Development of Bison Embryos Produced by Parthenogenesis, Interspecies Somatic Cell Nuclear Transfer, and Ooplasm Transfer

Development of Bison Embryos produced by Parthenogenesis, Interspecies Somatic Cell Nuclear Transfer, and Ooplasm Transfer

Leslie Antonio González Grajales

A Thesis presented to The University of Guelph

In partial fulfillment of requirements for the degree of Doctor of Philosophy in Biomedical Sciences

Guelph, Ontario, Canada

ABSTRACT

DEVELOPMENTAL OF BISON EMBRYOS PRODUCED BY

PARTHENOGENESIS, INTERSPECIES SOMATIC CELL NUCLEAR TRANSFER

AND OOPLASM TRANSFER

Leslie Antonio González Grajales Advisors: University of Guelph, 2014 Dr. Gabriela F. Mastromonaco Dr. W. Allan King

The success of reproductive biotechnologies including in vitro embryo production in bison species has been low although several techniques, such as interspecies somatic cell nuclear transfer (iSCNT) hold promise of efficacy in this species. However, interactions between cattle ooplasmic components and bison nuclear structures influence early development in bison iSCNT embryos. Differences in ATP content, incidence of apoptosis, and gene expression between cattle SCNT and bison iSCNT embryos suggest altered interactions in nuclear and mitochondrial genomes, although no differences in developmental rates were observed. A cattle IVM maturation and IVC system were used to determine the developmental potential of plains bison oocytes as cytoplasts. Maturation and developmental rates were similar between cattle and plains bison. The majority of oocytes in both groups reached metaphase meiosis II at 24 hours post maturation and the 8-16 cell stage on day 4. Although total blastocyst rate was similar between cattle and plains bison, embryos in the latter group consistently reached the blastocyst stage 24 to

48 hours after cattle parthenogenic embryos. Modifications to previously described ooplasm

transfer techniques were performed to decrease the number of micromanipulations needed when combined with SCNT. The effects of ooplasm transfer into cattle SCNT and iSCNT plains bison embryos was investigated and demonstrated that supplementation of ooplasm matching the genetic origin of the somatic cell in reconstructed SCNT or iSCNT embryos had no effects on development, ATP content, and gene expression profiles at the 8-16 cell stage. Finally, embryo quality between vitrified-thawed wood bison blastocysts produced in vivo and wood bison iSCNT blastocysts generated in vitro was investigated. The percentage of in vivo-derived embryos showing normal morphology was only 20% of that in vitro. Although in vivo embryos expanded, initiated the hatching process, and had higher total cell number, no differences were found in apoptosis incidence when compared to iSCNT embryos. Delayed development at the blastocyst stage was observed in iSCNT wood bison embryos when compared to in vivo embryos. The present study demostrated developmental alterations in iSCNT bison embryos, no effects on development by ooplasm supplementation, and compromised competence of in vivo- derived wood bison embryos.

DECLARATION OF WORK PERFORMED

I declare that all work presented in this thesis was performed by me with the exception of the procedures mentioned below.

Wood bison and plains bison skin biopsy samples were collected by Dr. Carl Lessard

(University of Saskatchewan). Cattle skin fibroblasts were collected, cultured, and frozen by Liz

St. John. Cattle ovaries were collected by Pradeep Balaraju, Steven Huang, and Heather Smale.

A majority of plains bison ovaries were collected by Melissa Filice. Media used for oocyte in vitro maturation, in vitro fertilization and in vitro culture were prepared by Liz St. John. ATP analyses were performed with the assistance of Dr. Gabriela Mastromonaco. Extraction of mRNA and real time qPCR experiments in IVF, SCNT, and iSCNT embryos were performed by

Dr. Laura Favetta.

ACKNOWLEDGEMENTS

First and foremost, I would like to thank my advisors Dr. Gabriela Mastromonaco and Dr. Allan

King. I am very thankful for all the support and trust I received from you during this wonderful journey as a graduate student. I have accomplished a very important goal in my life with the help of two incredible mentors. Four aspects were crucial to successfully complete my PhD degree: permanent guidance, motivation, continuous exposure to your immense knowledge, and patience. Thank you Gaby and Allan!

I would like to thank members of my advisory committee, Drs. Cathy Gartley and Pavneesh

Madan for their great contributions to develop this project. I am very privileged to have your scientific assistance. I also want to thank Dr. Laura Favetta for her amazing help with PCR analyses, Dr. Monica Antenos, Ed Reyes, Melissa Filice, Jeff Caudle, and Liz St. John for their technical support. Special thanks go to Liz for your support, countless learning experiences and friendship. On the other side of the world, I would like to express my gratitude to Drs. Otavio

Ohashi and Moyses Miranda, who received me in their laboratory (Universidade Federal do

Pará, Brazil) and helped me in acquiring important skills at the beginning of my PhD. I have to acknowledge all support and assistance provided by undergraduate and graduate students at

Universidade Federal do Pará.

This research would have not been possible without the financial support from NSERC, Toronto

Zoo Foundation, Federal Development Agency, and Canada Research Chairs Program. Thank you for your generous support!

I will never forget important colleagues at the University of Guelph and friends who shared wonderful moments with me. Thank you Jairo Melo and Jill Graydon (and the Mini-Melos),

Khaled Gohary and Heba Eldemnawy, Christina and Dave Smith, Aleja and Ivan, Rasa Levstein and from our school Jacky, Graham, Tim, Anja, Ahn, Tobi, Faz, Kim, Karla, Candice and

Nayoung.

Every day during my PhD and previous studies, I was very lucky to have support from my special angel on earth, Laura Pieper, who helped me numerous times in several aspects of my daily life .Thank you for your patience, love, and care.

“Muchas gracias” (Thanks in Spanish) to my parents Sergio González and Jilma Grajales, siblings Jessica, Sigrid and Sergio, my brothers in law Luis Solis and Orlando Kiewit, and all nephews (Orlando, Sergio, Pablo) and nieces (Hillary, Mary, Diane), who have tolerated me for so many years and in one way or another have been very important in my life. I am very thankful for your inconditional help, guidance, love, and the final push to finish on time.

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TABLE OF CONTENTS

INTRODUCTION ...... 1 LITERATURE REVIEW ...... 3 Taxonomy of bison species ...... 3 Historical range of the American bison...... 4 Bison current conservation status ...... 4 Conservation measures in endangered species...... 5 Assisted reproductive technologies in bison species...... 6 Applications of interspecies somatic cell nuclear transfer in species conservation ...... 14 Problems associated with SCNT and iSCNT technologies ...... 20 Nuclear and mitochondrial genome interaction ...... 25 Mitochondria functions during pre-implantation stages ...... 27 Mitochondrial inheritance in embryos produced by assisted reproductive techniques ...... 29 Mitochondrial replication during the preimplantation stages ...... 32 RATIONALE ...... 33 CHAPTER ONE ...... 36 In vitro production of bison parthenogenic embryos using a cattle protocol ...... 36 INTRODUCTION ...... 37 MATERIALS AND METHODS ...... 40 Sample collection ...... 40 Study animals ...... 40 Experimental design ...... 40 Chemicals ...... 41 Oocyte collection and IVM ...... 41 Parthenogenic embryo production ...... 42 TUNEL staining of embryos ...... 43 Statistical analysis ...... 44 RESULTS...... 45 Polar body extrusion of cattle and plains bison oocytes ...... 45 In vitro development of cattle and plains bison parthenogenic embryos ...... 45

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Apoptosis assessment ...... 49 DISCUSSION ...... 52 CHAPTER TWO ...... 57 Developmental competence of 8-16 cell stage bison embryos produced by interspecies somatic cell nuclear transfer ...... 57 INTRODUCTION ...... 58 MATERIALS AND METHODS ...... 61 Experimental design ...... 61 Chemicals ...... 61 Culture and preparation of donor somatic cells ...... 61 Oocyte collection and in vitro maturation (IVM) ...... 62 Oocyte reconstruction by micromanipulation techniques and in vitro culture (IVC) ...... 62 TUNEL staining of embryos ...... 63 ATP determination ...... 63 RNA extraction and reverse transcription ...... 63 Gene expression analysis ...... 64 Statistical analysis ...... 66 RESULTS...... 67 In vitro development of cattle SCNT and bison iSCNT embryos ...... 67 ATP quantification ...... 67 Apoptosis assessment ...... 68 Expression profiles of nuclear and mitochondria-encoded genes ...... 70 DISCUSSION ...... 73 CHAPTER THREE ...... 78 Effects of ooplasm transfer on early embryo development in domestic cattle SCNT and iSCNT plains bison embryos ...... 78 INTRODUCTION ...... 79 MATERIALS AND METHODS ...... 82 Experimental design ...... 82 Chemicals ...... 82 Culture and preparation of donor somatic cells ...... 82

Oocyte collection, IVM, and IVC of reconstructed embryos ...... 83 In vitro fertilization (IVF) of cattle oocytes ...... 83 Embryo reconstruction and ooplasm transfer ...... 83 Fusion of reconstructed embryos ...... 84 ATP determination ...... 85 RNA extraction, reverse transcription, and gene expression analysis ...... 85 Statistical analysis ...... 87 RESULTS...... 88 Fusion rates ...... 88 In vitro development of reconstructed embryos ...... 88 ATP quantification ...... 89 Expression profiles of nuclear and mitochondria-encoded genes ...... 89 DISCUSSION ...... 92 CHAPTER FOUR ...... 100 Quality assessment of in vivo-derived and interspecies somatic cell nuclear transfer wood bison embryos ...... 100 INTRODUCTION ...... 101 MATERIALS AND METHODS ...... 104 Experimental design ...... 104 Chemicals ...... 104 Sample collection ...... 104 In vitro fertilization (IVF) of cattle oocytes ...... 105 Thawing procedure ...... 105 Wood bison iSCNT embryo production ...... 105 TUNEL staining ...... 105 Differential staining for TE and ICM cells ...... 106 Statistical analysis ...... 106 RESULTS...... 107 In vitro development of wood bison embryos ...... 107 Apoptosis assessment ...... 107 Differential staining ...... 113

DISCUSSION ...... 115 GENERAL DISCUSSION ...... 121 FUTURE DIRECTIONS ...... 130 SUMMARY AND CONCLUSIONS ...... 131 REFERENCES ...... 133 APPENDIX ...... 166

LIST OF TABLES

Table 1. Implementation of ARTs (endocrinology, ovarian superstimulation, embryo transfer, cryopreservation, gamete analysis) in species with conservation status in the tribe Bovini (family Bovidae, Sub-family Bovinae)...... 9 Table2. Molecular biology, Intra-cytoplasmic sperm injection, tissue xenografting, in vitro embryo production, and contraception in species with conservation status in the tribe Bovini (family Bovidae, Subfamily Bovinae)...... 12 Table 3. Interspecies somatic cell nuclear transfer in threatened and endangered species...... 16 Table 4. Development of parthenogenic embryos ...... 46 Table 5. Median [25%; 75%] total cell number and proportion of TUNEL labeling (apoptotic) nuclei in cattle and plains bison parthenogenic blastocysts ...... 50 Table 6. List of primers used for qPCR ...... 66 Table 7. Embryo development in reconstructed embryos (%)...... 67 Table 8. Median [25%; 75%] number and proportion of condensed and TUNEL positive (apoptotic) nuclei in reconstructed embryos ...... 69 Table 9. Number of reconstructed embryos developed (%) to the 2, 4, and 8-16 cell stage...... 89 Table 10. Embryo assessment of in vivo wood bison embryos based on recommendations by the International Embryo Transfer Society...... 108 Table 11. Median [25%; 75%] total cell number and proportion of TUNEL labeling (apoptotic) nuclei in wood bison blastocsyts...... 111 Table 12. Cell number and allocation in cattle IVF and wood bison iSCNT embryos...... 113

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LIST OF FIGURES

Figure 1. Phase contrast micrographs from cattle and plains bison oocytes before IVM (A-B), 24 hours post IVM (C-D), and matured oocytes (E-F). Arrows denote polar bodies...... 47 Figure 2. Phase contrast micrographs of cattle and bison parthenogenic blastocyst on day 9 of development at 20X (A). Images in B area magnified (40X)...... 48 Figure 3. Cattle and plains bison parthenogenic embryos showing nuclei TUNEL positive labeling (arrows)...... 51 Figure 4. ATP quantification in reconstructed embryos at the 8-16 cell stage. Different letters denote significant differences between groups (p<0.05)...... 68 Figure 5. Cattle SCNT embryos showing no signs of nuclear condensation and/or TUNEL positive labeling. Wood bison iSCNT embryos showing condensed red nuclei without TUNEL labeling (filled arrows). Plains bison iSCNT embryos with condensed orange nuclei depict positive TUNEL labeling (thin arrows)...... 71 Figure 6. Gene expression profiles of mt-COX2 (A), NRF-2(B), and TFAM (C) in reconstructed embryos at the 8-16 cell stage. (*) indicates significant differences, p<0.05...... 72 Figure 7. Representation of iSCNT and OT procedures. A) Large blue and yellow circles represent plains bison and cattle oocytes, respectively. Red diamonds denote a nucleus. B) A portion of plains bison ooplasm (small blue circle) and plains bison somatic cell (small brown circle) are introduced in the perivitelline space. C) Reconstructed embryos + OT after first electropulse (note how the portion of plains bison ooplasm has been fused (scattered blue pattern). D) Reconstructed embryo followed by a second electro pulse showing successful fusion of the somatic cell...... 86 Figure 8. Phase contrast micrograph of A) cattle oocyte + cattle ooplasm and somatic donor cell nucleus, arrow represents the cattle somatic cell while the arrow head denotes the portion of ooplasm being transferred into the perivitelline space. B) Micrograph of a different oocyte taken 5 minutes after first DC pulse. Note fusion process between cattle oocyte and the portion of cattle ooplasm (arrow head) with presence of the somatic cell in the back (thin arrow)...... 87 Figure 9. ATP quantification in reconstructed embryos at 8-16 cell stage with or without OT. .. 90

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Figure 10. Gene expression profiles of mt-COX-2(A), NRF-2(B), and TFAM (C) in IVF and reconstructed embryos at the 8-16 cell stage...... 91 Figure 11. Phase contrast micrographs (day 7 and 8) from in vivo-derived wood bison embryos. Fluorescent micrographs denote total cell number 48 hours after thawing. Expanded blastocyst are observed in A) and B). Hatching blastocyst is indicated in C. Embryo identification in A, B, and C corresponds to 7a, 11a, and 7b, respectively...... 109 Figure 12. Phase contrast and fluorescent micrographs of grade 4 embryos at different stages of development. Note abnormalities in shape of embryonic cells (morula and early blastocyst), and nuclear condensation (13-cell embryo)...... 110 Figure 13. A) Wood bison in vivo-derived blastocyst displaying numerous cells TUNEL positive labeling (filled arrow). B) Wood bison iSCNT blastocyst with isolated apoptotic cells (thin arrow)...... 112 Figure 14. Differential staining of IVF cattle and iSCNT wood bison embryos. The two cell lineages are stained in blue (ICM) and pink (TE)...... 114

INTRODUCTION

Worldwide biodiversity is being severely jeopardized by an alarming reduction in species. In the last decade, more than 20000 species, including mammals, birds, amphibians, and corals, have been incorporated to the IUCN’s list of threatened species (International

Union for Conservation of Nature, 2014). Some of the measures that have been proposed for species conservation aim at the propagation of genetic material through application of assisted reproductive technologies (ARTs). Gametes, embryos, and somatic cells have been successfully obtained, and preserved in domestic species by ARTs. However, implementation of these technologies in threatened species has been limited due to the small number of animals accessible for research and the lack of knowledge regarding their reproductive physiology.

