Formation of the acrosome during spermiogenesis is an essential process for creating fertilization-competent sperm. Of the numerous aspects required for acrosome biogenesis, adherence of the acrosomal outer membrane to the nuclear surface is mediated by the subacrosomal perinuclear theca. However, the cellular dynamics and congruent functions pertaining to these acrosomal anchoring factors are not well understood despite many of them being implicated as potential causes for human male infertility. Actin-like 7A (ACTL7A) is one such factor for which deleterious polymorphisms have recently been shown to cause human male infertility. It is thought that acrosomal attachment is coordinated by cytoskeletal associations between the acrosome and nucleus via the acroplaxome. To further illuminate the mechanistic underpinnings of ACTL7A for essential acrosome associations, in this study, we investigated its dynamic localization in the developing germline, molecular associations with other cytoskeletal components, and the cellular consequences of ablation. Our intracellular localization data show ACTL7A to be dynamically present within the nucleus and subacrosomal space and later associated with postacrosomal regions of developing spermatids. Through the generation of an Actl7a knock-out mouse model, we consistently observed disruption of acrosomal biogenesis with abnormal migration of the acrosomal granule and peeling acrosomes during spermatid elongation. Significantly, we found a complete loss of subacrosomal filamentous actin (F-actin) structures in knock-out spermatids suggesting a regulatory role for subacrosomal F-actin. Considering our reported data together with existing literature, we propose a mechanistic model explaining the essential role of ACTL7A for acroplaxome-associated F-actin, acrosomal attachment integrity, and male fertility.

Spermatid

Spermiogenesis

10.1093/molehr/gaad005

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Testis Expressed Actin-Like 7b (Actl7b) Is Required for Mouse Spermatid Morphogenesis and Male Fertility.

Biology of Reproduction (2012)

Tracy M. Clement Chris Geyer William H. Willis Eugenia H. Goulding Mitch Eddy

During spermiogenesis round spermatids differentiate into elongated and then condensed spermatids followed by spermiation. Although the structural components and morphological changes in this complex process have been described in detail, relatively little is known about the mechanisms that drive these structural changes. The involvement of filamentous actin (F-actin) has been suggested for several aspects of spermatid differentiation, including acrosome formation and attachment to the nucleus, formation of tubulobulbar complexes and cytoplasmic removal, and spermiation. In addition, human male infertility is often associated with a high incidence of abnormally shaped sperm heads, suggesting that cytoskeletal regulation is important for male fertility. Actin-like 7b (Actl7b) is an intronless gene expressed in spermatids and conserved in mammals. Immunohistochemistry and indirect immunofluorescence were utilized to investigate the expression of ACTL7B. It is localized in the cytoplasm of round and elongating spermatids and co-localizes with phalloidin labeled F-actin) in or around the forming acrosome, suggesting a role in this process. To determine the functional relevance of (Actl7b), knockout mice were generated from targeted ES cells in which the (Actl7b) coding region was replaced with a LacZ reporter sequence, obtained from the KnockOut Mouse Project (KOMP) Repository. X-gal staining of tissues from heterozygous animals revealed that (Actl7b) is expressed in the testis and, unexpectedly, the brain. While (Actl7b) knockout mice develop to adulthood and appear normal, breeding studies revealed that (Actl7b) knockout males mate and produce vaginal plugs, but are infertile. Testis and epididymal weights and sperm counts are lower in knockout males than in wild type males. In addition, sperm heads are misshapen with a rounded appearance. Most of these sperm are immotile, with less than three percent showing minimal flagellar movement. These results indicate that ACTL7B is required for spermatid morphogenesis, sperm motility, and male fertility. They also suggest that ACTL7B is either the F-actin recognized by phalloidin or is required for F-actin assembly in spermatids. This research was supported by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences (EME).

Spermatid

Spermiogenesis

Knockout mouse

Phalloidin

10.1093/biolreprod/87.s1.140

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Alteration of the Developing Testis Transcriptome Following Embryonic Vinclozolin Exposure.

