DEVELOPMENT AND APPLICATION OF BOVINE IN VITRO FERTILIZATION SAKSIRI SIRISATHIEN. (Under the direction of BENJAMIN G. BRACKETT) ABSTRACT

Transcription

1 DEVELOPMENT AND APPLICATION OF BOVINE IN VITRO FERTILIZATION by SAKSIRI SIRISATHIEN (Under the direction of BENJAMIN G. BRACKETT) ABSTRACT Improved understanding of in vitro fertilization, in vitro oocyte maturation, and embryo culture will allow laboratory production of bovine blastocysts to become a well-established procedure. However, data related to production of bovine blastocysts in chemically defined media, i.e., no component of animal-origin included, are limited. Bovine blastocyst production in chemically defined media is an important technology that remains in need of improvement. The objective of this study was to examine the influence of growth factors or cytokines supplemented to embryo culture media on bovine embryonic development. A better means for handling of ovaries to maximize the efficiency of this procedure was also evaluated. Experimentation was also conducted to assess the feasibility of using bovine spermatozoa as a gene transfer vector to produce transgenic bovine embryos through in vitro fertilization or through intracytoplasmic sperm injection. Results presented here demonstrated that the way in which bovine ovaries were handled had a great impact on developmental competence of the harvested oocytes. Two hours of postmortem delay prior to oocyte aspiration benefited in vitro bovine embryo production.

2 An effectect of adding leukemia inhibitory factor to embryo culture media was shown to increase numbers of inner cell mass nuclei without improving blastocyst yields or survival after cryopreservation. Also, this treatment stimulated in vitro hatching of blastocysts from their surrounding zonae pellucidae. Both epidermal growth factor and insulin-like growth factor-i improved rates of blastocyst development (from inseminated oocytes) when added to defined embryo culture media at a concentration of 5 and 50 ng/ml, respectively. No significant interaction resulting from combined treatment with those growth factors was detected. Insulinlike growth factor-i also increased numbers of inner cell mass nuclei and reduced numbers of DNA fragmented nuclei while epidermal growth factor had no effect either on numbers of inner cell mass nuclei or numbers of DNA fragmented nuclei when compared to untreated controls. Only bovine epididymal spermatozoa, not ejaculated spermatozoa, possess an ability to take up exogenous DNA. However, in contrast to recent findings with murine spermatozoa, there remains a barrier in efforts to adopt this approach for bovine spermatozoa as gene transfer vectors either in conjunction with in vitro fertilization or intracytoplasmic sperm injection. This body of research has contributed to a better understanding of requirements for laboratory production of bovine blastocysts and provides encouragement for realistic optimization of this reproductive biotechnology. INDEX WORDS: Bovine blastocyst, In vitro fertilization, Epidermal growth factor, Insulin-like growth factor-i, Leukemia inhibitory factor, Intracytoplasmic sperm injection, DNA uptake, Transgenic, Chemically defined media, Nuclear DNA fragmentation.

3 DEVELOPMENT AND APPLICATION OF BOVINE IN VITRO FERTILIZATION by SAKSIRI SIRISATHIEN D.V.M., Kasetsart University, Thailand, 1993 A Dissertation Submitted to The Graduate Faculty of The University of Georgia in Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY ATHENS, GEORGIA 2002

4 2002 Saksiri Sirisathien All Rights Reserved

5 DEVELOPMENT AND APPLICATION OF BOVINE IN VITRO FERTILIZATION by SAKSIRI SIRISATHIEN Major Professor: Benjamin G. Brackett Committee: Hugh D. Doowah Oliver W. Li Royal A. McGraw Steven L. Stice Electronic Version Approved: Maureen Grasso Dean of The Graduate School The University of Georgia December 2002

6 iv TABLE OF CONTENTS CHAPTER Page 1 INTRODUCTION 1 2 LITERATURE REVIEW A. EXPERIMENTAL FINDINGS LEADING TO IMPROVEMENT OF IN VITRO PRODUCTION OF BOVINE BLASTOCYSTS IN CHEMICALLY DEFINED CONDITIONS...7 B. GENE TRANSFER METHODOLOGIES FOR MAMMALS 39 3 EFFECT OF LEUKEMIA INHIBITORY FACTOR ON BOVINE EMBRYOS PRODUCED IN VITRO UNDER CHEMICALLY DEFINED CONDITIONS BENEFICIAL POSTMORTEM INFLUENCE ON PRODUCTION OF BOVINE BLASTOCYSTS IN VITRO INFLUENCES OF EPIDERMAL GROWTH FACTOR AND INSULIN-LIKE GROWTH FACTOR-I ON BOVINE BLASTOCYST DEVELOPMENT IN VITRO...131

7 v 6 TUNEL ANALYSES OF BOVINE BLASTOCYSTS AFTER CULTURE WITH EPIDERMAL GROWTH FACTOR AND INSULIN-LIKE GROWTH FACTOR BULL SPERM UPTAKE OF EXOGENOUS DNA AND EFFORTS TO OBTAIN TRANSGENIC EMBRYOS CONCLUSIONS APPENDICES...200

8 1 CHAPTER 1 INTRODUCTION In vitro production of bovine blastocysts is a multi-step procedure consisting of the maturation of immature oocytes, fertilization of mature oocytes, and culture of embryos until the blastocyst stage. The production of bovine blastocysts in vitro is a promising approach to maximize the use of bovine gametes. For in vivo bovine blastocyst production, it is conceivable that the average numbers of 4.5 to 5 blastocysts are expected from each superovulated donor (Hasler, 1992). Bousquet et al (1999) showed that, during a 60-day period, in vitro production of bovine blastocysts in conjunction with transvaginal oocyte recovery resulted in 18.8 transferable blastocysts whereas the in vivo production approach resulted in 4.3 transferable blastocysts. Using media containing serum and somatic cells, bovine blastocyst development in vitro at the rates above 70 % of oocytes has been achieved after proper ovarian stimulation procedures (Blondin et al., 2002, Rizos et al., 2002). Enormous progress in the production of bovine blastocysts in vitro has been made since the first calf was born from an in vitro fertilized embryo in 1981 (Brackett et al., 1982). Improved fertilization rates, as represented by the cleavage of over 80 % of oocytes followed the use of heparin treatment for sperm capacitation (Parrish et al., 1985). Improvement in maturation of immature oocytes in vitro has been made mainly by the introduction of gonadotropins, estradiol, and several growth factors into the media. Formulation of synthetic oviductal fluid (Tervit et al., 1972) for bovine embryo culture medium allows blastocyst development without using somatic cells support. Currently, it has become feasible to obtain in vitro produced bovine

