Somatic histones are components of the perinuclear theca in bovine spermatozoa

The perinuclear theca is a non-ionic detergent-resistant, electron-dense layer surrounding the condensed nucleus of mammalian sperm. The known proteins originating from the perinuclear theca have implicated the structure in a variety of important cellular processes during spermiogenesis and fertilization. Nonetheless, the composition of the perinuclear theca remains largely unexplored. We have isolated a group of low molecular mass (14-19 kDa) perinuclear theca-derived proteins from acrosome-depleted bovine sperm heads by salt (1 M KCl) extraction and have identified them as core somatic histones. N-terminal sequencing and immunoblotting with anti-histone antibodies confirmed the presence of both intact and proteolytically cleaved somatic histones H3, H2B, H2A, and H4. Identical proteins were isolated using 2% SDS or 1 N HCl extractions. Subsequent acid and SDS extractions of intact bovine sperm revealed the presence of all four intact histone subtypes, with minimal proteolysis. Two-dimensional acid/urea/Triton-SDS-PAGE, coupled with immunoblotting analysis, confirmed the somatic nature of these perinuclear theca-derived histones. Estimates of the abundance of perinuclear theca-derived histones showed that up to 0.2 pg per sperm of each histone subtype was present. Immunogold labeling at the ultrastructural level localized all four core somatic histones to the post-acrosomal sheath region of bovine epididymal sperm, when probed with affinity-purified anti-histone antibodies. Little immunoreactivity was detected in residual perinuclear theca structures following the extractions. Taken together, these findings indicate the unprecedented and stable localization of non-nuclear somatic histones in bovine sperm perinuclear theca.     

Cryopreservation alters membrane sulfhydryl status of bull spermatozoa: protection by oxidized glutathione.

Cryopreservation induces extensive biophysical and biochemical changes in the membrane of spermatozoa that ultimately decrease the fertility potential of the cells. Sulfhydryl groups of sperm proteins regulate a number of activities of the cells. Qualitative and quantitative analyses of sulfhydryl groups in the sperm membrane were performed by fluorescence microscopy, fluorimetry and electrophoresis. Fluorimetric analysis using 5-iodoacetamidofluoresceine indicated a two-fold increase in the content of sulfhydryl groups in sperm membrane after a freezing/thawing cycle. Electrophoresis of Triton-soluble sperm proteins after labeling with 3-(N-maleimidylpropionyl) biocytin indicated that proteins of 40-65 and 34 kDa range expose more sulfhydryl groups after cooling at 4 degrees C and freezing/thawing. Cryopreservation of spermatozoa changed the distribution pattern of sulfhydryl groups on sperm surface measured with fluorescence microscopy using 5-iodoacetamidofluoresceine. The percentage of spermatozoa labeled at the level of the mid-piece decreased by 50 and 90% after cooling and freezing/thawing, respectively. Spin labeling studies showed a 15% faster rotational diffusion (mobility) of sulfhydryl containing proteins in the membrane of frozen/thawed spermatozoa as compared to that of fresh spermatozoa. Addition of glutathione, reduced (GSH) or oxidized (GSSG), to the cryoprotectant partially prevented the effects of freezing/thawing, such as higher exposure of sulfhydryl groups, changes in the cellular distribution, and enhanced rotational diffusion of sulfhydryl containing proteins of sperm membrane. Addition of GSSG to the cryoprotectant reduced by 35% the loss of motility of spermatozoa undergoing a freezing/thawing cycle. We concluded that cryopreservation perturbs sperm membrane sulfhydryl containing proteins and that these modifications could be partially prevented by the addition of GSSG to the cryopreservation medium.     

Basic proteins of the perinuclear theca of mammalian spermatozoa and spermatids: a novel class of cytoskeletal elements

The nuclei of bovine spermatids and spermatozoa are surrounded by dense cytoplasmic webs sandwiched between the nuclear envelope and the acrosome and plasma membrane, respectively, filling most of the cytoplasmic space of the sperm head. This web contains a complex structure, the perinuclear theca, which is characterized by resistance to extractions in nondenaturing detergents and high salt buffers, and can be divided into two major subcomponents, the subacrosomal layer and the postacrosomal calyx. Using calyces isolated from bull and rat spermatozoa we have identified two kinds of basic proteins as major constituents of the thecal structure and have localized them by specific antibodies at the light and electron microscopic level. These are an Mr 60,000 protein, termed calicin, localized almost exclusively to the calyx, and a group of multiple-band polypeptides (MBP; Mr 56,000-74,000), which occur in both the calyx and the subacrosomal layer. The polypeptides of the MBP group are immunologically related to each other, but unrelated, by antibody reactions and peptide maps, to calicin. We show that these basic cytoskeletal proteins are first detectable in the round spermatid stage. As we have not detected any intermediate filament proteins and proteins related to nuclear lamins of somatic cells in sperm heads, we conclude that the perinuclear theca and its constituents, calicin and MBP proteins, are the predominant cytoskeletal elements of the sperm head. Immunologically cross-reacting polypeptides with similar properties have been identified in the heads of rat and human spermatozoa. We speculate that these insoluble basic proteins contribute, during spermiogenesis, to the formation of the perinuclear theca as an architectural element involved in the shape changes and the intimate association of the nucleus with the acrosome and the plasma membrane.     