The wood bison (Bison bison athabascae) has been considered an essential species for

North American ecosystems. Unfortunately, the population number has not been stable over the last few decades, which has promoted enforcement of conservation measures including ARTs.

To date, most technologies applied to bison have extrapolated information from cattle studies, despite the species belonging to a different genus, resulting in limited success with embryo production and pregnancy rates. Nevertheless, ARTs are valuable mechanisms to study the developmental potential of gametes and embryos.

Over the last decade, there has been a surge of interest in more advanced techniques, such as interspecies somatic cell nuclear transfer (iSCNT), to generate embryos from endangered species using oocytes derived from domestic animals. Therefore, iSCNT represents an advantageous technique to produce embryos from species where oocytes are very difficult to 1

obtain. Thus, well established techniques involving in vitro maturation and in vitro culture of domestic gametes and embryos would seem adequate to culture oocytes and embryos carrying genetic material from more distant species. However, evidence has demonstrated that iSCNT success rate is linked to the taxonomic distance between the genetic donor and the oocyte donor.

Developmental alterations caused by abnormal cell signaling between nucleus and cytoplasm components have been associated with compromised development in iSCNT embryos.

Important advances have been made in the field of bison reproduction in the last ten years but these efforts have been minimal when compared to the significant progress in domestic cattle. Therefore, more studies investigating in vitro development of gametes and embryos in bison species would facilitate a better understanding of cellular processes during preimplantation development. The aim of this thesis is to summarize the main assisted reproductive techniques that have been implemented in the American bison subspecies (wood bison and plains bison), while emphasizing potential applications of iSCNT in the field of species conservation. Some of the problems leading to development arrest observed in iSCNT embryos, including the impact of nuclear-cytoplasmic incompatibilities on iSCNT embryo development, will also be discussed.

Finally, special attention will be paid to the mitochondrion due to its essential roles in gametogenesis and embryogenesis, such as ATP production, cell signaling, and cell death.

LITERATURE REVIEW

Taxonomy of bison species

Bison species are grouped within the genus Bison which comprises two species: the

American bison (Bison bison, Linnaeus, 1758) and the European bison (Bison bonasus,

Linnaeus, 1758). The Bison genus belongs to the family Bovidae , subfamily Bovinae together with genera Bubalus, Pseudoryx, Bos, and Syncerus (McDonald, 1981). In the last decade, there has been some debate about dividing the American bison in two subspecies: the wood bison

(Bison bison athabascae) mainly found in territories across North West Canada while the plains bison (Bison bison bison) occupies various provinces and states within Canada and the United

States. However, mitochondrial DNA (mtDNA) analysis has revealed no significant differences despite the distinct phenotype that exists between the two subspecies (Douglas et al., 2011). On the other hand, two European bison subspecies have been recognized (Bison bonasus bonasus and Bison bonasus caucasicus), which became extinct in the wild approximately one century ago

(Sztolcman, 1924). Fortunately, in situ reproductive programs using pure Bison bonasus bonasus, and hybrid founders (Bison bison bonasus x Bison bonasus caucasicus) prevented the decay of the species (reviewed by Pucek et al., 2004). Currently, free-ranging populations have been reported in nine countries within Europe and Asia (European Bison Conservation Center,

2014).

Historical range of the American bison

More than two centuries ago, American bison herds occupied immense territories from

Northern Canada to Mexico. In Canada, early documentation in the nineteenth century localized important populations of wood bison in the northern region of the great plains, such as the

Missouri River and the Great Slave Lake (Hewitt, 1921). However, their historical range drastically changed as a result of indiscriminate hunting, human development, and spread of cattle diseases among bison herds (Soper, 1941; Freese et al., 2007). This adverse event was extensively described by Hewitt (1921) as “one of the greatest tragedies in animal life in historical times”. In his book, he summarizes numerous testimonies about American bison extirpation, from countless millions to only 1,091 individuals by the year 1889. Although this alarming situation was known for decades in the United States, no conservation measures were implemented until the end of the 19th century. During this time, only a few plains bison individuals remained in Yellowstone National Park as a free ranging population. Similarly, unrestricted slaughter practices reduced the population of wood bison in Canada. Fortunately, with the establishment of the Northwest Game Act in 1906, the government of Canada finally prohibited hunting of wood bison (Hewitt, 1921).

Bison current conservation status

Recent demographic studies have estimated the North American bison population in commercial herds at approximately half a million animals. However, this apparent high value is not proportional to the population residing in conservation and protected areas. In Canada, plains bison is categorized as “threatened” with 1,200 to 1,500 mature individuals distributed only in 5

segregated subpopulations. Similarly, wood bison is at lower risk with approximately 7,172 mature individuals located in nine fragmented subpopulations (COSEWIC, 2013).

Global conservation recognition has also been given to both bison subspecies. In the last report by the IUCN in 2013, plains and wood bison subspecies were grouped under the category of “near threatened” on the Red List of Threatened Species (IUCN, Red List, 2013). However, the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES,

2013) only included wood bison in Appendix II. Therefore, several conservation efforts were performed to restore its historical distribution. Essential governmental actions, such as establishment of protected areas and creation of regulations against uncontrolled hunting, facilitated the recovery of the species a century ago. Soon after, results from these two changes showed a rapid increase in the population size and by 1919, a total of 4,033 wood bison individuals were reported (Gates et al., 2001). However, the population of bison in the last decade has been challenged with the incorporation of cattle genes into the wild populations and the spread of infectious diseases, such as brucellosis, tuberculosis, and anthrax, decreasing significantly the number of individuals (Gates et al., 2001; Nishi et al., 2002).

Conservation measures in endangered species

In an attempt to increase the population size of endangered species, conservation strategies have focused on ex situ propagation of numerous species (Hanks, 2001; Kuhnen et al.,

2012). However, these protocols usually are complex and challenging (Hanks, 2001). The main challenges to successfully integrating ARTs in these populations include the lack of information regarding their reproductive physiology, and the difficulty in accessing and accurately

monitoring their reproductive status (Schwarzenberger et al., 2000; Ghosal et al., 2010).

Therefore, to properly recommend these technologies in endangered species, there are basic concepts of reproductive physiology and embryology that require intensive investigation. In contrast to domestic cattle and water buffalo which have been extensively studied in the last 30 years due to their economic value in agriculture, only a few scientific papers have been published in non-domestic bovids in areas related to reproduction and ARTs (Table 1-2).

Assisted reproductive technologies in bison species

Ovulatory cyclicity in the North American bison is restricted to a few months beginning towards the end of August and extending to the first weeks of December. Consequently, implementation of ARTs is limited to a short period (Kirkpatrick et al., 1991; Matsuda et al.,

1996). Estrus synchronization protocols followed by artificial insemination have been used successfully in bison species (Dorn, 1995). However, ovarian stimulation using different combinations of gonadotrophins yielded unpredictable results (Othen et al., 1999).

More recently, a series of experiments were conducted to determine the feasibility of superovulation protocols during the breeding and the non-breeding season. These studies successfully induced follicular growth in the non-breeding season but the high number of unfertilized oocytes retrieved was suggestive of oocyte incompetence (Palomino et al., 2013). In contrast, more transferable embryos have been obtained during the fall months (Toosi et al.,

2013). The efficacy of these protocols in terms of offspring production was difficult to determine because a large proportion of embryos was used for cryopreservation studies.

Despite great advances in manipulation of estrous cycles in bison species over the last decade, the efficiency remains low when compared to the domestic cow. Only few offspring produced from artificial insemination or embryo transfer have been reported in both American bison subspecies (Mastromonaco, personal communication; Wilmsen, 2012; Toosi et al., 2013).

On the other hand, no pregnancies have been reported from in vitro produced embryos (Seaby et al., 2012). It is also important to note that there might be external factors negatively affecting the reproductive outcomes in wild animals. For instance, the detrimental effects of stress hormones on reproductive performance including embryo survival and pregnancy rates in wildlife species, have been documented (Daley et al., 1999; Gobush et al., 2007).

The first attempt to produce bison embryos by in vitro procedures was performed by

Thundathill et al., (2007). The authors retrieved cumulus oocyte complexes (COC’s) from wood bison abattoir ovaries and used a domestic cattle protocol as a model. Although the protocol yielded blastocyst rates less than 10%, the authors emphasized the importance of the technique to preserve and amplify the genetic diversity, especially in wild diseased herds. However, this is the only study that has been published to date on wood bison in vitro embryo production. Difficult access of wood bison oocytes in addition to the poor outcomes observed by these authors has impeded the diffusion of this technology as a conservation tool. Whilst some studies have determined the proper conditions during in vitro fertilization for pure bison or hybrid (cattle x bison) embryos (McHugh and Rutledge, 1998; Seaby et al., 2012), there are no reports focusing on optimal metabolic demands for oocytes and embryos from bison species. Proper characterization of gametes and embryos using molecular diagnostic tools would elucidate some of the aspects associated with the limited success of ARTs found in bison.

As mentioned previously, gametes and embryos from endangered species are very scarce.

Therefore, the production of hybrid embryos using oocytes from domestic species offers a unique opportunity to analyze proper culture systems using these embryos. However, this approach alters the species’ genome, which limits its application in species conservation. On the other hand, studies on hybrid embryos have shown promising results. For example, it has been demonstrated that hybrid bison embryos, regardless of the subspecies, are able to reach the blastocyst stage (Seaby et al., 2012). Furthermore, Seaby’s study demonstrated that cattle in vitro embryo production systems are able to support embryo development of hybrid bison embryos; however, low embryo quality and development rates compared to in vitro produced cattle embryos was also documented. Similarly, other studies have documented poor blastocyst rates for Bos hybrid embryos. The authors highlighted the importance of heparin concentration, bull effect, and insemination dose (McHugh and Rutledge, 1998).

Preliminary findings on wood bison oocyte maturation have revealed interesting results.

A study found that a large proportion of wood bison oocytes matured in vivo and in vitro reached meiosis II at approximately 30 and 24 hours, respectively (Cervantes et al., 2013). Conversely, nuclear maturation in cattle oocytes peaks at approximately 21 to 24 hours (Sirard et al., 1989).

Cervantes’s article presents a valuable study to identify differences in embryo development among bovids. Thus, new insights into genomics, proteomics, and metabolomics might identify some of the causes associated with poor in vitro embryo production outcomes and recognition of markers for embryo viability in bison. However, it is also important to stress that basic reproductive studies must be prioritized in order to successfully incorporate current assisted reproductive technologies into species at risk.

Scientific CITES/ Endocrinology Ovarian Embryo Cryopreservation Gamete analysis name IUCN superstimulation transfer Female Male Embryo Oocyte Sperm Oocyte Sperm (Common Status Estrus name) synchronization/ Artificial insemination Bison bison II/NT McCorkell McCorkell et al., Toosi et al., Hussain et al., 2013 Pegge et al., 2011 athabascae et al., 2013 2013 2013a Hussain et al., 2011 Helbig et al., (Wood bison) Matsuda et Palomino et al., Krishnakumar et al., 2007a al., 1996 2013 2011 Palomino et al., Krishnakumar et al., 2014 2013 Adams et al., 2009 Bogle et al., 2010 Othen et al., 1999 Bison bison Not Vervaecke Mooring Dorn et al., 1990 Dorn et al., Krishnakumar et al., Toosi et al., 2013b bison listed/NT and et al., Barfield and Seidel, 1990 2013 Krishnakumar et (Plains bison) Schwarzenb 2004 2012a Krishnakumar et al., al., 2011 erger, 2006 2011 Pegge et al., 2011 Kirkpatrick Hussain et al., 2013 Helbig et al., et al., 1993 Hussain et al., 2011 2007a Kirkpatrick Aurini et al., 2009 Helbig et al., et al., 1992 2007b Kirkpatrick et al., 1991 Bison bonasus Not Borque et Sipko, 2012 Sipko, 2012 (Wisent) listed/VU al., 2011 Kozdrowski et al., Gizejewski et al., 2011 2004 Pérez-Garnelo et al., Sipko et al., 1993 2005 Pérez-Garnelo et al., 2001 Sipko et al., 1993 Bubalus I/EN Yusuf et al., 2012 Yudi et al., 2011 Yudi et al., 2011 depressicornis Yusuf et al., 2010 (Lowland Seifert et al., 1974 Anoa)

Bubalus I/EN quarlesi

Field of study

Scientific CITES/ Endocrinology Ovarian Embryo Cryopreservation Gamete analysis name IUCN superstimulation transfer Female Male Embryo Oocyte Sperm Oocyte Sperm (Common Status Estrus name) synchronization/ Artificial insemination (Mountain Anoa) Bubalus I/CE mindorensis Solis et al., 1996 (Tamaraw) Bubalus III/EN annee (Wild water buffalo) Bos javanicus Not listed/ Asa et al., Johnston et al., 2002 Sarsaifi et al., 2013 Sarsaifi et al., (Banteg) EN 1993 2013 San et al., 2013 Bos gaurus I, VU Loskutoff et al., Hammer et al., Armstro Janesch et al., 2001 MacLean Jr et al., (Gaur) 2000 2001 ng et al., 1998 Armstrong et al., Armstrong et 1995 Junior et al., 1990 1995 al., 1995 Godfrey Godfrey et al., 1991 Pope et al., et al., Junior et al., 1990 1988 1991 Pope et al., 1988

Bos mutus I/VU Safar- Graham et al., 1978 Usupov et al., (Wild yak) Hermann 1981 et al., 1987 Bos sauveli I/CE (Kouprey) Syncerus Not Brown et Gerber et al., 2000 Herold et al., 2006 Herrick et al., caffer listed/LC al., 1991 Herrick et al., 2004 2004 (African Safar- Herold et al., 2004 Gerber et al., buffalo) Hermann Janesch et al., 2001 2002 et al., Lambrechts et al., Gibbons et al., 1987 1999 2001 Morfeld et al., 2001 Syncerus Not Friedman et al., caffer listed/LC 2000

Field of study

Scientific CITES/ Endocrinology Ovarian Embryo Cryopreservation Gamete analysis name IUCN superstimulation transfer Female Male Embryo Oocyte Sperm Oocyte Sperm (Common Status Estrus name) synchronization/ Artificial insemination (African Bartels et al., buffalo) 1999 Bezuidenhout et al., 1995 Ackerman et al., 1994 Brown et al., 1991 Pseudoryx I/CE nghetinhensis (Saola) CR (critically endangered), EN (endangered), VU (vulnerable), NT (near threathened), LC (least concern)

