Characterization,expression dynamics,and potential function of OPA1 for regulation of mitochondrial morphology during spermiogenesis in Phascolosoma esculenta

Mitochondria undergo morphological changes during spermatogenesis in some animals.The mechanism and role of mitochondrial morphology regulation,however,remain somewhat unclear.In this study,we analyzed the molecular characteristics,expression dynamics and subcellular localization of optic atrophy protein 1(OPA1),a mitochondrial fusion and cristae maintenance-related protein,to reveal the possible regulatory mechanisms underlying mitochondrial morphology in Phascolosoma esculenta spermiogenesis.The full-length cDNA of the P.esculenta opa1 gene(Pe-opa1)is 3743 bp in length and encodes 975 amino acids.The Pe-OPA1 protein is highly conservative and includes a transmembrane domain,a GTPase domain,two helical bundle domains,and a lipid-interacting stalk.Gene and protein expression was higher in the coelomic fluid(a site of spermatid development)of male P.esculenta and increased first and then decreased from March to December.Moreover,their expression during the breeding stage was significantly higher than during the non-breeding stage,suggesting that Pe-OPA1 is involved in P.esculenta reproduction.The Pe-OPA1 protein was more abundant in components consisting of many spermatids than in components without,indicating that Pe-OPA1 mainly plays a role in the spermatid in coelomic fluid.Moreover,Pe-OPA1 was mainly detected in the spermatid mitochondria.Immunofluorescence experiments showed that the Pe-OPA1 are constitutively expressed and co-localized with mitochondria during spermiogenesis,suggesting its involvement in P.esculenta spermiogenesis.These results provide evidence for Pe-OPA1's involvement in the regulation of mitochondrial morphology during spermiogenesis.

Germ cell-specific deletion of Pex3 reveals essential roles of PEX3-dependent peroxisomes in spermiogenesis

Peroxisomes are organelles enclosed by a single membrane and are present in various species.The abruption of peroxisomes is correlated with peroxisome biogenesis disorders and single peroxisomal enzyme deficiencies that induce diverse diseases in different organs.However,little is known about the protein compositions and corresponding roles of heterogeneous peroxisomes in various organs.Through transcriptomic and proteomic analyses,we observed heterogenous peroxisomal components among different organs,as well as between testicular somatic cells and different developmental stages of germ cells.As Pex3 is expressed in both germ cells and Sertoli cells,we generated Pex3 germ cell-and Sertoli cell-specific knockout mice.While Pex3 deletion in Sertoli cells did not affect spermatogenesis,the deletion in germ cells resulted in male sterility,manifested as the destruction of intercellular bridges between spermatids and the formation of multinucleated giant cells.Proteomic analysis of the Pex3-deleted spermatids revealed defective expressions of peroxisomal proteins and spermiogenesis-related proteins.These findings provide new insights that PEX3-dependent peroxisomes are essential for germ cells undergoing spermiogenesis,but not for Sertoli cells.

Intramanchette transport during primate spermiogenesis： expression of dynein, myosin Va, motor recruiter myosin Va, VIIa-Rab27a/b interacting protein, and Rab27b in the manchette during human and monkey spermiogenesis

Aim： To show whether molecular motor dynein on a microtubule track, molecular motor myosin Va, motor recruiter myosin Va, VIIa-Rab27a/b interacting protein （MyRIP）, and vesicle receptor Rab27b on an F-actin track were present during human and monkey spermiogenesis involving intramanchette transport （IMT）. Methods： Spermiogenic cells were obtained from three men with obstructive azoospermia and normal adult cynomolgus monkey （Macacafascieularis）. Immunocytochemical detection and reverse transcription-polymerase chain reaction （RT-PCR） analysis of the proteins were carried out. Samples were analyzed by light microscope. Results： Using RT-PCR, we found that dynein, myosin Va, MyRIP and Rab27b were expressed in monkey testis. These proteins were localized to the manchette, as shown by immunofluorescence, particularly during human and monkey spermiogenesis. Conclusion： We speculate that during primate spermiogenesis, those proteins that compose microtubule-based and actin-based vesicle transport systems are actually present in the manchette and might possibly be involved in intramanchette transport.

