Spermatid Mitochondria Research Articles

Expression dynamics indicate the involvement of SPG7 in the reproduction and spermiogenesis of Phascolosoma esculenta

Spastic paraplegia 7 (SPG7) is an m-AAA protease subunit involved in mitochondrial morphology and physiology. However, its function in animal reproduction is yet to be evaluated. In this study, its molecular features, subcellular localization, and expression dynamics were investigated to analyze its potential function in the reproduction of male Phascolosoma esculenta, an economically important marine species in China. The full-length cDNA of P. esculenta spg7 (Pe-spg7) measures 3053 bp and encodes an 853-amino acid protein (Pe-SPG7). Pe-SPG7 includes two transmembrane domains, an AAA domain and a proteolytic domain. Amino acid sequence alignment revealed that SPG7 was conserved during evolution. The mRNA and protein expression of spg7 indicated its involvement in reproduction. Its expression was the highest in coelomic fluid, where spermatids develop, and it was significantly higher in the breeding stage than in the nonbreeding stage. SPG7 was mainly found in the mitochondria of spermatids in the coelomic fluid, indicating that it functions in this organelle in spermatids. Immunofluorescence experiments showed that SPG7 was expressed and colocalized in the mitochondria during spermiogenesis, suggesting its involvement in P. esculenta spermiogenesis. Therefore, SPG7 may participate in spermiogenesis by functioning in the mitochondria and regulate the reproduction of male P. esculenta. This study provided insights into the function of SPG7 in animal reproduction and P. esculenta gametogenesis.

Targeted mutation and inactivation of the kinesin light chain 3 protein-encoding gene have no impact on mouse fertility†.

The kinesin light chain 3 protein (KLC3) is the only member of the kinesin light chain protein family that was identified in post-meiotic mouse male germ cells. It plays a role in the formation of the sperm midpiece through its association with both spermatid mitochondria and outer dense fibers (ODF). Previous studies showed a significant correlation between its expression level and sperm motility and quantitative semen parameters in humans, while the overexpression of a KLC3-mutant protein unable to bind ODF also affected the same traits in mice. To further assess the role of KLC3 in fertility, we used CRISPR/Cas9 genome editing in mice and investigated the phenotypes induced by the invalidation of the gene or of a functional domain of the protein. Both approaches gave similar results, i.e. no detectable change in male or female fertility. Testis histology, litter size and sperm count were not altered. Apart from the line-dependent alterations of Klc3 mRNA levels, testicular transcriptome analysis did not reveal any other changes in the genes tested. Western analysis supported the absence of KLC3 in the gonads of males homozygous for the inactivating mutation and a strong decrease in expression in males homozygous for the allele lacking one out of the five tetratricopeptide repeats. Overall, these observations raise questions about the supposedly critical role of this kinesin in reproduction, at least in mice where its gene mutation or inactivation did not translate into fertility impairment.

Glycerol kinase-like proteins cooperate with Pld6 in regulating sperm mitochondrial sheath formation and male fertility

Spermatids undergo the final steps of maturation during spermiogenesis, a process that necessitates extensive rearrangement of organelles such as the mitochondria. Male infertility has been linked to mitochondrial disorder, for example, hypospermatogenesis and asthenozoospermia. However, the mechanisms that regulate mitochondrial dynamics during spermiogenesis remain largely unknown. We found the glycerol kinase (Gyk)-like proteins glycerol kinase-like 1 (Gykl1) and glycerol kinase 2 (Gk2) were specifically localized to the mitochondria in spermatids. Male mice deficient in either Gykl1 or Gk2 were infertile due to dysfunctional spermatozoa, which exhibited unregulated ATP production, disordered mitochondrial sheath formation, abnormal mitochondrial morphology, and defective sperm tail. We demonstrated that the unique C-terminal sequences found in Gykl1 and Gk2 mediated their targeting to the mitochondrial outer membrane. Furthermore, both Gykl1 and Gk2 could interact with Pld6 (MitoPLD) and induce Pld6 and phosphatidic acid (PA)-dependent mitochondrial clustering in cells. Taken together, our study has revealed previously unsuspected functions of Gyk-like proteins in spermiogenesis, providing new insight into the potential mechanisms that lead to spermatozoa dysfunction and male infertility.

