Activated bovine cytoplasts prepared by demecolcine-induced enucleation support development of nuclear transfer embryos in vitro

Demecolcine-induced enucleation (IE) of mouse oocytes has been shown to improve development to term of cloned mice. In this study, we characterized the kinetics and morphological progression of bovine oocytes subjected to IE, and evaluated their ability to support embryo development to the blastocyst stage after nuclear transfer (NT). In vitro matured bovine oocytes were parthenogenetically activated and subsequently exposed to demecolcine at various times post-activation. Onset and duration of demecolcine treatment significantly altered activation and IE frequencies, which varied from 7.1% to 100% and 33.3% to 91.7%, respectively, at 5 hr post-activation. A significant decrease in IE frequencies was observed at 17 hr post-activation (3.4%-46.1%), possibly due to reincorporation of chromosomes into the oocyte after incomplete second polar body (PB) extrusion. Oocytes were reconstructed by NT before (treatment 1) or after (treatment 2) activation and demecolcine treatment, and cultured in vitro. Cleavage (48.1%-54.2%) and blastocyst rates (15.7%-19%) were equivalent for the two treatments, as well as the total cell number in NT blastocysts. Furthermore, most of the blastocysts were completely diploid (treatment 2) or heteroploid but with a majority of diploid nuclei (treatment 1). Our results demonstrate that the IE method can be successfully used to produce enucleated bovine cytoplasts that are competent to support development to the blastocyst stage after NT. This technically simple approach may provide a more efficient method to enhance the success rate of NT procedures. Further studies are needed to improve the in vitro development efficiency and to expand our understanding of the mechanism(s) involved in demecolcine-induced enucleation.     

Effects of increased ambient temperature on the development of in vitro derived bovine zygotes

In this study, presumptive bovine zygotes were subjected to two consecutive 24-h cycles of heat treatment during the first 48 h (Experiment I) of in vitro culture (IVC) or 24h of heat treatment during the fourth day of IVC (Experiment II). In Experiment I, the percentage of heat treatment zygotes that developed to > or =8-cell stage embryos after 72 h IVC was 2.0% (n = 459) compared with 28.4% (n = 458) for the control zygotes (P<0.001). The subsequent yield of morulae or blastocysts after 144 h IVC for the heat treatment and control groups was 0.9% (n = 457) and 12.3% (n = 456) (P<0.001), respectively. These results demonstrate that heat treatment during the first 48 h of IVC significantly impaired embryo development. In Experiment II, the percentage of zygotes that developed into morulae and blastocysts following heat treatment during the fourth day of IVC was 4.5% (n = 468) compared to 10.5% (n = 456) for the control group (P<0.001). This study has demonstrated that in vitro heat stress during the critical stage of early embryo development significantly increases the incidence of early embryonic mortality.     

Effects of resazurin on bovine oocyte fertilization and embryo development in vitro

Resazurin is a redox dye (7-hydroxy-3H-phenoxazin-3-one-10-oxide) used for assessing potential fertility of spermatozoa and functional status of eukaryotic cells. In this study, the fertilizing capacity of spermatozoa treated with resazurin and effects of resazurin on bovine embryo development in vitro was examined. Abattoir-derived bovine oocytes were collected and subjected to in vitro maturation (IVM), fertilization (IVF) and culture (IVC). In Experiment 1, bovine oocytes (n=2767) were fertilized with spermatozoa exposed to resazurin (17.6 μg/ml) for 0, 15, 30, 60 min, respectively. There was no significant (P>0.05) difference with respect to oocyte cleavage, morula and blastocyst production between treatments. In Experiment 2, oocytes (n=1671) were treated with resazurin (1.8 μg/ml) during IVM, IVF, IVC, respectively, or during the entire IVM, IVF and IVC procedures. There was no significant (P>0.05) difference in cleavage rates. However, the proportion of embryos that developed into blastocysts, expanded and hatched blastocysts in those groups in which oocytes/embryos were treated with resazurin during IVC or IVM/IVF/IVC was significantly (P<0.05) less than those exposed to resazurin during IVM only, or during IVF only. We conclude that resazurin did not have significant adverse effects on fertilizing capability of bovine spermatozoa; however, extended treatment of embryos with resazurin may be detrimental to embryonic development.     

