DEVELOPMENTAL CHARACTERISTICS OF INTERSPECIFIC

A Thesis

Presented to

The Faculty of Graduate Studies of The University of Guelph

bv DAWN A. KELK

In partial fulfilLment of requkements for the degree of Doctor of Philosophy

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Dawn A. Kelk Advisor: University of Guelph, 1997 Dr. W. A. King

Establishment of an capable of development to term involves precisely regdated nudear and cytoplasmic events. Interspeefic hybrids provide unique emb ryo models with morpho logical, b iochemical and temporal markers of development which enable investigation of these interactions. This study explores the feasibility of producing and utilizing interspecific hybrid embryos of , sheep and goats as models to examine the respective roles of the maternd and patemal contributions to the embryo and the interactions of the nucleus and cytoplasm. Interspeafic hybrid embryos were created under in vivo and/or in uitro conditions. Ram spermatozoa penetrated intact bovine oocytes under in oitro conditions, but the resulting embryos arrested at the 4-8-ceil stage. This developmental =est was exhibited even when bovine X ovine hybrid zygotes were transferred to sheep oviducts for in oiuo culture, illustrating that the arrest was not an effect of the in oitro culture. Construction of nuclear transfer hybrids between ovine karyoplasts and bovine cytoplasts resulted in the same pattern of developmentd arrest as in the bovine X ovine hybrid embryos. This indicates that the developmental =est exhibited by the hybrid embryos is not an intragenomic incompatibfity between the bovine and ovine componentç of the nudeus but an incompatibility between nudeus and cytoplasm. Autoradiogaphic detection of 3~-ur~dineincorporation provided evidence for Iow levels of genomic activiv at the 2-ceIl stage of all types of embryos. ki ail but bovine X ovine hybrids, transcriptional activity inaeased through the 4-ceII stage and culminated in a burst of transcriptional activity by the laie &cd stage. Bovine X ovine hybrid embryos exhibited generous transcriptional activïty at the 4ceU stage but no burst of activity as the ernbryos arrested at the 48-cd stage. Ovine X caprine hybrid embryos develop to the stage, hatch in vitro and establish when transferred to sheep or goats. The pregnancies however, have retarded fetal devdopment, incompletely formed placentornes and ultimately fail during the second month of gestation. In summary, most interspecific hybrid embryos can be produced readily under in oioo or in vitro conditions. This study confirms the potential of producing and utilizing interspecific hybrid embryos from domestic as models to study embryonic devdopment. ACKNOWLEDGEMENTS

I wish to thank the following people: Dr. Wh. King for his support, guidance and willingness to help at any time. Man made the duration of this project an enjoyable and rewarding experience through his encouragement and direction. Dr. C.J. Gartley for enduring di of the fnistations and successes of the in vivo aspects of the projed Thanks for maintaining a postitive atmosphere during the times when nothhg seemed to be working and for appreciating my nuv enthusiasm over a few pregnant sheep when things worked wd. The members of my advisory cornmittee; Dr. A. Hahnd and Dr. K. Goodrowe for their advice and assistance throughout the course of this researdi and during preparation of this manuscript.

The staff at the Ponsonby Sheep and Eramosa Research Stations, the Veterinary Teadiing Hospital and the Animal Saence Meat Laboratory for their cooperative assistance. Cathy Britton and Pam Jordan for being partidarly accommodating to my requests. My fellow graduate students from the WAK lab and the department of Biomedical Sciences and all the faculty and staff for help in the laboratory as well as comments and constructive criticisms. Elizabeth St. John for help preparing culture media. United Breeders Inc. for supplying frozen bu11 semen. NSERC, OMAFRA and OGS for their suppoa of the research. FinaUy, 1 wiçh to thank my parents and family for th& constant support. DECLARATION OF WORK PERFORMED

1 declare that with the exception of the items Ïndicated below, ail the work reported in this thesis was perfonned by me. AU laparoscopic and surgical ernbryo transfers and surgical embryo flushes of ewes and does were performed by Cathy Gartley with my assistance. Ultrasound diagnosis of ewe and doe pregnancies was occassionally assisted and confirmed by Cathy Gartley. Bovine ovarïes were collected from the abattoir by Lawrence Afflu. AU media used were made by either myself or Elizabeth St. John. Progesterone assays were performed by Claudia Jiminez. Histological preparations of placentomes were performed by Mary John. Nudear transfer micromanipulations were performed by Marie-Cede Lavoir with my assistance. Photographs were printed by a commercial photo laboratory.

Methods ...... In uivo production of ovine, caprine and hybrid ernb ryoç ...... Experimental animals...... Synchronization and superovulation of sheep and goats .... Breedïng ...... Confirmation of breeding ...... Embryo coIleaion ...... Surgical embryo fi ush ...... Slaughter flush ...... In uitro production of embryos ...... In vitro oocyte maturation ...... Collection of ovaries ...... Isolation of cumulus-oocyte complexes...... Culture of cumulus-oocyte complexes...... Preparation of oocytes for fërtilization...... collection ...... Sperm preparation ...... In vitro fertilization ...... oviduct explant preparation ...... Buffalo rat liver cdpreparation ...... Culture of embryos ...... Fertiliza tion assay ...... Cytogenetic analysis of embryos ...... of in oifro produced bovine X ovine hybrid embryos ...... Experimental animals...... Synchroniza tion of sheep reapients ...... Transfer of embryos...... Recovery of embryos ...... Results ...... Isolation of cumulussocyte complexes...... In vivo production of ovine, cap~eand hybrid embryos ...... Penehation and development of in uïtro produced embryos ... Bovine and ovine crosses ...... *...... Caprine and ovine crosses ...... Discussion...... Isolation of cumulus-oocyte complexes...... 64 Caprine and ovine crosses ...... 65 Bovine and ovine crosses ...... 67

CHAPTER 2: INTERSPECIFIC HYBRID EMBRYO TRANSFERS AND PREGNANCIES...... Introduction...... Methods ...... Production of interspeafic hybrid ernbryos ...... Synchronization of &pient ewes and does ...... Embryo transfer ...... diagnosis and fetal monitoring ...... Progesterone assays ...... Ultrasonographic diagnosis ...... Examination of fetuses and reproductive tracts...... Cytogenetic methods ...... Resdts ...... Discussion...... Utrasound ...... Hybrid fetus ...... Placentornes ...... ,......

CHAPTER 3: RNA AND PROTEIN SYNTHESIS IN INTERSPEClFlC HYBRID EMBRYOS...... Introduction ...... Me thods ...... Embryo production...... Radiolabeling of RNA ...... Embryo fixation ...... Autoradiopphy ...... Radiolabelhg of proteins ...... Scintillation counting of embryo proteins ...... Electrophoresis ...... Resdts ...... RNA transcnphon...... Protein translation ...... Discussion ...... 117 .. RNA transcnption ...... 117 Protein translation...... 118

CHAPTER4: DEVELOPMENT OF BOVINE X OVINE NUCLEAR TRGNSFER EMBRYOS ...... *...... *...... Inboduction ...... Methods ...... btrumentation ...... Nuclear tramfer ...... ,...... Preparation of cytoplasts...... Preparation of donor blastomeres ...... * Reconstitution and fusion ...... Culture of nudear transfer embryos...... *...... Results ...... Discussion......

GENERAL DISCUSSION ...... 0....*...... 131 Bovine X ovine hybrid embryos ...... *...*...... 131 Hybrids of sheep and goab ...... 134

SUMMARY AND CONCLUSIONS ...... *...... *...... 139

APPENDIX k List of Media and Solutions Used ...... 166

APPENDIX II: List of Materials and Supplies ...... 170 LIST OF TABLES

Features of in cattle. sheep and goats ...... cc.... 25

Summary of embryo collections in ewes and does bred to rams or bucks ...... 58

Summary of penetration. cleavage and development rates . * of rn mtro produced embryos...... 59

Timing of developmental events in bovine. ovine and caprine embryogenesis ...... 100

vii LIST OF HGURES

IVF of a bovine oocyte with ram spm......

Bovine X ovine IVF hybrid &ceIl embryo......

Bovine and bovine X ovine hybrid IVM/TVF embryos following in oioo culture in sheep oviducts......

Pronuclei of an ovine X caprine hybrid zygote ......

A hatching. in vitro produced ovine X caprine blastocyst......

6 . Caprine. caprine X ovine. ovine X caprine and ovine ul trasonographs ......

7. A day 51 ovine X caprine hybrid fetus and utenis of reapient goat......

8. Chromosomes horn amniocentesis of a day 51 ovine X caprine hybrid fetus ......

9 . Photographs of normal sheep pregnancies at 41. 44 and 47 days gestation ......

10 . Photographs of ovine X caprine hybrid pregnancies following fetal death......

11 . Crown-rump lengths of normal control sheep fetuses and ovine X caprine hybrid fehws...... 86

12. Histological sections of placentornes from normal sheep gestations at 41. 44 and 47 days...... 87

v iii Histological sections of placentornes from ovine X caprine hybrid fetuses at the tirne of fetal death...... cc... 89

Autoradiographs of in uïuo produced 2-, 4- and &ceU caprine, caprine X ovine hybrid and ovine ernbryos following 8 hours of incubation with 3~-uridine...... 107

Autoradiographs of in uit-ro produced Z, 4- and &cd bovine, bovine X ovine hybrid and ovine embryos following 8 hours of incubation with 3~-uridine...... 108

Autoradiograph of SDS-PAGE of cellular proteins from in vitro matwed bovine, ovine and caprine oocytes...... 111

Autoradiograph of SDS-PAGE of cellular proteins from in uitro produced bovine, bovine X ovine hybrid and ovine 2- and 4-ce11 embryos, labeled at 40 hpi...... 112

Autoradiograph of SDS-PAGE of cellular proteins from in vitro produced bovine and bovine X ovine hybrïd 2-, 4 and &ce11 embryos, labeied at 58 hpi ...... 114

Autoradiograph of SDS-PAGE of cellular proteins from in uitro produced caprine, caprine X ovine hybrid, ovine X caprine hybnd and ovine 4cdembryos, labeled at 48 hpi ...... 115

Autoradiograph of SDS-PAGE of cellular proteins from in vitro produced caprine X ovine hybrid embryos, labeled at 32, 40 and 48 hpi...... 116

Bovine and bovine < ovine hybrid nudear transfer embryos.. 125

Histogram of rates of developmental arrest at 20, 4, and &cd stage for bovine, ovine and bovine X ovine in uitro fertilized and nudear transfer embryos ...... 126 LIST OF ABBREVLATIONS

Menezo's 82 medium nudear transfer of ovine nudeus to bovine cytoplast

bovine oviduct epithelid c& bovine protein-1 carbon dioxide cumulus-oocyte complex CRL aown-rump length DNA deoxyribonudeic aad ECS estrous cow serum FCS fetal calf serum FSH foKcle stimulating hormone F.S.H.-P.@ follide stimulating hormone-pituitary h hour hpi hours post-insemination ICM IU international units NC in vitro culture IVF in uif ro fertiliza tion IVM in vitro maturation kiloDalton leukemia uihibitory factor medroxyprogesterone acetate sponge messenger ribonudeic aud nudear transfer ovine trophob last pro tein-1 PBS ~ecco'sphosphate buffered saline PBSS PBS supplemented with bovine sem PMSG pregnant mares senun gonadotrophin RNA ribonucleic acid SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis SS steer serum TALP tyrodes albumin lactate pynivate medium

V/V volume per volume w/v weight pet volume INTRODUCTION

The study of early embryo~cdevdopment of domestic animak is of importance for refining new reproductive technologies for humans, domestic and wildlife speaes. Selection of breeding stock based on the success of

produchg large numbers of offspring by embryo transfer techniques, in uih.0 fertilization and embryo doning, is becoming increasingly important to

agriculture. Effective use of these tediniques requires understanding of

embryo development. Sirice embryos of domestic animals cm provide relevant information about human embryos as well as those of rare and endangered species, investigations in the area of assisted reproduction currently use domestic anirnals to perkct techniques for IVM and ni?: to salvage germ ce& from rare and endangered animalç otherwise unable to reproduce. The similarity in development of bovine and human embryos has been pohted out by a number of researchers. Bovine and ovine embryos are likely to provide a more suitable mode1 for humans than traditional laboratory species. Sirnilarities between human and bovine embryos indude the timing of the omet of embryonic genome activation and the transit time of the embryo through the female reproductive tract. Additionally, the similarity in size of various embryos used as models is an essential factor for developing cryopreservation techniques. Therefore, an understanding of the developmental parameters of embryos of domestic species may have relevance for human IVF programs. The low efficiency of nuclear transfer, a potentiaily powerful technology, is impeduig its development and use. The establishment of an embryo capable of development to term involves precisely regulated nudear and cytoplasmic events prior to fertikation which continue throughout gestation. Failtue of either component or failure of their interaction will lead to abnormal development and/or embryo death. For this reason, understanding the parental components of the ernbryo~cgenome and their activation will provide insight into embryo loss and reproductive failure. The relationship between the cytoplasm and nucleus and the extent to which the genetic background of each affects the sequence of events leading to hinctional genomic activity may be explored through the examination of the development of uiterspeafic hybrid embryos. Interspecific hybrids provide unique embryo models with distinct morphological, biochemical and temporal markers of development that can readily be examined in an in vitro system. To date, there is no efficient mode1 to examine the interaction and contribution of the nucleus and cytoplasm to the early embryo. Although nudear transfer can be used to constnrct desired embryos, the procedures used are currently too inefficient and costly to provide large numbers of embryos for study. In a previous investigation (Kek, 1992), attempts were made to produce interspecific hybrid emb ryos. The curent investigation involves continued refinement of production of these previously studied hybrid embryos as well as newly produced crosses. These interspecific hybrid embryos were then utilized as models for early embryorüc development and compared with control (non-hybrid) embryos. More specifically, autoradiography and polyacrylamide gel electrophoresis were utilized to examine transcriptional and handational activity within the embryos. In addition, the development of one type of interspeufic hybrid (bovine X ovine) was compared to nuclear transfer bovine cytoplasts fused to ovine karyoplasts to Merexamine the nudear and cytoplasmic interactions. Reproductive Biology of Cattle, Sheep and Goats

The domestic ruminants, cattie (Bos taurus), sheep (Ouis nries) and goats (Capra hircus) have many similar reproductive characteristics but Vary in subtle yet important aspects. TEiese differences profoundly affect the application of reproductive technologies such as artificial insemination, embryo transfer, in vitro fertilization and nudear transfer in each of these speaes.

The estrous cycle involves a complex interaction of gonadotrophins and steroid hormones which hction together with ovarian and uterine factors to control follidogenesis, sexuaI behaviour and ovulation (Dieleman et al., 1983; Walters et al., 1984; Ireland and Roche, 1987). Estrus, a period lasting between 18 and 48 hours for most domestic ruminants, in which the fernale will dow mating, begins several hours before ovulation (Hanse1 and Echternkamp, 1972; Larsson, 1987). The first day of behavioural esms, generaily designated as day O of the estrous cycle, is also used as a reference for gestationai age of an embryo or fetus. The average length of the estrous cycle reported for ewes is 16.5 days in contrast to 21 days for cattle and goats, although individu& of each species may range I two days of the reported means (Hafez, 1993; Swenson, 1977; Arthur et al., 2989).

At esm, cattle generdy odate a single oocyte while sheep and goats are more likely to ovulate two or three ova (Hafez, 1993). The length of gestation is similar for sheep and goats (143 to 151 days, depending on breed), while the gestation length for cattle is much longer (approxirnately 280 days), (Hafez, 1993). There is also variation between species in the endocruiology and recognition of pregnancy. Cattle, sheep and goats share similar yet uniquely different signals and endocrine patterns throughout pregnancy. The timing of similar events or signals often varies between these three species (Mossman, 1987). A critical aspect of matemal recognition of pregnancy in sheep, cattle and goats involves physiological mechanisms that result in the protection of corpora lutea from luteolysis (Bazer et al., 1991). The conceptuses of each of these three speties produce the protein interferon-tau (INF-t) (Bazer et al., 1994). Intrauterine injection of a recombinant ovine ENF-t bas been shown to extend the interestrous interval and maintain a functional corpora lutea in sheep (Ott et al., 1993). This protein (NF-t) was formerly referred to as trophoblast protein-1 and functions as an antiluteolysin (Bazer et al., 1991). The production of ovine trophoblast protein-1 (oTP-l), begins at day 13 of pregnancy (Godkin et al., 1982), whereas the corresponding caprine protein is not produced until day 16 (Gnatek et ai., 1989). Ovine trophoblast protein-1 is the main secretory product of the day 13 to 21 ovine conceptus (Godkin et al., 1982) while major synthesis of the corresponding bovine pro tein (bTP-1) is initiated around day 15 but may be present as late as day 36 (Godkin et al., 1982). Production of bTP-1 and oTP-I in bovine and ovine species respectively, is reported to be triggered at the time the blastocyst of each species begins to elongate (Farin et al., 1990). In sheep, oTP-1 has been shown to control synthesis of some endometrial proteins (Vallet et ai., 1987). The maintenance of pregnancy in sheep and goats depends on a functional corpus Iuteum during the first month. By 50 to 60 days, the placenta becomes the prirnary source of progesterone for the sheep while in the goat, placental progesterone is either not produced or is not sufficient to maintain a pregnancy. Therefore a hctional corpus luteum remains essentid to maintain the goat pregnancy (Robertson, 1977; Hafez, 1993). A phenornenon uniquely characteristic of does is pseudopregnancy or hydrometra. Pseudopregnancy, which may occur whether does are mated or not, is charaderized by aseptic fluid accumulation in utero and a persistent corpus luteum (Hesselink, 1993; Fielden, 1984; Swenson, 1977; Smith, 1986). The Buid may be clear or doudy, but the condition is not generally due to infection (Smith, 1986). Pseudopregnancy may be longer or shorter than the

normal five month gestation period. At the end of pseudopregnancy, ute~e fluids are frequently discharged vagindy and the animal rnay lactate (Smith, 1986; Swenson, 1977). Progesterone is elevated and administration of prostaglandin F2-a terminates the pseudopregnancy (Smith, 1986). The

incidence of pseudopregnancy in some herds of dairy goats may reach 20.8% but on average is estimaied to be approximately 9% (Hessehk, 1993).

Ooeenesis and Fertiliza tion The overd struct~~dcharacteristics of the early embryo are similar in most mammals although there is considerable variation between species in the size of the oocyte, raie of deavage and Iength of the pre-attachment period (Betteridge, 1977). Early work on the genetic and biochemicd aspects of embryo development centred around mice and rabbits. With the advent of embryo manipulation, information on developmental aspects is being generated on speües of agridtural importance such as pigs, cattle and sheep (Kidder, 1992; 1993). Evidence from examination of early cleavage stage cattle, sheep and goat embryos indicates that there are inherent differences beginning in oogenesis between these three species. Early observations that enucleated amphihian ova could undergo deavage led to the concept that matemal products in the cytoplasm could control early deavage development (for review see: Telford et al., 1990). Stores of RNAs and proteins were later discovered in unfeftilized oocytes, confirming the theory that matemd gene products were present in the ooplasm (TeLford et aI, 1990). The nature and function of mRNA moIecdes in the devdoping oocyte have been examined in species such as Xenopus but are not well explored in domestic speaes (Benbow and Ford, 1975; Woodland and Adamson, 1977). Some mRNA molecules (Vgl mRNA in Xenopus) have been found homogeneously distributed with the ooplasm but, by Iater stages, when oocyte maturation was nearly complete, they were sequestered to the vegetal cortex (Yisraeli and Melton, 1988). In addition, when Vgl mRNA was

injected into eggs, it localized app rop rïately, indicating tha t the mRNA itself contains the requisite information for Localization (Yisraeli and Melton, 1988).

Other mRNA molecules, such as those for fibronectin, remain essentially homogeneously distributed throughout the ooplasm (Wilkins, 1990). The early development of rnarnmalian embryos also proved to be regulated through proteins and mRNAs produced in the oocyte pnor to fertiiization. In the process of rnammalian oogenesis, populations of materna1 RNA and protein are acçumulated within the ooplasm as the oocyte grows and matures. When a Wly grown oocyte is hormonally stimulated, RNA synthesis stops, the germinal vesicle breaks down and the oocyte is arrested at meiotic metaphase II. During meiotic maturation, matemal mRNAs are deadenylated, adenylated and degraded wulting in a net loss of approximatdy 30% of the polyadenylated mRNA pool between a Mygrown mouse oocyte and an ovulated oocyte (Telford et al., 1990). The events of fertdkation are similar in the three speaes. Fertilization in uioo takes place within the oviduct at the ampdary-isthmic junction. Capaatated spermatozoa bind to the zona pelIucida of the oocyte where receptors in the spermatozoon plasma membrane and proteins of the zona pelluàda interact to induce the acrosome reaction (Florman and First, 1988a; 1988b). After approximately 4 h of sperm-oocyte contact. an acrosome- reacted spermatozoa penetrates the zona pellucida, binds to the vitelline membrane and passes into the ooplasm (Xu and Greve, 1988; Xu and King, 1990). Although the process of fertilization foUows a similar sequence of events in all three species, there must also be some molecules or receptors which are unique for each speties. Several experimentç indicate that it is not possible in most cases to adueve cross fertilization under in oioo conditions. Early studies with goats and sheep found that although fertilization of goat oocytes readily occurred after breeding with a ram, sheep oocytes were not penetrated by buck sperm following mating (Warwick and Berry, 1949; Bowerman and Hancock, 1963). Further s tudies demonstra ted that goat sperm reached the expected site of fertilization but did not penetrate sheep oocytes (Hancodc and McGovem, 1968). A similar lack of fertiluation has been reported in cattle inseminated with ram sperm. In this case, 77 uncleaved oocytes were recovered kom five cows inseminated with ram semen whereas 45.4% of cattle oocytes were penetrated by ram sperm in nitro (Kek et al., 1991a; Kek, 1992). These studies indicate that the mechanisrn of capacitation or penetration must indude a species-specific factor whidi may be overcome under in vitro conditions. Embrvonic Develo~ment Following penetration of the spermatozoon into the ooplasm, each set of patemal and maternai chromosomes decondenses, forming the male and female pronuciei which move into apposition within 19 hours. The first deavage to the 2-cd stage occurs by 24 h post-penetration (Hyttel et al., 1988). The bovine zygote undergoes four ceil cycles as it moves through the oviduct to the uterus (Newcornb et al., 1976). The developing 16-ceU embryo passes through the utero-tubal junction into the uterus on the fïfth day of the estrous cyde (Moore, 1975; Newcomb et al., 1976)- Embryos of this stage are referred to as modae. During the Fifth and sixth ceU cycles, the celis become tightiy apposed as the embryo undergoes compaction (Betteridg and Hechon, 1988; Plante and King, 1994). Compaction is followed by the formation of a fluid-filled cavity (the blastocoele) within the embryo, which denotes the transformation from compact morula to the blastocyst stage. The blastocyst continues to expand and between days 8 and 11 post-estrus, it hatches out of the zona peilucida and begins to elongate (Betteridge and Fiechon, 1988). During the pre-attachent penod of up to 19 days, the developing bovine embryo secretes proteins which interact with the uterine to signal the maternai system that the embryo is present (Thatcher et ai., 1989). Upon recognition of pregnancy, the maternai systern continues to secrete progesterone, interrupts the estrous cycle and maintains an embryotrophic environment within the uterus. Protein signals from day 15 to day 18 embryos appear to ad in luteai maintenance by regulating uterine production of prostaglandins (Thatcher et al., 1989); but it is not known if embryonic factors influence the materna1 system prior to this. The trophectoderm attaches to the endometrium between 19 and 27 days in cattle (King et al., 1982). For cattle, as with many species, pre-attachment development constitutes the period of highest mortality with approximately 25 to 30% of bovine zygotes being lost in this period (King, 1991).