9 2 blastocysts in chemically defined media beginning with the maturation of immature oocyte collected from the slaughterhouse (Keskintepe and Brackett, 1996, Holm et al., 1999, Dinkins and Brackett, 2000, Hernandez et al., 2002). In general, chemically defined media are formulated with the use of synthetic macromolecules such as polyvinyl alcohol (PVA) or polyvinyl pyrolidone (PVP) to replace serum, serum albumin, or other biological components. PVA, initially found to be an acceptable BSA replacement in hamster fertilization medium (Bavister, 1981), has been proven to be a suitable BSA substitute in a variety of chemically defined bovine embryo culture media (Carney and Foote, 1991, Pinyopummintr and Bavister, 1991, Takahashi and First, 1992, Kim et al., 1993, Rosenkrans and First, 1994, Lee and Fukui, 1996, Keskintepe and Brackett, 1996, Olsen and Seidel, 2000). Although the production of bovine blastocysts in chemically defined medium affords development of lower blastocyst yields (Pinyopummintr and Bavister, 1991, Takahashi and First, 1992, Holm et al., 1999, Krisher et al., 1999), in turn, it is a more suitable approach to elucidate essential factors for blastocyst development (Bavister, 1992). Chemically defined medium is also a more desirable approach regarding repeatability among laboratories and hygienic concerns pertaining to transmission of infectious pathogens (Stringfellow and Givens, 2000). Therefore, more studies are needed to improve blastocyst yields as well as to bring the viability of in vitro produced blastocysts up to that of blastocysts obtained in vivo if embryo production in chemically defined medium is to be widely used for both research and commercial applications. The ultimate goal in formulating embryo culture medium is to simulate the microenvironment resemble to that in female reproductive tract that these embryos experience. Among that, a variety of growth factors and cytokines are now widely recognized to be present in the female reproductive tract. For past few years, the roles of growth factors and cytokines in controlling preimplantational embryonic development have been a subject of great research area of interest not only for fundamental scientific merits but also in the relation to be used to improve the quality of embryos produced by in vitro fertilization. The latter is the primary goal in this

10 3 work. It is in agreement that the ultimate measurement of blastocyst quality is the ability to initiate pregnancy with resulting healthy offspring. However, in most circumstance, it is impractical to transfer every blastocyst produced for quality assessment. Additionally, the selection of the best blastocysts from the blastocyst pools for embryo transfer may not truly represent the overall quality of the entire population (Bavister, 1995). Therefore, in vitro assessment by indicators of embryo quality such as metabolic activity, ultrastructure, cell numbers, or freezability provides an affordable alternative methodology for in vitro viability assessment. To date, the benefits from in vitro produced blastocyst methodology have gone beyond the initial goals to provide low-cost embryos for the cattle industry or to obtain embryos from problem cows. Production of transgenic embryo technology is another area that the author hopes to expand by the application of knowledge of in vitro production of bovine blastocysts. Although transgenic bovine embryo production via nuclear transfer approach has been shown to be an outstanding improvement, several alternative transgenic methodologies have not been overlooked. Among those, sperm mediated gene transfer is arguably the most attractive due to its simplicity and less zygote manipulation required. The present investigation addressed three major subjects. In brief, the purpose was to: 1) Evaluate the putative benefits of supplementing specific growth factor or cytokine such as epidermal growth factor, insulin-like growth factor-i, and leukemia inhibitory factor to chemically defined embryo culture media; 2) Assess the quality of the resulting blastocysts by employing several in vitro techniques including survival after cryopreservation, counting of inner cell mass cells and trophoblast cells, and incidences of DNA fragmented nuclei; 3) Investigate the feasibility of using spermatozoa as an alternative gene transfer method to produce transgenic bovine embryos. A review of pertinent literature and original findings from this research follow.

11 4 REFERENCES Bavister BD. Substitution of a synthetic polymer for protein in a mammalian gamete culture system. J. Exp. Zool. 1981;217: Bavister BD. Co-culture for embryo development: is it really necessary? Hum Reprod. 1992;7(10): Bavister BD. Culture of preimplantation embryos: facts and artifacts. Hum. Reprod. Update 1995;(1): Blondin P, Bousquet D, Twagiramungu H, Barnes F, Sirard MA. Manipulation of follicular development to produce developmentally competent bovine oocytes. Biol Reprod. 2002;66(1): Bousquet D, Twagiramungu H, Morin N, Brisson C, Carboneau G, Durocher J. In vitro embryo production in the cow: an effective alternative to the conventional embryo production approach. Theriogenology. 1999;51(1):59-7. Brackett BG, Bousquet D, Boice ML, Donawick WJ, Evans JF, Dressel MA. Normal development following in vitro fertilization in the cow. Biol Reprod. 1982;27(1): Carney EW, Foote RH. Improved development of rabbit one-cell embryos to the hatching blastocyst stage by culture in a defined, protein-free culture medium. J. Reprod. Fertil. 1991;91: Dinkins MB, Brackett BG. Chlortetracycline staining patterns of frozen-thawed bull spermatozoa treated with beta-cyclodextrins, dibutyryl camp and progesterone. Zygote. 2000;8(3): Hasler JF. Current status and potential of embryo transfer and reproductive technology in dairy cattle. J Dairy Sci. 1992;75(10): Hernandez-Fonseca HJ, Sirisathien S, Bosch P, Cho HS, Lott JD, Hawkins LL, Hollett RB, Coley SL, Brackett BG. Offspring resulting from direct transfer of cryopreserved bovine embryos produced in vitro in chemically defined media. Anim Reprod Sci. 2002;69(3-4):151-8.

