Ultrastructure of oocyte maturation and fertilization in superovulated cattle.
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Biomedical subjects
T Greve
Publications and source records attributed to T Greve.
Cytogenetic study of parthenogenetically activated bovine oocytes matured in vivo and in vitro.
Three experiments were performed to evaluate the potential for parthenogenetic activation of bovine oocytes in in vitro fertilization systems and to determine the chromosome complement of the resulting parthenogenotes. In the first experiment, immature oocytes from slaughtered cattles were matured in vitro in Defined Medium (DM) for 24 h to simulate in vitro fertilization conditions. Subsequently, a portion was fixed, and the remainder were transferred to rabbit oviducts. Oocytes were then cultured for 6-8 h or for 24 h with Colcemid present during the last 6 to 8 h and fixed on slides and examined. In the second experiment, mature oocytes were collected from the preovulatory follicles, and the oocytes were subjected to the same culture as in experiment I. In the third experiment, oocytes were treated as in experiment II, except that instead of transfer to rabbit oviducts, they were cultured an additional 48 h in vitro. In experiment I, 131 oocytes were fixed after culture in DM. Of the 79 oocytes analyzed in the pre-rabbit group, 71 (90%) were at the second meiotic metaphase (MII), and 8 (10%) were at pre-MII stage; none were activated. After transfer to rabbits, 291 were fixed. Of these, 80 were analyzed; 37 (46.3%) were MII, 7 (8.6%) were pre-MII, and 36 (45%) were activated. Of the 36 activated oocytes, 26 (72.2%) were haploid, 4 (11.1%) were diploid, 1 (12.8%) was tetraploid, and 5 (13.8%) were in the process of endoreduplication. In experiment II, 51 oocytes were fixed after culture in DM of which 36 (70.6%) could be analyzed; 30 (83.3%) were MII, and 6 (16.7%) were pre-MII. After culture in the rabbit, 68 were fixed of which 27 (39.7%) could be analyzed. Of these 27, 20 (74.1%) were MII, and 7 (25.9%) were activated; 6 were haploid, and 1 was endoreduplicating. In experiment III, 30 oocytes were fixed at the end of the culture period; only 10 could be analyzed of which 8 (80%) were MII and 2 (20%) were pre-MII. In all, 46% of in vitro and 26% of in vivo matured oocytes were activated, based on chromosomal analysis. Of those activated, the majority (74.4%) were haploid, suggesting that activation occurs at or after completion of MII. Endoreduplication appears to be one of the mechanisms leading to the formation of diploid and polyploid parthenogenotes.
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Scanning electron microscopy of in vitro fertilization in cattle.
Cumulus-oocyte complexes were collected from cows at an abattoir by aspiration from small (1-6 mm) follicles. The complexes were matured in vitro for 28 h. Subsequently, the cumulus cells and the zona pellucida were removed by enzyme treatment in a proportion of the complexes (zona-free ova). Both cumulus-enclosed and zona-free ova were inseminated in vitro and processed for scanning electron microscopy after different periods of culture. In the cumulus-enclosed ova the number of spermatozoa attached to and penetrating into the cumulus investment increased with increasing culture time. Practically all spermatozoa displayed intact acrosomes. In the zona-free ova clusters of spermatozoa attached to the ovum surface, and at 5 h a proportion of the spermatozoa had undergone the acrosome reaction, and their internalization into the ooplasma was initiated. The acrosome reaction was characterized by an increasing fenestration of the membrane coverings of the acrosomal region of the sperm head. During the sperm head internalization, where the ovum microvilli appeared to contact especially the equatorial segment and the postacrosomal region, the sperm head gradually disappeared from the ovum surface, and the microvilli at the site of internalization became more bulbous. Simultaneous abstriction of the second polar body was seen in some ova.
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Ultrastructural abnormalities of in vitro fertilization of in vitro matured bovine oocytes.
Cumulus-oocyte complexes collected from cows at an abattoir by aspiration from small (1-6 mm) antral follicles were matured and inseminated in vitro. At different time intervals after insemination the ova were processed for transmission electron microscopy. Up to and including 6 h after insemination all ova were unfertilized, and their cortical granules were more or less clustered. At 6 h acrosome reaction of spermatozoa was observed on the surface of the zona pellucida. At 8 h the first fertilized ovum appeared and the first fully developed spherical pronucleus was observed, at 20 h the first apposition of pronuclei was seen, and at 40 h divisions were ongoing or completed. More than one third of the fertilized ova showed polyspermic penetration of the zona pellucida, and in most of these ova different developmental stages of supernumerary pronucleus formation were observed in the ooplasm. Abnormal cortical granule release was seen in approximately half of the fertilized ova, and it was more frequent in ova with polyspermic as opposed to monospermic penetration of the zona pellucida.