miR-199-5p regulates spermiogenesis at the posttranscriptional level via targeting Tekt1 in allotriploid crucian carp

Background:Sperm abnormalities are one of the primary factors leading to male sterility,but their pathogenesis is still unclear.Although miRNAs are suggested to exert important roles in the regulation of spermatogenesis at both transcriptional and posttranscriptional levels,little is currently known regarding the regulation of sperm flagella assembly by microRNAs(miRNAs).The role of miRNAs in the development of sperm abnormalities in sterile triploid fish has not been studied.Results:In this study,we found that miR-199-5p was widely expressed in all detected tissues of different-ploidy crucian carp.As one of the testis-specific candidate markers,Tekt1 was predominantly expressed in the testis.Quantitative real-time PCR(qRT-PCR)analyses showed that the expression trend of miR-199-5p was exactly opposite to that of Tekt1.Through bioinformatics analysis,we identified a putative miR-199-5p binding site in the Tekt1 mRNA.We further identified Tekt1 as a target of miR-199-5p using luciferase reporter assay.Finally,we confirmed that miR-199-5p was necessary for sperm flagellar assembly and spermatogenesis in vivo via intraperitoneal injection of miR-199-5p antagomir or agomir in diploid red crucian carp.Moreover,miR-199-5p gainof-function could lead to spermatids apoptosis and abnormal spermatozoa structure,which is similar to that of allotriploid crucian carp.Conclusions:Our studies suggested that abnormally elevated miR-199-5p inhibited the sperm flagella formation in spermiogenesis by negatively regulating the expression of Tekt1,thereby causing sperm abnormalities of male allotriploid crucian carp.

Evidence for the role of KIF17 in fish spermatid reshaping:expression pattern of KIF17 in Larimichthys polyactis spermiogenesis

The homodimeric kinesin-2 protein KIF17 functions in intracellular transport and spermiogenesis in mammals.However,its role in fish spermiogenesis has not been reported.Here,we aimed to clone full-length kif17 cDNA and determine the molecular characteristics and expression patterns of KIF17 in Larimichthys polyactis spermiogenesis.The full-length cDNA of L.polyactis kif17(Lp-kif17)was sequenced and found to contain a 332-bp 5′untranslated region,480-bp 3′untranslated region,and 2433-bp open reading frame encoding 810 amino acids.Bioinformatics analyses showed that L.polyactis KIF17(Lp-KIF17)shared high sequence similarity with homologs in other animals and possessed an N-terminal motor domain with microtubule-binding sites and adenosine triphosphate(ATP)hydrolysis sites,a stalk domain containing two coiled-coil regions,and a C-terminal tail domain.The Lp-kif17 mRNA was widely expressed in various tissues,with the highest level in the brain,followed by that in the testis.Fluorescence in situ hybridization(FISH)analysis revealed that Lp-kif17 was continuously expressed in spermiogenesis,showing that it had potential functions in this process.Using immunofluorescence(IF)analysis,we found that Lp-KIF17 colocalized with tubulin and was transferred from the perinuclear cytoplasm to the side of spermatid where the tail forms during spermiogenesis.These findings suggested that KIF17 is involved in L.polyactis spermiogenesis.In particular,it may participate in nuclear shaping and tail formation by interacting with perinuclear microtubules during spermatid reshaping.In addition to providing evidence for the role of KIF17 in fish spermatid reshaping,this study provides important data for studies of reproductive biology in L.polyactis.

Mitochondria: transportation, distribution and function during spermiogenesis

Spermiogenesis is a dynamic process which includes organelle reorganization and new structure formation. The morphology and distribution of the mitochondria in germ cells change to accommodate the cellular requirement. Multiple molecular motors and related proteins participate in carrying and anchoring mitochondria to the midpiece during spermiogenesis and this process is regulated precisely. Energetic metabolism provides energy for cellular activity and influences sperm survival and motility directly. Ubiquitination of mitochondria takes place during spermiogenesis, which has been implicated in sperm quality control and mitochondrial inheritance. In light of the essential roles of mitochondria in energy production, calcium homeostasis and apoptosis, mitochondria dysfunction cause severe human diseases, such as male infertility. The present study paves a way for a more detailed exploration of the biology of mitochondria during spermiogenesis.

