Effects of selenium deficiency on sperm morphology and spermatocyte chromosomes in mice

Sperm morphology and spermatocyte chromosomes were examined in mice maintained on a Torula yeast diet for 5 weeks. In the selenium-deficient group, the proportion of abnormal sperm was high, ranging from 6.8% to 49.6%, while in the control group it ranged from only 4.0% to 15.0%. The most frequently occurring abnormalities in sperm shape were in the sperm head. There was also a tendency for abnormalities in other regions (neck, midpiece and tail) to be increased. However, in metaphase-I spermatocytes, the frequencies of various types of abnormal chromosomes (univalent chromosomes, translocations and structural anomalies) did not differ between the selenium-deficient and control groups. These findings indicate that selenium may be an essential constituent for spermatogenesis in mice.     

KLC3 is involved in sperm tail midpiece formation and sperm function

Kinesin light chain 3 (KLC3) is the only known kinesin light chain expressed in post-meiotic male germ cells. We have reported that in rat spermatids KLC3 associates with outer dense fibers and mitochondrial sheath. KLC3 is able to bind to mitochondria in vitro and in vivo employing the conserved tetratrico-peptide repeat kinesin light chain motif. The temporal expression and association of KLC3 with mitochondria coincides with the stage in spermatogenesis when mitochondria move from the spermatid cell periphery to the developing midpiece suggesting a role in midpiece formation. In fibroblasts, expression of KLC3 results in formation of large KLC3 aggregates close to the nucleus that contain mitochondria. However, the molecular basis of the aggregation of mitochondria by KLC3 and its role in sperm tail midpiece formation are not clear. Here we show that KLC3 expression from an inducible system causes mitochondrial aggregation within 6h in a microtubule dependent manner. We identified the mitochondrial outer membrane porin protein VDAC2 as a KLC3 binding partner. To analyze a role for KLC3 in spermatids we developed a transgenic mouse model in which a KLC3ΔHR mutant protein is specifically expressed in spermatids: this KLC3 mutant protein binds mitochondria and causes aggregate formation, but cannot bind outer dense fibers. Male transgenic mice display significantly reduced reproductive efficiency siring small sized litters. We observed defects in the mitochondrial sheath structure in a number of transgenic spermatids. Transgenic males have a significantly reduced sperm count and produce spermatozoa that exhibit abnormal motility parameters. Our results indicate that KLC3 plays a role during spermiogenesis in the development of the midpiece and in the normal function of spermatozoa.     

Fine structure of the spermatozoon of the viviparous teleost, Cymatogaster aggregata

The approximately 50 μm long sperm of Cymatoguster aggregata is composed of an elongate head (4 μm), an elongate mitochondria1 midpiece (3.5 μm) and a tail flagellum (roughly 40 μm). The sperm lacks an acrosome. Contained within depressions on one surface of the compressed head are a proximal centriole and a distal centriole separated by an electron dense, intercentriolar body. The anterior portion of the tail flagellum originates at the basal body (distal centriole) and is contained within an extracellular, flagellar tunnel within the mitochondria1 midpiece. The morphological similarity of C. uggregutu sperm to sperm of other internally fertilizing fishes supports the hypothesis that spermatozoan morphology is related to the mode of fertilization and that an elongate head and midpiece are specializations for internal fertilization.     

