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As the expression of the LH receptor (LH-R) in granulosa cells is thought to be associated with later stages of folliculogenesis, this study was undertaken to evaluate the presence of LH-R mRNA as a suitable marker for developmental competence of oocytes. Granulosa cells and cumulus-oocyte complexes (COCs) were recovered from cows that had received ovarian stimulation. The COCs were subjected to embryo production procedures in vitro to assess the embryonic potential of the oocyte, and the corresponding granulosa cells were used to evaluate the presence of LH-R mRNA by RT-PCR. The presence of LH-R transcripts in granulosa cells is not a key characteristic of a follicle bearing a competent oocyte, although a higher proportion of oocytes reach the blastocyst stage when LH-R mRNA is detected in the granulosa cells. Different LH-R isoforms were cloned and sequence discrepancies among six of the isoforms enabled the design of specific oligonucleotides to study the presence of the isoforms in different follicular cells. All LH-R transcripts studied and the 80 kDa protein product corresponding to the full length receptor were found in granulosa cells of small (< 4 mm) and large (> 5 mm) follicles. When the granulosa cells were cultured, the transcripts were downregulated by the culture conditions; downregulation was more acute in granulosa cells from small follicles. The addition of LH to the culture media enhanced LH-R mRNA downregulation. The presence of several LH-R transcript isoforms was tissue specific and in the theca cells LH-R mRNA was restricted mainly to cells from larger follicles. This finding indicates that the expression and the splicing of LH-R mRNA are regulated in a cell-specific and follicular size-specific manner.

Superstimulation in donor cows increases the number of cumulus-oocyte complexes (COC), but when compared to in vivo maturation, in vitro maturation results in only half as many blastocysts after prolonged in vitro culture. The objective of this study was to establish a superstimulation protocol that would produce a maximal number of competent COC for standard in vitro embryo production. During experiment 1, eight cyclic Holstein heifers were superstimulated with four doses of FSH. Half the heifers received an injection of LH 6 h before ovum pick-up (OPU). The COC were collected following OPU either 33 or 48 h following the last FSH injection (coasting period). During experiment 2, six cyclic Holstein heifers were superstimulated with six doses of FSH, and in half the heifers, LH was administered 6 h before OPU. The COC were collected following ultrasound-guided transvaginal aspiration of both ovaries 48 h after the last FSH injection (coasting period). The COC originating from follicles with a diameter of 5 mm or more (n = 180 for experiment 1 and 57 for experiment 2) were subjected to standard in vitro maturation, fertilization, and development. When animals were administered four doses of FSH, 48 h of coasting resulted in significantly more 5- to 10-mm follicles (P < 0.01) than 33 h of coasting. If a 33-h coasting period was used, administration of LH 6 h before OPU resulted in a significant increase in both percentage of blastocysts and embryo production rate at Days 7 and 8 (P ≤ 0.05) of in vitro culture. If a 48-h coasting period was used, LH injection did not affect the rates of blastocyst production. When donors were administered six doses of FSH with a 48-h coasting period, the highest results, although not significant (P < 0.08), were obtained when animals received LH 6 h before OPU, with 80% ± 9% (mean ± SEM) blastocysts and 0.8 ± 0.09 embryo produced per COC retrieved per heifer at Day 8 of culture. Never has in vitro technology been so close to producing 100% developmentally competent COC.

The effect of reducing the amount of cytoplasm available for early embryonic development was investigated in embryos produced by in vitro fertilization (IVF) and nuclear transplantation. In Experiment 1, approximately 1/2 or 1/20 of the cytoplasm was removed from bovine embryos at the pronuclear-stage of development. The percentage of embryos developing to the compact morula or blastocyst stage was significantly higher in non-manipulated controls (26%) than in embryos with 1/20 of the cytoplasm removed (16%), and those with 1/2 of the cytoplasm removed (10%; P0.05). There was also a significant difference in the average number of cells between blastocysts in which 1/20 of their cytoplasm was removed (67), those with 1/2 of their cytoplasm removed (55), and nonmanipulated controls (77; P0.05). In Experiment 2, nuclear transfer embryos were produced in which approximately 1/2 or 1/20 of the cytoplasm was removed during oocyte enucleation. The percentage of embryos developing to the blastocyst stage was 17% for both groups of nuclear transfer embryos compared to 44% for control embryos (P0.05). The mean number of cells in blastocysts produced by nuclear transfer in which 1/20 of the cytoplasm was removed during oocyte enucleation (61) was no different than that in control embryos (66), but significantly higher than the mean number of cells in blastocysts produced by nuclear transfer in which 1/2 of the cytoplasm was removed (42; P0.05). There was no indication that altering the amount of cytoplasm available for early embryonic development of IVF embryos affected the timing of differentiation events, including those of embryo compaction and blastocyst formation.

