Artificial Activation of Porcine Oocytes Matured In Vitro R.S. PRATHER, P.A. EICHEN, D.K. NICKS, AND M.S. PETERS Department of Animal Sciences, University of Missouri-Columbia, Columbia, Missouri These studies were undertaken to ABSTRACT understand the biological basis of artificially induced activation of meiotic metaphase II oocytes and to develop a source of oocytes as recipients for cloning by nuclear transfer. In vitro matured porcine oocytes were pulsed with various voltages of electricity and evaluated for pronuclear formation. The percentage of eggs that activated was significantly greater for the higher voltages. The effect on activation of the temperature of the ovaries returning from the abattoir was evaluated and it was found that oocytes derived from ovaries returning at 29°C activated at lower rates (45.5%)than those returning at 36°C (78.9%). An experiment was designed t o evaluate the pH of electroporation medium (EM) and the duration of exposure to EM on activation. Oocytes were placed in EM at various pHs for 5 minutes, pulsed, and immediately removed t o TL-Hepes or allowed an additional 2 minutes in EM prior t o rinsing in TL-Hepes. The results indicate an optimum activation rate at a pH of 7.0 and allowing the additional 2 minutes in EM. Additional glucosamine (5 mM) had no affect on development of the oocyte to metaphase but reduced the percent pronuclear formation from 61%and 47%. A final experiment evaluated the developmental competence of oocytes subjected to a optimum combination of the above treatments and illustrated that a significant portion of the activated oocytes can show limited signs of cleavage. Thus in vitro matured pig oocytes can be induced to activate at high rates.

Key Words: Nuclear transfer, Pronuclear formation, Glucosamine

Prather et al., 1987, 1989; Stice and Robl, 1988). However, a recent paper using rabbit oocytes by Ozil (1990) illustrated that simple release from meiotic metaphase I1 and progression to the pronuclear stage was not the same as release from meiotic metaphase I1 and subsequent progression to midgestation. Thus the ability of a stimulus to allow progression to the pronuclear stage does not necessarily simulate the events that normally occur with fertilization. To achieve this remarkable degree of development Ozil (1990) simulated, with electric pulses, the normally occurring 22 Ca++releases within the fertilized rabbit oocyte. Although fewer pulses of electricity could result in progression to the pronuclear stage, it did not result in development to midgestation. Since the procedures for cloning embryos by nuclear transfer are tedious, it is important that the process of artificially induced activation be as simple as possible, and as normal (i.e., as a sperm would induce activation) as possible. Thus, for normal artificial activation in mammals, development should, as defined in the mouse and rabbit, continue to midgestation. If the procedures for cloning by nuclear transfer are to be commercially viable, then the oocytes used must be capable of normally induced activation and inexpensive. Thus, in vitro matured oocytes recovered from ovaries collected at slaughterhouses are the obvious choice. The experiments described below were designed to evaluate some of the parameters that may affect artificial activation of in vitro matured pig oocytes.

MATERIALS AND METHODS Oocyte Maturation INTRODUCTION Pig ovaries were collected at an abattoir and reArtificial activation of mammalian oocytes, i.e., re- turned to the laboratory in a thermos of saline. Follicles lease from the block at meiotic metaphase 11, can be were aspirated with an 18-gauge needle and syringe. induced by a variety of stimuli such as electric shock, Follicular fluid was pooled in 50 ml conical tubes and Ca+ , inositol trisphosphate, heat, alcohol, puromycin, allowed to settle. Follicular fluid was aspirated from etc. (reviewed by Whittingham, 1980). Some of these the bottom of the tubes and cumulus-oocyte complexes artificially activated eggs, if given the correct chromo- (COCs) were isolated and placed in Hepes-buffered somal complement, are capable of participating in Tyrode’s medium (HbT: Bavister et al., 1983) supplenormal development to term. Artificially activated mented with 3 mg/ml bovine serum albumin (BSA). mouse oocytes that receive, by nuclear transfer, a pronucleus derived from a sperm cell can develop to term (reviewed by Surani et al., 1987). When embry- Received September 5, 1990; accepted November 27, 1990. onic nuclei are transferred to simultaneously activated Address reprint requests to R.S. Prather, 164 Animal Science Reenucleated oocytes development to term can occur in search Center, Department of Animal Sciences, University of Missheep, cattle, pigs, and rabbits (Willadsen, 1986, 1989; souri-Columbia, Columbia, MO 65211. +