Intra-individual variation in sperm tail length in murine rodents

In eutherian mammals, there are marked interspecific differences in sperm head shape and tail length. In a few species, sperm head variability occurs but intra-individual variation in sperm tail length has rarely been investigated or commented upon. Here, we ask the question: Do murine rodent species that have variable sperm head shapes exhibit greater intra-individual variation in sperm midpiece and total tail lengths than closely related species where little, or no, sperm head variability occurs? From three separate lineages, we selected three pairs of murine rodents, one of which has monomorphic, and the other variable, sperm head shape. These were from southern Asia the bandicoot rats Bandicota bengalensis and Bandicota indica , from southern Africa the veld rats, Aethomys chrysophilus and Aethomys ineptus and from Australia the fawn hopping mouse Notomys cervinus and the spinifex hopping mouse Notomys alexis . Cauda epididymal sperm smears were prepared and sperm midpiece and total tail lengths were determined. A linear mixed-effects model was used to estimate intra-individual variance. The results showed that in all three species where there are variable sperm head shapes ( B. indica , A. ineptus and N. alexis ), statistically significantly greater intra-individual variability of sperm midpiece and total tail lengths occurs ( P <0.0001 in all cases). These species all have relatively smaller testes mass compared with the closely related species with monomorphic sperm populations. This suggests that depressed levels of intermale sperm competition may result in the occurrence of variability in not only the divergent sperm head shape but also in the length of the midpiece as well as that of the total length of the sperm tail.     

Fate of sperm tail components after incorporation into the hamster egg

The changes in the cytoplasmic organelles of sperm tail in golden hamster eggs fertilized in vivo were observed by electron microscopy. Eggs were obtained from oviducts of hamsters that had been superovulated and inseminated by injection of cauda epididymal spermatozoa into the uteri. In the egg cytoplasm 10 hours after insemination, some of the mitochondria of the spermatozoon midpiece had begun to swell, and a number of multivesicular bodies were observed surrounding the midpiece. The fibrous sheath of the principal piece quickly disappeared prior to the first cleavage, whereas the axoneme and outer dense fibers were unaltered. During the two-cell stage, numerous multivesicular bodies gathered around the midpiece and fused with the mitochondria. The heterophagic vacuoles thus formed then gradually separated from the axial fibers. The outer dense fibers were disarranged and partially torn into small segments; then they seemed to dissociate into substructural granular components. The axonemal microtubules had begun to swell but were still present in the two blastomeres. It is indicated from these observations that at least the mitochondria of the tail constituents carried into the oocyte are digested into small molecular elements by the multivesicular bodies and are possibly distributed as nutrients for the blastomeres during the early stage of development.     

Sperm mitochondria of patients with normal sperm motility and with asthenozoospermia: morphological and functional study

Studies were performed on ejaculated human spermatozoa (32 subjects with normal sperm motility and 25 subjects with low sperm motility). Morphology of sperm midpiece was evaluated in light, fluorescent and transmission or scanning electron microscope. Changes in mitochondrial membrane potential (delta(psi)m) and mass of mitochondria were analysed by flow cytometry using mitochondrial specific probes JC-1 and Mito Tracker Green FM. Moreover, oxidoreductive capability of sperm mitochondria was assessed using cytochemical reaction for NADH-dependent dehydrogenases. In flow cytometry analysis of JC-1-stained spermatozoa, two asthenozoospermic subpopulations were distinguished: patients with a high percentage (76 +/- 11%, 13 subjects) and patients with a low percentage (29 +/- 14%,12 subjects) of spermatozoa with functional-polarized mitochondria with high delta(psi)m. Our microscopic investigations of spermatozoa of seven asthenozoospermic patients reveal that the deformed and unusually thickened sperm midpieces (50-70% of cells), occasionally with persistent cytoplasmic droplet, contain supernumerary mitochondria with normal substructure, full oxidoreductive capability and high delta(psi)m. The midpiece deformations cause nonprogressive movement or immotility. They can also appear in smaller number of spermatozoa (5-35% of cells) in patients with normal sperm motility. Moreover, in three cases of asthenozoospermia midpiece malformations were accompanied by abnormal morphology of outer dense fibers and axoneme. The cytochemical, fluorescence and SEM studies showed the absence of midpieces in many (60-80%) spermatozoa in some other cases of asthenozoospermia. The morphological observations corresponded with flow cytometry analysis of Mito Tracker Green FM-stained spermatozoa. Our results suggest that in some cases of asthenozoospermia the sperm mitochondria can be functionally active and display high delta(psi)m in large number of cells. The results may suggest that asthenozoospermia does not necessarily result from energetic disturbances of sperm mitochondria. The low sperm motility may be associated with deformations of the mitochondrial sheath containing functional mitochondria. The combination of fluorescence microscopy and flow cytometry with electron microscopic investigations is a sensitive, precise and comprehensive examination which helps discover sperm abnormalities responsible for asthenozoospermia.     