Effects of low temperatures on in vitro produced bovine zygotes

The objective of this research was to investigate the effects of cooling on the development of bovine zygotes. One-cell bovine embryos were maintained at 39°C (control), 20°C, 10°C, or 0°C for 5, 10, or 20 minutes, then cultured in vitro for 7 days and the proportion of embryos developing to the compact morula or blastocyst stage compared between different treatments. Duration of exposure time had no effect on development. Development rates to the compact morula or blastocyst stage were 3.9%, 11.4%, 17.4%, and 24.4% for zygotes maintained at 0°C, 10°C, 20°C, and 39°C, respectively, with differences in embryo yield between every treatment (P < 0.05). In a second experiment, bovine pronuclei (karyoplasts) and cytoplasts were cooled at 0°C or maintained at 39°C for 5 minutes. Pronuclear transplantation was then utilized to create 4 types of reconstructed embryos, those with: 1) non-cooled pronuclei and non-cooled cytoplasm, 2) non-cooled pronuclei and cooled cytoplasm, 3) cooled pronuclei and non-cooled cytoplasm, and 4) cooled pronuclei and cooled cytoplasm. The proportion of embryos developing to the blastocyst stage was highest when non-cooled pronuclei were transferred into non-cooled cytoplasm (18.9%), and similar to that of non-cooled, non-manipulated control zygotes (13.2%, P > 0.05). No embryos developed to the blastocyst stage when pronuclei (cooled or non-cooled) were transferred into cooled cytoplasm. However, zygotes with cooled pronuclei transferred into non-cooled cytoplasm yielded 4.5% blastocysts (P < 0.05). More embryos developed to the compact morula or blastocyst stage when non-cooled vs. cooled cytoplasm was utilized, regardless of whether the pronuclei were cooled (P < 0.05). These data demonstrate that pronuclei are more tolerant to low temperature exposure than is ovum cytoplasm. Mol. Reprod. Dev. 47:435–439, 1997. © 1997 Wiley-Liss, Inc.     

Effects of cryopreservation of pronuclear-stage rabbit zygotes on the morphological survival, blastocyst formation, and full-term development after DNA microinjection

The objectives of this study were to examine the freezing sensitivity of pronuclear-stage rabbit zygotes and to produce transgenic rabbits using the cryopreserved zygotes. Zygotes were cryopreserved either by one of two vitrification protocols or by one of the two conventional freezing protocols. The morphological survival rates of zygotes subjected to two-step freezing in 1.5 M ethylene glycol and 0.1 M sucrose (74%) or to vitrification in 7.2 M ethylene glycol and 1.0 M sucrose (81%) were higher than those subjected to freezing in 1.5 M DMSO (46%) or to vitrification in a mixture of 2.0 M DMSO, 1.0 M acetamide, and 3.0 M propylene glycol (41%). But the in vitro development into blastocysts of zygotes cryopreserved by vitrification (17%) or to a lesser extent by freezing (52%) was impaired, when compared to that of fresh control zygotes (89%). Next, a fusion gene composed from bovine aS1-casein promoter and a human GH structural gene (2.8 kb) was microinjected into the pronucleus of rabbit zygotes frozen-thawed in ethylene glycol and sucrose. Then, the presence of exogenous DNA in the genome of newborn offspring was determined by PCR. The post-injection survival of frozen zygotes (97%) was the same as that of fresh control zygotes (96%). However, of 18 offspring derived from 414 frozen-thawed and DNA-injected zygotes, no transgenic rabbits were produced. Of 52 offspring derived from 403 DNA-injected fresh zygotes, 3 transgenic rabbits were found. Here we report the first rabbit offspring resulting from zygotes cryopreserved at the pronuclear-stage, although the cryopreservation procedure employed must be improved if zygotes are to be used for systematic production of transgenic rabbits.     

In vitro oocyte fertilization and subsequent embryonic development after cryopreservation of bovine ovarian tissue, using an effective approach for oocyte collection

