{"title":"Genetic engineering of animals: An agricultural perspective, edited by J. Warren Evans and Alexander Hollaender; Plenum Press, New York, 1986, 328 pp. $49.50","authors":"J. Conover","doi":"10.1002/MRD.1120160111","DOIUrl":"https://doi.org/10.1002/MRD.1120160111","url":null,"abstract":"","PeriodicalId":12668,"journal":{"name":"Gamete Research","volume":"59 1","pages":"93-93"},"PeriodicalIF":0.0,"publicationDate":"1987-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"90054793","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}
It was shown previously that the frequencies of fertilization and pre- and post-implantation embryonic development of mouse oocytes matured in vitro were similar to those of oocytes matured in vivo (Schroeder and Eppig, Dev Biol 102:493–497, 1984). The present study determined the developmental capacity of mouse oocytes after they had been maintained in meiotic arrest in vitro by substances thought to be important regulators of meiosis in vivo. Oocytes were maintained in meiotic arrest for 12 or 24 h in medium containing maturation inhibitor(s), washed free of inhibitor, and cultured 16 h in inhibitor-free (control) medium to permit meiotic maturation. Four different medium supplements were used to maintain meiotic arrest: (1) 100 μM dibutyryl cAMP plus 1 mM hypoxanthine; (2) 4 mM hypoxanthine plus 0.75 mM adenosine (H + AR); (3) 300 μM dibutyryl cAMP; and (4) 50 μM IBMX. Parallel groups of oocytes were treated to the same experimental protocol except that no inhibitory compounds were used; eg, oocytes were cultured a total of 28 or 40 h in control medium that permitted the resumption of maturation. These latter groups tested the effect of extended culture of mature oocytes on subsequent development. Control oocytes were cultured 16 h in control medium. Oocytes were inseminated and subsequently assessed for development to two-cell and blastocyst stages. When oocytes were first cultured 12 or 24 h in medium that maintained meiotic arrest, development to two-cells in all groups but one were within 10% of controls (70%). The 24 h H + AR group was the one exception (47% two-cells). By contrast, culturing oocytes for 28 or 40 h in inhibitor-free medium resulted in a precipitous decrease in development to two cells (27% and 7%, respectively). Blastocyst development followed the same pattern. When uridine (U) was added to H + AR medium, development to two cells was increased significantly. Also, the addition of FSH to the maturation medium significantly increased both two-cell and blastocyst development in the H + AR and H + AR + U groups. Transfer of compacted morulae from the H + AR + U/FSH group into pseudopregnant hosts produced live young 19 days postinsemination. These data demonstrate that prolonged culture of oocytes matured in vitro decreased their capacity to undergo normal development following insemination, but if oocytes were maintained in meiotic arrest during prolonged culture and then allowed to mature spontaneously, their developmental potential was significantly preserved. These results also lend support for a physiological role of cAMP and purines in the maintenance of meiotic arrest in vivo.
先前的研究表明，体外成熟的小鼠卵母细胞的受精频率和着床前和着床后的胚胎发育频率与体内成熟的卵母细胞相似(Schroeder和Eppig, Dev Biol 102:493-497, 1984)。本研究确定了小鼠卵母细胞在体外被认为是体内减数分裂的重要调节因子的物质维持在减数分裂停滞状态后的发育能力。卵母细胞在含有成熟抑制剂的培养基中处于减数分裂停滞状态12或24小时，洗涤不含抑制剂，在无抑制剂(对照)培养基中培养16小时，使减数分裂成熟。四种不同的培养基补充物用于维持减数分裂停滞:(1)100 μM二丁基cAMP加1 mM次黄嘌呤;(2) 4 mM次黄嘌呤+ 0.75 mM腺苷(H + AR);(3) 300 μM二丁基cAMP;(4) 50 μM IBMX。平行的卵母细胞组采用相同的实验方案，但不使用抑制性化合物;例如，卵母细胞在允许恢复成熟的对照培养基中共培养28或40小时。后一组测试了成熟卵母细胞延长培养对后续发育的影响。对照卵母细胞在对照培养基中培养16h。卵母细胞被授精，随后被评估发育到两细胞和囊胚阶段。当卵母细胞在减数分裂停止的培养基中第一次培养12或24小时时，除一组外，所有组的卵母细胞发育为两个细胞的比例都在对照组的10%以内(70%)。24 h h + AR组是唯一例外(47%为2细胞)。相比之下，卵母细胞在无抑制剂培养基中培养28或40小时，发育成两个细胞的几率急剧下降(分别为27%和7%)。囊胚发育遵循同样的模式。在H + AR培养基中加入尿苷(U)后，两种细胞的发育明显增加。此外，在成熟培养基中添加FSH显著提高了H + AR和H + AR + U组的双细胞和囊胚发育。将H + AR + U/FSH组压实的乳剂转移到假孕宿主中，在人工授精后19天产生活的幼崽。这些数据表明，在体外成熟的卵母细胞长时间培养会降低其在授精后正常发育的能力，但如果卵母细胞在长时间培养期间保持减数分裂停滞状态，然后让其自然成熟，则其发育潜力得到显著保留。这些结果也支持cAMP和嘌呤在维持体内减数分裂停止中的生理作用。
