- Email: [email protected]
Effect of human leukemia inhibitory factor on in vitro development of parthenogenetic bovine morulae
Theriogenology
42: 1133-l
139, 1994
EFFECT OF HUMAN LEUKEMIA INHIBITORY FACTOR ON IN VITRO DEVELOPMENT OF PARTHENtXENEI’IC BOVINE MORULAE Y .Fukui,i T.Saito,t A.Miyamoto,i H.Yamashinaz and Y.Okamot& 1Laboratory of Animal Genetics and Reproduction Obihiro University of Agriculture and Veterinary Medicine, Obihiro 080 Japan ZTokachi-Ikusei Ranch, Taiki-cho, Hokkaido 089-22, Japan
Received for publication: Accepted
February September
15, 1994 14, 1994
ABSTRACT The present study was conducted to investigate the effect of human leukemia inhibitory factor (hLIF) addition to synthetic oviduct fluid medium (SOFM) supplemented with human serum (HS) on the development of in vitro matured and parthenogenetically activated bovine oocytes. The oocytes matured for 30 h were exposured to ethanol (7% 7 min) and cytochalasin B (5 &ml. 5 to 6 h). The treated oocytes were cultured for 5 d in SOFM supplemented with HS, and Day-5 morulae were cultured for 2 d in SOFM supplemented with HS and with or without hLIF (5ooo U/ml) to investigate the subsequent in vitro development to the blastocyst stage. Of the 1531 oocytes that were parthenogenetically activated, 592 (37.5%) cleaved to the 2- to &cell stage and 174 (13.8%) developed to the morula stage. The addition of hLIF at the morula stage resulted in a significantly (P&01) higher rate of development to the blastocyst stage in the medium with hLIF (55.9%) than without hLIF (28.9%). The mean cell number per blastocyst developed in the medium with hLIF was also significantly (PcO.01) higher than that developed in the medium without hLIF. To evaluate the viability, 6 parthenogenetically developed blastocysts were transferred to 3 recipient heifers (2 embryos per heifer), while in 2 other recipient heifers estrus was prolonged after transfer. The plasma progesterone levels of the 2 recipient heifers at the 28th day after transfer were 8.1 nglml and 9.0 ng/ml, but pregnancy was not observed by ultrasonic scanning. The present results indicate that the addition of hLIF to in vitro-produced, Day-5 parthenogenetic bovine morulae significantly improves the subsequent development to the blastocysts stage; however, the present method still does not promote for development of parthenogenetic fetuses in cattle. Key words: hLIF, parthenogenetic development, oocyte, cattle INTRODUCTION Parthenogenetic activation of mammalian oocytes can be induced by various stimuli such as exposure to ethanol (3, 9, 19), calcium ionophore (22, 31) and electric current (10, 21, 22, 31). However, information is limited on the postactivation development of in vitro matured oocytes of farm animals (1, 9,23). In mouse (28) and rabbit (22), reconstituted parthenogenetic ova with female pronuclei resulted in Day 10 to 11 pregnancy with about 25 somites and a beating heart after transfer to recipients, although the parthenogenetic fetuses were of smaller size than the controls (22, 27). Acknowledgements The authors wish to thank Dr. S.Raiti and Dr. D.J.Bolt for the supply of bovine LH and FSH, respectively and the AMRAD Corporation, Ltd., Australia for donation of hLIF (batch HlH2) used in this study.
Copyright
(9 1994 Butterworth-Heinemann
1134
Theriogenology
Lavranosand Seamark (14) and Robertson et al. (25) found that blastocysts cultured in media ~n~i~ng leukemia inhibitory factor (1000 U/ml) had a larger area of trophectodenn than the controls. The addition of human leukemia inhibitory factor (hLIFf significantly improved the development of in vivo-produced ovine (4) and bovine (5) embryos, and in vitro-produced bovine embryos (6, 15). Therefore, it was hypothesized that the addition of hLIF to medium may contribute to improving the quality of parthenogenetic embryos developed in vitro. Our aim in the present study was to investigate the effect of the presence or absence of hLIF addition to synthetic oviduct fluid medium (SOFM) supplements with human serum (HS) on the development of in vitro-matured, parthenogenetically activated and cultured bovine morulae. MATERIALS AND METHODS In Vitro Maturation The ovaries of Holstein cows and heifers were obtained from a local slaughterhouse and were transported in saline (9 g NaClIl) at 39°C to the laboratory within 1 h. The cumulus-~cy~ complexes were collected from follicles of 1 to 5 mm in diameter with an 18-gauge needle attached to an S-ml disposable syringe. Only oocytes with an unexpanded cumulus oophours and evenly granulated cytoplasm were washed 3 times with TCM 199 (Whittaker M.A. Bioproducts, Walkersville, MD, USA; pH 7.4, Earle’s salt with sodium bicarbonate and L-glutamine) supplemented with 0.3% (w/v) bovine serum albumin (BSA: Fraction V, fattyacid-free, Sigma Chemical Co., St. Lois, MO, USA) and 10 mM Hepes, and 10 to 20 cumulus-enclosed oocytes were cultured in a 4-well dish (Nunclon, Inter-Med., Roskilde, Denmark) con~ning 0.5 ml TCM 199 supplement with 10% (v/v) heat-inactivated f56”C, 30 min) fetal calf serum (FCS) without Hepes. The medium was also supplemented with 5 pg bovine LH-B-S/ml (National Hormone and Pituitary Program, Baltimore, ML, USA), 2.5 pg bovine FSH-B-l/ml (US Development