Cytogenetic analysis of oocytes and early preimplantation embryos from XO mice.

The cytoplasm of early sea urchin embryos contains nonribosomal, high molecular weight RNA both associated with ribosomes in polysomes and free of ribosomes in particles termed free RNP. In a 1-hr labeling period, 50% of the newly synthesized RNA enters the pool of ribosome-free RNP particles during the cleavage stages, and this percentage decreases until less than 20% of the new RNA in the mesenchyme blastula stage is found in the free RNP. mRNA from both polysomes and free RNP contain poly(A)(+) and poly(A)(?) species. During the cleavage stages only 8–10% of the RNA from each fraction is polyadenylated; however, in the blastula, 40–50% of the nonhistone polysomal RNA is polyadenylated while only 22–30% of the free RNP RNA is polyadenylated. At any developmental stage, the poly(A)(+)RNA from the free RNA and polysomes have identical sedimentation profiles; this is also the case for the poly(A)(?)RNA except for the absence of the 9 S histone mRNA from the free RNP. Changes in poly(A)(+)RNA content and sedimentation profiles during development occur simultaneously in the free RNP and the polysomes. Kinetic studies of these two RNP populations as well as nuclear RNP show that the bulk of the free RNP are not unusually stable cytoplasmic components. The free RNP decay with a half-life of about 40 min while nuclear RNA and polysomal RNA display half-lives of about 12 and 65 min, respectively. Further, the rate of synthesis of the free RNP is not consistent with their being the only precursors for polysomes. Our estimates of the rates of synthesis for nuclear RNA, polysomes, and free RNP are, respectively, 1.1 × 10^?15, 2.2 × 10^?16, and 5.0 × 15^?17 g/min/nucleus. The data on free RNP is discussed in terms of translational regulation of protein synthesis in the developing sea urchin.     

Successful in vitro culture of early cleavage stage embryos recovered from superovulated red deer (Cervus elaphus)

Three separate embryo culture systems were evaluated for their ability to support development of early cleavage stage red deer (Cervus elaphus ) embryos: ligated sheep oviducts (Treatment A); cervine oviduct epithelial monolayer in TCM 199 + 10% deer serum (Treatment B); synthetic oviduct fluid + 20% human serum at 7% O(2) atmosphere (Treatment Q. In addition, 2 superovulation protocols were compared for their efficacy in producing early cleavage stage embryos. Twenty red deer (2 to 7 yr old) were synchronized in April with intravaginal CIDR devices for 12 d. All animals received a total of 0.4 units of ovine FSH administered in 8 equal doses, 12 h apart, beginning 72 h before removal of CIDR devices. The deer additionally received 200 IU PMSG, either with the first FSH injection (Group 1, n = 10) or with the last FSH injection (Group 2, n = 10). Hinds were placed with fertile stags following withdrawal of CIDR devices. Ova were collected by surgical recovery 63 h post CIDR removal. At the time of collection, animals in Group 2 had a significantly greater mean (+/- SEM) ovulation rate (11.2 +/- 2.4 vs 5.3 +/- 2.4), with more animals responding to treatment (>1 ovulation), than the animals in Group 1 (10/10 vs 4/10). Late in the breeding season (June), 10 additional red deer (Group 3, Experiment 2) were superovulated using the same protocol as for the deer in Group 2, with ova collection advanced by 24 h. Mean (+/- SEM) ovulation rate was 6.4 +/- 1.2 with 9 10 animals responding. Ova recovery did not differ among the groups (range 73 to 87%). Superovulation treatment did not affect cultured embryo development to the morula/blastocyst stage. Furthermore, there was no difference among the 3 culture systems in their support of development either to the morula (range 50 to 58%) or to the blastocyst (range 22 to 26%) stage. After laparoscopic transfer of 4 morula/blastocyst embryos to recipient red deer (2 from Treatment B and 2 from Treatment C) 2 live calves were born from embryos cultured in Treatment B.     

