The temperature coefficients for the oxygen consumption rate of sperm from the rat, mouse, bull, and sea urchin were measured to be within a range of 2.7 to 3.0. For a reduction in temperature from 37°C to 30°C, the respiration rate of mammalian sperm declined by a factor of approximately 2.1. Hence, the availability of oxygen to sustain these sperm substantially increased over this temperature range. In addition, the solubility of oxygen in cauda epididymidal fluid increased by 7.6% for a decline in temperature from 37°C to 30°C. The increased availability of oxygen to sperm at the lower temperature is related to the increased storage capacity of the cauda epididymidis in scrotal mammals. In this context, it is suggested that the evolutionary migration of the cauda epididymidis to a cooler, extra-abdominal location may have been influenced by the increased availability of oxygen to sperm and hence resulted in an increased capacity to sustain and thereby store more sperm per unit volume of duct.
{"title":"Lower temperature of the cauda epididymidis facilitates the storage of sperm by enhancing oxygen availability","authors":"D. Djakiew, R. Cardullo","doi":"10.1002/MRD.1120150305","DOIUrl":"https://doi.org/10.1002/MRD.1120150305","url":null,"abstract":"The temperature coefficients for the oxygen consumption rate of sperm from the rat, mouse, bull, and sea urchin were measured to be within a range of 2.7 to 3.0. For a reduction in temperature from 37°C to 30°C, the respiration rate of mammalian sperm declined by a factor of approximately 2.1. Hence, the availability of oxygen to sustain these sperm substantially increased over this temperature range. In addition, the solubility of oxygen in cauda epididymidal fluid increased by 7.6% for a decline in temperature from 37°C to 30°C. The increased availability of oxygen to sperm at the lower temperature is related to the increased storage capacity of the cauda epididymidis in scrotal mammals. In this context, it is suggested that the evolutionary migration of the cauda epididymidis to a cooler, extra-abdominal location may have been influenced by the increased availability of oxygen to sperm and hence resulted in an increased capacity to sustain and thereby store more sperm per unit volume of duct.","PeriodicalId":12668,"journal":{"name":"Gamete Research","volume":"10 1","pages":"237-245"},"PeriodicalIF":0.0,"publicationDate":"1986-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"73843221","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
We have examined living and fixed gametes and early embryos of surf clams, sea urchins, and hamsters stained with the supravital dyes Hoechst 33342 for DNA and 3,3′-dihexyloxacarbocyanine iodide (DIOC6) for mitochondria and endoplasmic reticulum. Hoechst staining (10 μM) was confined exclusively to egg and sperm chromatin and, in living marine specimens, did not interfere with sperm motility, fertilization, or nuclear activity during meiosis or early embryogenesis. Although Hoechst staining did not appear to affect the motility of hamster sperm, only zonae-free eggs inseminated. Because chromatin retained Hoechst 33342 stain during fertilization, the paternally and maternally derived chromosomes of living and fixed preparations fluoresced and their number, organization, and location within the zygote cytoplasm could be determined. Hence, polyspermy and other nuclear abnormalities were amenable to examination in these stained preparations. DIOC6 staining (8.7 μM) was restricted primarily to the mitochondria of spermatozoa. Eggs stained with DIOC6 (0.87 to 8.7 μM) were brightly fluorescent because of their size and the presence of large numbers of mitochondria and other DIOC6-positive organelles. Sea urchin and surf clam sperm stained with DIOC6 fertilized unstained eggs and the location of the incorporated sperm mitochondrion up to first cleavage was followed. Although hamster sperm stained with DIOC6 were less motile than unstained sperm, they were capable of inseminating only zonae-free eggs. These observations demonstrate that staining with supravital fluorochromes provides a rapid and useful method to analyze macromolecular and organelle changes in a variety of living and fixed gametes and embryos.