RNA Stores and Transcription Embryogeneçis begins after fertiiization, the event which triggers development following meiotic arrest. The earliest stages of embryogenesis are therefore regulated by matemdy inherited components within the oocyte (Dworkh and Dworkin-Rastl, 1990; Eyestone and First, 1991). This control of early embryo~cdevelopment is often referred to as the post-transaiptional control or materna1 conbol per-iod (Eyestone and First, 1991). As matemally derived molecdes decay, embryogenesis becomes dependent on expression of the embryo~cgenome (BiIodeau-Goeseels and Schultz, 1997). In the case of the mouse, 30 to 40% of matemal RNA and up to 70% of poly-(A)-containing mRNA are degraded by the 2-ceU stage (Clegg and Piko, 1983). There are indications., however, that certain proteins are still translated from matemal mRNA at the 4- to &ceIl stage and that some matemal mRNA synthesized during oogenesis remains stable up to the blastocyst stage (Petzoldt, 1990). Termination of the maternal control period is marked by initiation transcription of the embryo~cgenome or transition to embryonic control development. The timing of this event has been examined in a variety ways and varies between cattle, sheep and goats. The first and second cleavage divisions of the bovine embryo proceed without cellular growth, and reportedly under the sole control of stored maternal mRNA (Bames and First, 1991). Initial transcription of the embryo~cgenome was reported to occur during the t.dce11 cycle (Camous et ai., 1986; Kopecny et al., 1989; Barnes and First, 1991), with full activation occurring within the fourth cell cycle (King et al., 1988; Bames and First, 1991). It was later dernonstrated that a low level of transaiptional activity occurred as early as the 2-celI stage in bovine embryos (Viuff et al., 1992; Plante et al., 1994; Hyttel et al., 1996). Although goats and sheep have not been examined as rigorously as cattle, initial reports Ïndicated that the sheep embryo~cgenome becomes activated around the 8-16 cdstage while the goat genome is thought to be activated at the 2-cell stage (Telford et al., 1990; Pivko et al., 1995). More recently, it has been demonstrated by 3~-uridineincorporation that a low level of transaiptional activity may occur as early as the 2-cd stage in both sheep and goat embryos. Transcription levels inaease gradually through the kelI and early 8-cell stages with a drarnatic burst of activity during the û-cell stage (KeIk et al., 1994b). Several methods have been used to examine the transition from matemal to embryonic control of development. Actinomych D and the mushroom toxin a-amanitin inhibit RNA synthesis and have been used to examine timing of embryonic genome expression in a number of species including mouse (Levey et al., 1978; Braude et al., 1979; Flach et al., 1982), rabbit (Manes, 1973) and human (Braude et al., 1988). Developmental arrest in the presence of a-amanitin indicates the time at which products from the embryonic genome become essential for further development of the embryo. The presence of a-amanitin does not affect deavage or protein synthesis during the first three cell cycles of sheep embryos but blocks cleavage and protein synthesis thereafter (Crosby et al., 1988). In sheep, few embryos (8.3%) can proceed beyond the 8-ce11 stage when cultured in the presence of a- amanitin (Crosby et d., 1988). If however, sheep embryos are cultured in the presence of a-amanitin beginning at the 8-ceU stage, 95.7% will develop beyond the 8-ceU stage (Crosby et al., 1988). Bovine embryos also readily develop to the û- to 16-cell stage in the presence of a-amanitin (Barnes and First, 1991; Plante et al-, 1994). Although the two inhibitors exert sirnilar effects on transcription, they display distinctly different effects on development. Actinomycin D causes inunediate arrest of cleavage in the rabbit embryo while a-amanitin pennits the omet of cleavage (Manes, 1973). EIectron microscopie examination of the nudeolus has also been used to study the onset of activation of the embryonic genome- The nucleolus reflects the physiological state of the ce11 through alteration of its conformation according to the transcriptional activity. The form of the nucleolus depends on the ce11 cycle stage, state of differentiation, tissue type and pathological condition of the ce11 (Goessens, 1984)- There are three nucleolar components wtiich have been disthguished: 1) the granular component, consisting of distinct, nearly spherical granula about 15nm in diameter; 2) the fibrillar centres, consisting of a loose network of fibrils which are 4 to 8nm thick; and 3) the dense fibrillar component composed of tightly packed 3 to 5nm thick fibrils (Goessens, 1984). ïhese three components take on a variety of conformations which reflect the transcriptional activity of the cell (Goessens, 1984; Jordan, 1984). Four nucleolar conformations have been recognized and classed as: 1) compact, 2) reticulated, 3) segregated and 4) resting. Compact and retidated nucleoli are associated with actively trançcribing ceils, while segregated or resting nucleoli are found in quiescent cells. Reticulated nucleoli are generally observed in cancer cells, while normal, actively transcribing cells exhibit many large, darkly staining compact nucleoli (Goessens, 1984; Jordan, 1984). Small ring-shaped nucleoli have also been observed (Schwarzacher and Wachtler, 1983). These ~g-shapednucleoii consist of a small fibrillar centre, surrounded by dense fibrillar material and a small granular component. The ~g-shapednucleolus is indicative of a cell with low transcrip tional activity (Schwarzacher and Wachtler, 1983). In the case of the preimplantation embryo, nudeologenesis tends to go through charaderistic conformational changes, from a quiescent "nucleolar precursor body", progressing to a ring-shaped and finally to fully active nucleoli as the embryonic genome is activated. However, timing of occurrence of these characteristic nudeolar forms varies according to speaes, as does the timing of embryonic genome expression. Nucleolar transformation occurs at the 2-ceil stage in the goat (Chartrain et al., 1987), the 2-cell stage in cattle (King et al., 1989; Plante and King, 1994) and the &cd stage in sheep (Calarco and McLaren, 1976). An understanding of the regdation of the switch from matemal to embryonic control of development may prove significant, not only for the production of embryos in nitro and for the refinement of embryo transfer methods, but also for an understanding of early development of interspeafic hybrids.

Protein Stores and Translation

As with mRNA, proteins are also stored in the ooplasm prior to fertilization and are utilized in a similar manner during the eady deavage stages. One example is the stored pools of histones, present at concentrations adequate to form nucleosomes for 10 diploid nuclei in mouse (Wassarman and Mrozak, 1981) and 20,000 diploid nudei in Xenopus (Woodland and Adamson, 1977). The storage of histones is necessary because sperm Hl histone is completely replaced by a cleavage stage histone Hl variant immediately after sperm penetration of the ooplasm (Poccia et a1.,1981). In addition to histones, oocytes synthesize a wide range of proteins induding mitochondrial and ribosomal proteins, actin, tubulin, calmodulin, lactate dehydrogenase, creatùie kinase and glucose-6-phosphate dehydrogenase (Wassarman, 1988). The absolute rates of protein synthesis in mammaIian oocytes deaease by about 30% duruig meiotic maturation, as the amount of RNA in the oocyte dedines by 20% (Schultz et al., 1978). The rnajority of changes in protein synthesis are quantitative in growing oocytes but qualitative changes have been obsewed in both mouse (Sdiultz et al., 1979; 1990) and pig (McGaughey et al., 1979) oocytes as they gain meiotic cornpetence, Qualitative changes in protein synthesis have also been shown during or after GVBD (germinal veside breakdown) in maturing oocytes of cattle (Kastrop et al., 1990), sheep (Moor et al., 1981) and goat (LeGd et a1.,1992). Qualitative changes in protein synthesis continue af ter fertilization. In a cornparison of rodent species, (mouse, rat, hamster and gerbil) kom the 1- cell stage through to the blastocyst stage qualitative changes were obsenred. In addition, it was noted that at the 1-ceii stage the four speties exhibited marked species differences in protein profiles but remarkably similar profdes at the blastocyst stage (Norris, et al., 1985). Both qualitative and quantitative dianges in protein synthesis have been observed by the 8-16-cell stage in cattle (Frei, et ai., 1989). These unique quantitative and qualitative differences between species provide useful markers for the examination of interspeac hybrid embryos.

Im~rinting Imprinting is defined as the differential expression of matemally or paternaliy derived genes (Steenman et al., 1994). In the case of interspedfic hybrid embryo development, it is conceivable that irnprinting of a particular gene could prove Iethal to the embryo if it were necessary for the gene product to hction cooperatively with an altemately irnprinted gene. The difference in the activity between matemal and paternai alleles of imprinted genes is the result of gene modification (DeGroot and Hochberg, 1993). A paterndy inherited gene in one generation can be a matemdy ùiherited gene in the following generation and vice versa (DeGroot and Hochberg, 1993). Imprinting is therefore not merdy a resdt of differences in nudeotide sequence between the two deles but a modification which cm be erased and reprogrammed when passed from one generation to the next. MethyIation of DNA was previously recognized as an epigenetic DNA modification which could change the biological activity of the DNA (Shemer et al., 1996). This is one mechanism which cm moddate transcriptional activity of a gene in a tissue- or differentiation-spellfic manner (Cedar, 1988). Methylation may be one of perhaps several methods utilized to accomplish genomic imprinting. Although both parental sexes contribute equivalent nudear genetic information to the zygote, the information is not necessarily funaionally equivalent (Barton et al., 1984; Cattanach and Kirk, 1985). In kt, numerous cases have Uustrated that matemally and patemdy derived genomes are not FunctionaUy equivalent in a broad range of developmental processes. The initial understanding of imprinting came from observations that diploid parthenotes and hydatiform mores, whidi possess two materna1 or patemal genomes respectively, are not viable (Markert, 1982; Kajii and Ohama, 1977). Construction of androgenetic and gynogenetic mouse embryos demonsbated a deficiency in the paterndy-derived genome in development of normal embryonic structures and a deficiency in the maternaily-derived genome in produdion of normai placenta (Surani, 1986). It was also realized through observations of mouse zygotes with materna1 or paternal disomy for individual chromosomes, that not aIl of the genome is involved in these parental effects (Cattanach and Kirk, 1985). DiEferential activity of materndy and paternaiiy derived chromosome regions has been demonstrated in mice (Cattanach and Kirk, 1985; Ferguson-Smith et al., 1992). More recent studies have begun to elucidate imprinting at the Ievel of specific genes (Barlow et ai,, 1991; Bartolomei et al., 1991; DeChiara et al., 1991; Rappolee et al., 1992). Inappropriate imp~tingof specific genes can result in inviability of developing embryos or altemativeiy, diseased or deficient states throughout development. An example of this is the association between the Ioss of imprinting of the insulin-like growth factor-II gene and Wilms' tumour (Steenman et al., 1994). The selective expression of maternally- and patemally-derived genomes is recognized as a fundamental mechanism controiling mammalian biology. The importance of genomic imprinting in implantation and placental proliferation and invasiveness is one such example (DeGroot and Hochberg, 1993; Goshen et al., 1994). In addition, results from an examination of the DDK mouse strain suggest that differential imprinting of parental genomes is involved and is a prerequisite for normal blastocyst development (Babinet et al., 1990). The DDK mouse mode1 has characterized imprinting at the level of an incompatibiiity between the nuclear and cytoplasmic cornponents of the early embryo. A differential expression between paternal and maternal genomes was revealed by the cytoplasm of DDK embryos when a letha1 effect was manifested in the presence of a patemal gertome from a different strain of mouse. This lethal effect was not observed in the presence of a maternal genome in the correspondhg situation (Babinet et al., 1990). In Vitro Production of Embryoa Since the production of the first calf by in oitro fertilization in 1981 (Bradcett et al., 1982), ruminant ofkpring have been produced as a result of in uitro fertilization in both sheep (Kelk et al., 1992) and goats (Yuunis et al., 1991). In uibo production of ernbryos has allowed rapid advancement of knowledge of early embryonic development by providing large numbers of embryos from oocytes of slaughtered animals. The in vitro system aiso allows examination of the precise timing of developmental events such as spermatozoon penetration, male pronudeus formation and initial deavage (Xu and Greve, 1988). In oitm produced embryos appear to be a suitable mode1 for in vivo produced embryos in a number of respects. It has been shown that in uitro and in aioo produced oocytes and embryos have similar types and frequencies of chromosomal abnormalities (King, persona1 communication). The greater accessibility afforded by the in vitro system has allowed be tter resolution of developrnental events and their consequences. For example, studies have shown that as the intemal from insemination to first cleavage increases, viability drops and the incidence of chromosomal abnormalities increases suggesting that there is a wuidow which dows for selection of optimum development (King, 1991). The influence of chromosomaI abnormalities on bovine embryo development has also been documented (Kawarsky et al., 1996). It has also been noted that male embryos develop more rapidly than females during the first week of culture, suggesting a sex related early expression of genes that regdate development (Avery et al., 1991,1992; King, 1991; Xu et al., 1992b; Bredbacka and Bredbadca, 1996). Similarly, findings that the viability of repeatedly manipulated embryos may also be under the control of sex-dependent or sex-influenced genes, substantiates the concept of early expression of regdatory genes (King et al., 1992). The development of an in vitro production system for embryos of each species involved a stepwise process, overcoming barriers to in vitro maturation, fertikation and culture, independently. The first attempts at in vitro production of bovine embryos (Brackett et al., 1982) involved in nitro fertilization of in oiuo matured oocytes obtained at laparotomy although in vitro maturation foUowed by in uiuo fertilization in cattle had already proven successftd (Newcomb et al., 1978). The technique of obtaining in vivo matured oocytes is Limited by the number of donors available as well as by the time, cost and labour required. Subsequent bovine studies, successfully combined in nitro maturation and fertilization after obtauiing material from slaughter (Critser et al., 1986; Xu et al., 1987; First and Pa-rrish, 1987). In vitro maturation and fertilization was also extended to the dornestic as a model for exotic wild cab (Johnston et al., 1989). The technique of in oitro maturation continues to be refined through

studies examining the effects of gonadotropins, semm and amino acids on nuclear maturation, cumulus expansion and oocyte morphology (Kito and Bavister, 1997). Human in ~ibomaturation of slow or late-maturing and aged oocytes also continues to be explored (Saunders et al, 1997; Kek et al., 1997b). It has recently been demonstrated that bovine oocytes in which meiotic resumption has been inhibited for 24 hours by 6-methylaminopuMe (6-DMAP) or cyclohexamide, remain competent of development to the blastocyst stage (Lonergan et al., 1997). The final barrier to complete in vitro production of bovine embryos was that of culture of the zygote through the early deavage stages. Bovine embryos, cdtured under standard in vitro culture conditions arrest at the & celI stage (Eyestone and First, 1991). Initial studies used temporary recipientç such as the sheep (Eyestone et al., 1987) or rabbit (Sirard et al., 1985; Xu et al, 1988) oviduct to culture bovine embryos from zygote to blastocyst. Eventually, in vitro culture systems for bovine embryos were designed which induded CO-culture of embryos with trophoblastic vesides (Camous et al.,

1984) or oviduct ce& (Eyestone and First, 1989; Ellington et al., 1990a; 1990b) and finally culture in defined media (Bavister et al., 1992; 1995). The development of an in oiho system for producing ovine embryos, although lagging somewhat behind that for the bovine, has undergone a similar evolution. Lambs were fkst produced by in oiho maturation and fertiIization followed by surgical transfer to the oviducts of pseudopregnant rabbits (Crozet et al., 1987). Ultimately, lambs were produced following in vitro maturation, fertilization and coculture with oviductal cells (Czlonkowska et al., 1991; Kek et al., 1992). The development of an in vitro system for producing goat embryos has, and still is undergohg a similar evohtion to that of the bovine and ovine systems (Tarusharma et al., 1996). The initial report of full term development in the goat involved use of in uiuo matured oocytes (Hanada,

1985). The first to document pregnancy by embryo hansfer of 2- and 4cell embryos after in vitro maturation and fertilization of goat oocytes, was Younis et al. in 1991. Although no üve offspring were produced from this study, two reupient does resorbed their pregnancy at approximately two months gestation, while a third recipient doe aborted a 3.5 month fetus. The in vitro fertilization rates of in uiuo and in uiho matured goat oocytes have been compared and have proven similar (De Smedt et al., 1992). In the latter study, although there was no report of attempts at in oiho culture of embryos, blastoqsts were recovered after transfer of in oiho matured and fertilized oocytes to sheep oviducts. In nitro maturation, fertilization and culture affords unique advantages such as the production of some interspecific hybrids which do not occur in oivo. For example, the in oiho method dows penetration of intact bovine ova with ram spermatozoa (Slavik et al., 1990; 1997; Kek et al., 1991a; 1996). The preimplantation interspecific hybrid embryo formed by fertilization of a bovine oocyte with ram spermatozoa under in vitro conditions has been confirmed through cytogenetic analysis (Kelk et al., 1991a). The formation of this hybrid appears to be an in oitro phenornenon since no fertilization was observed under in oivo conditions (Kelk et al., 1991a). In vitro fertilization of sheep and cattle oocytes by goat sperm has ais0 been accomplished (Slavik and Fulka, 1992; Cox et al., 1994). Subsequent embryonic development and establishment of pregnancy folIowing in oiho maturation, fertilization and culture of sheep oocytes penetrated by goat sperm has also been demonstrated (Kek et al., 1994a) These findings indicate that in vitro maturation, fertilization and culture can be used as a powerfui tool in the production of some interspecific hybrids.

Nuclear Transfer Nuclear transfer is a potentially powerful technique/tool for the production of geneticalIy identical individuals, involving the fusion of a single nucleated blastomere or somatic cell (karyoplast) with an enudeated oocyte (cytoplast) (Willadsen, 1986; Wilmut et al., 1997). Therefore it is theoretically possible to create an infinite number of genetically identical offspring from a single embryo or individuai. The production of genetically identical offspring would be usefui for the study of fundamental aspects of development and to provide controlled populations of animais with Little to no individual variation for saentific experimentation. Unfortuna tely, the reality of nudear transfer is that the efficiency of production of live offspring is too low for the technique to be commeraally viable. The technique is labour intensive and the development of hsed bovine embryos to the blastocyst stage is lower, averaging approximately 10% cornpared to greater than 35% following normal fertilization (Westhusin et aL, 1992; Barnes et al., 1993a; 1993b; vanStekelenburg-Harners et al., 1993; Yang et al., 1993; Collas and Barnes, 1994; Campbell et al., 1996; Wilmut et al., 1997). Subsequent pregnancy rates after embryo transfer Vary from 8% to 50%, while 6% to 770h of these pregnancies result in spontaneous abortion (Willadsen, 1989; Bondioli et al., 1990; Willadsen et al., 1991; Westhusin et al., 1992; Yang et al., 1993; Heyman et al., 1994; Whutet al., 1997). Therefore only 1.6% to 5.3% of fused embryos achieve full term development. The success of the technique is mercomplicated by the observation that the gestation of nudear transfer fetuses may be prolonged and the calves cmbe larger than normal, frequently leading to dystocia (Wiuadsen et al, 1991; Keefer et al, 1994; Wilson et al, 1995; Walker et al., 1996). Aithough large birth weights of nuclear transfer offspring have been noted, in vitro culture has also been implicated in increased gestation length and birth weight elevation (Holm et al., 1996). Therefore, the large birth weight of nuclear hansfer offspring may not necessarily be caused by the nudear transfer procedure, but in fact mav be an effect of in uiho maturation and/or culture. It is now evident that Mer knowledge of basic embryo development at the tevel of the nucleus and cytoplasm is necessary to promote merimprovements in nuden transfer. NucIear transfer has been examined in several species induding moue (Czolowska et al., 1984; McGrath and Solter, 1983a; 1983b; 1984a; 1984b; Surani et aL, 1984), rabbit (Collas and Robl, 1991), pig (Prather et d., 1990), cow (Kanka et al., 2991; Moor et al., 1992), and sheep (WüIadsen, 1986; Wilmut et al., 1997). Multiple generational or serial nudear transfers have also been explored in both mouse (Kwon and Kono, 1996) and bovine (Stice and Keefer, 1993) as a method for potentially producing a large number of identical offspring from the micromanipulation of a single mammalian embryo.

However, in mice the proportion of reconstituted embryos developing to the blastocyst stage dedined from the first (44%) to the fourth (4%) generations (Kono, 1997). Although calves were bom as a result of first, second and third generation bovine nudear transfer, the efficiency of the technique dedined as

&ion rates generaily declined with each additional generation (Stice and Keefer; 1993). The morphological events which follow transfer of a donor nucleus to an enudeated oocyte have been surnmarized by CampbelI et al., (1993). These include 1) nuclear envelope breakdown, 2) premature chromosome condensation, 3) dispersal of nucleoli, 4) reformation of the nudear envelope, and 5) nudear swehg. The concept that differentiation of the embryonic nucleus may occur in response to interaction with cytoplasmic factors is the basis for nuclear reprogamming (Stice and Robl, l988). Nuclear reprogramming, which involves developmental reprogramming of the incorning nucleus to direct development on schedule with a zygote, is essentiai to the success of nuclear transfer. Nuclear reprogramming has been examined using a few select markers of stages of early embryo development. One such study examined expression of the mouse antigen TEC-3 whidi is normally expressed on bovine morulae and but is absent From all sfages prior to the 32-cd stage in nuclear transfer embryos (Van Stekelenburg-Hamers et ai, 1994). The TEC-3 antigen was indeed present on blastomeres of monda stage embryos, disappeared after fusion and was expressed again when the nuclear transfer embryos developed to the morula and blastocyst stage. These observations indicate that the bovine embryo nucleus is able to revert to the equivalent of an earlier developmental stage when transferred to ooplasm, and is then capable of following the normal developmental pattern, at Ieast in the expression of one gene. However, reprogramming may not necessarily be complete, since embryos do not always develop to term following embryo bander to recipient females (Kono et al., 1996; Kono, 1997). The molecular mechanisms involved in reprogramming donor nudei remain unknown but it is proposed that DNA modifications such as methylation that regulate gene expression, are involved (Kono, 1997). Further examinations of nuclear and cytoplasmic interaction and the mechanism of nudear reprograrnming are currently being studied. Cytoplasmic transfer is a variation on the technique of nudear transfer which involves tusing cytoplasts with an oocyte or zygote (Levron et al., 1996). Levron et al. (1996) have shown that impiantation rates in mice were significantly reduced when germinal vesicle stage cytoplasts were kised to zygotes, but that implantation rates may be enhanced by transfer of a small amount of metaphase II cytoplasm. The technique is ultimately aimed at "rescuing" poor quality oocytes in the human to yield implantation and ongoing embryo development. However, the mechanisms responsible for these changes are not yet understood. Placentation The establishment of a funtional placenta is critical for exchange of gases, nutrients and waste between a deveioping fetus and mother. The development of this intimate relationship between embryo-fetal membranes and endometrium is not an isolated event but a progressive sequence of events which begins early and continues throughout gestation. In addition to ensuring close contact for exchange, development of a functional placenta involves alterations to the matemal immune and endocrine systems (Noden and delahunta. 1985). The process of establishing intimate contact between fetus and mother involves approximation of the outermost embryonic membrane and uterine epithelium, followed by p hysical attachment of these tissues, with actual invasion of chorionic cells across the interface and into the endometrial stroma, and matemal epithelial destruction in some speaes (King, 1993). in ruminants, chorionic buiudeate cells migrate auoss the matemal-conceptus interface and fuse with matemal cells in the luminal epithelium, fomiing a hybrid tissue on the materna1 side (Wooding, 1982, 1983). A more recent study found no sigruficant difference in the degree of this migration between sheep and goats (Wooding et al., 1993). The gross classification of the chorioallantoic placenta of cattle, sheep and goats is cotyledonary or multiplex (Mossman, 1987). A sumrnary of characteristics of bovine, ovine and caprine placentation is provided in Table 1. Placentas of sheep and goat are reported to be histologicaily similar (Lawn et al., 1969; Steven, 1975), although the sheep placenta has been examined more extensively. The non-pregnant uteri of al1 three species contain localized thickenings of sub-epithelial dense comective tissue, the caruncles, which are arranged in rows. The caruncles are non-glanddar regions and are surrounded by the uitercaruncdar region which contains the long tubular glands responsibIe for uterine secretions or uterine "milk". During pregnancy, the carundes and cotyledons interdigitaie and proMerate to form placentomes, which are responsible for the hemotrophic nutrition of the developing concep tus (Amoroso, 1952). Attachment begins by day 16 in sheep (Wooding, 1984) and between days 19 and 27 in cattie (King et al., 1982). Mthough intimate contact is established between fetal and matemal tissues between days 16 and 25 for sheep, evidence for the development of fetal villi or maternd crypis in the cadesis not obsemed before day 24 (Wooding, 1984). The interdigitation of caruncles and cotyledons to form placentomes begins at the region dosest to the fetus and extends outward to the peripherai extent of the fetal chorioallantois in the tips of the uterine horns. There are generally 70 to 120 placentomes in the cow, 90 to 100 in the ewe and 160 to 180 in the doe, although they Vary in size and distribution (Amoroso, 1952). In the ewe, placentornes continue to grow und about day 90 of gestation, &ter which the size remains relatively unchanged (Steven et al., 1981). Alexander (1964) suggested that placentornes form at a relatively constant proportion (70 to 80°') of the caruncles. The size and number of placentomes might be influenced by the number of fetuses camed in a given pregnancy, but there is little documentation of the change in sheep and goat placentorne size and histology throughout gestation. TABLE 1: Features of placentation in cattie, sheep and goats.