Biology of Reproduction (2009)

Tracy M. Clement Marina I. Savenkova Mathew Settles Mathew Anway Michael K. Skinner

The current study investigates the direct effects of in utero vinclozolin exposure on the developing rat testis transcriptome. Vinclozolin is a commonly used fungicide in agriculture and is an endocrine disruptor with anti-androgenic activity. Previous studies have demonstrated that exposure to vinclozolin during embryonic gonadal sex determination induces epigenetic modifications of the germ line and transgenerational adult onset disease states that include spermatogenic cell defects, prostate disease, kidney disease, and tumor development. An investigation of the molecular actions of vinclozolin was initiated through an analysis of direct actions on the F1 generation embryonic testis development. Microarray analyses were performed to compare control and vinclozolin treated testis transcriptomes at embryonic day 13, 14 and 16. A total of 567 differentially expressed genes were identified and the major cellular functions and pathways associated with these altered transcripts were examined. The sets of regulated genes at the different development periods were found to be transiently altered and distinct. Interestingly, genes previously shown to be regulated during normal male sex determination were not altered by vinclozolin treatment. Categorization by major known functions of all 576 genes altered by in utero vinclozolin exposure demonstrates transcription, signaling, cytoskeletal and extra cellular matrix associated transcripts are highly represented. Specific cellular process and pathway analysis suggests the involvement of Wnt and calcium signaling, vascular development and epigenetic mechanisms as potential mediators of the direct actions of vinclozolin. (poster)

Vinclozolin

10.1093/biolreprod/81.s1.644

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The REGγ-Proteasome Regulates Spermatogenesis Partially by P53-PLZF Signaling

Stem Cell Reports (2019)

Xiao Gao Hui Chen Jian Liu Shihui Shen Qingwei Wang

Development of spermatogonia and spermatocytes are the critical steps of spermatogenesis, impacting on male fertility. Investigation of the related regulators benefits the understanding of male reproduction. The proteasome system has been reported to regulate spermatogenesis, but the mechanisms and key contributing factors in vivo are poorly explored. Here we found that ablation of REGγ, a proteasome activator, resulted in male subfertility. Analysis of the mouse testes after birth showed there was a decreased number of PLZF+ spermatogonia and spermatocytes. Molecular analysis found that REGγ loss significantly increased the abundance of p53 protein in the testis, and directly repressed PLZF transcription in cell lines. Of note, allelic p53 haplodeficiency partially rescued the defects in spermatogenesis observed in REGγ-deficient mice. In summary, our results identify REGγ-p53-PLZF to be a critical pathway that regulates spermatogenesis and establishes a new molecular link between the proteasome system and male reproduction.

10.1016/j.stemcr.2019.07.010

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Citations (20)

Reviewer #3 (Public Review): Novel Nuclear Roles for Testis-Specific ACTL7A and ACTL7B Supported by In Vivo Characterizations and AI Facilitated In Silico Mechanistic Modeling with Implications for Epigenetic Regulation in Spermiogenesis