of Agriculture, Baltimore, ML, USA), 1 p(g estradiollml (E-8875, Sigma) and with 2 x 106 granulosa cells/ml. The granulosa cells were collected by dissection following the method of Moor and Trounson (18) and washed (500 g, 5 min), and were prepared as described elsewhere (8, 17). Oocytes were statically cultured for 30 h at 39°C under an atmosphere of 5% CO2 in air. Parthenogenetic Treatment The expanding cumulus-enclosed oocytes cultured for 30 h were treated with 7% (v/v) ethanol in SOFM (29) supplemented with 10% (v/v) FCS and 25 mM Hepes for 7 min at room temperature (19) and 5pglml cytochalasin B (CB: Sigma) in SOFM supplemented with 10% (v/v) heat-inactivated (56”C, 30 min) HS, but without Hepes for 5 to 6 h at 39°C under 5% CO2 in air. Following p~henogenetic activation, 5 to 10 oocytes removed cumulus cells by gentle pipetting, were cultured in a 24well dish (Falcon, USA) containing 0.5 ml SOFM supplemented with 10% (V/V) I-IS at 39°C for 5 d under an atmosphere of 5% CO2, 5% 02, 90% N2 (7). To investigate pronuclear formation of induced activation with ethanol and CB treatments, 161 activated oocytes were fixed with acetic-alcohol (1 : 3) after an additional culture in SOFM supplemented with 10% (v/v) HS for 10 h and stained with 1% (w/v) aceto-orcein. The number of oocytes with 2 pronuclei without the second polar body were recorded under a phase-contrast microscope. hLiFTreatment
and Viability
Cleavage (2- to 8-cell stage) and number of morulae were examined on the 3rd and 5th day, respectively, after parthenogenetic treatment (Day 0). Morula-stage embryos were cultured in a 24well dish containing 0.5 ml SOFM supplemented with 10% (v/v) HS and with or without hLIF (5ooo Ulml; AMRAD Corporation, Ltd., Melbourne, Australia) at 39°C for 2 d under an atmosphere of 5% COa. 5% 9, 90% Nz, and the development of blastocysts was recorded after
1135
Theriogenology
hLIF treatment. Some biastocysts were examined for the number of nuclei by the method of Ushijima et al. (30). Briefly, embryos were treated with 0.9% (w/v) sodium citrate for 10 to 20 min, and fixed with a solution of alcohol : acetic-acid : distilled water (3 : 2 : 1 by vol.) at 4 “C for 1 min. The fixed embryos were placed on slides with a small volume of fixative, dried for 1 h and stained by 10% (w/v) Giemsa solution for 15 min. The number of nuclei were determined under a phase-contrast microscope. To evaluate the viability of embryos developed in vitro, 6 blastocysts developed in the culture with hLIF were nonsurgically transferred into the uteri of 3 Holstein heifers (2 embryos per heifer) on Day 8 or 9 after es&us induced by an intramuscular injection of 250 jug prostaglandin Fza (Estrumate: Sumitomo Chemical Co., Osaka, Japan). Estrous behavior was observed every day following transfer. At 28 d after transfer, pregnancy diagnosis was performed using an ultrasonic scanning instrument with an ultrasound frequency of 5.0 MHz (Aloka Co., Tokyo, Japan) and a Kood sample was taken to examine plasma progesterone (P.+) levels. Plasma Pa concentrations were measured by enzyme immunoassay according to the method of Miyamoto et al. (16). The sensitivity of the assay was 1 pgiwell, and the in&a-assay vanation of Pe was 9.4%. Statistical Analysis Six replicates were carried out in this experiment. The proportions of moruiae developing to biastocysts in the medium with and without hLIF (5000 U/ml) were analyzed by a CATMOD procedure following logit transformation in the Statistical Analysis System (SAS). The results of the number of nuclei were analyzed by the Duncan’s new multiple range test. RESULTS On pronucleus formation after parthenogenetic treatment, 55 of 161 examined oocytes (34.2%) had 2 pronuclei without the second polar body (Table 1).
Table 1.
Examination of pronucleus formation of parthenogenetically-activated No. of oocytes examined
161
bovine oocytes
No. of oocytes with pronuclei (%) 0
1
82 (50.9)
20 (12.4)
2 55 (34.2)
23 4 (2.5)
Of 153 1 oocytes that were parthenogenetically activated, 592 (37.5%) cleaved to the 2- to 8ceil stage and 174 (13.8%) developed to the moruia stage (Table 2). The effect of hLIF (5000 U/ml) addition during the development to blastocysts resulted in significantly (PcO.01) higher rate of biastocyst formation in the presence of hLIF (55.9%) than in the absence of hLIF (28.9%; Table 3).
Theriogenolog
1136
Table 2.
In vitro development (cleavage and morula stage) of in vitromatured parthenogenetically-activated bovine oocytes No. of oocytes cultured (No. of trials) 1531 (n = 6)
Table 3.
No. of cleaved oocytes (mean % * SEM)
y
and
No. of oocytes developing to the morula stage (mean %* SEM) 174 (13.8% f 4.1)
592 (37.5% zt 3.6)
Effect of addition of hLIF (5000 U/ml) for 2-day culture of in vitro developed, parthenogenetic bovine morulae Addition of hLIF
No. of morulae cultured (No. of trials)
No. of morulae developing to blastocysts (mean % f SEM)
+ (n y6)
(55.9?* 8.3) a 26 (28.9% f 4.3) b
(n :6) a vs b: P&01.
There was significant difference (P&01) in the mean cell number of blastocysts formed in the presence (84.8) or absence (62.6) of hLIF (Table 4). The proportions of blastocysts with 2100 cells were 27.3% in the presence of hLIF and 0% in the absence of hLIF. Table 4.