{"title":"Developmental capacity of mouse oocytes following maintenance of meiotic arrest in vitro","authors":"S. M. Downs, A. Schroeder, J. Eppig","doi":"10.1002/MRD.1120150404","DOIUrl":"https://doi.org/10.1002/MRD.1120150404","url":null,"abstract":"It was shown previously that the frequencies of fertilization and pre- and post-implantation embryonic development of mouse oocytes matured in vitro were similar to those of oocytes matured in vivo (Schroeder and Eppig, Dev Biol 102:493–497, 1984). The present study determined the developmental capacity of mouse oocytes after they had been maintained in meiotic arrest in vitro by substances thought to be important regulators of meiosis in vivo. Oocytes were maintained in meiotic arrest for 12 or 24 h in medium containing maturation inhibitor(s), washed free of inhibitor, and cultured 16 h in inhibitor-free (control) medium to permit meiotic maturation. Four different medium supplements were used to maintain meiotic arrest: (1) 100 μM dibutyryl cAMP plus 1 mM hypoxanthine; (2) 4 mM hypoxanthine plus 0.75 mM adenosine (H + AR); (3) 300 μM dibutyryl cAMP; and (4) 50 μM IBMX. Parallel groups of oocytes were treated to the same experimental protocol except that no inhibitory compounds were used; eg, oocytes were cultured a total of 28 or 40 h in control medium that permitted the resumption of maturation. These latter groups tested the effect of extended culture of mature oocytes on subsequent development. Control oocytes were cultured 16 h in control medium. Oocytes were inseminated and subsequently assessed for development to two-cell and blastocyst stages. When oocytes were first cultured 12 or 24 h in medium that maintained meiotic arrest, development to two-cells in all groups but one were within 10% of controls (70%). The 24 h H + AR group was the one exception (47% two-cells). By contrast, culturing oocytes for 28 or 40 h in inhibitor-free medium resulted in a precipitous decrease in development to two cells (27% and 7%, respectively). Blastocyst development followed the same pattern. When uridine (U) was added to H + AR medium, development to two cells was increased significantly. Also, the addition of FSH to the maturation medium significantly increased both two-cell and blastocyst development in the H + AR and H + AR + U groups. Transfer of compacted morulae from the H + AR + U/FSH group into pseudopregnant hosts produced live young 19 days postinsemination. \u0000 \u0000 \u0000 \u0000These data demonstrate that prolonged culture of oocytes matured in vitro decreased their capacity to undergo normal development following insemination, but if oocytes were maintained in meiotic arrest during prolonged culture and then allowed to mature spontaneously, their developmental potential was significantly preserved. These results also lend support for a physiological role of cAMP and purines in the maintenance of meiotic arrest in vivo.","PeriodicalId":12668,"journal":{"name":"Gamete Research","volume":"2 1","pages":"305-316"},"PeriodicalIF":0.0,"publicationDate":"1986-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"75382425","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}
For optimal extraction and reactivation of ram sperm, glutamate, dithiothreitol, magnesium, and cyclic AMP were required in a medium of pH 7.9. On extraction with 0.01 % Triton X-100, ram sperm were only partially demembranated, and extensive areas of plasma membrane remained intact especially in the midpiece region. Treatment with 0.1% Triton X-100 removed all plasma membranes and extracted the mitochondrial membrane and matrix. In the absence of ATP, 16.6% ± 0.4 of the partially demembranated sperm were motile, but sperm extracted with 0.1% Triton X-100 were completely immotile. On adding ATP partially demembranated sperm reactivated better (81.6% ± 2.8) than sperm completely demembranated in 0.1 % Triton X-100 (39.5% ± 4.6). The release of intracellular LDH rose linearly with increasing concentrations of the detergent from 0.01 to 0.05%, at which it plateaued. There was a significant increase in beat frequency and forward velocity of partially demembranated sperm when treated with ATP. Partially demembranated sperm had intact mitochondria that presumably were still able to produce ATP, although the spermatozoan movement was stimulated by exogenous ATP.
