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Photoperiod is a pivotal factor in affecting spermatogenesis in seasonal-breeding animals. Transposable elements have regulatory functions during spermatogenesis. However, whether it also functions in photoperiodic spermatogenesis in seasonal breeding animals is unknown. To explore this, we first annotated 5,501,822 transposons in the whole genome of Brandt's voles (Lasiopodomys brandtii), and revealed that LINEs were the most abundant, comprising 16.61 % of the genome. Following closely, SINEs accounted for 10.13 %, LTRs for 7.54 %, and DNA transposons for 0.70 %. Subsequently, we exposed male Brandt's voles to long-photoperiod (LP, 16 h/day) and short-photoperiod (SP, 8 h/day) from their embryonic stages, and obtained testes transcriptome at 4 and 10 weeks after birth. Differential expression and Pearson analysis indicated strongly positive correlations between the expression of differentially expressed retrotransposons and the adjacent genes. KO, KEGG and GSEA results showed that sperm flagellar genes were most enriched nearby the retrotransposons such as Dnah1, Dnah2, Dnah17, Dnali1. RT-PCR results showed that SINE/Alu_1213291 co-transcripted with Dnali1 gene. Our findings first reveal the regulatory function of transposons in photoperiodic spermatogenesis, providing insights into the role of photoperiod in seasonal reproduction in wild animals., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Proper connection between the sperm head and tail is critical for sperm motility and fertilization. Head-tail linkage is mediated by the head-tail coupling apparatus (HTCA), which secures the axoneme (tail) to the nucleus (head). However, the molecular architecture of the HTCA is poorly understood. Here, we use Drosophila to investigate formation and remodeling of the HTCA throughout spermiogenesis by visualizing key components of this complex. Using structured illumination microscopy, we demonstrate that key HTCA proteins Spag4 and Yuri form a 'centriole cap' that surrounds the centriole (or basal body) as it invaginates into the surface of the nucleus. As development progresses, the centriole is laterally displaced to the side of the nucleus while the HTCA expands under the nucleus, forming what we term the 'nuclear shelf'. We next show that the proximal centriole-like (PCL) structure is positioned under the nuclear shelf, functioning as a crucial stabilizer of centriole-nucleus attachment. Together, our data indicate that the HTCA is a complex, multi-point attachment site that simultaneously engages the PCL, the centriole and the nucleus to ensure proper head-tail connection during late-stage spermiogenesis., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Dynein complexes are large, multi-unit assemblies involved in many biological processes via their critical roles in protein transport and axoneme motility. Using next-generation sequencing of infertile men presenting with low or no sperm in their ejaculates, we identified damaging variants in the dynein-related gene AXDND1. We thus hypothesised that AXDND1 is a critical regulator of male fertility. To test this hypothesis, we produced a knockout mouse model. Axdnd1 -/- males were sterile at all ages but presented with an evolving testis phenotype wherein they could undergo one round of histologically replete spermatogenesis followed by a rapid depletion of the seminiferous epithelium. Marker experiments identified a role for AXDND1 in maintaining the balance between differentiation-committed and self-renewing spermatogonial populations, resulting in disproportionate production of differentiating cells in the absence of AXDND1 and increased sperm production during initial spermatogenic waves. Moreover, long-term spermatogonial maintenance in the Axdnd1 knockout was compromised, ultimately leading to catastrophic germ cell loss, destruction of blood-testis barrier integrity and immune cell infiltration. In addition, sperm produced during the first wave of spermatogenesis were immotile due to abnormal axoneme structure, including the presence of ectopic vesicles and abnormalities in outer dense fibres and microtubule doublet structures. Sperm output was additionally compromised by a severe spermiation defect and abnormal sperm individualisation. Collectively these data identify AXDND1 as an atypical dynein complex-related protein with a role in protein/vesicle transport of relevance to spermatogonial function and sperm tail formation in mice and humans. This study underscores the importance of studying the consequences of gene loss-of-function on both the establishment and maintenance of male fertility., (© 2024. The Author(s).)

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., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Sperm flagella formation is a complex process that requires cargo transport systems to deliver structural proteins for sperm flagella assembly. Two cargo transport systems, the intramanchette transport (IMT) and intraflagellar transport (IFT), have been shown to play critical roles in spermatogenesis and sperm flagella formation. IMT exists only in elongating spermatids, while IFT is responsible for delivering cargo proteins in the developing cilia/flagella. Our laboratory discovered that mouse meiosis expressed gene 1 (MEIG1), a gene essential for sperm flagella formation, is present in the manchette of elongating spermatids. IFT complex components, IFT20 and IFT88, are also present in the manchette of the elongating spermatids. Given that the three proteins have the same localization in elongating spermatids and are essential for normal spermatogenesis and sperm flagella formation, we hypothesize that they are in the same complex, which is supported by co-immunoprecipitation assay using mouse testis extracts. In the Meig1 knockout mice, neither IFT20 nor IFT88 was present in the manchette in the elongating spermatids even though their localizations were normal in spermatocytes and round spermatids. However, MEIG1 was still present in the manchette in elongating spermatids of the conditional Ift20 knockout mice. In the sucrose gradient assay, both IFT20 and IFT88 proteins drifted from higher density fractions to lighter ones in the Meig1 knockout mice. MEIG1 distribution was not changed in the conditional Ift20 knockout mice. Finally, testicular IFT20 and IFT88 protein and mRNA levels were significantly reduced in Meig1 knockout mice. Our data suggests that MEIG1 is a key protein in determining the manchette localization of certain IFT components, including IFT20 and IFT88, in male germ cells., Competing Interests: Declaration of competing interest There is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Katanin microtubule-severing enzymes are crucial executers of microtubule regulation. Here, we have created an allelic loss-of-function series of the katanin regulatory B-subunit KATNB1 in mice. We reveal that KATNB1 is the master regulator of all katanin enzymatic A-subunits during mammalian spermatogenesis, wherein it is required to maintain katanin A-subunit abundance. Our data shows that complete loss of KATNB1 from germ cells is incompatible with sperm production, and we reveal multiple new spermatogenesis functions for KATNB1, including essential roles in male meiosis, acrosome formation, sperm tail assembly, regulation of both the Sertoli and germ cell cytoskeletons during sperm nuclear remodelling, and maintenance of seminiferous epithelium integrity. Collectively, our findings reveal that katanins are able to differentially regulate almost all key microtubule-based structures during mammalian male germ cell development, through the complexing of one master controller, KATNB1, with a 'toolbox' of neofunctionalised katanin A-subunits., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Defects in the structure or motility of cilia and flagella may lead to severe diseases such as primary ciliary dyskinesia (PCD), a multisystemic disorder with heterogeneous manifestations affecting primarily respiratory and reproductive functions. We report that CFAP61 is a conserved component of the calmodulin- and radial spoke-associated complex (CSC) of cilia. We find that a CFAP61 splice variant, c.143+5G>A, causes exon skipping/intron retention in human, inducing a multiple morphological abnormalities of the flagella (MMAF) phenotype. We generated Cfap61 knockout mice that recapitulate the infertility phenotype of the human CFAP61 mutation, but without other symptoms usually observed in PCD. We find that CFAP61 interacts with the CSC, radial spoke stalk and head. During early stages of Cfap61-/- spermatid development, the assembly of radial spoke components is impaired. As spermiogenesis progresses, the axoneme in Cfap61-/- cells becomes unstable and scatters, and the distribution of intraflagellar transport proteins is disrupted. This study reveals an organ-specific mechanism of axoneme stabilization that is related to male infertility., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Asthenoteratospermia is a common cause of male infertility. Recent studies have revealed that CFAP65 mutations lead to severe asthenoteratospermia due to acrosome hypoplasia and flagellum malformations. However, the molecular mechanism underlying CFAP65-associated sperm malformation is largely unclear. Here, we initially examined the role of CFAP65 during spermiogenesis using Cfap65 knockout (Cfap65-/-) mice. The results showed that Cfap65-/- male mice exhibited severe asthenoteratospermia characterized by morphologically defective sperm heads and flagella. In Cfap65-/- mouse testes, hyper-constricted sperm heads were apparent in step 9 spermatids accompanied by abnormal manchette development, and acrosome biogenesis was abnormal in the maturation phase. Moreover, subsequent flagellar elongation was also severely affected and characterized by disrupted assembly of the mitochondrial sheath (MS) in Cfap65-/- male mice. Furthermore, the proteomic analysis revealed that the proteostatic system during acrosome formation, manchette organization and MS assembly was disrupted when CFAP65 was lost. Importantly, endogenous immunoprecipitation and immunostaining experiments revealed that CFAP65 may form a cytoplasmic protein network comprising MNS1, RSPH1, TPPP2, ZPBP1 and SPACA1. Overall, these findings provide insights into the complex molecular mechanisms of spermiogenesis by uncovering the essential roles of CFAP65 during sperm head shaping, acrosome biogenesis and MS assembly., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

