PubMed Health⌕ Search

Biomedical subjects

N Crozet

Publications and source records attributed to N Crozet.

33 records · Page 2Linked to original sources
PubMed1989

In vitro fertilization in the sheep: effect of elevated calcium concentration at insemination.

Oocytes (n = 273), collected from superovulated ewes, were inseminated with in vitro capacitated spermatozoa from four rams [Crozet et al., 1987]. In each experiment, parallel insemination was performed using aliquants from a single ejaculate in either our standard fertilization medium DM-H-SS (a modification of Brackett'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 concentrations 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 improvement 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 incidence 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 tubal fluids from estrous ewes. Extended incubation in such a high calcium concentration is unnecessary for in vitro capacitation of ram spermatozoa.

Animals↗
PubMed1989

Microtubule and centrosome distribution during sheep fertilization.

The distribution of microtubules and centrosomes was studied during sheep fertilization by electron and immunofluorescence microscopy. Tubulin and centrosomal material was identified with monoclonal anti-alpha-tubulin and MPM-2 antibodies, respectively. In ovulated eggs, microtubules were exclusively found in the meiotic spindle and centrosomal material at each of its poles. At fertilization, sperm centrosomes were incorporated into the egg and organized the sperm astral microtubules. During pronuclear development and migration, the sperm aster increased in size; microtubules of the sperm aster extended from the male pronucleus to the egg center and towards the female pronucleus. The position of the sperm aster during pronuclear migration suggests that it plays a role in this process. When the pronuclei were in apposition in the egg center, a dense array of microtubules and the centrosomal material were present between the two pronuclei. The proximal centriole of the sperm was identified by electron microscopy, between the apposed pronuclei. The centrosomal material extending around the centriole and the sperm neck and proximal mid-piece, apparently contained several foci from which microtubules radiated. These data suggest that in sheep unlike in mice, centrosomal material originating from the sperm is involved in the fertilization events.

Alkaloids↗
PubMed1988

Fine structure of sheep fertilization in vitro.

The fertilization events of the sheep oocyte have been investigated by electron microscopy. Thirty-two oocytes recovered from the oviducts of superovulated ewes were fertilized in vitro by a procedure that has given rise to full embryonic development (Crozet et al.: Gamete Res 16:159-170, 1987) and were analyzed at different stages of fertilization. Sperm incorporation in the ooplasm occurred as early as 2 h postinsemination (PI). One hour later, a fertilization cone containing very few cytoplasmic organelles developed above the decondensing sperm chromatin. It was a transitory structure which disappeared when the male pronucleus (PN) was formed. The second meiotic division of the oocyte resumed soon after sperm incorporation; abstriction of the second polar body occurred by 3-4 h PI; the polar body constriction was associated with microfilaments. Both the male and female PN were formed at 5 h PI and were surrounded by nuclear envolopes containing pore complexes. The female PN was characterized by patches of condensed chromatin; the male PN was closely associated with the sperm neck and proximal midpiece. Thereafter the PN migrated toward the center of the egg, where the spindle of the first cleavage division was observed at 21 h PI. Dispermic ova analyzed at 2-4 h PI contained 2 decondensing sperm heads and 2 fertilization cones which have reached the same stage, thus suggesting that a few sperm may penetrate quite simultaneously. Multipronucleate eggs were characterized by disturbance of the PN migration.

Acrosome↗
PubMed1988

Ultrastructural aspects of in vitro fertilization in sheep.

Fertilization events of the sheep oocyte have been analyzed by electron microscopy. In the fertilization cone, near the decondensing sperm nucleus, several areas composed of electron-dense filamentous material (FA) were observed. Moreover aster-like structures were present in the vicinity of the sperm neck and proximal midpiece. During swelling of the pronuclei, redistribution of cytoplasmic organelles occurred near the male pronucleus in a region facing the center of the egg. In this region, containing the sperm midpiece, large aggregates of Golgi complexes and cisternae of endoplasmic reticulum were associated with FA, microtubule foci, and abundant microtubules, suggesting cytaster formation. This complex migrated with the male pronucleus and the proximal sperm midpiece toward the center of the egg. A potential role of these subcellular structures, in the process of pronuclear migration, is proposed.

