摘要:

AbstractCumulus cell processes remaining in the zona pellucida of mouse occytes mechanically isolated from the ovary have been indirectly visualized by labeling their actin microfilament core with rhodaminyl‐phalloidin. If the isolation of the oocytes is performed in Ca2+‐free medium, the preisence of such processes allows the entry into the cell of low molecular weight molecules (such as 5‐6 carboxyfluorescein) and contributes to the death of the cell in such experimental conditions.Following dissolution of the zona pellucida (by enzymatic or acidic treatment) the oocyte is no longer permeable to small molecules and becomes resistant to Ca2+‐free medium, probably as a consequence of the collapse of cumulus cell processes. The role of cumulus cell processes and gap junctions in the permeability of mechanically isolated ovarian oocytes is di

ISSN:0148-7280    

DOI:10.1002/mrd.1120230302

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

年代:1989

数据来源: WILEY

摘要:

AbstractProbable participation of sperm protease in the acrosome reaction was investigated using several inhibitors and substrates. Among those examined, L‐l‐tosylamide‐2‐phenylethyl chloromethyl ketone (TPCK) and chymostatin, chymotrypsin inhibitors, p‐nitrophenyl‐p′‐guanidinobenzoate (NPGB), a serine protease inhibitor, and N‐benzoyl‐L‐tyrosine ethyl ester (BTEE), a chymotrypsin substrate, inhibited the egg jelly‐induced acrosome reaction ofStrongylocentrotus intermedius. TPCK and BTEE, however, did not inhibit the reaction caused by ionophores, A23187, or nigericin. To know the mechanism of inhibition by chymotrypsin inhibitors and substrates of the egg jelly‐induced acrosome reaction, intraccllular Ca2+concentration ([Ca2+]i) and pH (pHi) were measured with fura‐2 and 2′,7′‐bis (carboxy‐ethyl)carboxyfluorescein (BCECF), respectively. Egg jelly caused increase of [Ca2+]iwhich was depressed by BTEE. Egg jelly also caused a transient rise of pHi, which was not depressed by BTEE. In the presence of verapamil, the acrosome reaction by egg jelly was significantly inhibited concomitant with depressed increase of [Ca2+]i. The rise of pHj was not depressed by verapamil. Thus, modes of action of BTEE and of verapamil are similar to each other. Bringing these findings together, the authors present a view that a chymotrypsin‐like protease of sea urchin sperm activates verapamil‐sensitive Ca2+channels, w

ISSN:0148-7280    

DOI:10.1002/mrd.1120230303

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

年代:1989

数据来源: WILEY

摘要:

AbstractThe present study examined the effects of gonadotropins and ovarian steroids during in vitro meiotic maturation of rat oocytes on their ability to undergo in vitro fertilization. Fully grown oocytes were isolated from antral follicles of immature rats and cultured as oocyte‐cumulus cell complexes (OCC) under conditions in which completion of meiotic maturation occurs spontaneously. They were then exposed to spermatozoa under conditions in which oocytes matured in vivo exhibit high fertilization rates. Compared with oocytes from pregnant mare's serum gonadotropin (PMSG) or follicle‐stimulating hormone (FSH)‐treated rats, a simiiar proportion of the oocytes (>80%) from untreated rats underwent germinal vesicle breakdown, but such oocytes had a lower rate of fertilization (70% vs. 20%). The presence of FSH during in vitro maturation restored the fertilization rate for oocytes from untreated rats, while a cytochrome P450 inhibitor, aminoglutethimide phosphate abolished this beneficial effect of FSH. The addition of progesterone during the in vitro maturation period duplicated the beneficial effect of FSH on fertilization rate of oocytes from untreated rats; oestradiol‐17β was less effective in this regard, and 5α‐dihydrotestosterone was ineffective. These findings indicate that FSH and progesterone, although having no apparent effect on nuclear maturation of the oocyte, play an important role during oocyte maturation in enabling normal fertilizati

ISSN:0148-7280    

DOI:10.1002/mrd.1120230304

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

年代:1989

数据来源: WILEY

摘要:

AbstractThe sperm head of many Australian hydromyine rodents has three curved hooks projecting from its anterior margin; the structure of the hooks has been characterized, but their function is unknown. In this study, we have investigated whether the hooks might have evolved to assist sperm penetration through more formidable egg vestments, particularly the zona pellucida. Cumulus‐oocyte complexes were obtained from two species that possess a three‐hooked sperm head (Pseudomys australisandP. nanus) and one species that does not (Notomys alexis) and examined by light and electron microscopy. After fixation in the presence of ruthenium red, the zona pellucida was found to consist of a fibrillar meshwork, but there were no interspecific structural differences. A corona radiata was absent, and the cumulus extracellular matrix was composed of filaments and electron‐dense granules in each species. Measurements of the zona thickness in freshly ovulated, unfixed oocytes revealed that it was thinnest (7.8 μm) inP. australis. Which has a three‐hooked sperm head, and thickest (11.4 μm) in N. alexis, the species in which the ventral hooks are absent. Hence, no correlation was found between the thickness of the zona pellucida or the structure of the cumulus‐oocyte complex, and the presence of three hooks on the sperm head. We conclude, therefore, that it is unlikely that the evolution of the three‐hooked sperm head is an adaptation for penetration of increased barriers arou