Development of early bovine embryos to the blastocyst stage in serum-free conditioned medium from bovine granulosa cells

Summary Bovine granulosa cell — conditioned medium (BGC-CM) was prepared in a serum-free medium consisting of TCM 199, 5μg/ml insulin, and 0.5μg/ml aprotinin (TCM 199 IAP). Granulosa cells surrounded with embryos were denuded 24 to 30 h after in vitro fertilization. The proportion of denuded granulosa cell-free embryos that developed to the blastocyst stage in BGC-CM (43/219; 20%) as well as in the co-culture system (43/178; 24%) was significantly greater (P<0.001) than in fresh TCM 199 IAP medium (FM: 10/191; 5%), whereas the proportion of embryos that developed to the eight-cell stage was similar (P>0.05) in all three culture systems (95/178; 53% in co-culture, 111/219; 51% in BGC-CM, and 86/191; 45% in FM, respectively). Higher rates of hatching and hatched blastocysts 8.5 days after in vitro fertilization were observed in co-culture (13/44; 29.5%) and in conditioned medium (8/39; 20.5%). On the other hand, no hatching or hatched blastocysts were obtained in the fresh medium (0.7; 0%). Cell numbers per blastocyst in BGC-CM (178.3 cells/blastocyst) were approximately two-fold higher than those in FM (97.1 cells/blastocyst). However, higher cell numbers (249.3 cells/blastocyst) were observed in co-culture with BGC than in BGC-CM. The embryotrophic activity in BGC-CM was stable upon freezing and thawing, lyophilization, and heating at 56° C whereas activity was reduced by dilution in fresh medium, dialysis, pronase digestion, and heating at 80° C. These results suggest that BGC cultured in a serum-free medium can synthesize and secrete an embryotrophic factor(s) that supports blastocyst formation in vitro beyond the 8- to 16-cell stage.     

Cytogenetic evaluation of in vitro matured bovine oocytes collected from ovaries of individual donors

Oocytes were collected after slaughter by aspiration from pairs of ovaries of individual donors. A total of 656 oocytes was selected for IVM from 74 pairs of ovaries (8.9 oocytes per pair, ranging between 1 and 25). The oocytes were matured in droplets of maturation medium (TCM-199 medium supplemented with 20% estrous cow serum (ECS), 50 microg/ml gentamycin, 10 microg/ml FSH, 1 microg/ml estradiol-17beta). Cytogenetic analysis of 348 oocytes showed 79 at the first metaphase (MI; 22.7%, 79 348 ), 11 at the first telophase (TI; 3.2%, 11/348 ), and 258 at the second metaphase (MII; 74.1% 258/348 ). Significant differences (P < 0.01) were shown among the donors regarding the number of oocytes selected for IVM and the number of oocytes matured for IVF.     

Cytogenetic analysis of diploidy in cloned bovine embryos using an improved air-dry karyotyping method

Of the few published studies on the cytogenetic analyses of bovine nuclear transferred (NT) embryos, results differ between air-dry and fluorescent in situ hybridization (FISH) procedures. A modified air-dry procedure is reported in this study that provides more metaphase plates for analysis. Day 5 and Day 7 bovine NT embryos were cultured in colcemid-containing CR1aa for 10-12 or 16-18 h, then treated in hypotonic sodium citrate for 3-5 min. The standard procedure of 5h in colcemid and 15-20 min in hypotonic solution was the control. A much higher (P<0.01) percent of mitotic nuclei was observed in the experimental groups. The 33 and 41% mitotic nuclei were obtained from 10 to 12 h and 16 to 18 h-colcemid-treated Day 5 embryos, respectively, which was higher (P<0.001) than the control (15%). The mitotic nuclei in Day 7 NT embryos were 24% in 10-12 h- and 28% in 16-18 h-colcemid-treated groups, which also was higher (P<0.05) than the control (10%). The majority of analyzable embryos were diploid. Analyses of mixoploid embryos showed on average that 70% of the cells were diploid. Day 5 mixoploid embryos contained numerically higher polyploid cells than Day 7 embryos, although statistically there were no differences. We concluded that the modified air-dry method provided a larger source of mitotic nuclei for chromosome analyses of cloned bovine embryos.     

