Effects of heterologous sera on fertilized rabbit ova

Undiluted blood serum of various species was used to culture two-celled rabbit ova for 24 hours. It was found that there is an ovocidal factor present in the serum of man, sheep, cattle, goat, and fowl. The factor is lethal rather than inhibitory; exposure to it for 10 minutes will cause the death of the ova. This factor is unstable, thermolabile (destroyed at 55 degrees C. in 30 minutes), and of large molecular size. The strength or concentration of this factor varies according to the origin of the serum, increasing in the order man, sheep, cattle, goat, fowl. The blood serum of rabbit, horse, dog, guinea pig, rat, and pig contains no ovocidal factor against rabbit ova. The ovocidal factor differs from the spermicidal factor, which is present in all the sera of the different species studied with rabbit spermatozoa. Immunization of the guinea pig against rabbit ova is possible. Normal development of young rabbits was obtained by transplantation of ova cultured in the heated or normal serum of other species after 24 hours.     

Uptake of exogenous DNA by mammalian spermatozoa: specific localization of DNA on sperm heads.

When mouse spermatozoa were briefly exposed in culture to radioactively labelled DNA (pSV2CAT plasmid), radioactivity could be detected by high-resolution autoradiography on the surface and within the nucleus of the spermatozoa. Spermatozoa from other mammalian species (boar, bull, man) could also bind foreign DNA. With the exclusion of human spermatozoa, which in most experiments showed very low labelling values, labelling percentages (evaluated by light microscope autoradiography) ranged between 39 and 78%. In all four species the DNA-binding ability was mainly confined to a specific region of the sperm head (equatorial segment and postacrosomal region), and the sperm-DNA association kinetics were rapid (maximum values were reached within 20-40 min). The data also indicate that factor(s) in seminal plasma might protect spermatozoa from accidental transfection by foreign DNA that may be present in the genital tracts from bacterial or viral sources.     

Freeze-dried sperm fertilization leads to full-term development in rabbits

To date, the laboratory mouse is the only mammal in which freeze-dried spermatozoa have been shown to support full-term development after microinjection into oocytes. Because spermatozoa in mice, unlike in most other mammals, do not contribute centrosomes to zygotes, it is still unknown whether freeze-dried spermatozoa in other mammals are fertile. Rabbit sperm was selected as a model because of its similarity to human sperm (considering the centrosome inheritance pattern). Freeze- drying induces rabbit spermatozoa to undergo dramatic changes, such as immobilization, membrane breaking, and tail fragmentation. Even when considered to be "dead" in the conventional sense, rabbit spermatozoa freeze-dried and stored at ambient temperature for more than 2 yr still have capability comparable to that of fresh spermatozoa to support preimplantation development after injection into oocytes followed by activation. A rabbit kit derived from a freeze-dried spermatozoon was born after transferring 230 sperm-injected oocytes into eight recipients. The results suggest that freeze-drying could be applied to preserve the spermatozoa from most other species, including human. The present study also raises the question of whether rabbit sperm centrosomes survive freeze-drying or are not essential for embryonic development.     

Synergistic cytotoxicity. I. Characterization of a heat labile plasma fraction that induces nonspecific cytotoxicity by human mononuclear cells.

The mechanism by which non-immune mononuclear cells recognize invading foreign material and are activated for cytotoxic attack was studied in a model system employing human mononuclear cells, fresh plasma, and 51Cr-labeled xenogeneic target erythrocytes. In these experiments, fresh antibody-depleted plasma or mononuclear leukocytes alone were poorly cytotoxic to xenogenic erythrocytes. However, these target cells were rapidly lysed when both fresh antibody-depleted plasma and mononuclear cells were present in the assay. The plasma factor could not be removed by extensive absorption with the target cells, was present in plasma from hypogammaglobulinemic patients, was heat labile, and was sensitive to incubation with zymosan and cobra venom factor. The "antigen" specificity of this reaction was directed by the serum factor inasmuch as target cells autologous to the effector cells could be killed in the presence of antibody-depleted xenogeneic plasma, but not autologous plasma. These data suggest that an important mechanism for the recognition of "foreigness" by non-immune mononuclear cells is via interaction with a plasma component, possibly a factor related to serum complement.     

