摘要:

AbstractComplete details are described for the first time of the procedures used in the author's laboratory for obtaining in vitro fertilization (IVF) of golden hamster eggs leading to the first cleavage division. These IVF procedures have been developed during the past 20 years and are very reproducible: IVF of at least 75% of eggs is routinely achieved, and on average 65% of inseminated eggs undergo the first cleavage division in vitro. These results can easily be obtained by inexperienced investigators. The ease and reproducibility of the hamster IVF procedures make them very suitable for studies of sperm:egg interaction and associated events. Studies in the author's laboratory have included analysis of sperm fertilizing ability under chemically defined conditions, the presence of sperm acrosome reaction stimulating factors in the egg investments, maturation of oocytes in vitro, the block to polyspermy, and the contribution of egg aging to fertilization anomalies. In addition, the motility of hamster sperm under chemically defined conditions is used in a routine screening protocol for detecting contaminants in the culture milieu. Golden hamster gametes of Ter several distinct advantages for IVF studies, including the large size of the sperm acrosome, the persistence of the very large sperm tail in the ooplasm for many hours following fertilization, and the translucence of the ooplasm, which facilitates observation of the sperm tail and pronuclei. The female golden hamster exhibits a regular 4 day estrous cycle, with distinctive indications of estrus and postestrus phases. Because of the advantages of using the golden hamster, the procedures described in this report may be useful to other investigators wishing to conduct research using IVF. Essentially the same IVF procedures can be used with monkey and bovine gametes.

ISSN:0148-7280    

DOI:10.1002/mrd.1120230202

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

年代:1989

数据来源: WILEY

摘要:

AbstractBy using a chemically defined (protein‐free) culture medium that supports sperm viability but not capacitation or the acrosome reaction, we have determined that hamster spermatozoa can be chemically capacitated in vitro by the divalent cation chelators D‐penicilla‐mine, L‐histidine, and L‐cysteine in the absence of bovine serum albumin (BSA). Washed cauda epididymal spermatozoa were preincubated (1–2 × 106sperm/ml) for 3, 4, or 6 hr at 37°C in 5% CO2in air. The basic culture medium used for sperm preincubation and for sperm:egg coincubation was a modified Tyrode's solution (protein‐free) containing 10 mM sodium lactate, 100 μM sodium pyruvate, and 1.0 mg/ml polyvinylalcohol (TLP‐PVA). Sperm viability was maintained in all preincubation and coincubation media with PHE (20 μM D‐penicillamine, 100 μM hypotaurine, and 1.0 μM epinephrine). The low control sperm preincubation medium consisted of TLP‐PVA. In some cases the high control preincubation medium also contained 3 mg/ml BSA (TALP‐PVA). The experimental preincubation medium was TLP‐PVA with additional D‐penicillamine (125 or 500 μM), or L‐histidine (10, 100, or 1,000 μM) or L‐cysteine (25, 75, or 125 μM). After preincubation, sperm were coincubated (2 × 104sperm/ml) with cumulus‐free hamster eggs in TALP‐PVA ± additional D‐penicillamine, L‐histidine, or L‐cysteine for 1.5 hr, fixed, and evaluated for percent egg penetration as an index of sperm capacitation. The results demonstrate that hamster spermatozoa can be chemically capacitated in vitro with D‐penicillamine (500 μM: range of mean penetration values, 53.6%–84.3%), L‐histidine (100 μM: range of mean values, 24.8%–56.3%) or L

ISSN:0148-7280    

DOI:10.1002/mrd.1120230203

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

年代:1989

数据来源: WILEY

摘要:

AbstractA dialysis unit was used to test whether direct physical contact between serum albumin and hamster spermatozoa is required for capacitation and/or the acrosome reaction. Sperm and bovine serum albumin (BSA) were incubated cither together (direct incubation) or separated by a dialysis membrane (indirect incubation). Sperm viability was supported with “sperm motility factors” (hypotaurine and epinephrine) and polyvinylalcohol (PVA). Spermatozoa became capacitated and underwent acrosome reactions when directly incubated in medium containing BSA (TALP‐PVA), but did not undergo acrosome reactions when indirectly incubated with BSA (medium TLP‐PVA). When sperm were first incubated for 4 hr indirectly with BSA, followed by 4 hr direct incubation with BSA, capacitation did not occur during indirect incubation. These findings indicate that an “intimate association” is necessary between serum albumin and spermatozoa to support capacitation under in vitro incubation conditions. The data are consistent with the concept of direct transfer of compounds from sperm to albumin and/or vice versa during sperm c

ISSN:0148-7280    

DOI:10.1002/mrd.1120230204

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

年代:1989

数据来源: WILEY

摘要:

AbstractThe “decapitated sperm” defect, found in both of two sterile brothers, may be assumed to have a genetic origin. The present material suggests that the term “decapitated spermatozoa” is not exact, because detached heads and tails were found in the brothers' ejaculate that could be regarded as “decapitated tails” and “decaudated heads.” The present report describes frequent, more or less advanced stages of detachment. Both heads and tails showed a normal structure in which only the postnuclear region was deficient, lacking basal plate and implantation fossa. A break at a different level of the midpiece, and therefore three kinds of separation, were observed. The defect, according to the present research, must originate in the testicular region, whereas the detachment occurs in

ISSN:0148-7280    

DOI:10.1002/mrd.1120230205

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

年代:1989

数据来源: WILEY

摘要:

AbstractThree approaches were investigated for improvement of in vitro maturation (IVM), in vitro fertilization (IVF), and early embryonic development in cattle. These were: 1) Selection of oocytes, 2) medium supplementation with fetal calf serum (FCS) and cow sera (DO, Dl, D10, and D20 to correspond with estrus, metestrus, diestrus, and proestrus, respectively), and 3) addition of follicle‐stimulating hormone (FSH), luteinizing hormone (LH), and estradiol‐17β (E2)during maturation. Greater proportions (percentage) of oocytes initially selected for their compact cumulus cells completed IVM and IVF when compared to unselected oocytes (P<.05). Proportions (percentage) of selected oocytes that matured and cleaved after in vitro insemination according to serum type used for IVM were: FCS: 110/175 (62.9%) and 37/110 (33.6%) and DO: 130/145 (89.7%) and 52/130 (40.0%); D1 127/130 (97.7%) and 41/127 (32.3%); D10 95/98 (96.9%) and 35/95 (36.8%); D20:113/116 (97.4%) and 49/113 (43.4%). A higher proportion (P<.05) of embryos resulting from the D20 group reached four‐ and eight‐cell stages. In FCS‐supplemented maturation media with no hormones added during maturation (control), results of IVM and IVF were 157/265 (59.2%) and 39/157 (24.8%), respectively. With E2(1 μg/ml) and FSH (5 μg/ml), comparable results were 189/215 (87.9%) and 71/189 (37.6%); with E2(1 μg/ml) plus LH (10 μml), 280/327 (85.6%) and 111/280 (39.6%). Added hormones improved IVM results (P<.05) and, when FSH or LH was added with E2, in vitro development to four‐ and eight‐cell stages was markedly enhanced (P<.05). Selection of oocytes, D20 serum, and added E2and FSH or LH for IVM improved in vitro development of bovine

ISSN:0148-7280    

DOI:10.1002/mrd.1120230206

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

年代:1989

数据来源: WILEY

摘要:

AbstractMammalian spermatozoa must undergo many changes to be able to fertilize the oocyte. One of these changes, the acrosome reaction, has been established as a requisite for gamete membrane fusion to occur; it consists of the fusion and vesiculation of the sperm plasma membrane with the outer acrosomal membrane of the principal segment of the acrosome. Reaction of the equatorial segment has occasionally been observed. The objective of the present work was to determine whether the presence of the sperm plasma membrane over the equatorial segment is necessary for gamete membrane fusion to occur.Golden hamster spermatozoa were capacitated in vitro in TAPL 10K, and the maximum possible percentage of acrosome reaction was determined at 82.79% + 1.69% SD (P= 0.27; r = 0.21). Ultrastructural studies showed that 93.6% of the reacted spermatozoa in this population had their principal and equatorial segments reacted. The fertilizing ability of these spermatozoa was assayed using zona‐free hamster oocytes. The percentage of fertilized ova obtained was 98.8% (308/312). Ultrastructural studies snowed the presence of spermatozoa with reacted equatorial segment inside the cytoplasm of immature oocytes. The evidence presented in this work demonstrates that the plasma membrane of spermatozoa with reacted equatorial segment retains its ability to fuse with the oocyt

ISSN:0148-7280    

DOI:10.1002/mrd.1120230207

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

年代:1989

数据来源: WILEY

摘要:

AbstractStudies have been carried out to 1) further characterize sperm specific plasma membrane polypeptides (33 and 35 kDa) that are recognized by a monoclonal antibody previously described (Longo, 1989) and 2) follow the incorporation and dispersal of these proteins within plasmalemmae of monospermic and polyspermic sea urchin (Arbacia punctuluta) eggs and oocytes utilizing immunocytochemical methods at the ultrastructural level of observation. Only sperm labeled when incubated with monoclonal antibody to the 33 and 35 kDa proteins followed by colloidal gold‐tagged second antibody. Colloidal gold label was observed on the egg plasma membrane immediately after gamete membrane fusion; the amount and extent of label, i.e., the distance from the site of sperm incorporation, increased with time postinsemination. By 20 min postinsemination approximately one hemisphere of the inseminated egg/oocyte was associated with label. The expanding distribution of colloidal gold label on inseminated eggs and oocytes vs. time reflects the free diffusion of 33 and 35 kDa sperm surface proteins among egg/oocyte plasma membrane components. Label was also found in forming endocytotic vesicles, suggesting that sperm plasma membrane proteins may be internalize

ISSN:0148-7280    

DOI:10.1002/mrd.1120230208

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

年代:1989

数据来源: WILEY

摘要:

AbstractA study of mouse gamete processing for in vitro fertilization (IVF) under various conditions showed that it is necessary to control the atmosphere if the temperature is raised from 22°C to 37°C. The data suggest that maximum IVF success is attained by processing the gametes at 37°C, under an atmosphere of 5% O2and 5% CO2, and overlaying the medium with silicone o

ISSN:0148-7280    

DOI:10.1002/mrd.1120230209

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

年代:1989

数据来源: WILEY

摘要:

AbstractMouse spermatozoa were capacitated and acrosome reacted by incubation for 30 min to 6 h in modified Tyrode's medium (T6) and in cGMP. The fertilization rates of eggs microin‐jected with a spermatozoon from samples incubated for 30 min, 2 h, and 6 h in T6 and in cGMP were 36%, 34%, 29%, and 43%, respectively. These rates were not correlated to the percentage of acrosome‐reacted spermatozoa identified after these treatments. The lack of association could be explained by the selection of an increased proportion of acrosomc‐reacted spermatozoa actually used for microinjection, particularly in samples incubated for 30 min and 2 h in T6. Both acrosome‐intact and acrosome‐reacted spermatozoa were identified under the zona in the eggs which failed to fertilize. Fertilized eggs developed at very high rates to the blastocyst stage

ISSN:0148-7280    

DOI:10.1002/mrd.1120230210

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

年代:1989

数据来源: WILEY

ISSN:0148-7280    

DOI:10.1002/mrd.1120230201

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

年代:1989

数据来源: WILEY