Effect of transportation stress on ovarian function in superovulated Hereford heifers

A study was conducted to determine the effect of transportation stress on ovarian function in superovulated heifers. Thirty cyclic Hereford heifers of similar age and weight and in good body condition were randomly assigned to control and stress-treated groups. All animals received two daily injections of 5 mg follicle stimulating hormone (FSH) for 4 d beginning on Day 10 to 12 of the estrous cycle. A blood sample was collected at each FSH injection. On the fourth day of injections, heifers were given 25 mg prostaglandin F(2)alpha (PGF(2)alpha) in the morning and a second injection of 15 mg PGF(2)alpha in the afternoon. During superovulation, the stressed heifers were transported to a different location every 12 h whereas control animals remained at the pretrial site. Following 4 d of intermittent transporting and FSH treatment, stress-treated heifers were recombined with control animals. Ovaries were examined 8 d following the onset of standing estrus to determine length, width, thickness, and number of corpora lutea (CL). Peripheral plasma levels of cortisol were higher in the stressed group (P< 0.1). Least squares means for numbers of CL were 20.4 +/- 2.1 and 15.4 +/- 1.7 for control and treated heifers, respectively (P< 0.1). There were no treatment differences (P> 0.1) between length, width, or thickness of ovaries when the number of CL was held constant. These data suggest that stress of the type, intensity, and duration imposed in this study increased plasma levels of cortisol and reduced ovulation rate as determined by CL formation in superovulated heifers.     

Calyculin-A improves chromosome condensation for cytogenetic analysis of blastomeres from bovine and murine eight-cell stage embryos

This study evaluated the serine/threonine phosphatase inhibitor calyculin-A for rapid, efficient induction of premature chromosome condensation (PCC) in blastomeres obtained from Day 3 bovine and Day 2 murine eight-cell stage embryos, and its potential for use in cytogenetic analysis. Experiment 1 tested calyculin-A duration (0, 60, 120, and 180min) to induce PCC in bovine blastomeres. More blastomeres that underwent PCC had chromosomes suitable for cytogenetic analysis if treated for 120 or 180min (P<0.005). Experiment 2 compared doses of calyculin-A (0, 10, 50, and 100nM) on bovine blastomeres; calyculin-A (50nM, 120min) induced PCC suitable for cytogenetic analysis in the greatest number of blastomeres when compared to other doses (52.5%; P<0.005). Effects of calyculin-A (50nM) on murine blastomeres at durations of 0, 60, 90, and 120min to induce PCC were tested in Experiment 3, with 90min inducing the highest frequency of condensed chromosomes suitable for cytogenetic analysis (34%; P<0.05). Finally, Experiment 4 evaluated calyculin-A treated bovine embryos under optimal conditions (50nM, 120min) for use in gender and cytogenetic analysis. Whole chromosome paint probes were successfully hybridized to chromosomes along with 4',6-diamidino-2-phenylindole dihydrochloride hydrate (DAPI) counterstaining, allowing detection of embryo gender (54% F:46% M) and ploidy of individual blastomeres within embryos (64% diploid:36% mixoploid embryos). In conclusion, we inferred that calyculin-A was useful for rapid induction of PCC, producing chromosome spreads suitable for cytogenetic analysis of blastomeres in G1 or G2/M phase of the cell cycle.     

Frequency of chromosomal abnormalities in embryos from superovulated merino ewes

Chromosomal analysis was carried out on 48 Day 2-7 embryos collected from superovulated Merino ewes. Three embryos had abnormal chromosome complements (1 X 1N, 1 X 1N/2N, 1 X 3N), yielding an incidence of 6.25% abnormal embryos. It is concluded that superovulation does not cause an increase in the incidence of chromosomal abnormalities in embryos of Merino sheep.     