The influence of electrical stimulation on the level of intracellular Ca2+ in bovine oocytes, as well as activation and extent of parthenogenetic development, was investigated. Mature oocytes were electrically stimulated at 29 hr of maturation, and intracellular Ca2+ concentration was determined with the Ca2+ indicator fura-2 dextran (fura-2 D). The Ca2+ response of oocytes to a given electrical pulse was variable. Oocytes responded with either no Ca2+ rise from baseline (approximately 12 nM), a short-duration Ca2+ rise (from 12 nM to 300 nM) that returned to baseline within 2 min of the pulse, or a long-duration Ca2+ rise (from 12 nM to 1,000-2,000 nM) that never returned to baseline during the 8 min period over which the oocytes were monitored. In these oocytes, Ca2+ level returned to baseline when oocytes were removed from 0.30 M mannitol and placed in an ionic medium. Increasing field strength or pulse duration tended to increase the proportion of oocytes displaying a Ca2+ rise, and at 1.0 kVcm-1 for 40 microseconds, all oocytes displayed a long-duration Ca2+ elevation. Direct transfer of oocytes from culture medium to mannitol also triggered a Ca2+ rise. Multiple stimulations, either electrical or by transferring to mannitol, produced multiple Ca2+ rises. This mannitol-induced Ca2+ rise could be inhibited by first washing the oocytes in medium containing equal parts of 0.30 M mannitol and phosphate buffered saline (PBS). The level of Ca2+ stimulation affected activation and development of oocytes. Insufficient, or, conversely, excessive Ca2+ stimulation impaired development. Optimum development was obtained with 1) three pulses of 0.2 kVcm-1 for 20 microseconds, each pulse 22 min apart, after direct transfer of oocytes from culture medium to mannitol (22% blastocysts) or 2) three pulses of 1.0 kVcm-1 for 20 microseconds after transfer of oocytes from culture medium to medium containing equal parts mannitol and PBS, then to mannitol (24% blastocysts). This procedure avoided induction of a Ca2+ rise prior to the pulse. The results indicate that the level of Ca2+ stimulation can be regulated by incubation conditions prior to the pulse and, to some extent, by field strength and pulse duration. The level of electrical stimulation influenced oocyte Ca2+ response, activation, and parthenogenetic development.

Superstimulation in donor cows increases the number of cumulus-oocyte complexes (COC), but when compared to in vivo maturation, in vitro maturation results in only half as many blastocysts after prolonged in vitro culture. The objective of this study was to establish a superstimulation protocol that would produce a maximal number of competent COC for standard in vitro embryo production. During experiment 1, eight cyclic Holstein heifers were superstimulated with four doses of FSH. Half the heifers received an injection of LH 6 h before ovum pick-up (OPU). The COC were collected following OPU either 33 or 48 h following the last FSH injection (coasting period). During experiment 2, six cyclic Holstein heifers were superstimulated with six doses of FSH, and in half the heifers, LH was administered 6 h before OPU. The COC were collected following ultrasound-guided transvaginal aspiration of both ovaries 48 h after the last FSH injection (coasting period). The COC originating from follicles with a diameter of 5 mm or more (n = 180 for experiment 1 and 57 for experiment 2) were subjected to standard in vitro maturation, fertilization, and development. When animals were administered four doses of FSH, 48 h of coasting resulted in significantly more 5- to 10-mm follicles (P0.01) than 33 h of coasting. If a 33-h coasting period was used, administration of LH 6 h before OPU resulted in a significant increase in both percentage of blastocysts and embryo production rate at Days 7 and 8 (Por = 0.05) of in vitro culture. If a 48-h coasting period was used, LH injection did not affect the rates of blastocyst production. When donors were administered six doses of FSH with a 48-h coasting period, the highest results, although not significant (P0.08), were obtained when animals received LH 6 h before OPU, with 80% +/- 9% (mean +/- SEM) blastocysts and 0.8 +/- 0.09 embryo produced per COC retrieved per heifer at Day 8 of culture. Never has in vitro technology been so close to producing 100% developmentally competent COC.

The developmental potential of bovine inner cell mass (ICM) and somatic differentiated (granulosa cell) nuclei was investigated using nuclear transplantation. ICM blastomeres were isolated after immunosurgery of day 7 in vitro produced blastocysts and cumulus granulosa cells recovered from in vitro matured oocytes. Nuclear transplantation was carried out by microinjection of the lysed donor cells into enucleated mature oocytes. Oocytes were activated by three 0.2 kVcm-1/20 microseconds pulses in mannitol containing 100 microM Ca2+, with each pulse 22 min apart. Embryos were cultured in vitro for 7 days and blastocysts were transferred into recipients. ICM and granulosa cell donor nuclei directed 7% (20/304) and 9% (19/213) development to blastocysts, respectively. Fifteen blastocysts from ICM donors resulted in four pregnancies (27%) and two births. No pregnancy was detected with granulosa cell donors. The results illustrate the totipotency of ICM nuclei and indicate that granulosa cell nuclei promote preimplantation development of nuclear transplant embryos.

Blastomeres from 2- to 32-cell bovine embryos were transferred to enucleated oocytes matured either in vivo or in vitro by micromanipulation and electrofusion. The percentage of donor cells fusing with the recipient oocytes was dependent on relative cell size or stage of development. Therefore, when smaller donor karyoplasts (17- to 32-cell vs. 2- to 8-cell) were transferred, the rate of fusion was significantly less (p less than 0.01). After fusion, nuclear transfer embryos were cultured either in vitro or in vivo (in a ligated ovine oviduct). Nuclear transfer embryos cultured in vitro developed to the 4- to 6-cell stage after 72 h (4-cell, 71%; 8-cell, 33%, 16-cell, 33%; p less than 0.30), whereas nuclear transfer embryos cultured in vivo developed to the morula or blastocyst stage (2- to 8-cell, 11.7%; 9- to 16-cell, 16.0%; 17- to 32-cell, 8.3%; p greater than 0.30) after 4 or 5 days. Freshly ovulated oocytes (collected 36 h after the onset of estrus), when used as recipients, resulted in morula/blastocyst-stage embryos more often than in vitro-matured oocytes or in vivo-matured oocytes collected 48 h after the onset of estrus (20% vs. 7.8% and 6.7%, respectively; p less than 0.02). After in vivo culture, nuclear transfer embryos were mounted and fixed or transferred nonsurgically to the uteri of 6- to 8-day postestrus heifers. Seven pregnancies resulted from the transfer of 19 embryos into 13 heifers; 2 heifers completed pregnancy with the birth of live calves.(ABSTRACT TRUNCATED AT 250 WORDS)