Pierre Ferrer Srijana Upadhyay James J. Cai Tracy M. Clement

A mechanistic role for nuclear function of testis-specific actin related proteins (ARPs) is proposed here through contributions of ARP subunit swapping in canonical chromatin regulatory complexes. This is significant to our understanding of both mechanisms controlling regulation of spermiogenesis, and the expanding functional roles of the ARPs in cell biology. Among these roles, actins and ARPs are pivotal not only in cytoskeletal regulation, but also in intranuclear chromatin organization, influencing gene regulation and nucleosome remodeling. This study focuses on two testis-specific ARPs, ACTL7A and ACTL7B, exploring their intranuclear activities and broader implications utilizing combined in vivo, in vitro, and in silico approaches. ACTL7A and ACTL7B, previously associated with structural roles, are hypothesized here to serve in chromatin regulation during germline development. This study confirms the intranuclear presence of ACTL7B in spermatocytes and round spermatids, revealing a potential role in intranuclear processes, and identifies a putative nuclear localization sequence conserved across mammalian ACTL7B, indicating a potentially unique mode of nuclear transport which differs from conventional actin. Ablation of ACTL7B leads to varied transcriptional changes reported here. Additionally, in the absence of ACTL7A or ACTL7B there is a loss of intranuclear localization of HDAC1 and HDAC3, which are known regulators of epigenetic associated acetylation changes that in turn regulate gene expression. Thus, these HDACs are implicated as contributors to the aberrant gene expression observed in the KO mouse testis transcriptomic analysis. Furthermore, this study employed and confirmed the accuracy of in silico models to predict ARP interactions with Helicase-SANT-associated (HSA) domains, uncovering putative roles for testis-specific ARPs in nucleosome remodeling complexes. In these models, ACTL7A and ACTL7B were found capable of binding to INO80 and SWI/SNF nucleosome remodeler family members in a manner akin to nuclear actin and ACTL6A. These models thus implicate germline-specific ARP subunit swapping within chromatin regulatory complexes as a potential regulatory mechanism for chromatin and associated molecular machinery adaptations in nuclear reorganizations required during spermiogenesis. These results hold implications for male fertility and epigenetic programing in the male-germline that warrant significant future investigation. In summary, this study reveals that ACTL7A and ACTL7B play intranuclear gene regulation roles in male gametogenesis, adding to the multifaceted roles identified also spanning structural, acrosomal, and flagellar stability. ACTL7A and ACTL7B unique nuclear transport, impact on HDAC nuclear associations, impact on transcriptional processes, and proposed mechanism for involvement in nucleosome remodeling complexes supported by AI facilitated in silico modeling contribute to a more comprehensive understanding of the indispensable functions of ARPs broadly in cell biology, and specifically in male fertility.

Spermiogenesis

10.7554/elife.95378.1.sa4

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Hmga2 deficiency is associated with allometric growth retardation, infertility, and behavioral abnormalities in mice

G3 Genes Genomes Genetics (2021)

Mi Ok Lee Jingyi Li Brian W. Davis Srijana Upadhyay Hadil M Al Muhisen

Abstract The high mobility group AT-hook 2 (HMGA2) protein works as an architectural regulator by binding AT-rich DNA sequences to induce conformational changes affecting transcription. Genomic deletions disrupting HMGA2 coding sequences and flanking noncoding sequences cause dwarfism in mice and rabbits. Here, CRISPR/Cas9 was used in mice to generate an Hmga2 null allele that specifically disrupts only the coding sequence. The loss of one or both alleles of Hmga2 resulted in reduced body size of 20% and 60%, respectively, compared to wild-type littermates as well as an allometric reduction in skull length in Hmga2−/− mice. Both male and female Hmga2−/− mice are infertile, whereas Hmga2+/− mice are fertile. Examination of reproductive tissues of Hmga2−/− males revealed a significantly reduced size of testis, epididymis, and seminal vesicle compared to controls, and 70% of knock-out males showed externalized penis, but no cryptorchidism was observed. Sperm analyses revealed severe oligospermia in mutant males and slightly decreased sperm viability, increased DNA damage but normal sperm chromatin compaction. Testis histology surprisingly revealed a normal seminiferous epithelium, despite the significant reduction in testis size. In addition, Hmga2−/− mice showed a significantly reduced exploratory behavior. In summary, the phenotypic effects in mouse using targeted mutagenesis confirmed that Hmga2 is affecting prenatal and postnatal growth regulation, male reproductive tissue development, and presents the first indication that Hmga2 function is required for normal mouse behavior. No specific effect, despite an allometric reduction, on craniofacial development was noted in contrast to previous reports of an altered craniofacial development in mice and rabbits carrying deletions of both coding and noncoding sequences at the 5′ part of Hmga2.

HMGA2

10.1093/g3journal/jkab417

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Regulation of the gonadal transcriptome during sex determination and testis morphogenesis: comparative candidate genes

Reproduction (2007)