How autophagy initiation is regulated and what the functional significance of this regulation is are unknown. Here, we characterized the role of yeast Vac8 in autophagy initiation through recruitment of PIK3C3-C1 to the phagophore assembly site (PAS). This recruitment is dependent on the palmitoylation of Vac8 and on its middle ARM domains for binding PIK3C3-C1. Vac8-mediated anchoring of PIK3C3-C1 promotes PtdIns3P generation at the PAS and recruitment of the PtdIns3P binding protein Atg18-Atg2. The mouse homolog of Vac8, ARMC3, is conserved and functions in autophagy in mouse testes. Mice lacking ARMC3 have normal viability but show complete male infertility. Proteomic analysis indicated that the autophagic degradation of cytosolic ribosomes was blocked in ARMC3-deficient spermatids, which caused low energy levels of mitochondria and motionless flagella. These studies uncovered a function of Vac8/ARMC3 in PtdIns3-kinase anchoring at the PAS and its physical significance in mammalian spermatogenesis with a germ tissue-specific autophagic function., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The calcium-binding protein spermatid-associated 1 (Cabs1) is a novel spermatid-specific protein. However, its function remains largely unknown. In this study, we found that a long noncoding RNA (lncRNA) transcripted from the Cabs1 gene antisense, AntiCabs1 , was also exclusively expressed in spermatids. Cabs1 and AntiCabs1 knockout mice were generated separately (using Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-Cas9 methods) to investigate their functions in spermatogenesis. The genetic loss of Cabs1 did not affect testicular and epididymal development; however, male mice exhibited significantly impaired sperm tail structure and subfertility. Ultrastructural analysis revealed defects in sperm flagellar differentiation leading to an abnormal annulus and disorganization of the midpiece-principal piece junction, which may explain the high proportion of sperm with a bent tail. Interestingly, the proportion of sperm with a bent tail increased during transit in the epididymis. Furthermore, Western blot and immunofluorescence analyses showed that a genetic loss of Cabs1 decreased Septin 4 and Krt1 and increased cyclin Y-like 1 (Ccnyl1) levels compared with the wild type, suggesting that Cabs1 deficiency disturbed the expression of cytoskeleton-related proteins. By contrast, AntiCabs1 -/- mice were indistinguishable from the wild type regarding testicular and epididymal development, sperm morphology, concentration and motility, and male fertility. This study demonstrates that Cabs1 is an important component of the sperm annulus essential for proper sperm tail assembly and motility.

Multiple morphological abnormalities of the flagella (MMAF) is a genetically heterogeneous disorder leading to male infertility. Recent studies have revealed that DNAH17 variants are associated with MMAF, yet there is no functional evidence in support of their pathnogenicity. Here, we recruited two consanguineous families of Pakistani and Chinese origins, respectively, diagnosed with MMAF. Whole-exome sequencing identified novel homozygous DNAH17 variants, which led to loss of DNAH17 proteins, in the patients. Transmission electron microscope analyses revealed completely disorganized axonemal structure as the predominant anomaly and increased frequencies of missings of microtubule doublet(s) 4-7 in sperm flagella of patients. Similar to those found in patients, Dnah17 -/- mice also displayed MMAF phenotype along with completely disorganized axonemal structures. Clusters of disorganized microtubules and outer dense fibers were observed in developing spermatids, indicating impaired sperm flagellar assembly. Besides, we also noticed many elongating spermatids with a deformed nuclear shape and abnormal step 16 spermatids that failed to spermiate, which subsequently underwent apoptosis in Dnah17-null mice. These findings present direct evidence establishing that DNAH17 is a MMAF-related gene in humans and mice, extend the clinical interpretations of DNAH17 variants, and highlight an essential and complex role of DNAH17 in spermatogenesis., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

In cilia and flagella, dyneins form complexes which give rise to the inner and outer axonemal arms. Defects in the dynein arms are the leading cause of primary ciliary dyskinesia (PCD), which is characterized by chronic respiratory infections, situs inversus, and sterility. While the pathological features associated with PCD are increasingly well characterized, many of the causative genetic lesions remain elusive. Using Drosophila, here we analyze genetic requirements for wampa (wam), a previously uncharacterized component of the outer dynein arm. While homozygous mutant animals are viable and display no morphological defects, loss of wam results in complete male sterility. Ultrastructural analysis further reveals that wam mutant spermatids lack the axonemal outer dynein arms, which leads to a complete loss of flagellar motility. In addition to a role in outer dynein arm formation, we also uncover other novel microtubule-associated requirements for wam during spermatogenesis, including the regulation of mitochondrial localization and the shaping of the nuclear head. Due to the conserved nature of dyneins, this study advances our understanding of the pathology of PCD and the functional role of dyneins in axoneme formation and other aspects of spermatogenesis., (Copyright © 2020. Published by Elsevier Inc.)

Sperm fibrous sheath, a unique cytoskeletal structure, is implicated in various sperm physiological functions, such as sperm maturation, motility and capacitation. AKAP4 has been described to be required for structural and functional integrity of the fibrous sheath. We generated Akap4-knockout mice line using CRISPR-Cas9 system. Cytomorphology and motility of sperm and testes were studied, confirming loss of Akap4 led to abnormal sperm morphology, motility and infertility. The proteomic components of testes were studied and Akap4 was found to be significantly decreased in the Akap4-knockout mice. Testis single-cell RNA sequencing and analysis revealed three genes with significant change in the general cell population, i.e., Akap4, Haspin, and Ccdc38. The single-cell RNA expression profiles also showed that the major difference between Akap4-knockout and wild-type testes existed in the elongating cell cluster, where in the Akap4-knockout testes, a subgroup of elongating cells with marker genes involved in cell adhesion and migration were increased, while a subgroup of elongating cells marked by mitochondrial sheath genes were decreased. Our results revealed the complex and well-coordinated procedures of spermatogenesis, and substantiated Akap4's indispensable roles in the integrity of sperm flagellum and the step-wise maturation of spermatozoa., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Gamete formation is key to survival of higher organisms. In male animals, spermatogenesis gives rise to interconnected spermatids that differentiate and individualize into mature sperm, each tightly enclosed by a plasma membrane. In Drosophila melanogaster , individualization of sister spermatids requires the formation of specialized actin cones that synchronously move along the sperm tails, removing inter-spermatid bridges and most of the cytoplasm. Here, we show that Combover (Cmb), originally identified as an effector of planar cell polarity (PCP) under control of Rho kinase, is essential for sperm individualization. cmb mutants are male sterile, with actin cones that fail to move in a synchronized manner along the flagella, despite being correctly formed and polarized initially. These defects are germline autonomous, independent of PCP genes, and can be rescued by wild-type Cmb, but not by a version of Cmb in which known Rho kinase phosphorylation sites are mutated. Furthermore, Cmb binds to the axonemal component Radial spoke protein 3, knockdown of which causes similar individualization defects, suggesting that Cmb coordinates the individualization machinery with the microtubular axonemes., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