Progressive defects observed in mouse sperm during the course of three generations of selenium deficiency

Three successive generations of mice were fed a Torula yeast based Se-deficient diet with or without 0.1 ppm Se in the drinking water. The Se-deficient mice, in the course of three generations, showed a decrease in body weight, testis weight, epididymal weight, and sperm production. The percentage of morphologically abnormal sperm increased in successive generations. The majority of sperm defects were found in the midpiece region of the tail. Many of these aberrant sperm were motile. A progressive decrease in fertility was noted during the first two generations of Se deficiency. This system confirms the essential role of Se in spermatogenesis and provides a model for the evaluation of the primary effect of Se deprivation on the structural development of sperm.     

Ultrastructure of spermiogenesis and spermatozoa of four Oriopsis species (Sabellinae, Sabellidae, Polychaeta)

The ultrastructure of sperm from four species of Oriopsis is described. Males of Oriopsis bicoloris produce sperm with an elongate nucleus divided into four rods, connected proximally, and four long mitochondria lying along the nucleus. The axoneme runs in the middle of the nuclear and mitochondria1 rods. Oriopsis brevicollaris males have sperm with an elongate nucleus and long midpiece comprised of four mitochondria wrapped around the axoneme. Males of O. mobilis have sperm with an elongate nucleus and long midpiece similar to that of O. brevicollaris , although the midpiece is much longer. Oriopsis dentata males havc sperm with a flattened head, similar to those of mammals, though the midpiece is simple and the axoneme free. The variability of reproductive mechanisms within the Polychaeta is discussed with reference to the elucidation of the function of various sperm morphologies. The implications for the taxonomy and systematics of Oriopsis , and the Sabellidae as a whole, are also discussed. It is concluded that Oriopsis is not monophyletic and examination of reproductive structures in other small sabellids is required. Study on reproduction in the type species, O. armandi , is needed to establish the reproductive method of Oriopsis and so allow revision of this genus.     

Influence of seminal plasma on fresh and post-thaw parameters of stallion epididymal spermatozoa

Fresh and post-thaw parameters (motility, morphology and viability) of stallion epididymal spermatozoa that have been and have not been exposed to seminal plasma were evaluated, and directly compared to fresh and post-thaw parameters of ejaculated spermatozoa. Six sperm categories of each stallion (n=4) were evaluated for motility, morphology and viability. These categories were fresh ejaculated spermatozoa (Fr-E), fresh epididymal spermatozoa that had been exposed to seminal plasma (Fr-SP+), fresh epididymal spermatozoa that had never been exposed to seminal plasma (Fr-SP-), frozen-thawed ejaculated spermatozoa (Cr-E), frozen-thawed epididymal spermatozoa that had been exposed to seminal plasma prior to freezing (Cr-SP+) and frozen-thawed epididymal spermatozoa that had never been exposed to seminal plasma (Cr-SP-). Results show that seminal plasma stimulates initial motility of fresh epididymal stallion spermatozoa while this difference in progressive motility is no longer present post-thaw; and that progressive motility of fresh or frozen-thawed ejaculated stallion spermatozoa is not always a good indicator for post-thaw progressive motility of epididymal spermatozoa. This study shows that seminal plasma has a positive influence on the incidence of overall sperm defects, midpiece reflexes and distal cytoplasmic droplets in frozen-thawed stallion epididymal spermatozoa while the occurance of midpiece reflexes is likely to be linked to distal cytoplasmic droplets. Furthermore, seminal plasma does not have an influence on viability of fresh and frozen-thawed morphologically normal epididymal spermatozoa. We recommend the retrograde flushing technique using seminal plasma as flushing medium to harvest and freeze stallion epididymal spermatozoa.     