A high activity carbonic anhydrase isoenzyme (CA II) is present in mammalian spermatozoa

Human and rat spermatozoa were stained for different carbonic anhydrase (CA) isoenzymes using specific antisera to human CA I, II and VI in conjunction with the immunofluorescence technique. The spermatozoa of both species were found to contain only CA II, which was located principally in the postacrosomal region of the human spermatozoa and in the acrosomal cap region of the rat spermatozoa. The presence of CA II could be confirmed by immunoblotting, which revealed a 29 K polypeptide in both the human and rat spermatozoa. No CA I or VI-specific fluorescence could be detected in the spermatozoa of either species. The immunoblottings were also negative. The results show mammalian spermatozoa to contain the high activity carbonic anhydrase isoenzyme II. Its presence is probably linked to hydration of CO2 produced by active energy metabolism and thereby to the maintaining of an adequate intraspermatozoal bicarbonate concentration as required for the maintenance of sperm motility.     

Freeze–thaw induced genotoxicity in buffalo (<Emphasis Type="Italic">Bubalus bubalis</Emphasis>) spermatozoa in relation to total antioxidant status

Use of cryopreserved semen has become an important tool in assisted reproduction but freezing and thawing cause sub-lethal damage to spermatozoa. This is detrimental to sperm because of the membrane damage including permeability and integrity. An excess generation of reactive oxygen species (ROS) creates oxidative stress due to reduced antioxidant status of the cryopreserved spermatozoa. In the present study fresh buffalo semen was collected and divided into two aliquots. One aliquot was used for fresh semen analysis and the other was cryopreserved in Tris-egg yolk-citrate extender. The semen samples were used to study different sperm quality parameters like motility, viability, membrane integrity and total antioxidant status. The DNA integrity in fresh and cryopreserved spermatozoa was also studied using comet assay. The sperm quality parameters like post-thaw sperm motility, viability, membrane integrity and total antioxidant status of cryopreserved spermatozoa were significantly lowered (P < 0.05) compared to fresh spermatozoa. The DNA fragmentation in cryopreserved spermatozoa was significantly higher (P < 0.01) as compared to fresh spermatozoa. The results show that the irreversible DNA damage occurs in spermatozoa during cryopreservation.     

Radicaux libres et spermatozoïdes humains: physiologie et physiopathologie

The role of reactive oxygen species in the physiopathology of human sperm function has been emphasized in recent years. Their production in semen has been associated with loss of motility, decreased capacity for spermoocyte fusion and loss of fertility. In semen preparations, there are two major sources of reactive oxygen species: leucocytes and spermatozoa themselve. It has been proposed that reactive oxygen species production by human spermatozoa was dependent upon a membrane-bound NADPH oxidase or a mitochondrial diaphorase. Hydrogen peroxide produced by the dismutation of superoxide anion has been recognized as the most toxic oxidizing species for human spermatozoa. Owing to their high content of polyunsaturated fatty acids, it has been proposed that lipid peroxidation of the sperm plasma membrane is largely responsible for defective sperm function. Reactive oxygen species also affect the sperm axoneme as a result of ATP depletion, inhibit mitochondrial functions, and synthesis of DNA, RNA and proteins, produce cytoskeletal modifications and inhibit sperm-oocyte fusion. Human spermatozoa possess enzymatic defence systems such as superoxide dismutase, glutathion peroxidas/reductase and catalase to counteract the toxic effects induced by reactive oxygen species. Correlations have been reported between their effectiveness and the duration of sperm motility. If the excessive production of reactive oxygen species is detrimental for human spermatozoa, they could also participate in the physiological function of the spermatozoa when present at low concentrations. Indeed, reactive oxygen species have been shown to be involved in the activation of several enzymes. Furthermore, sperm capacitation, acrosome reaction and sperm-zona interaction would be enhanced by reactive oxygen species.     