Formation of the perinuclear theca in spermatozoa of diverse mammalian species: relationship of the manchette and multiple band polypeptides

The perinuclear theca is a novel cytoskeletal consisting of a densely layered lamina that surrounds the nucleus of mammalian sperm. Using antibodies specific for the multiple band polypeptides present in the perinuclear theca of bull sperm, we show that a heterogeneous group of immunological related proteins are present in the sperm heads of other mammals with greatly different morphologies, including guinea pig, hamster, rat, and mouse. In none of the species were identical groups of immunoreactive polypeptides found, although immunoreactive proteins of molecular weights 65,000 to 80,000 were present in the sperm heads of all species examined. Immunoreactive proteins less than Mr 55,000 were prominent in rat sperm heads and mouse sperm: guinea pig, hamster, and rat sperm heads and mouse sperm had one band in common at approximately Mr 50,000. Different immunoreactive proteins were present in isolated sperm tails. The perinuclear theca first appeared in the subacrosomal space of round to elongating spermatids. Later, with the caudal movement of the manchette, the postacrosomal segment of the perinuclear theca was deposited in a cephalad to caudal direction along the sperm nucleus. Concomitantly, the cytoplasmic space between the nuclear envelope and the plasma membrane narrowed such that only the theca occupied this portion of the sperm head. Immunoreactivity accompanied the ultrastructural appearance of the subacrosomal layer and the postacrosomal segment. The periods of spermiogenesis, in which sub- and post-acrosomal components of the perinuclear theca are formed and the morphogenesis of sperm organelles with which these elements are associated, suggest that components of this cytoskeletal structure function to join the acrosome and the postacrosomal plasma membrane to the nucleus.     

Cryopreservation of mouse spermatozoa

Recently, it has become possible to freeze a large number of mouse spermatozoa immediately after collection from the epididymides of a small number of males. The cryopreservation of spermatozoa is simpler, less time-consuming, and less costly than that of embryos for maintaining various strains of mice with induced mutations. This chapter attempts to provide a realistic overview of the cryopreservation of mouse spermatozoa and to describe a detailed procedure for mouse sperm freezing. Received: 16 December 1999 / Accepted: 17 December 1999     

Cryopreservation of murine spermatozoa

Cryopreservation of mouse sperm provides an economic option for preserving the large number of mouse strains now being generated by transgenic and targeted mutation methodologies. The ability of a spermatozoan cell to survive cryobiological preservation depends on general biophysical constraints that apply to all cells, such as the avoidance or minimization of the formation of intracellular ice during cooling. This action is typically achieved by use of cryoprotectant substances and by controlled, slow rates of cooling. Superimposed on those general constraints may be special characteristics of mouse spermatozoa, such as more narrow, osmotically driven volume tolerance limits and the fact that relatively successful freezing can be obtained without the use of a permeating cryoprotective agent. The lack of important information regarding sperm cells fundamental cryobiological properties, including their osmotic and membrane permeability characteristics, has hindered progress in developing anything but empirically derived methods. Genetic differences between inbred mouse strains are reflected in motility and fertility characteristics of mouse sperm and contribute to the difficulty of developing successful cryopreservation methods. Recovery of live young from frozen sperm has been much more successful with sperm from hybrid mice than from most inbred strains. There have been no published reports of successful cryopreservation of rat sperm. Nevertheless, in mice, success in deriving live young from intracytoplasmic sperm injection using sperm frozen under suboptimal conditions raises the possibility of using this technique for the ultimate rescue of sperm regardless of the success of cryopreservation. This technique, however, requires additional development and verification of its efficacy before it will be suitable for general laboratory use. Although cryopreservation of mouse sperm is not yet universally successful, it can be used reliably to supplement cryopreservation of embryos and other germline cells or tissues for preserving biomedically important strains of mice for research.     

