Toluidine blue staining (a–g) and immunofluorescence labeling of MVI (h) and actin (i) of mouse seminiferous tubules during spermatogenis. aSpT spermatid at the acrosome phase, BV blood vessel, gSpT spermatid at the Golgi phase, Lc Leydig cell, mSpT spermatid at the maturation phase, Sc Sertoli cell, SE seminiferous epithelium, SpC spermatocyte, SpG spermatogonium, SpZ spermatozoa, STL seminiferous tubule lumen. Arrows or double arrows show MVI (red) and actin (green) staining in spermatids at maturation or acrosome phase, respectively; stars in i show actin localization in basal ectoplasmic specialization; dashed lines basement membrane. Nuclei are stained with DAPI (blue). Bars 50 μm (a), 20 μm (g–i), 5 μm (b-f)

Toluidine blue staining (a–g) and immunofluorescence labeling of MVI (h) and actin (i) of mouse seminiferous tubules during spermatogenis. aSpT spermatid at the acrosome phase, BV blood vessel, gSpT spermatid at the Golgi phase, Lc Leydig cell, mSpT spermatid at the maturation phase, Sc Sertoli cell, SE seminiferous epithelium, SpC spermatocyte, SpG spermatogonium, SpZ spermatozoa, STL seminiferous tubule lumen. Arrows or double arrows show MVI (red) and actin (green) staining in spermatids at maturation or acrosome phase, respectively; stars in i show actin localization in basal ectoplasmic specialization; dashed lines basement membrane. Nuclei are stained with DAPI (blue). Bars 50 μm (a), 20 μm (g–i), 5 μm (b-f)

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Myosin VI (MVI) is a versatile actin-based motor protein that has been implicated in a variety of different cellular processes, including endo- and exocytic vesicle trafficking, Golgi morphology, and actin structure stabilization. A role for MVI in crucial actin-based processes involved in sperm maturation was demonstrated in Drosophila. Because of...

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... These findings allow us to hypothesize that UBE2J1 and RNF133 are the first ER-localized E2 and E3 transmembrane proteins, respectively, that function in ERAD during spermiogenesis and for the formation of normal spermatozoa. During spermiogenesis, spermatozoa are dramatically remodeled architecturally into a morphology required for proper fertilization [16]. This includes mitochondrial rearrangement around the flagella at the midpiece of the tail, and proteins, organelles, and bulk cytoplasm that are no longer needed are discarded through the extrusion of cytoplasmic droplets, which are eventually removed from the sperm head and neck region [17,18]. ...
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Background Ubiquitination is a post-translational modification required for a number of physiological functions regulating protein homeostasis, such as protein degradation. The endoplasmic reticulum (ER) quality control system recognizes and degrades proteins no longer needed in the ER through the ubiquitin–proteasome pathway. E2 and E3 enzymes containing a transmembrane domain have been shown to function in ER quality control. The ER transmembrane protein UBE2J1 is a E2 ubiquitin-conjugating enzyme reported to be essential for spermiogenesis at the elongating spermatid stage. Spermatids from Ube2j1 KO male mice are believed to have defects in the dislocation step of ER quality control. However, associated E3 ubiquitin-protein ligases that function during spermatogenesis remain unknown. Results We identified four evolutionarily conserved testis-specific E3 ubiquitin-protein ligases [RING finger protein 133 ( Rnf133 ); RING finger protein 148 ( Rnf148 ); RING finger protein 151 ( Rnf151 ); and Zinc finger SWIM-type containing 2 ( Zswim2 )]. Using the CRISPR/Cas9 system, we generated and analyzed the fertility of mutant mice with null alleles for each of these E3-encoding genes, as well as double and triple knockout (KO) mice. Male fertility, male reproductive organ, and sperm-associated parameters were analyzed in detail. Fecundity remained largely unaffected in Rnf148 , Rnf151 , and Zswim2 KO males; however, Rnf133 KO males displayed severe subfertility. Additionally, Rnf133 KO sperm exhibited abnormal morphology and reduced motility. Ultrastructural analysis demonstrated that cytoplasmic droplets were retained in Rnf133 KO spermatozoa. Although Rnf133 and Rnf148 encode paralogous genes that are chromosomally linked and encode putative ER transmembrane E3 ubiquitin-protein ligases based on their protein structures, there was limited functional redundancy of these proteins. In addition, we identified UBE2J1 as an E2 ubiquitin-conjugating protein that interacts with RNF133. Conclusions Our studies reveal that RNF133 is a testis-expressed E3 ubiquitin-protein ligase that plays a critical role for sperm function during spermiogenesis. Based on the presence of a transmembrane domain in RNF133 and its interaction with the ER containing E2 protein UBE2J1, we hypothesize that these ubiquitin-regulatory proteins function together in ER quality control during spermatogenesis.