Mean cell numbers of bovine the addition of hLIF (5C00 U/ml)
blastocysts
developed
in vitro with or without
Addition of hLIF
No. of blastocysts examined
Mean cell numbers (rt SFM) per blastocyst (Min. - Max.)
+
33 13
84.8 zt 4.3a (34 - 148) 62.6 f 6.2b (30 - 93)
a vs b: P&01. To evaluate viability, 6 parthenogenetically developed embryos were transferred to 3 recipient heifers (2 embryos per heifer). Although the estrus of 1 recipient heifer was observed 14 d after transfer, estrus was also prolonged in 2 recipient heifers after transfer. The plasma P4 levels of 2 recipient heifers at 28 d after transfer were 8.1 and 9.0 ng/ml, respectively, but pregnancy was not observed by ultrasonic scarming. Thereafter, these 2 heifers returned to estrus on Days 35 and 36, respectively, after transfer.
Theriogenolog
1137
y
DISCUSSION Although Graham (12) reported that the majority of mammalian parthenogenetic embryos died soon after implantation, other studies of mouse (3, 13, 27, 28) and rabbit (21) embryo development following parthenogenetic activation have reported survival until Days 10 to 11 of pregnancy. The parthenogenetic fetuses tended to be smaller in size than the fertilized fetuses, although the external morphology was similar (22,27). The proportion (34.3%) of activated oocytes with 2 pronuclei in the ooplasma was lower in our present study than that (54.0%) in our previous study (9). Recently, it has been demonstrated that the combined treatment of calcium ionophore and a protein synthesis inhibitor, cycloheximide (CI-I), with or without electric stimulation, induced a high rate of activation (90 to 100%) of bovine oocytes cultured for 18 to 30 h (1, 10, 23,26). However, for the post-activation development of parthenogenetic embryos relies on an optimal protocol, with exact dosage and exposure times to various chemical substances (ethanol, calcium ionophore, CH and CB) used in parthenogenetic activation. LIF is a glycoprotein, first isolated in mice, that induces differentiation and inhibits the proliferation of Ml myeloid cell line (11). It has been reported that hLIF may play a significant role in the development of mouse (25), ovine (4) and bovine (5,6, 15) embryos. The site of the action of hLIF is most likely the trophectcderm in murine embryos (25). The addition of hLIF to the medium resulted in a significantly higher rate of development of 5 d old parthenogenetic morulae to blastccysts than without hLIF. The mean cell number per blastocyst developed in the medium with hLIF was also significantly higher than that developed in the medium without hLIF. The present results support the hypothesis that hLIF may contribute to improve the quality of parthenogenetic bovine embryos. However, the results are in contrast to the fact that hLIF added to SOFM supplemented with HS did not improve the development to the blastocyst stage of in vitro fertilized bovine embryos (6), although a stimulating effect of hLIF was observed in the embryos at morula and early blastocyst stages when BSA or polyvinil alcohol was added to the SOFM. The present results with parthenogenetic embryos are inferior to the previous results (6) with in vitrofertilized embryos, but hLIF had a beneficial effect in SOFM supplemented with HS in the case of parthenogenetic embryos. The parthenogenetic development of activated bovine oocytes remains lower (5 to 14% blastocysts; 9, 23) compared with the development capacity of in vitro-fertilized bovine embryos (24 to 48%; 7,24). Recently, however, Aoyagi et al. (1) reported that 94 and 51% of 298 bovine oocytes matured for 24 h and activated by the combined treatment with calcium ionophore (5 pg/ml, Smin), electric pulse (100 v/mm, 90 psec), CH (10 pglml, 6 h) and cytochalasin D (2.5 pglml, 12 h) cleaved and developed to blastocysts, respectively. Considering the results with mice (21), rabbits (21, 22) and cattle (l), electric stimulation appears to advance development of parthenogenetically activated oocytes to the blastocyst stage. Pregnancy was not established in the present study, although estrus was prolonged in 2 of the 3 recipients upto 28 d after transfer. Fukui et al. (9) also failed to observe heart beat in a Day-35 pregnant heifer. Recently, Boediono and Suzuki (2) aggregated parthenogenetic bovine embryos (8-cell) and transferred them after culture. They found that 3 of 5 recipients were diagnosed pregnant on Day 42, but the recipients returned to es&us at Days 57,62 and 67, respectively after the previous estrus. The transfer attempts of parthenogenetically activated and in vitro-developed bovine embryos indicate that either the activation methods or the culture conditions, or both are presently not optimal for the development of parthenogenetic fetuses in cattle. Nagy et al. (20) reported that paternally derived gene(s) might have a unique role in the development of tissues lacking parthenogenetic contribution. However, in mice (3, 13) and rabbits (22) embryo development following parthenogenetic activation until Day 10 to 11 of pregnancy has been reported. Further studies on fetuses obtained by artificial abortion may broaden our knowledge of the mechanism involving in survival of bovine parthenogenetic embryos.
1138
Theriogenology
In conclusion, the addition of hLIF (5000 U/ml) to Day-5 morulae cultured in SOFM supplemented with HS significantly improved the formation of blastocysts and their mean cell numbers compared with that of control embryos. However, after the transfer of the blastocysts, pregnancy was not established. REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
17. 18. 19.