A Splice Variant of Centrosomin Converts Mitochondria to Microtubule-Organizing Centers

Non-centrosomal microtubule organizing centers (MTOCs) direct microtubule (MT) organization toexert diverse cell-type-specific functions. In Drosophila spermatids, the giant mitochondria provide structural platforms for MT reorganization to support elongation of the extremely long sperm. However, the molecular basis for this mitochondrial MTOC and other non-centrosomal MTOCs has not been discerned. Here we report that Drosophila centrosomin (cnn) expresses two major protein variants: the centrosomal form (CnnC) and a non-centrosomal form in testes (CnnT). CnnC is established as essential for functional centrosomes, the major MTOCs in animal cells. We show that CnnT is expressed exclusively in testes by alternative splicing and localizes to giant mitochondria in spermatids. In cell culture, CnnT targets to the mitochondrial surface, recruits the MT nucleator γ-tubulin ring complex (γ-TuRC), and is sufficient to convert mitochondria to MTOCs independent of core pericentriolar proteins that regulate MT assembly at centrosomes. We mapped two separate domains in CnnT: one that is necessary and sufficient to target it to mitochondria and another that is necessary and sufficient to recruit γ-TuRCs and nucleate MTs. In elongating spermatids, CnnT forms speckles on the giant mitochondria that are required to recruit γ-TuRCs to organize MTs and support spermiogenesis. This molecular characterization of the mitochondrial MTOC defines a minimal molecular requirement for MTOC generation and implicates the potent role of Cnn (or its related) proteins in the direct regulation of MT assembly and organization of non-centrosomal MTOCs.

Effects of endocrine disruptors on reproduction in viviparous teleosts with intraluminal gestation

Many water systems worldwide are affected by pollutants, including potential endocrine disruptors (EDs). Most studies on the effects of EDs on fish reproduction have focused on oviparous species. However, some important groups of fishes are not oviparous and there is scarce information about how EDs affect species with alternative reproductive modes. Goodeinae is a viviparous matrotrophic subfamily with intraluminal gestation (IG), where transfer of nutrients occurs and embryos develop inside the ovarian cavity. Goodeinae is endemic to the Mexican Central Plateau, an area affected by potential EDs, including 2,4-dichlorophenoxyacetic acid (2,4-D). This review synthesizes the available information about EDs in viviparous teleosts with IG and performs a case study on the effects of 2,4-D on gonadal structure of two Goodeinae species. We hypothesized that individuals exposed to 2,4-D might show altered gonadal structure. The available information included effects on gene expression, sexual differentiation, gametogenesis, secondary sexual characteristics, and sexual behavior. Knowledge gaps persisted on the effects of EDs on viviparous teleosts with IG. Holistic approaches are needed to understand the mechanisms underlying endocrine disruption effects. Field studies are needed to evaluate the consequences of EDs on wild populations. The case study revealed histological alterations in oocytes, spermatogonia, and sperm cysts in fishes exposed to 2,4-D. Ultrastructurally, gonads exhibited alterations in oocyte mitochondrial and nuclear membranes, and in spermatid mitochondria. The observed changes could be related to 2,4-D exposure, which may affect species reproduction in their natural environment. Matrotrophic viviparous teleosts with IG may serve as models to explore endocrine disruption.

Failure of acrosome formation and globozoospermia in the wobbler mouse, a Vps54 spontaneous recessive mutant

The acrosome is a unique organelle that plays an important role at fertilization and during sperm morphogenesis and that is absent in globozoospermia, an inherited infertility syndrome in humans. At the light of recent experimental evidence, the acrosome is considered a lysosome-related organelle to whose biogenesis both the endocytic and biosynthetic pathways contribute. Vps54 is a vesicular sorting protein involved in the retrograde traffic; the recessive Vps54(L967Q) mutation in the mouse results in the wobbler phenotype, characterized by motor-neuron degeneration and male infertility. Here we have investigated the spatio-temporal occurrence/progression of the wobbler fertility disorder starting from mice at post-natal day 35, the day of the first event of spermiation. We show that the pathogenesis of wobbler infertility originates at the first spermiogenetic wave, affecting acrosome formation and sperm head elongation. Vps54(L967Q)-labeled vesicles, on the contrary of the wild-type Vps54-labeled ones, are not able to coalesce into a larger vesicle that develops, flattens and shapes to give rise to the acrosome. Evidence that it is the malfunctioning of the endocytic traffic to hamper the development of the acrosome comes out from the study on UBPy. UBPy, a deubiquitinating enzyme, is a marker of acrosome biogenesis from the endocytic pathway. In wobbler spermatids UBPy-positive endosomes remain single, scattered vesicles that do not contribute to acrosome formation. As secondary defect of wobbler spermiogenesis, spermatid mitochondria are misorted; moreover, with the progression of the age/disease also Sertoli-germ cell adhesions are compromised suggesting a derailment in the endocytic route that underlies their restructuring.