{"title":"Examination of living and fixed gametes and early embryos stained with supravital fluorochromes (Hoechst 33342 and 3,3′-dihexyloxacarbocyanine iodide)","authors":"S. Luttmer, F. Longo","doi":"10.1002/MRD.1120150308","DOIUrl":"https://doi.org/10.1002/MRD.1120150308","url":null,"abstract":"We have examined living and fixed gametes and early embryos of surf clams, sea urchins, and hamsters stained with the supravital dyes Hoechst 33342 for DNA and 3,3′-dihexyloxacarbocyanine iodide (DIOC6) for mitochondria and endoplasmic reticulum. Hoechst staining (10 μM) was confined exclusively to egg and sperm chromatin and, in living marine specimens, did not interfere with sperm motility, fertilization, or nuclear activity during meiosis or early embryogenesis. Although Hoechst staining did not appear to affect the motility of hamster sperm, only zonae-free eggs inseminated. Because chromatin retained Hoechst 33342 stain during fertilization, the paternally and maternally derived chromosomes of living and fixed preparations fluoresced and their number, organization, and location within the zygote cytoplasm could be determined. Hence, polyspermy and other nuclear abnormalities were amenable to examination in these stained preparations. DIOC6 staining (8.7 μM) was restricted primarily to the mitochondria of spermatozoa. Eggs stained with DIOC6 (0.87 to 8.7 μM) were brightly fluorescent because of their size and the presence of large numbers of mitochondria and other DIOC6-positive organelles. Sea urchin and surf clam sperm stained with DIOC6 fertilized unstained eggs and the location of the incorporated sperm mitochondrion up to first cleavage was followed. Although hamster sperm stained with DIOC6 were less motile than unstained sperm, they were capable of inseminating only zonae-free eggs. These observations demonstrate that staining with supravital fluorochromes provides a rapid and useful method to analyze macromolecular and organelle changes in a variety of living and fixed gametes and embryos.","PeriodicalId":12668,"journal":{"name":"Gamete Research","volume":"69 1","pages":"267-283"},"PeriodicalIF":0.0,"publicationDate":"1986-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"74127776","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
M. Cameo, Fernanda González Echeverría, J. Blaquier, M. Burgos
The localization of rat epididymal protein DE on cauda epididymis spermatozoa was studied with a specific antibody and the peroxidase antiperoxidase (PAP) immunocyto-chemical reaction. At the light microscopic level, all spermatozoa appeared to be labeled over the dorsal portion of the head, whereas tails were negative. This observation was confirmed using scanning electron microscopy. A large number of particles were seen on the external surface of the plasma membrane covering the acrosomal region and a smaller number on the postacrosomal portion. Flagella appeared free of particles. Sperm suspensions were incubated in conditions that induce capacitation and the acrosome reaction, and, in this instance, the permanence of protein DE on the vesicles and the postacrosomal region of the membrane were observed. The localization of this epididymal protein on the sperm surface is compatible with a role in the gamete interaction process.