Peroxidase activities in bull spermatozoa

The present study was carried out to determine the localization of peroxidase activity in bull spermatozoa. 3,3′-Diaminobenzidine (DAB) was used as a substrate for revealing peroxidase activity, and light and electron microscopic analysis of the results obtained was performed. Peroxidase activity was detected in the mitochondria of the middle piece and the outer acrosomal membrane. Catalase was excluded as an enzyme, catalyzing the detected peroxidase activity. Concerning the biochemical properties of bull sperm peroxidases, peroxidase activity was found to be manifested in a large pH range, 4–10.5. Bull sperm peroxidase activity appeared to be temperature sensitive and azide sensitive and could be readily inhibited by phenylhydrazine. Electrophoretic analysis of the proteins from bull sperm extracts separated in a Davis-Ornstein system of 7% polyacrylamide gel, followed by the determination of peroxidase activity on the polyacrylamide gels, revealed that all 14 sperm protein fractions available on the gel possessed peroxidase when benzidine was used as a substrate. The possible reasons for the electrophoretic heterogeneity of bull sperm peroxidases are discussed. © 1994 Wiley-Liss, Inc.     

Cryopreservation of mouse spermatozoa]

The spermatozoa of cauda epididymis of mature mice were suspended in 3% skim milk in distilled water supplemented with 12, 15, 18 or 21% (W/V) raffinose. The suspension of spermatozoa were frozen in liquid nitrogen gas for 10 min, then stored in liquid nitrogen (-196 degrees C). The frozen suspensions of spermatozoa were thawed by rapid warming in water bath at room temperature. For removing the cryopreservative solution, a pair of syringes connected with a three stop cock and a filter unit (pore size 0.45 mu) were used. Highest sperm motility was obtained after 1 hr of thawing from the cryopreservative solution containing 18% raffinose and 3% skim milk. These cryopreserved spermatozoa were used for fertilization in vitro. The proportion of pronuclear oocytes was 35.9% (74/206) 6 hr after insemination, and the proportion of 2-cell embryos was 33.6% (42/125) 28 hr after insemination. All 2-cell embryos were transferred to the oviducts of pseudopregnant recipients and 45.2% (19/42) developed to normal young.     

Cryopreservation of flow-sorted bovine spermatozoa

Experiments were designed to maximize sperm viability after sorting by flow cytometry and cryopreservation. Experiments concerned staining sperm with Hoechst 33342 dye, subsequent dilution, interrogation with laser light, and postsort concentration of sperm. Concentrating sorted sperm by centrifugation to 10 to 20 x 10(6) sperm/ml reduced adverse effects of dilution. Exposing sperm to 150 mW of laser light resulted in lower percentages of progressively motile sperm after thawing than did 100 mW. Sorted sperm extended in a TRIS-based medium had higher postthaw sperm motility after incubation for 1 or 2 h than sperm extended in egg-yolk citrate (EYC) or TEST media, and equilibrating sperm at 5 degrees C for 3 or 6 h prior to freezing was superior to an equilibration time of 18 h. For sorting sperm 4 to 7 h postcollection, it was best to hold semen at 22 degrees C neat instead of at 400 x 10(6)/ml in a TALP buffer with Hoechst 33342. Current procedures for sexing sperm using flow cytometry result in slightly lower postthaw motility and acrosomal integrity compared to control sperm. However, this damage is minor compared to that caused by routine cryopreservation. Fertilizing capacity of flow-sorted sperm is quite acceptable as predicted by simple laboratory assays, and sexed bovine sperm for commercial AI may be available within 2 years.     

Induced metachromasia in bull spermatozoa

Summary The availability of sequential DNA phosphates to bind toluidine blue molecules after acid hydrolysis was studied in normally shaped and misshaped spermatozoa from subfertile and highly fertile bulls. The aim was to associate induced spermatozoal metachromasia with infertility. Some few normally and abnormally shaped cells from highly fertile bulls exhibited an induced metachromasia after being treated with 4N HCl for 10–30 min at 25°C prior to staining. Subfertile bulls contained 12 times as many metachromatic spermatozoa as highly fertile animals. The induced toluidine blue metachromasia is suggested as a rapid and simple method for detecting bull spermatozoa bearing an anomalous DNA-protein complex. This nucleoprotein complex was found to be more frequent in subfertile bulls.     

Cryopreservation of wild mouse spermatozoa

Spermatozoa of wild mice from China, Czechoslovakia, Denmark, India, Japan and Switzerland were frozen and stored at -196 degrees C. After thawing, intact oocytes were inseminated in vitro with relatively high motility frozen-thawed mouse spermatozoa from Czechoslovakia, Denmark and India, while oocytes with a partially dissected zona were inseminated with low motility frozen-thawed spermatozoa from China, Japan and Switzerland. Embryos developing to the 2-cell stage from oocytes fertilized with frozen-thawed spermatozoa were transferred to the oviducts of female recipients on the first day of pseudopregnancy (day when a vaginal plug was confirmed). Successful embryo development to the 2-cell stage was 46 to 67%. Offspring resulted from 17 to 51% of these transferred 2-cell embryos.     