12 5 Holm P, Booth PJ, Schmidt MH, Greve T, Callesen H. High bovine blastocyst development in a static in vitro production system using SOFaa medium supplemented with sodium citrate and myo-inositol with or without serum-proteins. Theriogenology. 1999, 52(4): Keskintepe L, Brackett BG. In vitro developmental competence of in vitro-matured bovine oocytes fertilized and cultured in completely defined media. Biol Reprod. 1996;55(2): Kim JH, Niwa K, Lim JM, Okuda K. Effects of phosphate, energy substrates, and amino acids on development of in vitro-matured, in vitro-fertilized bovine oocytes in a chemically defined, protein-free culture medium. Biol. Reprod. 1993;48: Krisher RL, Lane M, Bavister BD. Developmental competence and metabolism of bovine embryos cultured in semi-defined and defined culture media. Biol Reprod. 1999;60(6): Lee ES, Fukui Y. Synergistic effect of alanine and glycine on bovine embryos cultured in a chemically defined medium and amino acid uptake by vitro-produced bovine morulae and blastocysts. Biol. Reprod. 1996;55: Olson SE, Seidel GE Jr. Reduced oxygen tension and EDTA improve bovine zygote development in a chemically defined medium. J Anim Sci. 2000;78(1): Parrish JJ, Susko-Parrish JL, First NL. In vitro fertilization of bovine oocytes using heparintreated and swim-up separated frozen thawed bovine semen is repeatable and results in high frequencies of fertilization. Theriogenology 1985;35:234 (Abstr). Pinyopummintr T, Bavister BD. In vitro-matured/in vitro-fertilized bovine oocytes can develop into morulae/blastocysts in chemically defined, protein-free culture media. Biol. Reprod. 1991;45: Rosenkrans CF Jr, First NL. Effect of free amino acids and vitamins on cleavage and developmental rate of bovine zygotes in vitro. J Anim Sci. 1994;72(2):434-7.

13 6 Stringfellow DA, Givens MD. Epidemiologic concerns relative to in vivo and in vitro production of livestock embryos. Anim Reprod Sci. 2000;60-61: Tervit HR, Whittingham DG, Rowson LE. Successful culture in vitro of sheep and cattle ova. J Reprod Fertil. 1972, 30(3): Takahashi Y, First NL. In vitro development of one-cell embryo: influence of glucose, lactate, pyruvate, amino acids and vitamins. Theriogenology 1992;37:

14 7 CHAPTER 2 LITERATURE REVIEW A. EXPERIMENTAL FINDINDS LEADING TO IMPROVEMENT OF IN VITRO PRODUCTION OF BOVINE BLASTOCYSTS IN CHEMICALLY DEFINED CONDITIONS IN VITRO OOCYTE MATURATION Early Reports on In Vitro Bovine Oocyte Maturation In vitro maturation is a process in which meiotically-arrested (prophaes, or germinal vesicle stage) oocytes from small to medium antral follicles are cultured in the laboratory to become ready for fertilization by reaching metaphase II with extrusion of the first polar body. Pincus and Enzman (1935) first observed that rabbit oocytes resume meiosis spontaneously after being removed from the follicular environment. Edwards (1965) reported similar findings with mouse, sheep, cow, pig, rhesus monkey and human ovarian oocytes. Successful and repeatable IVF results of bovine oocytes began with in vivo matured oocytes (Brackett et al., 1978, 1980) with appropriate sperm capacitation treatments, leading to the first bovine IVF offspring (Brackett et al., 1982). This was followed by reports of several more pregnancies (Brackett et al., 1984, Sirard and Lambert, 1985, Sirard et al., 1985, Lambert et al., 1986, Leibfried-Rutledge et al., 1987). The cost of retrieving in vivo matured oocytes is high, compared to collection of immature oocytes from small follicles, making in vivo matured oocytes less attractive as starting material than immature oocytes for IVF research and for large scale laboratory embryo production. Early IVF results of in vitro matured cow oocytes were extremely low and terminated at the pronuclear stage. Iritani and Niwa (1977) observed only 6 to 7% pronuclear

15 8 formation rates (i.e., percentages of inseminated oocytes that became zygotes) after IVF of cow oocytes matured in vitro. Fulka et al (1982) reported 45 % pronuclear formation rates after IVF of zona-free in vitro matured cow oocytes. Fukui et al (1983) capacitated frozen-thawed sperm with high ionic strength (HIS) medium (Brackett and Oliphant, 1975) and obtained 27 % pronuclear formation after insemination of in vitro matured oocytes. Ball et al (1983) observed the crucial role of cumulus cells during oocyte in vitro maturation for high pronuclear formation rates after IVF. Iritani et al (1984) capacitated bull sperm with isotonic medium (m-krb) and obtained up to 58 % fertilization rates. Hensleigh and Hunter (1985) matured cow oocytes for 48 h in vitro, inseminated with extended cooled sperm and obtained 15 % cleaved oocytes after IVF. Eighty percent male pronuclear formation rates were obtained after IVF with heparin-treated sperm (Leibfried-Rutledge et al., 1986, Parrish et al., 1986). Development to the blastocyst stage of in vitro matured and fertilized bovine oocytes was initially obtained after transfer of zygotes or early cleaved embryos into the oviducts which resulted in pregnancies after embryo transfer (Critser et al., 1986, Xu et al., 1987, Lu et al., 1987, Sirard et al., 1988, Fayrer-Hosken et al., 1989). This marked initiation of mass production of bovine embryos from immature oocytes. An Overview of In Vitro Bovine Oocyte Maturation and Factors Improving In Vitro Oocyte Maturation Although more than 80 % of immature oocytes usually reach the metaphase II stage after being incubated for a period of 20 h, in vitro fertilization following 24 h of in vitro maturation period has been shown to be optimal (Monaghan et al., 1993, Long et al., 1994, Ward et al., 2002). Dominko and First (1997) demonstrated that blastocyst development was higher when insemination was delayed for 8 h after first polar body extrusion. Tissue culture medium 199 (TCM-199) with Earle s salts is the most widely used basic medium for in vitro oocyte maturation. Rose and Bavister (1992) compared seven commercially available complex media and concluded that both TCM-199 and MEM were equally suitable for oocyte maturation while