Quantitative assessment of chromatin stability alteration in human spermatozoa induced by freezing and thawing. A flow cytometric study.

The authors examined the effect of cryopreservation on chromatin stability in human spermatozoa from 21 ejaculates. Each ejaculate was divided into four aliquots: (1) fresh aliquot, (2) frozen and thawed aliquot, (3) fresh aliquot subjected to an in vitro decondensation method, and (4) frozen and thawed aliquot subjected to the same in vitro decondensation method; all were then fixed with an ethanol fixative agent. Chromatin stainability was quantified by flow cytometric measurement of fluorochrome uptake by DNA. Study of 21 fresh aliquots showed that 37.9% of the DNA was accessible to propidium iodide. The comparative stainability between the 21 fresh and 21 frozen-thawed, undecondensed aliquots demonstrated a low but significant increase in accessibility of DNA by propidium iodide for the thawed samples: 38.7 +/- 1.7% (mean +/- SD) versus 37.9 +/- 1.3%. The biochemical action of the nuclear decondensation solution increased the accessibility of propidium iodide, but in different ways: 57.2 +/- 12.9% versus 54.7 +/- 13.7%, respectively, for fresh and frozen-thawed aliquots. Analysis of the flow cytometric histograms revealed an intermediate population of spermatozoa adjacent to the main germinal peak. This population increased significantly: 9.6 +/- 1.9% for the fresh versus 12.3 +/- 4.9% for the frozen-thawed undecondensed aliquots and 8.6 +/- 3.5% versus 12.3 +/- 4.9%, respectively, for fresh and frozen-thawed, decondensed aliquots. Because the chromatin stability of thawed spermatozoa may be a critical factor in assisted procreation, the authors discuss the effect of thermal denaturation on the nucleoprotein structures and the origin of the intermediate population of spermatozoa.     

A high activity carbonic anhydrase isoenzyme (CA II) is present in mammalian spermatozoa

Summary Human and rat spermatozoa were stained for different carbonic anhydrase (CA) isoenzymes using specific antisera to human CA I, II and VI in conjunction with the immunofluorescence technique. The spermatozoa of both species were found to contain only CA II, which was located principally in the postacrosomal region of the human spermatozoa and in the acrosomal cap region of the rat spermatozoa. The presence of CA II could be confirmed by immunoblotting, which revealed a 29 K polypeptide in both the human and rat spermatozoa. No CA I or VI-specific fluorescence could be detected in the spermatozoa of either species. The immunoblottings were also negative. The results show mammalian spermatozoa to contain the high activity carbonic anhydrase isoenzyme II. Its presence is probably linked to hydration of CO₂ produced by active energy metabolism and thereby to the maintaining of an adequate intraspermatozoal bicarbonate concentration as required for the maintenance of sperm motility.     

Vitrification induces critical subcellular damages in ram spermatozoa

The aim of the present study was to analyse morphological variations in ovine spermatozoa subjected to different cryopreservation protocols using high resolution imaging techniques. Ejaculates were pooled and diluted in Tris-based extender. Aliquots containing 300 × 10⁶ spz/ml were prepared and evaluated a) after the semen collection and pooling, b) after conventional freezing, c) after vitrification of samples maintained at room temperature (22 °C) prior to vitrification, and d) after vitrification of samples maintained at 5 °C prior to vitrification. Sperm motility, acrosome integrity, DNA fragmentation and morphology were assessed. Subcellular sperm changes were assessed and described by light microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The maintenance of spermatozoa at 5 °C prior to vitrification and the use of 0.4 M sucrose pointed out lower dimensions of area, length and width than fresh, frozen and sperm maintained at 22 °C prior to vitrification. It was observed that the head width and length are significantly higher (P < 0.0001) in fresh spermatozoa than in the vitrified sperm samples. It could be hypothesized that greater intracellular fluid loss during vitrification could prevent damages in the spermatozoon throughout the reduced ice crystals formation, but mainly by the reduction of extracellular ice crystals due to the physical properties modification obtained when high concentrations of sugars are added. This is the first ultramicroscopic study carried out in ovine vitrified spermatozoa, which confirms the functional sperm alterations previously detected.     

