Effects of different concentration and combinations of cryoprotectants on sperm quality,functional integrity in three Indian horse breeds

Traditionally Glycerol (Gly) is being used as major cryoprotectant and its toxicity could be a reason for the variation on stallion sperm freezability and fertility. In an effort to minimize Gly toxicity alternative cryoprotective agents like Di-methyl Formamide (DMF) have been investigated. The effect of the cryoprotectant and dose of cryoprotective agent varies from breed to breed and also from stallion to stallion within the same breed. Considering these factors a study was designed to study the effects of Gly and DMF at different concentrations and combinations on the plasma membrane, acrosome and DNA integrity as well as other post thaw seminal characteristics of semen of three Indigenous stallion breeds. In the current study, semen was collected from apparently healthy 4–6 years old 3 Marwari, 3 Manipuri and 3 Zanskari breed stallions. After semen collection and evaluation of fresh semen, each semen sample was extended with semen extender containing different concentrations and combinations of Gly and DMF cryoprotectants (i.e. 5% Gly, 5% DMF, 2% Gly, 2% DMF, 2.5% Gly +2.5% DMF and 1% Gly +1% DMF) and frozen. Post thaw semen evaluation was done on the basis of post thaw motility, live sperm count, hypo osmotic swelling test, acrosomal integrity and DNA integrity. Frozen thawed semen showed that the values of plasma membrane integrity, acrosome integrity and DNA integrity parameters were significantly higher (P < 0.05) with 5% DMF than the other cryoprotectants levels and combinations of Gly and DMF. From the present study, it was inferred that the combination of cryoprotectants at different concentrations (Gly and DMF @ 2.5 and 1%) also could not show better enhancement compared to the single cryoprotectant i.e DMF @5% in various post thaw seminal characteristics of Indigenous stallion semen. DMF at 5% concentration gave better protection to the plasma membrane and retained the acrosome and DNA integrity of the spermatozoa. Hence it can be concluded that DMF at 5% can be used for the cryopreservation of the Indigenous stallion with better preservation of the seminal quality.     

Sperm cryostorage in a dry tank: An accurate alternative

This prospective study aimed to determine the effects of dry nitrogen cryostorage on human sperm characteristics in comparison with liquid nitrogen cryostorage. For this purpose, 42 men undergoing routine semen analysis (21 normozoospermia and 21 with altered semen parameters) were analyzed. After slow freezing, half of the straws of each sample were randomly stored in liquid and dry tanks, at the top and bottom levels of the latter. After 6 months storage, thawed samples were treated by density gradient centrifugation and sperm characteristics were compared. There was no difference in sperm progressive motility (15.1% ± 14.2% vs. 15.1% ± 12.7%; p = 0.76), sperm vitality (25.5% ± 17.7% vs. 26.2% ± 19%; p = 0.71), percentages of acrosome-reacted spermatozoa (38% ± 8.5% vs. 38.5% ± 7.4%; p = 0.53) and DNA fragmentation spermatozoa (27.3% ± 12.4% vs. 28.5% ± 12.9%, p = 0.47) after cryostorage in the dry or the liquid nitrogen tank. Moreover, we did not observe differences between either cryostorage system for normal and altered sperm samples. This lack of difference was also observed whatever the floor level of cryostorage in the dry tank. The temperature measurement of the dry tank showed a stable temperature at −194 °C throughout storage whatever the storage floor level, guaranteeing the stability of the low temperatures suitable for human sperm storage. Because of its greater safety, dry storage without contact with the liquid phase should be preferred and can be a useful alternative for the cryostorage of human sperm samples.     