16 9 use of Waymouth s MB 752/1 and Ham s F-12 to support maturation resulted in significantly reduced cleavage rates and post-cleavage development of the mature oocytes after IVF to the blastocyst stage. Gliedt et al (1996) compared TCM-199 vs. RPMI-1640 for maturation and observed that blastocyst development from oocytes matured in TCM-199 was slightly higher than for those from RPMI Oocyte maturation conditions strongly affect not only fertilizing ability but also determine early embryonic developmental competence, this can be understood by considering the fact that mrna in early cleavage stage embryo predominantly arises from pools within the oocytes. Several simple media have been used successfully for oocyte maturation such as hamster embryo culture medium-6 (Rose-Hellekant et al., 1998), modified basic medium (Krisher and Bavister, 1999), and synthetic oviductal fluid (Hashimoto et al., 2000, Gandhi et al., 2000). The use of laboratory prepared media allows better understanding of factors that are important for in vitro oocyte maturation. Generally, TCM-199 is supplemented with a variety of substances that have been shown to contribute to some improvements. Ten to twenty per cent of various types of bovine sera are usually included in the maturation medium since early works uncovered advantages of including estrus cow serum, pro-estrous serum, steer serum, or fetal bovine serum (Leibfried-Rutledge et al., 1986, Parrish et al., 1986, Sirard et al., 1988, Younis et al., 1989, Sanbuissho and Threlfall, 1989). However, the presence of bovine sera during in vitro maturation is not crucial. Bovine oocytes have been matured in serum-free medium (Zuelke and Brackett, 1990, Saeki et al., 1991, Harper and Brackett, 1993, Lonergan et al., 1994) then fertilized, and cultured before resulting in healthy offspring after embryo transfer of either fresh (Keskintepe et al., 1995) or cryopreserved (Hernandez-Fonseca et al., 2002) embryos. Gonadotropins and Prolactin Gonadotropins are commonly included in maturation media to make mature oocytes more effective in sustaining high fertilization and developmental rates. Ball et al (1983) observed

17 10 that inclusion of FSH during in vitro maturation improved pronuclear rates after IVF. Inclusion of FSH also improved the degree of cumulus expansion over inclusion of hcg. In vitro maturation with a combination of FSH, LH, and estradiol increased morula plus blastocyst yields 28%, compared to 18% of oocytes in vitro matured without hormones (Sirard et al., 1988). Similarly, Younis et al (1989) founded that inclusion of FSH or LH improved cleavage and development to 4- to 8-cell stages from 6% (without hormone) to 15% (plus FSH) or 20% (plus LH) whereas inclusion of estradiol provided no improvement over maturation without hormones. Despite the fact that resumption of meiosis in oocytes is known to be triggered by the LH surge in vivo, benefits of including LH during vitro maturation has been controversial. Several reports from our lab showed that LH supplementation enhanced development. Brackett et al (1989) observed a positive dose dependent effect of LH and demonstrated that a high LH concentration (100 µg/ml) was beneficial for oocyte maturation as reflected in subsequent development to 6-to 8-cell stages. Zuelke and Brackett (1990) obtained 27% blastocysts when at least 50 µg/ml of LH was included in serum-free oocyte maturation medium. Harper and Brackett (1993) obtained both higher cleavage and blastocyst formation rates when LH (50 µg/ml) was included compared to serum-free maturation medium alone. Whether the positive effects of LH were caused by an impurity of the LH preparation may have been answered by the possibility that when thyroid stimulating hormone (TSH) was added at 0.5 µg/ml for maturation improved IVF results also followed whereas prolactin (up to 1000 µg/ml) had no effect (Younis and Brackett, 1992). Alternatively, some other contaminant in both of the biological preparations i.e., LH and for TSH derived from the same region of the anterior pituitary glands, may have been responsible for the positive effect. Although not a direct comparison, Harper and Brackett (1993) also observed that FSH at 1 µg/ml resulted in a comparable blastocyst yield to that of LH at 100 µg/ml. Gliedt et al (1996) included equine LH (up to 30 µg/ml) in maturation medium containing 20 % estrous cow serum and reported no positive effect of LH on either cleavage or

18 11 blastocyst formation rates. However, this was not surprising since serum most likely had a high LH content as shown for proestrous serum by Younis et al (1989). Bevers et al (1997) reported that hcg (0.05 IU/mL) during in vitro maturation had no positive effect whereas FSH (0.05 IU/mL) significantly improved blastocyst formation rates. Similarly, Martins et al (1998), following the lead of Harper and Brackett (1993), supplemented chemically defined maturation medium with recombinant gonadotropins to and concluded that FSH (10 ng/ml) significantly improved both cleavage and blastocyst development over maturation medium without hormone or with LH (1 ng/ml) alone whereas LH alone slightly improved blastocyst yields over those following maturation without any added hormone. Further there was no additive effect of FSH and LH supplementation for maturation. Unfortunately, only one concentration of LH was investigated at that moment. In apparent contrast, Anderiesz et al (2000) added recombinant gonadotropins in maturation medium containing 10 % fetal bovine serum and observed an additive effect of LH (10 IU/mL) plus FSH (1 IU/mL) over either FSH or LH alone. This result probably reflected a lower gonadotropin contribution to the basic medium by the included serum preparation selected by those investigators to cloud the issue A beneficial effect of including FSH during in vitro oocyte maturation is firmly established. Fukushima and Fukui (1985) observed that inclusion of FSH but not LH nor estradiol in maturation medium improved fertilization rates. Eyestone and Boer (1993) used serum-free maturation medium and demonstrated a positive effect of FSH on blastocyst development from 2- cell stage embryos. Martins et al (1998) employed recombinant FSH (10 ng/ml) in chemically defined oocyte maturation and obtained improvements in both cleavage and blastocyst formation rates. Ali and Sirard (2002) also reported similar findings of positive effects on blastocyst development but without affecting cleavage rates recombinant after using FSH (5 to 500 ng/ml) for maturation. Van Tol et al (1996) isolated oocytes that remaining connected to membrana granulosa to study oocyte maturation and demonstrated that membrana granulosa inhibited oocyte resumption of meiosis, i.e., germinal vesicle breakdown. The inhibitory effect produced from