Proacrosin/acrosin quantification as an indicator of acrosomal integrity in fresh and frozen dog spermatozoa

The scope of the present study was to evaluate the presence and activation of proacrosin/acrosin as a tool to determine the acrosomal status of fresh and frozen/thawed dog spermatozoa. Monoclonal antibody C5F11, directed against human acrosin, cross-reacted with dog spermatozoa and labeled the acrosome of both fresh and frozen/thawed dog spermatozoa. Frozen/thawed spermatozoa had a lesser proportion of labeled spermatozoa than fresh spermatozoa (P<0.05). When live spermatozoa were labeled with soybean trypsin inhibitor conjugated with Alexa 488 (SBTI-Alexa 488), the proportion of acrosome-labeled fresh spermatozoa was less than frozen/thawed spermatozoa (P<0.05). By using Western blots and enzymatic activity, frozen/thawed spermatozoa had a greater proportion of active acrosin than fresh spermatozoa. In addition, beta 1,4-galactosyl-transferase (GalT), a plasma membrane bound protein, remained attached to frozen/thawed spermatozoa. Proacrosin is activated during freezing/thawing of dog spermatozoa, and that proacrosin/acrosin may be a good indicator of acrosomal integrity of frozen/thawed spermatozoa.     

Sperm binding capacity and ultrastructure of the zona pellucida of stored canine oocytes

Sperm binding to the zona pellucida is a prerequisite for fertilization, and tests that evaluate this function have been described for several species. When carrying out such tests in the canine species, ovaries or oocytes have to be stored to obtain a sufficient number of oocytes at the time of testing. In the present study, the sperm binding capacities of salt-stored oocytes and oocytes from deep frozen ovaries were measured and compared with that of fresh oocytes. Two different procedures for washing the sperm-oocyte complexes (gentle and tough) were used before evaluating the number of bound spermatozoa. The total number of oocytes that bound spermatozoa was significantly lower for both salt-stored and deep frozen oocytes compared with fresh oocytes. Significantly fewer spermatozoa bound to stored oocytes than to fresh oocytes (P 相似文献   

Vitronectin is an intrinsic protein of human spermatozoa released during the acrosome reaction

Evidence has been presented that oolemmal integrins and their ligands on spermatozoa may play a role in gamete interactions leading to fertilization. We previously demonstrated that vitronectin (Vn) could be extracted from fresh human spermatozoa and detected in Western blots, and Vn was observed on the surface of living, capacitated sperm by indirect immunofluorescence. In the present experiments, messenger RNA encoding Vn was detected in human testis poly (A+) RNA using Northern analysis, and Vn was localized within the acrosomal region of ejaculated sperm by immunoperoxidase and immunofluorescence staining. During the acrosome reaction, induced in capacitated spermatoza by lonomycin, Vn was released into the medium in a calcium-dependent manner. Vn appears to be a specific product of intratesticular spermatozoa that is secreted during the acrosome reaction. These findings suggest that Vn is positioned to play a strategic role in gamete interactions leading to fertilization. © Wiley-Liss, Inc.     