Aoyagi Y, Konishi M, Wada T, Takedomo T. Unaged bovine oocytes successfully develop to blastocysts after parthenogenetic activation or nuclear transfer.Theriogenology 1994;41: 157 abstr. Boediono A, Suzuki T. Pregnancies after transfer of aggregated parthenogenetic bovine activated oocytes. Theriogenology 1994;41: 166 abstr. Cuthbertson KSR. Parthenogenetic activation in vitro with ethanol and benzyl alcohol. J Exp Zoo1 1983;226:311-3 14. Fry RC, Batt PA, Fairclough RJ, Parr RA. Human leukaemia inhibitory factor improves the viability of cultured ovine embryos. Biol Reprod 1992;46:470-474. Fry RC, Purdon TL, Squires TJ, Parr RA. The development of bovine embryos cultured in media containing hLIF. Proc Aust Sot Reprod Biol 1992;24:92 abstr. Fukui Y, Matsuyama K. Development of bovine embryos matured and fertilized in vitro in abstr. media containing human leukemia inhibitory factor. Theriogenology 1994;41: 199 Fukui Y, McGowan LT, James RW, Pugh PA, Tervit HR. Factors affecting the in- vitro development to blastocysts of bovine oocytes matured and fertilized in vitro. J Reprod Fertil 1991;92: 125-131. Fukui Y, Ono H. Effect of serum, hormones, and granulosa cells added to culture medium for in-vitro maturation, fertilization, cleavage and development of bovine oocytes. J Reprod Fertil 1989;86:501-506. Fukui Y, Sawai K, Furudate M, Iwazumi Y, Ohsaki K. Parthenogenetic development of bovine oocytes treated with ethanol and cytochalasin B after in vitro maturation. Molec Reprod Dev 1992;33:357-362. Goto K, Ishida M, Ookutsu S, Nakanishi Y. Activation of unaged bovine oocytes by various parthenogenetic stimuli. Theriogenology 1994;41:207 abstr. Gough NM, Gearing DP, King JA, Wilson TA, Hilton DJ, Nicola NA, Metacalf D. Molecular cloning and expression of the human homologue of the murine gene encoding for myeloid leukemia inhibitory factor. Proc Nat1 Acad Sci, USA 1988;85:2623-2627. Graham CF. The production of parthenogenetic mammalian embryos and their use in biological research. Biol Rev 1973;49:399-422. Kubiak J, PaIdi A, Weber M, Maro B. Genetically identical parthenogenetic mouse embryos produced by inhibition of the first meiotic cleavage with cytochalashin D. Development 1991;111:763-769. Lavranos TC, Seamark RF. Myeloid leukaemia inhibitory factor (LIF): an embryonic trophic factor. Proc Aust Sot Reprod Biol 1989;21:91 abstr. Marquant-Le Guienne B, Humblot P, Guillon N, Thibier M. Murine LIF improves the development of IVF cuItured bovine morulae. J Reprod Fertil, Abstr Ser 1993;12:61 abstr. Miyamoto A, Okuda K, Schweigert FJ, Schams D. Effects of basic fibroblast growth factor, transforming growth factor-& and nerve growth factor on the secretory function of the bovine corpus luteum. J Endocr 1992;135: 103-l 14. Mochizuki H, Fukui Y, Ono H. Effect of the number of granulosa cells added to the culture medium for in vitro maturation, fertilization, and development of bovine oocytes.Theriogenology 1991;36:973-986. Moor RM, Trounson AO. Hormonal and follicular factors affecting maturation of sheep oocytes in vitro and their subsequent developmental capacity. J Reprod Fertil 1977;49: lOl109. Nagai T. Parthenogenetic activation of cattle follicular oocytes in vitro with ethanol. Gamete Res 1987;16:243-249.
Theriogenology
1139
20. Nagy J, Sass M, Markkula M. Systematic non-uniform distribution of parthenogenetic cells in adult mouse chimaeras. Development 1989;106:321-324. 21. Onodera M, Tsunoda Y. Parthenogenetic activation of mouse and rabbit eggs by electric stimulation in vitro. Gamete Res 1989;22:277-283. 22. Ozil JP. The parthenogenetic development of rabbit oocytes after repetitive pulsatile electrical stimulation. Development 1990;109: 117- 127. 23. Presicce GA, Yong X. Development of 24h in vitro matured bovine oocytes following parthenogenetic activation by ethanol and cycloheximide treatment. Theriogenology 1994;41:277 abstr. 24. Pugh PA, Thompson JG, McGowan LT, McMillan WH, Tervit HR. Survival after transfer of fresh or frozen bovine embryos produced in vitro in a cell- and serum- free medium. Proc Aust Sot Reprod Biol 1993;25:86 abstr. 25. Robertson SA, Lavranos TC, Seamark RF. In vitro models of the maternal fetal interface. In: Wessmann T G, Nisbett-Brown E, Gill TJ (eds), The Molecular and Cellular Immunobiology of the Maternal Fetal Interface. Oxford University Press, New York, 1991;191-206. 26. Shi Z, Jiang S, Yang X. Synergistic effect of A23187 and cycloheximide allows effective activation of freshly matured bovine oocytes. Theriogenology 1993;39:309 abstr. 27. Surani MAH, Barton SC, Norris ML. Development of reconstituted mouse eggs suggests imprinting of the genome during gametogenesis. Nature 1984;308:548-550. 28. Surani MAH, Barton SC, Norris ML. Nuclear transplantation the mouse: heritable differences between paternal genomes after activation of the embryonic genome.Cell 1986;45: 127-136. 29. Tervit HR, Whittingham DG, Rowson LEA. Successful culture in vitro of sheep and cattle ova. J Reprod Fertil 1972;30:493-497. 30. Ushijima M, Okuda T, Nakayama A, Moji K, Ishida K, Murata H, Iguchi A, Etoh T. Relationship between the cell number and quality of Day 8-bovine blastocysts. Proc 3rd East Jpn Sot Anim Embryo Transfer 1988;9: 37-38, in Japanese. 3 1. Ware CB, Barnes FL, Maiki-Lauria M, First NL. Age dependence of bovine oocytes activation. Gamete Res 1989;22:265-275.