Table2. Molecular biology, Intra-cytoplasmic sperm injection, tissue xenografting, in vitro embryo production, and contraception in species with conservation status in the tribe Bovini (family Bovidae, Subfamily Bovinae). Field of Study Scientific name CITES/ Molecular biology Intra- Tissue xenografting In vitro embryo Contraception (common name) IUCN cytoplasmatic production status Female Male sperm injection Female Male Female Male

Bison bison II/NT Haigh et al., Cervantes et al., 2013 athabascae 1991 Thundathil et al., 2007 (Wood bison) Bison bison bison Not Kiewisz et Boisvert Abbasi and Krishnaku- (Plains bison) listed/NT al., 2008 et al., Honara- mar et al., Humblot., 2004 mooz., 2011 2013 2001 Bison bonasus Not listed/V Kozioro- Miller et al., (Wisent) wska et al., 2004 2013 Bubalus I/EN depressicornis (Lowland Anoa)

Bubalus quarlesi I-EN (Mountain Anoa)

Bubalus I,CE mindorensis (Tamaraw)

Bubalus annee III-EN (Wild water buffalo)

Bos javanicus NA, EN Kirkpatrick (Banteg) et al., 1995

Bos gaurus I, VU Dashtizad et al., Dashtizad et al., 2011 (Gaur) 2011 Hammer et al., 2001 Loskutoff et al., 2000 MacLean Jr et al., 1998 Armstrong et al., 1995 Jonhston et al., 1994 Bos mutus I, VU

Field of Study Scientific name CITES/ Molecular biology Intra- Tissue xenografting In vitro embryo Contraception (common name) IUCN cytoplasmatic production status Female Male sperm injection Female Male Female Male

(Wild yak) Bos sauveli I, CE (Kouprey)

Syncerus caffer Not listed, Raina et al., 2001 (African buffalo) LC Arlotto et al., 2000 Kidson et al., 2000 Smith et al., 1999 Shaw et al., 1995 Pseudooryx I, CE nghetinhensis (Saola) CITES: I(species that are the most endangered), II(species not necessarily now threatened with extinction but may become so unless trade is controlled), III(species included at the request of a Party that already regulates trade in the species and that needs the cooperation of other countries to prevent unsustainable or illegal exploitation) IUCN: CR (critically endangered), EN (endangered), VU (vulnerable), NT (near threatened), LC (least concern)

Applications of interspecies somatic cell nuclear transfer in species conservation

ARTs have played an important role in the perpetuation of genetic material of many species (Paris et al., 2007). Over the last fifteen years, domestic, companion, and wild animals have been generated by a technique known as somatic cell nuclear transfer (SCNT) (Wakayama et al., 1998; Shin et al., 2002; Galli et al., 2003; Gómez et al., 2004). The procedure involves the transfer of a somatic cell into an enucleated oocyte through micromanipulation techniques

(Campbell et al., 1996). However, oocytes from endangered species are rarely available which limits the use of this technique. Dominko et al. (1999) combined somatic cells and oocytes from different species, a technique referred to as interspecies or intergeneric somatic cell nuclear transfer depending on the taxonomic distance between the oocyte donor and the genetic donor.

One of the factors that contributed significantly to the development of iSCNT is the large availability of oocytes from domestic and laboratory species such as cattle, pigs and mice. While some studies have used oocytes from related species, others have tried to use more distant species yielding very little success in terms of blastocyst production (Murakami et al., 2005;

Kwon et al., 2011). However, few offspring produced by iSCNT have been reported. Only 6 studies have documented the birth of mammals using this technique, including mouflon, gaur, sand cat, Pyrenean ibex, wolf, and more recently coyote ( Loi et al., 2001; Kim et al., 2007;

Gómez et al., 2008; Folch et al., 2009; Srirattana et al., 2012; Hwang et al., 2013). Data analysis from more than 80 scientific reports and abstracts has suggested that better pregnancy and embryonic development rates are obtained when the taxonomic distance between species is decreased (Mastromonaco et al., 2014). Interestingly, higher pregnancy rates as well as number of offspring born per female have been reported in carnivores. Differences in in vivo and in vitro culture conditions of canine and feline gametes and embryos compared to ruminant species

might be attributable to higher developmental and pregnancy rates observed in these non- domestic species (Gómez et al., 2006; Kim et al., 2007; Hwang et al., 2013). However, the overall success of iSCNT has not improved in the last decade (Oback, 2008; Galli et al., 2012;

Mastromonaco et al., 2014). A summary of studies on iSCNT applied to endangered species is shown in Table 3.

Other iSCNT models have used domestic species as genetic and oocyte donors in order to understand the mechanisms that impair embryonic development during the pre-implantation stages (Wang et al., 2009; Ostrup et al., 2011). Thus, different combinations have been reported to produce inter-family bovine-pig (Dominko et al., 1999; Uhm et al., 2007), inter-order cat-and panda-rabbit (Wen et al., 2005), dog-pig (Sugimura et al., 2009), and inter-class chicken-rabbit embryos (Liu et al., 2008). Although these studies have revealed important structural and functional alterations in iSCNT embryos derived from very distant species, these changes might not occur in embryos derived from more related species. Conversely, few studies have investigated the factors leading to embryonic failure in the case of embryos reconstructed with somatic cells and oocyte donors belonging to the same genus which emphasizes the importance of elucidating these mechanisms to use this technology in species conservation.

Table 3. Interspecies somatic cell nuclear transfer in threatened and endangered species.

Cell donor Cell type CITES Recipient Reconst. Blastocyst Transferred No. Fetuses/ Offspring Outcome Reference Appendix Oocyte Embryos Rate (%)a embryos Pregnancies (n) (n) (n) (n) ~D30 ~D60 ~D120

Bovidae Bos gaurus Skin I Bos taurus 922 33.2-37.5 106 4 3 1 Dead <24 Srirattana et (Gaur) fibroblast hr al., 2012 Bos gaurus Ear fibroblast I Bos taurus 228 18.1 ND ND ND ND Mastromonaco (Gaur) et al., 2007 Bos gaurus Skin I Bos taurus 692 12.0* 44 6 2 1 Dead< 1 Lanza et al., (Gaur) fibroblast week 2000 Budorcas Ear fibroblast II Bos taurus 227 6.6 ND ND ND ND Li et al., 2006 taxicolor (Takin) Taurotragus Seminal Not listed Bos taurus 209 0 ND ND ND ND Nel-Themaat oryx epithelial cell et al., 2008 (Common Eland) Naemorhedus Skin I Bos taurus 506 0-5.0 ND ND ND ND Oh et al., 2006 goral fibroblast (Himalayan Goral) Bos javanicus Ear fibroblast Not listed Bos taurus or 319 20.0-28.0 38 2 0 0 0 Sansinena et (Banteng) Bos indicus al., 2005 Capra ibex Ear fibroblast Not listed Capra hircus 709 11.0 ND ND ND ND Wang et al., (Alpine Ibex) 2007 Capra Ear fibroblast Extinct Capra hircus 782 NA 184 7 1 Dead <24 Folch et al., pyrenaica hr 2009 pyrenaica (Pyrenean Ibex) Pantholops Ear fibroblast I Oryctolagus 1097 5.5-20.4 ND ND ND ND Zhao et al., hodgsonii cuniculus 2006 (Tibetan Antelope) Tragelaphus Skin III: Ghana Bos taurus 365 14.0-24.2* ND ND ND ND Lee et al., eurycerus fibroblast 2003 isaaci (Bongo)

Cell donor Cell type CITES Recipient Reconst. Blastocyst Transferred No. Fetuses/ Offspring Outcome Reference Appendix Oocyte Embryos Rate (%)a embryos Pregnancies (n) (n) (n) (n) ~D30 ~D60 ~D120

Ovis Skin II: Mexico Ovis aries 265 NA 223 5 0 0 0 Williams et canadensis fibroblast al., 2006 mexicana (Desert Bighorn Sheep) Ovis Granulosa I Ovis aries 23 30.4* 7 2 1 1 1 Live Loi et al., orientalis cell 2001 musimon (European Mouflon) Ovis ammon Skin II Ovis aries NA 0 28 1 0 0 White et al., (Argali fibroblast 1999 Sheep) Ovis Skin I Ovis aries 667 7.6 12 2 2 Dead <24 Hajian et al., orientalis fibroblast hr 2011 isphahanica (Esfahan Mouflon) Pseudoryx Skin I Bos taurus 312 35.1 ND ND ND ND Bui et al., nghetinhensis fibroblast 2002 (Saola Antelope) Bison bison Ear fibroblast II Bos taurus 226 19.2 ND ND ND ND Kumar et al., athabacae 2010 (Wood bison) Cervidae Pudu pudu Ear fibroblast I Bos taurus 89 0-7.4 ND ND ND ND Venegas et al., (Pudú) 2006 Felidae Pardofelis Muscle I Felis catus or 63/56 0-11.5 ND ND ND ND Thongphakdee marmorata fibroblast Oryctolagus et al., 2006 (Marbled Cat) cuniculus Pardofelis Skin I Felis catus 81 5.4 ND ND ND ND Thongphakdee marmorata fibroblast et al., 2010 (Marbled Cat) Felis Skin II Felis catus 485 6.0-43.0 1600 18 14 14 4 Dead 0 Gómez et al., margarita hr; 5 Dead 2008 (Sand cat) <24 hr; 5

Cell donor Cell type CITES Recipient Reconst. Blastocyst Transferred No. Fetuses/ Offspring Outcome Reference Appendix Oocyte Embryos Rate (%)a embryos Pregnancies (n) (n) (n) (n) ~D30 ~D60 ~D120

Dead < 60 d Felis nigripes Skin I Felis catus NR 0-3.3* 612 14 0 0 Gómez et al., ( Black fibroblast 2011 Footed Cat) Panthera Skin I Sus scrofa 675 0-1.6 ND ND ND ND Hashem et al., tigris altaica fibroblast domesticus 2007 (Siberian Tiger) Prionailurus Muscle and I Felis catus 561 8.3-8.6 384 0 0 0 Thongphakdee planiceps skin et al., 2010 (Flat-headed fibroblast Cat) Prionailurus Skin I or II Felis catus 185 5.7-20.7 409 6 0 0 Lee et al., bengalensis fibroblast 2010 (Leopard Cat) Prionailurus Skin I or II Felis catus 412 7.2-7.8* ND ND ND ND Yin et al., bengalensis fibroblast 2006 (Leopard Cat) Felis Skin II Felis catus 484 21.0-41.7 ND ND ND ND Gómez et al., silvestris fibroblast 2003 libica (African Wild Cat) Felis Skin II Felis catus 1552 NA 1552 24 17 17 7 Dead o Gómez et al., silvestris fibroblast hr; 7 2004 libica Dead< 36 (African Wild hr; 3 Live Cat) Panthera Ear fibroblast I Felis catus NR 8.8* NA 0 0 0 Hwang et al., tigris altaica 2001 (Siberian Tiger) Canidae Canis lupus Ear fibroblast I or II Canis 251 NA 251 4 2 2 Live Kim et al., (Gray familiaris 2007 Wolf) Canis lupus Skin I or II Canis 372 NA 372 6 6 6 3 Dead Oh et al., 2008 18

Cell donor Cell type CITES Recipient Reconst. Blastocyst Transferred No. Fetuses/ Offspring Outcome Reference Appendix Oocyte Embryos Rate (%)a embryos Pregnancies (n) (n) (n) (n) ~D30 ~D60 ~D120

(Gray fibroblast familiaris <24 hr; 3 Wolf) Live Ursidae Ailuropoda Muscle I Oryctolagus 612 10.9 ND ND ND ND Wen et al., melanoleuca fibroblast Cuniculus 2005 (Giant Panda) Ailuropoda Muscle I Oryctolagus 2510 NA 2510 2 0 0 Chen et al., melanoleuca fibroblast Cuniculus 2002 (Giant Panda) Ursus Skin I Bos Taurus 270 4.1* ND ND ND ND Ty et al., 2003 thibetanus fibroblast Ailuridae Ailurus Ear fibroblast I Oryctolagus 194 22.6 ND ND ND ND Tao et al., fulgens (Red Cuniculus 2009 Panda) Balaenopteri dae Balaenoptera Granulosa I Bos taurus / 1003 0 / 0 ND ND ND ND Ikumi et al., bonaerensis cell Sus scrofa 2004 (Minke Domesticus Whale) Balaenoptera Fetal I Sus scrofa 253 0 ND ND ND ND Lee et al., borealis fibroblast domesticus 2009 (Sei Whale) Balaenoptera Fetal I Bos Taurus 397 0 ND ND ND ND Bhuiyan et al., borealis fibroblast 2010 (Sei Whale) Hominidae Pan Skin I Bos Taurus 1224 0 ND ND ND ND Wang et al., troglodytes fibroblast 2009 (Chimpanzee) Macaca Skin II Oryctolagus 557 12.7 ND ND ND ND Yang et al., mulatta fibroblast Cuniculus 2003 (Rhesus Monkey) ND - procedure not done; NA - data not available, aBlastocyst development was calculated based on number of cleaved embryos unless otherwise indicated (*)

Problems associated with SCNT and iSCNT technologies

Despite the advantages that SCNT offers over many reproductive biotechnologies, its efficiency still remains very low. In the case of bovine embryos produced by SCNT, a large proportion is lost during the first trimester of gestation (Oback and Wells, 2007; Meirelles et al.,

2009). Additionally, a vast range of abnormalities have been observed in fetuses and offspring during pregnancy and at birth commonly associated with placental abnormalities and inability to adapt to extra-uterine life (Arnold et al., 2008; Meirelles et al., 2009; Brisville et al., 2011) resulting in less than 10% of the bovine pregnancies yielding healthy calves (Arnold et al.,

2008).

Pregnancy failure in females carrying fetuses produced by SCNT has been a major focus.

In bovid species, one of the most common causes linked to embryonic/fetal loss has been malfunction of placentomes (Hill et al., 2000; Constant et al., 2006). It has been hypothesized that placentome anomalies might be linked to epigenetic modifications in the trophectoderm. For instance, an association between placenta abnormalities and hypomethylation of some regions of imprinted genes involved in fetal growth and placenta formation such as SNRPN, H19, and

IGF2R in cattle SCNT embryos has been recently reported (Smith et al., 2012). Moreover, other studies have found differences in gene expression from trophoblastic tissues in SCNT embryos.

More specifically, abnormal profiles have been confirmed in the case of Mash2, hand1, and

VEGF-A, which are genes involved in mononucleate cell proliferation, binucleate trophoblast giant cells formation, vascular growth and permeability, respectively (Arnold et al., 2008).

In addition to the abnormalities observed at the fetal stages, numerous alterations during the preimplantation stages have also been documented in embryos produced by SCNT and

iSCNT (Balbach et al., 2012; Mastromonaco et al., 2014). Studies using in vitro fertilized embryos have revealed that early development is supported by stored mRNA (maternal transcripts) accumulated during ovarian follicular growth (reviewed by Li et al., 2010). Thus, embryos rely on maternal products during the first, second, and third mitotic divisions (Memili and First, 1999). However, through mechanisms not yet fully understood, maternal transcripts become less available coincidentally with embryo genome activation (EGA) (Gad et al., 2012).