... In early spermiogenesis, the Golgi apparatus secretes numerous small pro-acrosomal vesicles or pro-acrosomal granules that gradually migrate into the apical cytoplasm (Berruti and Paiardi 2011). Motor proteins such as kinesin (KIFC1, kinesin-7, KIF3A, and KIF3B) and myosin (myosin Va and myosin VI) have been reported to function in vesicle trafficking from the Golgi to the acrosome during acrosome biogenesis (Yang and Sperry 2003;Kierszenbaum et al. 2003;Zhao et al. 2017;Zakrzewski et al. 2017;She et al. 2020). Relevant evidence indicates that cytoplasmic dynein is involved in the vesicle transport of the Golgi apparatus (Papoulas et al. 2005;Horgan et al. 2010a, b), we speculated that dynein may function in acrosome biogenesis through vesicle transport, so we performed immunofluorescence analysis of Pt-DHC and the Golgi marker GM130. ...
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The mechanism of acrosome formation in the crab sperm is a hot topic in crustacean reproduction research. Dynein is a motor protein that performs microtubule-dependent retrograde transport and plays an essential role in spermatogenesis. However, whether cytoplasmic dynein participates in acrosome formation in the crab sperm remains poorly understood. In this study, we cloned the cytoplasmic dynein intermediate chain gene (Pt-DIC) from Portunus trituberculatus testis. Pt-DIC is composed of a p150glued-binding domain, a dynein light chain (DLC)-binding domain, and a dynein heavy chain (DHC)-binding domain. The Pt-DIC gene is widely expressed in different tissues, showing the highest expression in the testis, and it is expressed in different stages of spermatid development, indicating important functions in spermatogenesis. We further observed the colocalization of Pt-DIC and Pt-DHC, Pt-DHC and tubulin, and Pt-DHC and GM130, and the results indicated that cytoplasmic dynein may participate in nuclear shaping and acrosome formation via vesicle transport. In addition, we examined the colocalization of Pt-DHC and a mitochondrion (MT) tracker and that of Pt-DHC and prohibitin (PHB). The results indicated that cytoplasmic dynein participated in mitochondrial transport and mitochondrial degradation. Taken together, these results support the hypothesis that cytoplasmic dynein participates in acrosome formation, nuclear shaping, and mitochondrial transport during spermiogenesis in P. trituberculatus. This study will provide valuable guidance for the artificial fertilization and reproduction of P. trituberculatus.
... Several myosin actin-based motor proteins, including MYO6, have been linked to the Golgi complex and depletion of MYO6 results in changes in size of the Golgi complex and reductions in post-Golgi membrane trafficking in several cell types [54][55][56]. Recent study has further shown that MYO6 together with its binding partner TOM1/L2 is expressed in the mouse testes, where it is presented in actin-rich structures involved in acrosome biogenesis such as the Golgi complex throughout the Golgi stack from the CGN to the TGN [57]. Lacking of MYO6 in mice causes structural disruptions of the Golgi complex during early acrosome biogenesis. ...
Article
Sexual reproduction requires the fusion of two gametes in a multistep and multifactorial process termed fertilization. One of the main steps that ensures successful fertilization is acrosome reaction. The acrosome, a special kind of organelle with a cap-like structure that covers the anterior portion of sperm head, plays a key role in the process. Acrosome biogenesis begins with the initial stage of spermatid development, and it is typically divided into four successive phases: the Golgi phase, cap phase, acrosome phase, and maturation phase. The run smoothly of above processes needs an active and specific coordination between the all kinds of organelles (endoplasmic reticulum, trans-golgi network and nucleus) and cytoplasmic structures (acroplaxome and manchette). During the past two decades, an increasingly genes have been discovered to be involved in modulating acrosome formation. Most of these proteins interact with each other and show a complicated molecular regulatory mechanism to facilitate the occurrence of this event. This Review focuses on the progresses of studying acrosome biogenesis using gene-manipulated mice and highlights an emerging molecular basis of mammalian acrosome formation.