Cytogenetic analysis of Day-4 embryos from PMSG/hCG-treated prepuberal gilts

Prepuberal gilts were treated with pregnant mare serum gonadotropin (PMSG) to study the effects of its dosage on ovulation rate, fertilization rate after artificial insemination, embryo viability, and rate of development and incidence of chromosome abnormalities in Day-4 embryos. Gilts received 750 IU, 1250 IU or 1500 IU of PMSG, followed 72 h later by 500 IU human chorionic gonadotropin (hCG). Gilts were inseminated 28 to 30 h following the hCG injection, and resulting embryos were collected on Day 4 post ovulation. Ovulation rate was higher in the 1250 IU group than in the 1500 IU group or the 750 IU group. The 1500 IU dose caused excessive stimulation of the ovary, resulting in the occurrence of large (>10mm diameter) unovulated follicles, reduced fertilization rate and low embryo recovery rate. There was no difference in the incidence of chromosome abnormalities among the three groups, although the 1500 IU group had higher embryonic mortality than the two lower dose groups. A dose of 1250 IU PMSG increased ovulation rate above that achieved by 750 IU and, therefore, increased the number of oocytes or embryos available for transfer or for other studies, without sacrificing embryo viability or increasing the incidence of chromosome abnormalities.     

Immunocytochemical analysis of the association of bovine oviduct-specific glycoproteins with early embryos.

The bovine oviductal epithelium synthesizes and secretes a class of oviduct-specific glycoproteins that is present in the luminal fluid when fertilization and early embryonic development occur. The objective of this study was to determine if these characterized glycoproteins become associated with oviductal embryos. Ovarian ova and oviductal embryos were recovered from super-ovulated cows at 72 h after onset of estrus. Eggs were fixed in 3% paraformaldehyde-1% glutaraldehyde and subsequently embedded in Lowicryl K4M. Sections (1 micron) were processed for peroxidase-antiperoxidase immunocytochemistry. Immunolabeling was not detected in any region of ovarian ova. Oviductal embryos, regardless of cleavage stage, exhibited immunoperoxidase staining localized within their zona pellucidae. Sections (100 nm) obtained from a 4- and an 8-cell embryo were also subjected to colloidal gold immunoelectron microscopy to determine conclusively the subcellular distribution of the oviduct-specific glycoproteins. Gold particles were distributed uniformly throughout the width of the zona pellucida. Also, immunoreactivity was observed associated with flocculent material in the perivitelline space and with the vitelline membrane. These results indicate that the bovine oviduct-specific secretory glycoproteins become associated with oviductal embryos. This association may be biologically important to the developing embryo.     

Triglyceride content of bovine oocytes and early embryos

A microfluorescence technique was used to measure the triglyceride content of a minimum of two bovine oocytes or preimplantation embryos up to the hatched blastocyst stage. Embryos were produced in vitro from abattoir-derived ovaries and grown in medium containing synthetic oviductal fluid, amino acids and BSA (SOFaaBSA medium); 10% fetal calf serum was added to some of the embryos at the four-cell stage. Before maturation, the triglyceride content of oocytes was 59 +/- 1.37 ng and it decreased (P < 0.05) after maturation to 46 +/- 0.85 ng. A decrease in triglyceride content (P < 0.05) was also observed after fertilization with the formation of the two-cell embryo (34 +/- 1.80 ng). In the absence of serum, the triglyceride content remained relatively constant from the two-cell to the hatched blastocyst stage. The triglyceride content of blastocysts produced in vivo was similar (33 +/- 0.70 ng) to that of blastocysts produced in vitro in the absence of serum. In contrast, the triglyceride content of embryos grown with 10% fetal calf serum increased steadily from the 9-16-cell stage to a value in hatched blastocysts (62 +/- 1.14 ng) almost double that in serum-free conditions. These results indicate that triglyceride may act as energy source during bovine oocyte maturation and fertilization and that the presence of serum causes excessive synthesis or accumulation of triglyceride in early embryos.     