Capra hircus ongin chorioallantoic chorioallantoic chorioallantoic placentorne shape cotyledonary CO tyledonary cotyledonary convex concave concave villous

#: number of

Although information on pregnancy in the goat is scant, Dent (1973) reported transitory formation of a syncytium, including microvillus interdigitaiion of principal trophoblast and the syncytial layer, in the intercotyledonary placenta of the goat, beginning between days 25 and 28 and starting to be replaced by uterine columnar epithelium on day 42. Steven et al. (1981) suggested that, unlike the goat, no syncytial tissue is formed in the intercotyledonary regions of the sheep, although binucleate cell pseudopodia occasionally cm be seen extending into the materna1 epithelium. However, Wooding (1984) demonstra ted that much of the interplacentomal epithelium in the ewe was replaced by syncytial trophoblast very early in the implantation process, but that the interplacentomal epithelium was re- established by day 28 to 30. Histological cornparisons of several speaes with cotyledonary placenta have revealed a variable degree of branching and invasion of chorio~cvilli (Hradecky et al., 1988a). This variation in structure between species has significant implications for interspecific emb ryo transfer and interspecific hybrid development (Hradecky et al, 1987). Failure of proper branching and invasion of chorio~cva has been observed after transfer of a gaur (Bos ga ZL rus) embryo to a Holstein heifer, despite a macroscopically normal appearance of the placentorne (Mradecky et al., 1988b).

Interspecific Hybrids Bamers to Inters~ecificHvbridization For centuries, animal breeders have attempted to breed males and females from different speaes to combine the desirable traits of both speaes into hybrid offspring. Attempts at hybridization have uicluded such diverse speaes as chicken X pheasant (Basrur, 1969), rabbit X hare and ferret X mink (Chang and Hancock, 1967). There is some evidence that hybridization occurs occasionally in the natural environment, with males and females of closely related species, (goat and sheep: Pinheiro et al., 1989; white-tailed deer and mule deer: Carr et al., 1986; and donkey: Ryder et al., 1985)- Natural barriers or isolating mechanisms prevent breeding, fertilization and/or development of viable hybrids from animals of different species (McGovern, 1976). There are numerous barriers which may restrict the natural hybridization of two different species. These barriers may be relatively simple such as two species having different habitats, or physical differences between the two species which prevent than from mating. If two species are capable of mating and do indeed breed, there are further factors which may prevent the successful production of hybrid offspring. The zona pellucida may act as a species-specific block to ferülization. Failure of fertilization may result from the inability of the paternal species' spermatozoa to capacitate in the maternal species' reproductive tract, or the spermatozoa may not be capable of recognizing and/or penetrating into the ooplasm of the maternal species. This appears to be the barrier in fertilization of bovine oocytes with ram spermatozoa under in vivo conditions. The bamer to fertilization in this particular case cm be overcome by in mtro fertilization (Kelk et ai., 1991b; Slavik et al., 1997). Goat oocytes however, are readily fertilized under in vivo conditions when exposed to ram spermatozoa (Berry, 1938; Buttle and Hancock, 1966; Ilbery et al., 1967; McGovem, 1973; Basrur, 1986a; Kelk et al., 1997a). If fertilization ocms, a cytogenetic incompatibility may still prevent rnitotic pairing and appropriate segregation of the chromosomes of the two different speaes. Depending on the developmental stage at which a partïdar isolating mechanism operates, hybrid death may resdt, as generally seen with sheep X goat and ferret X mink hybnds. If interspecific fertilization produces an embryo, irrununological incompatibility between the mother and hybrid fetus, may cause the fetus to be aborted midway through gestation (Hancodc et al., 1968). This appears to be the usual case when goats are bred to sheep (McGovem, 1973). There may also be hormonal differences or a difference in gestation length between the two parental species which couid prevent the birth of live hybrid offsp ring. Embryo transfer procedures allow the establishment of true interspecific pregnancy in which the conceptus and female carrying the pregnancy, are of different species. Mechanisms that operate in interspecific pregnancy are often viewed as extensions of processes occurring in normal, intraspecific pregnancy. Thus, interspecific hybrid pregnanaes can be used to study the medianisms that allow the fetal allograft to survive in apparent violation of hansplantation laws (Anderson, 1988; MacLaren et al., 1992). In some instances, as with the goat X sheep hybrid pregnancy, death of the hybrid fetus generally results. Although the reason for the death of this particular hybrid fetus is not known, cytogenetic, immunologie and hormonal disparities between the two parental species have been considered as possible causes of fed mortality (Basnu, 1986a). Interspeafic chimeras, produced by combining cells from two or more embryos, have been useful for studying cellular distribution during development, because species-specific rnarkers indicate ce11 parentage (Siracusa et al., 1983; Rossant, 1985). The construction of interspecific chimeras between sheep and goat embryos has removed the reproductive barrier between these two speaes. A goat kid, camied by a sheep mother was produced after aggregation of one 4-cell sheep blastomere with two blastomeres of an 8-cell goat embryo (Meinecke-TdLmann and Meinecke, 1984). Cytogenetic analysis, haemoglobin and transfemïn typing, blood group serology, polyacqdamide gel electrophoretic analysis of blood and muscle proteins and breeding experiments gave no indication of diimerism in this particular animal. Chimeras produced by injecting goat X sheep hybrid inner ce11 masses into ovine embryos gave rise to chimeric offspring of sheep and goat X sheep hybrids (Roth et al., 1989). When hybrids do occasionally develop to terni, they are often partially or totally stede as exhibited by most equuie hybrids (Gray, 1972). GeneraIly, sex reshicted malfunctions tend to be pronounced in the heterogametic sex. There may also be a decrease in viability of backaoss progeny as found in cattle X bison hybrid crosses, which prevents the commercial potential for producing these animals (Basnu, 1969; 1986b). Many instances of hybrid sterility are characterized by a marked distubance in gametogenesis despite a relatively unaffected endocrine hction of the gonads, as evidenced by sexual behaviour (McGovem, 1976). Asynapsis at meiosis due to grossly divergent parental chromosome structure is held responsible for hybrid sterility in many cases. The sterility of the displayed a precoaous weight gain gives sorne hope for the future production of these hybrids. There are a few rare interspecific hybridizationç which have occurred nakirally. A cross between a male Polar bear (Ursus muritirnus) and a fernale Alaskan brown bear (Ursus nrcfos),produced a female hybrid which died at 6 months of age and two litter mates which suMved to produce cubs. Both the hybrid malsand the polar bear had diploid chromosome numbers of 74 and although the two animals exhibited very different phenotypic characteristics, the kqotypes were Wtually identicd (Benirschke, 1967). More recently, there has been evidence through analysis of mitochodrial DNA that there has been a natural hybridization between white-tailed deer and mule deer in west Texas. Cmet al., (1986)suggest that there has been interspecific hybridization primarily between mule deer bu& and white-tailed deer does and that the hybrid offspring have been absorbed preferentially into the mule deer gene pool. These findings have numerous implications, including the possibility of eventual extinction of a known species of animal which may inevitably result without human intervention. These findings also show that hybridization of two species is a natural occurrence which may lead to hybrid vigour.

Production of uiters~ecificHybrids Despite nurnerous barriers to interspecific fertilization some species have managed to overcome the difficulties of interspecific hybridizations but there is frequently a polarity in the success of the cross. Nearly all(96%) rabbit oocytes exposed to Snowshoe hare semen become fertilized; however, only 10% of hare oocytes are fertilized in the presence of rabbit spermatozoa. These hybrids develop to early blastocyst stage but do not implant (Chang and Hancock, 1967). It is generally believed that the failure of these hybrids iç due to chromosomal or cytogenetic incompatibility since they do not suvive beyond early cleavage divisions. In the case of ferret X mink crosses, dthough ferret oocytes have been fertilized by rnink spennatozoa, the reaprocai aoss has not yet been observed. The hybrids formed by this aoss develop to about midgestation, ùidicating that the fdure of these hybrids is not likely due to chromosornai pairing difficulties, but more ükely resdts from incompatibility between the mother and hybrid fetus (Chang et al., 1969). Chicken X pheasant crosses &O exhibit a polarity in success. Aithough there is a similar feertilization rate between reciprocal crosses, a higher hatdung rate is observed if a female dud

crosses, although there is variation in the success of the cross depending on the species used and there is often a polarity in the success of the aoss between two equidae. The best known interspecific hybrid is the mule which results from aossing a female hone, (Eqnus caballus; 2n=64; NF=94) and a male donkey, (Equus asinns; 2n=62; NF=104). The mule obtains 32 chromosomes from the mare and 31 chromosomes from the donkey, yielding a diploid complement of 63 chromosomes. The donkey and horse chromosomes are not similar enough however to allow normal homologous diromosorne pairing during prophase of the first meiotic maturation division. This generally results in meiotic arrest and no gametes are fomed, leading to the sterility exhibited by the mule. There has however, been a documented case of a fertile female mule (Ryder et al., 1985). nie female mule has proven to be endocrinologicaUy normal through carrying a horse fetus to term after embryo transfer at the blastocyst stage (Allen et al., 1985). The hybrid resulting from the reaprocd cross, between a female donkey and stallion, while somewhat less successful, produces an animal referred to as a hinn y. Microscopie examination was made of the male gonad of a hybrid (2n=53) produced from crossing a male donkey to a female Grant's zebra, (Equus burchelli; 2n=44). Examination of the seminiferous tubules of the testis showed that there was no development beyond the primary spermatoqrte stage of meiosis. Meiosis had ceased since the differences in the chromosomes between the two speues had prevented proper pairkg of chromosomes in the &st meiotic prophase (Hayes et al., 1991). Interspecific hybridization of sheep and goats is one of the most intriguing. The cross has been studied extensively over many years but an understanding of the causes of fetal mortality as welI as occasional full term development, continues to elude researchers. Sheep and goats are believed to have evolved from a common ancestor that, like the modem-day goat, camed 60 chromosomes. It is thought that the sheep species has undergone a series of Robertsonian translocations which has resulted in a progressive reduction in the number of chromosomes hom 60 to 54 (Hayes et al., 1991). This theory is supported by the existence of ovine species possessing intermediate diromosome numbers such as the Barbary sheep and Aoudad with 58 chromosomes induding one pair of metacentrics and the Afghan wild sheep which carry two pairs of metacentncs within their 56 chromosomes. Homology between chromosome arms of sheep and goats

has been revealed and the differences between the hospecies are iikely attributable to the accumulation of gene mutations which have formed a partial reproductive barrier (Dain, 1980; Hayes et al., 1991). There have been numerous attempb at aossing domestic goats (Capra hircus) and domestic sheep (Ovis aris) with the airn of producing a vigorous animal which could provide milk, meat and wool. Although the general trend with such matings is fetal mortality, occasionally hybrids have sunrived fetal life and even proven fertile (Bunch et al., 1976; Pinheiro et al., 1989). Rams and bu& will often mate indiscriminately with ewes and does if both are present. Fertilization in vivo readily occurs when goat oocytes are exposed to ram spermatozoa, but fertilization of sheep oocytes with goat spermatozoa is rare. Goat X sheep hybrid fetuses produced by does inseminated with ram semen generally fail to survive beyond the second month of pregnancy. Cytogenetic (Berry, 1938; Buttle and Hancock, 1966; Hancock and Jacobs, 1966; Ilbery et al., 1967; Bunch et al., 1976; Pinheiro et al., 1989), immunologie (McGovern, 1973) and hormonal differences between the two parental species were examined as possible causes of the fetal mortality. There has been no evidence of chromosomal nondisjunction in any cytogenetic studies of goat X sheep hybrids and more recently, embryonic mortality has been attributed primarily to "hemolytic disease" resulting from matemal antibodies against fetal red blood cells which cross the placenta from the matemal circulation, (McGovern, 1973). Berry (1938) was the first to examine the cytogenetic properties of amniotic ce& of goat X sheep hybrids. Berry found the majority of amniotic ceils of two, 30-day-old hybrid fetuses to contain 57 chromosomes, two of which were metacentric. With more advanced techniques for karyotypic examinations it was determined that goat X sheep hybrids cary 3 metacentric chromosomes within their total 57 chromosomes, (embryonic cells: Buttle and Hancock, 1966; fetal liver tissue: Hancock and Jacobs, 1966; fetal liver and skin ceus: Ilbery et al,, 1967). These studies also found no evidence of nondisjunction in the goat X sheep hybrids, indicating that this was not likely the cause of the observed embryonic mortality. A number of studies which involved crossing several different varieties of sheep and goats followed these preliminary investigations. Bunch et ai., (1976) bred a Spanish goat to a Barbados sheep ram which resulted in the birth of twin offspring, one of each sex. Although the male twîn was Iost before assessments were made, the femde twin exhibited three metacentric diromosornes within her total 57 diromosomes and dso proved to be fertile when backcrossed to a Barbados ram. This mating &O resdted in the birth of a male and female twin. The hybrïds produced in this study exhibited characteristics of both parental speaes: Barbados coloration and goat-like homs were prevalent in both female hybrïds. The animals resdting from the hybrid backaosses displayed five metacentric diromosomes within their total 55 chromosomes. Dain (1980) Iater crossed a Barbary ram, (2n=58)with a Saanen goat, (2n=60). These two speues have the same fundamental number (NF = 60) of chromosome armç and exhibit broadly similar G-banding patterns. The mating resulted in the stilIbirth of apparentiy normal male twins. Cells of one of the twins were karyotyped, 34.1% of which contained 58 chromosomes while only 74.6% of the ceLIs contained the expected 59 diromosomes. More recently, the natural occurrence of a goat X sheep hybrid was reported by Pinheiro et al. (1989). The caprine-like animal which was presented to the chic carried a 2n=57, XX chromosome makeup. Based on the Gbanding pattern, it was deduced that this animal was indeed the result of a ram and doe maüng. The animal was mated to a ram and proved fertile by delivering a male with a 2n=56, XY chrornosomaI constitution. Finally, goats have been bred in semi-arid areas with wild ibex to form an animal called a Yaez for the purpose of improvement of meat quaiity (Rattner et al., 1994). This cross Ieads to successful production of offspring but there are much higher abortion (6.7%), stillbirth (6.0%) and pre- (22.3%)and post- (15.5%) weaning mortality rates than are usually observed in goat herds (Rattner et al., 1994). A clear understanding of the polarity of in uiuo fertilization success between speues such as sheep and goat, remains to be found. There is still no adequate explanation for the htal mortality which generally results from hybridization of goats and sheep nor is there an explmation for the occasional full term deveIopment which may resdt in goat X sheep hybridization. RATIONALE

The aim of this investigation was to extend knowledge of early embryo development with specific emphasis on the roles and interactions of maternal and paternal contributions to the embryo. Little is known about how products which are specificdy maternally or paternally derived interact in a cooperative manner following fertilization to achieve full term development. Lmplicit within this investigation is the Fact that virtudy aii of the cytoplasm and organelles within the early embryo are maternally derived through the oocyte. Therefore, interactions or communication between the nuclear and cytoplasmic components of the embryo are considered, During the process of oogenesis, the femaie loads messenger RNA5 and proteins into developing oocytes. The stores of maternally derived gene products are critical for oocyte maturation, fertilization and the initial deavages of the embryo. This reliance continues until the embryo begins to express and accumulate products from its own genome. Since there is a switch from maternal (cytoplasmic) control of development to embryonic (nuclear) control, it is easy to conceive that there must be some fonn of communication and interaction between the nucleus and cytoplasm at th% early stage of embryonic development. An understanding of this interaction wouId have significant impact on the approach taken for techniques such as nudear transfer and provide insight into the incompatibiiity of two genomes which may in tm, result in infertility. Interspecific hybrid embryos provide unique genetic and morphoIogical markers of each parental species which aid in understanding the roles of the maternal and paternal genomes in early embryonic development. Interspeâfic hybrids have proven to be us& models for the study of a wide range of events including fertilization and fertilization failure, embryo development and embryo mortality and placenta1 development and fetal mortality. The paternal genome contributes prirnady to the nuclear component of a develaping zygote while the materna1 genome makes significant contributions to the deve1oping embryo through cytoplasmic inheritance as well as the nuclear cornponent, Examination of transcription and pro tein synthesis within interspeufic hybrid embryos provides some insight into the control and interaction of matemally and paternally derived components in embryo development. The production and developmental potential of some of the various interspecific hybrid enbryos of cattle, sheep and goats were previously established (bovine X ovine; cap~eX ovine; KeIk, 1992). It was hypothesized that these embryos would express or exhibit developmental, biochemical and/or morphological markers of development which could be used to hirther our understanding of the nucleus and cytoplasm. The cytoplasm was hypothesized to play a crucial role in regulating nudear activity in the early embryo. The specific objectives of this series of experiments were to 1) hther explore production and developmental potentiai of the various interspeafic hybrid embryos, particularly those which were not previously produced (ovine X caprine), and 2) begin examination of various developmental aspects of these embryos. Experiments were designed to examine transcriptional activity and protein synthesis in hybrid and control embryos in order to elucidate the roles and activities of maternai/ paternal and nuclear/cytoplasrnic factors. LN VITRO AND AT VIVO PRODUCTION OF INTERSPECIFIlC HYBRID EMBRYOS

INTRODUCTION The aim of this investigation was to extend the knowledge of early embryo development through the model of Ïnterspecies hybrid embryos which carry unique markers to indicate the roles of matemal and paternal contributions to the embryo. Implicit within this design, is the preference to establish a complete model which would include embryos from both reciprocal crosses (example: caprine X ovine and ovine X caprine) as well as control embryos (example: caprine X caprine and ovine X ovine). The fist step to establishing this mode1 was to examine both the in vivo and in vitro production of these embryos. The in uiuo and in dru production of interspecific hybrid and control embryos of cattle, sheep and goats wiU be discussed in this chapter. In each case, the species from which the oocyte was derived (matemal species) is kted first, followed by the speaes of sperm (paternai species) which was used to inseminate the oocytes. The in uiuo and in vitro embryo production of caprine X ovine and bovine X ovine interspecific hybrid embryos was previously established. For previous results and review of literature see Kek, 1992, This chapter expands upon these previously established crosses and details attempts at production of the additional, ovine X caprine cross whkh utilizes goat sperm. The ovine X caprine embryos discussed in this chapter, were traflsferred to retipient sheep and goats to evaluate their developmental potentid. Detailç of these embryo hansfers and subsequent pregnanàes are discussed Uyin Chapter 2. It has been well documented that female goats and rams will interbreed if housed together. The rnatings frequently result in conception and establishment of pregnancies which generally fail during the second month of a five month gestation (Warwick and Berry, 1949; Kek, 1992; Keik et al., 1997a). Although both sheep and goats wilI interbreed when housed together and female goats bred to rams readily conceive, the reciprocal cross of ewes bred to bucks has never led to fertilization. Results from attempts at in mm production of ovine X caprine hybrid embryos are discuçsed along with resdts of control ovine, caprine and caprine X ovine crosses. Since it was impossible create ovine X caprine hybrid embryos under in uivo conditons, the possibility of production under in vitro conditions was evaluated. Bovine oocytes fertilized by ram sperm in vitro develop up to, but not

beyond the 8-cell stage (Slavik et al., 1990; 1997; Kelk et al., 1991a; 1995; 1996). Since this is the stage ai wkich bovine embryos undergo developrnental arrest, (the 8-ce11 block) when cultured under inadequate conditions (Camous et al., 1984), it was important in this study to assess if this developmental arrest was a result of culture conditions. A short-tenn in oiuo control was therefore performed which involved transfer of bovine X ovine presumptive zygotes and 2-ceIl embryos at 24 hpi to the oviducts of reüpient ewes. The embryos were flushed 5 days later from the reproductive tracts after slaughter to determine if the embryos developed beyond the û-cell stage. In summary, the objectives of this study were: 1) to produce and improve the production of previously established crosses (in uifro bovine X ovine, control bovine and ovine) for use in Wercharacterization of their development (Chapters 3,4); 2) to determine if the previously established 4-& cell arrest of bovine X ovine hybrid embryos was an in uh-0 artifact; 3) to establish in oitro production of ovine X caprine and caprine control rrosses; 4) to evaluate the devdopmental potential of the interspedic hybrid embryos and define the limitations and polanty of the various crosses- Ali of the various types of embryos produced were utilized in Chapters 2, 3 and 4 to evaluate their developmental po tential, RNA transcription and pro tein translation.

MET'HODS In Vivo Production of Ovine, Caprine and Hybrid Embryos Exuerimental Anima 1s Sexually mature, mixed breed goats, including Saanen, Toggenburg, Alpine, Nubian and LaMancha were housed indoors at the Erarnosa Research Station under regular incandesent iighting from 08:00 h to 16:OO h. Sexually mature Arcott ewes were housed in outdoor shelters, exposed to naturd Iight conditions throughout the year at the Ponsonby Sheep Research Station. A total of 20 Arcott ewes and 12 crossbred does were synchronized for estrus, superovulated, and bred naturally to either an Alpine or Nubian buck or a

Suffolk, Lincoln or Arco kt ram.

Synchronization and Su~erovuIationof Shee~and Goats Estrus synchronization and superovulation were accomplished by one of two protocols as foUows. Anirnals destined for surgicd embryo recovery were treated with 60 mg medroxyprogesterone acetate (MAI?) vaginal pessaries (~eramixm,Appendix II) placed for 14 days. Beginnjng on day 12 after pessary insertion, a totai of 20 mg follicle-stimdating hormone (FSH- Appendix II) was injected intramuscularly at 1% intervals on three consecutive days (5,4,4,3,2,2 mg). Ewes and does given this treatment began to exhibit estrus behaviour within 24 hours of pessary removal. Animais destined for post-slaughter embryo recovery were given 3 intramuscular injections of 50 mg progesterone in an alcohol water base (Centra ~ro~estina,Appendix II) every fourth day followed by 4 daily intramuscular injections of 10 mg progesterone in a sesame oil base (Gesterol in 0d@,Appendix II) to synchronize estms. At the time of the third Gesterol in oïl@injection, a total of 20 mg follide-stimulating hormone (F.S.H.-PB, Appendix II) was injected intramuscularly at 12 hour intervals for three consecutive days on the same dose schedde: 5,4; 4,3; 2,2 mg. Ewes and does treated with this protocol began to demonstrate estrous behaviour between 36 and 48 hours following the final GesteroI in 0il@injection. For the purpose of monitoring a hybrid pregnancy, estrous was synchronized in those does to be bred to a ram by insertion of a MAP pessary for 14 days. At the time of pessary removal, 250 to 400 Iü (depending on the iime of year) of pregnant mares' semgonadotrophin (PMSG; ~~uinex@, Appendix II) were injected intramuscularly . Does synchronized with the

MAP pessaries began to exhibit estrous behaviour within 24 hours of pessary removal.

Breeding

Upon initiation of estnis, the ewes and does were bred or cross-bred naturally, to a ram or a buck. The ram/buck was penned with a group of up to 4 ewes/does for at least 24 hours. Five of the ewes induded in the data reported, underwent laparoscopie intrauterine insemination with buck semen (Keik, 1992; Kelk et al., 1997). Confirmation of Breeding Breeding of ewes and dues by a ram was confirmed by uçing a marking harness and crayon. BreedÏng by a buck was confkmed by swabbing the vagina of females withui 24 hours of expected omet of estrus. The swabs were smeared onto glass microscope slides and examined under phase contrast optics with a 40X objective for the presence of spermatozoa.

Embryo Collection Surkcal embrvo flush Food and water were withheld from ewes or does for 24 hours prior to scheduled surgery. Ewes were sedated with 15 mg (a dose ranging from 0.15 to 0.33 mg/kg body weight) xylazine (~orn~un@,Appendix II) administered intramuscularly. Does were anaesthetized with a mixture of 15 mg (0.35 to 0.45 mg/kg) xylazine (~orn~un@)and 150 mg (3.5 to 4.5 mg/kg) ketamine hydrochloride (~o~arsetic@,Appendix II) injected intramuscularly. The administration of sedative or anesthetic, was repeated if the surgical procedure lasted longer than 20 minutes. With both ewes and does, 10.0 ml of 2.0% Lidocaine (Appendix II) were administered locally as a line block. Each animal was placed in dorsal recumbency on a laparoscopy table raised to an - angle of about 30° from horizontal with the head dom and prepared for a ventral midline celiotomy. A midline incision was made anteriorly for 6 to 8 cm beginning 2 cm anterior to the udder. The uterus and ovarîes were exposed, corpora lutea were counted, and any pathological changes were noted. Heparinized saline (1000 IU heparin in 500 ml physiological saline) was frequently applied to the reproductive tract throughout the procedure to keep the tract moisi and aid in prevention of adhesions. For those animais fiushed on days 5 to 7, a modined antegrade uterine flush tedinique was employed (TeMt and Havik, 1976; Wilmut and Sales, 1981; Smith and Murphy, 1987; GartIey, 1988). At the base of each hom, distal to the bifurcation, a smd medhemostat was used to make a stab incision through the ute~ewall into the Lumen. An 8 Fr bdoon-tipped catheter (Appendix m) was inserted through this incision and direded towards the oviduct. The cuff was inflated ushg 4 to 6 ml of air. Great care was taken to ensure that the cuff was completely withÏn the uterine lumen, sealing it without causing overdistention and endometrial trauma. A blunt 20 gauge needle was used to punctwe the ute~ewall of the hom near the utero-tubal junction. A 20 an open-ended tom cal catheter (Appendix II) was uiserted through this hole and threaded proximally 2 to 3 an. A polypropylene syringe was used to inject warm flush medium through the tom cat catheter and the site of entry was held with a gauze swab saturated with heparVlized saline. Depending on the size of the uterus, 15 to 25 ml of flush medium were injected into the lumen until the hom was distended. The uterine wd was "milked" as necessary to initiate fluid 80w out through the balloon- tipped catheter into the polypropylene collecring vessel. F* to 60 ml of flush medium were used for embryo recovery from each hom. Following fluid recovery from one side, the Bush procedure was repeated in the opposite hom. For those ewes or does flushed on days 1 to 3.5, the procedure was essentially the same as just desaibed with the following exceptions. A sterile feeding needle was inserted through the firnbria into the distal tip of the oviduct. Ten to 20 ml of flush medium were passed retrograde through a tom cat catheter inserted at the utero-tubal junction. The flush medium whidi passed through the ovidua was recovered through the feeding needle into a polypropykne specimen container.