Drosophila mitoferrinis essential for male fertility: evidence for a role of mitochondrial iron metabolism during spermatogenesis

BackgroundMammals and Drosophila melanogaster share some striking similarities in spermatogenesis. Mitochondria in spermatids undergo dramatic morphological changes and syncytial spermatids are stripped from their cytoplasm and then individually wrapped by single membranes in an individualization process. In mammalian and fruit fly testis, components of the mitochondrial iron metabolism are expressed, but so far their function during spermatogenesis is unknown. Here we investigate the role of Drosophila mitoferrin (dmfrn), which is a mitochondrial carrier protein with an established role in the mitochondrial iron metabolism, during spermatogenesis.ResultsWe found that P-element insertions into the 5'-untranslated region of the dmfrn gene cause recessive male sterility, which was rescued by a fluorescently tagged transgenic dmfrn genomic construct (dmfrnvenus). Testes of mutant homozygous dmfrnSH115 flies were either small with unorganized content or contained some partially elongated spermatids, or testes were of normal size but lacked mature sperm. Testis squashes indicated that spermatid elongation was defective and electron micrographs showed mitochondrial defects in elongated spermatids and indicated failed individualization. Using a LacZ reporter and the dmfrnvenus transgene, we found that dmfrn expression in testes was highest in spermatids, coinciding with the stages that showed defects in the mutants. Dmfrn-venus protein accumulated in mitochondrial derivatives of spermatids, where it remained until most of it was stripped off during individualization and disposed of in waste bags. Male sterility in flies with the hypomorph alleles dmfrnBG00456 and dmfrnEY01302 over the deletion Df(3R)ED6277 was increased by dietary iron chelation and suppressed by iron supplementation of the food, while male sterility of dmfrnSH115/Df(3R)ED6277 flies was not affected by food iron levels.ConclusionsIn this work, we show that mutations in the Drosophila mitoferrin gene result in male sterility caused by developmental defects. From the sensitivity of the hypomorph mutants to low food iron levels we conclude that mitochondrial iron is essential for spermatogenesis. This is the first time that a link between the mitochondrial iron metabolism and spermatogenesis has been shown. Furthermore, due to the similar expression patterns of some mitochondrial iron metabolism genes in Drosophila and mammals, it is likely that our results are applicable for mammals as well.

Stage-specific enhanced expression of mitochondrial fusion and fission factors during spermatogenesis in rat testis

The mitochondrial fusion factors mitofusins 1 and 2 (Mfn1 and Mfn2) and the fission factor dynamin-related protein 1 (Drp1) were found to be highly expressed in the pubertal and adult rat testis by Northern blot analysis. Immunohistochemistry using specific antisera to Mfn2 and Drp1 revealed a pronounced expression of the fusion and fission factors in the round and elongating spermatids in the seminiferous tubules, suggesting that at precise steps of spermiogenesis (i.e., steps 8–12), spermatid mitochondria are rapidly homogenized by frequent fusion and division. Although physiological relevance of this phenomenon remains to be clarified, a role is proposed for it as an effective means of achieving complete and homogeneous ubiquitination of mitochondria, which has recently been demonstrated to be a mechanism for the elimination of paternal mitochondria during fertilization, based on the fact that the timing of expression of Mfn2 and Drp1 coincides well with that reported for a spermatid-specific ubiquitin-conjugating enzyme.

Complementary DNA cloning and characterization of rat spergen-1, a spermatogenic cell-specific gene-1, containing a mitochondria-targeting signal.

To elucidate the molecular mechanisms involved with spermiogenesis in testis, we performed differential display screening to isolate genes that are developmentally up-regulated during rat testis development. One of the cDNAs isolated by differential display was highly expressed in testis. Both reverse transcription-polymerase chain reaction and Northern blot analysis showed that the expression level of the gene developmentally increased. By screening the rat testis cDNA library, we successfully isolated rat cDNA clones encoding the entire open-reading frame of 462 base pairs coding a small protein of 154 amino acids. Because in situ hybridization revealed that the gene was specifically expressed in haploid spermatids in the rat seminiferous tubules, it was designated as spergen-1 (spermatogenic cell-specific gene-1). The recently opened database of the full-length mouse cDNA collection contains a mouse gene that is homologous to rat spergen-1. Subcellular fractionation followed by immunoblot analysis revealed that spergen-1 protein was associated with mitochondria. The transfection experiments performed in COS-7 cells suggested that spergen-1 has a N-terminal mitochondria-targeting signal. We suggest that spergen-1 might be involved in spermiogenesis by transiently associating with spermatid mitochondria.