{"title":"Effect of triton X‐100 on ultrastructure, reactivation, and motility characteristics of ram spermatozoa","authors":"R. Vishwanath, M. A. Swan, I. White","doi":"10.1002/MRD.1120150408","DOIUrl":"https://doi.org/10.1002/MRD.1120150408","url":null,"abstract":"For optimal extraction and reactivation of ram sperm, glutamate, dithiothreitol, magnesium, and cyclic AMP were required in a medium of pH 7.9. On extraction with 0.01 % Triton X-100, ram sperm were only partially demembranated, and extensive areas of plasma membrane remained intact especially in the midpiece region. Treatment with 0.1% Triton X-100 removed all plasma membranes and extracted the mitochondrial membrane and matrix. In the absence of ATP, 16.6% ± 0.4 of the partially demembranated sperm were motile, but sperm extracted with 0.1% Triton X-100 were completely immotile. On adding ATP partially demembranated sperm reactivated better (81.6% ± 2.8) than sperm completely demembranated in 0.1 % Triton X-100 (39.5% ± 4.6). The release of intracellular LDH rose linearly with increasing concentrations of the detergent from 0.01 to 0.05%, at which it plateaued. There was a significant increase in beat frequency and forward velocity of partially demembranated sperm when treated with ATP. Partially demembranated sperm had intact mitochondria that presumably were still able to produce ATP, although the spermatozoan movement was stimulated by exogenous ATP.","PeriodicalId":12668,"journal":{"name":"Gamete Research","volume":"11 1","pages":"361-371"},"PeriodicalIF":0.0,"publicationDate":"1986-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"81670863","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}
A. Sathananthan, S. Ng, R. Edirisinghe, S. Ratnam, P. C. Wong
The early events of gamete membrane fusion and sperm incorporation are portrayed, which are possibly the earliest pictures of human conception on record.Oocytes recovered at laparoscopy from an in vitro fertilization programme using clomiphene and human menopausal and chorionic gonadotrophin stimulation were zona-punctured and examined for polyspermic penetration 3–6 hours after insemination. Three eggs were penetrated, and one donor became pregnant in the same cycle.Fusion occurred between the oolemma and the midsegment of the sperm cell membrane extending from the equatorial vestige of the acrosome to the anterior region of the postacrosomal segment. Only acrosome-reacted sperm were capable of fusing with the egg. Fusion was followed by incorporation of the spermhead into the ooplasm by a process akin to phagocytosis. Sperm chromatin decondensation occurred by progressive inflation and disorganization of the original nuclear envelope.The mechanism of gamete fusion and sperm incorporation resembles that observed during human monospermic fertilization and generally conforms to that reported for eutherian mammals.