Cushioned versus noncushioned centrifugation: Sperm recovery rate and integrity

It was hypothesized that optimal sperm recovery rate (RR) without damage to the sperm would be obtained after centrifugation without a cushion solution. Semen collected three times from six light breed stallions was extended to 25 × 10⁶ sperm/mL and centrifuged at CON (noncentrifuged), 900NC (no-cushion), 900C (cushion), 1800NC, and 1800C × g for 10 minutes. Sperm concentration, motility (TM and PM), and intact plasma membranes (PLM) and acrosomes (ACR) pre- and postcentrifugation (D0) and after 24 hours (D1) of cooling were evaluated. The RR in the CON (100 ± 0.0), 900NC (93.7 ± 2.9), and 1800NC (96.7 ± 2.6) groups was significantly higher than the 900C (68.7 ± 4.6) and 1800C (79.6 ± 3.5) groups. The D0 TM and PM were not different between the CON, 900NC, 900C, and 1800C, but were lower for the 1800NC group. The D1 TM and PM of the 900NC (75.2 ± 3.8 and 71.1 ± 4.1) and 900C (76.2 ± 3.7 and 72.4 ± 4.0) groups were significantly higher than the 1800NC (71.7 ± 4.1 and 67.3 ± 4.4) and 1800C (71.6 ± 4.1 and 67.2 ± 4.4) groups, and the CON (66.2 ± 4.5 and 60.0 ± 4.8) group was significantly lower than the other groups. The D1 PLM of the CON, 900NC, 900C, 1800NC, and 1800C groups were not different. The ACR on D1 was significantly lower for the CON (93.0 ± 2.4) group compared with all other groups. Optimal RR preserving sperm integrity was obtained in the 900NC group.     

The subacrosomal granule and its evolution during spermiogenesis in a lizard

Summary Some aspects of spermiogenesis have been studied in the testis of the teiid lizard Cnemidophorus lemniscatus lemniscatus by electron microscopy. Shortly after the acrosomal vesicle is lodged in a nuclear concavity of the spermatid, a dense granule differentiates in the center of the subacrosomal space. It is cone-shaped and shows a longitudinal striation. Its base applies to the acrosomal membrane and, through this, to the acrosomal granule. Its rounded vertex causes a depression of the nuclear membranes which, initially juxtaposed, separates at this point to form a vesicle. The granule develops and becomes a rod when spermiogenesis is advanced and the subacrosomal space has taken the form of a secondary cap. The rod is cylindrical, retains its original striation and has a convex acrosomal end. It encloses the vesicle formed by the nuclear envelope in its base and follows the apex of the nucleus. Meanwhile, the acrosomal granule loses its identity and the acrosomal cap is filled with a dense substance, in which a fringe of translucent material differentiates. This fringe lies in the dorsal and apical margins of the acrosome and is incompletely divided by longitudinal crests of the dense acrosomal substance. A projection of the Sertoli cell forms an accessory cap which envelops the acrosome and is in turn covered by the cytoplasm of the spermatid, constituting an intricate association. Two reflex membranes underlie the plasmalemma in the outer surface of the projection of the Sertoli cell. They are continuous with one another at their ends and with the cell membrane in the edge of pores. In the peripheral cytoplasm of the spermatid facing the accessory cap, numerous microtubules run longitudinally. By means of thin membranes some are interconnected or connected with the plasmalemma, from which they seem to originate.This research forms part of project N. 31.26.S1-0244 supported by the Consejo Nacional de Investigaciones Científicas y Tecnológicas     

Les éléments cytoplasmiques au cours de la spermiogenèse du TritonPleurodeles waltlii Michah

Résumé L'origine et la morphogenèse des différents éléments de l'acrosome du spermatozode dePleurodeles waltlii ont été suivies et décrites depuis le tout début de la spermiogenèse. La formation de la vésicule acrosomienne et son évolution en une coiffe acrosomienne se fait selon le schéma classique. Son extrémité apicale se différencie tardivement en un bouton terminal et un crochet. Les trois parties de la coiffe diffèrent dans leur composition et leur structure fine.Les volumineux et complexes éléments situés sous la coiffe acrosomienne: axe, baguette puis manchon périphérique et manchon moyen, sont dépourvus de polysaccharides. Leur origine est envisagées. Ils sont comparés aux éléments situés dans l'espace sous-acrosomien des spermatozodes des autres vertébrés.