ISSN:0148-7280    

DOI:10.1002/mrd.1120230305

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

年代:1989

数据来源: WILEY

摘要:

AbstractOur previous study has shown that superovulatory treatment with pregnant mare serum gonadotropin (PMSG) in rats caused marked alterations in ovarian and serum steroid responses and coincidental increase in degenerate oocytes (Yun et al., 1987). This study examined the effects of superovulatory treatment (20 IU PMSG) on follicular steroid contents and oocyte maturation. Immature female rats aged 28‐30 days were injected with 4 or 20 IU PMSG and sacrificed at 24, 48, 60, and 72 hr. Compared to control regimen, follicular content of progesterone (P) in superovulated rats significantly (P<.05) increased at 48 hr. Androgen (A) content significantly (P<.01) decreased below control level at 24 hr but significantly (P<.05) increased above control level at 48 hr and 60 hr. There was no significant change in 17β‐estradiol (E) content between the two groups. In control regimen, the ratio of A/E sharply decreased and the ratios of P/Eand P/A increased steadily from 24 hr. However, superovulatory regimen showed a consistently steady state in the overall ratios of follicular steroids after 24 hr. Nuclear maturation of the majority of control oocytes recovered from oviducts at 72 hr was synchronized at metaphase II, whereas superovulated oocytes displayed different stages varying from prophase I to metaphase II at 24, 48, and 72 hr. The results provide direct evidence of atypical ovulations in superovulated oocytes with premature or asynchronous nuclear maturation and demonstrate a close relationship between meiotically aberrant oocytes and abnormal follicular steroidogenesis following superovulation with PMSG in

ISSN:0148-7280    

DOI:10.1002/mrd.1120230306

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

年代:1989

数据来源: WILEY

摘要:

AbstractGolden hamster eggs fused with human sperm were pulsed with bromodeoxyuridine to determine the timing of S‐phase and the length of the first ceil cycie in this hybrid cross. Fused eggs were fixed and pronuclei scored for incorporation of the thymidine analogue detected by indirect immunofiuorescence. Although S‐phase started synchronously 3–3.5 hr after coincuba‐tion of sperm and eggs, its duration was variable such that two‐cell stages appeared at 16 hr while a proportion of pronuclei was still engaged in DNA synthesis. Unlike rodent sperm chromatin, human sperrn chromatin was able to participate in DNA synthesis well before its maturation into a fully developed pronucleus. Human sperm chromatin appears able to function under conditions different in several respects from those in h

ISSN:0148-7280    

DOI:10.1002/mrd.1120230307

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

年代:1989

数据来源: WILEY

摘要:

AbstractMicrotubule assembly is required for the formation of the male and female pronuclei during mouse, but not sea urchin, fertilization. In mouse oocytes, 50 μM colcemid prevents the decondensation of the maternal meiotic chromosomes and of the incorporated sperm nucleus during in vitro fertilization. Nuclear lamins do not associate with either of the parental chromatin sets although peripherin, the PI nuclear peripheral antigen, appears on both. DN A synthesis docs not occur in these fertilized, colcemid‐arrested oocytes. This effect is limited to the first hours after ovulation, since colcemid added 4–6 hours later no longer prevents pronuclear development, lamin acquisition, or DNA synthesis. Neither microtubule stabilization with 10 μM taxol nor microfilament inhibition with 10 μM cytochalasin D or 2.2 μg/ml lalrunculin A prevent these pronuclear events; these drugs will inhibit the apposition of the pronuclei at the egg center. In sea urchin eggs, colcemid or griseofulvin treatment doe? not result in the same effect and the male pronucleus forms with the attendant accumulation of the nuclear lamins. The differences in the requirement for microtubule assembly during pronucleus formation may be related to the cell cycle: In mice the sperm enters a meiotic cytoplasm, whereas in sea urchin eggs it enters an interphase cytoplasm. Refertilization of mitotic sea urchin eggs was performed to test the possibility that this phenomenon is related to whether the sperm enters a meiotic/mitotic cytoplasm or one at interphase; during refertilization at first mitosis, the incorporated sperm nucleus is unable to decondense and acquire lamins. These results indicate a requirement for microtubule assembly for the progression from meiosis to first interphase during mouse fertilization and suggest that the cytoskeleton is required for changes in nuclear architecture necessary during fertilization and the cel

ISSN:0148-7280    

DOI:10.1002/mrd.1120230308

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

年代:1989

数据来源: WILEY

摘要:

AbstractHypoxanthine and adcnosine are present in preparations of mouse ovarian follicular fluid, and these purines maintain mouse oocytes in meiotic arrest in vitro (Eppig et al.:Biology of Reproduction33:1041–1049, 1985). The first hypothesis tested in this study is that purines which maintain meiotic arrest act by maintaining meiosis‐arresting levels of cyclic adenosine monophosphatc (cAMP) in the oocyte. Oocyte‐cumulus cell complexes were incubated in control medium (no added purines), or medium containing 0.75 mM adenosine, 4 mM hypoxanthine, or both for 3 hr and the percentage of the oocytes that underwent germinal vesicle breakdown (GVB) and the cAMP content of the intact complexes and the oocytes were determined. Adenosine alone had little inhibitory effect on GVB at this time point but sustained higher levels of cAMP in the oocytes. Hypoxanthine maintained 80% of cumulus cell‐enclosed oocytes in meiotic arrest and also sustained higher cAMP levels in the oocytes. The additon of adenosine to hypoxanthine‐containing medium increased the percentage of oocytes maintained in meiotic arrest, and increased the amount of cAMP in the oocytes above that maintained by either hypoxanthine or adenosine alone. Neither hypoxanthine, adenosine, nor hypoxanthine plus adenosine altered the cAMP content of intact complexes when assayed after 3 hr culture. Microinjection of an inhibitor of the catalytic subunit of cAMP‐dcpendent protein kinase induced GVB in denuded oocytes cultured in medium containing hypoxanthine. This purne, therefore, maintained meiotic arrest by sustaining elevated cAMP levels within the oocytes.The second hypothesis tested in this study is that purines maintain meiosis‐arresting levels of cAMP, at least in part, by inhibiting cAMP phosphodiestcrase activity. In descending order of potency, 3‐isobutyl‐l‐methylxanthine (IBMX), guanosine, hypoxanthine, adenosine, and xanthosine inhibited cAMP phosphodiesterase in oocyte lysates. Moreover, like the potent phosphodiesterase inhibitor IBMX, hypoxanthine augmented the cAMP meiotic arrest and cAMP accumulation mediated by follicle‐stimulating hormone (FSH) in intact complexes. Therefore, inhibition of oocyte phosphodiesterase appears to be one mechanism by which the purines could maintain meiosis‐a

ISSN:0148-7280    

DOI:10.1002/mrd.1120230309

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

年代:1989

数据来源: WILEY

摘要:

AbstractMembrane alterations accompanying in vitro capacitation of hamster spermatozoa were examined using the freeze‐fracture technique with or without use of filipin, a sterol‐binding probe. In the spermatozoa prior to or at 10 min after start of incubation in capacitating medium, large (about 11 nm) and small (8–9 nm) intramembranous particles (IMPs) were present in the periacrosomal region of the sperm plasma membrane (PAPM). Filipin sterol complexes (FSCs) were densely (about 500/μ2) distributed in the PAPM prior to incubation. The density of FSCs in the PAPM was reduced by 70–80% of the original density by 2 hr of incubation. At the same time, small patches of IMP‐free areas appeared in the plasma membrane above the equatorial and middle segments of the acrosome. By the end of 3 hr of incubation, the majority of small IMPs had disappeared from the PAPM. Remaining large and small IMPs tended to aggregate in the PAPM. During incubation in capacitation medium, “cords,” or linear arrangements of closely packed IMPs, appeared near the posterior ring of the sperm head. These observations strongly suggest that the acrosome reaction of the hamster spermatozoa is preceded by the removal (deletion) of filipin‐reactive sterols (FRSs) and the disappearance of small IMPs from the lipid

ISSN:0148-7280    

DOI:10.1002/mrd.1120230310

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

年代:1989

数据来源: WILEY

摘要:

AbstractIn the fully grownBufo arenarumoocyte, carbohydrate breakdown during the autumn‐winter season is accomplished mainly through the glycolytic pathway followed by the krebs cycle. During the breeding season (spring‐summer), carbohydrates are used mainly through the pentose phosphate cycle and through the variant of the Krebs cycle known as the glutamic aspartic cycle.The metabolism operating in the oocytes was determined using the following paramenters: 1) the capacity of isolated mitochondria to oxidize citrate and fumarate; 2) the enzymatic activities of phosphofructokinase (PEK) and glucose‐6‐phosphate dehydrogenase (G‐6‐PDH); and 3) cirate and ATP compartmentalization.The present paper shows that follicle‐stimulating hormone (FSH) would be one of the factors responsible for summer metabolism, since ovary fractios obtained from winter specimens treated with the hormone acquired the metabolic characteristics corresponding to oocytes obtained from breeding‐season animals from dose‐response, and response in the function of time curves, it could be assumed that the optimum doses and times were 0.1 μg FSH/ml of incubation medium and 30 min treatment, respecitively.The metabolic effect of FSH upon oocytes could be mediaated by the adenylate cyclase cAMP system, since treatment of ovric fractions with cAMP 10‐3M reproduced the effects obtained with the hormone. In addition, 0.02 mg/ml tetracyline proved to block the effect of FSH. A direct metabolic action of FSH on body cavity oocytes (without follicle cells) was observed when submitting these oocytes to the sam

ISSN:0148-7280    

DOI:10.1002/mrd.1120230311

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

年代:1989

数据来源: WILEY