42: 1133-l
139, 1994
EFFECT OF HUMAN LEUKEMIA INHIBITORY FACTOR ON IN VITRO DEVELOPMENT OF PARTHENtXENEI’IC BOVINE MORULAE Y .Fukui,i T.Saito,t A.Miyamoto,i H.Yamashinaz and Y.Okamot& 1Laboratory of Animal Genetics and Reproduction Obihiro University of Agriculture and Veterinary Medicine, Obihiro 080 Japan ZTokachi-Ikusei Ranch, Taiki-cho, Hokkaido 089-22, Japan
Received for publication: Accepted
February September
15, 1994 14, 1994
ABSTRACT The present study was conducted to investigate the effect of human leukemia inhibitory factor (hLIF) addition to synthetic oviduct fluid medium (SOFM) supplemented with human serum (HS) on the development of in vitro matured and parthenogenetically activated bovine oocytes. The oocytes matured for 30 h were exposured to ethanol (7% 7 min) and cytochalasin B (5 &ml. 5 to 6 h). The treated oocytes were cultured for 5 d in SOFM supplemented with HS, and Day-5 morulae were cultured for 2 d in SOFM supplemented with HS and with or without hLIF (5ooo U/ml) to investigate the subsequent in vitro development to the blastocyst stage. Of the 1531 oocytes that were parthenogenetically activated, 592 (37.5%) cleaved to the 2- to &cell stage and 174 (13.8%) developed to the morula stage. The addition of hLIF at the morula stage resulted in a significantly (P&01) higher rate of development to the blastocyst stage in the medium with hLIF (55.9%) than without hLIF (28.9%). The mean cell number per blastocyst developed in the medium with hLIF was also significantly (PcO.01) higher than that developed in the medium without hLIF. To evaluate the viability, 6 parthenogenetically developed blastocysts were transferred to 3 recipient heifers (2 embryos per heifer), while in 2 other recipient heifers estrus was prolonged after transfer. The plasma progesterone levels of the 2 recipient heifers at the 28th day after transfer were 8.1 nglml and 9.0 ng/ml, but pregnancy was not observed by ultrasonic scanning. The present results indicate that the addition of hLIF to in vitro-produced, Day-5 parthenogenetic bovine morulae significantly improves the subsequent development to the blastocysts stage; however, the present method still does not promote for development of parthenogenetic fetuses in cattle. Key words: hLIF, parthenogenetic development, oocyte, cattle INTRODUCTION Parthenogenetic activation of mammalian oocytes can be induced by various stimuli such as exposure to ethanol (3, 9, 19), calcium ionophore (22, 31) and electric current (10, 21, 22, 31). However, information is limited on the postactivation development of in vitro matured oocytes of farm animals (1, 9,23). In mouse (28) and rabbit (22), reconstituted parthenogenetic ova with female pronuclei resulted in Day 10 to 11 pregnancy with about 25 somites and a beating heart after transfer to recipients, although the parthenogenetic fetuses were of smaller size than the controls (22, 27). Acknowledgements The authors wish to thank Dr. S.Raiti and Dr. D.J.Bolt for the supply of bovine LH and FSH, respectively and the AMRAD Corporation, Ltd., Australia for donation of hLIF (batch HlH2) used in this study.
Copyright
(9 1994 Butterworth-Heinemann
1134
Theriogenology
Lavranosand Seamark (14) and Robertson et al. (25) found that blastocysts cultured in media ~n~i~ng leukemia inhibitory factor (1000 U/ml) had a larger area of trophectodenn than the controls. The addition of human leukemia inhibitory factor (hLIFf significantly improved the development of in vivo-produced ovine (4) and bovine (5) embryos, and in vitro-produced bovine embryos (6, 15). Therefore, it was hypothesized that the addition of hLIF to medium may contribute to improving the quality of parthenogenetic embryos developed in vitro. Our aim in the present study was to investigate the effect of the presence or absence of hLIF addition to synthetic oviduct fluid medium (SOFM) supplements with human serum (HS) on the development of in vitro-matured, parthenogenetically activated and cultured bovine morulae. MATERIALS AND METHODS In Vitro Maturation The ovaries of Holstein cows and heifers were obtained from a local slaughterhouse and were transported in saline (9 g NaClIl) at 39°C to the laboratory within 1 h. The cumulus-~cy~ complexes were collected from follicles of 1 to 5 mm in diameter with an 18-gauge needle attached to an S-ml disposable syringe. Only oocytes with an unexpanded cumulus oophours and evenly granulated cytoplasm were washed 3 times with TCM 199 (Whittaker M.A. Bioproducts, Walkersville, MD, USA; pH 7.4, Earle’s salt with sodium bicarbonate and L-glutamine) supplemented with 0.3% (w/v) bovine serum albumin (BSA: Fraction V, fattyacid-free, Sigma Chemical Co., St. Lois, MO, USA) and 10 mM Hepes, and 10 to 20 cumulus-enclosed oocytes were cultured in a 4-well dish (Nunclon, Inter-Med., Roskilde, Denmark) con~ning 0.5 ml TCM 199 supplement with 10% (v/v) heat-inactivated f56”C, 30 min) fetal calf serum (FCS) without Hepes. The medium was also supplemented with 5 pg bovine LH-B-S/ml (National Hormone and Pituitary Program, Baltimore, ML, USA), 2.5 pg bovine FSH-B-l/ml (US Development of Agriculture, Baltimore, ML, USA), 1 p(g estradiollml (E-8875, Sigma) and