{"title":"Human sperm‐egg interaction in vitro","authors":"A. Sathananthan, S. Ng, R. Edirisinghe, S. Ratnam, P. C. Wong","doi":"10.1002/MRD.1120150405","DOIUrl":"https://doi.org/10.1002/MRD.1120150405","url":null,"abstract":"The early events of gamete membrane fusion and sperm incorporation are portrayed, which are possibly the earliest pictures of human conception on record.Oocytes recovered at laparoscopy from an in vitro fertilization programme using clomiphene and human menopausal and chorionic gonadotrophin stimulation were zona-punctured and examined for polyspermic penetration 3–6 hours after insemination. Three eggs were penetrated, and one donor became pregnant in the same cycle.Fusion occurred between the oolemma and the midsegment of the sperm cell membrane extending from the equatorial vestige of the acrosome to the anterior region of the postacrosomal segment. Only acrosome-reacted sperm were capable of fusing with the egg. Fusion was followed by incorporation of the spermhead into the ooplasm by a process akin to phagocytosis. Sperm chromatin decondensation occurred by progressive inflation and disorganization of the original nuclear envelope.The mechanism of gamete fusion and sperm incorporation resembles that observed during human monospermic fertilization and generally conforms to that reported for eutherian mammals.","PeriodicalId":12668,"journal":{"name":"Gamete Research","volume":"14 1","pages":"317-326"},"PeriodicalIF":0.0,"publicationDate":"1986-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"89202168","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. Brito, J. Figueroa, J. Vera, P. A. Cortés, R. Hott, L. Burzio
To isolate the bull sperm outer dense fibers, the cells were treated at room temperature with 0.05% cetyltrimethylammonium bromide and 7 mM 2-mercaptoethanol. This treatment solubilized most of the sperm structure except for the sperm head and the fibrillar complex. The latter was purified by discontinuous sucrose gradient centrifugation. Electron microscopy showed that in the isolated complex each fiber remains attached to the corresponding segmented column of the connecting piece. Analysis of the fibrillar complex by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed three major components with molecular weights of 85,000, 33,000, and 11,000. These were purified, and their amino acid composition was found to be similar to that of the polypeptides of rat sperm outer dense fibers. Furthermore the components of 85,000 and 33,000 daltons contain about 2 mol of phosphoserine per mole of polypeptide, which indicates that they are phosphoproteins.
用0.05%十六烷基三甲基溴化铵和7 mM 2-巯基乙醇在室温下处理，分离公牛精子外致密纤维。这种处理溶解了大部分精子结构，除了精子头和纤维复合体。后者通过不连续蔗糖梯度离心纯化。电子显微镜显示，在分离的复合体中，每根纤维仍然附着在连接片的相应分段柱上。在十二烷基硫酸钠的存在下，用聚丙烯酰胺凝胶电泳对纤维复合物进行分析，发现其分子量分别为85,000,33,000和11,000。对其进行了纯化，发现其氨基酸组成与大鼠精子外密纤维的多肽相似。此外，85,000道尔顿和33,000道尔顿的成分每摩尔多肽含有约2mol磷酸丝氨酸，这表明它们是磷酸化蛋白。
{"title":"Phosphoproteins are structural components of bull sperm outer dense fiber","authors":"M. Brito, J. Figueroa, J. Vera, P. A. Cortés, R. Hott, L. Burzio","doi":"10.1002/MRD.1120150406","DOIUrl":"https://doi.org/10.1002/MRD.1120150406","url":null,"abstract":"To isolate the bull sperm outer dense fibers, the cells were treated at room temperature with 0.05% cetyltrimethylammonium bromide and 7 mM 2-mercaptoethanol. This treatment solubilized most of the sperm structure except for the sperm head and the fibrillar complex. The latter was purified by discontinuous sucrose gradient centrifugation. Electron microscopy showed that in the isolated complex each fiber remains attached to the corresponding segmented column of the connecting piece. \u0000 \u0000 \u0000 \u0000Analysis of the fibrillar complex by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed three major components with molecular weights of 85,000, 33,000, and 11,000. These were purified, and their amino acid composition was found to be similar to that of the polypeptides of rat sperm outer dense fibers. Furthermore the components of 85,000 and 33,000 daltons contain about 2 mol of phosphoserine per mole of polypeptide, which indicates that they are phosphoproteins.","PeriodicalId":12668,"journal":{"name":"Gamete Research","volume":"74 1","pages":"327-336"},"PeriodicalIF":0.0,"publicationDate":"1986-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"76524244","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}
Freshly ejaculated human spermatozoa exposed to 10 μM calcium ionophore A23187 for 30 minutes quickly bound to and penetrated into zona-free hamster eggs. When these eggs were cultured for 13 hours in the presence of low concentrations of vinblastin and podophyllotoxin, then fixed and spread with the use of a cold fixation technique, haploid sets of sperm chromosomes were demonstrated. The number of spermatozoa that were attached to the egg surface at insemination influenced the success rate of chromosome demonstration. The best results (an average of about 0.7 chromosome set per egg) were obtained when insemination was controlled so that an average of seven spermatozoa attached to each egg at the end of a 15–30-minute insemination period. This improved method for demonstrating human sperm chromosomes would be useful in the analysis of paternal genetic contributions in reproduction as well as in assessing the effects of environmental genotoxic agents or conditions on spermatozoa.