In the bovine embryo, EGA takes place at the 8-16 cell stage which has been described as one of the most important processes in mammalian embryos (Barnes and First, 1991). It has been estimated that approximately 4489 genes belonging to the categories of transcription initiation, purine nucleotide metabolic process, protein ubiquitination, and RNA metabolic process are activated at the 8-cell stage (Graf et al., 2014). Consequently, much attention has been paid to this particular time not only because major transcriptional events occur but also because a large number of in vitro produced bovine embryos undergo developmental block (Lequarre et al.,

2003). Therefore, failure to inactivate essential keys or inability to eliminate maternal transcripts has been correlated with embryo demise at the time of EGA (Meirelles et al., 2004, Biase et al.,

2014). Interestingly, a majority of studies involving iSCNT experiments have reported a developmental blockage coinciding with the stage of EGA (Wen et al., 2005; Tecirlioglu et al.,

2006; Zhao et al., 2006; Jiang et al., 2011).

One of the most intriguing properties of the ooplasm environment involves silencing of genes present in the somatic cell (Ng and Gurdon, 2005). Although mechanisms have not been elucidated yet, abnormal gene expression during normal pre-implantation stages has been documented (Pfister-Genskow et al., 2005). Therefore, the inability to turn off certain somatic genes has been associated with development failures in iSCNT and SCNT embryos leading to

embryonic arrest. For instance, an iSCNT study revealed that domestic cattle ooplasm was unable to properly silent more than 800 rhesus monkey somatic genes (Wang et al., 2011). The embryonic genome not only must undergo inactivation of somatic genes at the beginning of development, but also requires activation of essential genes (EGA) to reach further stages. Errors during these critical steps might cause increased embryo arrest and developmental abnormalities as reported by several authors (Amarnath et al., 2011; Gómez et al., 2011; Jiang et al., 2011;

Wang et al., 2011).

Generation of mammalian embryos by SCNT has been considered a milestone in reproductive biotechnology in the last century (Campbell et al., 1996). Although studies have revealed significant findings in nuclear reprogramming, it is not clear how the mechanisms by which either matured oocytes or enucleated one-cell embryos confer pluri-and totipotency attributes to the nucleus of the somatic cell (Esteves et al., 2010). Thus, successful development has been linked to high reprogramming rates leading to increased expression of pluripotent genes

(Staszkiewicz et al., 2013). Failure to reactivate pluripotent and developmental genes has been reported as a cause of embryonic demise in SCNT and iSCNT. Chung et al., (2009) found significant differences in gene expression profiles of Oct-4, Sox-2, and Nanog. Their human model identified down-regulation of these three genes when the donor oocyte was bovine or rabbit origin compared to increased levels of expression in SCNT human embryos. Despite this fact, the study showed that the majority of iSCNT early cleavage embryos successfully replicated the human DNA in the blastomeres, however all embryos failed to continue embryonic development.

Further mechanisms leading to low cloning efficiency include epigenetic modifications.

It has been reported that embryos from different species are predisposed to epigenetic defects

caused by disruption of genomic imprinting (reviewed by Weaver et al., 2009). Loss of imprinting during in vitro early mouse development has been associated with retardation reaching the blastocyst stage (Market and Velkert et al., 2010). In SCNT models, it has been reported that aberrant methylation patterns can cause altered expression of imprinted genes in mouse embryos (Mann et al., 2003). Furthermore, it has been reported that disruption of the imprinting might compromise further embryonic development (Suteevun et al., 2006; Zheng et al., 2012; Smith et al., 2012). DNA methylation and histone modification have been proposed as the main epigenetic mechanisms regulating chromatin status. Thus, unmethylated DNA and histone hyperacetylation occur concurrently with activation of embryonic genes and at the morula stage in cattle embryos (Hou et al., 2007). Changes in DNA methylation have also been reported in iSCNT black-footed cat embryos. Gómez et al., (2011) found that despite the related taxonomic distance between a wild-type cat and the domestic cat, iSCNT embryos showed altered epigenetic marks in histones H3K6ac and H3K9me2, specifically hypermethylation of the former histone when compared to IVF-derived embryos.

Many studies have suggested that iSCNT embryos fail to progress due to incompatibilities between the nucleus and cytoplasm at structural and functional levels. In pig- cattle iSCNT embryos, it has been demonstrated that the transition into activation of the embryonic genome might be blocked by a significant reduction in the activity of RNA Pol II as determined by accumulation of polyadenylated transcripts in the nuclei (Lagutina et al., 2010).

Similar models have also linked iSCNT embryonic arrest with improper nucleoli formation due to structural differences and aberrant expression of proteins necessary for nucleoli assembly

(Hamilton et al., 2004; Song et al., 2009; Lagutina et al., 2011). The Lagutina et al., 2011 study suggested that iSCNT embryos were unable to properly translate genes involved in the synthesis

of rRNA. Additionally, they observed partial nucleoli formation in embryos derived from related species, such as water buffalo-bovine and ovine-bovine, indicating a degree of compatibility between nucleus and cytoplasm compared to species taxonomically more distant.

Incompatibilities between nucleus and cytoplasm documented in nuclear transfer studies have been further explored by other researchers (Lloyd et al., 2006; Mastromonaco et al., 2007).

Transfer of a somatic cell into an enucleated oocyte results not only in incorporation of nuclear

DNA, but also cytoplasmic factors and organelles, such as the mitochondria (Campbell et al.,

1996). As a result, nuclear and mitochondrial DNA derived from a divergent donor somatic cell must interact with several ooplasmic components to successfully drive complex cellular processes during early development. However, it has been hypothesized that iSCNT embryos fail to develop to the blastocyst stage and subsequent post implantation stages due to impaired mitochondria function as a result of incompatibilities between the nuclear and mitochondrial genomes (Mastromonaco et al., 2007). However, only few studies have properly addressed this hypothesis. Mitochondria play essential roles during embryo development including homeostasis, cell signalling, and energy production via oxidative phosphorylation (OXPHOS)

(Nelson and Cox, 2004). Energy production via OXPHOS requires precise interplay between mitochondrial and nuclear genomes to transcribe and translate some of the subunits of the electron transfer chain (Attardi, 1986; Clayton, 1991). The role of nuclear-mitochondrial incompatibility was illustrated by Amarnath et al. (2011) who showed aberrant gene expression profiles of mitochondrial and nuclear encoded factors in pig-mouse iSCNT embryos. More details regarding the intimate communication between genomes will be discussed in the next section.

Nuclear and mitochondrial genome interaction

Mitochondria possess their own DNA (mtDNA) and it accounts for approximately 1% of the total DNA present in the cell. Genes encoded by the mtDNA include: 13 structural subunits of the electron transfer chain (complex I, III, IV, and V), 22 transfer RNAs, and only 2 ribosomal

RNAs all located within a portion of the genome known as coding region (Anderson et al.,

1981). A different section of the mtDNA is called the non-coding region, which plays important roles in mitochondrial transcription and replication, as well as species identification through phylogenetic studies (Clayton, 1991; Gill et al., 1994).

Although mitochondria are able to drive their inherent processes (e.g. translation of mitochondrial mRNA), mitochondria depend on nuclear genes for adequate function. Synthesis of the different proteins that constitute four of the complexes of the electron transport chain

(ETC) require participation of genes from both the nuclear and mtDNA genomes, except complex II whose subunits have a nuclear DNA origin (Hagerhall, 1997). Therefore, energy production through OXPHOS is dependent on synchronous communication between the mtDNA and nuclear DNA. However, when the two genomes are not compatible, such as in xenomitochondrial cybrids, abnormal respiratory rates have been reported (Kenyon and Moraes,

1997). These authors observed compatible interactions among cellular hybrids with less divergent distance compared to those with greater evolutionary distance as a result of sequence variation in the mtDNA and some of the mtDNA-encoded polypeptides.

Transcription of mitochondrial mRNA also requires complex interactions between the two genomes. Products encoded by the nucleus, such as transcription mitochondrial factor

(TFAM) (Fisher and Clayton, 1985), TFB1M and TFB2M (Falkenberg et al., 2002; McCulloch

et al., 2002) and mTERF (Daga et al., 1993; Fernández-Silva et al., 1997) are essential for mitochondrial transcription. TFAM is capable of inducing conformational changes in the promoter of the mtDNA that enables a specific RNA polymerase to transcribe the mtDNA

(Shadel and Clayton, 1997).

Other mitochondrial functions are also dependent on nuclear signaling. Mitochondrial genome biogenesis requires the activation of two nuclear factors known as nuclear respiratory factor 1 and 2 (NRF1, NRF2). Both genes can activate all ten nuclear genes responsible for encoding the subunits of the complex IV of the ETC. However, the mitochondrial genes (COX-I,

COX-II, COX-III) are also indirectly regulated by NRF1 and 2 (Ongwijitwat and Wong-Riley,

2005; Dhar et al., 2007). Although these interactions have been documented in few types of cells, studies performed in murine neurons have demonstrated some transcriptional regulatory functions of NRF1 and NRF2 on COX nuclear encoded genes, which emphasize the mechanisms responsible for regulation, function, and control of the bi-genomes (Machatkova, 2012).

Furthermore, TFAM has been implicated in mtDNA maintenance and replication by mediating the packing of specific binding sites of the mitochondrial genome (Guivizzani et al., 1994;

Ekstrand et al., 2004). However, this bi-genomic coordination of factors can lead to complex interactions. For instances, NRF1 and NRF2 also regulate the expression of nuclear genes encoding subunits of the terminal enzyme of ETC (Ongwijitwat et al., 2006), and activate transcription factors such as TFAM (Virbasius and Scarpulla, 1994). Thus, these experiments have shown that mitochondria function is highly dependent on nuclear encoded products.

Mitochondria functions during pre-implantation stages

Mitochondria not only play important roles in cell aging and apoptosis (Voet et al., 2006;

Van Blerkom, 2008), but also during the initial stages of embryonic development in vertebrate embryos. Mitochondria have been mainly postulated to play a role in cell metabolism through the generation of ATP via OXPHOS (Attardi and Schatz, 1998). Thus, this energy-supplying organelle is fundamental for oocyte maturation, fertilization, early cleavage division, and formation of a blastocoele, among other functions. Additionally, it stores and regulates intracytoplasmatic calcium concentrations (reviewed by Van Blerkom, 2008).

Acquisition of cytoplasmic maturation in competent oocytes has been associated with changes in mitochondrial distribution and morphology in many species (Bavister and Squirell,

2000; Katayama et al., 2006; Van Blerkom., 2011). Specific patterns of mitochondrial distribution have been associated with high metabolic demands at various sites within the ooplasm resulting in significantly higher mitochondria number (Torner et al., 2004). Thus, perinuclear organization has been observed during progression to meiosis II in the oocyte, after fertilization, and at early cleavage stages in the mouse (Van Blerkom, 1991), hamster (Bavister and Squirrell, 2000), and human embryo (Van Blerkom et al., 2000). Mitochondrial remodeling at different stages of development has highlighted the importance of this organelle to support key processes occurring in various cytoplasmic compartments (Barnett et al., 1996).

It has been shown that mitochondrial dysfunction can lead to embryonic demise as a result of aging. Studies have demonstrated that mitochondria derived from aged cattle oocytes produced significantly lower amounts of ATP (Iwata et al., 2011). Moreover, functional and morphological changes in oocyte mitochondria of human patients older than 35 years of age have

been linked to infertility problems (Wu et al., 2000; Ottolenghi et al., 2004). Additionally, mitochondrial DNA copy number has been associated with oocyte and embryonic developmental competence. However, there is enormous variability in terms of mtDNA copy number per oocyte or embryo (Jansen, 2000; Cummins, 2002). In the case of human oocytes, higher mitochondria

DNA copy number has been correlated with higher fertilization rates. Reynier et al., (2001) described a relationship between fertilization failure and low mitochondrial DNA copy number as a result of decreased mitochondrial biogenesis during oocyte maturation. However, a large proportion of these findings are difficult to correlate to embryo competence due to a lack of standardized methodology for mtDNA copy number quantification which leads to great variability. On the other hand, this type of analysis is useful to confirm the presence of more than one mitochondrial genome when mutations are suspected or to determine the presence of more than one mtDNA genome, commonly reported in SCNT and other ARTs.

OXPHOS not only drives most of the energy production in the eukaryotic cells but also generates reactive oxygen species, which in turn might contribute to higher mutation rates in the mitochondrial genome rather than the nuclear DNA (Cummins, 2001). Furthermore, absence of histones in mtDNA may predispose it to DNA mutations by a direct effect of reactive oxygen species, such as O2-and OH-, although some studies have demonstrated the existence of mtDNA repair mechanisms (Vermulst et al., 2008). One of the most common mtDNA mutations has been identified as a 4977-bp deletion. It has been reported in many cells including oocytes and embryos (Hsieh et al., 2004; Salahi et al., 2013). The 4977-bp deletion has been associated with removal of structural genes, such as ATPase 6 and 8, cytochrome oxidase III, and various subunits of the NADH-CoQ oxidoreductase complex (Wang and Lü, 2009). As a result, aberrant gene expression profiles of mitochondrial encoded genes might explain some of the anomalies

observed in oocytes and embryos presenting this mutation including fertilization failure and embryonic arrest (Hsieh et al., 2004).

Mitochondrial inheritance in embryos produced by assisted reproductive techniques

During fertilization of mammal oocytes, the newly formed zygote inherits 99% of the mitochondria from the oocyte while only 1% of the mitochondria are inherited from the sperm.

As embryonic development progresses, mitochondria originating from the sperm (around 75 to

100 organelles) are targeted by the early embryo through a process known as ubiquitin recognition. This mechanism is carried out by proteolytic enzymes which facilitate elimination of paternal derived-mitochondria before EGA. It has been postulated that the high levels of free radicals produced within the sperm mitochondria might lead to mtDNA mutations resulting in deleterious effects for proper cell function (Aitken and Baker, 2004). Consequently, mammalian embryos only carry mitochondria inherited from the maternal lineage (Sutovsky et al., 2004).

Interestingly, sperm-mitochondria transmission resulting in healthy offspring has been reported in some invertebrates (Kondo et al., 1992; Zourus et al., 1992), and vertebrate species. Examples of sperm-mitochondria inheritance has been documented in inter-specific crossing in mice, and sheep (Shitara et al., 2000; Zhao et al., 2004). Human cases have also been reported, and unfortunately the presence of mutated paternal mtDNA (e.g. 2 base pair deletion in the MTND2 gene) was associated with muscle pathologies (Schwarts and Vissing, 2002).