19 12 membrana granulosa could be overcome by adding recombinant FSH to the maturation medium but could not be overcome by adding hcg (which was predominantly LH with some FSH-like activity). The authors concluded that in vitro resumption of meiosis of oocytes, originating from antral follicles between 2 to 8 mm, was triggered by FSH and not by LH. FSH and LH receptor mrna expression in small antral follicles, i.e., smaller than 9 mm, has been characterized. LH receptor mrna was detectable exclusively in the theca cells whereas FSH receptor mrna was present in both granulosa and cumulus cells; LH receptor in granulosa cells was only detectable in follicles larger than 9 mm (Xu et al., 1995, van Tol et al., 1996). However, Baltar et al (2000) employed 125 I-LH to demonstrate that cumulus oocyte complexes were able to specifically bind LH, indicating the presence of LH receptors. Interestingly, the authors also showed that small antral follicles (2-3 mm) had higher LH binding capacity than larger follicles (> 5 mm). Nonetheless, the positive effect of gonadotropins is not a definitive finding. Several investigators have failed to observe any positive effect of supplementing gonadotropins in oocyte maturation medium either in serum-containing media (Fukui and Ono, 1989, Goto and Iritani, 1992, Keefer et al., 1993) or serum-free medium (Lonergan et al., 1994, Choi et al., 2001). There are few documentations regarding any effect of prolactin. Early reports showed no benefit of supplementing prolactin in either serum-free (Saeki et al., 1991) or serum-containing maturation medium (Younis and Brackett, 1992). A recent report showed that prolactin had some regulatory role on intracellular stored calcium during bovine oocyte maturation (Kuzmina et al., 1999), whether that role can affect IVF result remains to be demonstrated. Estradiol and Progesterone Estradiol is widely added to maturation media at a level of 1 µg/ml based on found in preovulatory follicles (Dieleman et al., 1983, Fortune and Hansel, 1985). Several investigators showed no benefits of including estradiol in maturation medium containing fetal bovine serum (Fukushima and Fukui, 1985, Younis et al., 1989, Saeki et al., 1991) or estrous cow serum

20 13 (Brackett et al., 1989, Gliedt et al., 1996). Data on benefit of including estradiol during oocyte maturation remain controversial. Beker et al (2002) confirmed no benefit of adding estradiol (1 µg/ml) to serum-free maturation medium. In contrast, Ali and Sirard (2002) observed a positive effect of estradiol (1 µg/ml) in serum-free maturation medium on cleavage rates while having no effect on post-cleavage development to the blastocyst stage. Guler et al (2000) reported a higher blastocyst development when sheep oocytes were matured with estradiol (0.1 µg/ml) in serumfree medium and showed no benefit of estradiol when maturation medium was supplemented with 10% follicular fluid that had been treated to contain only 0.06 ng/ml of estradiol. Mingoti et al (2002) detected a significant increase in estradiol concentration (120 ng/ml) in maturation medium after a 16 h culture period for bovine cumulus oocyte complexes matured with FSH and hcg (10 oocytes in 3 ml medium). This indicates that exogenous estradiol might not be required owing to the ability of cumulus oocyte complexes to secrete estradiol while being cultured with gonadotropins. Data on the effect of progesterone during oocyte maturation on fertilization and embryonic development are limited. Progesterone has been included in maturation medium due to the fact that follicular fluid of follicles approaching ovulation contain increasing levels of progesterone when luteinzation of granulosa cells occurs (Dieleman et al., 1983). Fukushima and Fukui (1985) observed a slight reduction in fertilization rates when progesterone was included in maturation medium. Silva and Knight (2001) observed a significant reduction in blastocyst development when oocytes were matured in maturation medium supplemented with 300 nm progesterone compared to control medium containing 10% estrous cow serum and this negative effect was partially reversed by adding RU486 (an anti-progestin) to the maturation medium while RU486 by itself had no effect either fertilization of blastocyst formation rates.

21 14 Activin A Activin A, measured from dominant follicles, is present in follicular fluid at approximately 3 µg/ml (Knight et al., 1996). Activin A is one of the ligands reported to have a positive effect. Izadyar et al (1996) observed positive effect of activin A (10 ng/ml) in conjunction with gonadotropins on cleavage rates without a further effect on blastocyst development while activin alone produced no effect. Stock et al (1997), however, observed higher blastocyst formation rates from 2-cell stage embryos when activin A alone (1 and 10 ng/ml) was included compared to serum-free maturation medium alone. Similarly, Silva et al (1998) employed 500 ng/ml activin A in serum-included maturation medium and obtained higher blastocyst development. Activin A also produced a similar effect on cumulus-free oocytes indicating the presence of activin receptors on oolemma which was later confirmed by Izadyar et al (1998). Growth hormone (GH) Several reports, mostly from one laboratory, have shown that supplementation of growth hormone in serum-free maturation medium improved IVF outcomes. Izadyar et al (1996) found that GH at 0.1 and 1 µg/ml accelerated the nuclear maturation process by increasing the proportions of oocytes reaching metaphase II stage by 16 h while total oocytes reaching the metaphase II stage at 24 h remained similar to controls. Growth hormone also improved both cleavage rates (from 50-60% to 73-78%) and blastocyst formation rates (from 18-20% to 25-30%). These findings support the report of Dominko and First (1997) who found that the sooner oocytes reach metaphase II, the higher the blastocyst formation rates that would be obtained. Oocytes matured in the presence of GH (0.1 µg/ml) had more cortical granules evenly disperse in the cortical cytoplasm aligning the oolemma than oocytes matured in maturation medium alone (Izadyar et al., 1998). Recently, Moreira et al (2002) added GH (10 µg/ml) to control maturation