{"title":"Human sperm chromosomes in zona-free hamster eggs: pretreatment of spermatozoa with calcium ionophore facilitates later examination of chromosomes","authors":"H. Wramsby, H. Wramsby, R. Yanagimachi","doi":"10.1002/MRD.1120150403","DOIUrl":"https://doi.org/10.1002/MRD.1120150403","url":null,"abstract":"Freshly ejaculated human spermatozoa exposed to 10 μM calcium ionophore A23187 for 30 minutes quickly bound to and penetrated into zona-free hamster eggs. When these eggs were cultured for 13 hours in the presence of low concentrations of vinblastin and podophyllotoxin, then fixed and spread with the use of a cold fixation technique, haploid sets of sperm chromosomes were demonstrated. The number of spermatozoa that were attached to the egg surface at insemination influenced the success rate of chromosome demonstration. The best results (an average of about 0.7 chromosome set per egg) were obtained when insemination was controlled so that an average of seven spermatozoa attached to each egg at the end of a 15–30-minute insemination period. This improved method for demonstrating human sperm chromosomes would be useful in the analysis of paternal genetic contributions in reproduction as well as in assessing the effects of environmental genotoxic agents or conditions on spermatozoa.","PeriodicalId":12668,"journal":{"name":"Gamete Research","volume":"133 1","pages":"295-303"},"PeriodicalIF":0.0,"publicationDate":"1986-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"86224875","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}
Direct isolation of the sea urchin egg vitelline envelope with intact sperm receptors is difficult because the envelope is firmly attached to the egg plasma membrane. We now report a method for producing an inseminated egg preparation in Strongylocentrotus purpuratus (using soybean trypsin inhibitor [STI] and Ca2+, Mg2+-free seawater) that contains an elevated vitelline envelope (VE*-STI). The VE*-STI is devoid of cortical granule material, and supernumerary sperm do not detach postinsemination, suggesting that the VE*-STI contains active sperm receptors. VE*-STIs contain a 305-kD polypeptide and additional components that range from 225 to 31 kD, whereas the 305-kD polypeptide was considerably reduced in VE*s. Electrophoresis of sperm receptor hydrolase digests of VE*-STIs showed that the 305-kD polypeptide and several other envelope polypeptides are protease substrates. Univalent Fab fragments against VE*s, VE*-STIs, and 305 and 225-kD polypeptides blocked sperm binding and fertilization in an Fab concentration-dependent manner. The 305 and 225-kD polypeptides were localized in the VE*-STI using indirect immunofluorescence. Enzyme-linked immunosorbent assays showed that the 305 and 225-kD polypeptides share determinants, suggesting that the 225-kD polypeptide may be derived from the 305-kD polypeptide by the proteolysis that occurs at the cell surface during fertilization. Fab fragments against S purpuratus VE*-STI antigens neither bound to nor blocked homologous sperm binding and fertilization of Lytechinus variegatus eggs. Cross fertilizability occurred to the extent of 5% or less between L variegatus and S purpuratus, therefore, we conclude that the 305 kD-polypeptide isolated from S purpuratus is a species-specific vitelline envelope sperm receptor.
{"title":"Immunological evidence that a 305‐kilodalton vitelline envelope polypeptide isolated from sea urchin eggs is a sperm receptor","authors":"M. Acevedo-Duncan, E. Carroll","doi":"10.1002/MRD.1120150407","DOIUrl":"https://doi.org/10.1002/MRD.1120150407","url":null,"abstract":"Direct isolation of the sea urchin egg vitelline envelope with intact sperm receptors is difficult because the envelope is firmly attached to the egg plasma membrane. We now report a method for producing an inseminated egg preparation in Strongylocentrotus purpuratus (using soybean trypsin inhibitor [STI] and Ca2+, Mg2+-free seawater) that contains an elevated vitelline envelope (VE*-STI). The VE*-STI is devoid of cortical granule material, and supernumerary sperm do not detach postinsemination, suggesting that the VE*-STI contains active sperm receptors. VE*-STIs contain a 305-kD polypeptide and additional components that range from 225 to 31 kD, whereas the 305-kD polypeptide was considerably reduced in VE*s. Electrophoresis of sperm receptor hydrolase digests of VE*-STIs showed that the 305-kD polypeptide and several other envelope polypeptides are protease substrates. Univalent Fab fragments against VE*s, VE*-STIs, and 305 and 225-kD polypeptides blocked sperm binding and fertilization in an Fab concentration-dependent manner. The 305 and 225-kD polypeptides were localized in the VE*-STI using indirect immunofluorescence. Enzyme-linked immunosorbent assays showed that the 305 and 225-kD polypeptides share determinants, suggesting