Animals↗
PubMed1988

In vitro maturation of sheep ovarian oocytes.

An in vitro system has been developed which induces full meiotic maturation in 98% ovarian sheep oocytes isolated from follicles 2-6 mm in diameter. 45.7% of these were fertilized, determined by the presence of two pronuclei, extrusion of the second polar body and the presence of the sperm flagellum. This culture system was used to describe the morphological changes during meiotic maturation, examining the nucleus, the cytoplasm and cumulus (corona)-oocyte relationship. 24 h are required for maturation of sheep oocytes. The culture medium must contain FSH, LH (10 micrograms/ml of each), estradiol-17 beta (1 micrograms/ml) and coculture of 10(6) mural granulosa cells in suspension (Crozet et al., 1987). Nuclear changes were the first evident transformations, showing that chromatin condensation leads to nuclear deformation, to germinal vesicle breakdown and to formation of the first and second meiotic metaphases. The axis of both spindles are oriented perpendicularly to the egg membrane. At each pole a bent disc composed of filamentous material represents the microtubule organizing centers (MTOC). The key event may be the initiation and control of chromosome condensation. Cytoplasmic changes include the development of a cortical layer of 1-4 microns thickness poor in cell organelles. Golgi complexes are localized in three distinct areas with possibly different functions: (1) around the germinal vesicle; (2) in the oocyte cortex, of regular distance; (3) in the central part of the oocyte. Cortical granules (CG) of different maturation stages (condensation) form clusters near the peripheral Golgi complexes while at Meta I they form a nearly continuous single layer. At Meta II the CGs are apparently anchored to the cell membrane by means of small spokes. The cumulus (corona) cells are attached by junctional complexes to each other and to the oocyte. Foot processes cross the zona and indent the oocyte. The termini are gradually exteriorized and contacts must be broken to isolate the oocyte. The sum of all the above changes represent meiotic maturation.

Animals↗
PubMed1988

Inhibitory effect of homologous solubilized zona pellucida on rabbit in vitro fertilization.

Rabbit zonae pellucidae were isolated using a modified technique of Dunbar et al. (1980). Zonae pellucidae were solubilized in saline phosphate buffer (pH 7.8) for 30 min at 70 degrees C. One dimensional SDS-PAGE analysis showed that zona pellucida is essentially composed of three major proteins with apparent molecular weights of 200 (275-165) kd, 100 (135-96) kd and 75 (96-51) kd. Preincubation of sperm with heat-solubilized zonae pellucidae (SZP) (5 to 8 SZP/microliters) did not reduce sperm binding ability. By contrast, it significantly decreased the percentage of penetrated eggs (20 versus 73% for the control) and significantly reduced the fertilization rate (10 versus 55% for the control). Ultrastructural analysis of several oocytes in the two groups demonstrated that inhibition of fertilization was not due to the inhibition of sperm-zona binding ability but essentially resulted from the impairment of sperm penetration through the zona pellucida.

Acrosome↗
PubMed1987

In vitro fertilization with normal development in the sheep.

Ovine tubal (n = 87) and ovarian in vitro matured oocytes (n = 99) were fertilized in vitro with ejaculated spermatozoa capacitated for 8 h in modified defined medium buffered with Hepes. High levels of fertilization were obtained as assessed by development to two- to six-cell stage within 40 h (75.8% for ovulated and 62.6% for in vitro matured oocytes). Electron microscope analysis of oocytes 20-22 h after insemination indicated that in vitro fertilization approximated the in vivo events. Embryos (two- to six-cell) were transferred surgically to the oviducts of pseudopregnant rabbits. Three days later, 42 (from ovulated oocytes) and 15 (from in vitro matured oocytes) embryos were recovered; 26 (61.9%) and 10 (66.6%), respectively, had cleaved at least once. Embryos incubated in vivo (n = 20 from ovulated oocytes; n = 9 from in vitro matured oocytes) were transferred surgically to the uteri of seven and four recipient ewes resulting in four and two pregnancies, respectively, from which three and one, respectively, have been maintained (greater than 3 months). The first lamb resulting from the in vitro fertilization of an ovulated oocyte was born. In addition, six embryos (two- to four-cell) from tubal oocytes and ten embryos (two- to six-cell) from in vitro matured oocytes were directly transferred to the oviducts of two and three ewes, respectively. Two pregnancies resulting from in vitro matured fertilized oocytes are in progress (greater than 3 months).

