摘要:

AbstractPorcine morulae were bisected by a glass needle after softening of zonae pcllucidae by pronase followed by a treatment with 0.05% trypsin/0,02% EDTA for decreasing intercel lular junction of blastomeres. Transfer of 49 half‐morulae to three recipients resulted in one pregnancy. The blastocysts were bisected symmetrically so as to leave a cellular bridge between the sister half‐embryos after the softening of zonae followed by or without the trypsin/EDTA treatment. Transfer of 47 monozygotic (MZ) pairs of half‐blastocysts to nine recipients resulted in four pregnancies. A litter of nine fetuses was obtained from seven MZ pairs of half‐blastocysts, demonstrating that at least two pairs of MZ twin fetuses were produced. It is thought that the procedure for bisecting blastocysts developed in this study is one of the potential methods of producing porcine M

ISSN:0148-7280    

DOI:10.1002/mrd.1120230102

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1989

数据来源: WILEY

摘要:

AbstractThe influence of the source of pregnant mares' serum gonadotropin (PMSG) on the num ber, quality, and in vitro development of mouse embryos before and after freezing was evaluated among three genotypes: N:NIH(S), C57BL/6N, and C3H/HeN‐MTV−. Immature females were given PMSG from one of five commercial sources. Following col lection ( 116 hr later), embryos were evaluated for stage of development, and four‐to eight‐cell embryos were pooled within genotype and assigned to standardized fresh or freeze‐thaw culture trials. Different PMSG sources stimulated the production of different num bers of total embryos (P<0.05) but not necessarily more embryos suitable for freezing. Differences in embryo production among genotypes indicated that absolute embryo num bers using a single mouse genotype may not accurately reflect the potency of a specific gonadotropin source. The PMSG source also affected the ability of an embryo to survive in culture either immediately after collection or after frozen storage. The effect, however, was genotype specific, with some mouse strains being relatively insensitive to PMSG source, whereas gonadotropin source played a major role in determining in vitro viability in others. Development rates for freshly collected embryos differed, often inconsistently, from those of thawed embryos regardless of the PMSG source used, demonstrating that fresh embryo development cannot be used to estimate expected post‐thaw survival. In vitro development of thawed embryos is influenced not only by genotype, but also the source of the gonadotropin used to promote follicular development and oocyte maturation. These findings may explain, in part, the wide variation in embryo viability and culture rates reported among laboratories and intraspecies animal

ISSN:0148-7280    

DOI:10.1002/mrd.1120230103

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1989

数据来源: WILEY

摘要:

AbstractDue to the central role the acrosomal region plays in sperm‐egg interactions, monoclonal antibodies (mAbs) were used to identify components of this domain in mouse sperm. Sev eral sperm proteins that localize specifically to the anterior acrosomal region are described here in terms of electrophoretic mobility, susceptibility to proteolytic degradation, and post‐translational modification during epididymal transit. Six different mAbs were used, each recognizing a distinctive antigen (Ag) or set of Ags in cauda epididymal mouse sperm: a doublet of 185/200 Kd (M42 mAb); 150‐160 Kd (M5 mAb); 105 Kd (W71 mAb); 21, 35, and 60 Kd (M41 mAb); 27 and 33 Kd (W33 mAb); and 57 and 86 Kd (W108 mAb). Previously reported work implicates two of these, M42 Ag and M5 Ag, as participants in sperm‐zona interaction (Saling and Lakoski:Biol Reprod33:527‐536, 1985; Saling:Dev Biol117:511‐519, 1986; and Lakoski et al.:Biol Reprod38:221‐233, 1988).Recognition of some (M42, M5, W108), but not all (W33), of the Ags by their corre sponding mAbs was affected by sperm incubation with proteases (trypsin or collagenase). Evidence of post‐translational modification during epididymal maturation was suggested by altered electrophoretic mobility of several of the Ags (M42, M5, W33, and W108) accompanying sperm transit from proximal to distal epididymis. Retention of sperm within the caput epididymis prevented structural alterations for the four proteins exam ined, indicating that spatial rather than temporal factors are critical for Ag modification in matur

ISSN:0148-7280    

DOI:10.1002/mrd.1120230104

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1989

数据来源: WILEY

摘要:

AbstractIt has been proposed that reduced glutathione (GSH) or other thiol reagents may partici pate in the basic mechanism by which sperm‐decondensing activity is accomplished. How ever, in vitro, these reagents seem to be inactive and require the presence of other chemi cals, usually detergents. Heparin binds specifically to the sperm membrane and provokes the decondensation of human sperm and the activation of DNA transcription and synthe sis. However, the concentrations at which these effects occur seem to be higher than those expected under physiological conditions. In the present study, thiol reagents at 10 mM concentration, either alone or combined, were completely ineffective in inducing any sig nificant nuclear decondensation after prolonged exposures (24 hr) of incubation. Heparin, 153.8 μM, was capable of inducing only a small increase in nuclear swelling. However, GSH at concentrations as low as 0.1 mM in combination with heparin induces deconden sation of human sperm nuclei in vitro. When GSH concentration was kept constant at 5 mM, nuclear decondensation was induced with heparin at concentrations as low as 11.6 μM, and a maximal decondensation (90%) was obtained with only 21.6 μM of heparin. The latter is more than ten times less than the minimal active concentration of heparin used a