Molecular Characterization, Tissue Distribution and Localization of Larimichthys crocea Kif3a and Kif3b and Expression Analysis of Their Genes During Spermiogenesis

KIF3A and KIF3B are two N-terminal motor proteins belonging to the kinesin-II superfamily that play essential roles in spermiogenesis.To understand the roles played by KIF3 A/3B during spermatogenesis of large yellow croaker Larimichthys crocea,we studied the testis characteristics at different developmental stages of L.crocea,and determined the spatiotemporal expression patterns of kif3a and kif3b during spermiogenesis.Quantitative real-time PCR(qR T-PCR)showed that the overall trends of kif3 a/3 b m RNA abundance during testis development are similar.From stage Ⅱ to stage V,kif3a/3b m RNA abundances first increased and then fell after reaching a peak at stage IV.Interestingly,the m RNA abundances of both genes at stage V were higher than those at stages Ⅱ and Ⅲ.In addition,it is worth of noting that kif3 b m RNA abundance was higher than that of kif3a at all stages.Fluorescence in situ hybridization results revealed that kif3a/3b m RNA abundance dynamics were consistent with the migration of mitochondria,the deformation of nucleus,and the formation of tail.The m RNA hybridization signals of both genes first appeared either around the nuclear periphery or on the side of the nuclei,then appeared at one side of nuclei,and finally were mainly on the tail during spermiogenesis.Our findings contributed to better understanding the molecular mechanisms of spermiogenesis in fish;and suggested that KIF3A and KIF3B may participate in the intracellular transport of mitochondria,nuclear deformation,and the formation of tail during the spermiogenesis in L.crocea.

Molecular Cloning and Putative Functions of KIFC1 for Acrosome Formation and Nuclear Reshaping during Spermiogenesis in Schlegel’s Japanese Gecko Gekko japonicus

Spermiogenesis, occurring in the male testis, is a complicated and highly-ordered developmental process resulting in the production of fertile mature sperm. In Gekko japonicus, this process occurs in 7 steps during which the spermatids undergo dramatic changes in the cytoskeleton and nucleus. Here, we cloned and sequenced the cDNA of the mammalian KIFC1 homologue in the testis of G. japonicus. The 2 344 bp full-length cDNA sequence contained a 191 bp 5’-untranslated region, a 134 bp 3’-untranslated region and a 2 019 bp open reading frame encoding a protein of 672 amino acids. Tissue expression analysis revealed the highest expression of kifc1 mRNA was in the testis. Fluorescence in situ hybridization revealed that the kifc1 mRNA signal was hardly detected in step 1 spermatids but became concentrated at the acrosome of step 2 spermatids and abundant in the nucleus of step 5 spermatids where the nucleus then undergoes dramatic elongation and compression. The kifc1 mRNA signal then gradually disappears in mature sperm. This expression of KIFC1 at specific stages of spermiogenesis in G. japonicus implies its important role in the major cytological transformations such as acrosome biogenesis and nucleus morphogenesis.

Expression Characteristics and Putative Functions of KIF3A/KIF3B During Spermiogenesis of Phascolosoma esculenta

The microtubule(MT)-associated proteins KIF3A and KIF3B are ubiquitously expressed in a wide range of taxa.This study investigated the functions of these proteins in spermiogenesis,which involves various MT-dependent processes,in Phasco-losoma esculenta.We cloned the complete cDNA of Pe-KIF3A/3B.Structural predictions showed that Pe-KIF3A/3B are composed of a highly conserved motor domain,a coiled-coil domain,and a tail domain.Real-time quantitative PCR showed that Pe-kif3a/3b are expressed in all tissues evaluated,with the highest levels in sperm masses.Fluorescence in situ hybridization and immunofluo-rescence were employed to analyze the dynamic expression patterns of KIF3A/3B during spermiogenesis.Pe-KIF3A/3B consistently co-localized with MTs at all stages of spermiogenesis,indicating their potential functions in cargo trafficking.Pe-KIF3A/3B co-localized with mitochondria,suggesting that they may mediate mitochondrial movement.In late-stage spermatids and mature sperm,co-localization was detected in the midpiece,suggesting that Pe-KIF3A/3B could facilitate midpiece formation.The co-localization of Pe-KIF3A and Pe-KIF3B at all stages of spermiogenesis suggests their function as the heterodimeric KIF3AB.Basing on the ob-served temporal and spatial expression patterns of Pe-KIF3A/3B,MTs,and mitochondria in our study,we suggest that heterodimer KIF3AB has a potential role in acrosome biogenesis,sperm head remodeling,enflagellation,and mitochondrial migration during spermiogenesis in P.esculenta.In addition,our study on the morphological characteristics of spermatogenic cells provided fundamental data on the reproductive biology of P.esculenta.

