Relationships between follicular fluid steroid hormone concentrations, oocyte maturity, in vitro fertilization and embryonic development in the rhesus monkey

Oocytes and matched samples of follicular fluid (FF) were obtained from 70 follicles of five rhesus monkeys stimulated with either pregnant mare serum gonadotropin or human menopausal gonadotropin. Follicular aspiration was performed 30-32 h after human chorionic gonadotropin administration. The concentrations of estradiol (E2), progesterone (P), testosterone (T), and dihydrotestosterone (DHT) in FF were measured. Twenty-six percent of oocytes were classified as mature (M), 41% matured in vitro (Miv), 13% were dysmature, and 20% atretic. M oocytes were associated with significantly higher levels of P and a higher P:E2 ratio. There were no differences in hormone levels associated with fertilized and nonfertilized oocytes. Thirty-five embryos developed to the six- to eight-cell stage in vitro, of which 13 exhibited optimal cleavage rates. Significantly lower levels of E2 and higher P:E2 ratios were associated with the more rapidly cleaving embryos. Proportionally more embryos showing optimal cleavage rates developed from M compared to Miv oocytes, and only embryos derived from M oocytes developed to blastocysts in culture. Optimal cleavage rates to the six- to eight-cell stage in vitro, rather than fertilization rates, are a better indicator of (subsequent) developmental capacity, and, in this study, embryonic development was closely associated with the maturity of the oocyte at recovery.     

Preimplantation development of rabbit embryos after transfer of embryonic nuclei into different cytoplasmic environment

The development of nuclear-transfer oocytes and zygotes was tested in the rabbit. Metaphase II oocytes and zygotes in the early pronuclear stage were treated with a cytoskeletal inhibitor (cytochalasin D), enucleated, and subsequently fused either with single blastomeres from eight- and 16-cell stages (oocytes and zygotes) or with pronuclei-containing karyoplasts (zygotes only). Also, nonenucleated zygotes were fused with 1/8 blastomeres. Fusion was performed by means of an electric field. Development of reconstituted embryos was monitored mainly in vitro, but a certain number of embryos developed from oocytes and zygotes receiving nuclei from eight-cell stages were also transferred into pseudopregnant does. Development of nuclear-transfer oocytes was distinctly better than that of nuclear-transfer zygotes, since 16.9% and 9.5% oocytes vs. 8.1% and 3.7% zygotes carrying eight- and 16-cell nuclei, respectively, developed to the blastocyst stage. Two advanced but already dead fetuses were found after transfer of 27 four-cell embryos obtained after fusion of oocytes with 1/8 blastomeres. No implantations were observed after transfer of 25 four-cell embryos developed from enucleated zygotes receiving eight-cell nuclei. These findings indicate that, in the rabbit, some nuclei from 16-cell embryos are still capable of promoting at least preimplantation development. Comparison between the developmental abilities of oocyte- and zygote-derived nuclear-transfer embryos also suggests that the cytoplasmic environment of recipient cell is more crucial for the development of reconstituted embryos than the stage of introduced nuclei (at least up to the 16-cell stage). The majority of pronuclear exchange embryos (69.9%) and 40% of nonenucleated zygotes receiving eight-cell nuclei were able to develop to the blastocyst stage. This latter observation indicates, similarly as with mouse, a supporting role of residual pronuclei for participation of an eight-cell nucleus in the development of reconstituted zygotes.     