22 15 medium consisting of steer serum and FSH and obtained higher cleavage rates but without improvement on blastocyst development from 2-cell stage embryos. Growth Factors Additions of epidermal growth factor (EGF) at concentrations of ng/ml are frequently included in maturation medium. EGF has been shown to stimulate nuclear maturation of bovine oocytes in vitro (Lorenzo et al., 1994). Coskun et al (1991) improved fertilizing and developmental ability of in vitro matured bovine oocytes by adding EGF to the maturation medium. Harper and Brackett (1993a) observed positive effects of EGF (1-100 ng/ml) on morula and blastocyst development when added in combination with FSH (10 µg/ml) while EGF by itself or in combination with LH produced no effect. The positive effect of EGF was confirmed in a subsequent report of Harper and Brackett (1993b). Kobayashi et al (1994) obtained higher cleavage rates and blastocyst development when either EGF or Transforming growth factor-α (TGF-α), a growth factor that is closely related to EGF, alone were included in maturation medium but no additive effect of EGF was detected when it was combined with FSH plus LH. Lonergan et al (1996), however, observed that EGF alone (1-100 ng/ml) improved only cleavage rates without any improvement on blastocyst development. Park et al (1998) obtained significantly higher cleavage and blastocyst formation rates when EGF (10-50 ng/ml) was included compared to serum-free maturation medium alone. Similarly, Watson et al (2000) reported that EGF alone (100 ng/ml) supplemented in serum-free maturation medium improved both cleavage rates and blastocyst formation rates. Palasz et al (2000) obtained a higher cleavage rate but found no benefit on blastocyst development of EGF (20 ng/ml) compared to that of serum-free maturation medium alone. Recently, Sakaguchi et al (2002) observed that EGF significantly accelerated the proportions of oocytes extruding their first polar bodies after 16 h of maturation when compared to controls.

23 16 Levels of Insulin-like growth factor-i (IGF-I) in bovine follicular fluid from either small or perovulatory follicles were reported to be at approximately 100 ng/ml (Funston et al., 1995). Herrler et al (1992) observed no benefit of adding IGF-I (50 ng/ml) into serum-included maturation medium except improved degree of cumulus expansion. Martins and Brackett (1998) obtained significantly higher blastocyst yielded, without observing any effect on cleavage rates, by supplemented IGF-I (1-100 ng/ml) in conjunction with gonadotropins into chemically defined maturation medium. Reiger et al (1998) observed an additive effect of combining IGF-I (100 ng/ml) and EGF (50 ng/ml) on cleavage and blastocyst formation rates while EGF and IGF-I alone produced no improvement compared to chemically defined maturation medium alone. Makarevich and Markkula (2002), however, found no benefit of supplementing IGF-I (100 ng/ml) into control medium consisting of fetal bovine serum and gonadotropins. Positive effect of platelet-derived growth factor (PDGF) at a level of 10 ng/ml in conjunction with FSH on blastocyst formation rates has been demonstrated in serum-free maturation medium (Harper and Brackett, 1993). Vascular endothelial growth factor (VEGF) at a level of 100 ng/ml has been shown to be benefit to blastocyst production in vitro compared to control maturation medium alone (Einspanier et al., 2002). Importance of Increased Glutathione During Oocyte Maturation An inability of in vitro matured oocytes to support male pronuclear formation was apparent in early experiments. The majority (85%) of in vitro matured golden hamster oocytes were unable to cause decondensation of sperm nuclei after 6 h of sperm/egg incubation while 98% of ovulated oocytes were fertilized normally (Leibfried and Bavister, 1983). The same problem also occurred with bovine in vitro matured oocytes. Leibfried-Rutledge et al (1987) reported that frequencies of sperm penetration were not different for in vitro matured oocytes vs. in vivo matured oocytes. However, formation of male pronuclei was reduced for oocytes matured in vitro compared to in those matured vivo. Only 3% of in vitro matured oocytes developed to

24 17 the 2-cell stage whereas 40% of oocytes matured in vivo showed normal development to the 4- cell stage after in vitro fertilization. The role of a disulfide bond reducing agent for sperm nuclear decondensation has long been recognized (Perreault et al., 1984, Perreault et al., 1987). Oocyte glutathione has been shown to play an important role for the sperm decondensation process in forming male pronuclei. Sperm nuclear decondensation was prevented or delayed by blocking glutathione synthesis with buthionine sulfoximine during the early stages of oocyte maturation in the hamster (Perreault et al., 1988) and cow (Sutovsky and Schatten, 1997). In vitro matured hamster oocytes were found to contain significantly lower amounts of intracellular glutathione than in vivo matured oocytes (Kito and Bavister, 1997). The need to increase oocyte glutathione was reported in porcine oocyte. The rates of male pronuclear formation were higher in porcine oocytes matured in Waymouth MB 752/l, a cysteine rich medium, than in TCM 199 (Yoshida et al., 1992). However, this finding was in contrast to a report by Rose and Bavister (1991) who found bovine oocytes that were matured in Waymouth's medium MB 752/l had a significantly reduced incidence of cleavage and blastocyst development compared to TCM 199. As somatic cells, mammalian oocytes are unable to take up free glutathione. Glutathione is synthesized during maturation from three amino acid precursors, i.e. glutamine, cysteine, and glycine. Therefore, concentrations of glutathione within oocytes depend on the availability of the precursors. Male pronuclear formation rates were improved when compounds like cysteamine or β-mercaptoethanol were added to maturation media. Supplementation of oocyte maturation medium with 0.1 mm cysteamine significantly increased oocyte glutathione content and subsequently improved blastocyst yields (De Matos et al., 1995). Similarly, improvement could be achieved by adding cystine or cysteine as well as glutamine to the IVM medium (De Matos et al., 1996, De Matos and Furnus, 2000). In vitro fertilization of bovine oocytes matured in the presence of 2 mm glutamine resulted in the highest cleavage and blastocyst development rates compared to those matured in medium supplemented with 0, 1, or 3 mm glutamine (Furnus et al.,