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The cytoplasmic elements during spermiogenesis in the triturusPleurodeles waltlii MichahI. Acrosome genesis

Summary The origin and the morphogenesis of the acrosome different parts ofPleurodeles spermatozoon, have been investigated and described from the early beginning spermiogenesis process. The acrosomal vesicle and acrosomal cap formation take place according to the classical scheme. The acrosomal anterior tip cap late differentiate in a blunt terminal knob and a hook. The three cap parts differ in their composition and fine structure.The large and complicated structure stretching under the acrosomal cap: axis, peripheral muff and middle muff, are devoided of polysaccharides; their origin is discussed. They are compared with the subacrosomal components lying in the other vertebrates spermatozoon subacrosomal space.

Equipe de Recherche Associée au C.N.R.S., E.R.A. n^o 129.     

Simultaneous evaluation of viability and acrosome integrity of mouse spermatozoa using light microscopy

Determination of the percentage of live cells with intact acrosomes and no morphologic aberrations could be a practical index of semen quality. We applied viability and acrosome staining techniques, originally described for bull, boar and rabbit sperm, to mouse spermatozoa. The viability stain was either trypan blue or Congo red. The stain was precipitated by neutral red in the fixative. The acrosome was stained by Giemsa. Sperm morphology, including cytoplasmic droplets, could be evaluated as well. The staining method described here is a useful routine tool for simultaneous evaluation of the plasma membrane integrity of different sperm subdomains, the status of the acrosome, and cellular morphology.     

Relationship between the chromatoid body and the acrosomal system in early spermatids of Myxine glutinosa L.

Summary A transient close relationship between the chromatoid body and the developing acrosome is demonstrated in early spermatids of Myxine glutinosa.This work was supported by the Norwegian Research Council for Science and Humanities (NAVF, Grant Nr. D 61.44) and the Austrian Fonds zur Förderung der wissenschaftlichen Forschung, Projekt 2183     

Rho-kinase (ROCK) in sea urchin sperm: its role in regulating the intracellular pH during the acrosome reaction

Sperm must undergo the acrosome reaction (AR) in order to fertilize the egg. In sea urchins, this reaction is triggered by the egg jelly (EJ) which, upon binding to its sperm receptor, induces increases in the ion permeability of the plasma membrane and changes in protein phosphorylation. Here, we demonstrated that the sperm expresses ROCK (∼135 kDa), which is a serine/threonine protein kinase. ROCK localized, as RhoGTPase (Rho), in the acrosomal region, midpiece and flagellum. H-1152, a ROCK antagonist, inhibited the two cellular processes defining the AR: the acrosomal exocytosis and the actin polymerization. The ionophores nigericin and A23187 reversed the AR inhibition induced by H-1152, suggesting that ROCK functions at the level of the EJ-induced ion fluxes. Accordingly, H-1152 blocked 70% the intracellular alkalinization induced by EJ. These results indicate that EJ activates a Na⁺-H⁺ exchanger (NHE) in the sperm through a Rho/ROCK-dependent signaling pathway that culminates in the AR.     

Lead chloride affects sperm motility and acrosome reaction in mice

Lead is highly toxic and persistent in the environment and, thus, a major concern for public health. In this study, the effects of lead chloride (PbCl₂) on mouse epididymal sperm were evaluated. Male mice were subcutaneously injected with 74 and 100 mg PbCl₂/kg body weight for four consecutive days. Sperm was collected from the epididymis and several parameters of sperm function, such as sperm density, motility, viability, mitochondrial function, acrosome integrity and morphology, were evaluated. Furthermore, DNA fragmentation was assessed by the terminal deoxylnucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick-end labelling (TUNEL) assay and chromatin integrity was evaluated by sperm chromatin structure assay (SCSA). In order to assess direct effects on existing sperm population, we sacrificed one group for each condition at day 5. The effects of lead upon one entire spermatogenic cycle were evaluated on day 35. Both lead concentrations used in this work affected sperm motility, although no significant differences were observed in sperm viability, mitochondrial function and DNA/chromatin integrity. However, a decrease in the percentage of intact acrosomes was also observed, mirroring a lead-induced premature acrosome reaction. Thus, the results obtained indicate that, together with impaired motility, the effect of lead toxicity on acrosome integrity, leading to premature reaction, may compromise the ability of sperm to fertilize the oocyte.