The mitochondrial sheath is composed of mitochondria that coil tightly around the midpiece of sperm flagellum. These mitochondria are recruited from the cytoplasm to the flagellum late in spermatogenesis. Initially, recruited mitochondria are spherical-shaped but then elongate laterally to become crescent-like in shape. Subsequently, crescent-like mitochondria elongate continuously to coil tightly around the flagellum. Recently, disorganization of the mitochondrial sheath was reported in Glycerol kinase 2 (Gk2) disrupted mice. To analyze the disorganization of the mitochondrial sheath further, we generated Gk2-deficient mice using the CRISPR/Cas9 system and observed sperm mitochondria in testis using a freeze-fracture method with scanning electron microscopy. Gk2-disrupted spermatids show abnormal localization of crescent-like mitochondria, in spite of the initial proper alignment of spherical mitochondria around the flagellum, which causes abnormal mitochondrial sheath formation leading to exposure of the outer dense fibers. These results indicate that GK2 is essential for proper arrangement of crescent-like mitochondria to form the mitochondrial sheath during mouse spermatogenesis.

Sperm cells are highly specialized mammalian cells, and their biogenesis requires unique intracellular structures. Perturbation of spermatogenesis often leads to male infertility. Here, we assess the role of a post-translational modification of tubulin, glutamylation, in spermatogenesis. We show that mice lacking the tubulin deglutamylase CCP5 (also known as AGBL5) do not form functional sperm. In these mice, spermatids accumulate polyglutamylated tubulin, accompanied by the occurrence of disorganized microtubule arrays, in particular in the sperm manchette. Spermatids further fail to re-arrange their intracellular space and accumulate organelles and cytosol, while nuclei condense normally. Strikingly, spermatids lacking CCP5 show supernumerary centrioles, suggesting that glutamylation could control centriole duplication. We show that most of these observed defects are also present in mice in which CCP5 is deleted only in the male germ line, strongly suggesting that they are germ-cell autonomous. Our findings reveal that polyglutamylation is, beyond its known importance for sperm flagella, an essential regulator of several microtubule-based functions during spermatogenesis. This makes enzymes involved in glutamylation prime candidates for being genes involved in male sterility., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Spermatogenesis involves a series of cellular transformations and thousands of regulated genes. Previously, we showed that the triploid fish (3nBY) cannot produce mature spermatozoa. In the present study, evaluation of the testis microstructure revealed that germ cells of 3nBY could develop into round spermatids, but then degenerated, resulting in male infertility. In this study we comparatively analysed the testis transcriptomes from 3nBY and its diploid parent YB and identified a series of differentially expressed genes (DEGs) that were enriched in the Wnt signalling pathway and the apoptotic and ubiquitin-mediated proteolysis processes in 3nBY. Gene ontology functional analyses revealed that some DEGs in 3nBY were directly associated with the process of gamete generation, development and sperm flagellum assembly. In addition, the expression of a number of genes related to meiosis (Inhibitor Of DNA Binding 2 (ID2), Ovo Like Transcriptional Repressor 1 (OVOL1)), mitochondria (ATP1b (ATPase Na+/K+ Transporting Subunit Beta 1), ATP2a (ATPase, Ca++ Transporting, Cardiac Muscle, Slow Twitch 2), ATP5a (ATP Synthase F1 Subunit Alpha), Mitochondrially Encoded Cytochrome C Oxidase I (COX1), NADH Dehydrogenase Subunit 4 (ND4)) and chromatin structure (Histone 1 (H1), Histone 2a (H2A), Histone 2b (H2B), Histone 3 (H3), Histone 4 (H4)) was lower in the testes of 3nBY, whereas the expression of genes encoding ubiquitin (Ubiquitin Conjugating Enzymes (UBEs), Ring Finger Proteins (RNFs)) and apoptosis (CASPs (Caspase 3, Caspase 7,Caspase 8), BCLs (B-Cell Lymphoma 3, B-Cell CLL/Lymphoma 2, B Cell CLL/Lymphoma 10)) proteins involved in spermatid degeneration was higher. These data suggest that the disrupted expression of genes associated with spermatogenesis and the increased expression of mitochondrial ubiquitin, which initiates cell apoptosis, may result in spermatid degeneration in male 3nBY. This study provides information regarding the potential molecular regulatory mechanisms underlying male infertility in polyploid fish.

Background: The mechanism of intramanchette transport is crucial to the transformation of sperm tail and the nuclear condensation during spermiogenesis. Although few dysfunctional proteins could result in abnormal junction between the head and tail of spermatozoon, little is known about the genetic cues in this process., Objective: Based on patients with severe decapitated and decaudated spermatozoa (DDS) syndrome, the study aimed to validate whether new mutation exists on their Hook microtubule-tethering protein 1 ( HOOK1 ) genes and follow their results of assisted reproduction treatment (ART)., Methods: 7 severe teratozoospermia patients with DDS (proportion >95%) and three relative members in one pedigree were collected to sequence the whole genomic DNA. The fertilisation rates (FRs) of these patients were followed. Morphological observation and interspecies intracytoplasmic sperm injection (ICSI) assays were applied., Results: A novel missense mutation of A to G (p.Q286R) in patients with DDS (n=3/7) was found in the HOOK1 gene, which was inherited from the mother in one patient. This variant was absent in 160 fertile population-matched control individuals. Morphological observation showed that almost all the DDS broke into decaudated heads and headless tails at the implantation fossa or the basal plate. The clinical studies indicated that the mutation might cause reduced FRs on both ART (FR=18.07%) and interspecies ICSI (FR=16.98%)., Conclusions: An unreported mutation in HOOK1 gene was identified, which might be responsible to some patients with DDS. Further studies need to uncover the molecular mechanism of spermiogenesis for genomic therapy., Competing Interests: Competing interests: None declared., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

Intraflagellar transport (IFT) is a conserved mechanism essential for the assembly and maintenance of most eukaryotic cilia and flagella. However, little is known about its role in sperm flagella formation and male fertility. IFT140 is a component of IFT-A complex. In mouse, it is highly expressed in the testis. Ift140 gene was inactivated specifically in mouse spermatocytes/spermatids. The mutant mice did not show any gross abnormalities, but all were infertile and associated with significantly reduced sperm number and motility. Multiple sperm morphological abnormalities were discovered, including amorphous heads, short/bent flagella and swollen tail tips, as well as vesicles along the flagella due to spermiogenesis defects. The epididymides contained round bodies of cytoplasm derived from the sloughing of the cytoplasmic lobes and residual bodies. Knockout of Ift140 did not significantly affect testicular expression levels of selective IFT components but localization of IFT27 and IFT88, two components of IFT-B complex, was changed. Our findings demonstrate that IFT140 is a key regulator for male fertility and normal spermiogenesis in mice. It not only plays a role in sperm flagella assembling, but is also involved in critical assembly of proteins that interface between the germ cell plasma and the Sertoli cell., (© 2017 Wiley Periodicals, Inc.)