Tracy M. Clement Matthew D. Anway Mehmet Uzumcu Michael K. Skinner

Gene expression profiles during sex determination and gonadal differentiation were investigated to identify new potential regulatory factors. Embryonic day 13 (E13), E14, and E16 rat testes and ovaries were used for microarray analysis, as well as E13 testis organ cultures that undergo testis morphogenesis and develop seminiferous cords in vitro. A list of 109 genes resulted from a selective analysis for genes present in male gonadal development and with a 1.5-fold change in expression between E13 and E16. Characterization of these 109 genes potentially important for testis development revealed that cytoskeletal-associated proteins, extracellular matrix factors, and signaling factors were highly represented. Throughout the developmental period (E13-E16), sex-enriched transcripts were more prevalent in the male with 34 of the 109 genes having testis-enriched expression during sex determination. In ovaries, the total number of transcripts with a 1.5-fold change in expression between E13 and E16 was similar to the testis, but none of those genes were both ovary enriched and regulated during the developmental period. Genes conserved in sex determination were identified by comparing changing transcripts in the rat analysis herein, to transcripts altered in previously published mouse studies of gonadal sex determination. A comparison of changing mouse and rat transcripts identified 43 genes with species conservation in sex determination and testis development. Profiles of gene expression during E13-E16 rat testis and ovary development are presented and candidate genes for involvement in sex determination and testis differentiation are identified. Analysis of cellular pathways did not reveal any specific pathways involving multiple candidate genes. However, the genes and gene network identified influence numerous cellular processes with cellular differentiation, proliferation, focal contact, RNA localization, and development being predominant.

Sexual Differentiation

10.1530/rep-06-0341

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Citations (34)

Novel Nuclear Roles for Testis-Specific ACTL7A and ACTL7B Supported by In Vivo Characterizations and AI Facilitated In Silico Mechanistic Modeling with Implications for Epigenetic Regulation in Spermiogenesis

Pierre Ferrer Srijana Upadhyay James J. Cai Tracy M. Clement

A mechanistic role for nuclear function of testis-specific actin related proteins (ARPs) is proposed here through contributions of ARP subunit swapping in canonical chromatin regulatory complexes. This is significant to our understanding of both mechanisms controlling regulation of spermiogenesis, and the expanding functional roles of the ARPs in cell biology. Among these roles, actins and ARPs are pivotal not only in cytoskeletal regulation, but also in intranuclear chromatin organization, influencing gene regulation and nucleosome remodeling. This study focuses on two testis-specific ARPs, ACTL7A and ACTL7B, exploring their intranuclear activities and broader implications utilizing combined in vivo , in vitro , and in silico approaches. ACTL7A and ACTL7B, previously associated with structural roles, are hypothesized here to serve in chromatin regulation during germline development. This study confirms the intranuclear presence of ACTL7B in spermatocytes and round spermatids, revealing a potential role in intranuclear processes, and identifies a putative nuclear localization sequence conserved across mammalian ACTL7B, indicating a potentially unique mode of nuclear transport which differs from conventional actin. Ablation of ACTL7B leads to varied transcriptional changes reported here. Additionally, in the absence of ACTL7A or ACTL7B there is a loss of intranuclear localization of HDAC1 and HDAC3, which are known regulators of epigenetic associated acetylation changes that in turn regulate gene expression. Thus, these HDACs are implicated as contributors to the aberrant gene expression observed in the KO mouse testis transcriptomic analysis. Furthermore, this study employed and confirmed the accuracy of in silico models to predict ARP interactions with Helicase-SANT-associated (HSA) domains, uncovering putative roles for testis-specific ARPs in nucleosome remodeling complexes. In these models, ACTL7A and ACTL7B were found capable of binding to INO80 and SWI/SNF nucleosome remodeler family members in a manner akin to nuclear actin and ACTL6A. These models thus implicate germline-specific ARP subunit swapping within chromatin regulatory complexes as a potential regulatory mechanism for chromatin and associated molecular machinery adaptations in nuclear reorganizations required during spermiogenesis. These results hold implications for male fertility and epigenetic programing in the male-germline that warrant significant future investigation. In summary, this study reveals that ACTL7A and ACTL7B play intranuclear gene regulation roles in male gametogenesis, adding to the multifaceted roles identified also spanning structural, acrosomal, and flagellar stability. ACTL7A and ACTL7B unique nuclear transport, impact on HDAC nuclear associations, impact on transcriptional processes, and proposed mechanism for involvement in nucleosome remodeling complexes supported by AI facilitated in silico modeling contribute to a more comprehensive understanding of the indispensable functions of ARPs broadly in cell biology, and specifically in male fertility.

Spermiogenesis

10.7554/elife.95378.1

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