Only oocytes with intact, unexpanded cumulus cells were selected for in vitro maturation. After an additional rinse in HbT, COCs were placed in maturation medium [TCM 199, supplemented with 10% heattreated fetal calf serum (Sigma Chemical Co., St. Louis, MO), 0.02 unitsiml pFSH (Sigma Chemical Co., St. Louis, MO), 0.02 unitsiml eLH (Sigma Chemical Co., St. Louis, MO) and 1 ygiml estradiol-17 (Sigma Chemical Co., St. Louis, MO)]. Ten COCs were cultured under paraffin oil in 50 pl drops of maturation medium at 39°C under an atmosphere of 5% C02 in air. After 45 hours the oocytes were stripped of their cumulus cells by vortexing for 5 minutes in HbT containing 1 mgiml hyaluronidase (Sigma Chemical Co., St. Louis, MO). Oocytes were then washed in HbT. For experiments 1, 4, and 5 grossly abnormal appearing oocytes (i.e., oocytes that were lysed or had uneven cytoplasm) were discarded before allocation to electroporation conditions. In Experiments 2 and 3 all oocytes introduced into maturation medium were allocated to the treatments.

Experiment 4: Time in activation medium and pH dependence on oocyte activation. Matured oocytes were placed in EM at a pH of 6.5, 7.0, 7.5, 8.0, or 8.5. Oocytes were allowed a 5 minute equilibration period prior to electroporation (120 Vimm) and either removed and placed in HbT immediately or allowed a 2 minute recovery period in EM prior to placing in HbT. Experiment 5: In vitro development of activated pig oocytes. Matured oocytes were placed in EM (pH 7.0) for 5 minutes stimulated with 10 seconds of 4.0 Vimm followed by 120 Vimm for 30 psec. After an additional 2 minutes in EM the oocytes were transferred to HbT and cultured for 4 days at 39°C (Hagen et al., 1990). The oocytes were then mounted and evaluated for nuclear number as described below. The control for this experiment was nonelectroporated oocytes, i.e., matured oocytes were placed in EM for 7 minutes and then cultured in HbT for 4 days prior to evaluation.

In Vitro Culture, Evaluation, and Statistics Experimental Conditions After electroporation and a rinse in HbT the oocytes were placed in 50 y1 drops of HbT under oil in a Experiment 1: Voltage dependence of oocyte activation. In vitro matured oocytes were placed in humidified chamber in air at 39°C. After 24 hours electroporation medium (EM: 0.3 M mannitol, 1 mgiml (except for experiment 5) of culture the oocytes were BSA, and 5% HbT a t pH 7.5: Hagen et al., 1990) for 5 mounted under posted coverslips, fixed in methanol: minutes and pulsed for 10 seconds with 4.0 Vimm AC, acetic acid (3:1), and stained with aceto-orcein (Prather followed by a 30 ysec pulse at 0,70,80,90, 100,110, or et al., 1987). The oocytes were subsequently evaluated, 120 Vimm DC. The chamber used in this and the under Hoffman Modulation Contrast Optics on an following experiments was composed of two (200 ym inverted Nikon Diaphot at 400 x , for the presence of a diameter) platinum electrodes spaced 1 mm apart on a germinal vesicle, metaphase chromosomes, or pronuglass slide. After the electric pulse the oocytes were clei. The presence of single or multiple pronuclei in immediately placed into HbT and cultured as described oocytes was used to classify those oocytes as being activated. Statistical methods were used to estimate below. Experiment 2: Temperature dependence prior to 95% confidence intervals for each mean percentage maturation on oocyte activation. In this experi- activation (Snedecor and Cochran, 1982) and development ovaries were placed in saline at either 25°C or mental rate. 39°C for the 1hour return trip to the laboratory. Upon RESULTS reaching the laboratory the temperature of the saline Experiment 1 was 29°C and 36"C, respectively (apparently the warm The percentage (mean t 95% confidence intervals) ovaries caused the saline in the 25°C group to increase). Cumulus-oocyte complexes were isolated, processed as activation (Fig. 1) progressively increased from described above, and electroporated for 30 psec at 120 15.7% ? 7.9" at 0 V ( arthenogenic activation) to Vimm. The oocytes were then cultured and evaluated 34.5% 10b,43.2%? 11g c ,57.5% ? ll", 64.6% -t lod, as described below. Since there was a significant dif- and 75.0% 2 9.4d (different superscripts are different, ference in the rate of activation between these two P 0.101, whether 5 mM glucosamine was oocytes were immediately placed into HbT medium and present (90.0%)or absent (94%)(N = 146, two replications). The morphology of the resulting cumulus cells cultured as described below.