{"title":"Immunochemical localization of epididymal protein DE on rat spermatozoa: Its fate after induced acrosome reaction","authors":"M. Cameo, Fernanda González Echeverría, J. Blaquier, M. Burgos","doi":"10.1002/MRD.1120150306","DOIUrl":"https://doi.org/10.1002/MRD.1120150306","url":null,"abstract":"The localization of rat epididymal protein DE on cauda epididymis spermatozoa was studied with a specific antibody and the peroxidase antiperoxidase (PAP) immunocyto-chemical reaction. At the light microscopic level, all spermatozoa appeared to be labeled over the dorsal portion of the head, whereas tails were negative. This observation was confirmed using scanning electron microscopy. A large number of particles were seen on the external surface of the plasma membrane covering the acrosomal region and a smaller number on the postacrosomal portion. Flagella appeared free of particles. Sperm suspensions were incubated in conditions that induce capacitation and the acrosome reaction, and, in this instance, the permanence of protein DE on the vesicles and the postacrosomal region of the membrane were observed. The localization of this epididymal protein on the sperm surface is compatible with a role in the gamete interaction process.","PeriodicalId":12668,"journal":{"name":"Gamete Research","volume":"24 1","pages":"247-257"},"PeriodicalIF":0.0,"publicationDate":"1986-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"84021448","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Sperm-oocyte membrane fusion has been observed during monospermic fertilization of a human oocyte in vitro. Women were stimulated with both clomiphene citrate and human menopausal gonadotropin and were given human chorionic gonadotropin before a LH-surge. Twelve oocytes, collected at laparoscopy from six women who became pregnant by IVF, were allowed to mature for 7–14 hours in vitro and inseminated with preincubated sperm, fixed between 1–3 hours after insemination, and examined by transmission electron microscopy. Membrane fusion had occurred in one ovum 2 hours after insemination, and the oocyte had resumed maturation and was at anaphase II of meiosis. Cortical granules had been exocytosed, and some of their contents were visible at the surface close to the oolemma all around the oocyte. The sperm that fused with this oocyte was acrosome-reacted and had been partly incorporated into the ooplasm, while the anterior two-thirds of its head was phagocytosed by a tongue of cortical ooplasm. Membrane fusion had occurred between the oolemma and the plasma membrane overlying the postacrosomal segment of the sperm head, posterior to the equatorial vestige. Sperm chromatin had not decondensed, and serial sections revealed a midpiece attached to the basal plate and a tail located deeper in the ooplasm, all devoid of plasma membrane. Supplementary sperm penetrating the inner zona, approaching the perivitelline space, had undergone the acrosome reaction but had a persistent vestige of the equatorial segment of the acrosome with intact plasma membrane. Evidence of sperm chromatin decondensation was seen in other oocytes, 3 hours after insemination, which were at telophase II of meiosis. Eight oocytes penetrated by sperm were monospermic, while four were unfertilized. The general pattern of sperm fusion and incorporation appears to conform to that seen in most other mammals. The study also reveals that sperm have to complete the acrosome reaction before fusing with the egg.
{"title":"Sperm-oocyte membrane fusion in the human during monospermic fertilization","authors":"A. Sathananthan, Christopher Chen","doi":"10.1002/MRD.1120150208","DOIUrl":"https://doi.org/10.1002/MRD.1120150208","url":null,"abstract":"Sperm-oocyte membrane fusion has been observed during monospermic fertilization of a human oocyte in vitro. \u0000 \u0000 \u0000 \u0000Women were stimulated with both clomiphene citrate and human menopausal gonadotropin and were given human chorionic gonadotropin before a LH-surge. Twelve oocytes, collected at laparoscopy from six women who became pregnant by IVF, were allowed to mature for 7–14 hours in vitro and inseminated with preincubated sperm, fixed between 1–3 hours after insemination, and examined by transmission electron microscopy. \u0000 \u0000 \u0000 \u0000Membrane fusion had occurred in one ovum 2 hours after insemination, and the oocyte had resumed