This study was undertaken to assess dissection/puncture combined technique for collecting large number of oocytes from bovine ovaries and to determine the effect of ovarian tissue cryopreservation on the oocytes capability to undergo in vitro maturation, fertilization and subsequent embryonic development. Ovaries (n=31) of slaughtered cows were cut into small fragments using a scalpel blade and the ovarian tissues were randomly assigned to cryopreserved by slow freezing and vitrification and non cryopreserved (fresh) groups. Oocytes were collected from non-atretic follicles from fresh and post-thawing ovarian tissue by the puncture method. The advantage of this technique appeared through morphologically good quality cumulus-oocyte complex (COC) recovery rate from fresh tissue (31.7±2.0 oocytes/ovary). However, the cryopreservation affected the post thawing total and good quality COC recovery rates from slow freezing (26.6±2.0 and 23.5±2.3 oocytes/ovary, respectively) and vitrification groups (21.7±1.1 and 17.6±1.8 oocyte/ovary, respectively). The maturation rate resulted in significant differences between the fresh tissue (94.1±1.1%) and the two cryopreservation groups. Moreover, this rate was significantly higher in the slow freezing group (80.1±1.3%) than in the vitrification group (73.0±1.9%). No statistical differences were observed in the cleavage and the embryonic developmental rates between fresh tissue group and cryopreservation groups. Furthermore the number of embryos produced per animal was statistically higher for fresh tissues than for slow freezing and the vitrification groups (34.4±1.4, 27.8±3.1 and 22.0±0.7, respectively). In conclusion, dissection method followed by puncture of bovine ovaries greatly maximizes the number of good quality oocytes recovered, as well as the number of embryos obtained per animal. Ovarian tissue can be successfully cryopreserved by slow freezing and vitrification.     

Effect of oocyte maturation medium on in vitro development of in vitro fertilized bovine embryos.

The objective of this study was to examine the effects of different culture media used for maturation of bovine oocytes on in vitro embryo development following in vitro fertilization. Oocytes were aspirated from 2-5 mm follicles of ovaries collected at a local abattoir. The oocyte-cumulus complexes (OCCs) were cultured for 23-25 h in one of seven commercially available media supplemented with 6 mg/ml bovine serum albumin (BSA), 0.25 mM pyruvate, 10 micrograms/ml luteinizing hormone (LH), 0.5 microgram/ml follicle-stimulating hormone (FSH), and 1 microgram/ml estradiol. After maturation for 23-25 h, all eggs were subjected to the same in vitro fertilization protocol using modified TALP medium and subsequently cultured in the same serum-free embryo culture medium (HECM-1/BSA) for 8 days, after which embryo development was assessed. Five media (SFRE, MEM alpha, TCM199, MEM alpha/+, RPMI:MEM alpha) better supported normal oocyte maturation as determined by embryo development to the two-cell (76-82%), morula/blastocyst (25-32%), and blastocyst (12-19%) stages. Oocytes that were matured in Waymouth's medium MB 752/l or Ham's F-12 had a significantly reduced incidence of cleavage to the two-cell stage (52% and 37%, respectively), which was not attributed to failure of fertilization. Of the eggs that did cleave to the two-cell stage in these two media, 27% and 9% developed to morulae/blastocysts but only 6% and 3%, respectively, developed into blastocysts.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro and in vivo survival of frozen-thawed bovine oocytes after IVF,nuclear transfer,and parthenogenetic activation

Cryopreservation of bovine oocytes would be beneficial both for nuclear transfer and for preservation efforts. The overall objective of this study was to evaluate the viability as well as the cryodamage to the nucleus vs. cytoplasm of bovine oocytes following freezing-thawing of oocytes at immature (GV) and matured (MII) stages using in vitro fertilization (IVF), parthenogenetic activation, or nuclear transfer assays. Oocytes were collected from slaughterhouse ovaries. Oocytes at the GV, MII, or MII but enucleated (MIIe) stages were cryopreserved in 5% (v/v) ethylene glycol; 6% (v/v) 1,2-propanediol; and 0.1-M sucrose in PBS supplemented with 20% (v/v) fetal bovine serum. Frozen-thawed oocytes were subjected to IVF, parthenogenetic activation, or nuclear transfer assays. Significantly fewer GV oocytes survived (i.e., remained morphologically intact during freezing-thawing) than did MII oocytes (47% vs. 84%). Subsequent development of the surviving frozen-thawed GV and MII oocytes was not different (58% and 60% cleavage development; 7% and 12% blastocyst development at Day 9, respectively, P > 0.05). Parthenogenetic activation of frozen-thawed oocytes resulted in significantly lower rates of blastocyst development for the GV than the MII oocyte groups (1% vs. 14%). Nuclear transfer with cytoplasts derived from frozen-thawed GV, MII, MIIe, and fresh-MII control oocytes resulted in 5%, 16%, 14%, and 17% blastocyst development, respectively. However, results of preliminary embryo transfer trials showed that fewer pregnancies were produced from cloned embryos derived from frozen oocytes or cytoplasts (9%, n = 11 embryos) than from fresh ones (19%, n = 21 embryos). Transfer of embryos derived by IVF from cryopreserved GV and MII oocytes also resulted in term development of calves. Our results showed that both GV and MII oocytes could survive freezing and were capable of developing into offspring following IVF or nuclear transfer. However, blastocyst development of frozen-thawed oocytes remains poorer than that of fresh oocytes, and our nuclear transfer assay suggests that this poorer development was likely caused by cryodamage to the oocyte cytoplasm as well as to the nucleus. Mol. Reprod. Dev. 51:281–286, 1998. © 1998 Wiley-Liss, Inc.     