Hypercholesterolemia is a marker for several adult chronic diseases. Recently we demonstrated that sub/infertility is also associated to Hypercholesterolemia in rabbits. Seminal alterations included: abnormal sperm morphology, decreased sperm number and declined percentage of motile sperm, among others. In this work, our objective was to evaluate the effects of hypercholesterolemia on testicular efficiency and spermiogenesis, as the latter are directly related to sperm number and morphology respectively. Tubular efficiency was determined by comparing total number of spermatogenic cells with each cell type within the proliferation/differentiation compartments. We found lower testicular efficiency related to both a decrease in spermatogonial cells and an increase in germ cell apoptosis in hypercholesterolemic rabbits. On the other hand, spermiogenesis-the last step of spermatogenesis involved in sperm shaping-was detaily analyzed, particularly the acrosome-nucleus-manchette complex. The manchette is a microtubular-based temporary structure responsible in sperm cell elongation. We analyzed the contribution of actin filaments and raft microdomains in the arrangement of the manchette. Under fluorescence microscopy, spermatocyte to sperm cell development was followed in cells isolated from V to VIII tubular stages. In cells from hypercholesterolemic rabbits, abnormal development of acrosome, nucleus and inaccurate tail implantation were associated with actin-alpha-tubulin-GM1 sphingolipid altered distribution. Morphological alterations were also observed at electron microscopy. We demonstrated for the first time that GM1-enriched microdomains together with actin filaments and microtubules are involved in allowing the correct anchoring of the manchette complex. In conclusion, cholesterol enriched diets promote male fertility alterations by affecting critical steps in sperm development: spermatogenesis and spermiogenesis. It was also demonstrated that hypercholesterolemic rabbit model is a useful tool to study serum cholesterol increment linked to sub/infertility.

The last phase of spermatogenesis involves spermatid elongation (spermiogenesis), where the nucleus is remodeled by chromatin condensation, the excess cytoplasm is removed and the acrosome and sperm tail are formed. Protein transport during spermatid elongation is required for correct formation of the sperm tail and acrosome and shaping of the head. Two microtubular-based protein delivery platforms transport proteins to the developing head and tail: the manchette and the sperm tail axoneme. The manchette is a transient skirt-like structure surrounding the elongating spermatid head and is only present during spermatid elongation. In this review, we consider current understanding of the assembly, disassembly and function of the manchette and the roles of these processes in spermatid head shaping and sperm tail formation. Recent studies have shown that at least some of the structural proteins of the sperm tail are transported through the intra-manchette transport to the basal body at the base of the developing sperm tail and through the intra-flagellar transport to the construction site in the flagellum. This review focuses on the microtubule-based mechanisms involved and the consequences of their disruption in spermatid elongation., (© 2016 Society for Reproduction and Fertility.)

A previous study indicated that genetic information encoded on the mouse Y chromosome short arm (Yp) is required for efficient completion of the second meiotic division (that generates haploid round spermatids), restructuring of the sperm head, and development of the sperm tail. Using mouse models lacking a Y chromosome but with varying Yp gene complements provided by Yp chromosomal derivatives or transgenes, we recently identified the Y-encoded zinc finger transcription factors Zfy1 and Zfy2 as the Yp genes promoting the second meiotic division. Using the same mouse models we here show that Zfy2 (but not Zfy1) contributes to the restructuring of the sperm head and is required for the development of the sperm tail. The preferential involvement of Zfy2 is consistent with the presence of an additional strong spermatid-specific promotor that has been acquired by this gene. This is further supported by the fact that promotion of sperm morphogenesis is also seen in one of the two markedly Yp gene deficient models in which a Yp deletion has created a Zfy2/1 fusion gene that is driven by the strong Zfy2 spermatid-specific promotor, but encodes a protein almost identical to that encoded by Zfy1. Our results point to there being further genetic information on Yp that also has a role in restructuring the sperm head.

Drosophila sperm are unusual in that they do not require the intraflagellar transport (IFT) system for assembly of their flagella. In the mouse, the IFT proteins are very abundant in testis, but we here show that mature sperm are completely devoid of them, making the importance of IFT to mammalian sperm development unclear. To address this question, we characterized spermiogenesis and fertility in the Ift88(Tg737Rpw) mouse. This mouse has a hypomorphic mutation in the gene encoding the IFT88 subunit of the IFT particle. This mutation is highly disruptive to ciliary assembly in other organs. Ift88(-/-) mice are completely sterile. They produce ∼ 350-fold fewer sperm than wild-type mice, and the remaining sperm completely lack or have very short flagella. The short flagella rarely have axonemes but assemble ectopic microtubules and outer dense fibers and accumulate improperly assembled fibrous sheath proteins. Thus IFT is essential for the formation but not the maintenance of mammalian sperm flagella., (© 2015 San Agustin et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

The septin gene belongs to a highly conserved family of polymerizing GTP-binding cytoskeletal proteins. SEPTs perform cytoskeletal remodeling, cell polarity, mitosis, and vesicle trafficking by interacting with various cytoskeletons. Our previous studies have indicated that SEPTIN12+/+/+/- chimeras with a SEPTIN12 mutant allele were infertile. Spermatozoa from the vas deferens of chimeric mice indicated an abnormal sperm morphology, decreased sperm count, and immotile sperm. Mutations and genetic variants of SEPTIN12 in infertility cases also caused oligozoospermia and teratozoospermia. We suggest that a loss of SEPT12 affects the biological function of microtublin functions and causes spermiogenesis defects. In the cell model, SEPT12 interacts with α- and β-tubulins by co-immunoprecipitation (co-IP). To determine the precise localization and interactions between SEPT12 and α- and β-tubulins in vivo, we created SEPTIN12-transgene mice. We demonstrate how SEPT12 interacts and co-localizes with α- and β-tubulins during spermiogenesis in these mice. By using shRNA, the loss of SEPT12 transcripts disrupts α- and β-tubulin organization. In addition, losing or decreasing SEPT12 disturbs the morphogenesis of sperm heads and the elongation of sperm tails, the steps of which are coordinated and constructed by α- and β-tubulins, in SEPTIN12+/+/+/- chimeras. In this study, we discovered that the SEPTIN12-microtubule complexes are critical for sperm formation during spermiogenesis.

Cystatin-related epididymal spermatogenic protein (CRES, also CST8) is expressed in both the testis and epididymis and found associated with spermatozoa. It appears as nonglycosylated (14 and 12 kDa) and glycosylated isoforms (19 and 17 kDa). The role of CRES remains enigmatic and is dependent on localization of its isoforms, which is the objective of this study. Our initial approach was to investigate testicular origins of these isoforms by immunohistochemistry and immunogold electron microscopy. We confirmed previous reports that CRES was expressed in the cytoplasm of elongating spermatids from step 8 to step 16. However, we noticed that the pattern of testicular expression was reminiscent of outer dense fiber (ODF) and fibrous sheath (FS) proteins. Western blot analysis of extracts of cauda epididymal sperm revealed a nonionic, detergent-insoluble 14-kDa CRES isoform. To further pinpoint and confirm CRES localization we separated sperm heads and tails and extracted the tails with progressively harsher protein solubilizing agents. Western blots of these sequential extracts, designed to progressively remove the mitochondrial sheath and the ODFs or FS, detected a CRES-immunoreactive 14-kDa band associated with the accessory fibers of the tail. Immunogold labeling was concentrated over growing ODFs in the testes and persisted in spermatozoa. This study discovers a CRES isoform that assembles as part of the ODFs during the elongation and maturation phases of spermiogenesis and is retained as a covalently bound component of the ODFs in spermatozoa.

We recently used three XO male mouse models with varying Y short-arm (Yp) gene complements, analysed at 30 days post partum, to demonstrate a Yp gene requirement for the apoptotic elimination of spermatocytes with a univalent X chromosome at the first meiotic metaphase. The three mouse models were i) XSxr(a)O in which the Yp-derived Tp(Y)1Ct(Sxr-a) sex reversal factor provides an almost complete Yp gene complement, ii) XSxr(b)O,Eif2s3y males in which Tp(Y)1Ct(Sxr-b) has a deletion completely or partially removing eight Yp genes - the Yp gene Eif2s3y has been added as a transgene to support spermatogonial proliferation, and iii) XOSry,Eif2s3y males in which the Sry transgene directs gonad development along the male pathway. In this study, we have used the same mouse models analysed at 6 weeks of age to investigate potential Yp gene involvement in spermiogenesis. We found that all three mouse models produce haploid and diploid spermatids and that the diploid spermatids showed frequent duplication of the developing acrosomal cap during the early stages. However, only in XSxr(a)O males did spermiogenesis continue to completion. Most strikingly, in XOSry,Eif2s3y males, spermatid development arrested at round spermatid step 7 so that no sperm head restructuring or tail development was observed. In contrast, in XSxr(b)O,Eif2s3y males, spermatids with substantial sperm head and tail morphogenesis could be easily found, although this was delayed compared with XSxr(a)O. We conclude that Sxr(a) (and therefore Yp) includes genetic information essential for sperm morphogenesis and that this is partially retained in Sxr(b).