maturation and was at anaphase II of meiosis. Cortical granules had been exocytosed, and some of their contents were visible at the surface close to the oolemma all around the oocyte. The sperm that fused with this oocyte was acrosome-reacted and had been partly incorporated into the ooplasm, while the anterior two-thirds of its head was phagocytosed by a tongue of cortical ooplasm. Membrane fusion had occurred between the oolemma and the plasma membrane overlying the postacrosomal segment of the sperm head, posterior to the equatorial vestige. Sperm chromatin had not decondensed, and serial sections revealed a midpiece attached to the basal plate and a tail located deeper in the ooplasm, all devoid of plasma membrane. Supplementary sperm penetrating the inner zona, approaching the perivitelline space, had undergone the acrosome reaction but had a persistent vestige of the equatorial segment of the acrosome with intact plasma membrane. Evidence of sperm chromatin decondensation was seen in other oocytes, 3 hours after insemination, which were at telophase II of meiosis. Eight oocytes penetrated by sperm were monospermic, while four were unfertilized. \u0000 \u0000 \u0000 \u0000The general pattern of sperm fusion and incorporation appears to conform to that seen in most other mammals. The study also reveals that sperm have to complete the acrosome reaction before fusing with the egg.","PeriodicalId":12668,"journal":{"name":"Gamete Research","volume":"56 1","pages":"177-186"},"PeriodicalIF":0.0,"publicationDate":"1986-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"79832019","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Depeptidyl peptidase (DPP II) was partially purified from guinea pig testes by (NH4)2SO4 precipitation, Con A-Sepharose 4B chromatography, and Sephadex G-200 chromatography to a specific activity of 27.4 μmol Ala3 hydrolyzed min−1 mg−1 protein. Chromatography on a calibrated G-200 column yielded a molecular weight of 135,000 daltons for the enzyme. Sodium dodecyl sulfate polyacrylamide electrophoresis showed an enrichment of a broad doublet at 64–66,000 daltons. The enzyme had optimal activity toward hydrolysis of L-alanyl-alanyl-alanine at pH 4.5 and showed sensitivity to cations of increasing size with Tris producing the most inhibition of those tested. The enzyme was moderately inhibited by serine proteinase inhibitors. Thin-layer chromatography revealed the dipeptidase nature of the enzyme's activity on tripeptides and dipeptidyl arylamides. A doublet of activity occurred when nitrocellulose electroblots of nondenaturing gel electrophoresis of the (NH4)2SO4 fraction were reacted with the specific DPP II substrate, lysyl-alanyl-4-methoxy-2-napthylamide. Analytical isoelectric focusing of the G-200 fraction followed by fluorescent enzyme activity detection that used cellulose triacetate overlay membranes impregnated with the specific DPP II substrate, lysyl-alanyl-7-amino-4-trifluoromethylcou-marin, revealed multiple isoforms focusing at pI = 4.8–5.6. Two prominent bands focused at pI = 4.9 and pI = 5.1. The properties of guinea pig testicular DPP II are compared and contrasted with similar dipeptidyl peptidases from other sources.
{"title":"Partial purification and characterization of dipeptidyl peptidase II (DPP II) from guinea pig testes","authors":"G. Dicarlantonio, P. Talbot, E. Dudenhausen","doi":"10.1002/MRD.1120150207","DOIUrl":"https://doi.org/10.1002/MRD.1120150207","url":null,"abstract":"Depeptidyl peptidase (DPP II) was partially purified from guinea pig testes by (NH4)2SO4 precipitation, Con A-Sepharose 4B chromatography, and Sephadex G-200 chromatography to a specific activity of 27.4 μmol Ala3 hydrolyzed min−1 mg−1 protein. Chromatography on a calibrated G-200 column yielded a molecular weight of 135,000 daltons for the enzyme. Sodium dodecyl sulfate polyacrylamide electrophoresis showed an enrichment of a broad doublet at 64–66,000 daltons. The enzyme had optimal activity toward hydrolysis of L-alanyl-alanyl-alanine at pH 4.5 and showed sensitivity to cations of increasing size with Tris producing the most inhibition of those tested. The enzyme was moderately inhibited by serine proteinase inhibitors. Thin-layer chromatography revealed the dipeptidase nature of the enzyme's activity on tripeptides and dipeptidyl arylamides. A doublet of activity occurred when nitrocellulose electroblots