ISSN:0148-7280    

DOI:10.1002/mrd.1120230105

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1989

数据来源: WILEY

摘要:

AbstractCalcium‐binding proteins (CBPs) of boar spermatozoa and boar seminal plasma were identified by using a45Ca overlay technique to detect these proteins on transblots of PAGE‐separated proteins. A single CBP (Mr ∼ 300 kDa) was detected in seminal plasma. This protein binds specifically to the plasma membrane overlying the principal segment and is removed from sperm during capacitation. The protein was purified for further charac terization by anion exchange chromatography and gel filtration. In addition, six major proteins (30, 35, 38, 42, 52, and 66 kDa) which do not originate from accessory gland secretions were found to be strongly associated with the plasma membrane. Most of these proteins are not integral to the membrane and appear to develop an association with the plasma membrane during cpididymai maturation. Similarly, calmodulin‐binding proteins appear to develop strong associations with the plasma membrane during epididymal

ISSN:0148-7280    

DOI:10.1002/mrd.1120230106

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1989

数据来源: WILEY

摘要:

AbstractA study of spermatozoa in the isthmus of the oviduct and of the surrounding epithelial cells in the dasyurid marsupial,Sminthopsis crassicaudata, was carried out. At least 10% of the ejaculated spermatozoa probably populate the isthmus region, where many come to reside in crypts until around the time of ovulation. Ultrastructural observations of spermatozoa in this region indicated that they had intact acrosomes and were identical in their morphology with those in the cauda epididymidis. After ovulation spermatozoa rapidly disappeared, some of which may be phagocytosed by the cells lining the crypts. These epithelial cells were also found to have many large, electron‐dense granules at the time of sperm storage, but the contents did not appear to be released until the zygotes passed along the tract. The secretory activity of these cells may thus relate more to the production of the shell membrane that comes to surround the zygote than to the cells performing a nutritive or protective function for the spermatozoa during their period of storage within the female reproductive trac

ISSN:0148-7280    

DOI:10.1002/mrd.1120230107

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1989

数据来源: WILEY

摘要:

AbstractMouse and human spermatozoa, but not rabbit spermatozoa, have long been known to be sensitive to loss of motility induced by exogenous H2O2. Recent work has shown that loss of sperm motility in these species correlates with the extent of spontaneous lipid peroxidation. In this study, the effect of H2O2on this reaction in sperm of the three species was investi gated. The rate of spontaneous lipid peroxidation in mouse and human sperm is markedly enhanced in the presence of 1‐5 mM H2O2, while the rate in rabbit sperm is unaffected by H2O2. The enhancement of lipid peroxidation, the rate of reaction of H2O2with the cells, the activity of sperm glutathione peroxidase, and the endogenous glutathione content are highest in mouse sperm, intermediate in human sperm, and very low in rabbit sperm. Inac tivation of glutathione peroxidase occurs in the presence of H2O2due to complete conver sion of endogenous glutathione to GSSG: No GSH is available as electron donor substrate to the peroxidase. Inactivation of glutathione peroxidase by the inhibitor mercaptosucci nate has the same effect on rate of lipid peroxidation and loss of motility in mouse and human sperm as does H2O2. This implies that H2O2by itself at 1‐5 mM is not intrinsically toxic to the cells. With merceptosuccinate, the endogenous glutathione is present as GSH in mouse and human sperm, indicating that the redox state of intracellular glutathione by itself plays little role in protecting the cell against spontaneous lipid peroxidation. Mouse and human sperm also have high rates of superoxide production. We conclude that the key intermediate in spontaneous lipid peroxidation is lipid hydroperoxide generated by a chain reaction initiated by and utilizing superoxide. Removal of this hydroperoxide by gluta thione peroxidase protects these sperm against peroxidation; inactivation of the peroxidase allows lipid hydroperoxide to increase and so increases the peroxidation rate. Rabbit sperm have low rates of superoxide reaction due to high activity of their superoxide dismutase; lack of endogenous glutathione and low peroxidase activity does not affect their rate or lipid peroxidation. As a result, these sperm are not affected by either H2O2or mercapto‐succinate. These results lead us to postulate a mechanism for spontaneous lipid peroxida tion in mammalian sperm which involves reaction of lipid hydroperoxide and O2as the rate‐determini

ISSN:0148-7280    

DOI:10.1002/mrd.1120230108

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1989

数据来源: WILEY

摘要:

AbstractThe polypeptide composition of unfertilized, fertilized, and protease‐treated zona‐free mouse eggs was evaluated in this study. Zona‐free eggs were radioiodinated by an Iodogen‐catalyzed reaction. Light microscopic autoradiography of egg sections revealed that labeling was restricted to the cell surface. Labeled eggs were solubilized, and cell surface polypeptides were identified by one‐dimensional SDS polyacrylamide gel electro‐phoresis and autoradiography. The unfertilized egg demonstrated 8–10 peptides that incorporated125I, with major bands observed at approximately 145–150, 94, and 23 kilo‐daltons (kD). Zona‐free eggs fertilized in vitro and then radiolabeled demonstrated several new bands in comparison to unfertilized eggs, with a major band appearing at approxi mately 36 kD. Treatment of radiolabeled unfertilized eggs with either trypsin or chymo‐trypsin (1 mg/ml for 5–20 min) caused enzyme‐specific modifications in labeled polypep tides. Trypsin (T) treatment resulted in time‐dependant modification of the three major peptides at 145–150,94, and 23 kD. Chymotrypsin (CT) treatment, in contrast, was asso ciated with loss or modification of the 94 kD band, with no apparent effect on either the 145–150 or 23 kD band. Taken together with previous data indicating that T or CT egg treatment interferes with sperm‐egg attachment and fusion (Boldt et al.:Biol Reprod39:19–27, 1988), these results suggest a possible role for the 94

ISSN:0148-7280    

DOI:10.1002/mrd.1120230109

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1989

数据来源: WILEY

摘要:

AbstractBoar sperm plasma membranes contain an integral protein (Mr 55 kDa) that apparently functions in the adhesion of sperm to the zona pellucida (Peterson and Hunt:J Cell Biol105:170a, 1987.) In experiments described in this report, the protein is identified after additional steps of purification involving lectin affinity chromatography and preparative PAGE. An active form of the adhesion protein (APz) develops or becomes first exposed in the corpus epididymis and is fully active in the cauda epididymis; a significant portion of this conformationally labile protein, while integral to the plasma membrane, cannot be solubilized by nonionic detergents and may be associated with the membrane skeleton. APzdoes not exhibit enzymatic properties thought possibly to be involved in sperm‐zona interaction in this and other species. Galactosyltransferase substrates and inhibitors and anliproteases including soybean trypsin inhibitor, pepstatin, leupeptin, and p‐aminoben‐zamidine failed to block sperm from binding to porcine eggs. Boar sperm proacrosin and antiproacrosin antibody failed to inhibit sperm‐egg binding. When plasma membranes or fractions containing APzthat bind to dextran sulfate agarose were chromatographed on L‐fucose agarose, a sugar which binds proacrosin, plasma membrane proteins that bound to the column failed to absorb anti‐APzantibody, Anti‐APzwas absorbed by fractions that did not contain proacrosin. These data indicate that APzis not proacrosin. Since anti‐APzmonovalcnt antibody raised from whole cauda or corpus sperm plasma membranes or from chromatographic fractions containing APzcompletely block capacitated sperm from binding to eggs, and since the ability of this antibody to be absorbed develops as sperm become capable of binding to eggs, we view AP, to be the major and perhaps onlyplasma membraneprotein involved in the adhesion of capacitated boar sperm to eggs prior to the ac

ISSN:0148-7280    

DOI:10.1002/mrd.1120230110

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1989

数据来源: WILEY

摘要:

AbstractOocytcs (n = 273), collected from superovulated ewes, were inseminated with in vitro capacitated spermatozoa from four rams [Crozet et al., 1987]. In each experiment, paral lel insemination was performed using aliquants from a single ejaculate in either our stan‐dard fertilization medium DM‐H‐SS (a modification of Bracken's defined medium, buffered with HEPES and containing 20% v/v sheep serum) or in the same medium supplemented with calcium lactate (DM‐H‐SS + Ca). The measured total calcium con centrations were Ca++T= 2.74 mM in DM‐H‐SS medium and Ca++T= 8.74 mM in DM‐H‐SS + Ca; the ratio of free to total calcium in DM‐H‐SS was Ca++F/Ca++T= 0.85. Fertilization was assessed at 17 hours postinsemination.Variations in the ejaculates were observed for each of the four rams tested. When DM‐H‐SS + Ca was used, the percentages of fertilized (75% vs. 50%) and monospermic (58% vs. 41%) eggs were significantly enhanced compared with use of DM‐H‐SS. No improve ment was observed in control medium DM‐H‐SS + lac containing neutralized lactic acid. Supplementing the fertilization medium with calcium had no apparent effect on the inci dence of polyspermy.These experiments show that the fertilization rate achievable in vitro by individual ejaculates from various rams can be increased by raising the calcium concentration in the fertilization medium to a value much higher than that present in tuba! fluids from estrous ewes. Extended incubation in such a high calcium concentration is unnecessary for in vitr

ISSN:0148-7280    

DOI:10.1002/mrd.1120230111

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1989

数据来源: WILEY