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Fig. 1. Electrically induced porcine oocyte activation: voltage dependence. In vitro matured oocytes were electropulsed for 30 p e c at the indicated voltages (Vimm). Different superscripts indicate significant (P < 0.05) differences.

ACTlVATlON 1 0 0 V+ 2 mn

Fig. 2. Electrically induced porcine oocyte activation: pH and time dependence. In vitro matured oocytes were pulsed with 100 Vimm in EM a t the indicated pHs and immediately removed to HbT or moved to Hbt after a n additional 2 minutes. Different superscripts indicate significant ( P < 0.05) differences.

TABLE 1. Activation and In Vitro Development of In Vitro Matured Porcine Oocytes Number of 1 (pro) 2 3to4 5to6 Multiple Percent 2 3 Nuclei Treatment nuclei Nucleus Nuclei Nuclei Nuclei micronuclei activation development Porated 15 3 12 11 2 17 75% (45/60)" 50% (30/60)" Control 32 9 22% (9/41)b 0 (0/41)b a,bDifferent superscripts within a c o l u m n are d i f f e r e n t (P< 0.05).

electroporated porcine oocytes. In contrast to others (Ozil, 1990; Collas et al., 1989) who have employed multiple pulses of electricity for the activation of oocytes, we have chosen to concentrate our efforts on providing a single pulse of electricity. The rationale behind such a decision is to increase the ease and convenience of nuclear transfer. Thus, it would be desirable to provide a single pulse of electricity that would facilitate both the fusion of the donor blastomere and the artificial activation of the recipient oocyte. The timing for applying the pulses of electricity was determined from Hagen et al. (19901, who found that Experiment 4 optimum activation rates could be achieved after 41 There was a trend (P < 0.05) for higher rates of hours of maturation and that these were not different activation (Fig. 2) to be achieved at the lower pHs, if the from the rates of activation at 48 and 65 hours of oocytes were allowed an additional 2 minutes in EM maturation. Thus the first experiment was designed to evaluate a variety of different voltages at 45 to 48 hours (N = 1087, five replications). of in vitro maturation. Preliminary experiments sugExperiment 5 gested that once the voltage is increased above 120 Development to the 6-cell stage was noted in the V/mm for 30 psec a significant amount of lysis occurs; electroporated oocytes cultured for 4 days in vitro thus an upper limit of 120 Vimm was imposed. Inter(Table 1). Activation rate and developmental rate was estingly, 120 V/mm also yielded the highest rate of significantly greater in the electroporated oocytes than activation, although not significantly different from 90, for the nonelectroporated oocytes (N = 101, P < 0.05). 100, or 110 V/mm. In the preliminary experiments it was noted that the DISCUSSION results were not very repeatable and sometimes the Here we report a variety of experiments designed to thermos was cold upon arrival in the laboratory; thus optimize the activation rates of in vitro matured and an experiment evaluating the temperature of the re-