The reproductive trad was rinsed with heparinized saline, returned to the abdomen and the linea alba, subcutaneous layer and the skin were sutured. The time required for the surgicd procedure for each animal was 20 to 30 minutes. Following each flush, 175 pg cloprostenol (~strumate@, Appendix II) were given intramuscularly to initiate Iuteolysis. The flushed fluid was taken to the Iaboratory and transferred from the specimen containers to sterile 100 X 15 mm petri dishes for embryo searching.

Slauhter flush Ewes or does that were to be culled from the herd were sent to a local abattoir on days 1 to 3.5 and the reproductive tracts were recovered and hansported to the Iaboratory. The number of corpora lutea on each ovqwas recorded and any pathologicd dianges to the reproductive trad were noted. The entire Length of each oviduct was dissected hom the tract and a feeding needle was inserted into the fimbria. Twenty ml of medium were slowly flushed through the feeding needle and oviduct into an etdied 100 X 15 mm petri dish for embryo seardung. Embryos and ova were located using a stereomicroscope at 25X power and were dassified as fertilized or unf-ed depending on morphology. A chi-squared test was used to compare mean fertilization rates. In Vitro Production of Embryos In Vitro Oocvte Maturation Collection of ovaries Groups of up to 100 ovaries from each of bovine, ovine and caprine were colleded on any given day From local abattoirs and held i to 4 hours in a thermos containing warm (33 to 37OC) saline (0.9% (w/v) NaCl) until amvai at the laboratory. Al1 grossly normal appearing ovaries were collected, regardless of the stage of estrous cycle or pregnancy. When possible, an oviduct from a cow which had ovulated within the past 1 to 4 days (as assessed by the presence of a corpus haemoragiçum), was aiso coilected in saline. In the laboratory, all ovaries were rinsed with fresh 37OC saline.

Isolation of cumulus-oocyte complexes Cumulus-oocyte-complexes (COC) were collected by either a slashing or an aspiration technique in a room held at 30 to 33OC. Both methods were explored on each type of ovary and it was found to be most practical to use the aspiration technique for isolation of bovine cumulus-oocyte complexes, whiie the slashing technique was more convenient for collecting ovine and caprine cumulus-oocyte complexes. Each bovine ovary was held in a gloved hand and every visible follicle was punctured with an 18 gauge needle. Cumdus-oocyte complexes were aspirated through this needle into a 125 ml Erlenmeyer flask containing 20 ml of collection medium (Appendix I). The flask was attached to a smd suction pump, adjusted to a flow rate of approximately 50 ml/min. The COC's from approximately 20 ovaries were collected into the flask before the contents were divided between two or more 100 X 15 mm petri dishes. After the cellular material had sedimented to the bottom of the petri dishes, the overlying, excess medium was aspirated off and discarded. The COC's were then located under a stereomicroscope at 12X power and transferred with a pded gIass embryo-handling pipette into 35 mm pehi dishes containing 3.0 ml of maturation medium (Appendix I). Each ovine and caprine ovary was held in a pair of hemostats above a 250 ml beaker and the entire ovarian surface slashed in a cross-wise manner to a depth of 2 to 5 mm with number 22 scapel blades. The surface of the ovary was then Bushed with collection medium (Appendix I) to wash COC's into the collection vessel. The COC's from approximately 20 ovaries were collected into a single beaker. Mer the cellular material had sedimented to the bottom of the beaker, the overlying medium and contaminating erythrocytes were decanted or aspirated off and discarded. The cellular material from the beakers was then divided between two 100 X 15 mm petri dished and flooded with fresh collection medium to dilute contaminating blood. Most of this medium was removed by aspiration to dow COC's to be located under a stereomicroçcope and transferred with a pded glass embryo- handling pipette into 35 mm pehi dishes containing 3.0 ml of maturation medium (Appendix 1).

Culture of cumulus-oocyte complexes Recovered COC's were selected or diçcarded according to the criteria of Liebfried and First (1979). Groups of 100 selected bovine, ovine or caprine COC's were transferred into each wd of Nunc 4-well plates containing 500 pl of maturation medium. Maturation medium to be used for culture of ovine and caprine COC's was supplemented with 5.0 pg/ml FSH. Maturation medium was prepared weekly and held at 4OC until use. On the morning of the day on which in vitro maturation (W)was to be initiated, FSH was added and the maturation medium was aliquoted and equiübrated to 37OC and 5% CO2 for at least 2 hours before the addition of COC's. Bovine, ovine and caprine oocytes were cultured for 22 to 25, 24 to 26 and 26 to 27 hous, respectively. When more than one type of ovary was processed on the same day, caprine COC's were colIected and placed into culture before ovine and/or bovine ovaries were processed. This sequence of processing provided the longer period of maturation for caprine COC's, and aUowed aIl ova to be fertilized simdtaneously.

Preparation of oocytes for fertilization Following culture, cumulus-oocyte complexes were removed from the maturation wells and pooled into one or two wells of a 4-well plate containing 1.0 ml of Hepes-TALP medium (Appendix 1). With a glass embryo-handling pipette, the COC's were gently rinçed to avoid disrupiion of the cumulus celI mass, and pipetted through two additional washes in 1.0 ml Hepes-TALP and a hal wash of 1.0 ml of TVF-TALP medium (Appendix 1). Approximately 100 matured COC's were placed in each weU of 4-well plates containing 500 p1 of fresh pre-equilibrated IVF-TALP. AU oocytes, regardless of speaes, which were to be fertilized by goat sperm were placed in wells with

IVF-TALP supplemented with 20°h estrous sheep serum and 7.75 mM calatm lactate.

Semen collection Frozen 0.25 ml straws of bull semen diluted in a rnilk extender were supplied by Gencor Inc. (Guelph, Canada). Frozen bdI sperm yielded acceptable fertilization rates (approximately 80%) and was used primarily for convenience. Fresh ovine and caprine semen were coilected from a Suffolk rarn and a Saanen bu&, as required. Although inconvenient, kesh sheep and goat sperm were previously shown to yield better fertilization rates than frozen sperm (Kelk, unpublished). Female goats (generally non-estnis) were used as teasers for the ram or bu& to mount and semen was couected via a 38 to 4S0C,lubricated artifiaal vagina into a semen collection tube hdd in a 35 to

37OC water jacket. The semen was then diluted with a 3 to 5 fold dilution of a warm (34 to 37OC) Tris-egg yolk extender and sperm motility was quiddy assessed under a compound microscope with a 40X objective. The extended semen was hansferred to 15 ml polystyrene centrifuge tubes and transported to the laboratory in a thermos containing 37OC water. Each ejaculate was generally divided in half with one half being used immediately upm rsmm to the laboratory. The other half was returned to the thermos and the entire thermos containing water and semen tubes was placed at 4OC to afford gentle cooling of the extended semen. This semen was frequently used within 1-2 days for subsequent in vitro fetilization runs and gave constant feertilization rates.

S~erm~re~aration

For each experimental replication, 8-12 straws (2.0-3.0 ml) of frozen bd semen and/or half an ejaculate of fresh ram or buck semen were used. Straws of hozen buU semen were thawed immediately followuig removal from liquid nitrogen by a 30 second immersion in a 37OC water bath. After thorough drying, the aimped end of each straw was removed and the straws were placed, open end down, into a 15 ml conical centrifuge tube. The plugged ends were then severed, the semen drained into the tube and diluted with 10 ml of Sperm-TALP medium (Appendix 1). The khram and bu& semen were also diluted with 10 ml of Sperm-TALP medium. The tubes were gently mixed and centrifuged at 300 xg for 5 minutes and all but 0.5 ml of medium overlying the sperm pellet was removed and discarded. An additional 10 ml of Sperrn-TALP medium were added to each tube and following gentle mixing the tubes were centrifuged again at 300 xg for 5 minutes. The overlying medium was removed and discarded and the sperm pellet was resuspended in 1.0 ml W-TALP medium. Each sperm suspension was loaded onto a pre-washed column of glass wool loaded into a 1 ml syringe whidi was held in a 15 mI tube.

In vitro fertilization Approximately 2 to 10 p1 of Wtered sperm were added to each 500 pl well of M-TALP containing up to 100 matured COC's at a concentration of approximately I X 106 rnotile sperm/mI (ie. approximately 5 X 103 motile sperm were combined with 100 COC's). Oocytes and sperm were CO-dtured for 24 hours at 3g°C in a humidified atmosphere of 5% CO2 in air.

Oviduct exdant ~re~aration Bovine oviducts were selected hom Mmals which had ovulated within 1 to 4 days of slaughter. The enüre length of an oviduct was dissected from the surrounding tissue and blood vessels, and rinsed in maturation medium. A pair of tweezers was then used to squeeze down the length of the oviduct, stripping sheets of lumenal celIs which were dropped into a 35 mm petri dish containing 2.0 mI of maturation medium. The sheets of celIs and medium were drawn up into a 1 ml syringe through a 25 gauge needle three times and transferred to a 15 ml conical tube. The resulting explants were rinsed three times with 5.0 ml maturation medium after dowing the ceUs to settle to the bottom of the tube by gravity. The supernatant was removed and 100 pI of oviduct explants were hansferred into 3.0 mI of WC medium

(Appendix 1) in each of three, 35 mm petn dishes. The viability of the ovidud ceIls was checked by examination for the preçence of rapidly beating cilia using an inverted microscope with a 20X objective under phase contrast optics. The ce& were cultured at 3g°C with 5% CO2 and within 6 to 12 hours of culture, the explants would curl and take on the appearance of "woms". Bovine oviduct explants to be used for embryo CO-culturewere rïnsed twice with 1.0 ml of NC medium (Appendix 1) and approximately 50 "worms" were added to each 500 p1 weIl of Menezo B2 medium the evening before ernbryos were to be added.

Buffalo rat liver ce11 ~re~aration Frozen buffalo rat liver (BRL) cells (Appendix II) were thawed by placing the ayovial in a 37OC waterbath. The vial was wiped with alcohol and the cells were washed in 10 ml of RPMI medium (Appendix II) supplemented with 20% fetal calf sem(FCS) and 1%antibiotic-anhycotic solution (Appendix II). The ceus were centsifuged for 5 minutes at 250 rpm and the supernatant removed before 5 ml of fresh RPMI were added and the cells plated in a small culture Bask. The ce& were grown to confluence with regular replacement of medium before trypsinization and 3 passages into large flasks. Each of the final Basks was ~rpsinizedwith 20 ml 1X trypçin (Appendix II) to disperse the cells and 2 ml of FCS were added to arrest enzyme activity. The ceus were centrifuged for 5 min at 250 rpm and the supernantant discarded. AU ceIls were pooled and resuspended in 100 ml of 20% DMSO in RPMI medium (approximately 1 X 107 cells/ml). This mixture was then pipetted in 1.5 ml aliquots into cryovials which were packed in a styrofoam insulated box and put into a -70°C freezer. If fresh bovine oviduct explants were not available for embryo culture, a cryovid of BRL ceils was thawed, washed in 10 ml of fresh RPMI medium and plated out in Nunc 4-well plates containing 500 pl of IVC medium. Culture wells of BRL cells were prepared 24-96 hours before embryo culture was required. Wells to be used 24, 48, 72 and 96 hours after platutg were seeded with approximately 200,000, 100,000, 50,000 and 25,000 cells/well

respectively. Fresh NC medium (500 pl) was added to each well of cells on the evening before ernbryos were to be added.

Culture of embrvos Foliowing 20-24 hours of CO-dtureof COC's and sperrn, presumptive zygotes were removed from the IVF-TALP wells and placed into 1.0 ml wells of fresh Hepes-TALP medium. With a glass embryo-handling pipette, each presumptive zygote was vigorously aspirated and repeatedly expelled to strip any remaining cumulus cells from the zona pellucida. Stripped presumptive zygotes were then rinsed through two additional washes in IVC medium and placed into 500 pl wells of the same medium containing previously prepared bovine oviduct ceh or BRL cells. An additional 250 pl of IVC medium were added to each well on the fourth day of embryo culture. Embryonic development was monitored regularly with an inverted rniaoscope (phase contrast, 20X objective), and embryos were removed at various stages, as required (Chapters 2-4). Embryos were classified as 1-, 2-, 4-, 8-cd, or as moda (>16-tek and showing signs of compaction), blastocyst (cavitating embryo), hatching blastocyst (embryo herniating through a niptured zona pellucida) or hatched blastocyst (blastocyst which has hatched free of the zona pducida).

Fertilization assav In preparation for zygote evaluation, 1.0 g of glass microcarrier beads (90-220 mm in diameter) was mixed into 50 mi of medical grade petroleum jeily (~aselineB]. The mixture of petroleum jelly and glass beads was appiied sparingly to two opposite edges of a 22 X 22 mm giass coverslip. A total of 473 presumptive zygotes were removed from culture 18-24 hours after sperm addition for evaluation. These presumptive zygotes were placed in groups of 5 to 10 on a pre-deaned microscope slide and a prepared coverslip was then placed over the presumptive zygotes and pressed into place so as to compress but not crush them. A small amount of methanokacetic acid 3:l (v/v) was added slowly through an open edge of the coverslip until aU air bubbles between the slide and coverslip were dispersed. Rubber cernent glue was applied to the two vaseline coated edges of the coverslip to affix it permanentiy to the slide. The siides were then placed in methano1:acetic acid 3:l (v/v) for at least 24 hours before staining with 1%aceto-orcein (Appendix r>- Presumptive zygotes were examined microscopically under Nomarski optics to assess the presence and number of pronuclei and sperm tails. Meiotic metaphase II figures were also noted as an indication of oocyte maturation but lack of fertilization. The criteria used in assessing the meiotic stage and deterrnining fertilization rates have been described by Xu et al. (1988)- Polyspermy was defined as the presence of any multiple and/or combination of sperm structures (whole sperm, sperm head, male pronucleus and assouated sperm taii) within the ooplasm of a single oocyte. Since rates of polyspermy were relatively low (4%)in all experiments, oocytes were classified into two groups: penetrated (those with evidence of sperm penetration) and unfertilized (those with no evidence of sperm within the ooplasm and generally containing a meiotic figure).

Cytogenetic Analysis of Embryos Embryos were prepared and examined to confirm both cytogenetic composition and number of nudei. The number of nudei was detennined to cobcelI numbers and indicate whether cytoplasmic fragmentation had occurred without nudear division. Embryos were placed in 500 pl wek of culture medium containing 10 pg/d nocodozole and incubated up to 24 hours at 3g°C in a humidified, 5% CO2 environment. Each embryo was then placed in 1.0% sodium citrate for 3 minutes before being placed on a pre-deaned microscope slide. The excess fluid was removed and the position of the embryo encirded on the back of the slide with a diamond tipped pend A drop of 1:l (v/v) methano1:acetic acid was dropped onto the embryo, and the sIide was blown dry by mouth under a 60 watt lamp and placed in 3:1 (v/v) methanol:acetic aad for 24 hours at 4OC (King et al., 1979; Tarkowski, 1966). The slides were then air-dried, stained in 4.0% Giemsa for 4 minutes, rinsed with distilled water, air-dried again and examïned under oil at lOOOX usuig a Leitz Anstoplan microscope.

Embryo Transfer of In Vitro Produced Bovine X Ovine Hybrid Embryos Ex~erimentalAnimals SexuaUy mature Arcott ewes were housed in outdoor shelters at the Ponsonby Sheep Research Station and were exposed to natural light conditions throughout the year. In total, 6 ewes were synchronized and used as recipients for embryo tramfer.

Syndironization of Shee~Reci~ients Ewes were synchronized for the onset of estrus using the following treatment. Medroxyprogesterone acetate (MAP) 60 mg (~erarnix@,Appendix II) vaginal sponges were inserted for 14 days. At the time of pessq removal, 300 IU of pregnant mares' semm gonadotrophin (PMSG; ~~uinex@, Appendix II) were injected intrarnuscularly. Ewes synchro~zedin this manner began to exhibit estrous behaviour within 36 hours of pessary removal, which was therefore defhed as day O of gestation.

Transfer of Embnros

Food and water were withheld from ewes for 24 hours pnor to scheduled surgery. Ewes were sedated with 15 mg (a dose ranging from 0.15 to 0.33 mg/ kg body weight) xylazine (~orn~un@,Appendix II) administered intramuscularly. A total of 10.0 ml of 2% lidocaine (Appendix II) was administered locally as a line block. Each animal was placed in dorsal recumbency on a laparoscopy table raised to an angle of about 30° from horizontal with the head down and prepared for a ventral midhe celiotomy. A midline incision was made anteriorly for 6 to 8 cm beginning 2 cm anterior to the udder. The utenis and ovaries were exposed for examination of corpora lutea, and pathological changes. Hepariwed saline (1000 Iü heparin in 500 ml physiological saline) was frequently applied to the reproductive tract throughout the procedure to keep the tract moist and to reduce adhesions. A tom cat catheter attached to a 1.0 ml syringe was wdto draw up approximately 50 embryos in about 20 J of PBS supplemented with 10% fetd calf senun which was isolated between two air bubbles. The tom cat catheter was fed through the fimbria and into the oviduct where the embryos were expeued before removal of the catheter. The reproductive tract was rinsed with heparuiized saline, returned to the abdomen and the linea alba, subcutaneous layer and the skin were sutured. The time required for the surgical procedure for each animal was 15 to 20 minutes.

Recovery of Embryos Ewes were slaughtered at the University of Guelph on day 5 of gestation (4 days after embryo transfer) and the reproductive tracts were recovered and transported to the laboratory. The number of corpora lutea from each ovary was recorded and any pathoiogical changes were noted. The entire length of each oviduct was dksected fiom the tract and a feeding needle was inserted into the fimbria. Twenty ml of flush medium were slowly flushed through the feeding needle and oviduct into a 100 X 15 mm petri dish for embryo seardUng. Similady, 50 ml of flush medium were slowly flushed through each ute~ehom into 100 X 15 mm petri dishes. The embryodova were located using a stereomicroscope and classified according to developmental stage or atresia (dark, shrunken and degenerating cells).

RESULTS Isolation of cumulus-oocyte complexes Although optirnizing the collection of cumulus-oocyte complexes was not a specific goal of the study, some general cornparisons and observations were made. The aspiration technique yielded fewer cumulus-oocyte complexes per ovmy (9-12COC's/bovine ovary) than the slashing technique (15-20 COC1s/bovineovary) but the quality of the COCrs obtained was grossly better with the aspiration method. Virtually all bovine COC's obtained by aspiration were of good quality and were induded in the maturation dture, while only 75-80% of bovine COC's obtained by slashing were selected for maturation. Since the number of bovine ovaries avdable was not a Limiting factor, it was faster and more convenient to aspirate bovine COC's. Since there had been no previous comparable examination of oocyte collections for other species, a similar cornparison was made with ovine ovaries. An average of 18 ovine COC1s/ovary couid be obtained using the slashing technique but only 50% (9.0) were acceptable quality for maturation. The aspiration teduiique yielded only 6 COC's/ovary with 63% (3.8) of those recovered acceptable for mahiration. The aspiration technique also seemed to strip the cumulus cells from the ovine and particularly caprine oocytes to a greater extent than was observed with bovine oocytes.

In Vivo Production of Ovine, Caprine and Hybrid Embryos High fertilitation rates were obtained from control crosses of ram-bred ewes (90%) and buck-bred does (96''). The fertilization rate among rarn-bred does was also high (72%) and did not differ sigruficantly (p<0.05) from those obtained in ram-bred ewes and buck-bred does. None of the ova collected from ewes inserninated with bu& semen or mated to bucks showed signs of fertilization. A chi-squared analysis showed that the in vivo fertilization rate observed for the sheep X goat cross was significantly different from dl other crosses. Even laparoscopie insemination of bu& semen into the utem of 5 superovulated ewes did not lead to fertilization. Seven of the ova collected from one bu&-bred ewe were fragmented and showed signs of degeneration. The complete redis are summarized in Table II.

TABLE II Summary of embryo coilections 1 to 4 days after observed estrus in ewes and does bred to rams or bucks-

sheep X sheep X goat X goat X goat goat i sheep 14" 8 4 # corpora lutea tolal ova/embrvos recovered

# unfertilized ova # fragmented ova

indudes 5 ewes that were inserninated laparoscopically ** accurate counts of corpora lutea were not available for all ewes a,b fertihation rates wiCh different superscripts dSer significantly (~~0.05)

Penetration and Development of In vitro Produced Embryos The penetration, cleavage and development rates for bovine, ovine and caprine conhol embryos and their respective hybrids are given in Table m. These data are a summary from TVF trials performed on 31 different days throughout the year but al1 three types of ovaries were not necessarily available on each day. Whenever possible, all three types of ovaries were obtained and processed on the same day such that the same sample of each type of sperm could be used on eadi type of oocyte. Due to the nature of the study, embryos were removed from culture at various times for other studies (Chapters 2-4). The crosses between bovine and caprine were not examined

due to the Iimited availability of caprine ovaries/oocytes.

TABLE III Summary of penetration, deavage and development rates of in oiho produced ernbryos

CROSS PENETRATION CLEAVAGE DEVELOPMENT materna1 X paternal moda/blastocyst bovine X bovine 80.4% (n=97) 70.1% (n=1272) 44.4% (n=606)

bovine X ovine 58.3% (n=48) 50.3% (n=1774) 4-&ceIl arrest ovine X bovine 8.2% (n=49) 1 22.3% (n=533) 1 fragmentation ovine X ovine 64.8% (n=88) 62.1% (n=1673) 35.0% (n=1114) ovine X caprine 58.1% (n=62) 67.9% (n=2850) 45.6% (n=2377) caprine X ovine 61.0% (n=41) 46.1% (n=479) 16.2% (n=235) 1 caprine X caprine ( 67.0% (n=88) 1 27.6% (n=326) 1 9.8Y0 (n=183) n = the number of presumptive zygotes/embryos which were evaiuated

% penetration/deavage/ development = the percentage of oocytes examined cleavage = the appearance of normal cell division fragmentation = la& of compaction, non-uniform cell size

Bovine and Ovine Crosses Ram sperm readily bound to the zona pelluuda of bovine oocytes under in vitro conditions (Figure 1). The proportion of bovine oocytes penetrated by ram sperm in vitro was 58.3% while bovine and ovine control crosses had penetration rates of 80.4% and 64.8% respectively. Bovine X ovine hybrid embryos cleaved at a rate of 50.3% but did not develop beyond

the €4-cell stage. Of those embryos whidi cleaved, 43.3% and 47.1% arrested at the 4- and 8-ceil stage (Figure 2) respectively while the remaining embryos arrested at the 2-ce11 stage. The cleavage and development (to moniIa/blastocyst) rates for bovine controls were 70.1% and 44.4% and for ovine controls; 62.1% and 35.O0/0, respectively . To determine if the 4-8-cd arrest obse~edin the bovine X ovine hybrid embryos was a function of the culture system, bovine control and bovine X ovine hybrid 1- or 2-cell oocytes/embryos were produced in aitru and transferred to sheep oviductç at 24 hpi. A total of 100 bovine control and 300 bovine X ovine, 1- or 2-ce11 embryos were transferred for 4 days before recovery while 50 control oocytes/embryos of each type were maintained in culture. Only 46 of the 300 bovine X ovine group were recovered. Of these, 14 and 13 were degenerating 4- and 8-cell embryos, respectîvely (Figure 3a), whde the remainder were degenerating oocytes or empty zonae. Only 6 of the 100 bovine controI group were recovered, including one early blastocyst (Figure 3b) and 5 degenerating oocytes or ernpty zonae. The reaprocal cross (between ovine oocytes and bull spermatozoa) was also examined under in vitro conditions. There was evidence of penetration of ovine oocytes b y buII spenn in only 4 of 49 (8.2%) oocytes examined from four different iVF runs. Out of a total of 533 in vitro matured ovine oocytes which were exposed to buil sperm, 119 (22.3%)cleaved. Ail of these embryos appeared to be of poor quality and most appeared to result from cytoplasmic fragmentation rather than true ceIl division.