淡水鱼类精子的冷冻与保存

养殖鱼类精子的保存具有深刻的研究意义和良好的应用前景,关于鱼类精子的冷冻保存国内外已经有开展了不少研究。对淡水鱼类精子的冷冻保存,特别是淡水鱼类精子的超低温冷冻保存研究进展进行综述。     

Oxidative stress alters membrane sulfhydryl status, lipid and phospholipid contents of crossbred cattle bull spermatozoa

Ferrous ascorbate (FeAA: FeSO4+ascorbic acid) has been used in the past by different investigators to induce oxidative stress. The optimum dose of FeAA for inducing oxidative stress by affecting thiols [total thiols (TSH), glutathione reduced (GSH), glutathione oxidized (GSSG), redox ratio (GSH/GSSG)], total lipids and phospholipids has been ascertained in the local crossbred cattle bull spermatozoa. The fractions of spermatozoa suspended in 2.9% sodium citrate were subjected to three doses of FeAA (100 microM:500 microM, 150 microM:750 microM, 200 microM:1000 microM; FeSO4:ascorbic acid), and were assessed for various parameters. On increasing the concentration of FeAA, a gradual decrease in TSH, GSH, GSH/GSSG, lipid and phospholipid levels, but increase in GSSG content were observed. It is concluded that thiol groups play an important role in antioxidation and detoxification of ROS as well as maintaining intracellular redox status. Thiol groups, thus, serve as defense mechanisms of sperm cells to fight against oxidative stress. In addition, all doses of FeAA cause leakage of lipids and phospholipids from the bull sperm membranes.     

Dynamics of microRNAs in bull spermatozoa

Background

Degenerative effects of critical regulators of reproduction, the kisspeptin peptides, on cellular aspects of sexually immature male gonads are known but similar information on accessory sex glands remain elusive.

Methods

Prepubertal laboratory rats were injected kisspeptin-10 at three different dosage concentrations (10 pg, 1 ng and 1 microgram) for a period of continuous 12 days at the rate of two doses per day. Control rats were maintained in parallel. The day following the end of the experimental period, seminal vesicles were removed and processed for light and electron microscopic examination using the standard methods. DNA damage was estimated by DNA ladder assay and DNA fragmentation assay.

Results

The results demonstrated cellular degeneration. Epithelial cell height of seminal vesicles decreased significantly at all doses (P < 0.05). Marked decrease in epithelial folds was readily noticeable, while the lumen was dilated. Ultrastructural changes were characterized by dilatation of endoplasmic reticulum and Golgi complex, heterochromatization of nuclei, invagination of nuclear membranes and a decreased number of secretory granules. Percent DNA damage to the seminal vesicle was 19.54 +/- 1.98, 38.06 +/- 2.09 and 58.18 +/- 2.59 at 10 pg, 1 ng and 1 microgram doses respectively.

Conclusion

The study reveals that continuous administration of kisspeptin does not lead to an early maturation but instead severe degeneration of sexually immature seminal vesicles.     

Cryopreservation of spermatozoa in cyprinid fishes

The present study investigated semen cryopreservation in cyprinid fish using computer-assisted sperm motility analysis for viability control. Spermatozoa of the bleak, Chalcalbumus chalcoides, were used as a basic model to describe the toxic and cryoprotective effects of internal and external cryoprotectants, their most effective concentrations and combinations, the freezing and thawing conditions, and the effects of equilibration. We also used these data to develop a cryopreservation protocol for Barbus barbus, Chondrostoma nasus, Ctenopharyngodon idella, Cyprinus cario, Hypohtalmichthys molitrix, Leuciscus cephalus, Rutilus meidingerii, and Vimba vimba. For all investigated species the optimal extender composition was a buffered physiological sperm motility-inhibiting saline solution containing 10% DMSO and 0.5% glycin. The optimal sperm equilibration period in the extender was < or = 5 min. Freezing was performed in an insulated box in liquid nitrogen vapor and it was optimal at 4 to 5 cm above the surface of the liquid, depending on the species. Thawing was optimal in a 25 degrees C water bath whereby the thawing time ranged depending on species from 15 to 45 sec. This cryopreservation protocol resulted in frozen-thawed semen with 35 to 65% motile and 5 to 25% locally motile spermatozoa depending on the quality of fresh semen.