Penetration of the zona-free or intact eggs by foreign spermatozoa and the fertilization of deer mouse eggs in vitro

Zona-free eggs were introduced to fresh or preincubated sperm suspensions and the penetration of eggs by foreign spermatozoa was examined, as evidenced by enlargement of the sperm head and formation of the male pronucleus. It was found that zona-free hamster eggs can be penetrated by guinea-pig, deer mouse and rabbit spermatozoa but zona-free rat, mouse and rabbit eggs cannot be penetrated by guinea-pig spermatozoa. Furthermore, zona-free rat and mouse eggs cannot be penetrated by spermatozoa from two species of deer mice and the Mongolian gerbil. The zona pellucida of a few intact rat eggs can be penetrated by mouse (6%) and by P. leucopus spermatozoa (14%) but enlargement of the sperm head and formation of pronuclei were observed in the former but not in the latter. It seems that (1) sperm capacitation is required for the penetration of zona-free eggs, (2) the attachment of foreign spermatozoa to eggs may indicate their potential ability of penetration in some cases, (3) there is a certain affinity between the vitellus of one species and spermatozoa from another species, (4) the block to the entry of foreign spermatozoa is not only in the zona pellucida but also in the vitelline membrane, (5) zona-free hamster eggs can be penetrated by spermatozoa of six species, (6) mouse spermatozoa can penetrate zona-free eggs of three species, and (7) fertilization of intact P. maniculatus eggs can be achieved in vitro.     

Maintenance of motility of fowl spermatozoa in vitro is prolonged by a low molecular weight factor derived from cultured chick embryo cells.

Gel filtration of a conditioned medium composed of the supernatant fluid removed from a 5-day culture of skeletal muscle cells from 9-day-old chick embryos with Bio-Gel P-2 revealed one peak of motility-prolonging activity (about 0.3 kDa), which was not present in fresh medium. Spermatozoa incubated in this fraction of the conditioned medium maintained their motility for at least 36 h at 37 degrees C. Both the formation of lipid peroxide and the leakage of lactic dehydrogenase of spermatozoa incubated in the conditioned medium fraction were lower than those incubated in the corresponding fresh medium. Initial rate of oxygen consumption of the spermatozoa incubated in the conditioned medium fraction increased compared with that of the fresh medium fraction. These results suggest that a low molecular weight factor(s) supplied by cultured cells effectively prolongs the motility of fowl spermatozoa, and that the effect could result from inhibition of the structural damage to the sperm membrane.     

Identification and characterization of a sperm motility promoting glycoprotein from buffalo blood serum

Early investigators reported the occurrence of unidentified protein factors in biological fluids that may regulate sperm motility essential for fertility potential. This study reports for the first time purification of a forward motility stimulating protein (FMSF-I), to apparent homogeneity, from a biological fluid (buffalo blood serum) and its characterization. FMSF-I is the major motility protein of buffalo serum: a rich source of the factor. FMSF showed high protein specificity and affinity for activating forward motility of goat cauda epididymal spermatozoa. The motility promoter at 0.5 microM level showed maximal activity when nearly 60%-70% of spermatozoa expressed forward motility. It is a 66 kDa monomeric acidic protein rich in aspartate, glutamate, and leucine with isoelectric point of 3.7. FMSF: a Mg2+ -dependent protein binds to concanavalin A-agarose and the glycoprotein nature of FMSF has been confirmed by PAS staining. The factor lost activity completely when treated with alpha-mannosidase showing that the sugar part of the protein is essential for its biological activity. FMSF has no species specificity for its motility-activating potential. Sperm surface has specific receptors of FMSF, which is strongly immunogenic. The factor is present in testis and epididymis although liver is its richest source. Motility promoting efficacy of FMSF is markedly higher than the well-known non-protein motility activators: theophylline and bicarbonate or their combination. FMSF is a physiological activator of sperm motility and as a slaughterhouse byproduct it has potentiality for solving some of the problems of animal breeding, conservation of endangered species, and human infertility: a global social problem.     