In vivo development of microinjected embryos from superovulated prepuberal slaughter lambs

This study was performed to investigate the developmental potential of microinjected embryos recovered from superovulated prepuberal lambs. Fifty-nine mixed-bred lambs (about 3 mo old) were superovulated either with 18 mg FSH-P with (Group FSH/+S) or without (Group FSH/-S) progestagen treatment, or with 10 mL Ovagene following progestagen treatment (Group OVA/+S). All animals received hCG to induce ovulation. Ovulation rates and the number of ova recovered per animal for the different groups were 8.7 and 4.7 (55%, FSH/+S); 7.3 and 3.2 (42%, FSH/-S); and 6.4 and 4.0 (65%, OVA/+S), respectively. No significant differences were seen in the ovulation and the recovery rates, but animals without progestagen treatment showed a significantly lower fertilization rate (44%) when compared with progestagen-treated groups (87%; P<0.001). Foreign DNA was microinjected into the pronuclei of fertilized ova (n = 155), which were transferred (n = 98) into synchronized recipient ewes (n = 21). Two animals were detected pregnant and both gave birth to a single lamb. Results of superovulation and embryo recovery from prepuberal lambs were promising, but the low rate of development to term indicates that ova recovered from prepuberal lambs have reduced developmental competence in vivo. Although 2 lambs were born, it seems that this is not a successful method for use in future gene transfer programs.     

Cleavage beyond the block stage and survival after transfer of early bovine embryos cultured with trophoblastic vesicles

Early bovine embryos (1- to 8-cell stages) were recovered from superovulated heifers at slaughter on Days 2 or 3. Embryos were cultured for 3-4 days in Medium B2 supplemented with 15% (v/v) fetal calf serum in the absence (B2SS, 106 embryos) or presence of trophoblastic vesicles (B2SS + TV, 190 embryos). At the end of culture, there were more (P less than 0.001) morulae (greater than or equal to 16 cells) in B2SS X TV (46%) than in B2SS alone (18%) irrespective of the initial cell stage. More 8-cell embryos reached the 16-cell stage than did embryos with less than 8 cells (30% vs 15% in B2SS, P greater than 0.05; 70% vs 41% in B2SS + TV, P less than 0.005). After culture, 102 morulae were transferred non-surgically to temporary recipient heifers (84 embryos cultured in B2SS + TV and 18 in B2SS). After 2 or 3 days, 14 out of 58 embryos from the B2SS + TV group and 3 out of 10 embryos from the B2SS group were recovered as blastocysts. Most blastocysts were deep-frozen and stored for several weeks. After thawing, 10 apparently normal embryos from the B2SS + TV group were transferred non-surgically into 10 recipient heifers. Four pregnancies were induced, but only one embryo survived to term (birth of a normal male calf). It is concluded that trophoblastic vesicles release one or several unknown compound(s) normally present in vivo, promoting the cleavage of early bovine embryos.     

The effect of co-treatment with recombinant bovine somatotrophin on plasma progesterone concentration and number of embryos collected from superovulated Holstein heifers

Mature Holstein heifers were induced to superovulate with twice-daily injections of porcine follicle-stimulating hormone (FSH), and were given either 20 mg i.m. of recombinant bovine somatotrophin (rBST) or saline with each FSH injection. The animals were artificially inseminated and the embryos were collected nonsurgically at Day 7. There was no significant difference in the mean (+/-S.D) total number of embryos collected from rBST-treated animals (8.3+/-5.3) when compared with that of the controls (7.2+/-6.6), or in the mean number of transferable embryos (5.3+/-4.0 vs 5.2+/-4.5). However, co-treatment with rBST tended to increase the ovulatory response, and it significantly increased plasma progesterone concentrations at Day 6 (P = 0.04). Based on these latter observations, we conclude that treatment with rBST enhanced the superovulatory response in heifers.     

Failure to isolate Brucella abortus from embryos or ova from culture-positive superovulated cows

Nine, Brucella abortus culture positive 2-yr-old cows were used to test the hypothesis that embryos and ova collected from such cows are not infected. Superovulation was induced at varying times postpartum or postabortion with intramuscular injections of follicle stimulating hormone (FSH). The cows were artificially inseminated with B. abortus-negative semen. Superovulations and nonsurgical embryo collections nonsurgical embryo collections were attempted twice for each cow. Jugular blood, udder secretions, cervical swabs, uterine collections, embryos and ova were cultured bacteriologically from the nine cows simultaneously at nonsurgical embryro collections, and B. abortus was isolated only from the udder secretions of seven cows. Brucella abortus was not isolated from 15 uterine collections, 21 embryos, or 18 ova from the culture-positive cows. It was concluded that B. abortus was not present at the detection limits of the culture method employed, which supports the finding or view that embryos and ova collected from donor cows at 100 days or greater post partum or post abortion are not likely to harbor Brucella.     