蓝尾石龙子精子的超微结构

蓝尾石龙子(Eumeces elegans)附睾以2．5％戊二醛和1％锇酸双重固定,按常规制作超薄切片,用H-600透射电镜研究观察精子的超微结构。精子由头部和尾组成,头部由顶体复合体和核组成,尾由颈段、中段、主段和末段组成。头部的顶体囊前部扁平,分为皮质和髓质,顶体下锥由类结晶状的顶体下物质组成,穿孔器顶端尖,、穿孔器基板塞子状,细胞核延长,核内小管缺,核伸展部前端具一电子透明区,核肩圆,核陷窝锥形。颈段具片层结构,近端中心粒和远端中心粒的长轴呈直角,9束外周致密纤维与远端中心粒相应的9束三联微管相联,向后与轴丝相应的9束双联微管相联,中央纤维与2个中央单微管相联。中段短,含有线状嵴的柱状线粒体,由连续的规则小卵状或小梯形致密体组成线粒体间的环状结构,纤维鞘伸入中段,终环紧贴于细胞膜的内表面。线粒体与环状结构的模式为：rs1／mi1,rs2／mi2,rs3／mi3,rs4／mi4,横切面上每圈线粒体数目为10个。主段前面部分具薄的细胞质颗粒区。纤维3和8至主段前端消失。轴丝复合体呈“9 2”型。蓝尾石龙子精子超微结构与已描述的石龙子科种类比较发现,与蜓蜥群和胎生群的石龙子相似;但没有发现石龙子科精子的独征。     

Ultrastructural observations of cryoinjury in kangaroo spermatozoa

Macropod spermatozoa have proven difficult to cryopreserve such that empirical studies using high concentrations of glycerol and/or DSMO have resulted in only 10% post-thaw motility. We examined the ultrastructure and freeze-fracture of caput and cauda epididymal macropod spermatozoa at 35, 4 degrees C and following cryopreservation with and without 20% glycerol. The addition of 20% glycerol resulted in significant damage to the sperm plasma membrane and mitochondria compared to no glycerol at the same temperatures (P<0.05). Following cryopreservation, 20% glycerol significantly improved the preservation of the cauda epididymal sperm plasma membrane and mitochondria and reduced the incidence of axonemal damage and axonemal spaces. For caput epididymal spermatozoa, glycerol only improved the preservation of the plasma membrane following cryopreservation (P<0.05). Freeze fracture microscopy revealed a pattern of helically wound intramembranous particles in the plasma membrane over the fibre network of the mid piece of the sperm tail. The fibre network is an interconnecting cytoskeletal structure found underneath the plasma membrane of the kangaroo sperm midpiece and is thought to add rigidity to the proximal portion of the sperm tail. After thawing, the plasma membrane was damaged such that this structure was missing in patches, and the helical rows of particles were mal-aligned. On the principal piece, particles were arranged randomly at physiological temperatures; however, upon cooling to 4 degrees C with 20% glycerol, the particles become aggregated. Once rewarmed (35 degrees C), particles over the principal piece resumed their random organisation. This finding is further evidence of a reversible phase transition of the macropod sperm plasma membrane during cooling that is not associated with a loss of motility or membrane integrity.     

Further studies on knockout mice lacking a functional dynein heavy chain (MDHC7). 2. A developmental explanation for the asthenozoospermia

Male mice had been previously generated in which the inner dynein arm heavy chain 7 gene (MDHC7) was disrupted. MDHC7-/- animals show asthenozoospermia and are sterile. Very few of their spermatozoa can achieve forward progression, but for those that can, we add here the information (1) that the three-dimensional aspects of their movement are normal; (2) that their maximum velocity is less than that of wild-type controls; and (3) that they are entirely unable to penetrate media of raised viscosity (25-4,000 cP). However, the large majority of the spermatozoa can achieve only a low amplitude vibration. In these sperm we find, using electron microscopy, that the outer dense fibres retain attachments to the inner surface of the mitochondria. Such attachments are present in normal epididymal mouse spermatozoa but are broken down as soon as the sperm become motile on release from the epididymis. The attachments are presumed to be essential during midpiece development and, afterwards, to require a threshold level of force to loosen them and so permit the sliding displacements necessary for normal bending. We presume that the disruption of the inner dynein arm heavy chain gene, MDHC7, means that there is insufficient force to overcome the attachments, for all but a few spermatozoa.     