Influence of somatotropin and nutrition on bovine oocyte retrieval and in vitro development

The objective of this study was to determine the effects of supplemental bovine somatotropin (bST) and limit feeding on follicular growth and oocyte competence in yearling beef heifers. Sixteen growing heifers (424+/-4 kg) were randomly assigned to 1 of 4 treatments in a 2 x 2 factorial arrangement, with main effects of bST (0 or 33 microg/kg BW/d) and feeding regimen (ad libitum or 0.75 ad libitum intake). Animals were treated for 100 d prior to follicular aspiration, and treatments continued for the 42-d period that follicles were aspirated. Follicles were observed ultrasonically then aspirated, and recovered oocytes were matured, fertilized and developed in vitro. The number of follicles observed ultrasonically was greater with bST treatment (P<0.01) but was unchanged by plane of nutrition. The number and quality of recovered oocytes were similar among treatments, as was the number of oocytes resulting in blastocyst formation.     

Effects of EGTA and slow freezing of bovine oocytes on post‐thaw development in vitro

Experiments were conducted to assess the morphological viability and in vitro developmental potential of bovine oocytes after exposure to Ethylene Glycol‐bis(‐aminoethyl Ether) N,N,N,N‐Tetra‐acetic Acid (EGTA) prior to slow freezing. Different concentrations of EGTA (0, 1, 5 and 10 mM) and exposure intervals (5, 10 and 15 min) were tested on immature (GV) and in vitro matured (IVM) oocytes equilibrated in 1.5 mM propylene glycol (PG) without (experiment 1) or with slow freezing (experiment 2). In addition, PG and ethylene glycol (EG) were compared for cryoprotective efficacy. In vitro maturation (IVM), in vitro fertilization (IVF) and embryo culture (IVC) were performed in defined conditions. Pretreatment of both types of oocytes with 1 mM EGTA for 5 min without freezing yielded morphological and functional results comparable to those obtained for controls while results from higher concentrations of EGTA were lower (P < 0.05). Higher rates of freeze‐thaw survival and embryonic development were obtained after pretreating GV oocytes with 1 or 5 mM EGTA for 5 min. Similarly, better results were obtained when IVM oocytes were pretreated with 1 mM EGTA for either 5 or 10 min. When pretreated with 1 mM EGTA for 5 min and frozen with PG IVM oocytes exhibited higher survival rates (P < 0.05) than those frozen with EG. However, no significant differences were observed in the in vitro development of surviving GV or IVM oocytes frozen with either PG or EG. Results suggest that a prefreeze treatment with 1 mM EGTA for 5 min can enhance oocyte viability. Conditions described enabled blastocyst development of 2.9% of GV oocytes and 8.0% of IVM oocytes after cryopreservation and IVF. Mol. Reprod. Dev. 52:86–98, 1999. © 1999 Wiley‐Liss, Inc.     

Effects of different cryopreservation methods on post-thaw culture conditions of in vitro produced bovine embryos

The aim of this work was to evaluate the effect of cryopreservation protocols on subsequent development of in vitro produced bovine embryos under different culture conditions. Expanded in vitro produced blastocysts (n = 600) harvested on days 7-9 were submitted to controlled freezing [slow freezing group: 10% ethylene glycol (EG) for 10 min and 1.2°C/min cryopreservation]; quick-freezing [rapid freezing group: 10% EG for 10 min, 20% EG + 20% glycerol (Gly) for 30 s]; or vitrification [vitrification group: 10% EG for 10 min, 25% EG + 25% Gly for 30 s] protocols. Control group embryos were not exposed to cryoprotectant or cryopreservation protocols and the hatching rate was evaluated on day 12 post-insemination. In order to evaluate development, frozen-thawed embryos were subjected to granulosa cell co-culture in TCM199 or SOFaa for 4 days. Data were analyzed by PROC MIXED model using SAS Systems for Windows?. Values were significant at p < 0.05. The hatching rate of the control group was 46.09%. In embryos cultured in TCM199, slow freezing and vitrification group hatching rates were 44.65 ± 5.94% and 9.43 ± 6.77%, respectively. In embryos cultured in SOFaa, slow freezing and vitrification groups showed hatching rates of 11.65 ± 3.37 and 8.67 ± 4.47%, respectively. In contrast, the rapid freezing group embryos did not hatch, regardless of culture medium. The slow freezing group showed higher hatching rates than other cryopreservation groups. Under such conditions, controlled freezing (1.2°C/min) can be an alternative to cryopreservation of in vitro produced bovine embryos.     