Spermatogenesis in all animal species occurs within a syncytium. Only at the very end of spermatogenesis are individual sperm cells resolved from this syncytium in a process known as individualization. Individualization in Drosophila begins as a membrane-cytoskeletal complex known as the individualization complex (IC) assembles around the sperm heads and proceeds down the flagella, removing cytoplasm from between the sperm tails and shrink-wrapping each spermatid into its own plasma membrane as it travels. The mulet (mlt) mutation results in severely disrupted ICs, indicating that the mlt gene product is required for individualization. Inverse PCR followed by cycle sequencing maps all known P-insertion alleles of mlt to two overlapping genes, CG12214 (the Drosophila tubulin-binding cofactor E-like homolog) and KCNQ (a large voltage-gated potassium channel). However, since the alleles of mlt map to the 5'-UTR of CG12214 and since CG12214 is contained within an intron of KCNQ, it was hypothesized that mlt and CG12214 are allelic. Indeed, CG12214 mutant testes exhibited severely disrupted ICs and were indistinguishable from mlt mutant testes, thus further suggesting allelism. To test this hypothesis, alleles of mlt were crossed to CG12214 in order to generate trans-heterozygous males. Testes from all trans-heterozygous combinations revealed severely disrupted ICs and were also indistinguishable from mlt mutant testes, indicating that mlt and CG12214 fail to complement one another and are thus allelic. In addition, complementation testing against null alleles of KCNQ verified that the observed individualization defect is not caused by a disruption of KCNQ. Finally, since a population of spermatid-associated microtubules known to disappear prior to movement of the IC abnormally persists during individualization in CG12214 mutant testes, this work implicates TBCE-like in the removal of these microtubules prior to IC movement. Taken together, these results identify mlt as CG12214 and suggest that the removal of microtubules by TBCE-like is a necessary pre-requisite for proper coordinated movement of the IC.

The lipid kinase phosphatidylinositol 4-phosphate 5-kinase (PIP5K) produces a versatile signaling phospholipid, phosphatidylinositol 4,5-bisphosphate. Three PIP5K isozymes, PIP5K1A, PIP5K1B, and PIP5K1C, have been identified in mammals so far. Although the functions of these three PIP5K isozymes have been extensively studied in vitro, the in vivo physiological roles of these PIP5K isozymes remain largely unknown. In this study, we examined the functions of PIP5K1A and PIP5K1B in spermatogenesis, using Pip5k1a-knockout (KO), Pip5k1b-KO, and Pip5k1a/Pip5k1b double (D)-KO mice. Pip5k1a-KO and D-KO males were subfertile and completely sterile, respectively. F-actin in the seminiferous epithelium was disorganized in the D-KO mice, although F-actin bundles at the apical ectoplasmic specialization was not affected. D-KO seminiferous tubules contained a greatly decreased number of elongated spermatids. Flagella of sperm from Pip5k1a-KO and D-KO mice remarkably underwent morphological change, whereas Pip5k1b-KO sperm were morphologically normal. Notably, the flagellar shape of D-KO sperm was more severely impaired than that of Pip5k1a-KO sperm. These results suggest that PIP5K1A and PIP5K1B may coordinately and/or redundantly function in the maintenance of sperm number and morphology during spermatogenesis.

During spermiogenesis, haploid round spermatids undergo dramatic cell differentiation and morphogenesis to give rise to mature spermatozoa for fertilization, including nuclear elongation, chromatin remodeling, acrosome formation, and development of flagella. The molecular mechanisms underlining these fundamental processes remain poorly understood. Here, we report that MNS1, a coiled-coil protein of unknown function, is essential for spermiogenesis. We find that MNS1 is expressed in the germ cells in the testes and localizes to sperm flagella in a detergent-resistant manner, indicating that it is an integral component of flagella. MNS1-deficient males are sterile, as they exhibit a sharp reduction in sperm production and the remnant sperm are immotile with abnormal short tails. In MNS1-deficient sperm flagella, the characteristic arrangement of "9+2" microtubules and outer dense fibers are completely disrupted. In addition, MNS1-deficient mice display situs inversus and hydrocephalus. MNS1-deficient tracheal motile cilia lack some outer dynein arms in the axoneme. Moreover, MNS1 monomers interact with each other and are able to form polymers in cultured somatic cells. These results demonstrate that MNS1 is essential for spermiogenesis, the assembly of sperm flagella, and motile ciliary functions., Competing Interests: The authors have declared that no competing interests exist.

The sperm surface is covered with a dense coating of carbohydrate-rich molecules. Many of these molecules are involved in the acquisition of fertilising ability. In the present study, eight lectins (i.e. Arachis hypogae (peanut) agglutinin (PNA), Lens culimaris (lentil) agglutinin-A (LCA), Pisum sativum (pea) agglutin (PSA), Triticum vulgari (wheat) germ agglutinin (WGA), Helix pomatia agglutinin (HPA), Phaseolus vulgaris (red kidney bean) leucoagglutinin (PHA-L), Glycine max (soybean) agglutinin (SBA) and Ulex europaeus agglutinin I (UEA-I)) were investigated to identify changes in the nature and localisation of glycoproteins in boar spermatozoa migrating along the epididymal duct. Complementary procedures included measurement of global lectin binding over the surface of the viable sperm population by flow cytometry, analysis of lectin localisation on the membrane of individual spermatozoa using fluorescence microscopy and the electrophoretic characterisation of the major sperm surface glycoprotein receptors involved in lectin binding. A significant increase was found in sperm galactose, glucose/mannose and N-acetyl-d-glucosamine residues distally in the epididymis. Moreover, the sperm head, cytoplasmic droplet and midpiece were recognised by most of the lectins tested, whereas only HPA and WGA bound to the principal piece and end piece of the sperm tail. Fourteen sperm surface proteins were observed with different patterns of lectin expression between epididymal regions. The sperm glycocalyx modifications observed in the present study provide an insight into the molecular modifications associated with epididymal maturation, which may be correlated with the degree of maturation of ejaculated spermatozoa.

Previous work showed that rat germ cells and spermatozoa contain ceramides and sphingomyelins with high proportions of nonhydroxy and 2-hydroxy (2-OH) polyunsaturated fatty acids (PUFA) with very long chains (VLCPUFA). The aim of this study was to assess how these lipids are distributed between the heads and tails of mature spermatozoa in comparison with other membrane lipid classes. In addition to quantitative differences due to the fact that these gametes have a long, voluminous tail and a minute head, several compositional dissimilarities emerged between these two regions. The total cholesterol/total phospholipid ratio, the choline/ethanolamine glycerophospholipid (ChoGpl/EtnGpl) ratio, and the proportion of plasmalogens within these two classes, were much larger in the head than in the tail. Whereas EtnGpl was rich in 22:5n-6 in both regions, ChoGpl had plenty of 22:4n-9, especially in the heads. An important proportion of the head EtnGpl- 22:5n-6 and ChoGpl 22:4n-9 was in plasmenyl- (rather than in phosphatidyl-) subclasses. The heads concentrated all of the sphingomyelin species with nonhydroxy- and 2-OH VLCPUFA, and the tails most of the saturated fatty acids that are present in total sperm sphingomyelin. Unexpectedly, virtually all of the abundant spermatozoal ceramides, predominantly made up by species with 2-OH VLCPUFA, was located in the tail. The fact that intact rat spermatozoa constitutively have much more VLCPUFA-containing ceramide than sphingomyelin is explained by the present findings, since the former are mostly lipids of the large tail while the latter mostly collect in the small head.