Developmental ability of porcine ova matured in porcine follicular fluid in vitro and fertilized in vitro

Developmental ability of porcine ova matured in porcine follicular fluid (pFF) with FSH in vitro and fertilized in vitro was examined by culturing in BMOC-2. Forty-eight hours after insemination, 35.6% of ova cleaved normally, and this rate was significantly higher (13.0%) than that of the ova matured in a modified Krebs-Ringer bicarbonate solution. Twenty-four percent (29 120 ) of ova matured in pFF with FSH developed to the four-cell stage and two of them developed to the eight-cell stage 66 h after insemination. Most cleaved embryos stopped developing at the four-cell stage and neither the morula nor blastocyst stage was observed throughout the culture period as reported in the in vivo matured ova. In culture at 37 degrees C, the appearance of two-cell and four-cell embryos was delayed from that of in vivo embryos, but their development was significantly accelerated by culturing at 39 degrees C. These results show that pFF is an excellent maturation medium for porcine oocytes, and the developmental capacity of the ova matured in pFF seems to be similar to that of in vivo matured ova. Culturing at 39 degrees C was found to be more suit-able for the development of ova than 37 degrees C.     

Parthenogenetic activation and subsequent development of rat oocytes in vitro.

Studies were undertaken to determine whether electrical stimulation, or ethanol treatment alone or in combination with 6-dimethylaminopurine (6-DMAP) influenced the rate of parthenogenetic activation of rat oocytes. The percentages of activated oocytes with pronuclei (89-91%) and those developed to the two-cell stage (68-72%) were significantly higher after electrical stimulation with direct current (DC) at 100 V/mm, 99 microsec once or twice, than when other DC voltages (75, 150, and 200) were applied or when ethanol or 6-DMAP treatment was given alone. However, none of the activated oocytes developed beyond the four-cell stage. The percentages of activated oocytes with pronuclei (100%) that developed to the two-cell (100%), eight-cell (89%) and blastocyst stages (50%) were significantly higher when electrical stimulation was followed by treatment with 2 mM 6-DMAP for 4 hr than when other combined procedures were applied. In conclusion, the results of the present study clearly showed that combined treatment of electrical stimulation or ethanol with 6-DMAP induces parthenogenetic activation and subsequent development of rat oocytes in vitro.     

In vitro fertilization of bovine follicular oocytes recovered by laparoscopy

Thirty-three laparoscopies were performed on 16 follicle stimulating hormone (FSH)-superovulated cows (one to four laparoscopies per cow). Two hundred and four follicles were aspirated (6.18/cow) and 157 oocytes (77% recovery rate) were isolated. Sixty percent of the oocytes found were considered to be of good to excellent quality. In cows with ongoing ovulations at laparoscopy only 32% of the oocytes were suitable for fertilization. Twenty-four percent of the oocytes from cows with no ovulation and 16% from cows with ovulations were fertilized. Higher proportions of oocytes were fertilized in cows showing estrous behavior vs cows with no signs of estrus (25 vs 15%). Using lysolecithin-treated spermatozoa, 28% of the oocytes with good to excellent quality were fertilized, and 35% of the fertilized eggs cleaved in vitro to two- to four-cell stages.     

Development of bovine in vitro-matured follicular oocytes activated with ethanol

Bovine follicular oocytes cultured in vitro for 30 hours were activated by ethanol and then the developmental ability of the oocytes was assessed. When the activated oocytes were treated with cytochalasin B at concentrations of 0, 1.25, 2.5, 5 and 7.5 mug/ml, the percentages of oocytes having two pronuclei were 8, 18, 54, 66 and 60%, respectively. The percentage of the activated oocytes with two pronuclei also increased with increased exposure time to cytochalasin B. When the activated oocytes were treated with cytochalasin B (5mug/ml) for 10 hours, 20% of them cleaved after 36 hours and 5% of them developed to the eight-cell stage after 60 hours of culture. These results indicated that bovine in vitro matured oocytes can be activated by ethanol and develop to the eight-cell stage following treatment with cytochalasin B.     