Animals↗
PubMed1987

Ultrastructure of in vivo fertilization in the goat.

In vivo fertilization of goat eggs has been studied by electron microscopy. Eggs were recovered from superovulated or natural cyclic goats, 32 to 52 hours after the onset of oestrus; only eggs recovered between 46 and 52 hours were fertilized. Spermatozoa penetrated the zona pellucida tangentially leaving vesiculated products of the acrosome reaction at the zona surface. As sperm penetrated into the ooplasm, the second meiotic division completed and cortical granule exocytosis occurred. However a few unreacted cortical granules usually remained in the cortex of the fertilized eggs, adjacent to the plasma membrane. After swelling the two pronuclei presented similar ultrastructural morphology: they contained small, compact, agranular nucleoli and unevenly distributed chromatin. The cytoplasm in close vicinity to the apposed pronuclei contained large stacks of annulate lamellae, smooth endoplasmic reticulum, prominent Golgi complexes, as well as dense areas of unidentified material. The abundance of cytoplasmic organelles near the pronuclei might be the expression of intensive metabolic activity. Conversely, in the cortex of fertilized ova several large organelles-free cytoplasmic areas were randomly distributed.

Animals↗
PubMed1986

Labelling of living mammalian spermatozoa with the fluorescent thiol alkylating agent, monobromobimane (MB): immobilization upon exposure to ultraviolet light and analysis of acrosomal status.

Living spermatozoa of seven mammalian species were treated with the thiol-alkylating fluorescent labelling compound, monobromobimane (MBBR). MB-labelling alone had no effect on sperm motility, nor on the time course or ability of golden hamster spermatozoa to undergo the acrosome reaction when capacitated in vitro. Exposure of MB-labelled spermatozoa to ultraviolet (UV) light and excitation of the MB fluorochrome resulted in virtually immediate immobilization of the spermatozoa without affecting acrosomal status. UV exposure of unlabelled spermatozoa for up to 30 sec had no effect upon motility. Immobilization of MB-labelled spermatozoa depended on the midpiece being irradiated, as irradiation of the head alone, or of the more distal parts of the principal piece, had little or no effect upon motility. Labelling with MB followed by immobilization of individually selected spermatozoa was most useful for detailing the course and site of occurrence of the acrosome reaction during penetration of the cumulus oophorus by golden hamster spermatozoa in vitro. In these often hyperactivated spermatozoa, precise determination of the acrosomal status could not often otherwise be made due to the difficulty in visualizing the acrosomal region of a vigorously thrashing, hyperactivated spermatozoon. This technique should prove valuable in a variety of studies on sperm motility, capacitation and fertilization, and could also be extended to other cell systems.

Acrosome↗
PubMed1986

Activity of maturation promoting factor in mammalian oocytes after its dilution by single and multiple fusions.

Mouse and porcine fully grown oocytes at metaphase I(MI) were fused to one or more fully grown oocytes of the same species that contained an intact germinal vesicle (GV). In fused cells containing one GV, premature chromosome condensation (PCC) was observed. In fused cells containing more than one GV, germinal vesicle breakdown (GVBD) and PCC were delayed. Fusion of an MI fully grown oocyte with a growing oocyte resulted in rapid PCC, whereas, fusion of an MI fully grown oocyte with more than one growing oocyte resulted in neither PCC nor GVBD. Moreover, MI chromosomes formed a clump of chromatin. Results of these experiments suggest that the delay in GVBD in fusions of MI oocytes with multiple GV-intact oocytes was due to dilution of maturation promoting factor (MPF) by the cytoplasm of the GV-intact oocytes and that the cytoplasm of growing oocytes can inhibit MPF present in MI oocytes.

Animals↗
PubMed1985

Inhibition of nuclear maturation in fully grown porcine and mouse oocytes after their fusion with growing porcine oocytes.