Caprine and Ovine Crosses A total of 62 in uibu matured ovine oocytes which had been exposed to goat spenn under in vitro conditions were removed lrom culture at 18-22 hpi to assess the fertilization rate. Of the presumptive zygotes removed from culture 36, (58.1%) showed evidence of fertilization (Table III). Penetration FIGURE 1. In oiho fertilization of a bovine oocyte with ram sperm. The ram sperm readily binds to the bovine zona pducida. Phase contrast (X8OO).

FIGURE 2- An 8-ceU bovine X ovine hybrid embryo derived from in vitro fertilization. Phase contrast (X800).

FIGURE 3. Phase contrast images of bovine and bovine X ovine hybrid in oitro matured and fertilized embryos which underwent in vivo culture in sheep oviducts.

3a. Degenerate 4- and 8-ceil bovine X ovine hybrid embryos retrieved following temporary in vivo culture in sheep oviduds. (X800).

3b. A controI bovine early bIastocyst embryo, retrieved foilowing ternporary in vivo culture in a sheep oviduct. (X1600).

FIGURE 4- Pronudei (p) of an ovine X caprine hybrid zygote. Phase contrast (Xl6OO).

FIGURE 5. A hatching, in vitro produced ovine X caprine blastocyst. Phase contrast (XSOO). was confirrned by the presence of two or more pronuclei (Figure 4) and evidence of spmstnictures such as an associated sperm tail. Penetration rates for control ovine and caprine IVF were 64.8% and 67.0°h, respectively. The reciprocal cross between caprine oocytes and ram sperm yielded a comparabIe penetration rate of 61.0%. Cleavage and morula/blastocyst development rates for caprine X ovine were 46.1% and 16.2% respecüvely, and for the control caprine cross, 27.6% and 9.8% respectively. Ovine oocytes exposed to goat spenn had a cleavage rate of 67.9% and 45.6% of the presumptive zygotes developed to the morula or blastocyst stage. These embryos aiso demonstrated the ability to hatch from the zona pellucida in uiho (Figure 5). The developmental potential of these embryos is discussed further in the foliowing chapter.

DISCUSSION Isolation of cumulus-oocyte complexes Cornparison and optimization of both the slashing and aspiration techniques has been examined by a number of researchers. Each group however, has utilized vastly different methods in each of these two approaches. Hamano and Kuwayama (1993) slashed the surface of Japanese Black cattle ovaries with a series of ten razor blades held at 2 mm intervals and ob tained significantly more oocytes compared to an aspiration technique which utilized a syringe and 18 gauge needle. in another cornparison, surface dissection of bovine ovaries yielded sigdicantly more oocytes compared to aspiration with a 5 ml syringe and 18 gauge needle (Carolan et al., 1994). More recent examinations of the aspiration technique utilize a pump instead of a syringe to ailow consistent vacuum pressures (Fry et al., 1997). This group showed that bovine oocyte recovery rates were higher and the quality of cumulus-oocyte complexes was better when utilizing a 17 gauge needle compared with a smder bored, 20 gauge needle. The recovery rate increased as vacuum pressure inaeased but the percent of viable oocytes began to decrease at 55 mmHg when utiliung a 17 gauge needle (Fry et al., 1997). Passage through smaller gauge needles and increased vacuum pressures likely strip many of the cumulus celIs from good quality COC'ç,yielding fewer apparently viable oocytes. None of these groups stated details about the type of bevel on the needles which were used. The number of ovine ovaries avaüable was somewhat limited while caprine ovaries were very limited. For this reason, ovine and caprine ovaries were slashed to obtain as many COC's as possible. In addition, there was less selection of ovine and caprine oocytes for maturation and fertilization, since the goal of the project was to produce embryos and not to optimize developmental rates. The success with production of the various interspecific hybrid embryos was largely through technical modifications of the previous production system (Kelk, 1992) which enabled efficient sperm capacitation, fertiliza tion and dture conditions.

Caprine and Ovine Crosses In light of the high in oivo fertilization and pregnancy rates in the caprine X ovine crosses, the total lack of in vivo fertilization in the ovine X caprine crosses was surprising. Polarity in the success or failure in interspeues crosses has been identified in several speties. However, with the exception of the ferret X mink cross (Chang et al., 1969), polarity in reaprocal crosses of other species is usually manifest as differences in the rate of fertilization (rabbit and hare: Chang and Hancock, 1967) or development (chidcen and pheasant: Basnir, 1969) rather than the inabiiity to fertilize. Although in oilro crosses utilizing goat sperm were previously reported to have either poor deavage rates or a la& of penetration of the oocyte (Kek, 1992), the addition of 20% estrus sheep senun and 7.75mM calcium lactate in the present study yidded penetration rates comparable to those obsenred for ram sperm. Under in vitro conditions, both combinations of sheep and goat crosses are possible and occur with simiiar rates of fertikation (58.1%of ovine oocytes were penetrated by goat sperm and 61.0% of caprine oocytes were penetrated by ram spem). These hybrid penetration rates are slightly lower, but compare favorably, with those obsemed for caprine and ovine control crosses (67.0% and 64.8%, respectively). This conhlms the ability of sheep and goat gametes to successtully interad and suggests that the bamer to fertifization in oioo is at the level of sperm hansport or capacitation. By-passing the cervix and inseminating directIy into the uterus did not promote fertilization, suggesting that the capautation of buck spermatozoa in the reproductive tract of the ewe may be disadvantaged (Kelk, 1992). It is additionally interestkg that one of the agents used to faditate capaatiation of bu& sperm for in aiho fertilization of both goat and sheep oocytes was 20% semfrom estrous sheep (De Smedt et al., 1992; Kelk et al., 1997a), despite the la& of fertilizing ability of the goat sperm in the sheep reproductive tract itself. One possible reason for this la& of fertilizing ability of the goat sperm in the sheep reproductive tract is Mat the sperm were prematurely capacitated in the sheep reproductive tract before they ever reached the oocytes in the oviduct. This hypothesis is &O supported by the fact that other researchers have performed laparoscopic, intrauterine insemination of sheep with capaatated goat sperm whkh had been subjected to a Percoll density gradient (MacLaren, personal communication). None of the four ewes inserninated with Percoll-treated goat sperm produced pregnmcies while five of six does inserninated with Percoll-treated sheep sperm produced pregnanaes. This modification in the treatment of goat sperm in the IVF system was sipifkant in that it allowed the production of ovine X caprine hybrid

embryos. There have been numerous attempts to produce these embryos in oioo by cross breeding ewes and bucks which readily mate, but do not conceive (Berry, 1938; Buttle and Hancock, 1966; Kek, 1992). These in uifro produced, ovine X caprine embryos were capable of development to the blastocyst stage and even demonstrated the abiLity to hatch from the zona pellucida. The developmental potential of these embryos foLlowing embryo transfer to recipient sheep and goats is discussed in the following chapter. Due to the limited availability of ovine and caprine oocytes, only those oocytes which were obviously abnormal or degenerating were exduded from culture. That is, ovine and caprine cumulus-oocyte complexes were not as highly selected as those of the bovine. This would account, at least in part, for the somewhat lower rates of penetration, cleavage and development for

ovine and caprine crosses compared to those utilking bovine oocytes.

Bovine and Ovine Crosses In vitro production of viable bovine (Brackett et al., 1982; Xu et al., 1988), ovine (Crozet et al., 1987; Czlonkowska et al., 1991; Kelk et al, 1992) and caprine (Hanada, 1985; DeSmedt et al., 1992) embryos has proven successful under various conditions in several different Iaboratories. However, it remains crucial to establish a base for cornparison within each laboratory and if possible within each experirnental trial. Bovine, ovine and caprine crosses were examined in oitro in this study to ver* whether the culture system uçed was compahile with each species and to provide a basis for cornparison of penetration, deavage and development rates. The abundant supply of bovine ovaries dowed numerous replicates of crosses involving the bovine oocyte. This &O allowed for the bovine cumulus-oocyte complexes (COC) and oocytes to be more stringently selected before and after maturation. As a result, the bovine cross yielded the highest penetration and deavage rates (80.4% and 70.1% respectively). These numbers are nearly 10% better than those previously reported (72.4% and 61.0%; Kek, 1992). This is likely due to better COC/oocyte seledion as well as development of a more efficient culture system which alIowed faster processing of oocytes/embryos at each stage of culture. In this study, al1 culture steps were performed with 100-125 oocytes/embryos in 500 pl wek of medium with no oil overlay, in contrast to the microdroplet system used in the previous report (Kelk, 1992). The bovine control cleavage rates are comparable to those observed by other researchers which range between 72% and 87% (Hamano and Kuwayama, 1993; Carolan et al., 1994; Keskintepe and Brackett, 1996). Fertilization of bovine oocytes by rarn sperm is intriguing since bovine oocytes are readily penetrated by ram sperm under in uitro conditions (58.3%) but it was not possible to achieve fertikation during in oioo trials (Kelk, 1992). The penetration rates observed in this study were higher than those reported in previous studies (58.3% versus 45.4% Kek, 1992; 41.2% Slavik et al., 1990). This again suggests that irnprovements to the culture system over that previously used Ied to higher penetra tion and development rates. Development of in vitro fertilized bovine X ovine hybrid embryos arrests at the 4 to 8-ce11 stage whether cultured under in vitro or in vivo conditions. Of those embryos that deaved in vitro, nearly equal numbers of embryos arrested at the 4- and 8-cell stages (43.3% and 47.1% respectively).

This was &O true of in vivo control embryos recovered after temporary transfer to sheep oviducts. These embryos confirmed that the devdopmental block observed was not due to insufficient cuiture conditions but is more likely due to a mechanism within the embryos themselves. Since thiç is the time at which expression of the bovine embryonic genome becomes essential to Merdevelopment of the embryo (Barnes and First, 1991), it is possible that there is either a genornic incompatibility between the ovine and bovine components of the nucleus or that there is an incompatibility between the hybrid nucleus and the bovine cytoplasm. Both possibilities are examhed in Chapters 3 and 4. INTERSPECIFIC HYBRID EMBRYO TRANSFER AND PREGNANCY

INTRODUCTION The previous chapter outiined the developmental potential of various interspecific hybrid embryos under in vitro conditions. Aithough it was previously known that goat X sheep hybrid embryos could be formed in oivu by cross breeding does and ramç and that these embryos could establiçh pregnancies in the cross bred does, the reciprocai cross could not be adiieved in vivo. However, in the case of ovine oocytes fertdized by buck sperm in oitro, the resulting embryos developed to the blastocyst stage and would even hatch. Further developrnental potential of these embryos was evahated by transferring these embryos to recipient ewes and does to examine whether ovine X caprine interspecific hybrid embryos could establiçh a pregnancy. Ln this study, the in vitro produced ovine X caprine hybrid embryos were transferred to both recipient does and ewes to examine whether the recipient species had any effect on the success of the pregnancy. Since genomic imprinting is known to play a sigmficant role in pregnancy and placental development, it was suspected that there might be some difference in the success of hybrîd pregnancies if the embryo was hansfered to a recipient species which matched the patemal species of the embryo. The established pregnancies were monitored closely by serum progesterone analysis and ultrasonographic evaluation and compared with normal, gestational age- matched goat and sheep pregnancies. The primary objective for this study was to determine if in vitro produced ovine X caprine hybrid embryos codd establish a pregnancy and carry to term, in either a goat or a sheep recipient. When pregnancies were established, the pregnancies were monitored dosely since it was suspeded that fetal loss might occur. The secondary objective was to attempt to characterize the nature of the fetal failue through gross and cytogenetic examinations of the fetuses as welI as gross and histological examinations of the placentae. Cytogenetic analysis was uçed to confirm that the fehws were indeed hybrids and that there were no chromosomal anomalies. Histologicd examinations of the hybrid placentomes were performed and compared with age-matched ovine control placentomes to detennine the nonnalcy of the hybrid placentae. The final objective was to determine if there was any polarity in the succesç of the pregnanûes between different types of recipients to draw inferences with respect to genomic imprinting and matemal or paternal contributions to embryo / fetd develoment.

METHODS Production of Interspecific Hybnd Embryos Ovine oocytes were isolated and fertilized with goat sperm and the resulting embryos were cultured for 5, 6 or 7 days as described in Chapter 1 before embryo hansfer to reapient ewes and does.

Synduonization of Recipient Ewes and Does Embryo tramfers were performed to both sheep and goats on two different days in September and November. There were 2 or 3 moda or blastocyst stage embryos transferred to each of 17 ewes and 16 does. Retipient ewes and does were synchronized with medroxyprogesterone acetate vaginal pessaries and PMSG as described in Chapter 1. The pessaries were removed 6 to 8 days before embryo transfer to best synchronize embryos and reapients. Emb ryo Transfer Food and water were withheid from ewes for 24 hours prior to scheduled surgery. Ewes were sedated with 15 mg (a dose ranging from 0.15 to 0.33 mg/kg body weight) xylazine (~orn~un@,Appendix 11) administered intramuscularly. A total of 10.0 ml of 2% lidocaine (Appendix II) was administered locally as a heblock. Reapient does were also prepared in this manner except that they were

anaesthetized with a mixture of 3 mg (a dose ranging Crom 0.07 to 0.09 mg/kg body weight) xylazine (~om~m@,Appendix II) and 150 mg (3.5 to 4.5 mg/kg) ketamine hydrodilo ride (~o~arsetic@, Ap pendix II) injected intramuscularly. Each ewe/doe was restrained at about 45O from horizontal, with the head down, for laparoscopy. A Veres pneumoperitoneum needle (Appendix II) attached to a carbon dioxÎde (CO2) cyLinder was inserted into the left caudal abdomen and the abdominal cavity was inflated with C02. A stab inasion was made, 6 to 10 an anterior to the udder, 4 cm laterd to the midline on each side. A pyramidal trocar and cannula were inserted into the abdominal cavity via the left stab incision in a horizontal manner, pointing dorsomedially. The endoscope, attached to a 150 watt light source, was inserted through the cannula. Schroeder uterine tenacuhm forceps (Appendix 11) were inserted through the slightly larger stab incision on the right side. The ovaries were examined for the presence of a corpus luteum (CL) by using the forceps to manipulate the uterine horns as necessary. The horn ipsilateral to a CL was grasped with the Schroeder forceps and the tip of the uterine hom was exteriorized. A tom cat catheter attached to a 1 ml syringe was used to draw up two or three morula or blastocyst stage embryos in about 10 pl of PBS supplemented with 10% fetal calf serum (FCS)which was isolated between two air bubbles. A hoIe was made in the tip of the uterine horn with a Hunt 20 ga needle and the tip of the tom cat catheter was passed through this hole into the uterine lumen. The embryos were expelled into the ute~elumen and the tom cat catheter was removed. The cannulae and instruments were removed from the abdomen and abdominal CO2 escaped via the -pet valve of the trocar cannula. The ewe/doe was lowered into a horizontal position and the three stab wounds were sutured.

Pregnancy Diagnosis and Fetal Monitoring Pro~esteroneAssavs Blood samples (5-20ml)- were drawn From the jugular vein of all recipient ewes and does on day O and on the day of embryo transfer as weli as every 2-3 days until approximately day 40 when an absolute pregnancy diagnosis could be made using ultrasonographic analysis. The senun was collected, aliquoted and held at -20°C until a progesterone assay was performed. Progesterone was extracted with ethyl ether for assay and concentrations of progesterone were measured using previously described RIA methods (Abraham et al., 1971; Vighio and Liptrap, 2990). In cross- reactivity assays, the antibody to progesterone cross-reacted 1.5% with hydroxyprogesterone and less than 0.3% with other gonadal and adrenal steroids. Sensitivity of the progesterone assay was 12.6 pg/ml and the intra- and interassay coefficients of variation were 4.6 (n=7) and 10.3 (n=6), respectively. -?hic Diagnosis Reapient ewes/does were examined for pregnancy at 30 days post- fertilization using real time B mode ultrasonography with a 5 MHz transducer inserted rectally. An initial positive or negative diagnosis of pregnancy was made, but there was no attempt to estimate the number of fetuses present The recipient ewes/does were scanned again every 3-4 days until day 40 of gestation and daiIy therea€ter, to confirm the initial diagnosis, evaluate the number of fetuses present and obseme the timing of fetal death. Fetal death was evident by a Iadc of independent fetal movement and Iack of a fetal heart beat.

Examination of Fetuses and Reproductive Tracts Un the day in which a feuwas found dead, the recipient ewe/doe was humanely euthanized by an intravenous injection of 30 ml of sodium phenobarbitol utha han sol@, Appendix II). The entire reproductive tract, including fetus and fetal membranes was removed and transported to the laboratory. The utems was carefully opened with a small pair of surgical scissors such that the diorio-allantoic membrane was not ruptured. The uterus was opened to reveal the fetus(es) and placentomes before photographing the entire structure. Amniotic Buid was aspirated from the aminotic cavity for cytogenetic analysis, the fetuses were removed and measured, and the placentomes counted and measured. Fetal cells were collected from both the amniotic fluid and the fetus for cytogenetic analysis.

Cytogenetic Methods Approximately 10 ml of amniotic Buid were aspirated into a syringe through an 18 gauge needle. The amniotic fluid was centrihged at 1000 xg for 10 minutes and the supernatant was removed. The resulting cdpellet was

resuspended in 12 ml of cdculture medium and was used to seed two 75 ml plastic culture vessels. The cells were cultureci for 4 days at 3g°C in a humidified 5% CO2 environment at which time the medium was discarded

and fresh culture medium was added, The c& were then cultured under the same conditions until a monolayer formed approximately 3-4 weeks later. Mer the crown-nimp length of the fetus(es) was measured, a piece of fetd tissue was removed from a hind limb, rinsed in ceU culture medium and transferred to a petn dish with approximatdy 0.5 ml of hesh cell culture medium. The tissue was cut into srnall pieces which were transferred to 75 ml plastic culture vesseIs to initiate fetal explant cultures. The pieces of tissue were alIowed to adhere to the culture surface for 10 minutes before 8.0 ml of fresh cell culture medium were added to each vessel. The explants were cultured for 4 days at 3g°C in a humidified 5% CO2 environment at which time the medium was discarded and fresh culture medium was added. The cells were then cultured under the same conditions until a monolayer formed approximately 4 to 6 weeks later. When monolayers had formed for both types of cultures, the cells were passaged as follows. The medium was discarded and 7 ml of 1X trypsin were added to the culture vessel for 5 to 10 minutes, as required to suspend the ceb. The -sin solution was transferred to a 15 ml conical centrifuge tube containing 1.0 ml of FCS to inactivate the -sin. The tube was centrifuged for 10 minutes ai ZOO0 xg to pellet the cells. The inactivated tsrpsin medium was removed and the cells were resuspended in 2.0 ml of fresh culture medium. Fifty pl of this ceU suspension were then used to seed a new 75 ml plastic culture vessel containing 8 mI of kesh culture medium. The seeded cek were dtured for 2 to 5 days at 3g°C in a humidified CO2 environment until the cells had grown to approximately 50% confluence. The medium was discarded, 8 ml of fresh cuiture medium were added and the ce& were cultured for 18 to 24 hours before 50 pl of colcemid were added to each culture vessel. The cells were cuitured for 2 hours in the presence of colcemid to block mitotic spindle formation and accumulate cells in metaphase of the ceil cycle. The c& were then trypsinized and coilected as described for the previous passage, but the pellet of cells was resuspended in 10 ml of hypotonic solution (0.075M KI). The cells were Ieft for 10 to 15 minutes at room temperature in the KC1 solution before centrifugation at 1000 xg for 10 minutes. The KCl solution was discarded, 10 mI of 3:l (v/v) methano1:acetic aud fixative were added and the conical centrifuge tube was centrihiged for 10 minutes at 1000 xg. The cell peiiet was resuspended in 15 ml of f-resh fixative and stored overnight at 4OC. The foilowing day the cells were rinsed twice with fresh fixative and were resuspended in 100 to 400 ml of fixative. The cell suspension was then dropped ont0 wet 4OC glass microscope slides and air dned. 'Ilte slides were stained with 4.0% Giemsa as described in Chapter 1, and examined for the presence of metaphase chromosome spreads using a Leih Aristoplan microscope.

RESULTS

Of the 17 ewes which were recipients of ovine X caprine embryos, six (35.3%)maintained elevated prugesterone Ievels between days 15 and 20 of gestation. Five (29.4%) of these ewes were confirmed pregnant by real-time ultrasound by day 32. Of the 16 goats which were reapients of ovine X caprine embryos, four (25.0%) maintained elevated progesterone Ievels between days 19 and 24 of gestation. One (6.3%) of these goats was confhned pregnant by red-üme ultrasound. Each day of ultrasound monitoring of the pregnancies was recorded on video tape and later converted to the photographs displayed in Figure 6. In each case, the Iargest placentomes which codd be found were photographed, since the range of sizes of placentomes hom any one pregnancy varies dong with the specific freeze frame used during the ultrasound monitoring. The ultrasound records indicate that the hybrid pregnanues had consistentiy smaller placentomes, ranging from 4 to 12 mm compared with age-matched sheep (19 mm) and goat (14 mm) placentomes. In addition, the hybrid placentomes ladced the weii defined, concave shape of those from control pregnancies. It is evident from Figures 6a and 6b that placentomes of both normal goat and sheep pregnancies assume a discernable concave or donut shape by day 42 of gestation. In contrast, the placentomes of ail respective age- matched hybrid prepancies (goat X sheep in goat at day 43; sheep X goat in sheep at day 42; sheep X goat in goat at day 43) remain as srnalier, Bat buttons of tissue (Figures 6c-e respectively). In addition to evaluating the progression of placental development, the ultrasound monitoring allowed precise determination of the time of fetal death in the hybrid pregnancies. The single pregnancy of a sheep X goat hybrid fetus carried by a goat recipient died on day 51 of gestation, The crown-nimp length of the fetus (Figure 7)was 7.9 cm and the placentomes measured up to 20 mm whidi is smder than the average 25 mm pIacentomes found 4 days eariier, in day 47 sheep pregnancies (Figure 9c). The placentomes, despite measuring up to 20 mm in diameter, continued to lack the characteristic concave shape of normal sheep and goat placentornes. The karyotypic composition of this fehis was confirmed by a chromosome spread (Figure 8), prepared from a fibroblast FIGURE 6. Caprine, caprine X ovine, ovine X caprine and ovine dhasonographs. Note the centimetre marks dong the top or lefi side of each ultrasonopph.

6a. Ultrasonograph of a placentorne (p) hom a normal day 42 goat pregnancy. Note the size (1.4 an diameter) and the concave nature indicated as a donut shape.

6b. Ultrasonograph of a placentomes (p) from a normal day 42 sheep pregnancy. Note the size (1.9 cm diameter) and concave shape.

6c. Ultrasonograph of placentomes (p) of a day 43 goat X sheep hybrid pregnancy carried in a goat reapient. Note the very small size (0.4 an diameter) of the placentomes.

6d. Ultrasonograph of placentomes (p) of a day 42 sheep X goat hybrid pregnancy carried in a sheep recipient. Note the srnd size of the placentomes (1.0 cm diameter).

6e. Ultrasonograph of placentomes (p) of a day 43 sheep X goat hybrid pregnancy camed Ïn a goat recipient. Note the size (1.2 cm diameter) and lack of concave shape of the placentomes.

FIGURE 7. A day 51 ovine X caprine hybrid fetus and uterus of reapient goat. Note the fetal crown-nimp Iength of 7.9 cm and matemal carundes (matemal portion of the placentomes) which measure up to 2.0 cm in diameter but la& the characteristic concave shape of normal goat pregnancies. The fetal membranes and cotyIedons have been removed.