Laparoscopic oviductal transfer of in vitro matured and in vitro fertilized bovine oocytes

The objective of this study was to obtain normal pregnancy following laparoscopic oviductal transfer of in vitro matured and fertilized bovine oocytes. Methods for in vitro maturation and in vitro fertilization were similar to those previously reported (1). Primary oocytes judged to be potentially viable were cultured for 26 h in modified TCM 199 supplemented with heat-treated fetal calf serum (20% v/v), 5mug/ml FSH (USDA-bFSH-B-1), and 1mug/ml estradiol 17-beta. Oocyte cumulus complexes were microscopically evaluated for maturation (first polar body formation) following a brief treatment with hyaluronidase. Mature oocytes were inseminated with heparin-treated spermatozoa and incubated at 39 degrees C under paraffin oil and moist 5% CO(2), 5% O(2), 90% N(2). In this work, 450 oocytes were recovered at slaughter from ovaries of 42 random cows of unknown reproductive status and 336 oocytes (74.7%) with compact cumulus were selected for culture. Of these, 322 (95.4%) matured in vitro. Of 218 inseminated oocytes, 198 (90.8%) were penetrated by sperm and 83 (38.1%) cleaved, with 102 (46.6%) of the embryos reaching four- to eight-cell stages. None of 40 oocytes not exposed to sperm and none of 30 oocytes inseminated with untreated sperm showed signs of activation. In a control experiment with hormones added, 105 of 115 (91.3%) oocytes matured in vitro and 20 of 105 (19.5%) cleaved following in vitro insemination. Laparoscopy was performed on four synchronized recipients under local anesthesia. A catheter containing three embryos in the two to four cell stages was passed through the operating channel of a direct viewing bronchoscope for deposition in the oviduct ipsilateral to the recipients developing corpus luteum while the fimbria and the mesovarium were manipulated with Semm's forceps. A normal term pregnancy confirmed in vitro fertilization and provides feasibility data for use of laparoscopic methodology developed in this work for testing viability of bovine oocytes and embryos. These results are encouraging for the application of in vitro maturation and in vitro fertilization for overcoming infertility in domestic and endangered species.     

Effects of co-culture, medium components and gas phase on in vitro culture of in vitro matured and in vitro fertilized bovine embryos

To develop an in vitro culture system for bovine oocytes and early embryos, we examined the effects of co-culture of in vitro matured and in vitro fertilized embryos with trophoblastic vesicles and cumulus cells. We also studied the effects of culture medium components and oxygen gas pressure by modifying TCM-199 medium and using a gas-tight chamber. We found that co-culture with trophoblastic vesicles or cumulus cells promoted early embryos to develop beyond the eight-cell block; 17 to 19% of the initial oocytes developed to the morula stage. The effects of removing glucose and other energy sources from the medium, adding EDTA to the medium, reducing the concentration of serum, and reducing the oxygen gas pressure on the development of embryos were also examined. These modifications during the initial phase of co-culture greatly increased the rate of embryo development to the morula (36 to 38% of oocytes developed to morulae) and blastocyst stages.     

In vitro fertilization of Siberian hamster oocytes

Siberian hamsters were superovulated and various media were tested in an effort to fertilize the recovered oocytes in vitro. The highest percentage of fertilized ova was achieved by using a modified Tyrode's medium, designated MT (Bavister, J. Reprod. Fertil., 18:544-545, '69), previously formulated to fertilize Syrian hamster ova in vitro. Spermatozoa incubated in this medium in a concentrated state overnight (14 hr) and then diluted (1 hr) fertilized 39% of the ova. Similar results (40%) were obtained with this medium by adding 20% human follicular fluid to fresh concentrated sperm for 30 min and then diluting the sperm for 2-3 hr prior to the addition of ova. Ova fertilized in vitro cleaved to the two-cell stage but failed to develop any further in culture. Two-cell embryos recovered from mated hamsters and cultured did not undergo additional cleavage. Four-cell embryos collected from mated females and cultured cleaved to the six- to eight-cell stage and stopped. Techniques and media used for fertilizing large numbers of Syrian and Chinese hamster ova in vitro will have to be modified to achieve the same degree of success in the Siberian hamster.     