of nondenaturing gel electrophoresis of the (NH4)2SO4 fraction were reacted with the specific DPP II substrate, lysyl-alanyl-4-methoxy-2-napthylamide. Analytical isoelectric focusing of the G-200 fraction followed by fluorescent enzyme activity detection that used cellulose triacetate overlay membranes impregnated with the specific DPP II substrate, lysyl-alanyl-7-amino-4-trifluoromethylcou-marin, revealed multiple isoforms focusing at pI = 4.8–5.6. Two prominent bands focused at pI = 4.9 and pI = 5.1. The properties of guinea pig testicular DPP II are compared and contrasted with similar dipeptidyl peptidases from other sources.","PeriodicalId":12668,"journal":{"name":"Gamete Research","volume":"15 1","pages":"161-175"},"PeriodicalIF":0.0,"publicationDate":"1986-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"84141883","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Methods for the investigation of cell-associated calcium and intracellular calcium were studied in washed ejaculated human spermatozoa. Experiments using 45Ca2+ indicated that human spermatozoa were permeant to calcium and that a significant proportion of the cellassociated calcium (approximately 50%) was accumulated in the mitochondrion. This necessitated the use of alternative procedures to measure cytoplasmic free calcium. The ability of human spermatozoa to accumulate and de-esterify the intracellular fluorescent calcium indicator Quin-2 was established. Using this technique, the resting level of free intracellular calcium in human spermatozoa was found to be 146.0 ± 19.9 nM, and was significantly elevated upon addition of the divalent cation ionophore ionomycin. In further experiments designed to illustrate the applications of the Quin technique, data was obtained suggesting that the mechanisms controlling intracellular calcium in human spermatozoa are temperature dependent but do not involve voltage-sensitive calcium channels.
{"title":"Measurement of intracellular calcium in human spermatozoa","authors":"D. Irvine, R. Aitken","doi":"10.1002/MRD.1120150107","DOIUrl":"https://doi.org/10.1002/MRD.1120150107","url":null,"abstract":"Methods for the investigation of cell-associated calcium and intracellular calcium were studied in washed ejaculated human spermatozoa. Experiments using 45Ca2+ indicated that human spermatozoa were permeant to calcium and that a significant proportion of the cellassociated calcium (approximately 50%) was accumulated in the mitochondrion. This necessitated the use of alternative procedures to measure cytoplasmic free calcium. The ability of human spermatozoa to accumulate and de-esterify the intracellular fluorescent calcium indicator Quin-2 was established. Using this technique, the resting level of free intracellular calcium in human spermatozoa was found to be 146.0 ± 19.9 nM, and was significantly elevated upon addition of the divalent cation ionophore ionomycin. In further experiments designed to illustrate the applications of the Quin technique, data was obtained suggesting that the mechanisms controlling intracellular calcium in human spermatozoa are temperature dependent but do not involve voltage-sensitive calcium channels.","PeriodicalId":12668,"journal":{"name":"Gamete Research","volume":"1 1","pages":"57-71"},"PeriodicalIF":0.0,"publicationDate":"1986-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"79832012","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
The time taken to dissolve the zona pellucida was compared with fertilizability as well as the meiotic maturation rate of the oocytes from the same (KE × CBA) F2 females. The presence of granular material in oocyte cytoplasm was also examined. It was found that for F2 females in which the zona pellucida digestion was fast, the number of fertilized oocytes was high; for F2 females with zonae pellucidae more resistant to enzyme, the number of fertilized oocytes was low. The correlation between the two characters was significant, indicating their common genetic and/or physiological control. The low or high solubility of zona pellucida did not correlate with the rate of meiotic maturation of the oocyte. This suggests separate factors controlling these two characters. A separate factor seems to control the appearance of granules in cytoplasm since their presence interfered neither with zona pellucida solubility nor with maturation rate of the oocyte.