with 2 x 106 granulosa cells/ml. The granulosa cells were collected by dissection following the method of Moor and Trounson (18) and washed (500 g, 5 min), and were prepared as described elsewhere (8, 17). Oocytes were statically cultured for 30 h at 39°C under an atmosphere of 5% CO2 in air. Parthenogenetic Treatment The expanding cumulus-enclosed oocytes cultured for 30 h were treated with 7% (v/v) ethanol in SOFM (29) supplemented with 10% (v/v) FCS and 25 mM Hepes for 7 min at room temperature (19) and 5pglml cytochalasin B (CB: Sigma) in SOFM supplemented with 10% (v/v) heat-inactivated (56”C, 30 min) HS, but without Hepes for 5 to 6 h at 39°C under 5% CO2 in air. Following p~henogenetic activation, 5 to 10 oocytes removed cumulus cells by gentle pipetting, were cultured in a 24well dish (Falcon, USA) containing 0.5 ml SOFM supplemented with 10% (V/V) I-IS at 39°C for 5 d under an atmosphere of 5% CO2, 5% 02, 90% N2 (7). To investigate pronuclear formation of induced activation with ethanol and CB treatments, 161 activated oocytes were fixed with acetic-alcohol (1 : 3) after an additional culture in SOFM supplemented with 10% (v/v) HS for 10 h and stained with 1% (w/v) aceto-orcein. The number of oocytes with 2 pronuclei without the second polar body were recorded under a phase-contrast microscope. hLiFTreatment
and Viability
Cleavage (2- to 8-cell stage) and number of morulae were examined on the 3rd and 5th day, respectively, after parthenogenetic treatment (Day 0). Morula-stage embryos were cultured in a 24well dish containing 0.5 ml SOFM supplemented with 10% (v/v) HS and with or without hLIF (5ooo Ulml; AMRAD Corporation, Ltd., Melbourne, Australia) at 39°C for 2 d under an atmosphere of 5% COa. 5% 9, 90% Nz, and the development of blastocysts was recorded after
1135
Theriogenology
hLIF treatment. Some biastocysts were examined for the number of nuclei by the method of Ushijima et al. (30). Briefly, embryos were treated with 0.9% (w/v) sodium citrate for 10 to 20 min, and fixed with a solution of alcohol : acetic-acid : distilled water (3 : 2 : 1 by vol.) at 4 “C for 1 min. The fixed embryos were placed on slides with a small volume of fixative, dried for 1 h and stained by 10% (w/v) Giemsa solution for 15 min. The number of nuclei were determined under a phase-contrast microscope. To evaluate the viability of embryos developed in vitro, 6 blastocysts developed in the culture with hLIF were nonsurgically transferred into the uteri of 3 Holstein heifers (2 embryos per heifer) on Day 8 or 9 after es&us induced by an intramuscular injection of 250 jug prostaglandin Fza (Estrumate: Sumitomo Chemical Co., Osaka, Japan). Estrous behavior was observed every day following transfer. At 28 d after transfer, pregnancy diagnosis was performed using an ultrasonic scanning instrument with an ultrasound frequency of 5.0 MHz (Aloka Co., Tokyo, Japan) and a Kood sample was taken to examine plasma progesterone (P.+) levels. Plasma Pa concentrations were measured by enzyme immunoassay according to the method of Miyamoto et al. (16). The sensitivity of the assay was 1 pgiwell, and the in&a-assay vanation of Pe was 9.4%. Statistical Analysis Six replicates were carried out in this experiment. The proportions of moruiae developing to biastocysts in the medium with and without hLIF (5000 U/ml) were analyzed by a CATMOD procedure following logit transformation in the Statistical Analysis System (SAS). The results of the number of nuclei were analyzed by the Duncan’s new multiple range test. RESULTS On pronucleus formation after parthenogenetic treatment, 55 of 161 examined oocytes (34.2%) had 2 pronuclei without the second polar body (Table 1).
Table 1.
Examination of pronucleus formation of parthenogenetically-activated No. of oocytes examined
161
bovine oocytes
No. of oocytes with pronuclei (%) 0
1
82 (50.9)
20 (12.4)
2 55 (34.2)
23 4 (2.5)
Of 153 1 oocytes that were parthenogenetically activated, 592 (37.5%) cleaved to the 2- to 8ceil stage and 174 (13.8%) developed to the moruia stage (Table 2). The effect of hLIF (5000 U/ml) addition during the development to blastocysts resulted in significantly (PcO.01) higher rate of biastocyst formation in the presence of hLIF (55.9%) than in the absence of hLIF (28.9%; Table 3).
Theriogenolog
1136
Table 2.
In vitro development (cleavage and morula stage) of in vitromatured parthenogenetically-activated bovine oocytes No. of oocytes cultured (No. of trials) 1531 (n = 6)
Table 3.
No. of cleaved oocytes (mean % * SEM)
y
and
No. of oocytes developing to the morula stage (mean %* SEM) 174 (13.8% f 4.1)
592 (37.5% zt 3.6)
Effect of addition of hLIF (5000 U/ml) for 2-day culture of in vitro developed, parthenogenetic bovine morulae Addition of hLIF
No. of morulae cultured (No. of trials)
No. of morulae developing to blastocysts (mean % f SEM)
+ (n y6)
(55.9?* 8.3) a 26 (28.9% f 4.3) b
(n :6) a vs b: P&01.
There was significant difference (P&01) in the mean cell number of blastocysts formed in the presence (84.8) or absence (62.6) of hLIF (Table 4). The proportions of blastocysts with 2100 cells were 27.3% in the presence of hLIF and 0% in the absence of hLIF. Table 4.
