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1.
Theriogenology ; 64(2): 261-74, 2005 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-15955352

RESUMO

At the time of fertilization, release of inositol 1,4,5-trisphosphate (IP3) into the cytoplasm of oocytes is said to be induced by hydrolysis of phosphatidylinositol bis phosphate (PI2) via activation of phospholipase C and is responsible for the Ca2+ oscillation in oocytes immediately after sperm penetration. On the other hand, cumulus cells have been reported to play an important role in cytoplasmic maturation of mammalian oocytes and to affect embryonic development after fertilization. To obtain more information on the role of cumulus cells in cytoplasmic maturation of oocytes, the effects of cumulus cells on the rise in [Ca2+]i and the rates of activation and development of porcine mature oocytes induced by IP3 injection were investigated. Mature porcine oocytes that had been denuded of their cumulus cells in the early stage of the maturation period had a depressed rise in [Ca2+]i (4.0-6.0) and reduced rates of activation (31.4-36.8%) and development (10.0-24.4%) induced by IP3 injection compared with those of their cumulus-enclosed counterparts (7.3, 69.1% and 43.8%; P < 0.05). The [Ca2+]i rise and the rates of activation and development depressed by the removal of cumulus cells were restored by adding pyruvate to the maturation medium. Furthermore, the IP3 injection-induced depression of [Ca2+]i rise in mature oocytes derived from cumulus-denuded oocytes (DOs) was restored when they were cultured in a medium with pyruvate (3.9-6.3, P < 0.05). Also, mature oocytes from cumulus-oocyte complexes (COCs) cultured in a medium without glucose had a lower rise in [Ca2+]i than that in mature oocytes from COCs cultured with glucose (7.4-6.0, P < 0.05). Cumulus cells supported porcine oocytes during maturation in the rise in [Ca2+]i induced by IP3 and the following activation and development of porcine oocytes after injection of IP3. Moreover, we inferred that a function of cumulus cells is to produce pyruvate by metabolizing glucose and to provide oocytes with pyruvate during maturation, thereby promoting oocyte sensitivity to IP3.


Assuntos
Cálcio/metabolismo , Inositol 1,4,5-Trifosfato/farmacologia , Oócitos/fisiologia , Folículo Ovariano/citologia , Suínos , Animais , Células Cultivadas , Técnicas de Cocultura , Citoplasma/metabolismo , Feminino , Glucose/metabolismo , Oócitos/efeitos dos fármacos , Oócitos/ultraestrutura , Folículo Ovariano/fisiologia , Ácido Pirúvico/metabolismo
2.
Fertil Steril ; 34(1): 55-7, 1980 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-6772474

RESUMO

An inhibin-like substance, a follicle-stimulating hormone (FSH) inhibitor, was isolated from porcine follicular fluid and purified. Serum FSH levels of unilaterally ovariectomized mice which received injections of the inhibitor were lower than those of unilaterally ovariectomized mice which received injections of saline. The inhibitor also suppressed FSH binding to granulosa cells in vitro. These results suggest that this protein is an FSH inhibitor and that it has two modes of action: suppression of FSH levels in serum and suppression of FSH binding to granulosa cells.


Assuntos
Hormônio Foliculoestimulante/metabolismo , Células da Granulosa/metabolismo , Folículo Ovariano/análise , Proteínas/farmacologia , Animais , Castração , Feminino , Hormônio Foliculoestimulante/antagonistas & inibidores , Hormônio Foliculoestimulante/sangue , Células da Granulosa/efeitos dos fármacos , Hormônio Luteinizante/metabolismo , Camundongos , Tamanho do Órgão/efeitos dos fármacos , Ovário/anatomia & histologia , Ovário/efeitos dos fármacos , Proteínas/isolamento & purificação , Suínos
3.
Fertil Steril ; 53(6): 1049-54, 1990 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-2351227

RESUMO

Approximately one fourth of all human oocytes collected for in vitro fertilization are of immature origin. Even when these oocytes undergo nuclear maturation, fertilization, and cleavage in vitro, transfer of such embryos rarely results in pregnancy reaching delivery. We hypothesized that human embryos derived from prophase I oocytes were developmentally incompetent because they lacked a factor(s) found in in vivo matured oocytes. Using micromanipulation techniques in monkeys, we removed ooplasm from metaphase II oocytes and injected it into prophase I oocytes. After nuclear maturation, oocytes were transferred to the fallopian tube for fertilization. After ooplasmic transfusion, prophase I oocytes resulted in a delivery rate of 13%. When metaphase II ooplasm was heated or exposed to ribonuclease A before microinjection into prophase I oocytes, it lost effectiveness in conferring developmental competence.