Ultrastructural changes in spermatozoa of the Brush-tailed possum,Trichosurus vulpecula (Marsupialia), during epididymal transit

Summary During epididymal transit, a fibre network and an array of vesicles develop in the posterior two-thirds of the midpiece in sperm of the Brush-tailed possum, Trichosurus vulpecula. The fibre network is developed by the time the sperm reach the corpus epididymidis, and is composed of evenly spaced, helically arranged fibres lying immediately beneath the plasma membrane. The angle of these fibrous helices is always counter to that of the underlying mitochondrial helix. Separating the fibre network from the mitochondria is a layer of granular material which develops at the same time, and over the same length of the midpiece as the fibre network.A somewhat tenuous fibre network is found between the fibrous sheath and plasma membrane in the anterior principal piece of sperm from all regions of the epididymis.The array of vesicles is developed by the time sperm reach the cauda epididymidis. The vesicles resemble pinocytotic vesicles; some appear as invaginations of the plasma membrane, and are open to the medium surrounding the spermatozoon by a narrow neck, while others are entirely enclosed within the spermatozoon, and lie at varying distances between the plasma membrane and the layer of granular material.The authors would like to acknowledge the assistance of Miss Dianne Higginbotham of the Electron Microscope Unit, University of Sydney for expert assistance with the scanning electron microscopy. Also to Dr. D.J.H. Cockayne, Director of the Electron Microscope Unit, for use of the transmission electron microscope facilities.     

Characterization of a 23 kDa sperm-binding polypeptide of the golden hamster epididymis

A 23 kDa polypeptide has been identified on the flagellum of sperm obtained from the cauda epididymis of the golden hamster. A monospecific antiserum to the 23 kDa hamster polypeptide was prepared and used to study its distribution on sperm, in the epididymis, and in epididymal fluid. In the cauda, the polypeptide is found on the midpiece and endpiece of the sperm tail, in detergent extracts of sperm, and in epididymal luminal fluid-enriched fractions. It is not present on sperm or in luminal fluid-enriched fractions from the caput epididymis. Immunocytochemical staining of epididymal tissue has demonstrated the 23 kDa polypeptide in the Golgi region of the principal cells of the proximal cauda and on sperm in the tubules of this segment and in tubules distal to it. Antiserum to the 23 kDa golden hamster polypeptide cross-reacts with sperm from rats and Chinese hamsters, but not with sperm from rabbits, cattle, mice, and guinea pigs. The antigen is localized to the tail of sperm obtained from the cauda of the rat and from the distal caput of the Chinese hamster. Immunoblots of detergent extracts of sperm and luminal fluid-enriched fractions from these two species reveal a 26 dKa polypeptide that is immunologically related to the golden hamster polypeptide.     

Sperm abnormalities in the PL/J mouse strain: A description and proposed mechanism for malformation

A high frequency (42%) of sperm from the inbred homozygous mouse strain PL/J are abnormal. Head shape abnormalities occur in 15% of the total sperm; and 27% of the sperm are headless, with the mitochondria condensed into a mass at the caudal end of the midpiece region. The sperm without heads exhibit relatively normal motility. Electron microscopy of the testes indicates that some of the abnormal sperm in PL/J males result from a failure of the paired centrioles to attain a normal position on the nucleus opposite the acrosome prior to implantation, or to attach at all. The centrioles that are not attached to the nuclear envelope can differentiate to form the principal piece and midpiece region. The frequency of headless variants in heterozygous F₁ indicates that the trait is mainly recessive. The offspring from the backcross of the F₁ to homozygous PL/J parents did not give a clear-cut segregation pattern. The frequency of abnormal sperm in the F₁ and the backcross is higher when the female parent is a PL/J.     