Nuclear transfer of porcine embryos using cryopreserved delipated blastomeres as donor nuclei

Nuclear transfer protocol for the pig using cryopreserved delipated four- to eight-cell and morula stage embryos as nucleus donors was developed. Donor embryos, which had been delipated by micromanipulation following centrifugation for polarizing cytoplasmic lipid droplets, were cryopreserved with 1.5 M 1,2-propanediol and 0.1 M sucrose. Recipient cytoplasts were prepared from ovulated oocytes. Activation of oocytes could be induced more efficiently when electric stimulation was given 53 hr after the hCG injection or later (66–83%), compared with 52 hr or earlier (11–16%, P < 0.05), suggesting that aging after ovulation may be required for in vivo matured porcine oocytes to be activated by electric stimuli. Membrane fusion rates between donor blastomeres and enucleated oocytes were 88% (127/144) and 97% (56/58, P > 0.05) for the four- to eight-cell and morula stage embryos, respectively. In vitro developmental rates to the two-cell (53/100 vs. 35/65), four-cell (34/100 vs. 26/65), and morula stage (17/100 vs. 18/65) were the same between the nuclear transfer embryos with four- to eight-cell and morula nuclei. However, more embryos reconstituted with morula nuclei developed to blastocysts (15% vs. 6%, P < 0.05). These data demonstrated that blastomeres of cryopreserved, delipated porcine embryos can be used as donor nuclei for nuclear transfer. Frozen-thawed, delipated blastomeres can be efficiently isolated and fused, and therefore provide a useful source of donor nuclei. Mol. Reprod. Dev. 48:339–343, 1997. © 1997 Wiley-Liss, Inc.     

Developmental ability of enucleated bovine oocytes matured in vitro after fusion with single blastomeres of eight-cell embryos matured and fertilized in vitro

Single blastomeres from eight-cell stage bovine embryos matured and fertilized in vitro were electrically fused with enucleated oocytes matured in vitro. In experiment 1, The percentage of these reconstituted embryos developed to the two- to eight-cell stage 48 hr after electrofusion was increased when both the eight-cell embryos and the enucleated oocytes were derived from oocytes cultured with granulosa cells (14% vs. 38%). In experiment 2, the relationship between activation of oocytes and developmental ability of reconstituted embryos was examined. Although both ethanol and electrical stimulation efficiently induced parthenogenetic activation of oocytes matured in vitro for 26–28 hr (ethanol, 89%; electrical stimulation, 73%), the ratio of the second polarbody extrusion differed (80% vs. 22%). Ethanol-treated enucleated oocytes, however, were not significantly different from the early cleavage of the reconstituted embryos 48 hr after electrofusion (nontreated, 38%; treated, 43%). In experiment 3, reconstituted embryos at the two- to eight-cell stage 48 hr after the electrofusion were cocultured with granulosa cells for 6–7 days. Of 69 embryos, one developed to a morula and three developed to blastocysts. © 1993 Wiley-Liss, Inc.     

In vitro techniques of bovine oocyte maturation, fertilization and embryo culture resulting in the birth of a calf

Oocyte cumulus complexes were aspirated from 3 to 5 mm follicles of cows prestimulated with 2.000 IU PMSG 24 h before slaughter. Oocytes matured in culture were fertilized in vitro by heparinized freshly ejaculated or epididymal spermatozoa. The cultivation procedure for fertilized eggs was the same as that used for cultivation of oocytes. From 163 matured oocytes, 109 cleaved to the 2-cell stage 24 h after fertilization and after 6 days of cultivation, 18 developed to the late morula and 18 to the blastocyst stages. Eleven blastocyts and 1 late morula were transferred surgically to the uteri of 7 recipient heifers. Two heifers became pregnant: one delivered a bull-calf at term, while the other pregnancy resulted in abortion at the 3rd month. The examination of some embryos by transmission electron microscopy showed an almost normal morphology for most cells. The degenerated cells contained mostly electron-dense residual bodies of unknown origin.     