Mean cell numbers of bovine the addition of hLIF (5C00 U/ml)
blastocysts
developed
in vitro with or without
Addition of hLIF
No. of blastocysts examined
Mean cell numbers (rt SFM) per blastocyst (Min. - Max.)
+
33 13
84.8 zt 4.3a (34 - 148) 62.6 f 6.2b (30 - 93)
a vs b: P&01. To evaluate viability, 6 parthenogenetically developed embryos were transferred to 3 recipient heifers (2 embryos per heifer). Although the estrus of 1 recipient heifer was observed 14 d after transfer, estrus was also prolonged in 2 recipient heifers after transfer. The plasma P4 levels of 2 recipient heifers at 28 d after transfer were 8.1 and 9.0 ng/ml, respectively, but pregnancy was not observed by ultrasonic scarming. Thereafter, these 2 heifers returned to estrus on Days 35 and 36, respectively, after transfer.
Theriogenolog
1137
y
DISCUSSION Although Graham (12) reported that the majority of mammalian parthenogenetic embryos died soon after implantation, other studies of mouse (3, 13, 27, 28) and rabbit (21) embryo development following parthenogenetic activation have reported survival until Days 10 to 11 of pregnancy. The parthenogenetic fetuses tended to be smaller in size than the fertilized fetuses, although the external morphology was similar (22,27). The proportion (34.3%) of activated oocytes with 2 pronuclei in the ooplasma was lower in our present study than that (54.0%) in our previous study (9). Recently, it has been demonstrated that the combined treatment of calcium ionophore and a protein synthesis inhibitor, cycloheximide (CI-I), with or without electric stimulation, induced a high rate of activation (90 to 100%) of bovine oocytes cultured for 18 to 30 h (1, 10, 23,26). However, for the post-activation development of parthenogenetic embryos relies on an optimal protocol, with exact dosage and exposure times to various chemical substances (ethanol, calcium ionophore, CH and CB) used in parthenogenetic activation. LIF is a glycoprotein, first isolated in mice, that induces differentiation and inhibits the proliferation of Ml myeloid cell line (11). It has been reported that hLIF may play a significant role in the development of mouse (25), ovine (4) and bovine (5,6, 15) embryos. The site of the action of hLIF is most likely the trophectcderm in murine embryos (25). The addition of hLIF to the medium resulted in a significantly higher rate of development of 5 d old parthenogenetic morulae to blastccysts than without hLIF. The mean cell number per blastocyst developed in the medium with hLIF was also significantly higher than that developed in the medium without hLIF. The present results support the hypothesis that hLIF may contribute to improve the quality of parthenogenetic bovine embryos. However, the results are in contrast to the fact that hLIF added to SOFM supplemented with HS did not improve the development to the blastocyst stage of in vitro fertilized bovine embryos (6), although a stimulating effect of hLIF was observed in the embryos at morula and early blastocyst stages when BSA or polyvinil alcohol was added to the SOFM. The present results with parthenogenetic embryos are inferior to the previous results (6) with in vitrofertilized embryos, but hLIF had a beneficial effect in SOFM supplemented with HS in the case of parthenogenetic embryos. The parthenogenetic development of activated bovine oocytes remains lower (5 to 14% blastocysts; 9, 23) compared with the development capacity of in vitro-fertilized bovine embryos (24 to 48%; 7,24). Recently, however, Aoyagi et al. (1) reported that 94 and 51% of 298 bovine oocytes matured for 24 h and activated by the combined treatment with calcium ionophore (5 pg/ml, Smin), electric pulse (100 v/mm, 90 psec), CH (10 pglml, 6 h) and cytochalasin D (2.5 pglml, 12 h) cleaved and developed to blastocysts, respectively. Considering the results with mice (21), rabbits (21, 22) and cattle (l), electric stimulation appears to advance development of parthenogenetically activated oocytes to the blastocyst stage. Pregnancy was not established in the present study, although estrus was prolonged in 2 of the 3 recipients upto 28 d after transfer. Fukui et al. (9) also failed to observe heart beat in a Day-35 pregnant heifer. Recently, Boediono and Suzuki (2) aggregated parthenogenetic bovine embryos (8-cell) and transferred them after culture. They found that 3 of 5 recipients were diagnosed pregnant on Day 42, but the recipients returned to es&us at Days 57,62 and 67, respectively after the previous estrus. The transfer attempts of parthenogenetically activated and in vitro-developed bovine embryos indicate that either the activation methods or the culture conditions, or both are presently not optimal for the development of parthenogenetic fetuses in cattle. Nagy et al. (20) reported that paternally derived gene(s) might have a unique role in the development of tissues lacking parthenogenetic contribution. However, in mice (3, 13) and rabbits (22) embryo development following parthenogenetic activation until Day 10 to 11 of pregnancy has been reported. Further studies on fetuses obtained by artificial abortion may broaden our knowledge of the mechanism involving in survival of bovine parthenogenetic embryos.
1138
Theriogenology
In conclusion, the addition of hLIF (5000 U/ml) to Day-5 morulae cultured in SOFM supplemented with HS significantly improved the formation of blastocysts and their mean cell numbers compared with that of control embryos. However, after the transfer of the blastocysts, pregnancy was not established. REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
17. 18. 19.
