnormal spermatids were degraded and levels of autophagy markers were increased. Co-immunoprecipitation with mass spectrometry experiments identified an interaction between ACTL7B and the LC8 dynein light chains DYNLL1 and DYNLL2, which are first detected in step 9 spermatids and mislocalized when ACTL7B is absent. Our data unequivocally establish that mutations in ACTL7B are directly related to male infertility, pressing for additional research in humans.

Aberrant histone methylation in mouse early preimplantation embryos derived from round spermatid injection.

Round spermatid injection (ROSI) is the last resort and recourse for men with nonobstructive azoospermia to become biological fathers of their children. However, the ROSI-derived offspring rate is lower than intracytoplasmic sperm injection (ICSI) in mice (20% vs. 60%). This low success rate has hindered the spread of ROSI in ART (Assisted Reproductive Technology). However, the cause of the ROSI-zygote-derived low offspring rate is currently unknown. In the previous studies, we reported that H3K9me3 and H3K27me3 exhibited ectopic localizations in male pronuclei (mPN) of ROSI-zygotes, suggesting that the carried over histone to zygotes conveys epigenetic information. In this study, we analyzed other histone modifications to explore unknown abnormalities. H3K36me3 showed an increased methylation state compared to ICSI-derived embryos but not for H3K4me3. Abnormal H3K36me3 was corrected until 2-cell stage embryos, suggesting a long window of reprogramming ability in ROSI-embryos. Treatment with TSA of ROSI-zygotes, which was reported to be capable of correcting ectopic DNA methylation in ROSI-zygotes, caused abnormalities of H3K36me3 in male and female PN (fPN) of the zygotes. In contrast, round spermatid TSA treatment before ROSI, which was reported to improve the preimplantation development of ROSI-zygotes, showed beneficial effects without toxicity in fPN. Therefore, the results suggest that TSA has some negative effects, but overall, it is effective in the correction of epigenetic abnormalities in ROSI-zygotes. When attempting to correct epigenetic abnormalities, attention should be paid to epigenomes not only in male but also in female pronuclei.