Background: CABYR is a polymorphic calcium-binding protein of the sperm fibrous sheath (FS) which gene contains two coding regions (CR-A and CR-B) and is tyrosine as well as serine/threonine phosphorylated during in vitro sperm capacitation. Thus far, the detailed information on CABYR protein expression in mouse spermatogenesis is lacking. Moreover, because of the complexity of this polymorphic protein, there are no data on how CABYR isoforms associate and assemble into the FS., Methods: The capacity of mouse CABYR isoforms to associate into dimers and oligomers, and the relationships between CABYR and other FS proteins were studied by gel electrophoresis, Western blotting, immunofluorescence, immunoprecipitation and yeast two-hybrid analyses., Results: The predominant form of mouse CABYR in the FS is an 80 kDa variant that contains only CABYR-A encoded by coding region A. CABYR isoforms form dimers by combining the 80 kDa CABYR-A-only variant with the 50 kDa variant that contains both CABYR-A and CABYR-B encoded by full length or truncated coding region A and B. It is proposed that this step is followed by the formation of larger oligomers, which then participate in the formation of the supramolecular structure of the FS in mouse sperm. The initial expression of CABYR occurs in the cytoplasm of spermatids at step 11 of spermiogenesis and increases progressively during steps 12-15. CABYR protein gradually migrates into the sperm flagellum and localizes to the FS of the principal piece during steps 15-16. Deletion of the CABYR RII domain abolished the interaction between CABYR and AKAP3/AKAP4 but did not abolish the interaction between CABYR and ropporin suggesting that CABYR binds to AKAP3/AKAP4 by its RII domain but binds to ropporin through another as yet undefined region., Conclusions: CABYR expresses at the late stage of spermiogenesis and its isoforms oligomerize and bind with AKAPs and ropporin. These interactions strongly suggest that CABYR participates in the assembly of complexes in the FS, which may be related to calcium signaling.

The annulus is a higher order septin cytoskeletal structure located between the midpiece and principal piece regions of the sperm tail. The annulus has been hypothesized to generate the diffusion barrier that exists between these two membrane domains. We tested this premise directly on septin 4 knockout mice, whose sperm are viable but lack an annulus, by following the diffusing membrane protein basigin. Basigin is normally confined to the principal piece domain on testicular and caput sperm, but undergoes relocation into the midpiece during sperm epididymal transit. On Sept4(-/-) sperm, domain confinement was lost, and basigin localized over the entire plasma membrane. Both immunofluorescence and immunoblotting further revealed reduced levels of basigin expression on sperm from the knockout. Testicular immunohistochemistry showed similar basigin expression and tail targeting in wild-type (WT) and Sept4(-/-) tubules until step 15 of spermatid development, at which point basigin was redistributed throughout the plasma membrane of Sept4(-/-) spermatids. The basigin outside of the tail was subsequently lost around the time of sperm release into the lumen. The redistribution in the knockout coincides with the time in WT sperm when the annulus completes its migration from the neck down to the midpiece-principal piece junction. We posit that basigin may not diffuse freely until after the annulus arrives at the midpiece-principal piece junction to restrict lateral movement. These results are the strongest evidence to date of a mammalian septin structure establishing a membrane diffusion barrier.

SPEF2 is expressed in all ciliated cells and is essential for correct sperm tail development and male fertility. We have previously identified a mutation within the SPEF2 gene as the cause for infertility because of immotile and malformed sperm tails in pigs. This mutation in pigs alters the testis-specific long SPEF2 isoform and exclusively affects the sperm tail development. In infertile boars, axonemal and all accessory structures of the sperm tail are affected; thus, SPEF2 seems to participate in the organization of these structures. In the present study, we have investigated the expression of SPEF2 during mouse spermatogenesis. SPEF2 mRNA and protein products appear to be localized both in germ cells and in Sertoli cells. In differentiating germ cells, SPEF2 protein is localized in the Golgi complex, manchette, basal body, and midpiece of the sperm tail. In mature murine sperm, SPEF2 is present in the distal part of the sperm tail midpiece. Using yeast two-hybrid assay and coimmunoprecipitation experiments, we identified an interaction between SPEF2 and the intraflagellar transport protein IFT20 in the testis. Furthermore, these two proteins colocalize in differentiating male germ cells. These results support the crucial importance of SPEF2 in sperm differentiation and involvement of SPEF2 in structuring of the sperm tail.

Trypsin is well known as a pancreatic enzyme that is typically secreted into the intestine to digest proteins. We show in our current study, however, that trypsin is also a key factor in the control of spermatogenesis. A progestin in teleost fish, 17alpha, 20beta-dihydroxy-4-pregnen-3-one (DHP), is an essential component of the spermatogenesis pathway, particularly during the initiation of the first meiotic division. In the course of our investigations into the mechanisms underlying progestin-stimulated spermatogenesis, we identified that eel trypsinogen is upregulated in eel testis by DHP treatment. Trypsinogen is expressed in the Sertoli cells surrounding spermatogonia and in the membranes of spermatids and spermatozoa. Using an in vitro eel testicular culture system, we further analyzed the roles of trypsin in spermatogenesis. The inhibition of trypsin using specific antibodies or serine protease inhibitors was found to compromise DHP-induced spermatogenesis. A low dose of trypsin induces DNA synthesis and the expression of Spo11, a molecular marker of meiosis, in germ cells. By comparison, a higher dose of trypsin partially induced spermiogenesis. Furthermore, trypsin was detectable in the membranes of the spermatozoa and found to be associated with fertilization in fish. Our results thus demonstrate that trypsin and/or a trypsin-like protease is an essential and multifunctional factor in spermatogenesis.

The hypodactylous (hd) locus impairs limb development and spermatogenesis, leading to male infertility in rats. We show that the hd mutation is caused by an insertion of an endogenous retrovirus into intron 10 of the Cntrob gene. The retroviral insertion in hd mutant rats disrupts the normal splicing of Cntrob transcripts and results in the expression of a truncated protein. During the final phase of spermiogenesis, centrobin localizes to the manchette, centrosome, and the marginal ring of the spermatid acroplaxome, where it interacts with keratin 5-containing intermediate filaments. Mutant spermatids show a defective acroplaxome marginal ring and separation of the centrosome from its normal attachment site of the nucleus. This separation correlates with a disruption of head-tail coupling apparatus, leading to spermatid decapitation during the final step of spermiogenesis and the absence of sperm in the epididymis. Cntrob may represent a novel candidate gene for presently unexplained hereditary forms of teratozoospermia and the "easily decapitated sperm syndrome" in humans.

Two isoforms of phosphoprotein phosphatase 1, PPP1CC1 and PPP1CC2, are translated from alternatively spliced transcripts of a single gene, Ppp1cc, and differ only at their extreme C-termini. While PPP1CC1 expression is almost ubiquitous, PPP1CC2 is largely restricted to testicular germ cells and mature spermatozoa. Targeted deletion of Ppp1cc leads to sterility of -/- males due to a combination of gross structural defects in developing spermatids resulting in apoptosis and faulty spermiation. Because PPP1CC2 is the only PP1 isoform that demonstrates high-level expression in wild-type meiotic and postmeiotic male germ cells, we have tested whether its loss in Ppp1cc-/- males is largely responsible for manifestation of this phenotype by expressing PPP1CC2 transgenically in the testis of Ppp1cc-/- mice (rescue mice). Herein, we demonstrate that PPP1CC2 expression in the Ppp1cc-/- testis is antiapoptotic, thus reestablishing spermatid development and spermiation. However, because aberrant flagellar morphogenesis is incompletely ameliorated, rescue males remain infertile. Because these results suggest that expression of PPP1CC2 in developing germ cells is essential but insufficient for normal spermatogenesis to occur, appropriate spatial and temporal expression of both PPP1CC isoforms in the testis during spermatogenesis appears to be necessary to produce structurally normal fertility-competent spermatozoa.