Aoyagi Y, Konishi M, Wada T, Takedomo T. Unaged bovine oocytes successfully develop to blastocysts after parthenogenetic activation or nuclear transfer.Theriogenology 1994;41: 157 abstr. Boediono A, Suzuki T. Pregnancies after transfer of aggregated parthenogenetic bovine activated oocytes. Theriogenology 1994;41: 166 abstr. Cuthbertson KSR. Parthenogenetic activation in vitro with ethanol and benzyl alcohol. J Exp Zoo1 1983;226:311-3 14. Fry RC, Batt PA, Fairclough RJ, Parr RA. Human leukaemia inhibitory factor improves the viability of cultured ovine embryos. Biol Reprod 1992;46:470-474. Fry RC, Purdon TL, Squires TJ, Parr RA. The development of bovine embryos cultured in media containing hLIF. Proc Aust Sot Reprod Biol 1992;24:92 abstr. Fukui Y, Matsuyama K. Development of bovine embryos matured and fertilized in vitro in abstr. media containing human leukemia inhibitory factor. Theriogenology 1994;41: 199 Fukui Y, McGowan LT, James RW, Pugh PA, Tervit HR. Factors affecting the in- vitro development to blastocysts of bovine oocytes matured and fertilized in vitro. J Reprod Fertil 1991;92: 125-131. Fukui Y, Ono H. Effect of serum, hormones, and granulosa cells added to culture medium for in-vitro maturation, fertilization, cleavage and development of bovine oocytes. J Reprod Fertil 1989;86:501-506. Fukui Y, Sawai K, Furudate M, Iwazumi Y, Ohsaki K. Parthenogenetic development of bovine oocytes treated with ethanol and cytochalasin B after in vitro maturation. Molec Reprod Dev 1992;33:357-362. Goto K, Ishida M, Ookutsu S, Nakanishi Y. Activation of unaged bovine oocytes by various parthenogenetic stimuli. Theriogenology 1994;41:207 abstr. Gough NM, Gearing DP, King JA, Wilson TA, Hilton DJ, Nicola NA, Metacalf D. Molecular cloning and expression of the human homologue of the murine gene encoding for myeloid leukemia inhibitory factor. Proc Nat1 Acad Sci, USA 1988;85:2623-2627. Graham CF. The production of parthenogenetic mammalian embryos and their use in biological research. Biol Rev 1973;49:399-422. Kubiak J, PaIdi A, Weber M, Maro B. Genetically identical parthenogenetic mouse embryos produced by inhibition of the first meiotic cleavage with cytochalashin D. Development 1991;111:763-769. Lavranos TC, Seamark RF. Myeloid leukaemia inhibitory factor (LIF): an embryonic trophic factor. Proc Aust Sot Reprod Biol 1989;21:91 abstr. Marquant-Le Guienne B, Humblot P, Guillon N, Thibier M. Murine LIF improves the development of IVF cuItured bovine morulae. J Reprod Fertil, Abstr Ser 1993;12:61 abstr. Miyamoto A, Okuda K, Schweigert FJ, Schams D. Effects of basic fibroblast growth factor, transforming growth factor-& and nerve growth factor on the secretory function of the bovine corpus luteum. J Endocr 1992;135: 103-l 14. Mochizuki H, Fukui Y, Ono H. Effect of the number of granulosa cells added to the culture medium for in vitro maturation, fertilization, and development of bovine oocytes.Theriogenology 1991;36:973-986. Moor RM, Trounson AO. Hormonal and follicular factors affecting maturation of sheep oocytes in vitro and their subsequent developmental capacity. J Reprod Fertil 1977;49: lOl109. Nagai T. Parthenogenetic activation of cattle follicular oocytes in vitro with ethanol. Gamete Res 1987;16:243-249.
Theriogenology
1139
20. Nagy J, Sass M, Markkula M. Systematic non-uniform distribution of parthenogenetic cells in adult mouse chimaeras. Development 1989;106:321-324. 21. Onodera M, Tsunoda Y. Parthenogenetic activation of mouse and rabbit eggs by electric stimulation in vitro. Gamete Res 1989;22:277-283. 22. Ozil JP. The parthenogenetic development of rabbit oocytes after repetitive pulsatile electrical stimulation. Development 1990;109: 117- 127. 23. Presicce GA, Yong X. Development of 24h in vitro matured bovine oocytes following parthenogenetic activation by ethanol and cycloheximide treatment. Theriogenology 1994;41:277 abstr. 24. Pugh PA, Thompson JG, McGowan LT, McMillan WH, Tervit HR. Survival after transfer of fresh or frozen bovine embryos produced in vitro in a cell- and serum- free medium. Proc Aust Sot Reprod Biol 1993;25:86 abstr. 25. Robertson SA, Lavranos TC, Seamark RF. In vitro models of the maternal fetal interface. In: Wessmann T G, Nisbett-Brown E, Gill TJ (eds), The Molecular and Cellular Immunobiology of the Maternal Fetal Interface. Oxford University Press, New York, 1991;191-206. 26. Shi Z, Jiang S, Yang X. Synergistic effect of A23187 and cycloheximide allows effective activation of freshly matured bovine oocytes. Theriogenology 1993;39:309 abstr. 27. Surani MAH, Barton SC, Norris ML. Development of reconstituted mouse eggs suggests imprinting of the genome during gametogenesis. Nature 1984;308:548-550. 28. Surani MAH, Barton SC, Norris ML. Nuclear transplantation the mouse: heritable differences between paternal genomes after activation of the embryonic genome.Cell 1986;45: 127-136. 29. Tervit HR, Whittingham DG, Rowson LEA. Successful culture in vitro of sheep and cattle ova. J Reprod Fertil 1972;30:493-497. 30. Ushijima M, Okuda T, Nakayama A, Moji K, Ishida K, Murata H, Iguchi A, Etoh T. Relationship between the cell number and quality of Day 8-bovine blastocysts. Proc 3rd East Jpn Sot Anim Embryo Transfer 1988;9: 37-38, in Japanese. 3 1. Ware CB, Barnes FL, Maiki-Lauria M, First NL. Age dependence of bovine oocytes activation. Gamete Res 1989;22:265-275.