Aberrant mitochondrial transmission has also been reported in embryos produced by

ARTs such as SCNT, ooplasm transfer, and spindle transfer (Cibelli et al., 2002; Cheng et al.,

2009; Amato et al., 2014). The presence of two of more mitochondrial genomes (heteroplasmy) has been implicated as one of the causes of poor survival rates of fetuses and offspring produced

by either SCNT or iSCNT (St John et al., 2004). Interestingly, the fate of mitochondrial replication reported by most SCNT studies does not follow a specific pattern. Moreover, three possible outcomes have been documented in the literature: 1) only mitochondria derived from the oocyte donor is maintained and then replicated (homoplasmy), 2) mitochondria present in the donor cell and in the oocyte donor are selected to undergo replication and thus, two mitochondria populations are transmitted to the next cell population (heteroplasmy), and 3) mitochondria derived from the oocyte and the somatic cell are segregated unevenly among blastomeres during the pre-implantation stages resulting in recombination of two populations in some blastomeres while others only inherit one (Steinborn et al., 2002; Burgstaller et al., 2007, Imsoonthornruksa et al., 2012). Many studies have properly described the proportion of mitochondria inherited into the different tissues of several species reproduced by SCNT techniques. However, the mechanisms favoring a particular pattern and the incidence of this phenomenon are not fully understood (Meirelles et al., 2001; Takeda et al, 2003; Jiang et al., 2006).

In the last decade, SCNT experiments have revealed important aspects regarding mitochondrial inheritance. Several studies have suggested that the presence of two mitochondria genomes can negatively impact early embryonic stages leading to developmental abnormalities and embryo failure (Barritt et al., 2001, John et al., 2005, Meirelles et al., 2009). However this hypothesis requires further investigation since healthy heteroplasmic offspring have been generated (Meirelles et al., 2009; Ferreira et al., 2010). Additionally, other authors argue that heteroplasmy might be tolerated by the cells as long as respiratory capacity is not negatively compromised (Van Blerkom., 2004). Although factors leading to heteroplasmy are not fully understood, some have suggested that mtDNA inheritance patterns might be affected by the type of somatic cell used during the SCNT procedures, the existence of two pre-existing

mitochondrial genomes, the distribution of blastomeres during the pre-implantation period, and the proximity of the mitochondria to the nucleus (Meirelles and Smith, 1998, Takeda et al.,

2005).

Other essential techniques, such as ooplasm transfer (OT) have also been used to understand the interactions between genomes and the effects on embryo and fetal development

(Takeda et al., 2005; Acton et al., 2007). In order to address these questions, researchers have generated heteroplasmic mice through ooplasm transfer and analysed a series of physiological variables (Acton et al., 2007; Sharpley et al., 2012). It was found that heteroplasmic mice had cardiovascular abnormalities, such as high blood pressure, increased weight and fat mass, as well as hematopoietic disturbances (Acton et al., 2007). In human reproduction, OT was first attempted to overcome chronic infertility in female patients (Cohen et al., 1997). Early studies hypothesized that addition of ooplasm from competent oocytes into less competent gametes would increase the developmental potential of the future embryo. Several cytoplasmic factors, mRNAs, proteins, and mitochondria present in the portion of transferred ooplasm are thought to be responsible for improving implantation rates in human patients (Levy et al., 2004). However, there has been constant debate about the use of this technique since predictable mitochondria transmission patterns have not yet been addressed. In addition, chromosomal abnormalities, psychological disorders, and spontaneous abortions have been associated with the use of this technique (Barrit et al., 2001). Consequently, the use of this technique in human reproduction was banned.

Mitochondrial replication during the preimplantation stages

The total number of mtDNA copies present in the mature metaphase II oocyte (~100,000 copies) are responsible not only to guarantee normal development but also to populate the mitochondrial genome in the entire organism (Jansen et al., 2000; Steuerwals et al., 2000).

However, mitochondrial replication is prevented during the early stages of development.

(Sutovsky et al., 1999, Sutovsky et al., 2004). In the mouse embryo, mtDNA copy number undergoes minimal variations (Thundathil et al., 2005), whilst in the cattle embryo there is a reduction (May-Panloup et al., 2005) coincidentally with every mitotic division, a process known as mitochondrial segregation. Thus, mtDNA is diluted into the blastomeres at each division in the absence of replication (Spikings et al., 2007). In mammalian embryos (e.g. bovine, porcine, and human) mtDNA replication is initiated at the blastocyst stage in the trophectoderm cells

(McCulloch et al., 2002; Asin-Cayuela and Gustafsson, 2007) and accompanied by mitochondrial morphological changes which are suggestive of acquisition of higher oxidative capacity (Wilding et al., 2001). It is believed that mtDNA replication is prevented at earlier stages because the level of expression of mitochondrial replication factors, such as TFAM and

PolgA, is very low or almost inexistent (May-Panloup et al., 2005). Bowles et al. (2007) indicated that high expression of these factors during early development might explain the levels of heteroplasmy found in iSCNT and SCNT embryos. However, the mechanisms driving elimination or replication of donor mtDNA during the preimplantation and post-implantation stages have not yet been elucidated.

RATIONALE

Implementation of assisted reproductive technologies in threatened and endangered species has been challenging. Restricted access to gametes and embryos from small populations has become one of the main obstacles. In wood bison, production of embryos by in vitro techniques has been inefficient (Thundathil et al., 2007). Although few bison embryos have been generated in vitro, there are no reports assessing the feasibility of these techniques to produce viable offspring which highlights the problems commonly affecting preimplantation stages. iSCNT has been reported as an alternative biotechnology to produce embryos and offspring from endangered species by using oocytes from related domestic species (Lanza et al., 2000).

Therefore, embryos from species whose gametes and embryos are very difficult to obtain can be reconstructed through iSCNT. Production of bison iSCNT embryos might provide valuable information regarding differences in developmental competence when compared to in vivo, in vitro IVF, or SCNT embryos. Multiple reports have indicated that one of the causes leading to poor developmental outcomes is attributed to incompatibilities between components present in the reconstructed embryo, such as nuclear and mitochondrial genomes. Evidence gathered in the last fifteen years has suggested that a large proportion of iSCNT embryos arrest at different stages during the pre-implantation period, more commonly observed at the time of embryonic genome activation (Yang et al., 2010), but only few reports have investigated the causes leading to embryo death at early stages. Investigating the potential causes leading to embryo demise in bison iSCNT studies is crucial to estimate the feasibility of iSCNT as an aid in preservation of scarce genotypes.

Ooplasm transfer has provided relevant information about complex nuclear-ooplasmic interactions taking place during embryo development and identified potential causes of embryo

demise. However, the number of studies involving this technique is very small. Only one report has combined iSCNT and ooplasm transfer simultaneously (Sansinena et al., 2010). Thus, essential information can be generated by application of these protocols in iSCNT studies; especially to understand the degree of compatibility that a reconstructed embryo might tolerate due to the presence of species-specific cytoplasmic factors and/or organelles.

The hypothesis of this study is as follows: cattle ooplasmic components influence mitochondrial function and related gene expression in iSCNT bison embryos, thereby affecting pre-implantation embryonic stages. This hypothesis was addressed by the following objectives:

Objective 1: to assess the developmental potential of parthenogenic plains bison embryos using domestic cattle in vitro protocols

Objective 2: to investigate the developmental potential and mitochondrial function of domestic cattle, plains bison, and wood bison embryos produced by iSCNT using domestic cattle oocytes as cytoplasts

Objective 3: to examine the effects of ooplasm transfer on in vitro embryo development, mitochondrial function, and gene expression of cattle SCNT and plains bison iSCNT embryos

Objective 4: to determine embryo viability of in vivo wood bison blastocysts and in vitro wood bison blastocyst produced by iSCNT

The general objective of this study was to investigate nuclear and cytoplasmic interactions in bison embryos taking place at the 8-16 cell stage through techniques such as iSCNT and ooplasm transfer. Firstly, a protocol to mature, culture, and chemically activate plains bison oocytes was implemented to determine the developmental potential of parthenogenic embryos and assess oocyte competence as potential ooplasm donor. Therefore, a co-culture system was

implemented to mature plains bison oocyte obtained from abattoir ovaries. Using previous protocols established in our laboratory, cattle SCNT and iSCNT bison embryos were generated to further investigate their developmental potential at earlier stages and to identify differences that lead to embryo demise, commonly reported at the time of embryonic genomic activation.

Evidence has suggested that there are communication failures in iSCNT embryos due to altered genetic divergence. By using a technique to successfully mature and culture cattle and plains bison oocytes, the effects of plains bison ooplasm transfer into plains bison iSCNT on early development were investigated. Finally, in vivo wood bison blastocysts were compared to in vitro iSCNT wood bison embryos to determine differences in embryo development.

CHAPTER ONE

In vitro production of bison parthenogenic embryos using a cattle protocol

INTRODUCTION

In vitro embryo production techniques in non-domestic bovids have progressed slowly when compared to domestic cattle. Important advances have been achieved in species, such as

African buffalo (Syncerus caffer; Arlotto et al., 2000; Kidson et al., 2000; Raina et al., 2001), and gaur (Bos gaurus; Armstrong et al., 1995; Hammer et al., 2001; Loskutoff et al., 2000;

Mastromonaco et al., 2007). Only four scientific reports have been published regarding in vitro embryo production in the North American bison including both subspecies (Thundathil et al.,

2007; Barfield and Seidel, 2012b; Seaby et al., 2012; Cervantes et al., 2013). Limited access to gametes and embryos from these species, few ovulatory cycles per year, and lack of information regarding proper conditions for transporting and culturing oocytes in vitro have adversely affected implementation of in vitro culture techniques (IVC) in bison species. Therefore, cattle in vitro production (IVP) protocols are usually extrapolated to species that have not been meticulously investigated in an attempt to support embryonic development; such is the case for wood bison (Thundathill et al., 2007).

Cattle IVP routinely uses abattoir-derived ovaries with blastocyst development rates of

30-40% (Lonergan et al., 2006; Mastromonaco et al., 2004). Although oocytes collected post mortem from bison females have been used for in vitro maturation (IVM) studies, the results have been modest for maturation rates and impaired embryo development post in vitro fertilization (IVF) (Seaby, 2010 M.Sc.; Mastromonaco et al., unpublished). Factors negatively affecting oocyte competence, including increased length of transportation of ovaries, temperature fluctuations during ovary transportation and storage, and presence of concomitant diseases

(Schernthaner, 1998; Sirard and Blondin, 1998; Fair, 2009), might explain abnormal morphological findings observed in immature bison oocytes under light microscopy prior to IVM

that result in impaired development due to decreasing oocyte quality (Mastromonaco et al., unpublished).

Oocytes isolated from abattoir ovaries have been used to study different aspects of early embryo development in vitro. Embryo production involves three fundamental steps: maturing oocytes to acquire fertilizing capability (IVM), incorporation of the paternal genome through the zona pellucida and plasma membrane (IVF), and supporting embryo development by supplementing specific components in a controlled-artificial environment (IVC) (Critser et al.,

1986; Stubbings et al., 1988). Despite the great advances in IVP of embryos in the last two decades, determining the causes leading to poor development in non-domestic bovids, such as bison, might require extensive analysis during each stage of embryo production to rule out technical aspects and optimize each step. Parthenogenesis, a reproductive mechanism employed by reptiles (e.g. snakes and lizards) and other organisms, results in the development of embryos carrying only the maternal genome (reviewed by Lampert, 2008). In mammalian species, parthenotes can be generated in vitro by artificial activation of the oocyte using ionophore derivatives and protein kinase inhibitors, which mimic the effects of the sperm during fertilization triggering intracellular calcium oscillation (Presicce and Yang, 1994). Thus, parthenogenesis in mammals not only can be used to assess appropriate conditions for IVC while omitting the IVF stage, but is also an essential step for the preparation of recipient cytoplasts for somatic cell nuclear transfer embryo production (Campbell et al., 1997, Yin et al., 2000).

The plains bison, a subspecies of the North American bison, is found across North

Mexico, the United States, and Canada on many farms where they are raised for meat production. In Canada, it is estimated that there are more than a quarter of a million bison raised for this purpose (Canadian Bison Association, 2014). Therefore, higher availability of gametes

from plains bison, in comparison to wood bison, represents a valuable opportunity to test and compare proper culture conditions between domestic cattle and bison. The use of plains bison as on experimental model instead of a taxonomically distant species (domestic cattle) might provide accurate information regarding IVP conditions for gametes and embryos in threatened species, such as the wood bison. Thus, successful development of IVP techniques in plains bison might contribute to the implementation of advanced assisted reproductive techniques, such as somatic cell nuclear transfer in bison species. The objectives of this study were to: 1) determine the maturation rates of plains bison oocytes based on polar body extrusion using a cattle co-culture system, and 2) assess the developmental potential of parthenogenic plains bison embryos using a cattle oocyte chemical activation protocol. The tests used to determine the first objective include morphological assessment and cell number and apoptosis rate for the latter objective.

MATERIALS AND METHODS

Sample collection

All samples were obtained from Canadian Food Inspection Agency approved abattoirs

(cattle, plains bison). Cattle ovaries were obtained from an abattoir within 30 minutes from the laboratory. Plains bison producers were contacted through the Canadian Bison Association member listing. The farms were selected based on distance time and travel time to our laboratory at the University of Guelph, Ontario. Farms located outside a 3 hour radius from the university were excluded to decrease transport time of the excised bison ovaries. Cumulus-oocyte complexes (COCs) were obtained from plains bison ovaries (n=12) between June and

September, 2014, and used for this study. A small number for COCs were used for experiments involving ooplasm transfer (described in chapter 3).

Study animals

Information regarding age of cattle females and reproductive status was not available. All plains bison females used in this study were nulliparous ranging in age from 2 to 3 years. Post mortem examination of the bison reproductive tract revealed no abnormalities. Bison ovaries were collected during the non-breeding season and beginning of the breeding season.

Experimental design

Groups consisted of plains bison oocytes with domestic cattle oocytes as the control group. Maturation rates were calculated based on the number of oocytes with extruded polar body at 24 hours post IVM. Cleavage divisions at 2 and 8-16 cell stages were estimated at 36 and

80 hours post oocyte chemical activation (hpa), respectively. Blastocyst rates were determined

every 24 hours starting on day 7 (~172 hpa post activation) until day 10 (~244 hpa). Blastocysts were assessed for cell number and apoptosis index. Cleavage and blastocyst rates were estimated based on the total number of oocytes in culture.

Chemicals

All chemicals were obtained from Sigma-Aldrich Chemical Co. (St Louis, MO) unless otherwise stated.

Oocyte collection and IVM

Domestic cattle and plains bison ovaries were washed and transported in 0.9% sodium chloride solution (Baxter Corporation, Ontario, Canada) at 37°C. Cattle and plains bison ovaries were collected from abattoirs located in Guelph Ontario and Dashwood Ontario, respectively.