In vitro fertilization and cleavage of in vivo matured bovine oocytes

The effect of the interval between onset of estrus and oocyte collection on in vitro fertilization (IVF) rates has been investigated. The oocytes were surgically collected 6-18 h (Group I), 19-24 h (Group II), 25-29 h (Group III) and 30-36 h (Group IV) after the beginning of estrus. Recognizable stages of nuclear maturation were identified in 54.9% of the oocytes used for IVF (5.9% at germinal vesicle, 31.4% at metaphase I, 17.6% at metaphase II); the other 45.1% were degenerate. Considerable between- and within-cow variation in oocyte morphology, oocyte maturation and IVF results was observed. The cverall fertilization and cleavage rates (to four-cell stages) were 26.5 and 6.0%, respectively. The fertilization rate increased as the interval between onset of estrus and collection increased and was optimal 30-36 h after onset. Thus, onset of estrus proved an effective means of timing oocyte collection for IVF.     

Fertilization and early embryonic development in the blue fox (Alopex lagopus)

A total of 15 blue fox vixens aged 1–6 years were mated, 12 once on the first day of estrus and three a second time 48 hr after the first mating, and were killed 4 hr to 8 days following mating. Ova were collected from the oviducts, evaluated by stereomicroscopy, and studied by transmission (TEM; N = 49, 12 vixens) or scanning (SEM, N = 11, three vixens) electron microscopy. At 0–3 days after ovulation, the ova had not cleaved and were at different stages of meiotic maturation. In about one-half of these ova, representing all stages of meiotic maturation, a decondensing sperm head without nuclear envelope or a small pronucleus with partial nuclear envelope was observed. No clear relationship was found between maternal meiotic stage and the stage of paternal pronucleus formation. Sperm tails were never identified in the ooplasm. Cortical granules were released after sperm penetration at early stages of meiotic maturation. Thus the block against polyspermic penetration was activated during maturation of the oocyte. The first two-cell stage appeared 4 days after ovulation (3 days after mating), the first four-cell stage the following day (day 5), and the first eight-cell stage 6 days after ovulation (5 days after mating). In a single vixen mated late (7 days postovulation) two- to four-cell stages appeared the following day (day 8). This indicates that the time required for the first cleavage division decreases with increasing interval from ovulation to mating. The development of a functional nucleolus with fibrillar centers and fibrillar and granular components at the eight-cell stage indicates activation of embryonic RNA synthesis in fox embryos at the six- to eight-cell stage, suggesting that the embryonic genome is activated at this stage. © 1993 Wiley-Liss, Inc.     

Effect of follicle cells on the acrosome reaction, fertilization, and developmental competence of bovine oocytes matured in vitro

The role of follicle cells in the acrosome reaction of frozen-thawed bovine spermatozoa, in vitro fertilization, cleavage, and development in vitro was investigated. Cumulus-oocyte complexes were cocultured and matured in vitro with additional granulosa cells for 24 hr. Immediately before in vitro insemination, the oocytes were divided into three types with different follicle cells: denuded and corona- and cumulus-enclosed oocytes. The proportion of live, acrosome-reacted spermatozoa significantly increased at 3 and 6 hr after insemination in all types of oocytes. However, the mean proportion of live, acrosome-reacted spermatozoa that inseminated cumulus-enclosed oocytes at 6 hr after insemination was significantly higher than that of spermatozoa inseminating denuded oocytes (18.3% and 13.3%, respectively). The frequency of in vitro fertilization was significantly higher for cumulus-enclosed oocytes (65.4%) than for denuded and corona-enclosed oocytes (30.8% and 39.4%, respectively). Cumulus-enclosed oocytes when cocultured with oviduct epithelial cells also had significantly higher rates of cleavage (two- to eight-cell, 59.8%; eight-cell, 22.4%) and blastocyst formation (7.7%) than denuded and corona-enclosed oocytes. No eight-cell embryos or more advanced stages of embryonic development were observed in either denuded or corona-enclosed oocytes without the coculture. The present results indicate that cumulus cells at fertilization play an important role in inducing the acrosome reaction and promoting a high fertilization rate, cleavage, and development into blastocysts in vitro.     