... Myosin VIIa (MYO7a) has been implicated in the adhesion and transport of developing spermatids across the rat seminiferous epithelium and its depletion perturbs the spatiotemporal expression of different ABPs involved in spermiogenesis (Velichkova et al. 2002;Wen et al. 2019). Finally, gene expression profiling in rodent tissues has revealed that transcripts of the minus-directed myosin VI (MYO6) are present in mouse testes (Avraham et al. 1995) and that two different MYO6 isoforms are expressed in mouse and rat testes (Buss et al. 2001;Zakrzewski et al. 2017). ...
... MYO6 is broadly expressed in different animal tissues including the testes in humans, rodents, worms and Drosophila (Kelleher et al. 2000;Kellerman and Miller 1992;Hasson and Mooseker 1994;Avraham et al. 1995Avraham et al. , 1997. Moreover, PCR analysis demonstrated that two MYO6 isoforms (the SI and NI) are expressed in rodent testes (Buss et al. 2001;Zakrzewski et al. 2017) and are associated with several key actin-rich structures throughout sperm development and maturation in mice (Figs. 5 and 6); (Zakrzewski et al. 2017(Zakrzewski et al. , 2020a. During the Golgi phase, MYO6 is present at/around the Golgi complex adjacent to the acrosome-nuclear pole, including the trans-Golgi network and uncoated and as well as coated vesicles and at the inner acrosome membrane-acroplaxome interface (Fig. 5a-d); (Zakrzewski et al. 2017(Zakrzewski et al. , 2020a. ...
... MYO6 is broadly expressed in different animal tissues including the testes in humans, rodents, worms and Drosophila (Kelleher et al. 2000;Kellerman and Miller 1992;Hasson and Mooseker 1994;Avraham et al. 1995Avraham et al. , 1997. Moreover, PCR analysis demonstrated that two MYO6 isoforms (the SI and NI) are expressed in rodent testes (Buss et al. 2001;Zakrzewski et al. 2017) and are associated with several key actin-rich structures throughout sperm development and maturation in mice (Figs. 5 and 6); (Zakrzewski et al. 2017(Zakrzewski et al. , 2020a. During the Golgi phase, MYO6 is present at/around the Golgi complex adjacent to the acrosome-nuclear pole, including the trans-Golgi network and uncoated and as well as coated vesicles and at the inner acrosome membrane-acroplaxome interface (Fig. 5a-d); (Zakrzewski et al. 2017(Zakrzewski et al. , 2020a. ...
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Spermiogenesis is the final stage of spermatogenesis, a differentiation process during which unpolarized spermatids undergo excessive remodeling that results in the formation of sperm. The actin cytoskeleton and associated actin-binding proteins play crucial roles during this process regulating organelle or vesicle delivery/segregation and forming unique testicular structures involved in spermatid remodeling. In addition, several myosin motor proteins including MYO6 generate force and movement during sperm differentiation. MYO6 is highly unusual as it moves towards the minus end of actin filaments in the opposite direction to other myosin motors. This specialized feature of MYO6 may explain the many proposed functions of this myosin in a wide array of cellular processes in animal cells, including endocytosis, secretion, stabilization of the Golgi complex, and regulation of actin dynamics. These diverse roles of MYO6 are mediated by a range of specialized cargo-adaptor proteins that link this myosin to distinct cellular compartments and processes. During sperm development in a number of different organisms, MYO6 carries out pivotal functions. In Drosophila, the MYO6 ortholog regulates actin reorganization during spermatid individualization and male KO flies are sterile. In C. elegans, the MYO6 ortholog mediates asymmetric segregation of cytosolic material and spermatid budding through cytokinesis, whereas in mice, this myosin regulates assembly of highly specialized actin-rich structures and formation of membrane compartments to allow the formation of fully differentiated sperm. In this review, we will present an overview and compare the diverse function of MYO6 in the specialized adaptations of spermiogenesis in flies, worms, and mammals.