suggested that the inclusion of 5 mM glucosamine had a detrimental affect on complete cumulus cell expansion as compared to the controls. Although this was a very subjective evaluation, close attention was paid to the borders of the cumulus mass. Those without glucosamine had a more undefined border and appeared to be more similar to that obtained in vivo. In addition the presence of 5 mM glucosamine resulted in a decreased (P < 0.05) rate of activation (47.1% 9.7) when compared to the absence of glucosamine (61.3% 9.9) (N = 223, two replications).





turning thermos was designed. The observation of high activation rates when the thermos returned warm versus cold has not been reported for the pig but is consistent with a report by Moor and Crosby (1985), which showed that meiosis is temperature dependent in the sheep. Cumulus cell expansion in vitro is not as extensive as that achieved in vivo. This expansion has been suggested to be a result, at least in part, of the synthesis of hyaluronic acid. Glucosamine addition to the maturation of mouse oocytes, as a precursor to hyaluronic acid, has been suggested to result in cumulus expansion similar to that achieved in vitro (Salustri et al., 1989). A more “healthy” cumulus mass would presumably result in an oocyte that was more similar to an in vivo matured oocyte. It was found that the inclusion of 5 mM glucosamine, although not significantly affecting progression through germinal vesicle breakdown, resulted in lower levels of electroporation-induced activation. It may be argued that the oocyte matured in vitro in the presence of glucosamine is indeed more similar t o in vivo matured oocytes and that the electroporation pulse is not adequate. However, results using in vivo matured oocytes and very similar electroporation parameters resulted in 81% activated oocytes (Prather et al., 1989). Since glutamine and glucose can also serve as precursors to hyaluronic acid synthesis and they are present in the TCM 199 base medium used in this study, we may have overloaded the source of hyaluronic acid precursors with the additional 5 mM glucosamine. In addition to the effects of glucosamine on the oocyte, one-dimensional polyacrylamide gel electrophoresis showed that a qualitative change was observed in the 35S-methionine-labeled COC secreted proteins. The pattern of secreted proteins from oocytes matured in the presence of glucosamine was more similar to that secreted by COCs that had not been exposed to LH (unpublished observations), whereas the pattern of proteins secreted by COCs in the absence of 5 mM glucosamine was more similar to post-LH in vivo recovered COCs. Experiments by Cran et al. (1988) suggest that in the sheep and hamster the pH of the external medium is important to the release of cortical granules from oocytes injected with compounds that result in the rise of intracellular Ca’ . If the pH is important, then we also wanted to determine if moving the oocytes immediately to HbT was detrimental to activation. Therefore we evaluated the affect of various pHs (6.5,7.0,7.5,8.0, and 8.5) and immediate removal from EM versus a 2 minute period in EM for the oocyte to recover from the electroporation. It was determined, in contrast t o Cran et al. (1988) and cortical granule release, that lower pHs tended to result in higher rates of pronuclear formation. In addition, it was found that a 2 minute postelectroporation equilibration period in EM also tended to result in higher rates of pronuclear formation. This 2 minute period was chosen based on the observation that immediately after a large calcium release (induced +