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A semiquantitative and rapid radioimmunosorbent assay of luteinizing hormone for early prediction of time of ovulation in cattle.
A radioimmunoassay (RIA) for bovine luteinizing hormone (LH) is presented. It is based on the binding of the second antibody to polystyrene vials. Less than 4 h is required to estimate the hormone level in the plasma. LH content is determined by comparing unknown plasma samples to relevant control plasma to identify the preovulatory LH surge. Of 182 samples, 176 (97%) were found to contain either more or less LH than the control plasma, and 12 out of 13 (92%) LH surges were identified using the immunosorbent RIA. The method can be used for early prediction of ovulation in cattle, and it may be used to time aspiration of oocytes.
Journal Article↗
Dynamic changes of estradiol and progesterone concentrations during in vitro oocyte maturation in cattle.
Two culture techniques, an open system using 1.0-ml medium and a covered system using 50-mul droplets of medium covered by paraffin oil, used for in vitro bovine follicular oocyte maturation were compared. Estradiol-17beta (E2) was added to Ham's F-10 medium together with other supplementations. In the absence of oocytes, E2 concentration remained unchanged in the 1.0-ml open system, but it decreased gradually in the 50-mul covered system. In the presence of oocytes, E2 increased significantly in the 1.0-ml open system, but this increase could not be detected in the 50-mul covered system. Progesterone (P4) concentration increased in both systems, but it was much higher in the 1.0-ml culture than in the 50-mul culture. The two culture systems allowed an identical nuclear oocyte maturation rate of 88.6 vs 87.0%, a sperm penetration rate of 54.3 vs 59.6%, and a polyspermy rate of 6.8 vs 11.6% for 1.0-ml and 50-mul systems, respectively. The cleavage rate, however, differed significantly (78.3 vs 41.7% for 1.0-ml and 50-mul systems, respectively). It is concluded that diffusion of steroids into the paraffin oil occurs and may affect the cleavage rate but not the maturation or penetration rate.
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Preovulatory evaluation of the superovulatory response in donor cattle.
Dairy cows and heifers (n = 134) were induced to superovulate with exogenous gonadotrophins. In 103 animals, peripheral plasma concentrations of progesterone (P4) and luteinizing hormone (LH) were measured during the preovulatory period. On the basis of these measurements, normal and deviating profiles of P4 and LH were defined. A high degree of correlation existed between the normality of the two profiles; when the P4 profile was normal, the probability for the LH profile also to be normal was greater than 10:1. This relationship was utilized to evaluate donors based on four preovulatory measurements of P4. When used on 31 animals used for collection of eggs, a superior superovulatory response was encountered in animals with normal vs deviating P4 profiles (eggs recovered: 7.2 +/- 1.1 vs 0.5 +/- 0.3, P < 0.001; transferable embryos: 4.4 +/- 0.9 vs 0.3 +/- 0.2, P < 0.01). It is concluded that evaluation of donors by measurements of progesterone in plasma at four preovulatory sampling points allows for the early exclusion of donors with inferior embryo yield.
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Ultrastructure of in-vivo fertilization in superovulated cattle.
Heifers were induced to superovulate by treatment with PMSG or FSH. Subsequently, oestrus was induced with prostaglandins and artificial insemination was performed. Ova were collected from the oviducts and their ultrastructural features were related to an estimated time of ovulation based on the time of the LH peak. With the insemination schedule used, the estimated time of ovulation defined the time at which fertilization was expected to occur. The ova were characterized as unfertilized, fertilized or possibly fertilized, and a sequence of nuclear and cytoplasmic changes associated with fertilization was revealed. Within 4 h after the estimated time of ovulation formation of the female and male pronucleus was initiated, and at 5-7 h swelling of the pronuclei occurred. At 19 h the pronuclei were closely apposed and synkaryosis was seen, and at 23 h the first two-cell stage was obtained. Within 2-3 h after the estimated time of ovulation cortical granule release, development of conspicuous Golgi complexes, and transformation of the smooth endoplasmic reticulum occurred. At approximately 7 h parallel arrays of annulate lamellae appeared. In one third of the unfertilized ova deviant oocyte maturation was noticed.
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A detailed analysis of early events during in-vitro fertilization of bovine follicular oocytes.