FIGURE 8. Chromosomes of ce& derived from amniotic hid from a 51 day ovine X caprine hybrid fetus carried in a goat recïpient.. Note the diploid chromosome complement of 57 chromosomes, including three large bi-armed ovine chromosomes (1,2 and 3). (X4000).

culture. This hybrid fetus had the characteristic diploid chromosome count of 57 including 3 unpaired bi-armed dvomosomes from the sheep. Control, normal sheep pregnanaes were examined to compare fetd crown-rump length and placental development with those hybrid pregnanaes carried in reapient sheep. Photographs of these day 41,44 and 47 normal sheep pregnanaes are shown in Figure 9a-c respectively. Although the relative weight and volume of the placentomes codd not be accurately measured, a dramatic increase in placentome development is evident. Hacentorne size decreases with increasing distance from the htus as the placenta grows into the homs of the uterus. For this reason, the 10 largest placentomes, near to a htus were measured and averaged to estimate the relative size differences of placentomes, between pregnancies. The average diameter of sheep placentomes, which increased from 14 mm (day 41) to 19 mm (day 44) to 25 mm (day 47) gives some indication of the sigrufïcant growth which occurs within this window of tirne. The five pregnancies resulting from the transfer of sheep X goat embryos to sheep recipients ended with fetal death at days 35 (single), 40 (single), 43 (twin), 43 (single) and 47 (twin). The opened uterus displayhg the fetus(es) and placentomes of these pregnancies are shown in Figure 10a-d. The hybrid placentomes were all drasticdy smder (averaging 6.6 to 8.6 mm) than those of the normal sheep pregnancies (averaging 14 to 25 mm) and appeared as flat buttons, lacking the charaderistic concave shape as was previously indicated in the ultrasound observations. In each case the diameter of the largest placentomes was less than 1.0 cm. Upon dissection, the fetal cotyledons of all hybrid pregnancies separated readily from the matemal carunde. In the case of control sheep gestations, the finger-lüce fetal villi, even at 41 days of gestation, required FIGURE 9a. Photograph of a normal twin sheep pregnancy at 41 days gestation. Note the placentomes (p) up to 1.6 cm in diameter with a concave or donut shape.

9b. Photograph of a normal twin sheep pregnancy at 44 days gestation. Note the size of the placentomes (p) has inaeased significantly, up to 2.4 un in diameter, with only 3 days additional gestation than those shown above.

FIGURE 9c- Photograph of a normal triplet sheep pregnancy at 47 days gestation. Note the size of the placentornes (p) has mer inaeased, up to 2.9 an in diameter.

FIGURE 10a. Photograph of an ovine X caprine singleton pregnancy camed in a ewe recipient fooUowuig Çetal death at 40 days gestation. Note the placentomes (p) are merely small flat buttons of tissue, less than 1.0 cm in diameter.

lob. Photograph of an ovine X caprine twin pregnancy carried in a ewe recipient Çollowing fetai death at 43 days gestation. Note the placentomes (p) are merely small flat buttons of tissue, less than 1.0 cm in diameter.

FIGURE 10c Photograph of an ovine X caprine singleton pregnancy carried in a ewe reapient following fetal death at 43 days gestation. Note the placentomes (p) are merely srnaII flat buttons of tissue, less than 1.0 an in diameter.

10d. Photograph of an ovine X caprine twin pregnancy carried in a ewe recipient followuig fetal death at 47 days gestation. Note the placentomes (p) are merely smalI 8at buttons of tissue, less than 1.0 an in diameter. The fetus on the left side of the figure failed to have a detectable heart between days 41 and 43 of gestation. peeling ftom the matemal caruncle where they had interdigitated. By day 47 of gestation of a normal sheep pregnancy, it was not possibIe to separate the fetal and maternal components of the placentome without ripping the fetal viK which had brded and penetrated into the maternd carunde. The total number of placentomes ranged from 91 to 153 in hybrîd pregnancies and 97 to 132 in normal sheep pregnanues. There was no correlation between either the precise length of gestation (days 40 to 47) or number of fetuses (single, twin or triplet) and total placentome number. The crown-nimp lengths (CRL)of both the control sheep fetuses and sheep X goat hybrid fetuses carried in sheep recipients, according to gestational age are displayed in Figure 11. The CRL of normal control sheep fetuses are Uustrated by triangles, with the shaded area indicating the 95% confidence interval. Sheep X goat hybrid fetal CRL are indicated by the horizontal bars and squares. The width of the bars indicates the tirne period between ultrasound observations in which fetal cardiac activity ceased. This diagram illustrates that the CRL of hybrid fetuses fall below the 95% confidence interval for age-matched sheep control ktuses. The gross morphologicai observations of the la& of interdigitation of the hybrid pregnancies in cornparison to control sheep pregnanues was confirmed with histological examination of the placentomes. Normal sheep control placentomes at al1 three times (days 41, 44 and 47) all showed extensive interdigitation of fetal villi into the matemal caruncles (Figures 12a-d). Histological examination of the sheep X goat hybrid placentomes (Figure 13a-f) revealed that there was minimal invasion of fetal villi into the maternal caruncle. Crown- R-P Length 5 (cm)

Gestation (days)

Crown-rump lengths (CRL)of normal control sheep ktuses and sheep X goat hybrid fetuses carried in sheep retipients, accordhg to gestational age. The CRL of normal control sheep fetuses are flustrated by triangles, with the shaded area indicating the 95% confidence intemal. Sheep X goat hybrid fetal CRL are indicated by the horizontal bars and squares. The width of the bars indicaies the window of time in which fetd cardiac activity ceased. The open cirde represents the tirne at which a fetd heart beat was last obsenred and the dosed square, the time when no fetal heart beat was deteded. FIGURE 12a. Histological section of a placentorne hom a normal twin sheep pregnancy at 41 days gestation. Note the penetration of the fetal villi (f) into the matemal cmcle (m). (X400).

12b. Histological section of the same placentorne above at higher magnification. (Xl6OO).

FIGURE l2c. Histological section of a placentome from a normal twin sheep pregnancy at 44 days gestation. Note the extensive penetration and branching of the fetal villi (0 into the maternai caruncle (m), (X400)-

12d. Histological section of a placentome from a normal twin sheep pregnancy at 47 days gestation. Note the continued penetration and branching of the fetal villi (f) into the maternai caruncle (m). (X400).

FIGURE 13a. Histological section of a placentome from a singleton sheep X goat hybrid pregnancy at 40 days gestation carried in a sheep recipient. Note the la& of penetration of the fetal villi (f) into the matemal caruncle (m). (X400).

13b. Hiçtological section of the same placentome above at higher magnification. (Xl6OO).

FEGURE 13c. Histological section of a placentorne from a twin sheep X goat hybrid pregnancy at 43 days gestation carried in a sheep recipient. Note the lirnited penetration of the fetd villi (f) into the materna1 caruncle (m). (X400).

136. Histological section of the same placentorne above at higher magnification. (X1600).

FIGURE 13e. Histological section of a placentorne hom a twin sheep X goat hybrid pregnancy at 47 days gestation camed in a sheep recipient. Note the lîmited penetration of the fetal villi (f) into the matemal caninde (m). (X400).

13f. Histological section of the same ptacentome above at higher rnagmfication. (X1600).

DISCUSSION Interspecific pregnancies have been established and examined in a nuniber of species inciuding mouse (Crepeau, 1988), horse and donkey (Allen et al., 1985) and Dall's sheep in domestic sheep (BuckrelI et al., 1990). In many cases, interspecific mating or interspecific embryo transfer establish pregnancies, but these rarely proceed to full tenn development and birth of live offspring. It has been known for over 60 years that does conceive when interbred naturaily to rams (Berry, 1938). The resdtuig pregnancies generally result in fetal death during the second month of gestation (Warwick and Berry, 1949). Previous studies of the reciprocal cross between ewes and bucks have shown that despite natural mating, this cross invariably failed (Warwick and Berry, 1949). The failure of this cross was later shown to be due to failure of the goat sperm to fertilize sheep oocytes in the sheep reproductive tract (Kelk, 1992; Kelk et al., 1997a). Eppleston and Moore (1977) reported that although sheep oocytes were not fertilized by goat sperrn in the sheep oviduct, 27% to 75% of sheep oocytes were fertilized within the goat oviduct if they were transferred from a sheep to a goat within 36 hours of the onset of es tm. This study, dong with a study which shows that sheep oocytes cmbe fÊrtilized in vitro by goat sperm (Kelk et al., 1994a), indicates that the barrier to fertilization is not at the level of the zona pellucida but within the sheep reproductive tract. It is most likely that goat sperm capacitation does not occui: in the sheep reproductive tract, thereby preventing fertilization. This is the first report to show that sheep oocytes are readily fertilized by goat sperm under in vitro conditions and that the resdüng embryos are capable of initiating a pregnancy. Furthemore, pregnmaes were established in both sheep and goat retipients. The production of this particular hybrid allows for a complete experimental mode1 whereby both reciprocd hybrid embryos as well as control sheep and goat embryos can now be hansferred to either sheep or goat recipients. Further examination of the resulting combination of pregnancies WU aIIow for significant understanding of the matemal and patemal roles in placental development and pregnancy.

The ultrasonographic examination of the interspecific hybrid pregnancies was the fist indication that the pregnanaes were not progressing normally. The most stnkÏng difference between interspecific hybrid pregnancies and normal sheep and goat pregnanaes was prior to day 40 when the hybrid placentomes were very difficult to find. When found, these placentomes were drastically smaller than those of age matched, normal sheep and goat pregnariues. As the normal sheep and goat pregnanues progressed beyond day 40, the size of the placentomes increased rapidly and began to take on the more mature concave shape. This timing correlates gestationally with a hlly formed fetal heart, and may be indicative of an increased metabolic rate and increased nuhitional and respiratory need for the fetus. uiterestingly, thiç time also correlates with the approximate tune that the sheep placenta begins to replace the sheep corpus luteum as the primary source of p rogesterone (Robertson, 1977; Hafez, 1993). The placentomes of the interspecific hybrid pregnancies however, never developed a mature, concave shape and remained as smd Bat buttons of tissue until fetal death erisued- Fetal development was normal dtrasonographically. It was difficult to determine if fetal size was significantly compromised through simple dtrasound examination. Ultrasound however, could ako be used to monitor fetal heart rate which is a predidor of fetal distress prior to fetal death. One day 46 sheep X goat hybrîd fetus c&ed in a sheep reapient had a rdatively high heart rate of 172 beats per minute More fetal death occurred on day 47. The heart rates of these fetuses were not monitored dosely enough to give an absolute indication that they were abnormal but thiç measurement should be incorporated into future examinations of interspeafic pregnanaes. Ultrasound can also be used to diagnose hydrometra or pseudopregnancy in the goat (Hesselink, 1993; Hesselink and Taverne, 1994) and would be usefd to monitor hydrops uteri. Hydrops uten is an uncommon condition in goats caused by an abnormaI accumulation of fiuid (often greater than 10 L) in either the amniotic (hydramnios) or allantoic (hydrallantois) sacs (Jones and Fecteau, 1995). Hydrops uteri in a goat pregnant with goat X sheep hybrid fetmes (Jones and Fecteau, 1995) may also

give indication of the cause of Mure of hybrid pregnanaes. McGovem (1977) reported that the amniotic fluid volume was significantly lower and allantoic volume was significantly higher for goat X sheep hybrid conceptuses than control goat conceptuses. Cows pregnant with cow X bison hybrids also have a substantially greater chance of developing hydrops uteri than those pregnant with nonhybrid fetuses, and fetal and placental defects are the postulated cause (Basnir, 1986b). Real-time ultrasound cmbe used after day 40 of gestation to diagnose interspecific hybrid pregnancies in goats which could result from indiscriminate matuig with a rarn. This is of clinical significance since incidents of Mdvertent interspecific pregnancies between does and rams do occur on farms. It is impossible to estimate the frequency of these pregnancies because virtuaUy all goat X sheep interspecific pregnanaes are lost dbgthe second month of gestation and are not detected because the fetuses are either resorbed or aborted and lost in the animal's bedding- Ultrasound has been impIicated to have an adverse effect on reproduction in rats (Bologne et al., 1983) as well as humans (Deinoulin et al., 1985). These reports focus on foilicdar dynamics rather than the effect of ultrasound on fetal development. Heyner et al., (1990) however, reported that in mice, diagnostic levels of puIsed ultrasound did not affect either the number of embryos produced, or the ability of those preimplantation embryos to synthesize DNA and RNA, Regular use of ultrasound to monitor the hybrid pregnanaes may have caused a small degree of stress for the recipients but this is not the suspected cause of fetal loss nor reduction in fetai size. Sheep X goat pregnancies not monitored with ultrasound result in spontaneous abortion at approximately the same gestationai age (Kelk et al-, 1997a).

Hybrid Fetus Previous researchers performed reciprocal embryo transfers of both sheep embryos into goat reapients and goat embryos into sheep reapients (Warwick and Berry, 1949; Hancock and McGovern, 1968). Bot. groups found that goat embryos in sheep failed to survive much beyond 20 days after transfer, while sheep embryos in goats survived for at least 30 days. The sheep X goat and goat X sheep hybrid pregnancies produced in this study, showed development beyond day 40 of gestation regardless of the combination of hybrid and reapient, indicatirtg at least a smail advantage for embryos which had half of the genome which matched theïr recipients. h the case of sheep X goat hybrids carried in sheep recipients, the fetd crown- rump length was less than age matched sheep fetuses. The hybrid fetuses also appeared to be smaller than published estimates of fetal age for both sheep and goat (Sergeev et d.,1990; Gall et al., 1994; Sivachelvan et d., 1996). Interspecific chimeras between sheep and goats have also been produced to examine the nature of interspecific pregnancy. In the first attempts to produce interspeufic chimeras, one or more 4- or 8-ceII blastomeres from either sheep or goat embryos were combined with one or more 4- or û-ceU blastomeres from the opposite speaes (FehiIly et al., 1984; Meinecke-Tillmann and Meinecke, 1984). The resulting dllmeric embryos were different from interspeufic hybrids because they consisted of a combination of two unique ceU populations (sheep and goat). Interspecific hybrid embryos on the other hand consist of one uniform hybrid cell population. Nevertheless, the chimeric embryos in both of those studies produced pregnancies which developed to term to yield Iive offspring. It was proposed that the diimeric animals might finciion as successfu.I recîpients for both goat and sheep as weLl as goat X sheep hybrid embryos. Unfortunately, although the chimeras appeared to carry hybrid pregnancies longer than ewes and does generally carry hybrid pregnancies, none were carried to term (Anderson et ai., 1991; Gustafson et al., 1993). Subsequen t production of chimeric embryos utilized a modified blastocyst injection tedinique where an immunosurgicdy isolated inner ceU mass of one embryo was placed into the blastocyst of another embryo (Roth et al., 1989). This group showed that goat X sheep hybrid embryos could be rescued and develop to term when the trophoblast portion of the embryo was matched to the reapient (sheep) species. The inherent differences between chimeric, hybrid and interspecific pregnancies should be noted. In a chïmeric pregnancy a portion of the cells of the embryo would generally be of the same species as the recipient. The success or failure of these pregnanues generaIly seems dependent on the speaes of cells which colonize the fetal portion of the placenta (Roth et al., 1989). In a hybrid pregnancy, all of the cells of the fetus and fetal membranes carry haIf of each parental speues' genome, while interspecific pregnancies are a result of the transfer of an embryo of one speues to a recipient of a different speues. Interspeufic embryo transfers have been explored as a method to propagate endangered species with mixed success (BudcreIl et al., 1990; Flores-Foxworth et al., 1995; Summers et id., 1987). Despite failures of many interspecific pregnancies such as DalI's sheep in domestic ewes (BudcreU et al., 1990), successhil production of offspring has been achieved with transfers of Red sheep to domestic sheep (Flores-Foxworth et aL, 1995) and Przewalski's horse and Grant's zebra to domestic mares (Summers et al., 1987). The success of interspecific pregnancies where there is no direct genomic contribution to the embryo by the recipient gives hope for the success of hybrid pregnancies where there is genomic investment by the recipient / matemal species.

Placentornes The gross morphology of both the goat X sheep and the sheep X goat placentomes was invariably different from those of age matched control sheep and goat placentomes regardless of whether the hybrÏd was carried in a sheep or goat recipient. In each interspecific hybrid pregnancy, the placentomes were, as the ultrasonographic examination had indicated, small and flat compared with the larger, concave placentomes of age matched control sheep and goat pregnancies. The histologicd examination of these placentomes further confirmed that they were abnormal, There was virtually no penetration of fetal villi into the materna1 caruncle. A similar observation was made with histological examination of a heifer placentome der interspecies trançfer of a gaur embryo (Hradew et al., 1988) which ultimately ended in the delivery of a dead 9.5 month-old gaur calf. Although the heifer placentome appeared noma1 in size, miaoscopicaUy it was noted that the fetal villi had failed to brandi completely and entered only about one haIf of the available matemal crypt spaces (Hradecky et al., 1988). The lack of interdigitaiion of fetd and matemal surfaces in hybrid and interspecific pregnancies would result in a substantidy reduced surface area for oxygen and nutnent exchange. This reduced potentiaI for nutrient and gas exchange would likely cause respiratory, nutritional and/or toxin distress for the fetus and could account for the somewhat underdeveloped fetal size observed and ultimately fetal death.

The fetal heart beat ceased in 4 of the 5 sheep X goat hybrid pregnancies carried in sheep reapients with a narrow period of time (day 41 to day 47 of gestation). To examine the normal changes and development of the fetus and placenta at this tirne, nomal sheep pregnancies were examuied at days 41, 44 and 47. It was evident through gross examination of 7 control sheep pregnancies carryïng a total of 17 fetuses, that the placentornes were undergohg a significant growth phase between days 41 and 47 of gestation. This is the time interval during which the sheep placenta becomes the dominant source of progesterone (Robertson, 1977; Hafez, 1993). In contrast, goat pregnancies are dependent on a functional corpus Iuteum throughout gestation (Robertson, 1977; Hafez, 1993). It is not likely however, that the hybrid pregnancies failed due to a lack of appropriately controlled progesterone production since previous researchers have supplemented goats carrying caprine X ovine pregnancïes with progesterone with no prolongation of gestation (Hancock et al., 1968). The obsenration that the hybrid placentomes were not as devdoped as normal, control sheep placentomes was hrther confirmed through histological examination of the placentomes from these pregnancies. There was increased invasion and branching of the fetal villi into the matemal carunde between day 41 and day 47. The significant growth and fetal invasion of the sheep placentorne during this short penod of time is indicative of a shift in fetal pIacental gene expression. Genomic imprinting plays a significant role in placental development (DeGroot and Hochberg, 1993; Moore and Reik, 1996). An imp~tedshift in fetal placental gene expression may lead to miscommu~cationbetween the sheep and goat portions of the genome in the sheep X goat hybrid pregnancies. Considering the obviously complex dialogue which must occur between the matemal and Çetal components to placental development, it is not surprishg that interspecific hybrid pregnanues generdy fail to estabhh a suffïcient nutrient exchange mechanism to support a fetus. The sheep and goat in this case provide a unique research model through the creation of interspecific hybrid embryos which cm be transferred to either recipient species. This model allows for matching either the matemal or patemal species of the hybrid embryo to the recipient species in ail combhations and permutations. Careful his tological monitoring, dong with smtiny of the expression of irnprinted genes uivolved in placental development would yield insight into the mechanism by which a fetus can successfully eliut nutrients through the placenta. Potential causes of spontaneous fetal loss could be examined as well. RNA AND PROTEIN SYNTHESIS IN INTERSPECIES HYBRID EMBRYOS

INTRODUCTION During rnammalian oogenesis, proteins and &As are sequestered in the oocyte prior to fertilization, and the earliest stages of embryogenesis are

regulated by these matemally inherited components within the oocyte. This control is often referred to as the post-transcriptionai control or matemal control period. As ma temally derived molecules decay, embryogenesis becomes dependent on products derived frorn expression of the embryonic genome. Termination of the materna1 contror period is marked by initiation of hanscription of the embryonic genome or transition to embryonic conho1 of development. The timing of this event varies between cattIe, sheep and goats as indicated in Table IV.

TABLE N: Timing of developmental events in bovine, ovine and caprine emb ryogenesis.

Develo prnental Event 1 Bovine Ovine I RNA synthesis 2-cell 8-16-cell unknown I Nucleolar transformation 4-cell 8-cell

a-ammitin sensitivity 8-ce11 8-16-ce11 1 -1 protein profile shift 4-cell 8-16-ceLI unknown 1 in oitro block 1 8-16-cell 1 û-16-cd

There has been an increasing interest in understanding the molecular events which occur during early embryo development. The understanding of these events should aid not onIy in reducing ernbryonic mortality in vitro and potentially in vivo, but also in the refinement of new reproductive technologies such as doning by nudear transfer and production of transgenic animais. Oocyte maturation and fertitization as well as early cleavage development are subject to both genomic and extracellular regulations. The oocyte, zygote or embryo must maintain a balance of two unique genomic sources. One source is established during the process of oogenesis when matemdy derived RNA's and proteins are accumulated within the ooplasm and the other is from the nucleus of the oocyte/zygote/embryo itself. Normal accumulation of maternaIly derîved RNA's and proteins in oocyte development is essential for normal fertilization and continued embryonic development. Equally essential is the participation of the embryonic genome to sustain continued development. Although the bovine embryo can develop up to the 8-celI stage with contribution of the embryonic genome suppressed (Bames and First, 1991; Eyestone and First, 1991; Plante et al., 1994), the embryonic genome is not necessarily quiescent prior to this stage. It was previously thought that the first and second deavage divisions of the bovine embryo proceed without cellular growth, under the sole control of stored materna1 mRNA (Camous et al., 1986). Barnes and First (1991) compared protein profiles of large numbers of in vitro derived bovine embryos culhired in the presence or absence of a-arnanitin and were the first to report activity of the bovine embryonic genome at the 4ceU stage. Since that time, using a more sensitive method of detection involving relatively long exposure to 3~-uridinefollowed b y autoradiography of whole mounted embryos, low levels of RNA transcription have been detected as early as the 2- cdstage (Plante et al., 1994). The objective of this study was to utilize the in aivo and in nitro produced interspecific hybrid and control embryos (Chapter 1) for detailed examination of RNA transcription and protein translation with particdar attention to the influence of the matemal (cytoplasmic) and paternal (nudear) contributions to the early deavage stage embryo. The goal was to find species-specific markers (example: unique pro teins or level of transcription) which could be used to determine if activation of the embryonic genome, and ultimately control of embryonic development, was under the direction of the nudeus or cytoplasm.

MJ3HODS Embryo Production Bovine, ovine, caprine and interspedic hybrid embryos were produced in oitro as described in Chapter 1. Matured oocytes and 2-, 4 and 8-cell embryos were examined at varying times of development ranging fiom 32-58 hours post-insemination. In viuo produced caprine, caprine X ovine and ovine embryos, obtained by superovulation and surgical or slaughter flush (Chapter 1) were also examined.

Radiolabelhg of RNA The technique for detection of radiolabeled RNA was a modification of Plante et al., (1992). Both in vivo and in vitro produced embryos were incubated in 50 ~1 droplets of Ham's-FI0 supplemented with 20 PM 3~- uridine. Each droplet was placed in one weU of a Nunc four-well culture plate, under saline-equilibrated silicone oil. Droplets were equilibrated at 3g°C in a humid atmosphere of 5% CO2 in air for at least 2 hours before the embryos were added. Ali types and stages of embryos were radiolabeled for a period of 8 hours. This was to dow estimation of the relative amount of RNA synthesis among stages of embryos. In the case of in vivo produced sheep and goat 8-cell embryos, those which had disaete, round blastomeres were classified as early 8-cells and those which showed evidence of compaction were grouped as late û-cell embryos. For each replicate, the background was determined with unlabeled controls. For each age and stage of embryo which was examined, negative control embryos were nin in parallel încluding the same incubation the. These control embryos were incubated in 50 pl droplets of Ham's-FI0 supplemented with 20 pM 3~-uridineand an excess of unlabeled uridine (5.0 mg/&; 20 mM). At the end of the incubations, ali embryos were washed twice in cold (4OC) PBS supplemented with 10% fetal caU serum (PBSS). The embryos were then held for 30 minutes in PBSS supplemented with 2.5 mg/rnl of unlabeled uridine at 4OC and washed again in PBSS. Embryos were then fixed and spread on glass slides as described below.

Embryo fixation Embryos were spread on slides according to the technique of King et al., (1979). Embryos were placed in 3 ml of 1.0% (w/v) sodium citrate for 3 min. at room temperature and then spread on dean glass slides with a drop of glacial acetic aud:methanol (1:l; v/v). The slides were dried under an incandescent lamp while gently blowing to spread the embryo. The embryos were then fixed ovemight in glacial aceüc aad:methanol(1:3; v/v) at 4OC. The following day, the slides were air dried at room tempera-. Autoradiograp hy Autoradiography was performed in total darkness. Each slide was dipped into NTB2 photographie emulsion diluted with two parts of water. The slides were air dned for 3 to 4 hous at room temperature before being placed in black, plastic, light-tight boxes with a smd amount of desiccant wrapped in gauze. The boxes were then sealed with bladc eledrical tape and wrapped in alumïnum fol The spread embryos were exposed to the emulsion at 40C for 10 days before the emulsion was developed in Dl9

developer for 3 min. The embryos were subsequently counterstained with 4% Giemsa (v/v) for 4 min.