25 ). Glutathione has been shown to be synthesized predominantly within cumulus cells and transported into oocytes through gap junctions. Experimentally, the amount of glutathione in cultured oocytes tended to decrease as the concentration of heptanol, a gap junction inhibitor, in the maturation medium was increased. Although there were no differences in the rates of sperm penetration after porcine oocytes were matured in maturation medium with different concentrations of heptanol, proportion of porcine oocytes forming male pronuclei decreased significantly (by comparison to controls without heptanol) at all heptanol concentrations tested (Mori et al., 2000). IN VITRO FERTILIZATION An understanding of sperm capacitation was initiated by Chang (1951) and Austin (1951) who simultaneously observed that spermatozoa must be exposed to the female reproductive environment for a period of time before gaining the ability to penetrate oocytes. The term sperm capacitation was coined to describe the process by which sperm cells achieve the capacity to fertilize (Austin, 1952). This knowledge made efforts to fertilize mammalian oocytes feasible and led to in vitro fertilization (IVF) of rabbit oocytes (Chang, 1959). Efforts to fertilized bovine oocytes in vitro were first successfully in late 1970 s (Shea et al., 1976, Iritani et al., 1977, Brackett et al., 1977). Several approaches have been employed to capacitate bull spermatozoa in vitro. Iritani et al (1977) incubated washed spermatozoa in Kreb s-ringer bicarbonate modified to contain lactate and BSA (4 mg/ml) for h at 37 C prior to expose to ova in vitro. A brief high ionic strength treatment (Brackett and Oliphant, 1975) of bull sperm proved successful with embryos developing to 2-cell stage (Brackett et al., 1977, 1978), 4-cell stage (Brackett et al., 1980), and eventually, birth of live offspring (Brackett et al., 1982). The use of calcium ionophore to capacitate bull spermatozoa (Jiang et al., 1991, 1992) has been useful in several labs.

26 19 Flushings from bovine oviducts revealed a high concentration of glycosaminoglcans (Lee and Ax, 1984). Heparin has been shown to be the most potent glycosaminoglcan in its ability to induce the acrosome reaction of epididymal bull spermatozoa (Handrow et al., 1982). Based on that, Parrish et al (1986) employed tyrode albumin lactate pyruvate (TALP) medium and observed that pretreating cryopreserved bull spermatozoa with heparin (10 µg/ml) for 15 min prior to IVF increased fertilization rates from 40 % to 79 %; more than 70 % fertilization rates were achieved with four different bull semen. To date, heparin is most widely used, and probably is the most effective way currently known for capacitating bull spermatozoa. Other capacitation methods have been reported such as the use of progesterone (Dinkins and Brackett, 2000), cyclodextrins (Choi and Toyoda, 1998, Dinkins and Brackett, 2000), or xanthine-xanthine oxidase (O flaherty et al., 1999) but the results have not surpassed those provided by the use of heparin. Swim-up (Parrish et al., 1985, Keefer et al., 1985) and Percoll density gradient are the two most widely used sperm treatment, usually in conjunction with heparin-induced capacitation. A modified swim-up with the use of hyarulonic acid was shown to provide better results with cryopreserved bull spermatozoa (Shamsuddin et al., 1993). Either two discontinuous layers of 30 % and 45 % Percoll (Utsumi et al., 1991) or 45 % and 90 % Percoll (Saeki et al., 1991) have been used successfully. However, the 45 % and 90 % Percoll density gradient has gained the most popularity. Parrish et al (1995) obtained higher percentages of ova penetrated and cleavage rates but similar percentages of blastocysts using swim-up vs. Percoll for fractionating motile spermatozoa. However, the use of Percoll resulted in almost six-fold greater motile sperm recovery compared to the swim-up method. Several chemicals have been included to improve fertilization rates. Phosphodiesterase inhibitors, caffeine, are commonly used to increase sperm camp levels. Caffeine has been shown to improve sperm motility in poor ejaculates (Critser et al., 1984). Niwa et al (1991) reported a synergic effect of a high level of caffeine (5 mm) with heparin on sperm penetration rates. Lower

27 20 concentrations of caffeine ( µm) have been shown to be ineffective (Coscioni et al., 2001). Numabe et al (2001) obtained similar results when using pentoxifylline (5 mm) in conjunction with heparin as following caffeine plus heparin. Slaweta and Laskowska (1987) showed an improvement by using glutathione (5 mm) during IVF. Kim et al (1999), however, obtained positive effects as well as negative effects with glutathione (1 mm) supplementation of the IVF medium, depending on the bull semen. A PHE mixture, i.e., penicillamine (2 mm), hypotaurine (10 mm), and epinephrine (1 mm) in IVF medium first shown effective in hamster experiments (Leifried and Bavister, 1983) has been commonly used for bovine IVF. Reports involving PHE either show positive effect (Susko-Parrish et al., 1990, Miller et al., 1992) or no effect (Long et al., 1993, Palma et al., 1993) on bovine IVF. Penicillamine alone has been shown to improve IVF results (Keskintepe and Brackett, 1995). The sperm-oocyte ratios for IVF are far greater than those that occur in vivo which are close to 1:1 (Hunter, 1993). A ratio of 5,000 to 10,000 spermatozoa per oocyte are common for bovine IVF systems. A sperm: oocyte ratio below 500:1 significantly reduced cleavage rates (Long et al., 1994, Ward et al., 2002). IN VITRO EMBRYO CULTURE Reduced Oxygen During Embryo Culture The oxygen tension in the mammalian female reproductive tract is lower than in the atmospheric oxygen (Mastroianni and Jones, 1965, Mitchell and Yochim, 1968, Maas et al., 1976, Garris and Mitchell, 1979, Fischer and Bavister, 1993). Reduced tension condition was beneficial for murine embryonic development in vitro (Whitten 1957, Auerbach and Brinster, 1968, Brinster and Troike, 1979). Tervit et al (1972) first reported a low O 2 atmosphere to be beneficial for ovine and bovine embryo culture with birth of lambs following culture of in vivo fertilized embryos (Tervit and Rowson, 1974). The benefit of low O 2 was confirmed by their later work with bovine embryos (Wright et al., 1976). When bovine embryos at 2-to 4-cell and 8-cell stages were cultured in synthetic oviduct fluid previously equilibrated with one of several O 2