... In addition to myosin Va and VIIa, myosin VI (MYO6) is also expressed in mouse testes, where it is present in actin-rich structures during acrosome biogenesis such as the Golgi complex and the acrosome-acroplaxome complex [ [12]; summarized in Figure 1]. MYO6 is expressed in most cell types and tissues, and since it is the only pointed-end-directed actin-based motor, it participates in several cellular processes including endocytosis, Golgi organization and function, basolateral targeting and sorting in the secretory pathway, epithelial integrity, and cell adhesion and migration [13][14][15]. ...
... Our previous results have shown that in wild-type mice, MYO6 is present at actin-rich structures involved in acrosome formation, such as the Golgi complex and the acrosome-acroplaxome complex ( Figure 1) [12]. We therefore analyzed here whether this motor protein has a role in acrosomogenesis by testing whether MYO6 is important for maintaining the morphology of the testis-specific structures during acrosome biogenesis. ...
... MYO6 together with its binding partner TOM1/L2 are localized at the Golgi complex and developing acrosome Acrosome biogenesis involves the delivery of proacrosomal vesicles from either the Golgi complex or from late endosomes/lysosomes to the nascent acrosome [28]. We previously demonstrated that MYO6 localizes to the Golgi stacks during the acrosomogenesis in wildtype mouse spermatids [12]. Indeed, our present immunofluorescent analysis shows that MYO6 is associated with the Golgi complex including the cis-Golgi domain (Figure 4A.a-a') and the region corresponding to the trans-Golgi network (Figure 4A.a-a") in sv/+ spermatids, whereas no signal was observed in sv/sv cells confirming the specificity of our MYO6 antibodies ( Figure 4A.b-b"). ...
Article
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During spermiogenesis in mammals actin filaments and a variety of actin-binding proteins are involved in the formation and function of highly specialized testis-specific structures. Actin-based motor proteins, such as myosin Va and VIIa, play a key role in this complex process of spermatid transformation into mature sperm. We have previously demonstrated that myosin VI (MYO6) is also expressed in mouse testes. It is present in actin-rich structures important for spermatid development, including one of the earliest events in spermiogenesis-acrosome formation. Here, we demonstrate using immunofluorescence, cytochemical and ultrastructural approaches that MYO6 is involved in maintaining the structural integrity of these specialized actin-rich structures during acrosome biogenesis in mouse. We show that MYO6 together with its binding partner TOM1/L2 is present at/around the spermatid Golgi complex and the nascent acrosome. Depletion of MYO6 in Snell's waltzer mice causes structural disruptions of the Golgi complex and affects the acrosomal granule positioning within the developing acrosome. In summary, our results suggest that MYO6 plays an anchoring role during the acrosome biogenesis mainly by tethering of different cargo/membranes to highly specialized actin-related structures..
... In somatic cells, unconventional myosins function in organelle and vesicle transport and mechanotethering (Bar-Shira Maymon et al., 2000;Dolezel et al., 2008), cell migration and mitosis (Buss et al., 2004;Chibalina et al., 2009 anticipated that myosins will take part in a similar range of functions. Specifically, at least two myosin motor proteins play roles in haploid germ cell development (as detailed within this review) across several species: myosin Va in mice (Kierszenbaum et al., 2004), Drosophila melanogaster (Mermall et al., 2005), rats (Kierszenbaum et al., 2003b) and crabs (Sun et al., 2010;Ma et al., 2017) and myosin VI in mice (Zakrzewski et al., 2017), Drosophila (Rogat and Miller, 2002) and Caenorhabditis elegans (Hu et al., 2019). ...
... While largely unexplored in spermatids, it is anticipated that similar motor-based mechanisms regulate Golgi organisation and vesicle formation. The kinesin KIF1C, myosins VI and Va and the myosin Va adapter RAB27a/b have all been localised to the spermatid Golgi (Yang and Sperry, 2003;Kierszenbaum et al., 2004;Zakrzewski et al., 2017). As detailed below, however, these motor proteins also decorate the surface of Golgi-derived pro-acrosomal vesicles, and the expression of myosin Va and RAB27a/b within the Golgi appears to be restricted to the region wherein pro-acrosomal vesicles accumulate. ...
... Actin has also been detected at the manchette (Kierszenbaum et al., 2003b), but its role in protein/vesicle transport is poorly understood. Myosin Va-decorated vesicles have been detected in association with manchette MTs in the rat (Kierszenbaum et al., 2003b), and myosin VI is localised to the manchette in the mouse (Zakrzewski et al., 2017), implying actin-myosin networks function in the transport of vesicle and/or protein complexes along the manchette. ...
