[The occurrence of calmodulin in seminal plasma, sperm heads and sperm tails of cattle].
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Analysis of sperm quality in recombinant inbred mouse strains: correlation of sperm head shape with sperm abnormalities and with the incidence of supplementary spermatozoa in the perivitelline space.
Recombinant inbred strains were developed from reciprocal crosses between two inbred strains of mice (CBA and KE) differing in sperm head shape, proportion of normal sperm heads (CBA, 95%; KE, 78%) and fertilization efficiency (CBA, 100% of fertilized ova; KE, 72%), to determine whether the indices of sperm morphology and function were correlated. The following parameters were analysed in recombinant inbred and progenitor strains: index of sperm head shape (head width in the middle of its length/head length), percentage of abnormal sperm heads, percentage of spermatozoa with progressive movements, efficiency of penetration of hyaluronic acid polymer (Sperm Select) and percentage of fertilized ova after mating males from the tested strains with females from an outbred stock. For each investigated character, recombinant inbred strains, recombinant inbred EXCB and CBXE, could be divided into at least three categories: KE-like, CBA-like and intermediate, suggesting that in each case a minimum of two genes was involved. Recombinant strains derived from the reciprocal crosses of progenitor strains differed only with respect to the proportion of abnormal sperm heads, showing the involvement of the Y chromosome in determining this character. Penetration into Sperm Select was significantly correlated both with fertilization efficiency and sperm motility, while correlation with the proportion of normal spermatozoa did not reach the level of significance. However, there was a significant negative correlation of both sperm abnormalities and the incidence of supplementary spermatozoa in the perivitelline space with the index of sperm head shape.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Morphometric analysis of intact sperm heads and of sperm nuclei in the mouse.
A morphometric analysis of mouse sperm and of their nuclei was undertaken to investigate their respective post-testicular maturation. Sperm were collected from the testis, caput and cauda epididymidis, and their corresponding nuclei were isolated. Results indicate that the post-testicular maturation of sperm is distinct from that of nuclei. The size of intact sperm heads increases in the caput followed by a subsequent decrease in the cauda. In contrast, sperm nuclei decrease progressively in size. In general, a greater magnitude and number of alterations in intact heads and nuclei occur while in transit from the testis to the caput than during passage to the cauda epididymis. These results suggest that the period immediately following their release from the testis is crucial to the complete morphological maturation of sperm heads and nuclei.
Animals↗
Cryopreservation of human spermatozoa: correlations of ultrastructural sperm head configuration with sperm motility and ability to penetrate zona-free hamster ova.
Freezing and storage of human male gametes is associated with a reduction in the overall semen quality and establishment of pregnancy. This study was done to evaluate the integrity of sperm head ultrastructure (SHU) with computerized and vapor freezing. Comparisons were made between the effect of cryopreservatives glycerol (G) and dimethylsulfoxide (DMSO) on SHU. Twelve ejaculates from five proven fertile donors were studied with the use of routine semen analysis, zona-free hamster ova, and SHU. Both cooling processes, regardless of the preservative used, significantly reduced sperm function and the number of SH with intact plasma membranes. The staged cooling technique was substantially superior to vapor freezing in all parameters analyzed (P less than 0.01). G was less detrimental to the postthaw SHU than 1 M DMSO. A significant positive correlation (r = 0.98; P less than 0.01) was noted between the total number of intact SH and motile sperm. Computerized freezing in a G-diluted semen rendered a sperm environment that allowed the highest number of forms with intact SH membranes and with the best chances to penetrate zona-free hamster ova.
Animals↗
A quantitative study of sperm head ultrastructure in subfertile males with excess sperm precursors.
OBJECTIVE: To make an objective comparison between sperm head ultrastructure in fertile subjects and a subfertile cohort with an excess of immature germinal elements in the ejaculate. DESIGN: A quantitative analysis of ultrastructural features of the sperm head using transmission electron microscopy in the defined groups. PATIENTS: Ten men of proven fertility as controls and 10 subfertile subjects with a persistent excess of sperm precursors in the ejaculate were investigated. SETTING: The Infertility Clinic at Queen Charlotte's and Chelsea Hospital, London. MAIN OUTCOME MEASURES: Each individual in the study achieved a score for a range of previously defined features of sperm head ultrastructure. These scores provided the basis for comparison between fertile and subfertile subjects. RESULTS: Subfertile individuals were found to have motile sperm with significantly more hypoplastic, detached, and abnormally shaped acrosomes than fertile controls. Sperm nuclei in these subjects also contained significantly more intranuclear vacuoles and immature chromatin and were associated more commonly with cytoplasmic droplets than fertile controls. CONCLUSION: Men with an excess of sperm precursors in the ejaculate have motile sperm with a range of abnormalities involving the nucleus and acrosome to account for reduced functional competence.