Porcine ovarian oocytes, isolated from follicles of 5 mm in diameter (large oocytes), were fused either together or with oocytes isolated from follicles of 0.5 mm in diameter (small oocytes). In giant cells composed of two large oocytes (control) germinal vesicle breakdown (GVBD) occurred and two metaphase I chromosome sets (M I) were observed 24 to 30 h after fusion. By contrast, in giant cells composed of one large and one small porcine oocyte, both germinal vesicles (GVs) remained well conserved after 24-30 h of culture. An identical situation was observed after fusion and cultivation of small porcine and large mouse oocytes isolated from preovulatory follicles. The results demonstrate the presence of inhibiting activity in the ooplasm of small porcine oocytes that prevents nuclear maturation of large porcine and mouse oocytes fused to them. This maturation inhibiting activity can be overcome by preincubating large porcine oocytes for more than 14 h before fusion with small oocytes. During preincubation the ooplasm produces sufficient amount of maturation promoting factor (MPF) to overcome the inhibiting activity present in small porcine oocytes thus inducing GVBD and chromatin condensation both in small and large oocytes.

Animals↗
PubMed1984

Meiotic competence in vitro of pig oocytes isolated from early antral follicles.

Pig oocytes were isolated from early antral follicles of different sizes and their abilities to resume and complete meiotic maturation in vitro were compared. After 24 h of culture, more than 80% of the oocytes from follicles 0.3-0.7 mm in diameter remained at the germinal vesicle stage, while 66, 94.3 and 100% oocytes from follicles 0.8-1.6, 1.7-2.2 and 3-5 mm in diameter, respectively, completed germinal vesicle breakdown. After 48 h of culture, 35% of the oocytes in the smallest follicle class progressed to prometaphase and only 4% to metaphase I. Of the oocytes from follicles 0.8-1.6 mm in diameter, 23% reached metaphase I and 17.3% metaphase II. About 50 and 76% of the oocytes from follicles 1.8-2.2 mm and 3-5 mm in diameter, respectively, extruded the first polar body. The ability to resume meiosis (i.e. to undergo germinal vesicle breakdown) is reached by porcine oocytes when they approach their full size in antral follicles greater than 0.8 mm in diameter and before they are capable of completing it (i.e. reaching metaphase II). The ability to complete meiotic maturation acquired in antral follicles of about 2 mm in diameter coincided with a significant decrease in the nucleolar transcriptional activity of the oocytes.

Animals↗
PubMed1983

Effects of actinomycin D and cycloheximide on the nucleolar ultrastructure of porcine oocytes.

In early antral follicles 0.5 mm in diameter, pig oocytes were intensively engaged in rRNA synthesis. Actinomycin D caused degranulation of their nucleoli which became entirely compact; the fibrillar centers that remained adjacent to the nucleolar surface also increased in size. In contrast, oocytes from preantral follicles treated under the same conditions failed to complete nucleolar compaction and were engaged in a special process of vacuolation. The rate of [3H]-uridine incorporation, evaluated by light-optical autoradiography, indicated that, even at high concentrations, cycloheximide impaired nucleolar transcriptional activity of the oocytes without suppressing it. The nucleoli of such treated oocytes were either unchanged or incompletely transformed, fibrillo-granular regions remaining adjacent to large compact areas. This study provides evidence that the process of nucleolar compaction in pig oocytes depends directly on the inhibition of rRNA synthesis. This process, which only occurs in oocytes from antral follicles, might represent an important step in the acquisition of oocyte capacity to initiate meiotic maturation.

Animals↗
PubMed1981

Germinal vesicle breakdown in the Xenopus laevis oocyte: description of a transient microtubular structure.

During progesterone-induced meiotic maturation of Xenopus oocytes in vitro, 7 morphological stages were defined. Using cytological analysis, nuclear breakdown was divided into three stages. Stage 1 corresponded to basal germinal vesicle breakdown. Stage 2 was characterized by the advent and development of a fibrillar network formed by microtubules at the basal part of the nucleus. Below, a lamellar microtubule organizing center (MTOC) was present. Numerous vesicles of smooth endoplasmic reticulum were proximal to both of these structures. During its formation and modification (stages 2 and 3), the fibrillar network migrated towards the animal pole. A prometaphase 1 stage was observed before the formation of the metaphase 1 spindle.

Animals↗