Follicles were aspirated 2 to 2.5 hours after ovarian collection. Collection of cumulus-oocyte complexes (COCs) was performed by follicular aspiration into HEPES-buffered Ham’s F-10

(Sigma-Aldrich Canada, Oakville, ON, Canada), supplemented with 2% steer serum (Cansera;

Rexdale, ON, Canada). The cellular content was diluted in 10 ml of HEPES-buffered Ham’s F-

10 and immediately placed in a 60mm petri dish. Selection of COCs was performed following

Leibfried and First (1979) inclusion criteria. COCs were washed twice in IVM medium (TCM-

199®+ 10% Cansera) and once in IVM medium supplemented with FSH (National Hormone and

Peptide Program, Torrance, CA, USA), hCG (Chorulon®, Intervet, Ontario, Canada), and estradiol at a concentration of 2 μg/ml, 14 IU/ml, and 1 μg/ml, respectively. A co-culture system containing granulosa and cumulus cells was implemented for IVM. Cells were isolated from cattle ovarian follicles ranging from 4-8 mm and cultured using DMEM (Dulbecco's modified eagle's medium) supplemented with 2.4 mM L-glutamine (Life Technologies Inc, Burlington,

ON, Canada), 1% Penicillin/Streptomicin (P/S; Invitrogen Canada Inc., Burlington, ON,

Canada), and 10% fetal bovine serum (FBS; Life Technologies Inc, Burlington, ON, Canada).

Subsequently, cells in passage 2 were frozen in DMEM supplemented with 2.4 mM L-glutamine,

1% P/S, 20% FBS, and 10% dimethyl sulphoxide (DMSO) until needed. 24 hours prior to IVM, cells were thawed and cultured in 1.9cm2 wells (Fisher Scientific Company, Ottawa, ON,

Canada) at a final concentration of 3.23 x 105 cells/ml. Cell culture media (DMEM + 10% FCS) was replaced with 500 μl IVM medium containing hormones under 500 μl of silicone oil (Paisley

Products, Scarborough, ON, Canada) 3 hours prior placing the COCs in each well. The total number of COCs per well was 40 to 45. Co-culture was maintained in an atmosphere of 5% CO2 in air with maximum humidity at 38.5°C.

Parthenogenic embryo production

Domestic and plains bison cumulus cells were stripped from COCs by manual pipetting using hyaluronidase (1mg/ml) at 24 hours post-IVM for 3 minutes. Oocytes exhibiting a polar body and homogeneous cytoplasm were selected for parthenogenic activation. Chemical activation was performed in HEPES/Sperm TALP containing 5μM ionomycin for 5 minutes followed by incubation of an additional 4 hours in synthetic oviductal fluid (SOF) with 2mM 6- dimethylaminopurine (6-DMAP) at 38.5°C in 5% CO2 with maximum humidity.

Oocytes/embryos were cultured in 30 µl drops containing synthetic oviductal fluid (SOF)

(Chemicon-Millipore, Billerica, MA, USA) in a humidified atmosphere of 5% CO2, 5%O2, and

90% N2 for 10 days maximum (~244 hpa).

TUNEL staining of embryos

The terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling (TUNEL) assay (Roche Diagnostics, Indianapolis, IN, USA) was used to determine apoptosis in all treatment groups. Embryos at 80 hpa were stained as described previously (Favetta et al., 2004).

Briefly, embryos were washed three consecutive times in PBS containing 0.1% polyvinyl alcohol

(PVA) and fixed in 4% paraformaldehyde for one hour. Embryos were stored in 1% paraformaldehyde for up to two weeks, if not used immediately. Subsequently, embryos were permeabilized in 0.5% Triton X-100 supplemented with 0.5 % sodium citrate for 40 and 60 minutes in the case of positive control and test embryos, respectively. Positive control embryos were incubated at 38.5°C for 20 minutes in a solution containing 8μl of DNA buffer and 2.5 μl of

DNAse I (400 IU/ml; ZyGEM, New Zealand). Exposure of positive control and test embryos to fluorescein isothiocyanate (FITC)-conjugated dUTP and terminal deoxynucleotidyl transferase

(TdT) enzyme was performed for 1 hour at 38.5°C in a ratio of 9:1, while negative control embryos were incubated only with FITC. All groups were treated with 50 µg/ml RNAse (Roche

Diagnostics, Indianapolis, IN, USA) for 1 hour. The embryos were counterstained with 40 µg/ml propidium iodide (PI) for 45 minutes at 38.5°C. Embryos were then washed 5 times in 0.1%

PBS-PVA and placed onto a slide containing 13 μl of anti-fade solution (VECTASHIELD®,

Vector Laboratories, California) diluted in 0.1% PBS-PVA solution (1:1 ratio). TUNEL staining was detected using a Leica DM 5500 B fluorescence microscope at 20x magnification. A total of

13, 14, and 20 embryos were analyzed in cattle SCNT, plains bison iSCNT, and wood bison iSCNT, respectively.

Statistical analysis

Polar body extrusion rates, cleavage at the 2 and 8-16 cell, and blastocyst stage were compared using Chi-square. The proportion of TUNEL positive (apoptotic) nuclei and total cell number were assessed by Wilcoxon rank-sum test. A probability of <0.05 was considered significant. Data were analysed using the computer program RSTUDIO Version 0.97.551/IC

10.1

RESULTS

Polar body extrusion of cattle and plains bison oocytes

Presence of the first polar body post IVM has been used as an indicator of nuclear maturation status and progression of the oocyte from meiosis I to II. In this study, no significant differences were observed in the proportion of oocytes with extruded polar bodies between groups at 24 hours post IVM (p=0.12). The percentage of cattle and plains bison oocytes with polar bodies was 83.9 (59/71) and 72.3% (47/65), respectively. The presence of polar bodies in cattle oocytes and plains bison is shown in Figure 1.

In vitro development of cattle and plains bison parthenogenic embryos

To study the developmental potential of parthenogenic embryos from cattle and plains bison oocytes using a cattle IVC model, activated oocytes were cultured. The results showed no significant differences in the proportion of embryos at 2, and 8-16 cell stages between groups

(p>0.05). Developmental rates for 2, and 8-16 cell stage ranged between 70.8 to 87.5% (p=0.11) and 50 to 63% (p=0.64), respectively. Blastocyst rates differed depending on the assessment day with significant differences observed in the proportion of embryos reaching the blastocyst stage on days 7, 8, and 9 between groups (Table 4). However, no significant differences were found in the total blastocyst rate between cattle and plains bison parthenotes (p=0.08). Embryos at the blastocyst stage (day 9) are shown in Figure 2.

Table 4. Development of parthenogenic embryos

Stage of Cattle Plains Bison p -value development* [N=59] [N=47]

n (%) n (%)

2-cell stage 51(86.4) 35(74.5) 0.11

8-16 cell stage 34(57.6) 25(53.19) 0.64

Day 7* 5(8.5) 0(0) 0.04

Day 8* 6(10.1) 0(0) 0.02

Day 9* 3(5.1) 9(19.14) 0.02

Day 10* 0(0) 2(4.2) 0.11

Total blastocyst rate 14(23.7) 11(23.4) 0.96

* Determination of 2-cell stage and 8-16 cell stage were performed 36 and 80 hpa, respectively. Blastocyst rate calculation was performed every 24 hours from day 7 to day 10. To estimate total blastocyst rate, embryos were counted only once, as they were encountered.

Cattle Plains bison

)

A) B)

C) D)

E) F)

Figure 1. Phase contrast micrographs from cattle and plains bison oocytes before IVM (A-B), 24 hours post IVM (C-D), and matured oocytes (E-F). Arrows denote polar bodies.

Cattle Plains bison

Figure 2. Phase contrast micrographs of cattle and bison parthenogenic blastocyst on day 9 of development at 20X (A). Images in B area magnified (40X).

Apoptosis assessment

Cell number and apoptotic incidence was assessed to evaluate embryo quality in each treatment group. There were no significant differences in the proportion of TUNEL positive nuclei between groups (p=0.78). Similarly, total cell number per blastocyst did not differ between groups (p=0.49) (Table 5). The highest proportion of TUNEL positive nuclei per embryo was found in plains bison parthenotes while cattle parthenotes had the lowest incidence.

Fluorescence micrographs highlighting these findings are shown in Figure 3.

Table 5. Median [25%; 75%] total cell number and proportion of TUNEL labeling (apoptotic) nuclei in cattle and plains bison parthenogenic blastocysts

Parameter Cattle Plains Bison P-value

[n=14] [n=9]

Total cell 93 [70; 108] 66 [68; 103] 0.48 number/embryo

Proportion of 0.04 [0.02; 0,06] 0.02 [0,04; 0.08] 0.72

TUNEL positive nuclei/embryo

Red Channel Green Channel Merge

Cattle

Plains bison

Figure 3. Cattle and plains bison parthenogenic embryos showing nuclei TUNEL positive labeling (arrows).

DISCUSSION

The aim of this study was to assess the feasibility of applying techniques, commonly employed in domestic cattle, to mature and activate bison oocytes in vitro to produce parthenote embryos and cytoplasts. We evaluated progression from Germinal Vesicle stage oocytes to meiosis metaphase II stage and subsequently, development to the blastocyst stage in parthenotes.

The time at which the majority of plains bison oocytes reached meiosis metaphase II was 24 hours post IVM, similar to the work of Cervantes et al., 2013 using oocytes aspirated from super- stimulated follicles following FSH administration in vivo. Experiments conducted in our laboratory have reported low cleavage rates (< 53%), normal progression to the 8-16 cell stage

(45-53 % embryos advancing to this stage), and poor blastocyst rates (0%) when either plains bison or wood bison oocytes were matured and fertilized in vitro with bison sperm

(Mastromonaco et al., unpublished). Interestingly, the Mastromonaco study found similar results regardless of transport time, subspecies or individual age. Although the percentage of matured oocytes was not assessed, the low cleavage rates might be attributed to the inability of the oocytes to fully complete nuclear and cytoplasmic maturation resulting in compromised development at later stages (reviewed by Krisher, 2004). Conversely, our study showed that a large number of plains bison oocytes exhibited normal morphology characterized by homogeneous cytoplasm and expansion of cumulus cells post IVM similar to cattle oocytes.

Nuclear maturation and cleavage rates were 72 and 74%, respectively. The proportion of embryos progressing through the first cleavage divisions did not differ between groups.

Additionally, our study resulted in an increase in blastocyst rates (>20%) compared to previous studies, however delayed development at the blastocyst stage was observed in plains bison parthenotes. Cattle parthenogenic blastocysts were first found on day 7 and bison blastocysts on

day 9. Studies involving cattle models have indicated that delayed development in parthenogenic embryos can be caused by the absence of paternal genes (Van de Velde et al., 1999; Gómez et al., 2009) which could explain the slow progress through the next embryonic stages commonly observed at the time of embryonic genome activation (Caamaño et al., 2000). Barfield and Seidel

(2011) indicated that IVF plains bison embryos reach the blastocyst stage at day 7 while

Thundathil et al., (2007) reported the same development stage at day 8 in IVF wood bison embryos. Thus, species-specific differences leading to slower progression from the morula to the blastocyst stage in bison subspecies require further investigation. Applying techniques such as time-lapse imaging of bison development would be fundamental to provide explanations for this phenomenon.

Identification of optimal culture conditions for bison oocytes and embryos has not yet been documented. Variations in serum and hormones concentrations, addition of antioxidants, and presence of somatic cells during IVM have yielded different outcomes in cattle in vitro embryo production (Sirard and Blondin, 1998; Santana et al., 2014). Regarding the use of co- culture, evidence suggests that culturing COCs on a monolayer cell formed by granulosa cells improves blastocyst yield and embryo development (Dey et al., 2012). Oocytes denuded from cumulus cells have also shown an ability to complete cytoplasmic and nuclear maturation (Feng et al., 2013). The beneficial effects of culturing COCs on a monolayer have been linked to enhanced communication between cumulus cells and oocyte via gap junctions, which in turn stimulates secretion of paracrine factors essential for nourishment and cell signaling (Orsi and

Reischl, 2007, Gilchrist et al., 2008). Therefore, the use of a co-culture system in our study might explain the enhanced IVM and IVC outcomes obtained in this study compared to previous

attempts. However, this statement is difficult to confirm since culture of plains bison oocytes lacking granulosa cells was not performed.

Culture environment significantly influences embryo development (Rizos et al., 2002).

Delayed development has been associated with suboptimal culture conditions including metabolic deficiencies, increased oxidative stress, and epigenetic modifications (reviewed by

Fleming et al., 2004). Our findings indicate that bison parthenotes reached the blastocyst stage

24-48 hours later than cattle parthenotes suggesting deficiencies of the IVC system. A study conducted by Barfield and Seidel (2012b) investigated the effects of adding fetal calf serum

(FCS) at different concentrations on plains bison embryo development. The authors reported an improvement in the blastocyst rate (~16%) when 5% FCS was used during IVC. Interestingly, the blastocyst rates reported by Barfield and Seidel (2012b) were obtained on day 7 post IVF, a day earlier than the results reported by Thundathil et al., (2007) in wood bison. Thus, the use of serum at higher concentrations after the 8-16 cell stage might result in accelerated development as reported in other cattle studies (Van Langendonckt et al., 1997). Another factor that could compromise the transition to the blastocyst stage might be associated with decreased oocyte competence as a result of ovarian inactivity. In our study, the majority of bison oocytes were collected during the non-breeding season as evidenced by the presence of small ovaries (< 2 cm), absence of luteal structures, and follicle sizes less than 2 mm. It has been suggested that seasonality influences the developmental potential of IVP embryos in several species (Freistedt et al., 2001; Zheng et al., 2001; Colleoni et al., 2004). A study including more than 5000 ovine

IVP embryos found significantly higher blastocyst rates when oocytes were collected during the breeding season in comparison with the non-breeding season. However, no differences in pregnancy and lambing rates were observed when embryos were transferred into recipient ewes

(Mara et al., 2014). Nonetheless, further investigation of oocytes obtained during the breeding and non-breeding season in bison species might reveal interesting associations.

Apoptosis determination in preimplantation embryos has been used as a marker of proper development. Higher apoptotic incidence has been associated with DNA damage (Leidenfrost el at., 2011) while low apoptotic cell number might be indicative of competent embryos that are capable of eliminating altered cells causing no detrimental effects on further development (Byrne et al., 1999). The apoptotic assessment from our study revealed that the proportion of TUNEL- positive cells was similar between cattle and bison parthenogenic embryos. Experiments using cattle embryos have reported an apoptotic index of 9.1 in cattle parthenotes embryos using ionomycin in combination with 6-DMAP (Wang et al., 2008). On average, the apoptosis index found in our study in both groups was lower than the one reported by Wang’s study.

Interestingly, no significant differences were found in the proportion of TUNEL positive cells despite the delayed development observed in plains bison parthenotes.

Supplementation of in vitro oocyte maturation media with somatic cells has been used in the past to overcome the detrimental effects observed during IVC due to their beneficial properties including secretion of growth factors, removal of toxic metabolites from the culture medium, and progesterone synthesis among others (Simón et al., 1999; Bhushan et al., 2004;

Taniguchi, 2004). Therefore, the use of granulosa cells has been implemented in IVC systems in different species to improve embryo development outcomes. Goovaerts et al., (2011) found down regulation of GPX1, a gene involved in detoxification and protection against oxidative stress, in single embryos cultured without somatic cells, which could explain some of the positive effects observed when culturing embryos under the influence of somatic cells. In our study, granulosa cells were only used during IVM, however some cumulus cells were not fully

removed by the stripping process resulting in cumulus cell proliferation throughout development.