Bovine follicular oocytes collected at slaughter were matured and fertilized in vitro with in-vitro capacitated spermatozoa. Analysis of 621 penetrated ova fixed at various times after in-vitro insemination led to definition of 6 stages of early development. A time sequence for sperm penetration, sperm head decondensation, male pronucleus formation, the activation of second meiotic division, female chromosome decondensation and pronucleus development was established. First sperm penetration into the ooplasm was recorded 6 h after insemination; 1-2 h was required for the sperm head to decondense and another 4-6 h to develop into the opposing pronucleus stage. Synkaryosis and first cleavage occurred 28 h after fertilization. Examination of the early stages revealed four types of abnormalities, i.e. polyspermy, polygyny, asynchrony between male and female pronucleus development, and preactivation of cytokinesis.
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Fertilization in vitro of bovine oocytes: analysis of some factors affecting the fertilization rates.
Ovarian follicles were aspirated from superovulated heifers at different periods from the beginning of standing heat. The follicular fluid of these follicles was analyzed for progesterone, estradiol, testosterone and androstenedione concentrations and compared to the maturation stages of the oocytes and their ability to undergo fertilization in vitro. The results obtained suggest that high concentrations of progesterone, testosterone and androstenedione compared to a lower concentration of estradiol correspond to a high proportion of M-II oocytes and a high in vitro fertilization rate.
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Ultrastructure of the final nuclear maturation of bovine oocytes in vitro.
Cumulus-oocyte complexes were obtained from cow ovaries by aspiration from small (1-6 mm in diameter) antral follicles after slaughter. Complexes with a compact multilayered cumulus investment were cultured and subsequently processed for electron microscopy after various periods of culture. By morphological criteria the oocytes could be divided into the following sequence of meiotic stages. The oocyte nucleus I stage was characterized by a spherical nucleus located peripherally in the ooplasm while undulation of the nuclear envelope and initial chromatin condensation was seen at the oocyte nucleus II stage. The oocyte nucleus breakdown stage was characterized by formation of long slender projections from the nuclear envelope in which the envelope doubled back on itself, appearance of dense areas and haphazardly oriented microtubules in the nucleus, marked condensation of the chromatin, and dissolution of the nuclear envelope into irregular vesicles and tubules. The condensed chromatin I stage was characterized by the location of condensed chromatin configuration and uniformly oriented microtubules in a dense area peripherally in the ooplasm while the final condensed chromatin II stage was characterized by a gradual invasion of condensed chromatin configurations into a dense area combined with the presence of the first polar body in the perivitelline space.
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In vivo development of in vitro fertilized bovine oocytes matured in vivo versus in vitro.
In vivo developmental potentials of in vivo and in vitro matured oocytes fertilized in vitro were assessed in cattle. One-cell stages produced from in vivo matured oocytes developed into a pregnancy when transferred to the ampulla part of oviducts of synchronized heifers. In vitro matured oocytes achieved high penetration and cleavage rates but did not develop into pregnancies when transferred to synchronized heifers.
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Premature ovulations in superovulated cattle.
The incidence and consequences of premature ovulations in superovulated cattle were studied. These ovulations, presumably induced by the luteinizing hormone content of the gonadotrophin preparation used, were found in 17 of 133 (13%) stimulated cows and heifers used as oocyte donors. In general this group of donors exhibited deviating periovulatory patterns of plasma hormone profiles (progesterone, estradiol17beta and LH), estrus behaviour, follicular steroidogenesis of progesterone and estradiol-17beta, and oocyte meiosis. It is concluded that superovulated donor cattle exhibiting premature ovulations constitute a group of poor oocyte donors that also must be considered as inferior embryo donors.
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Pregnancy resulting from cattle oocytes matured and fertilized in vitro.
Follicular oocytes (n = 81) collected from cattle at a local slaughterhouse were matured and fertilized in vitro. Of 27 ova 19 (70%) were penetrated by spermatozoa and 40/54 (74%) inseminated ova transferred surgically to the oviducts of a synchronized heifer were recovered by non-surgical flushing of the uterine horns 6 days later. Of the 40 ova 15 (38%) were at the morula, early blastocyst or diminutive morula stages. Culture in vitro sustained further development of all embryos and 9 were expanding or expanded blastocysts. One pregnancy resulted from non-surgical transfer of 2 blastocysts. The results demonstrate that immature oocytes from cattle can be matured and fertilized in vitro, subsequently develop to the blastocyst stage, and develop into a normal pregnancy after non-surgical transfer.
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An automated direct-injection HPLC-method for the electrochemical/fluorimetric quantitation of monoamines and related compounds optimized for the screening of large numbers of animals.