Article
Background: 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. Objective and rationale: 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. Search methods: 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. Outcomes: 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. Wider implications: 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.
... Furthermore, spermiogenesis can be divided into the following main phases: Golgi, acrosome cap/elongation, and maturation phases [7]. The Golgi apparatus produces proacrosomal vesicles during the Golgi phase, which coalesce and result in the acrosomal vesicle adjacent to the nuclear membrane on the opposite side of the tail. ...
Article
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Globozoospermia (sperm with an abnormally round head shape) and asthenozoospermia (defective sperm motility) are known causes of male infertility in human patients. Despite many studies, the molecular details of the globozoospermia etiology are still poorly understood. Serine-rich single pass membrane protein 1 (Ssmem1) is a conserved testis-specific gene in mammals. In this study, we generated Ssmem1 knockout (KO) mice using the CRISPR/Cas9 system, demonstrated that Ssmem1 is essential for male fertility in mice, and found that SSMEM1 protein is expressed during spermatogenesis but not in mature sperm. The sterility of the Ssmem1 knockout (null) mice is associated with globozoospermia and loss of sperm motility. To decipher the mechanism causing the phenotype, we analyzed testes with transmission electron microscopy and discovered that Ssmem1-disrupted spermatids have abnormal localization of Golgi at steps eight and nine of spermatid development. Immunofluorescence analysis with anti-Golgin-97 to label the trans-Golgi network, also showed delayed movement of the Golgi to the spermatid posterior region, which causes failure of sperm head shaping, disorganization of the cell organelles, and entrapped tails in the cytoplasmic droplet. In summary, SSMEM1 is crucial for intracellular Golgi movement to ensure proper spatiotemporal formation of the sperm head that is required for fertilization. These studies and the pathway in which SSMEM1 functions have implications for human male infertility and identifying a potential target for non-hormonal contraception.
... As one of the important cytoskeleton, the microfilament network not only provides structural support for cell morphology and movement, but also participates in transporting intracellular molecules myosin (Zakrzewski et al., 2017;Li et al., 2017). The disruption of actin-based cytoskeleton during spermatogenesis may affect male fertility (Johnson, 2015;Li et al., 2016a). ...
Article
Spermatogenesis is a highly complex physiological process which contains spermatogonia proliferation, spermatocyte meiosis and spermatid morphogenesis. In the past decade, actin binding proteins and signaling pathways which are critical for regulating the actin cytoskeleton in testis had been found. In this review, we summarized 5 actin-binding proteins that have been proven to play important roles in the seminiferous epithelium. Lack of them perturbs spermatids polarity and the transport of spermatids. The loss of Arp2/3 complex, Formin1, Eps8, Palladin and Plastin3 cause sperm release failure suggesting their irreplaceable role in spermatogenesis. Actin regulation relies on multiple signal pathways. The PI3K/Akt signaling pathway positively regulate the mTOR pathway to promote actin reorganization in seminiferous epithelium. Conversely, TSC1/TSC2 complex, the upstream of mTOR, is activated by the LKB1/AMPK pathway to inhibit cell proliferation, differentiation and migration. The increasing researches focus on the function of actin binding proteins (ABPs), however, their collaborative regulation of actin patterns and potential regulatory signaling networks remains unclear. We reviewed ABPs that play important roles in mammalian spermatogenesis and signal pathways involved in the regulation of microfilaments. We suggest that more relevant studies should be performed in the future.
... The granule enlarges with Golgi-derived glycoprotein-rich contents and gradually flattens and spreads over the nucleus to form a cap structure, while the Golgi apparatus migrates toward the posterior pole of the nucleus. As the spermatid elongates, the acrosome contents condense and the cap continues to spread over the nucleus [192]. ...