{"title":"Comparison of some properties of oocytes segregating in F2 hybrids between KE and CBA/Kw inbred strains of mice","authors":"B. Wabik-Sliz","doi":"10.1002/MRD.1120150110","DOIUrl":"https://doi.org/10.1002/MRD.1120150110","url":null,"abstract":"The time taken to dissolve the zona pellucida was compared with fertilizability as well as the meiotic maturation rate of the oocytes from the same (KE × CBA) F2 females. The presence of granular material in oocyte cytoplasm was also examined. It was found that for F2 females in which the zona pellucida digestion was fast, the number of fertilized oocytes was high; for F2 females with zonae pellucidae more resistant to enzyme, the number of fertilized oocytes was low. The correlation between the two characters was significant, indicating their common genetic and/or physiological control. The low or high solubility of zona pellucida did not correlate with the rate of meiotic maturation of the oocyte. This suggests separate factors controlling these two characters. A separate factor seems to control the appearance of granules in cytoplasm since their presence interfered neither with zona pellucida solubility nor with maturation rate of the oocyte.","PeriodicalId":12668,"journal":{"name":"Gamete Research","volume":"38 1","pages":"87-94"},"PeriodicalIF":0.0,"publicationDate":"1986-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"90803365","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Dried smears prepared from vitally stained sperm were evaluated as a method of simultaneously determining sperm viability and acrosomal morphology. A combination Fast Green FCF-Eosin B stain was used. The stained smears were examined at × 1, 250 using differential interference contrast microscopy (DIC). For comparison, the percentage of sperm with intact acrosomes was also determined from wet smears using DIC. Acrosomal morphology was not altered by the staining procedure, as the percentage of intact acrosomes was similar whether quantitated from wet or stained smears. Absence of eosinophilic staining in the acrosome was used as an indication of sperm viability. The percentage of sperm with unstained acrosomes was highly correlated with the percentage of intact acrosomes quantitated from stained smears. Thus, vital staining provided an indication of sperm viability comparable to acrosomal integrity, a highly reliable technique. The major advantages of using dried stained smears were more thorough examination of individual sperm without sperm activity interference, simultaneous evidence of sperm viability and morphology, and the opportunity to delay evaluation. In addition, diluting spermatozoa in complex or simple media with or without egg yolk or follicular fluid did not interfere with subsequent staining or acrosomal evaluation.
从活力染色的精子制备的干涂片被评估为同时测定精子活力和顶体形态的方法。采用Fast Green FCF-Eosin B联合染色。用差示干涉对比显微镜(DIC)在× 1,250下检查染色的涂片。为了进行比较，使用DIC也可以从湿涂片中确定具有完整顶体的精子的百分比。染色过程没有改变顶体形态，因为无论从湿涂片还是染色涂片定量，完整顶体的百分比都是相似的。顶体嗜酸性染色的缺失被用作精子活力的指示。未染色顶体的精子百分比与染色涂片定量的完整顶体百分比高度相关。因此，生命染色提供了精子活力与顶体完整性相当的指标，这是一种高度可靠的技术。使用干燥染色涂片的主要优点是在没有精子活动干扰的情况下对单个精子进行更彻底的检查，同时提供精子活力和形态的证据，并且有机会延迟评估。此外，在含有或不含卵黄或卵泡液的复杂或简单培养基中稀释精子不会影响随后的染色或顶体评估。
{"title":"Vital staining and acrosomal evaluation of bovine sperm","authors":"E. Aalseth, R. G. Saacke","doi":"10.1002/MRD.1120150108","DOIUrl":"https://doi.org/10.1002/MRD.1120150108","url":null,"abstract":"Dried smears prepared from vitally stained sperm were evaluated as a method of simultaneously determining sperm viability and acrosomal morphology. A combination Fast Green FCF-Eosin B stain was used. The stained smears were examined at × 1, 250 using differential interference contrast microscopy (DIC). For comparison, the percentage of sperm with intact acrosomes was also determined from wet smears using DIC. Acrosomal morphology was not altered by the staining procedure, as the percentage of intact acrosomes was similar whether quantitated from wet or stained smears. Absence of eosinophilic staining in the acrosome was used as an indication of sperm viability. The percentage of sperm with unstained acrosomes was highly correlated with the percentage of intact acrosomes quantitated from stained smears. Thus, vital staining provided an indication of sperm viability comparable to acrosomal integrity, a highly reliable technique. The major advantages of using dried stained smears were more thorough examination of individual sperm without sperm activity interference, simultaneous evidence of sperm viability and morphology, and the opportunity to delay evaluation. In addition, diluting spermatozoa in complex or simple media with or without egg yolk or follicular fluid did not interfere with subsequent staining or acrosomal evaluation.","PeriodicalId":12668,"journal":{"name":"Gamete Research","volume":"40 1","pages":"73-81"},"PeriodicalIF":0.0,"publicationDate":"1986-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"86281285","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Fertilized mouse eggs were centrifuged at 5,000g, 10,000g, 15,000g, and 20,000g for 3 and 10 min or at 25,000g for 10 min. The pronuclei of centrifuged eggs became more distinctive than those of uncentrifuged eggs. The proportion of centrifuged and uncentrifuged eggs that developed to blastocysts was not significantly different. There was also no significant difference in the proportion of live fetuses obtained following the transfer of blastocysts developed from centrifuged and uncentrifuged eggs. This study demonstrated that there is no effect of centrifugation on the subsequent development of mouse eggs.