Assuntos
Citoplasma/transplante , Oócitos , Oogênese , Animais , Feminino , Fertilização in vitro/métodos , Macaca fascicularis , Microinjeções
4.
Theriogenology ; 35(4): 695-703, 1991 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16726938

RESUMO

Follicular oocytes were cultured for 28h in vitro and 91% of the oocytes reached the the second metaphase in culture. The penetration rate after insemination in vitro using frozen-thawed spermatozoa was 81%. After cultivation for 48h in vitro, 18% of the in vitro fertilized oocytes developed to the three- to four-cell stages and 21% of these developed to the six- to eight-cell stages. Following in vivo culture in the rabbit oviduct, 18% of six- to eight-cell and 5% of three- to four-cell embryos developed to the blastocyst stage. To confirm the full developmental competence, 11 blastocysts were transferred to recipient cows, and six (55%) cows became pregnant or delivered calves.

5.
Theriogenology ; 47(2): 541-8, 1997 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-16728006

RESUMO

We examined effects of medium volume and two different culture media (HECM-3 and HECM-4) on in vitro development of hamster embryos. Groups of 5 to 8 1-cell embryos were cultured for 72 h in either < or =100 or > or =100 microl volumes. In the first experiment, embryos were cultured in Petri dishes with 2, 5, 20, 50 or 100 microl of medium using the two media (2 x 5 factorial experiment). Optimal volumes for morula and blastocyst development were 100 microl of HECM-3 and > or =50 microl of HECM-4; in HECM-4, > or =20 microl volumes were suitable whereas in HECM-3 < or = 50 microl volumes were unsuitable. In the second experiment, embryos were cultured in 100, 200, 500 and 1000 microl of HECM-3 and HECM-4 using organ culture dishes. Controls were 100 microl drops in Petri dishes. In organ culture dishes, blastocyst development was < or =6% in HECM-3 and 33-41% in HECM-4, and suitable volumes for development to at least morulae were > or =200 microl of HECM-3, and > or =100 microl of HECM-4. In both experiments development to morula and blastocyst stages with 100 microl volume in Petri dishes was significantly higher with HECM-4 (96 and 85% in Experiment 1 and 2, respectively) than that with HECM-3 (52 and 40% in Experiment 1 and 2, respectively; P < 0.05). These results indicate that attention should be paid to both type and volume of medium and interaction with type of culture dish for optimizing development of embryos in vitro.

6.
Theriogenology ; 35(5): 1051-8, 1991 May.
Artigo em Inglês | MEDLINE | ID: mdl-16726971

RESUMO

Bovine follicular oocytes were matured and inseminated in vitro with spermatozoa capacitated in vitro. The first evidence of sperm penetration was observed at 3 h after insemination. The penetration rate increased until 5 h, and reached a maximum rate (92%) at 5 h. Decondensation of the sperm head and pronuclear formation were observed 4 h and 7 h after insemination, respectively. Female chromatins of all penetrated oocytes were activated at 3 h, and female pronuclei were formed at 7 h after insemination. Percentages of oocytes with male and female pronuclei at 9 h were 88 and 94%. Polyspermy (4, 7, 19 and 29% at 4, 5, 7 and 9 h after insemination, respectively) and abnormal development of male pronuclei (6 and 7% at 7 and 9 h after insemination, respectively) were also seen.