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Infertility is considered a global public health issue since it affects more than 50 million couples worldwide. Current assisted reproductive technologies (ARTs) have minimal requirements for gametes that are competent for fertilisation and subsequent embryo development. In cases where genetic abnormalities lead to arrested gametogenesis and the production of immature, defective or degraded gametes, treatment is not usually possible. Identifying the molecular causes of these types of infertility is crucial for developing new strategies to treat affected couples. Moreover, these patients represent a unique opportunity to discover new actors of oogenesis and spermatogenesis and to decipher the molecular pathways involved in the production of competent gametes.Genetic analysis of cohorts of infertile patients with shared ancestry can allow the identification of inherited genetic variants as possible causal factors. Using whole exome sequencing, we identified a homozygous pathogenic variant of the gene PATL2 in a cohort of patients with a phenotype of arrested oogenesis due to oocyte meiotic deficiency (OMD). OMD is a rare pathology characterised by the recurrent ovulation of immature oocytes. PATL2 encodes an oocyte ribonucleoprotein whose amphibian orthologue had been shown to be involved in oocyte translational control and whose function in mammals was poorly characterised. We also identified a pathogenic variant of the gene SPINK2 in a familial case of azoospermia. SPINK2 encodes a serine protease inhibitor essential for the neutralisation of acrosin activity during sperm acrosome formation.We showed, through generation of Patl2 and Spink2 knockout (KO) mice and Patl2 tagged mice (the latter using CRISPR-Cas9), that both corresponding proteins play essential respective roles in gametogenesis. We demonstrated that Patl2 is strongly expressed in growing mouse oocytes and that its absence leads to the dysregulation of numerous transcripts necessary for oocyte growth, meiotic maturation and preimplantation embryo development. This was accompanied by a phenotype of subfertility in KO females in natural mating, a large proportion of ovulated oocytes lacking a polar body (immature) and/or displaying spindle assembly defects in immunostaining, and high rate of oocytes with an aberrant response to fertilisation in IVF experiments. In Spink2 KO mice, we demonstrated that absence of Spink2 protein, which is located in the acrosome of maturing and mature spermatozoa, leads to arrested spermiogenesis and azoospermia due to autophagy at the round-spermatid stage. This is plausibly due to aberrant acrosin activity in the absence of its inhibitor, corroborated by fragmentation of the Golgi and absence of the acrosome in immunostaining.We have thus characterised two genetic subtypes of human infertility associated with mutation of these two genes. In doing so, we have furthered our understanding of the respective roles of these crucial actors of mammalian gametogenesis, potentially paving the way for improvement of current ARTs and development of new, personalised therapies.
... For example, elevated expression of MVI was linked with aggressive phenotypes of ovarian and prostate cancers in humans and mice [23,24] and association of MVI with active RNAPII was shown for HeLa cells [27]. Moreover, our observations that MVI is present within the nuclei of primary bovine adrenal medulla chromaffin cells, skeletal muscle, highly motile unicellular Amoeba proteus and developing mouse spermatids [16,26,47,48] indicate that MVI in the nuclei is not only associated with pathology but may also have physiological role(s) in non-transformed cells/tissues. The PC12 cell line was derived from rat pheochromocytoma, adrenal medulla tumor, and is a widely accepted model of neurosecretory cells [49]. ...
Article
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Myosin VI (MVI) is a unique actin-based motor protein moving towards the minus end of actin filaments, in the opposite direction than other known myosins. Besides well described functions of MVI in endocytosis and maintenance of Golgi apparatus, there are few reports showing its involvement in transcription. We previously demonstrated that in neurosecretory PC12 cells MVI was present in the cytoplasm and nucleus, and its depletion caused substantial inhibition of cell migration and proliferation. Here, we show an increase in nuclear localization of MVI upon cell stimulation, and identification of potential nuclear localization (NLS) and nuclear export (NES) signals within MVI heavy chain. These signals seem to be functional as the MVI nuclear presence was affected by the inhibitors of nuclear import (ivermectin) and export (leptomycin B). In nuclei of stimulated cells, MVI colocalized with active RNA polymerase II, BrUTP-containing transcription sites and transcription factor SP1 as well as SC35 and PML proteins, markers of nuclear speckles and PML bodies, respectively. Mass spectrometry analysis of samples of a GST-pull-down assay with the MVI tail domain as a “bait” identified several new potential MVI binding partners. Among them are proteins involved in transcription and post-transcriptional processes. We confirmed interaction of MVI with heterogeneous nuclear ribonucleoprotein U (hnRNPU) and nucleolin, proteins involved in pre-mRNA binding and transport, and nucleolar function, respectively. Our data provide an insight into mechanisms of involvement of MVI in nuclear processes via interaction with nuclear proteins and support a notion for important role(s) for MVI in gene expression.