{"title":"Effect of centrifugation of mouse eggs on their development in vitro and in vivo","authors":"K. Nakamura, Y. Tsunoda, T. Nagai, T. Sugie","doi":"10.1002/MRD.1120150109","DOIUrl":"https://doi.org/10.1002/MRD.1120150109","url":null,"abstract":"Fertilized mouse eggs were centrifuged at 5,000g, 10,000g, 15,000g, and 20,000g for 3 and 10 min or at 25,000g for 10 min. The pronuclei of centrifuged eggs became more distinctive than those of uncentrifuged eggs. The proportion of centrifuged and uncentrifuged eggs that developed to blastocysts was not significantly different. There was also no significant difference in the proportion of live fetuses obtained following the transfer of blastocysts developed from centrifuged and uncentrifuged eggs. This study demonstrated that there is no effect of centrifugation on the subsequent development of mouse eggs.","PeriodicalId":12668,"journal":{"name":"Gamete Research","volume":"39 1","pages":"83-86"},"PeriodicalIF":0.0,"publicationDate":"1986-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"73233766","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
In the ram, spermatozoa develop the ability to initiate pregnancy only after reaching the body of the epididymis. To determine the zona pellucida binding ability of ram spermatozoa collected from different levels of the epididymis, sufficient numbers of motile sperm cells of different epididymal origin were inseminated surgically below the uterotubal junction of ewes at the time of ovulation. Intratubal ova were recovered 24 hr later, and those having spermatozoa attached to the zona were examined by transmission electron microscopy to assess the characteristics of the bound spermatozoa. Data indicate that the ability of the capacitated spermatozoa to adhere to the zona pellucida depends on sperm egg binding sites that develop on the acrosomal membranes from the apex to equatorial segment during epididymal transit.
{"title":"Electron microscopic study of the in vivo zona pellucida binding ability of epididymal ram spermatozoa","authors":"S. Fournier-delpech, N. Crozet, M. Courot","doi":"10.1002/MRD.1120140306","DOIUrl":"https://doi.org/10.1002/MRD.1120140306","url":null,"abstract":"In the ram, spermatozoa develop the ability to initiate pregnancy only after reaching the body of the epididymis. To determine the zona pellucida binding ability of ram spermatozoa collected from different levels of the epididymis, sufficient numbers of motile sperm cells of different epididymal origin were inseminated surgically below the uterotubal junction of ewes at the time of ovulation. Intratubal ova were recovered 24 hr later, and those having spermatozoa attached to the zona were examined by transmission electron microscopy to assess the characteristics of the bound spermatozoa. Data indicate that the ability of the capacitated spermatozoa to adhere to the zona pellucida depends on sperm egg binding sites that develop on the acrosomal membranes from the apex to equatorial segment during epididymal transit.","PeriodicalId":12668,"journal":{"name":"Gamete Research","volume":"57 1","pages":"225-234"},"PeriodicalIF":0.0,"publicationDate":"1986-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"90948907","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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