Acrosome↗
Automated analysis of toxicant-induced changes in rat sperm head morphometry.
An automated sperm morphometry analysis (ASMA) instrument was developed to obtain measurements of toxicant-induced changes in rat sperm head morphometry. 1,3-dinitrobenzene (1,3-DNB), a testicular toxicant known to affect sperm parameters, was used. Twelve-week-old Sprague-Dawley rats were allocated to a control (C) and to two 1,3-DNB treatment groups (T1 = 15 mg/kg; T2 = 25 mg/kg). 1,3-DNB was administered as a single dose by gavage, and animals were sacrificed 22 days after exposure. Sperm were collected, and morphology smears were made by a standard method. One hundred sperm heads were digitized on each slide, and 8 metric measurements were automatically reported. All measurements tended to decrease in a dose-dependent manner with increasing doses of 1,3-DNB. All values for total width (Wa) and interior width (W(e)) were significantly different from one another. Wa/L was significantly larger in the control than in T1 or T2, and symmetry (S = Wb/Wa) was significantly smaller in the control than in T1 or T2. Multivariate cluster analysis revealed three subpopulations that were also visually distinct. Subpopulation no. 1 was normal, based on published descriptions of normal rat sperm; subpopulation no. 2 was abnormal with a flattened curvature and a normal length; subpopulation no. 3 was abnormal with a foreshortened length and a flattened curvature. T1 and T2 contained significantly more sperm from subpopulation no. 2 and no. 3 than C (T1 = 22% and T2 = 34% vs. C = 8% by cluster analysis). C had 93% normal sperm, while the treatments had 78% and 66%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Phagocytosis of sperm heads lacking the acrosomal process by unfertilized starfish oocytes.
In sperm of the starfish Asterina pectinifera, the acrosomal process and the flagellum were mechanically separated from the sperm head with a disperser. The sperm head fraction was then used to examine the direct interaction between the sperm head and the egg surface. Sperm heads lacking the acrosomal process and the flagellum did not fertilize oocytes, even after removal of the vitelline coat. Transmission electron microscopy showed that each denuded oocyte engulfed the sperm head without gamete membrane fusion. The sperm-engulfing response, similar to phagocytosis, was induced without the mediation of the acrosomal process. The present results suggest that the process of sperm incorporation consists of two independent events, acrosomal process-egg surface fusion and the phagocytotic movement of the egg surface.
Acrosome↗
Injection of sperm heads into immature rat oocytes.
When sperm heads are injected into rat oocytes at the germinal vesicle stage the sperm heads remain intact until the germinal vesicle breaks down. Then they decondense but do not form pronuclei. This observation agrees with the results reported for in vitro fertilization of immature oocytes from rats, mice and hamsters.
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Cholesteric organization of DNA in the stallion sperm head.
The fine structure of chromatin in sperm heads was investigated by different microscopic techniques: in vivo examinations in the polarizing microscope, thin sections and freeze-fracture replicas observed by transmission electron microscopy. The freeze-fractured chromatin appears to be formed of superimposed lamellae, each one 330 A thick. These lamellae are parallel to the flattening plane of the sperm head. This situation was already described in other mammal spermatozoa and in particular in the bull and the rabbit. This work presents a new interpretation of this lamellated aspect. The chromatin structure of these spermatozoa is that of a cholesteric liquid crystal. This structure resembles that of a plywood, made of superimposed layers of parallel filaments, but instead of having a right angle between two successive layers, there is a progressive rotation and similar orientation occurs at each 180 degrees rotation. The apparent lamellae result from cleavages due to freeze-fracture between levels of parallel filament orientation. The thickness of lamellae corresponds therefore to the half helicoidal pitch of the cholesteric liquid crystal. This model is consistent with our observations by polarizing microscopy. The lamellation is not visible in thin sections of stallion spermatozoa. There are however biochemical methods to decondense chromatin and we are able to observe this lamellation in sections normal to the flattening plane of sperm heads. The methods used classically to decondense the sperm chromatin lead to extremely varied aspects which are discussed, some of them being closely related to the structure of cholesteric liquid crystals.
Animals↗
Acrosomal reaction of thyone sperm. I. Changes in the sperm head visualized by high resolution video microscopy.