Selective and Continuous Elimination of Mitochondria Microinjected Into Mouse Eggs From Spermatids, but Not From Liver Cells, Occurs Throughout Embryogenesis

Exclusion of paternal mitochondria in fertilized mammalian eggs is very stringent and ensures strictly maternal mtDNA inheritance. In this study, to examine whether elimination was specific to sperm mitochondria, we microinjected spermatid or liver mitochondria into mouse embryos. Congenic B6-mt(spr) strain mice, which are different from C57BL/6J (B6) strain mice (Mus musculus domesticus) only in possessing M. spretus mtDNA, were used as mitochondrial donors. B6-mt(spr) mice and a quantitative PCR method enabled selective estimation of the amount of M. spretus mtDNA introduced even in the presence of host M. m. domesticus mtDNA and monitoring subsequent changes of its amount during embryogenesis. Results showed that M. spretus mtDNA in spermatid mitochondria was not eliminated by the blastocyst stage, probably due to the introduction of a larger amount of spermatid mtDNA than of sperm mtDNA into embryos on fertilization. However, spermatid-derived M. spretus mtDNA was eliminated by the time of birth, whereas liver-derived M. spretus mtDNA was still present in most newborn mice, even though its amount introduced was significantly less than that of spermatid mtDNA. These observations suggest that mitochondria from spermatids but not from liver have specific factors that ensure their selective elimination and resultant elimination of mtDNA in them, and that the occurrence of elimination is not limited to early stage embryos, but continues throughout embryogenesis.

Hst7: a male sterility mutation perturbing sperm motility, flagellar assembly, and mitochondrial sheath differentiation.

Hst7, a mouse hybrid sterility locus, has been mapped in close linkage to four other hybrid sterility loci, on proximal chromosome 17 within the t complex. When an allele (s) of Hst7 from the species Mus spretus is crossed into the Mus musculus domesticus (laboratory mouse) background, all male offspring are sterile. This occurs regardless of whether the Hst7 allele on the other chromosome 17 homolog is wild-type (+) or an allele (t) derived from the structurally variant homolog known as a t haplotype. Males of the Hst7 genotype s/+ produce sperm that, after release from the cauda epididymis, display moderate asthenospermia (straight line velocity = 49 +/- 4 microm/second, significantly lower than 102 +/- 7 microm/second for congenic wild-type controls) and normal morphology. However, males of the Hst7 genotype s/t produce sperm whose forward movement is below the detectable limit of the sperm motion analysis system. In addition, these sperm exhibit a variety of flagellar abnormalities, with about one third having normal heads attached to sacklike caudal regions. These sacks consist of membrane-delimited cytoplasm containing disorganized and/or misshapen axonemal elements. The remainder of the sperm from s/t mice have flagella with seemingly normal axonemes, although many exhibit enlarged areas of cytoplasm in their midpieces with extra layers of misaligned mitochondria. The s/t sperm mitochondria also display diffuse and vacuolated matrices reminiscent of meiotic germ cell and spermatid mitochondria. Observations of developing spermatids in the s/t testis reveal an unusual phenotype in which gaps of varying length occur in the mitochondrial wrapping of the midpiece. These data suggest that both the s and t alleles of Hst7 are defective alleles that contribute differentially to the severe asthenospermia phenotype and interact genetically to perturb flagellar development.

Sperm tail differentiation in the nudibranch mollusc Hypselodoris tricolor (gastropoda, opisthobranchia)

The sperm axoneme of Hypselodoris tricolor forms from a single centriole that is located initially beneath the plasma membrane and then migrates to the nuclear surface. A conspicuous centriolar adjunct-like formation is present in the neck of midspermatids, but it becomes very reduced at the end of spermiogenesis. In spermatocyte and spermatid mitochondria, intracristal bodies originate from the accumulation of a dense material in some cristae. From our observations and foregoing reports, it may be concluded that the process of sperm tail differentiation in opisthobranchs resembles that in pulmonates, whereas it differs in many respects from that occurring in prosobranchs. The appearance of intracristal bodies in modified mitochondria seems to be a special feature of spermatogenesis in the opisthobranchs that does not occur in the two other groups of gastropod molluscs.