that the 225-kD polypeptide may be derived from the 305-kD polypeptide by the proteolysis that occurs at the cell surface during fertilization. Fab fragments against S purpuratus VE*-STI antigens neither bound to nor blocked homologous sperm binding and fertilization of Lytechinus variegatus eggs. Cross fertilizability occurred to the extent of 5% or less between L variegatus and S purpuratus, therefore, we conclude that the 305 kD-polypeptide isolated from S purpuratus is a species-specific vitelline envelope sperm receptor.","PeriodicalId":12668,"journal":{"name":"Gamete Research","volume":"95 1","pages":"337-359"},"PeriodicalIF":0.0,"publicationDate":"1986-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"73572233","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 majority of the spermatozoa precapacitated in Ca2+-free medium underwent the acrosome raction rapidly when they were transferred to Ca2+-containing medium. The presence of Na+ and Ca2+ in the medium was essential for the acrosome reaction. The vast majority of spermatozoa failed to undergo the reaction in Ca2+ medium lacking monovalent ions, although they remained motile. At the concentration of 140 mM, Na+, K+, Rb+, and Cs+ all supported the reaction at the maximum level, but at 50 mM the latter three ions were not as effective as Na+. Li+ was least effective in supporting the reaction. Virtually no acrosome reactions took place when precapacitated spermatozoa were first exposed to Na+ medium (no Ca2+) and then to Ca2+ medium (no Na+). On the other hand, a considerably higher proportion of spermatozoa acrosome reacted when they were exposed to these media in the reverse order. The most efficient acrosome reactions took place when the medium contained both a monovalent ion (Na+) and Ca2+ simultaneously. Possible mechanisms by which monovalent and divalent cations participate in the acrosome reaction are discussed.
{"title":"Requirement of monovalent cations in the acrosome reaction of guinea pig spermatozoa","authors":"A. Bhattacharyya, E. Roldan, R. Yanagimachi","doi":"10.1002/MRD.1120150402","DOIUrl":"https://doi.org/10.1002/MRD.1120150402","url":null,"abstract":"The majority of the spermatozoa precapacitated in Ca2+-free medium underwent the acrosome raction rapidly when they were transferred to Ca2+-containing medium. The presence of Na+ and Ca2+ in the medium was essential for the acrosome reaction. The vast majority of spermatozoa failed to undergo the reaction in Ca2+ medium lacking monovalent ions, although they remained motile. At the concentration of 140 mM, Na+, K+, Rb+, and Cs+ all supported the reaction at the maximum level, but at 50 mM the latter three ions were not as effective as Na+. Li+ was least effective in supporting the reaction. Virtually no acrosome reactions took place when precapacitated spermatozoa were first exposed to Na+ medium (no Ca2+) and then to Ca2+ medium (no Na+). On the other hand, a considerably higher proportion of spermatozoa acrosome reacted when they were exposed to these media in the reverse order. The most efficient acrosome reactions took place when the medium contained both a monovalent ion (Na+) and Ca2+ simultaneously. Possible mechanisms by which monovalent and divalent cations participate in the acrosome reaction are discussed.","PeriodicalId":12668,"journal":{"name":"Gamete Research","volume":"4 1","pages":"285-294"},"PeriodicalIF":0.0,"publicationDate":"1986-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"87060375","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}
Hyperthermia-induced X-Y dissociation has been observed in diakinesis-metaphase I sper-matocytes but not in pachytene spermatocytes, which have been implicated as highly susceptible to heat stress. To determine X-Y dissociation in pachytene spermatocytes and to compare levels of dissociation between pachytene and diakinesis-metaphase I spermatocytes male ICR mice were exposed to 35°C ± 0.07°C and 65% ± 0.14% relative humidity for 2 or 4 days. Control mice were housed at 24°C ± 0.04°C and 43% ± 0.58% relative humidity. Mice were killed 0, 3, 5, 8, or 10 days after stress and the testes processed for meiotic chromosome display at diakinesis-metaphase I and synaptonemal complex display at pachynema. Twenty-five to thirty cells per mouse at both stages of meiosis were observed with light microscopy, and pachytene spreads with transmission electron microscopy to determine heat-stress effects on synaptonemal complex structure. Statistical analyses revealed significant linear increases in X-Y dissociation with prolonged exposure to heat at pachynema and diakinesis-metaphase I. Levels of pachytene dissociation were one-half the levels of dissociation at diakinesis-metaphase I. The resolvable structure of the lateral elements of the synaptonemal complex was not affected by heat stress.