Radiolabelhg of Pro teins Selected oocytes and embryos were washed in modified TALP medium and labeled by a 6 hour incubation in 50 pl droplets of 35s- methionine + cysteine (specific aclivity - 28 Ci/mmol) dissolved in modified TALP medium (Appendix III) at 38.5'C in a humidified atrnosphere. The embryos were then rinsed 4 times through PBS with 1 mg/d polyvinyl alcohol (PVP). Each embryo was picked up in a volume of 10 pl of PBS with PVP and piaced in an Eppendorf tube to be held at -70°C until being used for scintillation counting or electrophoresis.

Scintillation Counting of Embryo Pro teins Embryos to be used for scintillation counting were removed from the -70'~ freezer and lysed by addition of 10 p1 of 2x dissociation buffer (without bromophenol blue dye) to each Eppendorf tube containing one oocyte or embryo. Each tube was boiled for 3 minutes to denature the proteins and given a qui& pulse in a microcentnfuge to b~gail the contents to the bottom of the tube. The full volume of 20 pl was transferred to a scintillation vial with 5 ml of scintillation cocktail (EcoScint A) and counted using a scintillation counter.

Electrophoresis Embryos to be used for SDÇPAGE were removed from the -700C freezer and lysed by addition of lOml of 2x dissoaation buffer containing bromophenol blue dye to each eppendorf tube containing one oocyte or embryo. Each tube was boiled for 3 minutes to denature the proteins and given a qui& (2 sec.) puise in a microcentrifuge to bring aLI the contents to the bottom of the tube. Radiolabeled proteins were separated and resolved by one-dimensional

SDS-PAGE carried out on a BioRad mini gel apparatus attached to a BioRad power supply according to the method of Laemmli (1970). The entire 20 pl of SDSdissociated extracts derived from one embryo was loaded into a well on either a 12.5% or 4-15%, 0.5 mm thidc polyaaylamide slab gel. Low and high molecular weight markers were loaded into the outermost empty Left and right lanes respectively. Electrophoresis was carried out on two slab gels simultaneously for 18 min. at 30 mA, foUowed by 40 mA for 42 min. Following eiectrophoresis, the gels were fixed in gel fixative for at least 4 hours before being stained for at least 30 minutes in Coomassie Blue stain to indicate the molecular weight standards. The gels were then washed in several changes of destaining solution over 3-6 hours followed by two, 5 minute rimes in distilled water and a final treatment of 1M sodium salicylate for one hour to enhance the autoradiography. The gels were then dried ont0 filter papa and exposed to Kodak X-ray film for 1-7 days. RESULTS RNA Transcription Autoradiographs of in vivo produced 4, early 8- and Iate &cell caprine (CXC), caprine X ovine hybrid (CXO) and ovine (0x0) embryos following 8 hours of incubation with 3~-rilidineare shown in Figure 14. AU 4- and early 8-cell embryos (Figure 14a, b, d, e, g, h) show low levels of 3~-uridine incorporation with a localized concentration of silver grains over the nucleoli. Dense labehg of all nudei is prevalent as well as the presence of silver grains in the cytoplasm of aii late 8-cell embryos (Figure 14c, f, i)- There was no obvious difference between the caprine X ovine hybrid and control caprine and ovine embryos at any given cell stage, despite indications from the literature that sheep and goat embryos undergo nucleolar transformation at different stages (see Table TV for summary). Figure 15 displays the RNA autoradiographic patterns for in vitro produced 2-, 4- and 8-cell bovine (BXB), bovine X ovine hybrid (BXO) and ovine (0x0) embryos following 8 hours of incubation with 3~-uridine.Aü 2- and 4-cd embryos (Figure 15a, b, d, e, g, h) show low levek of 3~-uridine incorporation, although those embryos generated from bovine oocytes (ie. BXB and BXO) appear to exhibit a higher level of transcriptional advity, particularly at the 4-cell stage than the ovine control embryos. The 4-ceii BXO hybrid embryo (Figure 15e) indicates generous transcriptional activity prior to developmental arrest. There is dense labeling of the nuclei of 8-cell bovine and ovine ernbryos (Figure 15c, i) but the 8-cell BXO hybrid embryo (Figure 15f) does not indicate the continued increase of transcriptional activity exhibited by the control ernbryos. This is iikely due to the developmental arrest of BXO &ceil embryos. FIGURE 14. Autoradiographs of in uiao produced 4, early 8- and late 8- cell caprine (CXC), caprine X ovine hybrid (CXO) and ovine (0x0) embryos following 8 hours of incubation with 3~- uridine- Note that all4- and early &cd embryos (a, b, d, e, g, h) show low leveb of 3~-uridineincorporation with a localized concentration of silver grains over the nudeoIi. Aiso note the dense labehg of all nuclei and presence of silver grains in the cytoplasm of al1 late 8-ceil embryos (c, f, i).

14a. 4ceil caprine embryo.

14b. early 8-ceil cap~eembryo.

14c. late &ceU caprine embryo.

14d. 4-cell caprine X ovine hybrid embryo.

14e. early 8-ceil caprine X ovine hybrid embryo.

14f. late &ceil caprine X ovine hybrid embryo.

14g. 4-ceIl ovine embryo-

14h. early 8-ceil ovine embryo.

14i. late 8-ce1 ovine embryo. mGURE 15. Autoradiographs of in vitro produced 2-, 4- and $-ceil bovine (BXB), bovine X ovine hybrid (BXO) and ovine (0x0) ernbryos following 8 hours of incubation with 3~-uridine. Note that alI 2- and 4-cd embryos (a, b, d, e, g, h) show Iow Ieveis of 3~-~dineincorporation. The 4-cell BXO hybrid ernbryo (e) indicates generous transcriptional activity prior to developmentd arrest. Also note the dense labelhg of the nuciei of %ceLi bovine and ovine embryos (c, i). The 8-ceil BXO hybrid embryo (0 does not indicate the continued increase of transcriptional activity exhibited by the control bovine (c) and ovine (i) ernbryos. This is iikely due to the developmentd arrest of BXO 8-cell embryos.

1%. 4-cell bovine embryo.

15c. 8-ceIl bovine embryo.

15d. 2-cell bovine X ovine hybrid embryo.

15e. 4-ceIl bovine X ovine hybrid embryo.

15f. 8-celi bovine X ovine hybrid embryo.

1 2-cell ovine embryo.

15h. 4-celi ovine embryo.

15i. 8-celi ovine embryo.

FIGURE 15j. Autoradiograph of an in nitro produced 4-ce11 bovine (BXB), coId/negative control embryo following 8 hours of incubation with 3~-uridineand a 1000 times excess of udabeled uridine. This autoradiograph is representative of cold/negative control embryos for aIl species (bovine, ovine, and caprine, as weil as alI hybrids) from the 2-&cell stage-

The generaI pattern of incorporation of SH-uridine was similar for each speaes (bovine, ovine and caprine). There was generally a low level of incorporation in the early deavage stages (2- and 4celI) with a constant uicrease through to the early 8-celi stage. In each case a burst of hanscriptional activity was evident by the late 8-ceU stage. The patterns of labeiing were more consistent for in viuo produced embryos than for those embryos produced in vitro. VVtually all in viuo produced embryos of any particular stage displayed identical labelirig patterns. That is, all 4-ceIl embryos euhibited a low level of labeling which was primarily evident within the nudei. Early 8-ceU embryos showed an increase in this labeling pattern, while late 8-ceU stage embryos exhibited dense labehg of the nudei and a significant concentration of silver grains within the cytoplasm as weiI.

Protein TransIation Autoradiographs of SDS-PAGE of cellular proteins from in uitro matured bovine (B), ovine (0)and caprine (C) oocytes demonstrate a unique and distinctive protein profile for each of the three speues (Figure 16).

Bovine oocytes are more heavily Iabeled than those of ovine and caprine, indicating a higher level of protein synthesis. Autoradiographs of SDS-PAGE of cellular proteins from in vitro produced bovine (BXB), bovine X ovine hybrid (BXO)and ovine (0x0)2- and 4-cd embryos, labeled at 40 hpi (Figure 17) Uustrate that the protein profiles observed in hybrid embryos reflect those of normal bovine embryos and not those of ovine embryos. This figure also demonstrates the relatively low level of protein synthesis in ovine embryos compared to their age and stage matched bovine and hybrid counterparts. The difference in activity between FTGURE 16. Autoradiograph of SDS-PAGE of cellular proteins From in uitro mabed bovine (B), ovine (0)and caprine (C) oocytes. One oocyte was loaded into each lane of the gel. Note the difference in protein profiles between the three speaes and that the bovine is more heavily labeled than ovine and caprine.

FIGURE 17. Autoradiograph of SDS-PAGE of cellular proteins from individual in vitro produced bovine (BXB), bovine X ovine hybrid (BXO) and ovine (0x0)2- and 4-ceU embryos, all labeled at 40 hpi. Note the difference in the amount of labeling in BXB and BXO hybrid embryos compared with 0x0 embryos. The difference in activity between the bovine and ovine derived embryos was so great that it was not possible to achieve adequate exposure for the ovine embryos. Also note that the BXO hybrid embryos have comparable protein profiles to the bovine, oocyte/cytoplasrn-derived specieç. BXB BXO 0x0 BXB BXO 0x0 (24 (24 (2.~1 4 cecl M-CI the bovine and ovine derived embryos was so great that it was not possible to achieve adequate exposure for the ovine ernbryos. By 58 hours in culture post-insemination, many embryos had cleaved to the 8-cd stage but there were stdi some slower devdoping embryos which remain at the 2- or 4-cell stage- Autoradiographs of SDS-PAGE of cellular proteins from in vitro produced bovine and BXO hybrid 2-, 4- and 8-cell embryos, ail labeled at 58 hpi illustrate that all stages of embryos at a given time have the same protein profiIes, regardless of stage (Figure 18). This figure also confinns that BXO hybrid embryos have comparable protein profiles to the bovine which is the oocyte/cytoplasm-derived species of the hybrid embryos. Autoradiographs of SDS-PAGE of cellular proteins from in vifro produced caprine, caprine X ovine hybrid (CXO), ovine X caprine hybrid (OXC) and ovine 4-ce11 embryos, al1 labeled at 48 hpi (Figure 19) yield somewhat different results from the bovine X ovine hybrid embryos. Although the CXO hybrid exhibits a similar protein profile to that of the control caprine (oocyte/cytoplasm) species, the reciprocal OXC hybrid is obviously synthesizing significantly more protein than the control ovine (oocyte/cytoplasm) species. The protein profile is, however, different kom that of the age and stage matched cap~eembryo, indicating that at least some of the increased protein synthesis observed may be from the ovine portion of the hybrid genome. Autoradiographs of SDS-P AGE of cellular proteins from in vitro produced CXO hybrid 2-ceU embryos labeled ai 32,40 and 48 hpi (Figure 20a) show no evident shift in protein profiles from 32 to 48 hpi. Autoradiographs of SDS-PAGE of cellular proteins from in vifro produced CXO hybrid 2-, 4- and 8-ce11 embryos labeled at 32, 40 and 48 hpi, respectively (Figure 20b), FIGURE 18. Autoradiograph of SDS-PAGE of cellular proteins from individual in vitro produced bovine (BXB) and bovine X ovine (BXO) hybrid 2-, 4- and û-cell embryos, all labeled at 58 hpi. Note the similar protein profiles for all embryos, regardless of stage. Also note that BXO hybrid embryos have comparable protein profdes to the bovine, oocyte/cytoplasm- derived speaes.

FIGURE 19. Autoradiograph of SDS-PAGE of cellular proteins from individual in uitro produced caprine (CXC), caprine X ovine hybrid (CXO), ovine X caprine hybrid (OXC) and ovine (0x0) 4cdembryos, aii labeled at 48 hpi.

FIGURE 20a. Autoradiograph of SDS-PAGE of cellular proteins from individual in aiho produced caprine X ovine hybrid (CXO)2- cdembryos, labeled at 3540 and 48 hpi.

20b. Autoradiograph of SDS-PAGE of cellular proteins from in oitro produced caprine X ovine hybrid (CXO) 2-ceU embryo labeled at 32 hpi, 4-cell embryo labeled at 40 hpi and 8-cd embryo labeled at 48 hpi. illustrate a shift of proteins at the &ceIl stage which was not observed in the 2- ceU CXO hybrid embryos of 48 hpi (Figure 20a).

DISCUSSION RNA Transcription Previous studies have used a-amanitin to examine the timing of embryonic genome expression (mouse: Levey et al., 1978; Braude et al., 1979; Flach et al,, 1982; rabbit: Manes, 1973; human: Braude et al., 1988; sheep: Crosby et al., 1988). The developmental arrest observed in those studies indicates the time at whîch the products from the embryonic gemme become essential to further development of the embryo. The actual synthesis of the transcrïpts is assumed to be prior to this period. Previous studies, using techniques which were not as sensitive as the ones in this study, have noted the onset of embryonic transcription as the point at which the burst of embryonic transcription occurs (Barnes and First, 1991). The resdts from the present study demonstrate that Iow Ievels of embryonic transcriptional activity occur as early as the 2-cell stage in cattle, sheep and goats. These low Ievels of transcriptional activity increase through the next two cleavages, cuhinating in a burst of transcription of the embryonic genome during the 8-cd stage for all three species. These results suggest that transcription of the embryonic genome is dom-regdated, but not entirely suppressed until the 8-ceLl stage in these speaes. The low leveb of transcription observed may be a general or random low levd of activity or may be a specific transcript(s) which could be involved in further regdation of transcription or emb ryonic development. In the case of caprine X ovine interspecific hybrid embryos, the pattern of transcription was similar to both parental species indicating that the embryos do not seem to be compromised by the hybridization. This is further supported by the results from Chapter 2, that show these hybrid embryos are capable of establishing a pregnancy. In the case of cow oocytes fertilized by ram sperm the pattern of transcription deviates hom those of the parental speties. Although the initiai low, gradually increasing, level of transcriptional activity is similar to that of both cattle and sheep embryos, the hybrid embryos do not show the characteristic burst of transcriptional activity at the 8-celI stage. As discussed in Chapter 1 such hybrid embryos arrest during the 4 and $-cd stage. This arrest, therefore does not appear to be due to a la& of the characteristic low leveI transcription but may perhaps reflect an improper signahg for the burst of transcription. There must be one or more signals to evoke the burst of transcription of the embryonic genome. This as yet &own signal could originate hom either the cytoplasm or nucleus. The dedine in one or more rnaternally derived RNA's and/or proteins could directly or indirectly be involved in the signaling process. Although a low level of hanscriptional activity ocms in early cleavage stage embryos, the cytoplasm is crucial for initial cleavage development.

Pro tein Translation The protein synthesis levels, Iike the RNA synthesis levels, were highly variable in in vitro produced embryos. This variability is most likely due to the different states of viability of IVF embryos given the developmental potential (Chapter 1) in any given population as well as the efficiency of the in vitro culture system. The oocytes obtained for TVM and IVF are from abattoir ovaries which range from any stage of the estrous cycle and it is logical to expect that some cumulus-oocyte complexes obtained hom these ovaries wilI not have suffiuent maternally-derived tiranscripts and proteins to support early deavage development Siniilarly it is also expeded that some of these oocytes wodd have been nearly mature at the time of COC collection and would become overly or post-mature prior to M. In the in vivo situation, ovulation is a more pretisely controlled event whereby appropriately matured oocytes are ovdated. It wodd be valuable to examine in vivo produced embryos but unfortunately, as outlined in previous chapters, it is not possfble to aeate BXO and OXC hybrid embryos in uiuo. There are two main patterns which are exhibited throughout the autoradiographs of the SDS-PAGE of these control and hybrid ernbryos. The

first illustrates the concept of "age versus stage". In other words, the protein profiles observed, particularly within the bovine and BXO hybrid embryos, indicate that the protein profiles exhibited are a characteristic of the number of hours post-insemination or age of the embryo or cytoplasm rather than the ce11 stage of the embryo. The second illustrates that the developmental patterns in bovine X ovine hybrid embryos generally exhibit protein profiles which match the matemal speaes, (ie. speaes of oocyte or cytoplasm origYi). In the case of BXO hybrids, in effect, the bovine embryo represents the oocyte or cyiooplasm species while the ovine embryo reflects the paternd, sperm or a nuclear contribution to the embryo. Interspecific hybrid embryos therefore reflect the protein profiles of their matemalIy derived species at least at the level of resolution of single dimension electrophoresis. It is possible that small quantities of proteins derived from the patemal genome might be detected using two-dimensional electrop horesis. DJWELOPMENT OF BOVINE X OVINE NUCLEAR TRANSFER EMBRYOS

INTRODUCTION Nuclear transfer is a potentially powerful technique for the prodution of geneticdy identical individuals, involving the fusion of a single nucleated blastomere or somatic cell (karyoplas t) with an enucleated oocyte (cytoplast). Knowledge of basic ernbryo development at the level of the nucleus and cytoplasm can also be gained through the use of nuclear transfer techniques. Nuclear transfer has been examined in several species including niouse (Czolowska et al., 1984), rabbit (Collas and Robl, 1991), pig (Prather et al., 1990), cattle (Kanka et al., 1991), and sheep (Willadsen, 1986). The morphological events which follow transfer of a donor nucleus to an enucleated oocyte have been summarized by Campbeil et al., (1993). These include 1) nuclear envelope breakdown, 2) premature chromosome condensation, 3) dispersal of nucleoli, 4) reformation of the nuclear envelope, and 5) nuclear swelling. Interspecific nuclear transfer may yield some insight into the roles and importance of both the nucleus, and cytoplasm, Bovine X ovine hybrid embryos, which can be produced in vitro were shown to arrest at the 4-8-cell stage (Chapter 1). It was also dernonsbated through in uivo culture in sheep oviducts, that this developmental arrest did not appear to be a result of the in uitro culture system (Chapter 1). The bovine X ovine hybnd embryos expressed generous transcription of RNA at the 4cell stage, prior to developrnental arrest (Chapter 3), indicating that lack of generalized transcription of the embryonic genome did not appear to be the cause of developmental arrest. Finally, it was determined that bovine X ovine hybrid ernbryos express the same protein profiles as their bovine courtterparts (Chapter 3). There were no obvious protein ciifferences between the bovine X ovine hybrid embryos and bovine control embryos which might be suspect of causing the observed developmental arrest of the hybrid embryos. Despite al1 of the characterization of the bovine X ovine hybrid embryos, it was not possible to determine if the observed developmental arrest was caused by a nuclear-cytoplasmic or genomic incompatibility. The objective of this study was to examine if the developmental arrest exhibited by bovine X ovine hybrid embryos was indeed an incompatibility between the nuclear and cytoplasmic components of the hybrid embryo or an intragenornic incompa tibilty between the bovine and ovine components of the hybrid embryonic nucleus/genome+ The goal therefore was to construd nuclear transfer hybrid embryos by fusing ovine karyopIasts with bovine cytoplasts and determine if these bovine < ovine embryos were capable of development beyond the 8-ceU stage.

METHODS Instrumentation Hand-held and mouth-operated micropipettes were made from coagulation capillary tubes. To reduce adhesion of the c& to the wd, the tubes were fist soaked in a 0.5% (v/v) Photo-Ho solution, drained and dried for 2 hours in a 300°C oven. Depending on their desired size, micropipettes were either pded and cut by hand (12.5-2!50 pm) or pulled on an automated vertical pipette pder, cut at the desired diameter (20-60 p)and fie-polished on a microforge as described by Loskutoff and Kraemer (1990). The micropipettes were secured to tubing using a Unopipette. The four different glass tools made for micromanipulation induded, a needle, hansfer pipette, bevelled enudeation pipette and a holding pipette. AU tools, except the holding pipette were pulled on the pipette pulIer. All cutting and fite-polishing was done on the microforge. Silastic tubing was conneded to the micromanipulation pipettes which were mounted on Leica miaoma~puIatorsat least an hou, but preferably the evening before use, to allow the instruments to equilibrate in the manipulation medium. The tubing was Wed with air and a 3 cm column of oil was aspirated into the pipette after the calibration to obtain fine control over the aspiration and enudeation pipettes.

Nuclear Transfer The nuclear transfer procedure was based on the technique of Willadsen (1986) and subsequent modifications by Prather et al., (1987) and Bames et al., (1993a). Control bovine, ovine and bovine X ovine IVF embryos were produced as discussed in Chapter 1, as were the bovine and ovine modae that were used for donor blastorneres (karyoplasts). Interspecific nudear transfer embryos were created by fusing bovine cytoplasts (enudeated oocyte) with ovine karyoplasts (blastomeres). These interspecific nudear transfer hybrïd embryos are referred to as "bovine < ovine". That is, the oocyte species is listed first and the karyoplast specieç which was hsed is indicated second.

Pre~arationof cvto~lasts Bovine oocytes which were to be used as cytoplasts were matured for 20 hours before being vortexed for 2.5 min in Hepes-TALP (Appendix X) with 0.5 mg/rnl hyaluronidase to remove the cumulus cells. Oocytes with a polar body were seleded and enucleated in Ham's F-10 supplemented with 25 mM Hepes, 7.5 pg/d cytochalasin-b and 2 pg/ml Hoechst 33342 fluorochrome on a Zeiss inverted microscope equipped with a fluorescent fater. Enucleation was done by cutting the zona pelluuda with the needle and aspiratuig the cytoplasm with an aspiration pipette. The aspirated cytoplasm was then checked by epifluorescence for the presence of the meiotic spuidle and a polar body. The bovine cytoplasts were then activated at 24 hours by a 4 minute incubation in 5 pM ionomyàn followed by 3 hours of culture in 1.9 mM 6- dimethylaminopurine before fusion.

Pre~arationof donor blastomeres Blastomeres were isolated from 16-32 cell stage bovine and ovine embryos produced in vitro as described in Chapter 1. The zona pellucida of the embryos was removed by a short incubation in 0.1% (w/v) pronase E before the embryos were incubated in 0.5 mi ~a2+/~~2+-freeHanks medium und the blastomeres separated during gentle pipetting. The biastomeres were then selected and placed into the manipulation droplets.

Reconstitution and Fusion Donor cells and 2 cytoplasts were combined in 25 p1 droplets of manipulation medium (Appenck 1) under oil. Each 3.5 cm dish contained 4 manipulation droplets under 7 ml of silicone oil. One donor blastomere was inserted into each zona pellucida, adjacent to the cytoplast using the aspiration pipette. After one wash through fusion medium (Appendix 1), the oocyte-donor ce11 pairs were electrically fused using a BTX electrocell manipulaior 200, by exposing them to a 5 sec., 0.12 kV/an alignment pulse followed by a 30 msec. hsion pulse of 1.2 kV/an. The oocyte-donor cell complexes were then placed in the uicubator for 30-60 min. in culture-TAI2 (Appendix I) before being checked for &ion. If fusion had not occured, the process was repeated once.

Culture of nuciear transfer embryos The reconstructed embryos were CO-cultured with bovine oviduct explants in culture-TALP (10 embryos/25 pl culture droplet) for 3 days before transfer to Menezo's B2 medium with 10% senun. Cleavage and developrnent rates were checked and recorded at 24 hours and on day 8 and were compared with control NF data from Chapter 1.

RESULTS A total of 142 ovine and 42 bovine blastomeres were fused with mucleated bovine oocytes to produce nuciear transfer hybrid and control embryos. The cleavage rates for bovine < ovine nuclear transfer hybrids and bovine nuclear transfer control embryos were 52.1% and 71.4% respectively. None of the 74 cleaved nuclear transfer hybrid embryos developed beyond the 8-cell stage (Figure 21a). This is in contrast to the 63.3% of bovine nuclear transfer control embryos which developed to the morula or blastocyst stage (Figure 21b). When compared with bovine X ovine TVF hybrid embryos, the reconstructed hybrid embryos showed a similar ratio of developmental arrest at the 4 and û-celi stages as displayed in Figure 22. FIGURE 21- Bovine and bovine < ovine hybrid nuclear transfer embryos-

21a. Developmentdy arrested 4- and 8-celI bovine < ovine hybrid nuclear transfer embryos. These embryos were created by fusing a bovine cytoplast with an ovine karyoplast. Phase contrast (X800).

21b. A control bovine nuclear transfer blastocyst. Phase contrast (XSOO).

Y0 cleaved 40

BXB BXO

Embryo type 2-ce11 4ceU 8-ce11 morula/blastocyst

FIGURE 22. Histogram of rates of developmental arrest at 20, 4, and &ce11 stage for bovine, ovine and bovine X ovine in vitro fertïlized and nudear transfer (NT) embryos. Embryos constructed by nuclear transfer are indicated as B

For centuries, animal breeders have atternpted to breed males and females from different species to combine the desirable traits of both speaes into the hybrid offspring. Naturd barriers or isolathg mechanisms prevent breeding, fertilization and/or development of viable hybrids fiom animais of different species. These bamers to hybridization were examined in three

niminant speaes (cattle, sheep and goats). Understanding the medianisms by whkh these isolating bamers prevent interspecies hybridization yields information on normal fertilization, embryo, fetal and placental development. Our results have clearly demons tra ted three unique isolating mechanisms. A block to fertilization was obsemed in viuo when sheep were bred by goat bu&, fadure of embryonic development beyond the 8-ceU stage was observed foUowing bovine X ovine hybrid fertilizations, and fetal loss was observed in both goat X sheep and sheep X goat hybrid pregnancies and when both speües were used as recipients.