Unfortunately, the limited availability of plains bison oocytes impeded further investigation using oocytes completely devoid of cumulus cells.

The results of this study demonstrated that the use of a bovine-based protocol for oocyte maturation, chemical oocyte activation, and in vitro embryo culture can successfully be implemented in plains bison. To our knowledge, this is the first report showing the use of a chemical oocyte activation protocol to produce parthenogenic bison embryos in vitro. However, further research is necessary to determine the incidence of chromosomal abnormalities derived from these procedures and the causes of delayed development in plains bison parthenotes.

Although we were able to surpass blastocyst rates reported by other investigators (Thundathill et al., 2007; Seaby, 2010, M.Sc.), delayed development observed after the 8-16 cell stage might indicate suboptimal culture conditions. The data presented here demonstrated that acceptable maturation and development rates throughout preimplantation stages can be achieved by using a co-culture IVM and IVC system generally employed for cattle gametes. However, further studies are necessary to determine the causes of delayed development in plains bison parthenotes.

Additionally, the results of this study provided valuable information regarding implementation of oocyte activation protocols, which are essential steps in more advanced reproductive biotechnologies such as somatic cell nuclear transfer. Establishment of proper culture conditions for maturation of bison oocytes is also indispensable to conduct experiments involving transfer of a portion of competent bison cytoplast in interspecies plains bison nuclear transfer embryos.

CHAPTER TWO

Developmental competence of 8-16 cell stage bison embryos produced by

interspecies somatic cell nuclear transfer

INTRODUCTION

In vitro embryo production (IVP) in domestic species has become a standard reproductive biotechnology globally. A survey conducted by the International Embryo Transfer Society

(IETS) estimated that almost a half million bovine embryos were generated by in vitro techniques in 2012 (Perry, 2013). Conversely, the number of embryos produced using these procedures in non-domestic and endangered species is unknown (Wildt et al., 2003;

Mastromonaco et al., 2014). Although numerous scientific communications have recommended the implementation of IVP techniques as potential tools for species conservation, poor embryonic developmental rates have been obtained when compared to domestic species (Wildt et al., 1992; Pukazhenthi et al., 2006). There are several factors contributing to this low success, including the difficulty in obtaining gametes from endangered species and the lack of knowledge regarding optimal in vitro culture conditions of gametes and embryos (Monfort, 2014).

Further advanced embryo technologies have been proposed to perpetuate the genomes of endangered species. Dominko et al., (1999) introduced the concept of interspecies somatic cell nuclear transfer (iSCNT) by using enucleated cattle oocytes to reprogram the nuclei of four taxonomically distant mammalian species, and consequently, support early embryonic stages.

Close to 20,000 iSCNT embryos have been reconstructed from species categorized in a conservation status under CITES (Convention on International Trade in Endangered Species of

Wild Fauna and Flora) (reviewed by Mastromonaco et al., 2014). Despite this extensive amount of work, few healthy offspring have been born following iSCNT. Wild species with significant success include: canids [coyotes and wolves (Hwang et al., 2013; Kim et al., 2007)], and felids

[African wildcat and sand cat (Gómez et al., 2004; Gómez et al., 2008)].

While numerous studies have focused on the oocyte’s ability to reprogram and support early embryonic stages, few studies have investigated factors involved in embryonic failure during the preimplantation stages. iSCNT outcomes in non-domestic species suggest that higher blastocyst rates and successful establishment of pregnancy are obtained when the taxonomic distance between species is decreased (reviewed by Mastromonaco et al., 2014). Causes associated with early embryo demise include: incomplete nuclear reprogramming, downregulation of embryonic genes, epigenetic alterations, aberrant gene expression, and nuclear and cytoplasmic incompatibilities (Amarnath et al., 2011; Gómez et al., 2011; Jiang et al., 2011;

Wang et al., 2011). Regarding this last aspect, two cellular structures have been linked to poor embryo development: malfunctioning of the nucleoli in pig-cattle iSCNT embryos (Lagutina et al., 2011), and improper cross talk between the mitochondrial and nuclear genomes (Lloyd et al.,

2006). However, the majority of models have used very distant species resulting in several alterations. Consequently, it is not clear how these interactions between different genomes or structures within the foreign cytoplasm are causing deleterious effects in taxonomically divergent reconstructed embryos.

Previous iSCNT experiments in our laboratory have used domestic cattle cytoplasts and somatic cells from Bos gaur and Bison spp. to reconstruct embryos. These studies showed that a large proportion of gaur and bison iSCNT embryos arrest at the 8-16 cell stage. As development progresses, lower blastocyst rate, higher apoptosis index, and low cell number also were reported in comparison with cattle SCNT embryos. Additionally, gene expression profiles between cattle

SCNT and iSCNT bison embryos were very similar indicating that potential aberrant transcriptional interactions might occur at earlier stages in these embryos (Mastromonaco et al.,

2007, Seaby et al., 2013). Therefore, our main objective was to evaluate the developmental

potential of iSCNT embryos during the stage at which embryonic arrest occurred more frequently using two American bison subspecies as genetic donors: plains bison (Bison bison bison) and wood bison (Bison bison athabascae) included in the list of species protected by

CITES. Furthermore, the degree of incompatibility was assessed by ATP production, and gene expression of selected nuclear and mitochondrial encoded genes in embryos from both subspecies at the 8-16 cell stage was compared to cattle SCNT embryos.

MATERIALS AND METHODS

Experimental design

Treatment groups consisted of the following: 1) cattle SCNT, 2) plains bison iSCNT, 3) wood bison iSCNT. All reconstructed embryos were produced using domestic cattle oocytes.

Each experiment was repeated on 4 biological replicates. Cleavage divisions at 2, 4, and 8-16 cell stages were determined at 36, 50, and 80 hours post oocyte chemical activation (hpa), respectively. Reconstructed embryos at 80 hours hpa were randomly assigned to various analyses. Only embryos showing normal morphology under light microscopy were included.

Cleavage rates were estimated based on the total number of reconstructed embryos in culture.

Chemicals

All chemicals were obtained from Sigma-Aldrich Chemical Co. (St Louis, MO) unless otherwise stated.

Culture and preparation of donor somatic cells

Ear fibroblasts obtained from cattle, plains bison, and wood bison adult males were used as somatic cell donors. Punch biopsy samples from tissues were collected from Canadian Food

Inspection Agency approved abattoirs (cattle, plains bison) and the University of Saskatchewan.

(wood bison). Fibroblast cell cultures were produced as described previously (Mastromonaco et al., 2007). Cells used as the somatic cell donor were at passages 3-5 and confluent for 2-3 days.

Oocyte collection and in vitro maturation (IVM)

Procedures for collection and maturation, of cattle cumulus-oocyte complexes (COCs) were performed as described in chapter 1.

Oocyte reconstruction by micromanipulation techniques and in vitro culture (IVC)

The SCNT technique was carried out according to Mastromonaco et al., (2004). Briefly, oocyte stripping was performed by manual pipetting using hyaluronidase (1mg/ml) at 17.5 hours post IVM for 3 minutes. Oocytes exhibiting a polar body and homogeneous cytoplasm were selected and stained with 0.5 μg/ml bisbenzimide (Hoechst-33342) for 3 minutes. Enucleation and transfer of the somatic cell were carried out on an inverted microscope (Leica DM IRB).

Denuded oocytes were manipulated in 40 μl microdroplets containing HEPES/Sperm TALP medium and cytochalasin B (5 μg/ml). The fusion procedure was performed using an Electro

Square Porator ECM 830® (BTX Harvard Apparatus, USA). A single DC pulse was used at 2.3 kV/cm for 23 μs in a 0.5 mm electrofusion chamber. The fusion medium contained 0.28M mannitol solution supplemented with 0.01%BSA, 100μM MgCl2, and 50μM CaCl2. Chemical oocyte activation was performed as described in chapter 1. The same monolayer used for each run of IVM was then employed for IVC. Similar culture conditions described above were used for embryo co-culture, with substitution of IVM medium with 350 μl of SOF medium under 500

μl of silicone oil at 38.5 °C in 5% CO2 with maximum humidity. Half of the total SOF volume was replaced with new SOF at day 3 of co-culture. Cattle SCNT and bison iSCNT embryos were collected at 80 hours post activation (hpa; day 4) and randomly distributed for various analyses.

TUNEL staining of embryos

The terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling (TUNEL) assay (Roche Diagnostics, Indianapolis, IN, USA) was used to determine apoptosis in all groups.

All embryos at the 8-16 cell stage were treated as described in chapter 1.

ATP determination

Mitochondrial function was assessed using adenosine 5′-triphosphate quantification assay

(Bioluminescent somatic cell assay kit; Sigma-Aldrich, Oakville, ON, Canada) in single embryos from all experimental groups following the protocol by Tamassia et al., (2004) with some modifications. Embryos were washed four consecutive times in HEPES/Sperm TALP medium and stored in 10 μl of the same medium. Subsequently, embryos were snap frozen and kept at

-80C° until needed. Solution preparations and assay were performed according to the manufacturer’s instructions. Individual samples were thawed on ice and 10 μl of Somatic Cell

Releasing Agent was added and incubated for 5 minutes. The reaction took place by adding 30 μl of ATP Assay Mix Working Solution to each sample previously placed in a 96-well microplate

(Corning® Costar 96-well white clear-bottom plate, Fisher Scientific). The plates were read 3 minutes after the ATP Assay Mix Solution was added and samples were mixed. ATP content

(pmol/embryo) was determined using a standard curve, with values ranging from 0 to 5 pmoles.

Luminescence was determined using a Fluostar Optima plate reader (Fisher Scientific). A total of

10 embryos per group were used for this analysis.

RNA extraction and reverse transcription

Embryos from the three groups were collected in pools of 5 at the 8-16 cell stage and washed three times in PBS-PVA 0.1%, snap-frozen in liquid nitrogen and stored at -80°C until

needed. RNA was isolated from pooled embryos using the miRNeasy Micro Kit (QIAGEN, Inc,

Burlington, ON), following the manufacturer’s instructions with minor modifications. In brief, pellets were added of QIAzol Lysis reagent, RNA separated with chloroform, washed multiple times and eluted from the columns in RNAase-free water. RNA samples underwent column- based DNAase digestion during the extraction process according to the manufacturer’s instructions. RNA samples were reverse transcribed immediately following extraction using the one-step protocol with qScript cDNA Super Mix (Quanta Biosciences, Canada) following the manufacturer’s instructions. cDNA samples were stored at -20°C until needed.

Gene expression analysis

Quantitative real time PCR (qPCR) was used to measure mRNA expression profiles of selected genes in the three treatment groups (cattle SCNT, plains bison iSCNT, wood bison iSCNT). Each transcript analysis was performed on three biological replicates with 3 technical replicates for each of them, unless otherwise stated. Two of the chosen transcripts [mitochondrial transcription factor A (TFAM), and nuclear respiratory factor 2(NRF-2)] belong to the group of genes related to mitochondrial replication, respiration and mitochondrial DNA transcription, all of which are encoded by the nucleus. The third transcript is encoded by the mitochondria (mt-

COX2) and encodes the subunit 2 of cytochrome c oxidase (complex IV of the electron transport chain). GAPDH was used as a reference gene for all qPCR experiments. The primer sequences are listed in Table 1. NRF-2 and GADPH primers were previously and successfully used in domestic cattle (Mastromonaco et al., 2007, Seaby et al., 2013), while mt-COX2 and TFAM primers were designed using Primer 3 Input (version 0.4.)-MIT software (Rozen and Skaletsky,

2000), and specificity of the primers was checked by sequencing and BLAST analysis. qRT-PCR was carried out using the BIO-RAD CFX96 Real-Time PCR System and products were detected

with SsoFast TMEvaGreen® Supermix (Bio-RadLaboratories, Hercules, CA, USA) according to the manufacturer’s protocols. Each reaction contained 5 μl of the SsoFastTM EvaGreen®

Supermix reaction mix, and 1 μl of a mix of the forward and reverse primers (at a 0.5 μM concentration) adjusted to a total volume of 10 μl using H2O. A standard curve was established for each primer set using ovarian tissue cDNA template in five serial dilutions and efficiencies were calculated for each of the primer sets used. The amplification program was as follows: preincubation for EvaGreen® Supermix polymerase activation at 95°C for 10 minutes, followed by 50 amplification cycles of denaturation at 95°C for 10 seconds, annealing at 65°C for 10 seconds, elongation at 72°C for 10 seconds, and acquisition of fluorescence for 10 seconds. After the last cycle, fluorescence acquisition started at 72°C, and measurements taken every 0.5°C until 95°C to generate the melting curve. Amplification was performed on GAPDH gene, used as a reference gene, as it was proven to be stable among our samples. Relative expression to

GADPH was calculated for each target gene, taking into account the primers’ efficiency in the

CFX Manager software (Bio-Rad).

Table 6. List of primers used for qPCR

Gene Gen bank Species Primer sequence Product

accession (5’-3’ direction) size

number (bp)

GAPDH NP_001029206 Bos taurus 5’ttcctggtacgacaatgaatt-3’ 131

5’-ggagatggggcaggactc-3’ mt-COX-2 AY676871.1 Bos taurus 5’attctgcccgccatcatc-3’ 203

5’-cgtagctcccctggcttt-3’

TFAM NM_001034016.2 Bos taurus 5’-ccgaaaagacctcgctca-3’ 221

5’-tctcgtccaacttccatcatt-3’

NRF-2 AB162435 Bos taurus 5’-tccaacctttgtcgtcatca-3’ 174

5’-ttgcccgtagctcatctctt-3’

Statistical analysis

Cleavage rates at 2, 4, and 8-16 cell stage of development were compared using Chi- square. ATP quantification was analysed using One-way ANOVA. The proportion of condensed,

TUNEL positive (apoptotic) nuclei, and gene expression levels were assessed by Kruskal-Wallis test. When comparing only two groups, Wilcoxon rank-sum test was used accordingly. A probability of <0.05 was considered significant. Data were analysed using the computer program

RSTUDIO Version 0.97.551/IC 10.1.

RESULTS

In vitro development of cattle SCNT and bison iSCNT embryos

To study the developmental potential of interspecies embryos from different taxonomic distance but within the same tribe bovini, we produced embryos by iSCNT. The results from our study showed no significant differences in the proportion of developed embryos at 2, 4, and 8-16 cell stages among groups (p>0.05). Developmental rates for 2, 4, and 8-16 cell stage ranged between 56.3 to 63.1% (p=0.16), 50.9 to 53.4% (p=0.26), and 38.2 to 51.9% (p=0.34), respectively (Table 7).

Table 7. Embryo development in reconstructed embryos (%).