{"title":"X-Y chromosome univalency in the testes of hyperthermic mice. II: Light and electron microscopic analysis of synaptonemal complexes","authors":"G. Waldbieser, C. Chrisman","doi":"10.1002/MRD.1120150307","DOIUrl":"https://doi.org/10.1002/MRD.1120150307","url":null,"abstract":"Hyperthermia-induced X-Y dissociation has been observed in diakinesis-metaphase I sper-matocytes but not in pachytene spermatocytes, which have been implicated as highly susceptible to heat stress. To determine X-Y dissociation in pachytene spermatocytes and to compare levels of dissociation between pachytene and diakinesis-metaphase I spermatocytes male ICR mice were exposed to 35°C ± 0.07°C and 65% ± 0.14% relative humidity for 2 or 4 days. Control mice were housed at 24°C ± 0.04°C and 43% ± 0.58% relative humidity. Mice were killed 0, 3, 5, 8, or 10 days after stress and the testes processed for meiotic chromosome display at diakinesis-metaphase I and synaptonemal complex display at pachynema. Twenty-five to thirty cells per mouse at both stages of meiosis were observed with light microscopy, and pachytene spreads with transmission electron microscopy to determine heat-stress effects on synaptonemal complex structure. Statistical analyses revealed significant linear increases in X-Y dissociation with prolonged exposure to heat at pachynema and diakinesis-metaphase I. Levels of pachytene dissociation were one-half the levels of dissociation at diakinesis-metaphase I. The resolvable structure of the lateral elements of the synaptonemal complex was not affected by heat stress.","PeriodicalId":12668,"journal":{"name":"Gamete Research","volume":"1 1","pages":"259-265"},"PeriodicalIF":0.0,"publicationDate":"1986-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"84265273","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}
Sea urchin eggs secrete esteroproteolytic activity at fertilization. This enzyme has been shown to be proteolytic toward embryo protein and casein, but a systematic study of its substrate specificity has not been done. In this communication we present data that demonstrates for the first time that the cortical granule protease from Strongylocentrotus purpuratus eggs cleaves arginyl residues in a protein substrate, lysozyme. We have developed a sensitive reverse-phase high-performance liquid chromatography (RP-HPLC) assay that detects femtomole levels of trypsin and chymotrypsin protease activity [Green, 1986: Anal Biochem 152:83–88]. In the sea urchin system, we have detected protease activity from as few as 50 eggs. Correlating the RP-HPLC analysis with a spectrophotometric Nα-benzoyl-L-arginine ethyl ester assay, we have found that each egg secretes approximately 40 attomoles of trypsin-like activity. This general method should be quite useful in investigations into the natural substrate of the egg protease.
{"title":"Sea urchin egg-cortical granule protease has arginyl proteolytic specificity","authors":"Jeffrey D. Green","doi":"10.1002/MRD.1120150304","DOIUrl":"https://doi.org/10.1002/MRD.1120150304","url":null,"abstract":"Sea urchin eggs secrete esteroproteolytic activity at fertilization. This enzyme has been shown to be proteolytic toward embryo protein and casein, but a systematic study of its substrate specificity has not been done. In this communication we present data that demonstrates for the first time that the cortical granule protease from Strongylocentrotus purpuratus eggs cleaves arginyl residues in a protein substrate, lysozyme. We have developed a sensitive reverse-phase high-performance liquid chromatography (RP-HPLC) assay that detects femtomole levels of trypsin and chymotrypsin protease activity [Green, 1986: Anal Biochem 152:83–88]. In the sea urchin system, we have detected protease activity from as few as 50 eggs. Correlating the RP-HPLC analysis with a spectrophotometric Nα-benzoyl-L-arginine ethyl ester assay, we have found that each egg secretes approximately 40 attomoles of trypsin-like activity. This general method should be quite useful in investigations into the natural substrate of the egg protease.","PeriodicalId":12668,"journal":{"name":"Gamete Research","volume":"13 1","pages":"227-236"},"PeriodicalIF":0.0,"publicationDate":"1986-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"82467298","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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