Bovine X ovine hybrid embryos There are several possible mechanisms which could be operating to cause the developmentd arrest obse~edin the bovine X ovine interspeafic hybrid and nudear transfer embryos. The results from Chapter 1 showed that bovine oocytes could be fertilized by sheep sperm under in oitro conditions but that the resdting embryos did not develop beyond the 48-cd stage. A temporary in vivo culture control conhned that this developmental arrest was not the result of an inadequate culture environment but was a problem inherent within the hybrid embryo. The results fkom Chapter 3 showed that there was a low level of transcriptional aaivity in these bovine X ovine interspecinc hyb rid embryos before their developmentd arrest at the 4-&ceU stager just prior to the expected burst of transcriptional activity. In other words, the bovine cytoplasm appears to be capable of supporting appropriate development of the hybrid embryo through the first two deavage divisions, including support of the initial transcriptional activity of the hybrid embryonic genome. The isolating barrier in the case of the bovine X ovine hybrid therefore appears to be at the signaling of the burst of transcriptional activity of the embryonic genome. The first possible reason wodd be that the hybrid ernbryos experience difficulty in undergohg mitosis, resulting in aneuploid c& or that there is a genomic incompatibility between the ovine (2x1 = 54) and bovine (2n = 60) components of the genome. There is no evidence for this since other hybrid embryos like those between sheep (2n = 54) and goat (2n = 60) which also have a diploid chromosome number of 57 are capable of forming blastocysts and establishg pregnancies as shown in Chapter 2. Aneuploid cells were not previously found in any karyotypes of 4-cell bovine X ovine hybrid embryos (Kelk et al., 1991a). In each case the karyotypes exhibited a diploid chromosome number of 57, indicating that there is no apparent diffidty in mitosis. In addition, the nudear transfer hybrid embryos which would not have either of these complications, also exhibit the same pattern of developmental arrest as the IVF hybrid embryos, suggesting that another mechanism is the likely cause of developmental arrest. The second possibility is that there is a miscommunication between the ovine portion of the nucleus and the bovine cytoplasm. It is possible that as the matemally derived transcripts within the bovine cytoplasm are depleted, the usual "tum on" signal which triggers the burst of transcription of the embryo~cgenome, is not recognized by the ovine portion of the genome. If this were the case, the embryo would essentidy be in a haploid state since the

bovine portion of the genome shodd still recognize the usual "turn on" signal. However, there must be additional factors contributing to the

developmentd arrest of these embryos because hapIoid embryos have proven capable of development beyond the &cd stage (Kawarsky et al., 1996; Plante and King, 1996). A third possibility is that there is a gene which is essential for development which is patemally imprinted and the ovine equivalent of this gene, if there is one, is not hctional. Again the contraindication of this condusion is that haploid and parthenogrnetic embryos @ohsheep and cattle) are capable of development beyond the &cd stage (Kawarsky et al., 1996; Plante and King, 1996). The only possible way that a mechanism could theoretically function would be if the presence of a paternally derived pronucleus inhibited the expression of the homologous matemally derived allele. This seems a rather complicated and unlikely strategy for regdation of early embryonic development since it would apparently require gene products derived from the male pronucleus.

The fininal, and most likeIy cause of 48-ceU devdopmental arrest of the bovine X ovine IVF and nuclear transfer hybrid embryos, is that a factor derived from the ovine genome acts to repress transcription of both the ovine and bovine portions of the genome. When a-ammitin is utilized in culture to block transcription, development of both bovine and bovine X ovine hybrid embryos proceeds up to the 8-16-cell stage (Plante et al., 1994), an entire cell cycle further than without the presence of a-amanitin. It may be possible that the complete inhibition of RNA transcription by a-amanitin allows the embryos to deave at a faster rate such that they develop one ceU-cyde stage furthex in the same the fkne. This supports the concept that the age of the cytoplasm may be a more influentid factor or directive in early embryo devdopment than the ceII stage of the embryo. It is possible that the low Ievel of transcription which is observed as early as the 2-ce11 stage, yields a protein which could act by binding to the embryonic DNA to prevent, regulate or repress mertranscription of either Iocalized genes or the entire genome. in normal deveiopment of sheep embryos, this repression factor would be removed, perhaps by a cy-topIasmic signal to aUow the burst of transcriptional activity which is normaily observed during the 8-cell stage (Chapter 3). It is also known from Chapter 3 that the normal bovine embryo exhibits a slightly higher level of transcriptionai activïty, earlier than the normal ovine embryo. Tt is therefore possible that the repression factor is not removed earIy enough to allow the bovine cytoplasm to continue to support embryo development-

Hybrids of sheep and goats It has been hown for over 60 years that goats conceive when interbred naturally to rams (Berry, 1938). The resdting pregnancies generdy result in fetal death during the second month of gestation (Warwick and Berry, 1949). The barrier to hybridization in this cross is therefore at the leve1 of fetal or placental development.

In the reaprocal cross of sheep interbred naturaily to goat bu&, there is an isolating mechanism which functions to block fertilization (K& et al., 1997a). The fist chapter discussed how this isolating mechanism codd be overcome through in vitro fertïkation and Chapter 2 iiIustrated that these embryos could establish pregnancies in both sheep and goat recipients. These pregnancies however, were subject to the same isolating mechanism as the reciprocal goat X sheep hybridization. That is, a second bamer to hybridization of sheep X goat operates during fetal or placental development- Cornparisons of these hybrid pregnancies with age-matched control pregnancies, demonstrated sigruficantly reduced placental development in the hybrid pregnanaes. Genomic imprinting is defined as gene expression based on the gamete of origin (Goshen et al., 1994). Hence, it must be considered as a potentidy important parameter in the development of interspecific pregnanaes. It is a process by which the differential expression of matemal and patemd alleles at certain loa in mammalian embryos occurs. In other words, certain genes are expressed preferentially from the maternally or the paterndy inherited allele. Such genes are implicated in the control of fetal, placental and neonatd growth, and, more generally, in diverse aspects of fetal nutrient acquisition and matemal-fetal interactions (Moore and Reik, 1996). Research on the effect of imprinting on embryonic and placental development has gone through a series of developments, beginning with experiments which showed that both matemal and paternd genomes are absolutely required for the normal progress of embryogenesis in mice (DeGroot and Hochberg, 1993). Both parthenogenetic and gynogenetic embryos, which contain a diploid set of matemal chromosomes, cmundergo implantation but die soon after, at least in part due to the lack of functiond extraembryonic tissues (Surani et al., 1984). Androgenetic zygotes containhg two male pronudei cmalso be constructed after the fernale pronucleus has been removed (McGrath and Solter, 1984b). Although the resulting embryos showed rapid growth of extraembryonic tissues, there was Little postimplantation development of the embryo proper (Barton et al., 1984). The concept of genomic imprinting is furthex supported in other species such as the human, where pathological conditions such as the complete hydatidiform mole, a gestational tissue derived totally from the paternd genome, exhibits aggressive capabilities for implantation, tissue invasion and proliferation (Goshen et al, 1994).

The next phase of research on impriniing involved utilizing mouse strains with Robertsonian handoactions to iden* the dvomosomal regions responsible for the difference behveen the functions of matemally and patemally derived genomes to embryonic development (Cattanach and Kirk, 1985). Finally, researchers have begun to identify specific genes which are imprinted. For example, insulin-like growth factor-II, (IGF-II) has been shown to be patemally imprinted in both mouse (DeChiara, et al., 1991) and human (Ohlsson et al., 1993; Giannoukakis et al., 1993), while the IGF-II receptor is matemally inherited (Barlow et al., 1991). There is evidence that proper regdation of IGF-II as well as maternally imprinted, leukemia- hhibiting factor (LE), (Stewart et al., 1992) and Hl9 (Bartolomei et al., 1991; Rachmilewitz et al., 1992; Zhang and Tycko, 1992; Ferguson-Smith et al., 1993) are ail involved in normal embryo and placental development.

In essence placental development appears to be a carefully controlled invasion of fetal trophoblast. Marzusch et aI., (1995) noted that the extensive proliferation and invasive growth exhibited by human trophoblast is comparable to that of a malignant tumour. This group found immunochemically detectable expression of p53 protein (a tumour suppressor gene) in trophoblast tissue. It was suggested that this expression was not due to mutation of the gene, as in malignant tumours, but rather to up-regulation of the p53 tumour suppressor gene, which could be a mechanism for controhg trophoblast proliferation. It has been suggested that paternally derived fetal alleles are seleded to demand more resources from the mother than materndy derived alleles and thiç selection differential would favour the evolution of parental imprinting. This would result in conflidùig expression patterns of maternal and patemal alleles at fetal genetic loa (Haig, 1992). There appears to be an asymmetry between matemal and fetal optima which rnight even predict the existence of

matemal mechanisms for reducing the extent to which a fetus elicits nutrients through the placenta (Moore and Haig, 1991; Haig, 1993; 1996). It is also therefore, of interest to note the devdopmental progress of these hybrid fetuses and their placenta with respect to whether the matemal or patemal species of the hybrid embryo matched the recipient speaes. For example, of the pregnancies resulting from sheep oocytes fertilized by goat sperm, 5 were established in sheep recipient. and 1 was established in a goat recipient. In those pregnancies camed in sheep recipients, the maternal contribution of the embryo matched the recipient and the pregnancies failed between 41 and 47 days of gestation. In the pregnancy camed by the goat recipient, the paternd contribution of the embryo matched the recipient, the pregnancy survived until day 51 and the placentornes were more than twice the size of any of the pregnmcies c-ed in sheep. The observation that embryos in which the patemal contribution was matched to the reapient appear to progress better than those whidi were not, supports the concept that the paternal genome plays a signifiant role in the invasive component of placental development. However, the fact that the matched pregnancies did not develop to term to produce live offspring indicates that development of the conceptus involves a highly complex interaction of both matemal and paternal gene expression. Abnonnd gene dosages, such as trisomy 21 (Down's syndrome) or monosomy XO (Turner's syndrome) in humans are a cornmon cause of spontaneous abortions or bkth defects (Fisher and Scarnbler, 1994). Although the genes of sheep and goats are very sünilar, the two genomes are not entirely homologous, therefore an interspecific hybrid must experience a haploid (partial monosomy) state for at least a smaü number of genes. If these partidar genes were essential for normal fetal development, fetal death could result. Aside from the potential gene dosage difficulty that an interspeofic hybrid might encounter, it is also possible that a gene such as ZGF-II ligand which would be patemaiiy imprinted from one speaes does not recognize the matemdy imprinted receptor of the other species. Interspecies hybrids may be effectively used to examine the mechanism of fertilization, the roles of nucleus and cytoplasm in the early embryo, as well as fetal and matemal interactions in pregnancy and the role of genornic imprinting. The ment prospect of producing sheep X goat hybrid embryos by in vitro fertilization is exating because it ailows for creation of reciprocal hybrid embryos which can be transferred to either parent species, thereby matching or mgmaternal and patemal parental speaes with prospective reapient animals. Careful examination of placental development may yield a polarity in fetal invasion of the maternal caruncle which could elucidate specific roles and timing of genetic imprinting. SUMMGRY AND CONCLUSIONS

Interspeafic hybrids between cattle, sheep and goats were used as models for early embryo and fetal development to examine the roles of the nucleus and cytoplasrn as weU as materna1 and paternal contributions to the developing embryo/fetus. The occurrence of interspecific fertilization using the bovine, ovine and cap~espeaes was explored under both in uiuo and in oifro conditions. Ram spermatozoa proved capable of penetrating intact bovine oocytes under in uifro conditions, but the resulting embryos arrested at the 48-celi stage. This developmental arrest was exhibited even when bovine X ovine hybrid zygotes were transferred to sheep oviducts for in uiuo culture, illustrating that the arrest was not an effect of the in oitro culture system. Bovine X ovine hybrid embryos exhibited active RNA transcription as early as the 2-celi stage. Transcription continued to increase through the 4- cell stage until developmental arrest occurred. This level of transa-iption was similar to that exhibited by bovine 4-ceU control embryos, which was also greater than that of ovine 4-ce11 control embryos. This indicates that the bovine X ovine hybrid follows the developmental pattern of the cytoplasmic or matemal species. Protein translation in bovine X ovine embryos followed a similar pattern as the RNA transcription. Using single dimension SDSPAGE, the hybrid embryos displayed the same protein profiles as the bovine, cytoplasmic or matemal species, again, indicating the importance of the cytoplasm in early cleavage development. There may however be srnail quantities of unique proteins derived from the paternal genome which might be resolved using two-dimensional electrophoresis. Finally, nuclear transfer hybrids were constnicted between ovine karyoplasts and bovine cytoplasts to explore the possibility that the developmental arrest exhibited by the hybrid embryos was an intragenomic incompatibility between the bovine and ovine components of the nucleus.

The bovine c ovine nudear transfer hybrid embryos exhibited the same 4-8- cell arrest as the bovine X ovine hybrid embryos, indicating that the incompatibility withui the hybrid embryos must be between the nudeus and the cytoplasm. Goat sperm proved capable of penetrating intact sheep oocytes under in vitro conditions, despite the blodc to fertilization in vivo. Ovine X caprine embryos developed through to the blastocyst stage in vitro and would even hatch. When hansferred to reapient sheep or goats, these embryos were capable of establishing pregnanaes. Ali pregnanaes however, ended with fetal death during the second month of gestation. Hybrid fetuses were found to be significantly smaller than age matched sheep fetuses and the placentae failed to develop completely. The placentornes were grossly smaller and histological examination revealed Little interdigitation of fetal villi înto the materna1 carundes. This substantially reduced the fetal-maternai interface for nutnent and gas exchange, which is the most probable cause of fetal retardation and dtimately, fetal rnortality. Sheep, goat and goat X sheep hybrid early cleavage embryos were shown to exhibit similar levels of RNA transcription despite indications in the literature that goat embryos would be expected to exhibit higher levels of transcription in earlier deavage stages. The present study has show that interspecific hybrid embryos between cattle, sheep and goats cm be produced under in vitro conditions, which could not necessarily be achieved using in vivo techniques. Furthemore, this study provides information on the relative effiaency of each cross under in vivo and itz oitro conditions. Interspeofic hybrid embryos can be used as unique models to study the mechanisms of fertilization and embryo development as well as fetal and placental development. These embryos express unprecedented genetic and morphological markers of each parental speaes, which can be utilized to evaluate matemal and patemal contributions to the embryo as weIl as nudear and cytoplasmic interactions. Abraham, GE., R., Swerdloff, D. Tuldùnsky and W.D. OdeII. 1971. Radioimmunoassay of plasma progesterone. J. Clin. Endoc~ol. Metab. 32: 619-624.

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Culture Media

Medium Com~onent Ouantity Measure

1) Collection Medium Ham's-FI0 - Hanks salts 1 envelope (pH:7.40) H20 960-0 ml NaHC03 12 g HEPES 10.0 ml Heparin (2 W/d) 02 ml Penidlin-Strep tomycin 10.0 mI ECS or SS 20.0 ml

2) Maturation Medium TCM-199 - Hanks salts 1 envelope (pH:7-40) H20 1000 mI NaHC03 O -35 g Na-pyruvate 22.0 mg Gentamicin 5.0 ml ECS or SS 200 ml

3) NC Medium (pH: 7.40) TCM-199 - Hanks sdts 1 envelope ECS or SS 10 % (v/v) PeniciUin-Strep tomyciri 0.5 % (v/v) Sodium Pyruvate 5.0 mM L-glutamine 1.0 mM BSA 3 .O mg/ml

4) Menez0 82 Medium Ménézo B2 10.0 ml ECS or SS 1.2 ml

5) Manipulation Medium Ham's-FI0 - Hanks salts 1 envelope HEPES 25.0 mM SS 10 % (v/v) Penicillin-Strep tomycin 1.O % (v/v)

6) Fusion Medium Sucrose O .28 mM Ca-aceta te 0.1 mM Na@04 1.0 mM Mlw4.W 0.5 mM BSA 0.01 mghl TALP Solutions 1. Ion stock sohtions prepared, aüquoted and stored for up to 6 months at -200C- 2. TL solutions prepared weekly and stored at 40C. 3. TALP soIutions prepared on the day of use. 4. Sperm-TALP and IVF-TALP equilïbrated for 3 hours at 390C in a 5% CO2 atmosphere before use. 5. HEPES-TALP equili-brated for 3 hours at 3g°C in ai.before use. 6. Only Sigma water was used in the preparation of TALP solutions

Solution Com~onent Ouantity Measure

Primary Ion Stock Solutions TL Stock Solutions

1) Sperm TL (OsmoIarîty: 295) H20 NaCl KCI N~HCO~ NaH2P04-H20 Na-lactate HEPES CaU2-WO MgCl243320

2) HEPES TL (Osmolarity: 280) Hz0 NaCI KCl N~HCO~ NaH2P04-H20 Na-lactate HEPES CaC12-2H20 MgC12-6H2O

3) IVF TL (Osmolarity: 295) Hz0 NaCl KC1 NaHC03 NaH2P04-H20 Na-lacta te CaC12-0 MgC12-6H20

TALP Solutions

1) Sperm TALP (pH:7.40) Sperm TL BSA (fraction V) Na-p yruva te (2.2 mg/d) Gentamycin (50 mg/ml) 2) HEPES TALP (pH: 7-40) HEPES TL 50.0 BSA (fraction V) 150 Na-p yruvate 0.50 (2.2 mg/ml) Gentamycin 50.0 (50 mg/d)

3) IVF TALP (pE7.40) IVFTL 10.0 BSA (fraction V) 60.0 Na-p yruvate OS0 (2-2 mg/&) Gentamycin 10.0 (50 mg/ml) Heparin (2.0 mg/ml) 0.10

Electrophoresis Solutions

1) Dissociation buffer (2X) Water 100 TRIS 1.5 Sucrose 20 .O SDS 10.0 Bromophenol blue 5 (saturated aqueous)

2) Coomassie blue stain Coomassie brilliant 125 blue R-250 Ethano1 (95%) 400 Glacial acetic aad 530

3) Gel destainer Ethanol (95%) 1000 Glacial acetic acid 400 Water 2400

4) Gd fixative Ethanol (95%) 70 Glacial acetic acid 400 Water 530

Miscellaneous Solutions

1) Orcein Stain (1.0%) Orcein Acetic Atid H20 List of Materials and Supplies

Acetic acid (99.8%;Glaad) Fisher Scientific Co. Ltd., Don Mills, Ontario, Canada

Antibiotic-antimyco tic Canadian Life Technologies, Burlington, Ontario, Canada

Atraumatic grasping forceps WISAP, distributed by Trudell Medical, Canada

Balloon-tipped catheter Amencan Latex Co., Noraoss, GA, USA (8 Fr,3 cc)

Bovine serum albumin-fraction V Sigma, Si. Louis, MO, USA

Buffalo rat Liver (BRL) ceils ATCC, RockviUe, MD, USA

Cannula (100 mm) WISAP, distributed by Tnidell Medical, Canada

Centra Progestin Central Sales Ltd., Brampton, Ontario, Canada

Chromic 1 gut (metric size 5) Ethicon Ltd., Peterborough, Ontario, Canada

Coated Viayl (mehic size 3.5) Ethicon L td., Peterborough, Ontario, Canada

Colcemid (lyophylized) Canadian Life Technologies, Burlington, Ontario, Canada

Coming 15 ml conicd tubes Fisher Scientific Co. Ltd., Don Mills, Ontario, Canada

Coverslips Fisher Scientific Co. Ltd., Don Mills, Ontario, Canada

Cdture dish, Nunc (4-well) Canadian Life Technologies, Burlington, Ontario, Canada

Dl9 developer (Kodak) Pond's Camera, Guelph, Ontario, Canada Disposable specïmen container Fisher Scientific Co. Ltd., Don Mills, Ontario, Canada

Estrumate Coopers Agropharm Inc., Willowdale, Ontario, Canada

Ethanol (absolute) Consolidated Alcohols Ltd., Toronto, Ontario, Canada

Euthansol (pentobarbital sodiumshering Canada, PointeClaire, Quebec, injection) Canada

Falcon 50 ml conical tube Fisher Scientific Co. Ltd., Don Mills, Ontario, Canada

Fetal caIf serum Canadian Life Technologies, Burlhgton, Ontario, Canada

Filters (0.Zpm) Millipore, Mississauga, Ontario, Canada Pond's Camera, Guelph, Ontario, Canada

Folltropin Vetrepharm, London, Ontario, Canada

Schering Canada Inc., Pointe Claire, Quebec, Canada

Gentamycin Sigma, St. Louis, MO, USA

Gesterol in oil Steris Laboratories hc., Phoenix, AZ, USA

Giemsa stain Fisher Scientific Co. Ltd., Don Mills, Ontario, Canada

Glass wool Fisher Scientific Co. Ltd., Don Mills, Ontario, Canada

L-glutamine Sigma, St. Louis, MO, USA

Ham's F-10 Canadian Lîfe Technologies, Burlington, Ontario, Canada

Heparin sodium injection USP Allen and Hanburys, Glaxo Canada Ltd. Co., Toronto, Ontario, Canada Canadian Life Technologies, Burlington, Ontario, Canada

Leitz Microscope (Aristoplan) Wild-Leitz Canada L td., Willowdale, Ontario, Canada

Armitage Carroll, Guelph, Ontario, Canada

Luer sy~ge(30 mI, type A) Transcoject, Gennany

Magnesium diloride Fisher Scientific Co. Ltd., Don Mills, Ontario, Canada

Magnesium sulphate Fisher Scientific Co. Ltd., Don Mills, Ontario, Canada

Menezo-B2 medium IMV International Minneapolis, MN, USA

Fisher Scientific Co. Ltd., Don Mills, Ontario, Canada

Microscope slides (hosted end) Almedic Ltd., Montreal, Quebec, Canada

NTB2 emuision (Kodak) Terachem, RexdaIe, Ontario, Canada

PBS (Dulbecco's phosphate Canadian Life Technologies, Burlington, buffered saline) On tario, Canada

Penicillin-streptomycin Canadian Life Technologies, Burlington, Ontario, Canada

Petri dish (100 x 15 mm) Fisher Scientific Co. Ltd., Don Mills, Ontario, Canada

Petri dish (35 mm) Nunc Canadian Life Technologies, Burlington, Ontario, Canada

P.M.S.G. Equinex, Ayerst Laboratories, Montreal, Quebec, Canada

Polypropy lene cannula Argyle, division of Sherwood Medical, (1.1 x 45 mm) TuLIamore, Ireland Potassium chloride Fisher Scientific Co. Ltd., Don Mills, Ontario, Canada

ICI Pharmaceuticals, Mississauga, Ontario, Canada

Pyramidal-tip trocar and cannula R. Laborie and Associates hc-, Brossard, (7 x 90 mm) Quebec, Canada

Rogarsetic Rogar/STB hc., Montreal, Quebec, Canada

Haver, Bayvet division, Chemagro Ltd., Etobicoke, Ontario, Canada

RPMI medium Canadian Life Technologies, Burlington, Ontario, Canada

Silicone oil Dow Corning Medical fluid 200, Paisley Products, Scarborough, Ontario, Canada

Sodium bicarbonate Fisher Scientific Co. Ltd., Don Mills, Ontario, Canada

Sodium citrate Fisher Scientific Co. Ltd., Don Mills, Ontario, Canada

Sodium pynivate Canadian Life Technologies, BurIington, Ontario, Canada

Steer serum Cansera hc., Rexdde, Ontano, Canada

Sucrose Fisher Scientific Co. Ltd., Don Mifls, Ontario, Canada

Canadian Life Technologies, Burlington, Ontario, Canada

Tom cat catheter Monoject Division, Sherwood Medical, St. (3.5 Fr, 4.5 inch) Louis, Missouri, USA

Trypsin (lx solution) Canadian Life Technologies, Burlington, Ontario, Canada

Uridine Sigma, St. Louis, MO, USA 3~-uridine Amersham, OMe,Ontario, Canada Veramix sheep sponges Tuco Products Co., Division of Upjohn, Orangeville, Ontario, Canada

Veress pneumoperitoneumR. Laborie and Assoc. Inc., Brossard, needle Quebec, Canada

Wild-Leitz M8 Wild-Leitz Canada Ltd., Willowdale, Ontario, Canada TEST TARGET (QA-3)

APPLlED IWGE. Inc t 653 East Main Street --.- Rochester, NY t 4609 USA --- Phone: 716f482-0300 ------Fax: 716/28&5989