SPERMIOGENESIS IN THE YELLOWFIN PORGY (SPARUS LATUS HOUTTUYN), WITH EMPHASIS ON THE ASSOCIATED MITOCHONDRION

Transmission electron microscopy of the yellowfin porgy (Sparus latus Houttuyn) spermatozoa ultrastructure showed the spermatozoon as a primitive type made up of the acrosomeless head , the flagellum , and the midpiece , at the periphery of which was a relatively big mitochondrion with more complex structure . It was found that during spermiogenesis, only one relatively big mitochondrion occurred in both the spermatid and the spermatozoon . This is different from other teleost fishes . During spermiogenesis, the mitochondria number is one, and morphology did not change . All these are different fromthose of other fishes .

Cytosolic Ca^(2+)as a multifunctional modulator is required for spermiogenesis in Ascaris suum

The dynamic polar polymers actin fi laments and microtu-bules are usually employed to provide the structural ba-sis for establishing cell polarity in most eukaryotic cells.Radially round and immotile spermatids from nematodes contain almost no actin or tubulin,but still have the abil-ity to break symmetry to extend a pseudopod and initiate the acquisition of motility powered by the dynamics of cytoskeleton composed of major sperm protein(MSP)during spermiogenesis(sperm activation).However,the signal transduction mechanism of nematode sperm activation and motility acquisition remains poorly under-stood.Here we show that Ca^(2+) oscillations induced by the Ca^(2+) release from intracellular Ca^(2+) store through inositol(1,4,5)-trisphosphate receptor are required for Ascaris suum sperm activation.The chelation of cytosolic Ca^(2+) suppresses the generation of a functional pseudopod,and this suppression can be relieved by introducing ex-ogenous Ca^(2+) into sperm cells.Ca^(2+) promotes MSP-based sperm motility by increasing mitochondrial membrane potential and thus the energy supply required for MSP cytoskeleton assembly.On the other hand,Ca^(2+) promotes MSP disassembly by activating Ca^(2+)/calmodulin-depend-ent serine/threonine protein phosphatase calcineurin.In addition,Ca^(2+)/camodulin activity is required for the fusion of sperm-specifi c membranous organelle with the plasma membrane,a regulated exocytosis required for sperm mo-tility.Thus,Ca^(2+)plays multifunctional roles during sperm activation in Ascaris suum.

Rapid Evolution of the Mammalian HILS1 Gene and the Nuclear Condensation Process during Mammalian Spermiogenesis

Many genes associated with reproduction show rapid evolution across diverse animal groups, a result commonly due to adaptive evolution driven by positive selection （Swanson and Vacquier, 2002）. Different theories have been proposed to explain the elevated rates of evolution （Swanson and Vacquier, 2002）, including sperm competition, where sperm compete to fertilize eggs leading to the proteins in the sper- matozoa adaptively evolving to increase their ability to fertilize eggs; sexual conflict, where the egg experiences a loss of fitness when sperm are too abundant; sexual selection, where eggs bind sperm carrying adaptive alleles （Palumbi, 1999）; and cryptic female choice （reviewed in Swanson and Vacquier, 2002）.