Structural changes inside the head of Thyone sperm undergoing the acrosomal reaction were followed with a high-resolution, differential interference contrast (DIC) video microscope. The beating sperm, adhering by their midpiece to the cover slip of a wedge perfusion chamber, were activated by a calcium ionophore (20 microM A23187) suspended in sea water containing 50 mM excess CaCl2. Before activation of the sperm, the acrosomal region appears as a 1.1-microM diameter sphere, slightly less dense than the rest of the sperm head. Upon activation, the acrosome pops; the acrosomal region suddenly swells and its refractive index drops. After approximately 1 s, a crescent-shaped periacrosomal cup appears behind the acrosomal vacuole. In the next several seconds, the cup loses more refractive index and expands forward as the acrosomal process extends. The acrosomal vacuole becomes smaller, but without appreciable drop in refractive index. These observations, coupled with the behavior of the extending acrosomal process reported in the companion paper, and in electron microscopy (EM) and early physiological studies, suggest that the acrosomal process is extended by a combination of the explosive polymerization of actin and the osmotic swelling of the periacrosomal cup material. In this paper, we also consider the meaning of the enhanced DIC image seen in the high-resolution video microscope, and discuss the reliability of measurements on small linear dimensions made with the DIC microscope.
Acrosome↗
Formation of the ventral hooks on the sperm head of the plains mouse, Pseudomys australis.
The sperm head of the plains mouse, Pseudomys australis, has three curved hooks projecting from its anterior margin. The two ventral hooks have previously been shown to consist largely of an extension of the subacrosomal material. To characterize further the structure and composition of the ventral hooks, we have examined their formation during spermiogenesis using transmission electron microscopy, silver staining, and actin localization with NBD-phallacidin. The ventral hooks develop as an extension of the perinuclear space and postacrosomal dense lamina on the anteroventral margin of the sperm head. Bundles of 6-nm-thick filaments appear in the core of each hook; these are probably actin filaments based on staining of the hooks with NBD-phallacidin. Just prior to spermiation, electron-dense material condenses in the core of the ventral hooks and concurrently in the perinuclear space in the remainder of the sperm head. The two ventral hooks thus appear to consist of a core of perinuclear material and actin filaments, which is enclosed by a continuation of the postacrosomal dense lamina.
Actins↗
In vitro study on the increase of trypsin resistance in the rat testicular sperm head.
The alteration of trypsin resistance in rat sperm head was investigated after the sperm were incubated in the culture media (199-Earele, 1% BSA) containing the inhibitors of protein synthesis. The head of non-incubated testicular sperm was easily digested by trypsin (1 mg/ml) while the head of the incubated sperm showed resistance to trypsin digestion after 3 hr-incubation. Trypsin resistance in epididymal sperm head did not change after the sperm was incubated. Neither cycloheximide (100 micrograms/ml) nor chloramphenicol (100 micrograms/ml) affected the trypsin resistance in the testicular sperm head.
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A simplified method for the isolation of sperm heads from the caput epididymidis of rodents and from ejaculated rabbit seminal plasma.
The present report describes a simple and rapid procedure to isolate highly purified sperm heads from mouse, rat and rabbit. The isolation procedure involves mincing of the caput epididymidis (CPE) of mice and rats to obtain a crude CPE suspension. This suspension is filtered to remove tubular fragments. The filtrate containing sperms and cellular contaminants is trypsinized, followed by sonication. To obtain rabbit sperm head suspension, ejaculated spermatozoa are briefly exposed to the cationic detergent CTAB-D, followed by trypsinization. Trypsin treatment of the suspension yields sperm heads and tails. The suspension containing sperm heads and tails is separated by discontinuous sucrose density gradient centrifugation. Highly purified and enriched sperm heads are recovered from the bottom of 2.5 M sucrose of the gradient. Enriched sperm heads contain less than 1% sperm tails.
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Immunocytochemical demonstration of kinetochores in human sperm heads.
CREST sera have been used to identify kinetochores in mature mammalian sperm heads. It is necessary to decondense the sperm heads artificially to permit access of the reagents before the kinetochores can be demonstrated immunocytochemically. The distribution of kinetochores in the sperm heads appears to be random. These results show that the kinetochore antigen recognized by the CREST sera used here is retained during spermiogenesis and is passed on to the zygote at fertilization.
Autoantibodies↗
Molecular nature of calicin, a major basic protein of the mammalian sperm head cytoskeleton.