Telomerase activity in bovine embryos during early development

The telomere is the end structure of the DNA molecule. Telomerase is the ribonuclear enzyme that helps the cell's telomere to elongate; otherwise, the telomere will shorten with each cell division through conventional DNA replication. In most mammalian species, telomerase activity is present in germ cells but not in somatic cells. Recent research shows that telomerase activity is also present in early embryos, but to our knowledge, the dynamics of this enzyme during early embryo development have not been studied. In the present work, we conducted telomerase activity assays on bovine embryos fertilized in vitro and harvested at different stages from zygote to blastocyst. A polymerase chain reaction-based assay (Telomeric Repeat Amplification Protocol) was used to detect the telomerase activity in these embryos. We demonstrated that the telomerase activity is present in the early embryos, but that its level varies with the different developmental stages. The activity was relatively low in mature oocytes. It increased after in vitro fertilization and then decreased gradually until the embryo reached the eight-cell stage. After the eight-cell stage, the telomerase activity increased again and reached its highest level in the blastocyst stage. This study provides insight regarding how telomerase activity and, possibly, the length of the telomere are reprogrammed during early embryo development.     

The hyaluronic acid receptor (CD44) is expressed in bovine oocytes and early stage embryos

Hyaluronic acid (HA) is a high molecular weight polysaccharide found in the extracellular matrix of most animal tissues, that exerts a profound influence on cell behavior. HA is one of the most abundant glycosaminoglycans (GAGs) in the uterine, oviductal and follicular fluids in mouse, pig, human and cattle. CD44, the principal cell membrane receptor for HA, is expressed from the 1- to 8-cell stage in human embryos, during post-implantation mouse embryogenesis and on the surface of differentiated embryonic stem cells. In the present study, we have analyzed by immunofluorescence, whether CD44 is present in bovine oocytes, fertilized oocytes and early stage embryos. Bovine cumulus–oocyte complexes (COCs) were aspirated from follicles (2–5 mm) and were selected for IVM and incubated for 24 h. Oocytes showing an expanded cumulus (generally 90–95%) were used for IVF. Fertilized oocytes were separated for immunofluorescence assay after 16 h of sperm incubation in order to fix the eggs at the pronuclear stage. The embryos were cultured for 8 days and the different stages of development for immunofluorescence assay were separated every 24 h of culture. The CD44 receptor was detected at every observation time examined. Fluorescence-tagged HA for the internalization assay was prepared by mixing fluorescein amine, Isomer I and 1 mg of HA from umbilical cord. Fluorescence-tagged HA was internalized in 2-, 4-, 8- and 16-cell-stage embryos, morulae and blastocysts. CD44 is expressed on the surface and in the cytoplasm of bovine oocytes and embryos in different stages of development.     

Incidence of chromosomal anomalies in early bovine embryos derived from in vitro fertilization

The incidence of chromosomal anomalies in early bovine embryos derived from follicular oocytes fertilized in vitro using sperm separated by Percoll density gradient centrifugation was investigated. Overall, chromosomal anomalies were observed in 13.7% (138/1005) of embryos. There were 14 haploids (1.4%), 2 hypodiploids (0.2%), 6 hyperdiploids (0.6%), 101 triploids (10.0%), 12 tetraploids (1.2%), 2 diploid/triploid mosaics (0.2%), and 1 diploid/tetraploid mosaic (0.1%). The frequency of triploidy was caused mainly by polyspermy. There was a significant difference in the frequency of embryos with abnormal chromosomes between the two bulls used (P < 0.005), but Percoll centrifugation did not affect the observed incidence of anomalies. The frequency of chromosomal anomalies in embryos at each stage increased with delay or arrest of development. These results suggest that the incidence of chromosomal anomalies depended on the conditions of in vitro fertilization and the arrest of development.