7.
Theriogenology ; 41(7): 1435-45, 1994.
Artigo em Inglês | MEDLINE | ID: mdl-16727497

RESUMO

In Experiment 1, development of bovine embryos derived from in vitro-matured (IVM) and in vitro-fertilized (IVF) oocytes was examined under 4 culture conditions: 1) co-culture with mouse ampullae continuously for 8 d, 2) co-culture with mouse ampullae that were replaced with fresh ampullae at 48-h intervals, 3) co-culture with bovine granulosa cell monolayers, and 4) culture in medium alone. Culture medium consisted of tissue culture medium 199 (TCM-199) supplemented with 1% fetal calf serum (FCS). Inseminated oocytes were transferred to each of the culture treatment 24 h after insemination and were cultured for 8 d. The number of blastocysts per number of cleaved ova obtained after co-culture with mouse ampullae (42.9%) was significantly (P<0.05) higher than that obtained after co-culture with granulosa cell monolayers (28.3%) or culture without cells (4.2%). In Experiment 2, the developmental ability of bovine IVM/IVF embryos co-cultured with mouse ampullae supplemented with or without serum was examined. When serum was excluded from the culture medium, 26.4% (33 125 ) of the total number of embryos cultured were able to develop to the blastocysts stage using this co-culture system. This value was comparable to that obtained in a serum-supplemented co-culture system (30.7%; 39 125 ). In addition, the developmental ability of embryos that reached to the 4-cell stage or beyond at 46 to 48 h after insemination was not significantly different when the embryos were co-cultured with mouse ampullae with (38.5 vs 44.6%) or without (37.0 vs 33.8%) serum.

8.
Theriogenology ; 39(2): 485-98, 1993 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16727228

RESUMO

One-cell stage embryos, recovered from superovulated golden hamsters (8 to 12 weeks of age) 12 hours after egg activation, were cultured in HECM-1 medium at 37 degrees C and 5% CO(2) in air. The culture conditions investigated were the time and temperature required for embro recovery, the pH shift of the washing medium, and the oxygen concentration of the gas phase during and after embryo recovery. Each condition was assessed by the developmental efficiency of the embryo as determined by morphological criteria. As the time required for embryo recovery was reduced, the developmental rates of the embryos were improved: 2.3% (3 128 ) 26.9% (35 130 ) at 5 and 3 minutes, respectively, as determined by the number of embryos developed to the blastocyst stage. No blastocysts were obtained when more than 10 minutes were required for embryo recovery. As the oxygen concentration was reduced from 40 to 20% or to 5%, rather high developmental rates were obtained even when the time required for embryo recovery was prolonged: 6.9% (9 130 ) and 21.7% (28 129 ) of the embryos developed to the blastocyst stage when they were recovered under 5% oxygen within 10 and 5 minutes, respectively. Neither the temperature during embryo recovery (37 degrees C and 25 degrees C) nor the pH shift (pH 7.22 to 7.52) of the washing medium used in embryo recovery procedures influenced the development of the embryos. These findings suggest that the developmental block in hamster embryos may involve oxidative stress, which may result from exposure to high oxygen concentration and light during the manipulation of oocytes and embryos.

9.
Theriogenology ; 38(6): 1043-54, 1992 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-16727202

RESUMO

The effect of light exposure during collection and culture of hamster embryos on their subsequent development in vitro was examined. When embryos were collected under dark conditions (70 lux) within 10 minutes and then cultured in a HECM-1 medium in 5% CO2 in air, the developmental rates of 1-cell embryos to the 4- and 8-cell stages were 88.6% (93/105) and 66.7% (70/105), respectively. These rates were significantly higher than those under light conditions (1600 lux): 51.9% (56/108) and 34.3% (37/108). Light irradiation during the culture of 1-cell embryos suppressed subsequent development. The degree of suppression correlated inversely with duration of light irradiation, and light irradiation of 30 minutes or more completely blocked development to the 2-cell stage. When 1-cell embryos were irradiated through a yellow filter, cutting the light wavelengths to less than 500 nm, embryonic development was still suppressed. However, the degree of the suppression varied and 45.7% (53/116), 6.0% (7/116), and 0.9% (1/116) of the embryos developed to the 2-, 4-, and 8-cell stages, respectively, under 30 minute light irradiation. Inhibitory effects of light irradiation on the development of 2- and 8-cell embryos were also observed, showing an inverse correlation with duration; the developmental rates of 2-cell embryos to the 8-cell stage under 0, 10, and 30 minutes of irradiation were 85.6% (107/125), 1.6% (2/122), and 0% (0/129), respectively, and those of 8-cell embryos to the blastocyst stage were 79.8% (91/114), 74.8% (86/115), and 0% (0/110), respectively. These findings indicate that early-stage embryos are sensitive to light exposure; however, severe light exposure adversely affects the development of embryos at any stage. Thus, the protection of embryos from light exposure at all stages of embryo manipulation, from collection to culture, is essential.

