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.

Subzonal fertilization of mouse round spermatids

Mouse round spermatids were electrofused with homologous mature oocytes to examine the be-haviour of their nuclei within the ooplasm and the abilities of development. A single spermatid was injected in the perivitelline space of a mature oocyte and an electron fusion pulse was given. The best round spermatid-oocyte pairs (RS-O) fusion took place at 20-30 s AC (1 MHz, 50V/cm) followed by a single fusion DC pulse (3 700-3 800 V/cm, 25 μs) and another 30 s AC current. The total survival rate and fusion rate of RS-O were 89.0% (575/646) and 61. 9% (356/575), respectively. 49.2% (175/356) of fused oocytes developed to 2PN stage . The concentration of Ca2+ in the fusion medium produced no significant effect on the above targets. The 2PN development rate of the fused RS-O from the oocytes collected 14-16 h after hCG injection was higher than others. 32.6% (57/175) of the 2PN oocytes had fully developed spermatid (male) and oocyte (female) pronuclei. The rest spermatid-derived pronu-clei remained small in

Human protamines and the developing spermatid: their structure, function, expression and relationshipwith male infertility

<abstract>During spermiogenesis, the protamine proteins play an integral role in spermatid chromatin compaction. Recent research has focused on many facets of protamine biology, including protamine gene and protein structure/ function relationships, mechanisms of protamine expression regulation and involvement of the protamines in male fertility. In this paper, we review our current understanding of the structure and function of the protamine-1 (P1) and protamine-2 (P2) proteins and genes, the expression and regulation of these genes and the relationship between the protamines and male fertility. In addition, we offer a brief outlook on future investigation into protamine proteins.

Quantitative (stereological) study on the spermatozoal storage capacity of epididymis in rats and monkeys

Aim: To investigate the spermatozoal production rate of the testis and the spermatozoal storage capacity of the epi-didymis in monkeys and rats. Methods: The number of the late spermatids (steps 13 - 14 in the monkey or steps15 - 19 in the rat) per testis and the number of spermatozoa per epididymis were estimated in 6 normal adult monkeys(Macaca fascicularis) and 6 normal adult SD rats on 25 um-thick methacrylate-embedded sections using a contempo-rary unbiased and efficient stereological method-the optical disector. The diameter and length of the efferent ductulesand ductus epididymidis and the volume of the epididymal fluid in the tubules were also estimated. Results: The totalnumber of the late spermatids per testis was 2902 ±749 (million, x ±s) in the monkey, or 179 ±31 in the rat; thenumber of spermatozoa per epididymis was 3235 ±1835 in the monkey, or 241±76 in the rat. Conclusion: A largenumber of spermatozoa was densely packed and stored in the ductus epididymidis; the epididymal transit time for sper-matozoa was around 5 days in monkeys or 11 days in rats.

Colloid Centrifugation of Semen: Applications in Assisted Reproduction

Colloid centrifugation can be used to separate heterogeneous populations of cells particularly semen samples, which typically contain mixtures of spermatozoa at different stage of maturity together with dead and dying spermatozoa and also non-sperm cells. Colloids can be used to enrich the population of spermatozoa that are viable and functional;these are the spermatozoa that are needed for fertilization in Assisted Reproduction. Sperm samples obtained after colloid centrifugation may be enriched for motile, morphologically normal spermatozoa with intact chromatin. Insemination of mares with stallion sperm samples prepared by colloid centrifugation resulted in a higher pregnancy rate per cycle than controls, confirming that the good sperm attributes observed in the laboratory were indicative of functional spermatozoa. The method does not simply enrich for viable spermatozoa by inhibiting dead or dying spermatozoa from passing through the colloid since emerging evidence shows that colloid centrifugation can select for spermatozoa with certain properties such as metabolic activity. Other reproductive cells, such as spermatids or progenitor cells, may also be purified by colloid centrifugation. This review describes the history of colloid centrifugation for selecting spermatozoa, compares colloid centrifugation with alternative techniques, and finally describes some of the applications of the technique within the animal breeding industry.

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.

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.

Two New Selenoproteins Found in the Prostatic Glandular Epithelium and in the Spermatid Nuclei

After labeling of rats in vivo with 75Se and protein separation by sodium dodecyl sulfatepolyacrylamide gel electrophoresis more than 25 Se-containing bands could be distinguished.Of those proteins which were detected only in certain compartments and might therefore have tissue-specific functions, two were chosen for detailed investigation.A 15 kDa-protein was found in the prostatic epithelium where it accounted for about two thirds of the protein-bound 75Se. It was mainly present in the cytosol but was not released into the prostatic secretion. After gel chromatography it was found in the fraction which contained proteins with molecular masses of about 300 kDa. Using two-dimensional electrophoresis a plvalue of about 4. 5 was determined.In the testis a specific Se-containing 34 kDa-protein was observed which appeared after the onset of puberty. It was localized in the spermatid nuclei where it contained about 80% of the Se tracer present and was found to be bound to the DNA. After extraction it partly disintegrated into a 20 kDa-protein.Both compounds contain Se in the form of selenocysteine. The fact that their formation had priority over that of glutathione peroxidase during insufficient Se intake is an indication of their biological significance. Special interest in the prostatic epithelial selenoprotein derives from a possible inverse relationship between the Se status and the incidence of prostate cancer observed in epidemiological studies, whereas with the 34 kDa-selenoprotein its appearance during the condensation phase of the spermatid nuclei might suggest its participation in some processes of sperm maturation