Single intraperitoneal injection of busulfan at 20 mg/kg body weight to mature male mice induced the deletion of the spermatogenic cells, followed by the restoration of the spermatogenesis by the surviving undifferentiated spermatogonia. The changes of the protein contents in testis during these processes were analyzed by two-dimensional gel electrophoresis in order to identify the proteins expressed at the specific stages of spermatogenesis. An acidic protein that disappeared and recovered in the same time course as spermatids after the busulfan treatment was identified as CABS1 by mass spectrometry. It was found that CABS1 was specifically expressed in the elongate spermatids at steps 13 to 16 in stages I to VIII of the seminiferous epithelium cycle of the mouse, and then it localized to the principal piece of flagellum of the mature sperm in the cauda epididymis. We have found for the first time that CABS1 is a calcium-binding protein that binds calcium during the maturation in the epididymis.

In addition to their role in centrosome organization, the centrioles have another distinct function as basal bodies for the formation of cilia and flagella. Centriole duplication has been reported to require two alternate assembly pathways: template or de novo. Since spermiogenesis in the termite Mastotermes darwiniensis lead to the formation of multiflagellate sperm, this process represents a useful model system in which to follow basal body formation and flagella assembly. We present evidence of a possible de novo pathway for basal body formation in the differentiating germ cell. This cell also contains typical centrosomal proteins, such as centrosomin, pericentrin-like protein, gamma-tubulin, that undergo redistribution as spermatid differentiation proceeds. The spermatid centrioles are long structures formed by nine doublet rather than triplet microtubules provided with short projections extending towards the surrounding cytoplasm and with links between doublets. The sperm basal bodies are aligned in parallel beneath the nucleus. They consist of long regions close to the nucleus showing nine doublets in a cartwheel array devoid of any projections; on the contrary, the short region close to the plasma membrane, where the sperm flagella emerge, is characterized by projections similar to those observed in the centrioles linking the basal body to the plasma membrane. It is hypothesized that this appearance is in connection with the centriole elongation and further with the flagellar axonemal organization. Microtubule doublets of sperm flagellar axonemes are provided with outer dynein arms, while inner arms are rarely visible., ((c) 2009 Wiley-Liss, Inc.)

A-kinase anchor protein 4 (AKAP4) is an X-linked member of the AKAP family of scaffold proteins that anchor cAMP-dependent protein kinases and play an essential role in fibrous sheath assembly during spermatogenesis and flagellar function in spermatozoa. Marsupial spermatozoa differ in structural organization from those of eutherian mammals but data on the molecular control of their structure and function are limited. We therefore cloned and characterized the AKAP4 gene in a marsupial, the tammar wallaby (Macropus eugenii). The gene structure, sequence, and predicted protein of AKAP4 were highly conserved with that of eutherian orthologues and it mapped to the marsupial X-chromosome. There was no AKAP4 expression detected in the developing young. In the adult, AKAP4 expression was limited to the testis with a major transcript of 2.9 kb. AKAP4 mRNA was expressed in the cytoplasm of round and elongated spermatids while its protein was found on the principal piece of the flagellum in the sperm tail. This is consistent with its expression in other mammals. Thus, AKAP4 appears to have a conserved role in spermatogenesis for at least the last 166 million years of mammalian evolution.

Mitochondrial transcription factor A (TFAM) is responsible for stability, maintenance, and transcriptional control of mitochondrial DNA (mtDNA). We have studied the expression and distribution of TFAM in the gametes and preimplantation embryos of the domestic pig (Sus scrofa). We hypothesized that TFAM is not present in the boar sperm mitochondria to reduce the possibility of paternal mtDNA propagation in the progeny. In contrast, we anticipated that Tfam gene is expressed in a developmental stage-dependent manner in porcine oocytes and embryos. The appropriate TFAM band of 25 kDa was detected by Western blotting in ejaculated boar spermatozoa, as well as in porcine oocytes and zygotes. Boar sperm extracts also displayed several bands >25 kDa suggestive of post-translational modification by ubiquitination, confirmed by affinity purification of ubiquitinated proteins. TFAM immunoreactivity was relegated to the sperm tail principal piece and sperm head in fully differentiated spermatozoa. The content of Tfam mRNA increased considerably from the germinal vesicle to blastocyst stage and also between in vitro fertilized and cultured blastocysts compared to in vivo-derived blastocysts. TFAM protein accumulated in the oocytes during maturation and was reduced by proteolysis after fertilization. This pattern was not mirrored in parthenogenetically activated oocytes and zygotes reconstructed by SCNT, suggesting deviant processing of TFAM protein and transcript after oocyte/embryo manipulation. Thus, TFAM may exert a critical role in porcine gametogenesis and preimplantation embryo development. Altogether, our data on the role of TFAM in mitochondrial function and inheritance have broad implications for cell physiology and evolutionary biology., ((c) 2008 Wiley-Liss, Inc)

Male infertility is one possible consequence of a group of disorders arising from dysfunction of cilia. Ciliopathies include primary ciliary dyskinesia, polycystic kidney disease, Usher syndrome, nephronophthisis, Bardet-Biedl syndrome, Alstrom syndrome, and Meckel-Gruber syndrome as well as some forms of retinal degenerations. Mutations in the retinitis pigmentosa GTPase regulator gene (RPGR) are best known for leading to retinal degeneration but have also been associated with ciliary dysfunctions affecting other tissues. To further study the involvement of RPGR in ciliopathies, transgenic mouse lines overexpressing RPGR were generated. Animals carrying the transgene in varying copy numbers were investigated. We found that infertility due to aberrant spermatozoa correlated with increased copy numbers. In animals with moderately increased gene copies of Rpgr, structural disorganization in the flagellar midpiece, outer dense fibers, and fibrous sheath was apparent. In contrast, in animals with high copy numbers, condensed sperm heads were present, but the flagellum was absent in the vast majority of spermatozoa, although early steps of flagellar biogenesis were observed. This complexity of defects in flagellar assembly suggests a role of RPGR in intraflagellar transport processes.

Aim: To characterize mouse capping protein alpha3 (CPalpha3) during spermatogenesis and sperm maturation., Methods: We produced rat anti-CPalpha3 antiserum and examined the expression of CPalpha3 in various mouse tissues using Western blot analysis and the localization of CPalpha3 in testicular and epididymal sperm using immunohistochemical analyses. We also examined how the localization of CPalpha3 and beta-actin (ACTB) in sperm changed after the acrosomal reaction by performing immunohistochemical analyses using anti-CPalpha3 antiserum and anti-actin antibody., Results: Western blot analysis using specific antiserum revealed that CPalpha3 was expressed specifically in testes. Interestingly, the molecular weight of CPalpha3 changed during sperm maturation in the epididymis. Furthermore, the subcellular localization of CPalpha3 in sperm changed dynamically from the flagellum to the post-acrosomal region of the head during epididymal maturation. The distribution of ACTB was in the post-acrosomal region of the head and the flagellum. After inducing the acrosomal reaction, the CPalpha3 and ACTB localization was virtually identical to the localization before the acrosomal reaction., Conclusion: CPalpha3 might play an important role in sperm morphogenesis and/or sperm function., ((c) 2008, Asian Journal of Andrology, SIMM and SJTU. All rights reserved.)