An automated, highly stable HPLC system is described allowing the quantitation of monoamines and related compounds with virtually online data processing and statistical evaluation of the mean values of various differently treated groups of animals. The system is highly suited to the screening of drugs involving large numbers of samples. To ensure long-term, uninterrupted performance, two robust chromatographic systems were developed optimized to separate neighboring peaks as widely as possible. This was achieved by using mobile phases of relatively low pH to retard acidic compounds, and optimal concentrations of the ion-pairing reagent to manipulate the retention times of amines on the RP-18 column, resulting also in clearcut separations from the solvent/tissue peak. Direct injection of deproteinized tissue samples, requiring no clean up procedures is used. One faster system allows measurement of the main biogenic amines, metabolites and TRP, whereas another system allows the simultaneous quantitation of DOPA, 5HTP, NA, MOPEG, NMN, A, MN, VMA, DA, 3MT, DOPAC, HVA, 5HT, 5HIAA and TYR. By pretreatment of animals with a decarboxylase inhibitor the latter system offers the possibility of detecting drug effects in the same animal on either dopamine and serotonin turnover or noradrenaline and serotonin turnover, depending on the brain area chosen. The system described has been running for over a year without major disturbance and with minimal technician attendance.
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Progesterone and luteinizing hormone profiles in heifers used as oocyte donors.
Progesterone (P(4)) and luteinizing hormone (LH) profiles were analyzed throughout the estrous cycle in 11 superovulated heifers that had follicular oocytes aspirated at different times after standing heat. It was found that high P(4) during estrus was incompatible with normal LH release, oocyte maturation and subsequent in vitro fertilizing capability. However, an LH peak was not a prerequisite for initiation of meiosis, since both metaphase I (MI) and metaphase II (MII) stages were observed in animals without an LH surge. Following follicular aspiration, the progesterone levels and the length of luteal phase were similar to those of superovulated animals that had no follicular intervention. We concluded that aspiration per se does not interfere with normal corpora lutea (CL) development in heifers when aspiration occurs after the LH surge.
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Ultrastructure of bovine embryos frozen and thawed by a two-step freezing method.
An ultrastructural evaluation of a rapid tow-step freezing method, by which 6-7-day-old bovine embryos equilibrated in 1.4 M glycerol in Dulbecco's phosphate-buffered saline were frozen and thawed, was undertaken. In all non-frozen control embryos trophoblastic and embryonic cells formed a spherical structure enclosed by an intact zona pellucida. The spacial arrangement of the cells of the frozen embryos was less regular and the surrounding zona pellucida was damaged in approximately half of the cases. Some embryonic cells had increased electron density and lysosomal content showing reaction sites for acid phosphatase. In all frozen embryos, cytoplasmic defects appearing as non-membrane-bound 'empty spaces' were observed more frequently in the trophoblastic cells than in the embryonic cells. Culture of frozen embryos for 24 h revealed that cells appearing nondefective after culture may have the capability of organizing a viable embryonic structure. It was found that the most commonly used freezing method is associated with certain morphological changes. However, no additional cryoinjuries were observed in comparisons with the more complicated freezing procedures using dimethylsulfoxide as cryoprotectant.
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Ultrastructural features of preovulatory oocyte maturation in superovulated cattle.
Cows and heifers were induced to superovulate by treatment with PMSG or FSH. The ultrastructural features of the oocytes were related to the time of the LH peak and the progesterone/oestradiol-17 beta ratios in the follicular fluid. At 0-2 h after the LH peak the perivitelline space developed; at 9-12 h there was disconnection of the junctions between cumulus cell projections and oolemma, and the concomitant breakdown of the oocyte nucleus; at approximately 15 h there were spatial rearrangements in the ooplasm of (a) mitochondrial clusters from a peripheral to an even distribution and (b) vesicles from an even distribution to a more central location; at approximately 19 h there was abstriction of the first polar body with dislocation of mitochondrial clusters and vesicles towards the site of polar body formation; at 21-22 h there was migration of cortical granules to solitary positions along the oolemma and decrease in the sizes of Golgi complexes and, on some occasions, the smooth endoplasmic reticulum. These ultrastructural changes were accompanied by an increase in progesterone/oestradiol ratios in the follicular fluids. It is concluded that preovulatory oocyte maturation in gonadotrophin-stimulated cattle comprises nuclear as well as cytoplasmic changes accompanied by steroidogenic changes in the follicle, each of which are closely related to the time of the LH peak. However, some variation existed between animals, between follicular and oocyte maturation and even within oocytes between nuclear and cytoplasmic maturation.
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