The Effect of Tripterygium Wilfordii Monomer T4 on Rat Spermatid Nuclear Protein Transition

Rat testis elongating spermatids and epididymal sperms were collected after 7 weeks of treatment with Tripterygium wilfordii monomer T4.Total nuclear basic protein(TNBP)was extracted from the elongating spermatid nuclei and the sperm nuclei isolated by sonication.Polyacrylamide gel electrophoresis has been used to separate the TNBP and individual proteins were quantified by scanning microdensitometry.It was found that the content of protamine was reduced and the TH(Total Histones)/RP(Rat Protamine)ratios were increased following treatment in the testis elongating spermatids, and same result was found in the epididymal sperms.These results suggest that the interruption of nuclear protein transition of testis spermatids induced by T4 might cause aberrant epididymal sperm nuclear protein and lead to infertility.The relationship between protamine and fertility was discussed.

The PIWI-specific insertion module helps load longer piRNAs for translational activation essential for male fertility

PIWI-clade proteins harness pi RNAs of 24–33 nt in length.Of great puzzles are how PIWI-clade proteins incorporate pi RNAs of different sizes and whether the size matters to PIWI/pi RNA function.Here we report that a PIWI-Ins module unique in PIWIclade proteins helps define the length of pi RNAs.Deletion of PIWI-Ins in Miwi shifts MIWI to load with shorter pi RNAs and causes spermiogenic failure in mice,demonstrating the functional importance of this regulatory module.Mechanistically,we show that longer pi RNAs provide additional complementarity to target m RNAs,thereby enhancing the assembly of the MIWI/e IF3f/Hu R super-complex for translational activation.Importantly,we identify a c.1108C>T(p.R370W)mutation of HIWI(human PIWIL1)in infertile men and demonstrate in Miwi knock-in mice that this genetic mutation impairs male fertility by altering the property of PIWI-Ins in selecting longer pi RNAs.These findings reveal a critical role of PIWI-Ins-ensured longer pi RNAs in fine-tuning MIWI/pi RNA targeting capacity,proven essential for spermatid development and male fertility.

Putting things in place for fertilization： discovering roles for importin proteins in cell fate and spermatogenesis

Importin proteins were originally characterized for their central role in protein transport through the nuclear pores, the only intracellular entry to the nucleus. This vital function must be tightly regulated to control access by transcription factors and other nuclear proteins to genomic DNA, to achieve appropriate modulation of cellular behaviors affecting cell fate. Importin-mediated nucleocytoplasmic transport relies on their specific recognition of cargoes, with each importin binding to distinct and overlapping protein subsets. Knowledge of importin function has expanded substantially in regard to three key developmental systems： embryonic stem cells, muscle cells and the germ line. In the decade since the potential for regulated nucleocytoplasmic transport to contribute to spermatogenesis was proposed, we and others have shown that the importins that ferry transcription factors into the nucleus perform additional roles, which control cell fate. This review presents key findings from studies of mammalian spermatogenesis that reveal potential new pathways by which male fertility and infertility arise. These studies of germline genesis illuminate new ways in which importin proteins govern cellular differentiation, includ ng v a d rect ng proteins to d st nct ntrace ular compartments and by determining cellular stress responses.

BET bromodomain inhibitor JQ1 regulates spermatid development by changing chromatin conformation in mouse spermatogenesis

As a BET bromodomain inhibitor, JQ1 has been proven have efficacy against a number of different cancers. In terms of male reproduction, JQ1 may be used as a new type of contraceptive, since JQ1 treatment in male mice could lead to germ cell defects and a decrease of sperm motility, moreover, this effect is reversible. However, the mechanism of JQ1 acting on gene regulation in spermatogenesis remains unclear. Here, we performed single-cell RNA sequencing (scRNA-seq) on mouse testes treated with JQ1 or vehicle control to determine the transcriptional regulatory function of JQ1 in spermatogenesis at the single cell resolution. We confirmed that JQ1 treatment could increase the numbers of somatic cells and spermatocytes and decrease the numbers of spermatid cells. Gene Ontology (GO) analysis demonstrated that differentially expressed genes which were down-regulated after JQ1 injection were mainly enriched in “DNA conformation change” biological process in early developmental germ cells and “spermatid development” biological process in spermatid cells. ATAC-seq data further confirmed that JQ1 injection could change the open state of chromatin. In addition, JQ1 could change the numbers of accessible meiotic DNA double-stranded break sites and the types of transcription factor motif that functioned in pachytene spermatocytes and round spermatids. The multi-omics analysis revealed that JQ1 had the ability to regulate gene transcription by changing chromatin conformation in mouse spermatogenesis, which would potentiate the availability of JQ1 in male contraceptive.