Development of single blastomeres from four- and eight-cell mouse embryos fused into the enucleated half of a two-cell embryo

Single blastomeres from four- and eight-cell mouse embryos were fused into the enucleated halves of two-cell embryos, and the ability of these reconstituted embryos to develop in vitro and in vivo was examined. The proportion of these reconstituted embryos developing to blastocysts was 74% (60/81) when four-cell embryo blastomeres were used as nuclei donors and 31% (57/182) when eight-cell embryo blastomeres were used. Eight complete sets of the quadruplet-reconstituted embryos developed to blastocysts, and five live young (9%, 5/57) were obtained after transfer; however, none of the live young were clones. Although when using blastomeres from eight-cell embryos no complete set of eight developed to blastocysts, sextuplets were obtained. The blastocysts, however, failed to produce live young after transfer. In assessing the outgrowths, it was found that 43% of those derived from reconstituted embryos using blastomeres from four-cell embryos had an inner cell mass (ICM); however, outgrowths derived from reconstituted embryos using blastomeres from eight-cell embryos lacked an ICM. These results suggest that the genomes of four- and eight-cell nuclei introduced into the enucleated halves of two-cell embryos are reversed to support the development of the reconstituted embryo.     

Development to live young from bovine small oocytes after growth, maturation and fertilization in vitro

Bovine oocytes (90 to 99 microns in diameter) were isolated from early antral follicles (0.5 to 0.7 mm in diameter). Cumulus-oocyte complexes (COC) with pieces of parietal granulosa were embedded in collagen gels and cultured for 14 d. After in vitro growth culture, oocytes recovered from the collagen gels were further matured, fertilized and cultured in vitro, and then were transferred to recipient cows. After 14 d of growth culture, 37% of the oocytes (203/556) showed normal morphology in the collagen gels. The mean diameter of the oocytes was 110.1 +/- 6.0 microns, significantly larger (P < 0.01) than before growth culture (94.8 +/- 2.7 microns), and 77% were at the germinal vesicle stage while 23% had undergone germinal vesicle breakdown. After 24 h of maturation culture followed by insemination, 27% of in vitro-grown oocytes reached the second metaphase, and 42% of the oocytes were normally fertilized. After insemination, 18.2% of in vitro-grown oocytes cleaved and 3.7% developed to the blastocyst stage. Three blastocysts obtained from in vitro-produced 90- to 99-micron oocytes were transferred to 3 recipients. One recipient subsequently became pregnant and delivered a live calf on Day 277. These results demonstrated for the first time that 90 to 99-micron oocytes from early antral follicles can complete growth and acquire full developmental competence in vitro so that live young can be produced after maturation, fertilization, subsequent culture in vitro, and transfer to recipient cows.     

Production of live piglets following cryopreservation of embryos derived from in vitro-matured oocytes

We have successfully produced healthy piglets following cryopreservation of embryos derived from oocytes matured and fertilized in vitro. The appropriate timing of cryopreservation pretreatment (removal of cytoplasmic lipid droplets [delipation] and vitrification) was initially determined using parthenogenetic embryos derived from in vitro-matured (IVM) oocytes. Viable embryos were obtained at the highest rate when embryos were delipated at the four- to eight-cell stages (Day 2 of embryo culture) and were vitrified approximately 15 h later (Day 3) by means of the minimum volume cooling method. After cryopreservation of embryos derived from oocytes matured and fertilized in vitro under the most appropriate conditions, 401 embryos were transferred to five recipient gilts, and the recipients all became pregnant. At autopsy of one of the recipients, which had received 47 embryos, eight fetuses (17.0%) were found. Three recipients each gave birth to two to four piglets (1.4%-6.0%). These results demonstrate that normal offspring can be produced from vitrified porcine embryos derived from IVM oocytes by a strategic combination of delipation and vitrification at the early cleavage stages. This approach has great potential in the reproduction of micromanipulated porcine embryos, such as cloned and sperm-injected embryos, produced from IVM oocytes.