Aquaporins in spermatozoa and testicular germ cells： identification and potential role

Mammalian spermatozoa have relatively high water permeability and swell readily, as in the hypo-osmotic swelling test used in the andrology clinic. Physiologically, spermatozoa experience changes in the osmolality of the surrounding fluids in both the male and the female tracts on their journey from the testis to the ovum. Sperm volume regulation in response to such osmotic challenges is important to maintain a stable cell size for the normal shape and function of the sperm tail. Alongside ion channels for the fluxes of osmolytes, water channels would be crucial for sperm volume regulation. In contrast to the deep knowledge and numerous studies on somatic cell aquaporins （AQPs）, the understanding of sperm AQPs is limited. Among the 13 AQPs, convincing evidence for their presence in spermatozoa has been confined to AQP7, AQP8 and AQP 11. Overall, current findings indicate a major role of AQP8 in water influx and efltux for sperm volume regulation, which is required for natural fertilization. The preliminary data suggestive of a role for AQP7 in sperm glycerol metabolism needs further substantiation. The association of AQP 11 with the residual cytoplasm of elongated spermatids and the distal tail of spermatozoa supports the hypothesis of more than just a role in conferring water permeability and also in the turnover and recycling of surplus cellular components made redundant during spermiogenesis and spermiation. This would be crucial for the maintenance of a germinal epithelium functioning efficiently in the production of spermatozoa.

Immunolocalization assessment of metastasis-associated protein I in human and mouse mature testes and its association with spermatogenesis

Aim： To investigate the stage-specific localization of metastasis-associated protein 1 （MTA1） during spermatogenesis in adult human and mouse testis. Methods： The immunolocalization of MTA1 was studied by immunohistochemistry and Western blot analysis. The distribution pattern of MTA1 in mouse testis was confirmed by using quantitative analysis of purified spermatogenic cells. Results： The specificity of polyclonal antibody was confirmed by Western blot analysis. MTA1 was found expressed in the nucleus of germ cells, except elongate spermatids, and in the cytoplasm of Sertoli cells; Leydig cells did not show any specific reactivity. MTA1 possessed different distribution patterns in the two species： in humans, the most intensive staining was found in the nucleus of round spermatids and of primary spermatocytes while in mice, the most intense MTA 1 staining was in the nucleus of leptotene, zygotene and pachytene spermatocytes. In both species the staining exhibited a cyclic pattern. Conclusion： The present communication initially provides new evidence for the potential role of MTA1 in mature testis. In addition, its distinctive expression in germ cells suggests a regulatory role of the peptide during spermatogenesis.

Studies on Expression of P1 Protamine Gene in Rat and Mouse Testis

Protamine is a kind small, basic protein rich in arginine residues and found to be complexed with DNA in spermatozoa. We have cloned a 150 bp cDNA encoding the rat protamine (rP) by RT PCR technique. Dig labelled cDNA for rP was used for Northern blot analysis to study the expression of P1 protamine gene in rat and mouse. P1 protamine mRNA was detected only in rat testis, no hybridization signals were detected in rat brain and lever. In addition, the presence of P1 protamine mRNA was detected not only in rat testis, but also in mouse testis. Dig labelled cDNA for mouse protamine 1 (mP1) was used to study the expression of mP1 gene during the process of sexual maturation of mouse. 7～8 d after birth, no mP1 mRNA could be detected. At d 24～26, mP1 mRNA was detectable migrating as a homogeneous band at 580 nucleotides, whereas in sexually mature animals, a heterogeneous mixture of RNAs ranging from 450～580 bases in length was observed. Histological studies revealed that in the testis of 7～8 day old mouse, spermatogenesis has developed to the spermatocyte stage, whereas round spermatids (Rs) were present in the testis of the mice with 24～26 d age and elongating spermatids (Es) were present in the testis of sexually mature animals. Electrophoresis of total nuclear basic proteins (TNBP) revealed that the Rs could possess the somatic histones, while Es was found to have protamine and less histone. These results indicate that the P1 protamine gene is tissues specifically expressed and the P1 protamine is showing to be conservative in evolution. During the process of sexual maturation, along with morphological changes, mP1 gene was transcribed in Rs and translated in Es. The mechanism of protamine gene expression was discussed.

Analysis of gene expression patterns with cDNA microarray during late stage of spermatogenesis in mice

The differentiation process of round spermat-ids to spermatozoa during the late stage of spermatogenesis is called spermiogenesis. To explore spermiogenesis-related genes, cDNA microarray was used to study expression patterns of 1176 genes in pachytene spermatocytes, round spermatids and elongating spermatids of Balb/c mice. The results showed that 208 genes were detected in all the three cell types. Most of them were down-regulated from pachytene spermatocytes to round spermatids and elongating spermatids. However, up-regulation of 7 genes expression in round spermatids and 3 genes in elongating spermatids were found. Expression of 7 differentially expressed genes in cDNA arrays was further confirmed by semi-quantitative RT-PCR study. The RT-PCR results indicated that the expression of 6 genes was consistent with that in cDNA arrays, only one gene did not show differential expression by RT-PCR. These results may provide important clues for studying of expression, regulation, and function of

Transformation:how do nematode sperm become activated and crawl?