Culture of in vitro produced bovine zygotes in vitro vs in vivo: implications for early embryo development and quality

The objectives of this study were to examine the effect of culture system on bovine blastocyst formation rates and quality. Presumptive IVM/IVF bovine zygotes were cultured either in vitro in synthetic oviduct fluid (SOF, 25 embryos/25 microL in 5% CO2, 5% O2, 90% N2 at 39 degrees C) or in vivo in the ewe oviduct (approximately 100 embryos per oviduct). The recovery rate after in vivo culture was 53% (813/1,530). The blastocyst rate on Day 7 was significantly higher for the in vitro system (28%, 362/1,278 vs 17%, 37/813; P< 0.0001). However, after culture in vitro for a further 24 h, there was no difference in Day 8 yields (36%, 457/1,278 vs 32%, 258/813, for in vitro and in vivo culture, respectively). There was no difference in blastocyst cell number between treatments (Day 7: 96 vs 103; Day 8: 78 vs 85 for in vitro and in vivo culture, respectively). Irrespective of culture system, Day 7 blastocysts had a significantly higher cell number than those appearing on Day 8. There was no difference in pregnancy rate at Day 35 after fresh transfer of a single Day 7 blastocyst (37.5%, 21/56 vs 45.3%/, 24/53 for in vitro and in vivo culture, respectively). After cryopreservation by freezing in 10% glycerol, VS3a vitrification or solid surface vitrification, the survival of in vitro cultured embryos was significantly lower than survival of embryos cultured in the ewe oviduct or those produced by superovulation of donors. In conclusion, these findings demonstrate that while bovine zygotes cultured in vitro are capable of rates of development similar to those of their in vivo cultured counterparts (in terms of Day 8 blastocyst yield, cell number and early pregnancy rate), there are significant differences in embryo cryosurvival. This suggests that current in vitro culture systems need to be improved to optimize embryo quality and pregnancy rates.     

Effects of bovine spermatozoa preparation on embryonic development in vitro

Background

The changes occurring in the rodent uterus after parturition can be used as a model of extensive tissue remodeling. As the uterus returns to its prepregnancy state, the involuting uterus undergoes a rapid reduction in size primarily due to the degradation of the extracellular matrix, particularly collagen. Membrane type-I matrix metalloproteinase (MT1-MMP) is one of the major proteinases that degrades collagen and is the most abundant MMP form in the uterus. Matrix metalloproteinase-2(MMP-2) can degrade type I collagen, although its main function is to degrade type IV collagen found in the basement membrane. To understand the expression patterns of matrix metalloproteinases (MMPs) in the rat uterus, we analyzed their activities in postpartum uterine involution.

Methods

We performed gelatin zymography, northern blot analysis and immunohistochemistry to compare the expression levels of MT1-MMP, MMP-2, matrix metalloproteinase-9 (MMP-9) and the tissue inhibitors of MMPs-1 and 2 (TIMP-1 and TIMP-2) in the rat uterus 18 h, 36 h and 5 days after parturition with their expression levels during pregnancy (day 20).

Results

We found that both MT1-MMP and MMP-2 localized mainly in the cytoplasm of uterine interstitial cells. The expression levels of MT1-MMP and MMP-2 mRNAs and the catalytic activities of the expressed proteins significantly increased 18 h and 36 h after parturition, but at postpartum day 5, their mRNA expression levels and catalytic activities decreased markedly. The expression levels of MMP-9 increased 18 h and 36 h after parturition as determined by gelatin zymography including the expression levels of TIMP-1 and TIMP-2.

Conclusion

These expression patterns indicate that MT1-MMP, MMP-2, MMP-9, TIMP-1 and TIMP-2 may play key roles in uterine postpartum involution and subsequent functional regenerative processes.