In the mammalian sperm head the nucleus is tightly associated, in many species in its posterior part, with a large and dense nonfilamentous cytoskeletal structure, the calyx, whose major proteins are basic, representing a novel category of cytoskeletal element. Using specific antibodies, biochemical methods, and cDNA cloning we have characterized one of these calyx proteins, previously termed calicin, in bull and man. The polypeptide of 588 amino acids (Mr of 66,889; IEP 8.1) is very similar in the two species and is encoded by a approximately 2.2-kb mRNA that has been detected only in testis but not in any other tissue or cell culture examined. Sequence analysis has revealed that calicin is homologous to the kelch protein of the ring canal structure of Drosophila ovaries. In particular, it contains three consecutive repeating units of 48 amino acids each which are homologous to the so-called "beta-strand folds" occurring in proteins of the kelch family, including the actin cross-linking protein scruin of Limulus sperm and a series of other eukaryotic, bacterial, and viral proteins. Moreover, the amino terminal domain of calicin contains a region of about 100 amino acids homologous to an extended motif shared by the kelch protein as well as various zinc finger and poxvirus proteins. The possible role of calicin as a morphogenic cytoskeletal element in spermiogenic differentiation is discussed, also in relation to the demonstrated absence or altered arrangement of calicin in frequent forms of human teratozoospermia such as "round-headed" or other "postacrosomal sheath defect" sperm malformations.
Amino Acid Sequence↗
Alteration of free sulphydryl content of rat sperm heads by suppression of intratesticular testosterone.
Subcutaneous injections of testosterone propionate to adult male rats at a dose of 2.5 or 10 mg/kg body weight, 3 times per week for 7 weeks, resulted in a 75% reduction in serum LH and more than 50% reduction in intratesticular testosterone concentration, but serum FSH levels remained unchanged. The free -SH content, measured as iodo[14C]acetamide binding, increased by 70-100% in testicular sperm heads after suppression of testicular testosterone, and by 25-30% in caput epididymal sperm heads but was decreased by 70-80% in cauda epididymal sperm heads. These results demonstrate an alteration in the oxidative state of sperm nuclear basic proteins, suggesting incomplete nuclear maturation. These changes may be specific for the suppression of intratesticular testosterone, thus illustrating the androgen dependency of sperm head maturation. The contrast effects noted between the iodo[14C]iodoacetamide binding by the caput and the cauda epididymal sperm heads indicate that testosterone propionate treatment may affect the mechanisms regulating the oxidation of the sulphydryl residues in sperm heads during epididymal transit. This alteration may not directly relate to the tissue androgen concentrations.
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Proteins of demembraned mouse sperm heads. Characterization of a major sperm-unique component.
A 15-kilodalton protein has been identified as a major component of the residual protein fraction of mouse epididymal/vas spermatozoal heads, demembraned by treatment with Triton X-100 and sequentially extracted with 1 M NaCl/2-mercaptoethanol/DNase I. Two-dimensional electrophoresis of that protein before and after treatment with alkaline phosphatase indicated that it is present in epididymal/vas spermatozoa as a series of five differentially phosphorylated molecules with pI 6.0-7.0. Cyto-immunofluorescence with an affinity-purified antibody to the 15-kDa protein localized that protein to a circumscribed region of the demembraned mouse sperm head mediad from the dorsal margin. By radioimmunoassay, the 15-kDa protein was shown to be sperm-unique and species-specific. The antibody was nonreactive with homogenates of meiotic spermatogenic cells and round spermatids (stages 1-11) but was reactive with a non-phosphorylated 15.5-kDa protein of elongating spermatids (stages 12-16) and testicular spermatozoa. Following alkaline phosphatase treatment, the spermatozoal 15-kDa protein migrated to the position of the spermatidal 15.5-kDa protein on a sodium dodecyl sulfate gel. Thus, we conclude that the 15-kDa protein of mouse spermatozoa is synthesized during the elongation phase of spermiogenesis (stages 12-16) and is phosphorylated in the terminal period of that phase and/or after excursion of spermatozoa from the seminiferous tubules.
Alkaline Phosphatase↗
Cell surface structure of rodent sperm heads.
Replicas of critical point dried rodent sperm were examined by transmission electron microscopy. The surface of rat sperm heads appeared to be coated with regularly spaced 90A lamellar material. The plasma membrane overlying the acrosomal region of guinea pig spermatozoa displayed a regular scalloped array of lamellar structures. In replicas, the surface of Chinese hamster spermatozoa appeared coated by an array of small tubles and vesicles in the region overlaying the acrosome. It was possible to obtain replicas of the outer acrosomal membrane by removing the plasma membrane of mouse spermatozoa by brief treatment with Hank's balanced salt solution containing 0.2-0.5% MgC12. Replicas of the surface of the outer acrosomal membrane reveal evenly spaced, hexagonally-packed 90A particles similar to those which have been observed by other workers in replicas of freeze fractured outer acrosomal membranes. The finding of hexagonally arranged structures on the surface of the outer acrosomal membrane which appear very similar to those which have been observed in the plane of the membrane suggests that protein molecules in the plane of the membrane may protrude through or in some other manner distort the membrane surface. The post-acrosomal region of mouse sperm displays parallel lamellae with 100A spacing. If surface changes occur on sperm heads during maturation or capacitation, it should be possible to detect them with this technique.
Animals↗