by electroporation) a longer sustained calcium influx occurs. This influx is dependent upon the extracellular calcium concentration (see Houslay, 1987). Since HbT has -1 mM calcium and EM has -50 pM, excessive levels of extracellular calcium, resulting in high levels of intracellular calcium, may be detrimental to development. Alternatively, the plasma membranes may be sensitive post-electroporation and any ion changes may be deleterious. The final experiment evaluated the development of these electroporated oocytes. A portion of these oocytes formed multiple micronuclei, similar to that described by Ozil (1990). The cause of these is unknown but is likely due to an inadequate activation stimulus. The culture system used has resulted in 60% to 70% formation of morula or blastocyst stage embryos (Hagen et al. 1991; unpublished observations) from one- and two-cell stage embryos. Although development did not continue to the blastocyst stage, these results show that the conditions employed do allow limited development of in vivo matured oocytes following electroporation. The results presented here suggest an inadequate method of activation. However, since there is currently no routinely successful method of in vitro maturation or in vitro fertilization reported for the pig, it is not possible to test the developmental competence of the oocytes. Thus, the oocytes may not be capable of continued development. Current experiments are designed to incorporate second messengers, such as inosito1 trisphosphate into the EM, followed by transfer of the activated oocytes to recipient gilts.

ACKNOWLEDGMENTS This research would not have been possible without the collaboration of B.N. Day and his laboratory personnel or the generous donation of ovaries from Montfort Pork, St. Joseph, MO and Wilson Foods, Marshall, MO. This manuscript was prepared while supported by the Cooperative State Research Service, U.S. Department of Agriculture under agreement number 8837240-3755, Food for the 21st Century and is a contribution from the Missouri Agriculture Experiment Station Journal Series number 11,276. REFERENCES Bavister BD, Leibfried ML, Liebermann G (1983): Development of preimplantation embryos of the golden hamster is a defined culture medium. Biol Reprod 28935-247. Collas P, Balise JJ, Hoffman GA, Rob1 JM (1989):Electrical activation of mouse oocytes. Theriogenology 32:835-844. Cran DG, Moor RM, Irvine RF (1988): Initiation of the cortical reaction in hamster and sheep oocytes in response to inositol trisphosphate. J Cell Sci 91:139-144. Hagen DR, Prather RS, First NL (1990):Response of porcine oocytes to electrical and chemical activation during maturation in vitro. Mol Reprod Dev 28:70-73. Hagen DR, Prather RS, Sims MM, First NL (1991): Development of one-cell porcine embryos to the blastocyst stage in simple media, J Anim Sci (in press). Houslav MD (1987): EE -- activation unscrambles a ootential role for IP,. TIBS 12:133. Moor RM, Crosby IM (1985): Temperature-induced abnormalities in sheep oocytes during maturation. J Reprod Fertil 75:467-473.

ACTIVATION OF PORCINE OOCYTES Ozil J P (1990): The parthenogenetic development of rabbit oocytes after repetitive pulsatile electrical stimulation. Development 109:117-127. Prather RS, Barnes FL, Sims ML, Robl JM, Eyestone WH, First NL (1987):Nuclear transfer in the bovine embryo: Assessment of donor nuclei and recipient oocyte. Biol Reprod 37959-866. Prather RS, Sims MM, First NL (1989): Nuclear transplantation in early pig embryos. Biol Reprod 41:414-418. Salustri A, Yanagishita M, Hascall VC (1989): Synthesis and accumulation of hyaluronic acid and protoglycans in the mouse cumulus cell-oocyte complex during follicle-stimulating hormone-induced mucification. J Biol Chem 264:13840-13847. Snedecor GW, Cochran WG (1982): “Statistical Methods,” 7th edition. Ames IA, The Iowa State University Press, pp 54-59.


Stice SL, Robl JM (1988): Nuclear reprogramming in nuclear transplant rabbit embryos. Biol Reprod 39:657-664. Surani MAH, Barton SC, Norris ML (1987):Experimental reconstruction of mouse eggs and embryos: An analysis of mammalian development. Biol Reprod 36:l-16. Whittingham DG (1980): Parthenogenesis in mammals. Oxford Rev Reprod Biol 2:205-231. Willadsen SM (1986): Nuclear transplantation in sheep embryos. Nature, London 320:63-65. Willadsen SM (1989): Cloning of sheep and cattle embryos. Genome 31:95&962.

Artificial activation of porcine oocytes matured in vitro.

These studies were undertaken to understand the biological basis of artificially induced activation of meiotic metaphase II oocytes and to develop a s...
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