Flow cytometric sorting of fresh and frozen-thawed spermatozoa in the western lowland gorilla (Gorilla gorilla gorilla)

We adapted flow cytometry technology for high-purity sorting of X chromosome-bearing spermatozoa in the western lowland gorilla (Gorilla gorilla gorilla). Our objectives were to develop methodologies for liquid storage of semen prior to sorting, sorting of liquid-stored and frozen-thawed spermatozoa, and assessment of sorting accuracy. In study 1, the in vitro sperm characteristics of gorilla ejaculates from one male were unchanged (P > 0.05) after 8 hr of liquid storage at 15 degrees C in a non-egg yolk diluent (HEPES-buffered modified Tyrode's medium). In study 2, we examined the efficacy of sorting fresh and frozen-thawed spermatozoa using human spermatozoa as a model for gorilla spermatozoa. Ejaculates from one male were split into fresh and frozen aliquots. X-enriched samples derived from both fresh and frozen-thawed human semen were of high purity, as determined by fluorescence in situ hybridization (FISH; 90.7%+/-2.3%, overall), and contained a high proportion of morphologically normal spermatozoa (86.0%+/-1.0%, overall). In study 3, we processed liquid-stored semen from two gorillas for sorting using a modification of methods for human spermatozoa. The sort rate for enrichment of X-bearing spermatozoa was 7.3+/-2.5 spermatozoa per second. The X-enriched samples were of high purity (single-sperm PCR: 83.7%) and normal morphology (79.0%+/-3.9%). In study 4 we examined frozen-thawed gorilla semen, and the sort rate (8.3+/-2.9 X-bearing sperm/sec), purity (89.7%), and normal morphology (81.4%+/-3.4%) were comparable to those of liquid-stored semen. Depending on the male and the type of sample used (fresh or frozen-thawed), 0.8-2.2% of gorilla spermatozoa in the processed ejaculate were present in the X-enriched sample. These results demonstrate that fresh or frozen-thawed gorilla spermatozoa can be flow cytometrically sorted into samples enriched for X-bearing spermatozoa.     

Immunodetectable galactosyltransferase is associated only with human spermatozoa of high buoyant density

Human ejaculated spermatozoa are heterogeneous and can be separated into two distinct populations according to their respective buoyant densities. In order to investigate the functional differences between these two types of spermatozoa, we have searched for the presence of galactosyltransferase. A Western blot of sperm proteins following their electrophoresis was probed with an anti-galactosyltransferase serum revealing that this enzyme is present in human spermatozoa. Furthermore, galactosyltransferase is detectable only in those proteins isolated from the head of high density spermatozoa. These results suggest that ejaculated spermatozoa consist of two populations that are functionally different.     

Labile Serum Factor and Its Effect on Arbovirus Neutralization

Enhancement of neutralization of Sindbis, Venezuelan equine encephalitis, Eastern equine encephalitis, Western equine encephalitis, and St. Louis encephalitis viruses by labile serum factor (LSF) in human serum and plasma was demonstrated. Human serum and plasma could be diluted 1:8 and 1:16 and still retain some LSF activity. Satisfactory storage temperatures for retention of LSF activity were −20 or −56 C. Repeated freeze-thaw cycles of serum did not alter LSF activity, but the activity was completely eliminated by heating at 56 C for 5 min. LSF of human serum equally enhanced neutralization by Sindbis immune mouse and rabbit sera; these results suggest a lack of species specificity. Rehydrated lyophilized gunea pig complement did not restore LSF activity to heated human plasma. Serum components responsible for LSF activity were not dialyzable. Discovery of fresh serum without LSF activity established the need to pretest all sera used as LSF sources.     

Inhibition by serum of autoimmune aspermatogenic orchitis.

Inhibition by serum of experimental autoimmune orchitis (EAO) was studied in guinea-pigs. It was found that the capacity of an autoantigen, antigen P, purified from guinea-pigs' spermatozoa, to produce lesions of EAO could be inhibited by mixing antigen P with a small amount of normal human serum before injection into guinea-pigs. In protected animals, cell-mediated immunity and humoral antibodies to antigen P were also significantly suppressed.