Unusual structural changes in mitochondria during spermiogenesis in the oribatid mite, Hafenrefferia gilvipes (Acari).

During spermiogenesis in the oribatid mite, Hafenrefferia gilvipes, striking alterations in the structure and location of mitochondria in spermatids were observed. The structural changes include formation of a single, distended annular crista, which subsequently becomes tubular. Initially, the mitochondria are located in central regions of the cytoplasm, they then move first to the plasmalemma and subsequently to the nuclear envelope, and finally are engulfed in the nuclear material.

The spermatozoa and spermiogenesis of Perophora formosana (ascidia) with special reference to the striated apical structure and the filamentous structures in the mitochondrion

The spermatozoon of Perophora formosana is approximately 140 μ m long. It contains an extremely elongated filiform nucleus (ca. 90 μ m in length) and a single long mitochondrion (ca. 80 μ m in length) which is loosely wound around the nucleus as a lateral body. The anterior third of the head is coiled in a helical configuration. Filamentous structures approximately 10 nm thick are observed exclusively in the mitochondrial matrix. They are arranged parallel to one another along the long axis of the mitochondrion (lateral body). During spermiogenesis, they appear in the mitochondria of spermatids as the mitochondrion begins to elongate into the lateral body. They occur regularly aligned in a row in the matrix just beneath the inner membrane in the elongating region of the mitochondrion. They are first recognized as an accumulation of moderately electron-dense material. Possible functions for these filamentous structures in producing and maintaining mitochondrial shape changes are discussed. A striated apical structure, about 2 μ m in length, is found at the anterior end of the spermatozoon. It is composed of an electron-opaque substance in the form of dense striations. Each striation is approximately 7 nm thick. During spermiogenesis, an electron-dense material is deposited to form striated structures in the finger-like process at the apex of spermatids at almost the same stage that the filamentous structures appear in the mitochondrion. The role of the apical structure in the process of fertilization has not been determined.

The Cytoplasmic Inclusions of the Germ-Cells

(1) In the germinal epithelial cell of Limnæa stagnalis a Golgi apparatus is present. It is excentric and lies around the archoplasm, consisting of a number of rods (chondrioplasts or dictyosomes, dittosomi). (2) In the progerminative oöcyte mitochondria appear at a very early stage, but it is not known whether they exist in the indifferent germinal epithelial cell. The mitochondria lie at first in the zone of the Golgi apparatus. (3) The rods of the Golgi apparatus divide by binary fission and keep growing in number. The archoplasm upon which they repose gradually becomes divided into regions; these regions again subdivide till each Golgi rod is discrete and provided with a small part of the archoplasm, which it partly embraces. As each Golgi rod divides transversely the urchoplasm does not divide. The latter only divides by binary fission after it has become studded with a number of rodlets. (4) The Golgi apparatus gradually, from its excentric position, spreads completely throughout the egg cytoplasm, and in the full-grown oocyte is evenly distributed here and there in all parts of the egg cytoplasm. No segregation into special regions was noticed. (5) The mitochondria, from their excentric position near the Golgi apparatus, grow, divide, and spread evenly throughout the cytoplasm. The mitochondria are not all the same size; this is apparently due to the fact that some granules grow larger and more quickly than others. (6) While the egg mitochondria grow much larger than the spermatid mitochondria, it has been shown that the individual Golgi batonette or rodlet never grows beyond a certain size. The difference between the Golgi apparatus of a young oöcyte and a full-grown ovum lies, not iu the fact that the Golgi rods of the latter are individually very much larger (if at all) than those of the former, but mainly in the fact that the rods have increased enormously in number by binary fission. The individual Golgi rodlet of spermatid, young and old oöcyte are approximately subequal in size. (7) Deutoplasmagenesis, or the formation, of yolk, does not begin very early; the first yolk-discs make their appearance after the Golgi elements and mitochondria have progressed far in the process of spreading throughout the growing oöcyte. The yolk-discs do not appear in any special region of the cytoplasm, but eventually become evenly spread out. The discs at first are very small, and later grow some two or three times larger than the largest mitochondria. In Flemming-without-acetic (overnight) + iron-alum hæmatoxylin, yolk goes dark brownish-green, mitochondria black. (8) Towards the end of oögenesis the cytoplasm gradually becomes filled with vacuoles of a fluid nature. These leave a coagulum on fixation, but most of the vacuole is empty. The granules in the cytoplasm only abnormally lie in these vacuoles; yolk, Golgi elements and mitochondria lie in the trabeculas between the vacuoles.