10.
J Anim Sci ; 55(4): 873-7, 1982 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-6292155

RESUMO

The purification and action sites of a follicle stimulating hormone (FSH) inhibitor obtained from bovine follicular fluid were examined. Bovine follicular fluid was salted out with ammonium sulfate as a concentration of 14.5 to 18.5%, and the salted out fraction was separated into two peaks by Sephadex G-200 column chromatography. The second peak, detectable as a single band by polyacrylamide gel disc electrophoresis, contained all the inhibitory activity to compensatory ovarian hypertrophy in mice. Serum FSH levels of unilaterally ovariectomized mice given injections of the inhibitor were lower than those of unilaterally ovariectomized mice given injections of saline. The inhibitor also suppressed FSH binding to granulosa cells in vitro. These results suggest that this inhibitor has two modes of action: suppression of FSH levels in serum and suppression of FSH binding to granulosa cells.


Assuntos
Bovinos/fisiologia , Inibidores do Crescimento/isolamento & purificação , Folículo Ovariano/análise , Animais , Cromatografia em Gel , Feminino , Hormônio Foliculoestimulante/antagonistas & inibidores , Células da Granulosa/efeitos dos fármacos , Inibidores do Crescimento/farmacologia , Camundongos , Receptores de Superfície Celular/efeitos dos fármacos , Receptores do FSH
11.
J Anim Sci ; 69(8): 3343-7, 1991 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-1894571

RESUMO

There is evidence that mucus of the female bovine genital tract contains pheromones that induce physiological and behavioral responses in other animals. To study these pheromones, vaginal mucus was collected from heifers either at estrus or during diestrus. The mucus was then applied to the hindquarters of the same animal during diestrus or to the hindquarters of herdmates during diestrus. The behaviors of the treated animal and its herdmates were then observed. To attempt to isolate the mounting-inducing substance, mucus was dialyzed or separated on ion-exchange resins. Diestrous heifers to which their own estrual mucus has been applied were nearly always mounted by herdmates (P less than .01). But, heifers to which another's estrous mucus had been applied were not mounted. This suggests that vaginal mucus contains not only estrus-related pheromones, but also individual distinctive odors. The dialyzable fraction of vaginal mucus and the neutral fraction, prepared by ion-exchange chromatography of the dialyzable solution of vaginal mucus, had a mounting-inducing activity on the herdmates, as did the application of an animal's own vaginal mucus. These findings suggest that mounting-inducing pheromones are relatively low molecular weight, neutral substances.


Assuntos
Bovinos/fisiologia , Estro/fisiologia , Muco/química , Atrativos Sexuais/análise , Vagina/química , Animais , Cromatografia por Troca Iônica , Diestro/fisiologia , Feminino , Atrativos Sexuais/farmacologia , Comportamento Sexual Animal/efeitos dos fármacos
12.
J Vet Med Sci ; 60(4): 523-6, 1998 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-9592729

RESUMO

This study examined the timing of completion of meiosis I of bovine oocytes in which meiotic resumption had been inhibited by cycloheximide (CHX), and also determined the optimum interval of maturation in culture for subsequent fertilization and development. Most oocytes treated with CHX reached metaphase II at 16 hr in the maturation culture, while control oocytes did at 20 hr. CHX-treated oocytes cultured for 16 hr were normally fertilized but failed to develop into blastocysts. Maturation in culture for 20 hr resulted in comparable development for control oocytes. The results indicate that nuclear maturation of CHX-treated oocytes was completed 4 hr faster than for control oocytes, however the same interval of maturation as that of control oocytes is necessary for subsequent development to blastocysts.


Assuntos
Cicloeximida/farmacologia , Oócitos/citologia , Animais , Blastocisto/citologia , Blastocisto/efeitos dos fármacos , Bovinos , Desenvolvimento Embrionário e Fetal/efeitos dos fármacos , Feminino , Fertilização in vitro/métodos , Fertilização in vitro/veterinária , Técnicas In Vitro , Masculino , Meiose/efeitos dos fármacos , Oócitos/efeitos dos fármacos , Oócitos/fisiologia , Folículo Ovariano , Espermatozoides/fisiologia , Fatores de Tempo
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