Nematode sperm undergo a drastic physiological change during spermiogenesis(sperm activation).Unlike mammalian flagellated sperm,nematode sperm are amoeboid cells and their motility is driven by the dynamics of a cytoskeleton composed of major sperm protein(MSP)rather than actin found in other crawling cells.This review focuses on sperm from Caenorhabditis elegans and Ascaris suum to address the roles of external and internal factors that trigger sperm activation and power sperm motility.Nematode sperm can be activated in vitro by several factors,including Pronase and ionophores,and in vivo through the TRY-5 and SPE-8 pathways.Moreover,protease and protease inhibitors are crucial regulators of sperm maturation.MSP-based sperm motility involves a coupled process of protrusion and retraction,both of which have been reconstituted in vitro.Sperm motility is mediated by phosphorylation signals,as illustrated by identification of several key components(MPOP,MFPs and MPAK)in Ascaris and the characterization of GSP-3/4 in C.elegans.

MORN3：MEIG1的分子伴侣在小鼠生精细胞中的表达

Mammalian spermatogenesis is a well-organized process of cell development and differentiation. Meiosis expressed gene I （MEIG 1） plays an essential role in the regulation of spermiogenesis. To explore potential mechanisms of MEIGI＇s action, a yeast two-hybrid screen was conducted, and several potential binding partners were identified; one of them was membrane occupation and recognition nexus repeat containing 3 （MORN3）. MORN3 mRNA is only abundant in mouse testis. In the testis, Morn3 mRNA is highly expressed in the spermiogenesis stage. Specific anti-MORN3 polyclonal antibody was generated against N-terminus of the full-length MORN3 protein, and MORN3 expression and localization was examined in vitro and in vivo. In transfected Chinese hamster ovary cells, the antibody specifically crossed-reacted the full-length MORN3 protein, and immunofluorescence staining revealed that MORN3 was localized throughout the cytoplasm. Among multiple mouse tissues, about 25 kDa protein, was identified only in the testis. The protein was highly expressed after day 20 of birth. Immunofluorescence staining on mixed testicular cells isolated from adult wild-type mice demonstrated that MORN3 was expressed in the acrosome in germ cells throughout spermiogenesis. The protein was also present in the manchette of elongating spermatids. The total MORN3 expression and acrosome localization were not changed in the Meig/-deficient mice. However, its expression in manchette was dramatically reduced in the mutant mice. Our studies suggest that MORN3 is another regulator for spermatogenesis, probably together with MEIG1.

Delayed sexual maturity in males of Vespa velutina

Vespa velutina var nigrithorax （Lepelletier, 1835） is an invasive predator of bees accidentally introduced in France in 2004, and it is having a serious impact on apiculture and ecosystems. Studying the reproduction of an invasive species is key to assess its population dynamic. This study explores the sexual maturation of IT. velutina males and the evolution of their fertility. The main studied parameters were physiologic （spermiogenesis, spermatogenesis） and anatomic （testes size and structure, head width）. Two populations of males were described based on their emergence period： early males in early summer or classic males in autumn. Each testis has an average of 108 testicular follicles. Spermatogenesis is synchronous, with only 1 sperm production wave, and completed, on average, at 10.3 d after emergence with the degeneration of the testes. The sperm counts in seminal vesicles of mature males are 3 x 106 in October/November and 0.8 ~ 106 in June. In com- parison, females store 0.1 x 106 sperm in their spermathecae. The early males emerged from colonies made by fertilized queens. The reproductive potential of these early males seemed limited, and their fimction in the colony is discussed. The sperm stock evolution in autumn males suggests the occurrence of a reproductive pattern of male competition for the access to females and a single copulation per male. The synchronicity of male and foundress emergences and sexual maturation is of primary importance for the mating success and the future colony development.