Meichroacidin (MCA) is a highly hydrophilic protein that contains the membrane occupation and recognition nexus motif. MCA is expressed during the stages of spermatogenesis from pachytene spermatocytes to mature sperm development and is localized in the male meiotic metaphase chromosome and sperm flagellum. MCA sequences are highly conserved in Ciona intestinalis, Cyprinus carpio, and mammals. To investigate the physiological role of MCA, we generated MCA-disrupted mutant mice; homozygous MCA mutant males were infertile, but females were not. Sperm was rarely observed in the caput epididymidis of MCA mutant males. However, little to no difference was seen in testis mass between wild-type and mutant mice. During sperm morphogenesis, elongated spermatids had retarded flagellum formation and might increase phagocytosis by Sertoli cells. Immunohistochemical analysis revealed that MCA interacts with proteins located on the outer dense fibers of the flagellum. The testicular sperm of MCA mutant mice was capable of fertilizing eggs successfully via intracytoplasmic sperm injection and generated healthy progeny. Our results suggest that MCA is essential for sperm flagellum formation and the production of functional sperm.

Males of the genus Drosophila produce sperm of remarkable length. Investigation of giant sperm production in Drosophila melanogaster has demonstrated that specialized actin and microtubule structures play key roles. The gene yuri gagarin (yuri) encodes a novel protein previously identified through its role in gravitaxis. A male-sterile mutation of yuri has revealed roles for Yuri in the functions of the actin and tubulin structures of spermatogenesis. Yuri is a component of the motile actin cones that individualize the spermatids and is essential for their formation. Furthermore, Yuri is required for actin accumulation in the dense complex, a microtubule-rich structure on the sperm nuclei thought to strengthen the nuclei during elongation. In the yuri mutant, late clusters of syncytial nuclei are deformed and disorganized. The basal bodies are also mispositioned on the nuclei, and the association of a specialized structure, the centriolar adjunct (CA), with the basal body is lost. Some of these nuclear defects might underlie a further unexpected abnormality: sperm nuclei occasionally locate to the wrong ends of the spermatid cysts. The structure of the axonemes that grow out from the basal bodies is affected in the yuri mutant, suggesting a possible role for the CA in axoneme formation.

The outer dense fiber protein ODF2 is the major component of the sperm tail cytoskeleton and a critical component of the mature centriole of the centrosome. Centriole maturation involves the formation of appendages and the recruitment of ODF2/Cenexin. ODF2 and Cenexin are alternative splice variants that differ in a short stretch of amino acids at their N-terminal regions encoded by exon 3b. Whereas Cenexin is ubiquitously expressed, Odf2 is the predominant transcript of testes [Hüber, D., Hoyer-Fender, S., 2007. Alternative splicing of exon 3b gives rise to ODF2 and Cenexin. Cytogenet. Genome Res. 119, doi:10.1159/000109621]. Here, we show that testicular expression of Odf2 correlates with spermiogenesis and ongoing sperm tail formation thus implicating functional differences between ODF2 and Cenexin. By generation of a series of ODF2/Cenexin deletion constructs fused to GFP and inspection of their subcellular localization in transfected NIH3T3 cells we found that a peptide of 42 amino acids specific for Cenexin is necessary for targeting ODF2/Cenexin to the centrosome and the primary cilium. Additionally, this region is also necessary for the formation of ODF2/Cenexin fibers that are associated with acetylated microtubules. Centrosomal targeting of ODF2/Cenexin does not depend on dynein-mediated transport further supporting an alternative targeting mechanism. However, part of the C-terminal coiled-coil region of ODF2 is also important in centrosomal/ciliary targeting and fiber formation presumably by supporting self-association and the formation of higher-order structures.

We report characterization of a novel testis- and sperm-specific protein, FSCB (fibrous sheath CABYR binding), that is expressed post-meiotically and localized in mouse sperm flagella. FSCB was identified as a binding partner of CABYR, a calcium-binding protein that is tyrosine-phosphorylated during capacitation. Orthologous genes of FSCB are present in other mammals, including rat and human, and conserved motifs in FSCB include PXXP, proline-rich and extensin-like regions. FSCB is phosphorylated by protein kinase A as shown by in vitro phosphorylation assay and also by determining phosphorylation sites in native FSCB from mouse sperm. Calcium overlay assay showed that FSCB is a calcium-binding protein from sperm. FSCB is a post meiotic protein first expressed at step 11 of mouse spermatogenesis in the elongating spermatids, and it subsequently incorporates into the flagellar principal piece of the sperm. Ultrastructurally, FSCB localized to a cortical layer of intermediate electron density at the surface of the ribs and longitudinal columns of the fibrous sheath. Due to its temporal appearance during spermiogenesis and location at the cortex of the fibrous sheath, FSCB is postulated to be involved in the later stages of fibrous sheath assembly.

The fibrous sheath (FS) is a novel structural feature in the principal piece region of the sperm flagellum in marsupial and eutherian mammals. It begins anteriorly at the annulus, is composed of two longitudinal columns connected by circumferential ribs, and surrounds the outer dense fibers in the principal piece. The formation of the FS was described eloquently nearly twenty-five years ago, but the identification of its components has occurred only during the last few years. Most of the genes encoding the known FS components are expressed during the postmeiotic period of spermatogenesis, are expressed only in spermatogenic cells, and are either novel genes or paralogues of genes expressed in somatic cells. The proteins of the FS can be classified in nine categories based on their functional or structural roles. Most of these are resistant to solubilization and remain in the FS during rigorous isolation procedures. Mouse sperm with a knock out of the gene encoding GAPDHS are immotile, which demonstrates that most of the ATP needed for sperm motility is generated by glycolysis. Most of the glycolytic enzymes have been localised to the principal piece and some are tightly associated with the FS. However, the nature of the protein-protein interactions involved in assembly and maintenance of FS are known for only a few of the component proteins.

Sperm are motile cells. Thus, a significant component of the spermatogenic cycle is devoted to the formation of flagellum, a process that must be coordinated to insure proper construction. To document the temporal pattern of flagellar gene expression, we employed real-time PCR to assess changes in accumulation of a cohort of genes encoding axoneme, outer dense fibre (ODF) and fibrous sheath (FS) proteins during the first wave of spermatogenesis in the mouse. Axoneme genes were expressed first at the pachytene spermatocyte stage, followed by expression of transcripts encoding ODF and FS components. However, there were differences among these families with respect to the time of initial expression and the rate of mRNA accumulation. To gain understanding of factors that determine these patterns of expression, we cloned the promoters of three axoneme central apparatus genes (Pf6, Spag6 and Pf20). These promoters shared common features including the absence of a TATA box, and putative binding sites for several factors implicated in spermatogenesis (CREB/CREM, SOX17 and SPZ1) as well as ciliogenesis (FOXJ1). Collectively, our findings demonstrate a sequential pattern of expression of flagellar component genes, differential times of expression or rates of transcript accumulation within each class and shared promoter features within a class.

The heterodimeric hypoxia-inducible factor (HIF)-1 is a transcriptional master regulator of several genes involved in mammalian oxygen homeostasis, including erythropoietin, vascular endothelial growth factor, and factors involved in glucose transport and metabolism. The mouse Hif1a gene is expressed from two distinct promoter/first exon combinations resulting in tissue-specific (mHIF-1alphaI.1) and ubiquitous (mHIF-1alphaI.2) mRNA isoforms. By in situ hybridization, we detected mHIF-1alphaI.1 mRNA exclusively in the elongated spermatids of the testis. In vitro studies indicated that the switch from mHIF-1alphaI.2 to mHIF-1alphaI.1 mRNA expression does not occur at the premeiotic stages of mouse spermatogenesis. Exposure of mice to hypoxic conditions induced mHIF-1alphaI.2 protein in spermatocytes and probably in Sertoli cells but not in spermatogonia. In contrast, expression of the putative mHIF-1alphaI.1 protein in spermatozoa of the testis and epididymis was oxygen independent and located to the midpiece of the spermatozoal flagellum. Both the switch in transcript expression during spermiogenesis and the unexpected protein localization in mature sperm cells suggest a so far unrecognized function of HIF-1alpha.