28 21 concentrations ranging from 0 to 20 % the proportions of embryos reaching at least the morula stage were higher at 4% and 8 % O 2, compared to those at 20 % or without O 2 (Thompson et al., 1990). Their results confirmed that, under lowered oxygen levels, development of bovine embryos can occur through the 8- to 16-cell block in a simple medium without somatic cell support. Clearly, a reduced O 2 atmosphere from approx 20% in air to 5% positively influences numbers of zygotes developing to the blastocyst stage in ruminants (Batt et al., 1991, Voelkel and Hu, 1992, Liu et al., 1995, Lonergan et al., 1999) as well as for other mammalian species including the rabbit (Li and Foote, 1993), goat (Berthelot and Terqui, 1996), and human (Dumoulin et al., 1999). Culture under a low O 2 atmosphere also eliminates the need for coculture (Xu et al., 1992, Watson et al., 1994, Carolan et al., 1995, Rizos et al., 2001). In contrast, reduced oxygen tension may not benefit the discrete events of bovine oocyte maturation and fertilization. Thus, lower percentages of bovine oocytes reached the metaphase II stage after in vitro maturation under 5% oxygen tension compared to that under 20% oxygen tension (Pinyopummintr and Bavister, 1995, Hashimoto et al., 2000). Similarly, in vitro fertilization rates of bovine in vitro matured oocytes have been shown to be higher at 20% oxygen tension than that of 5% oxygen tension under certain culture conditions (Pinyopummintr and Bavister, 1995, Watson et al., 2000). Inclusion of Amino Acids in Culture Media In 1972, Tervit et al (1972) reported the first somatic cell-free medium formulated for ruminant embryo culture based on the composition of sheep oviductal fluid and named the medium synthetic oviductal fluid (SOF). The use of SOF (supplemented with BSA) failed to reproduce results reported by Tervit et al (1972). Efforts to improve results of embryo culture led to a variety of co-culture systems including cumulus cells (Goto et al., 1988), trophoblastic vesicles (Camous et al., 1984, Heyman et al., 1987), and oviductal cell co-culture or conditioned

29 22 medium (Gandolfi and Moor, 1987, Rexroad and Powell, 1988, Eyestone and First, 1989). However, those approaches might be considered as a draw back in efforts toward developing defined media. Several factors such as the estrous cycle stage from which oviducal tissue was obtained, and the conditioning period of medium must be better defined for repeatable results (Eyestone et al., 1991). More important, exactly how co-culture or conditioned media benefit embryonic development remains unknown. Some investigators have suggested that some peptide growth factors or cytokines were secreted into the medium (Gandolfi et al., 1994, Gandolfi, 1995) while other investigators argued that co-culture and conditioned medium simply reduced some inhibitory components such as high oxygen (Bavister, 1992, Watson et al., 1994) or high glucose (Reiger et al., 1995, Edward et al., 1997). The complex nature of these approaches makes it impossible to identify specific factors critical for embryonic development. The successful use of SOF re-emerged when BSA was replaced with human serum (McLaughlin et al., 1990), still a few steps away from being a defined medium. It was found that the need for serum could be replaced with amino acids. Takahashi and First (1990) reported benefits of adding a pool of amino acids (both essential and non-essential) to embryo culture media (SOF) supplemented with BSA in improving blastocyst development while additions of vitamins had no such effect. Kim et al (1993) also reported the benefit of adding amino acids in chemically defined medium (TALP-PVA). Rosenkrans and First (1994) confirmed these findings with amino acids incorporated into their CR1 culture medium. Interestingly, amino acids have long been known to be abundant in female reproductive fluids (Fahning et al., 1967, Leese et al., 1979, Miller and Schultz, 1987, Casslen, 1987). Why the need for amino acids in culture media was overlooked for so many years is not clear. This may be due to some early findings (Brinster, 1965, Whitten and Biggers, 1968, Cholewa and Whitten, 1970) in which murine 2-cell embryos developed to blastocysts in culture media without amino acid other than those provided by inclusion of BSA. The effects of each individual amino acid on bovine embryonic development has not been extensively investigated. In hamster embryonic development, Bavister and Arlotto

30 23 (1990) found 6 amino acids (phe, val, isoleu, tyr, trp, and arg) to inhibit whereas 3 amino acids (gly, cys, and lys) were found to stimulate embryonic development. Addition of non-essential amino acids has been found to be superior to addition of essential amino acids for bovine embryo culture (Thompson et al., 1992, Gardner et al., 1994, Lui and Foote, 1995, Keskintepe et al., 1995, Pinyopummintr and Bavister, 1996, Lee and Fukui, 1996), especially for the early cleavage stages (Steeves and Gardner 1999). In studies with bovine embryos, uptake of individual amino acids from the culture medium has been reported (Lee and Fukui, 1996, Partridge and Leese, 1996, Jung et al., 1998). Threonine was the only amino acid found to be depleted from medium at all stages of development and glutamine was depleted from the presumptive zygote stage until the 4-cell stage (Partridge and Leese, 1996) whereas alanine was highly increased in the culture media (Lee and Fukui, 1996, Partridge and Leese, 1996, Jung et al., 1998) indicating that alanine was secreted into culture medium. Interestingly, alanine is also the most abundant amino acid found in bovine oviductal and uterine fluid (Elhassan et al., 2001). Metabolism (Reiger et al., 1992) and uptake (Partridge and Leese, 1996) of glutamine was high in 2- and 4-cell embryos. Those findings agreed with the developmental results of Steeves and Gardner (1999) on the beneficial effect of glutamine for early cleavage stages. Energy Substrates for Culture Media Glucose, pyruvate, and lactate are the most common energy substrates present in culture media. Bovine embryos, unlike somatic cells, do not use glucose as a major source of energy substrates. Bovine zygotes were able to develop to the blastocyst stage in a glucose free medium (Holm et al., 1999, Van Langendonckt et al., 1997, Gomez and Diez, 2000, Holm et al., 2002) and also resulted in healthy calves born at a 50% pregnancy rate (Holm et al., 1999). In fact, the plasma-like concentrations of 5-6 mm were found to inhibit early embryonic development, reducing zygotes reaching the morula stage compared to culture without glucose or lower concentrations e.g. 1.5 or 0.5 mm (Takahashi and First, 1990, Kim et al., 1993, Lim et al., 1993,