低硒小鼠模型的建立及低硒对心肌的影响

目的建立低硒实验动物模型,观察低硒对心肌的影响。方法利用黑龙江地产酵母配制低硒小鼠饲料,使用配制的小鼠饲料喂养BALB/c幼鼠,经过4个月的喂养,测定血清、肝脏、心肌细胞的硒含量,观察心肌超微结构的变化,测定血清心肌酶的变化。结果利用黑龙江地产酵母配制的低硒饲料,硒含量为0.016 mg/kg,符合低硒标准。BALB/c鼠用该饲料喂养4个月,心肌、肝脏、血清硒含量分别为0.187 mg/kg、0.219 mg/kg、0.241mg/kg,符合低硒诊断标准。观察低硒鼠心肌超微结构,可见心肌细胞线粒体肿胀,细胞核出现了异型性,血清心肌酶较常硒鼠升高。结论利用黑龙江地产酵母成功配制了低硒饲料。经过低硒饲料饲养可建立低硒鼠模型。低硒可以引起BALB/c鼠心肌细胞损伤。     

Phospholipid hydroperoxide glutathione peroxidase: expression pattern during testicular development in mouse and evolutionary conservation in spermatozoa

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is a selenoprotein belonging to the family of glutathione peroxidases and has been implicated in antioxidative defense and spermatogenesis. PHGPx accounts for almost the entire selenium content of mammalian testis. In an attempt to verify the expression pattern of PHGPx, testes of mouse mutants with arrest at different stages of germ cell development and testes of mice at different ages were subjected to immunostaining with a monoclonal anti-PHGPx antibody. PHGPx was detected in Leydig cells of testes in all developmental stages. In the seminiferous tubuli, the PHGPx staining was first observed in testes of 21-day-old mice which is correlated with the appearance of the first spermatids. This result was confirmed when the testes of mutant mice with defined arrest of germ cell development were used. An immunostaining was observed in the seminiferous tubuli of olt/olt and qk/qk mice which show an arrest at spermatid differentiation. In Western blot analysis of proteins extracted from testes of mutant mice and from developing testes, two signals at 19- and 22-kDa were observed which confirm the existence of two PHGPx forms in testicular cells. In mouse spermatozoa, a subcellular localization of PHGPx and sperm mitochondria-associated cysteine-rich protein (SMCP) was demonstrated, indicating the localization of PHGPx in mitochondria of spermatozoa midpiece. For verifying the midpiece localization of PHGPx in other species, spermatozoa of Drosophila melanogaster, frog, fish, cock, mouse, rat, pig, bull, and human were used in immunostaining using anti-PHGPx antibody. A localization of PHGPx was found in the midpiece of spermatozoa in all species examined. In electronmicroscopical analysis, PHGPx signals were found in the mitochondria of midpiece. These results indicate a conserved crucial role of PHGPx during sperm function and male fertility.     

Variation in sperm morphology among Afrotropical sunbirds

Birds show considerable variation in sperm morphology. Closely related species and subspecies can show diagnostic differences in sperm size. There is also variation in sperm size among males within a population, and recent evidence from passerine birds suggests that the coefficient of inter‐male variation in sperm length is negatively associated with the level of sperm competition. Here we examined patterns of inter‐ and intra‐specific variation in sperm length in 12 species of sunbird (Nectariniidae) from Nigeria and Cameroon, a group for which such information is extremely limited. We found significant variation among species in sperm total length, with mean values ranging from 74 μm to 116 μm, placing these species within the short to medium sperm length range for passerine birds. Most of this variation was explained by the length of the midpiece, which contains the fused mitochondria and is an important structure for sperm energetics. Relative midpiece length was negatively correlated with the coefficient of inter‐male variation in sperm total length across species, suggesting that sperm competition may have selected for greater midpiece length in this group. We also mapped sperm lengths onto a time‐calibrated phylogeny and found support for a phylogenetic signal in all sperm length components, except head length. A test of various evolutionary or tree transformation models gave strongest support for the Brownian motion model for all sperm components, i.e. divergences were best predicted by the phylogenetic distance between lineages. The coefficients of inter‐male variation in sperm total length indicate that sperm competition is high but variable among sunbird species, as is the case with passerine birds at large.