Stage of Cattle SCNT Plains Bison iSCNT Wood Bison iSCNT p- value development* [N=110] [N=102] [N=103]

n (%) n (%) n (%)

2-cell stage 62(56.4) 67(65.7) 65(63.1) 0.16

4-cell stage 56(50.9) 57(55.9) 55(53.4) 0.28

8-16 cell stage 42(38.2) 53(52.0) 44(42.7) 0.34

ATP quantification

Mitochondrial function was determined by estimating the amount of ATP accumulated in single 8-16 cell stage embryos (pmol/embryo). Significantly higher ATP levels were found in

cattle SCNT embryos compared with iSCNT bison, regardless of the subspecies (p<0.05). ATP values in cattle SCNT, plains bison iSCNT and wood bison embryos were 2.03± 0.71, 1.39±

0.25, 1.45± 0.39, pmol/embryo. ATP content in embryos from all groups is shown in Figure 4.

a b b

Figure 4. ATP quantification in reconstructed embryos at the 8-16 cell stage. Different letters denote significant differences between groups (p<0.05).

Apoptosis assessment

The incidence of apoptosis in single embryos was assessed through TUNEL staining.

There were significant differences in the proportion of condensed and TUNEL positive nuclei among groups (Table 8). The highest proportion of condensed and TUNEL positive nuclei per

embryo were found in plains bison iSCNT, while wood bison iSCNT and cattle embryos had the lowest incidence. Few wood bison iSCNT embryos (n=2) and cattle SCNT (n=1) showed

TUNEL positive labelling. Fluorescence micrographs highlighting these findings are shown in

Figure 5.

Table 8. Median [25%; 75%] number and proportion of condensed and TUNEL positive (apoptotic) nuclei in reconstructed embryos

Parameter Cattle SCNT Plains Bison iSCNT Wood Bison iSCNT p-value

[n=13] [n=14] [n=20]

Number of 10 [8; 12] 9 [7.25; 11.75] 10 [7.75; 13.5] 0.647 blastomeres/embryo

Proportion of 0 [0; 0.13]a 0.41 [0.16; 0.63]b 0.13 [0.04; 0.31]c <0.001 condensed nuclei/embryo

Proportion of 0 [0; 0]a 0.4 [0; 0.14]b 0 [0; 0]a 0.01 TUNEL positive nuclei/embryo a, b, c different letters indicate significant differences p<0.05

Expression profiles of nuclear and mitochondria-encoded genes

The level of expression of each transcript was determined by qPCR. The expression profiles for mt-COX2 showed no significant difference among treatment groups (p=0.32).

However, a numerical difference was found between the two bison subspecies. Higher levels of expression were found in wood bison iSCNT embryos while the lowest expression occurred in the plains bison iSCNT group. In the case of nuclear encoded genes, similar expression levels were found for NRF2 in all groups (p=0.60), but expression of TFAM was overall significantly different among groups (p=0.03). However, significant differences in the level of expression of

TFAM were not observed when comparing only two groups (p>0.05). Higher levels of expression were found in wood bison iSCNT embryos with the lowest being observed in the plains bison iSCNT group. Expression profiles of all genes are shown in Figure 6.

Red Channel Green Channel Merge

Cattle

Wood Bison

Plains Bison

Figure 5. Cattle SCNT embryos showing no signs of nuclear condensation and/or TUNEL positive labeling. Wood bison iSCNT embryos showing condensed red nuclei without TUNEL labeling (filled arrows). Plains bison iSCNT embryos with condensed orange nuclei depict positive TUNEL labeling (thin arrows).

5 A 4.5 4

3.5

3 2.5

2 COX2 mRNA - 1.5 1

0.5 abundance vs vs abundanceGAPDH

Relative mt Relative 0 Cattle Plains B Wood B 1.2 B 1

0.8

0.6

0.4

0.2 Relative NRF2 Relative mRNA abundance vs vs abundanceGAPDH 0 Cattle Plains B Wood B

30 C *

25 20 15 10

5 Relative TFAM TFAM Relative mRNA abundance vs vs abundanceGAPDH 0 Cattle Plains B Wood B Species

Figure 6. Gene expression profiles of mt-COX2 (A), NRF-2(B), and TFAM (C) in reconstructed embryos at the 8-16 cell stage. (*) indicates significant differences, p<0.05.

DISCUSSION

To date, the majority of studies on iSCNT have focused on determining the feasibility of an oocyte to reprogram a nucleus from a different species and support the first cleavage divisions. However, fewer reports have thoroughly investigated factors contributing to embryonic failure during the pre-implantation stages in species taxonomically less divergent

(Mastromonaco et al., 2007). Even less information is available at later stages of development.

Failure to reprogram the somatic cell as well as incompatibilities between nucleus and cytoplasm are some of the main research interests in iSCNT (Lagutina et al., 2011; Wang et al, 2011).

Studies on xenocytoplasmic and nuclear hybrid embryos have shown that incompatibilities between nucleus and cytoplasm can lead to detrimental cellular events as a result of species divergence. More specifically, it has been hypothesized that larger distances in species divergence might alter the communication between the nucleus and cytoplasmic elements, such as the mitochondrial genome (Kenyon and Morales, 1997; Narbonne et al., 2011). Here we report no differences in embryo development rates but significantly lower ATP accumulation, higher apoptosis incidence, and altered gene expression (TFAM) in bison iSCNT embryos compared to cattle SCNT.

Many reports on oocyte quality and embryo production have focused on the importance of mitochondria due to their involvement in ATP synthesis, cell signalling, and apoptosis among other functions (Nelson and Cox, 2004; Van Blerkom and Davis; 2007). Measuring the amount of ATP accumulated within the embryo is a valid approach to estimate the ability of the mitochondrion to properly generate energy and support in vitro embryo development (Van

Blerkom et al., 2000). A strong correlation between oocyte and/or embryo competence and ATP concentration in different species has been previously shown (Sturmey and Leese, 2003;

Tamassia et al., 2004; Wakefield et al., 2011). The results of our study revealed that embryos containing bison nuclear DNA and cattle mitochondrial genome accumulated significantly less

ATP than cattle SCNT embryos. Similarly, Wang et al., (2009) reported less ATP content for bovine-chimpanzee iSCNT embryo at the 8-16 cell stage compared to domestic cattle SCNT.

Embryos from species such as rat, sheep, cow, and human depend on ATP mainly produced through aerobic mechanism via oxidative phosphorylation during the first days of development. Progression through the morulae into the blastocyst stage is characterized by a switch to ATP synthesis through glycolytic pathways in response to low oxygen concentration in the uteri of these species (reviewed by Harvey et al., 2002). Since all samples in this study were collected at a stage where embryos preferably use aerobic mechanisms to produce ATP, we hypothesized that the low ATP content present in bison iSCNT embryos is suggestive of impaired mitochondria function. Consequently, processes such as embryonic genome activation, occurring at the 8-16 cell stage (Rozell et al., 1992) might be compromised due to inability of the embryo to maintain sufficient energy levels indispensable for cell function. In our study, embryos from the control and treatment groups were assessed daily to determine the proportion of embryos progressing after each cleavage division. We were unable to find differences in developmental rates in bison iSCNT embryos at the 2, 4 and 8-16 cell compared to cattle SCNT embryos which might indicate adequate ATP reserves to complete the first mitotic divisions.

However, the significant low ATP accumulation observed at the 8-16 cell stage in bison iSCNT embryos might reflect suboptimal energy values contributing to embryo arrest beyond this phase or decreased embryo viability at the blastocyst stage; a phenomenon documented in previous iSCNT studies performed in our laboratory (Mastromonaco et al., 2007; Seaby et al., 2013).

Cell death by apoptosis determination has been used as a reliable indicator of developmental potential in embryos from several species (Byrne et al., 1999; Kamjoo et al.,

2002; Mateusen et al., 2005). Evidence suggests that the proportion of blastomeres with apoptotic nuclei at the 8-16 cell stage is low. In vitro produced cattle embryos at the 8-16 cell exhibit low incidence (<5%) of TUNEL labelling compared to expanded and hatched stages

(Matwee et al., 2000). In our study, a significant increase in apoptosis index was observed in plains bison iSCNT compared to the other groups, which had values close to 0. Interestingly, bison embryos from both subspecies had a significant proportion of blastomeres with condensed nuclei compared to cattle SCNT embryos, which also is suggestive of morphological changes occurring during early stages of apoptosis (Hardy, 1997). Nevertheless, nuclear condensation can also be observed before cell replication and it should be interpreted carefully since cell cycle length in bison embryos has not been determined yet. Many of the biochemical processes leading to apoptosis are regulated by the mitochondria (reviewed by Dumollard et al., 2009). Therefore, abnormal mitochondrial function has been associated with elevated apoptosis in many cells and mammalian embryos (Thouas et al., 2004; Maloyan et al., 2005; Wang et al., 2010). Thus, mitochondrial dysfunction determined by low ATP accumulation in bison iSCNT embryos might lead to higher apoptosis incidence.

One of the main alterations found in SCNT and iSCNT embryos is aberrant gene expression during the pre-implantation stages including failure to reactivate pluripotent and developmental genes, and inability to properly silence somatic transcripts (Chung et al., 2009;

Wang et al., 2011; Esteves et al., 2012). Previous studies in our laboratory investigated differences in gene expression at the blastocyst stage in iSCNT embryos (Mastromonaco et al.,

2007; Seaby et al., 2013). However, no differences were reported in genes responsible for

mitochondrial transcription and replication. Therefore, we analysed iSCNT embryos at earlier stages to identify dissimilarities that could lead to developmental failure during this or later stages. Cytochrome c oxidase or complex IV forms an essential protein essential for oxidative phosphorylation in eukaryotes. The complex comprises thirteen different subunits which are encoded by the nuclear and mitochondrial genome (reviewed by Capaldi, 1990). Thus, adequate communication between the two genomes is imperative to guarantee proper assembly and functioning of the different subunits of the electron transfer chain including mt-COX-1, 2, and 3, which are encoded by the mitochondrial genome (Anderson et al., 1981). TFAM is encoded by the nucleus and it is involved in mitochondria transcription and replication, which takes place only at the blastocyst stage in cells derived from the trophectoderm of bovine embryos

(McCulloch et al., 2002; Asin-Cayuela and Gustafsson, 2007). Consequently, expression of

TFAM and genes encoding proteins of the electron transport chain is very low or undetectable at earlier stages (8-16 cell stage) (May-Panloup et al., 2005). Interestingly, it has been reported that upregulation of TFAM in SCNT embryos during the 8-16 cell stage promotes donor cell mtDNA to be targeted and ultimately replicated (Bowles et al., 2007). Based on these findings, we propose two possible mechanisms to explain the higher expression levels of TFAM and mt-

COX2 in wood bison iSCNT, although no significant differences among groups were found in the case of the latter gene. Firstly, improper silencing of these genes by the oocyte soon after the transfer of the somatic cell might favour propagation of somatic mitochondria at a stage where only oocyte-derived mitochondria must be maintained (Sutovsky et al., 2004). A second explanation might involve the expression of these transcripts as a compensatory response to overcome inadequate ATP reserves, resulting in premature mitochondrial biogenesis. This theory has been mentioned previously by Chiaratti et al. (2010b), who reported an increase in transcripts

such as TFAM and NRF-1 (nuclear respiratory factor 1) in bovine one-cell parthenote embryos depleted of approximately 50% of their mitochondria. The authors concluded that embryos carrying significant less mitochondria DNA might modify mitochondria replication events during the early development stages. In addition, we also speculate that the low levels of TFAM and COX-2 shown in cattle SCNT and plains bison iSCNT might be explained by cattle gene introgression in the plains bison genome when crossbreeding between the two species occurred in the 20th century (Hedrick, 2010). Additional DNA analysis of the cell line used in our study would determine the degree of cattle ancestry in both nuclear and mitochondrial genomes and its effect on gene expression.

Studies have elucidated molecular mechanisms leading to embryonic failure in embryos produced by iSCNT (Amarnath et al., 2011; Lagutina et al., 2011; Jiang et al., 2011). However, the majority of studies have used models where the species of interest are very distant. In these cases, more reprogramming errors as well as incompatibilities between cytoplasmic and nuclear components are expected. In contrast, our study used two species that have been known to produce embryos and offspring by interspecies crosses indicating some compatibility between the two species. The findings from this study provide insight regarding some of the alterations that could lead to embryonic arrest in bison iSCNT embryos during the pre-implantation stages.

Variations in ATP content, higher apoptosis incidence, and altered gene expression are suggestive of impaired mitochondrial function potentially enhancing embryo demise following the 8-16 cell stage in iSCNT bison embryos. Subsequent detrimental effects observed at the blastocyst stage, such as low development and decreased embryo viability, might also be associated with alterations at earlier stages in taxonomically divergent iSCNT embryos.

CHAPTER THREE

Effects of ooplasm transfer on early embryo development in domestic cattle SCNT

and iSCNT plains bison embryos

INTRODUCTION

Ooplasm transfer from an oocyte donor into the cytoplasm of a recipient oocyte allowed the discovery of maturation promoting factor in frog oocytes (Masui and Market., 1971). A decade later, similar experiments performed on mouse embryos provided fundamental evidence about the ooplasm control on embryonic arrest at the 2-cell stage (Muggleton-Harris et al.,

1982). In human reproduction, ooplasm transfer (OT) has been used to influence the outcomes of in vitro produced embryos mainly in patients with chronic infertility undergoing repeated embryo failure (Cohen et al., 1997) and idiopathic infertility (Dale et al., 2001). It was hypothesized that a portion of ooplasm from a competent oocyte might correct the unidentified problems in the recipient oocyte resulting in improved development during the pre and post implantation stages (Cohen et al., 1997; Huang et al., 1999). Despite the lack of experiments carried out on animal models, OT was implemented in human fertility clinics resulting in healthy offspring. However, fetuses and individuals carrying chromosomal abnormalities and persistence of donor mtDNA have been reported (Hawes et al., 2002; Yabuuchi et al., 2012). Consequently, the use of this technique in humans was discouraged due to safety and ethical concerns, including the possibility to carry DNA from three parents (Baylis, 2013; Amato et al., 2014).

Application of OT as a therapeutic alternative to treat infertility has received constant criticism due to lack of evidence linking specific factors or cytoplasmic components to apparent benefits. Currently, mechanisms leading to increased developmental rates and pregnancy outcomes have not been fully understood. Only little evidence has suggested that ooplasm isolated from young women and transferred into postmature oocytes has a positive effect on microtubule architecture (Goud et al., 2004). Animal models, on the other hand, can be used to investigate the effects of ooplasm-derived components on embryo development and the

interactions occurring between ooplasmic and nuclear genomes, as in the case of iSCNT embryos. Sansinena et al. (2010) documented the use of this technique to further determine the effects of ooplasm transfer in iSCNT embryos and explored its potential applications to overcome embryonic arrest observed in a large proportion of these embryos. Studies on iSCNT have postulated that incompatibilities between nuclear and mitochondrial genomes might negatively influence embryo development (reviewed by Mastromonaco et al., 2014). Among these aberrant interactions, one theory proposes that proteins encoded by the nuclear genome are unable to recognize specific sequences of the mtDNA due to different taxonomic mtDNA origin

(Smith et al., 2005). Furthermore, other incompatibilities between components residing in the somatic cell and the ooplasm have been demonstrated (e.g. embryonic centrosome dysfunction due to the lack of specific proteins to form properly this complex) (Zhong et al., 2007).

Therefore, supplementing ooplasm from the same species as the somatic cell might influence embryo development by transferring species-specific components and organelles (Mastromonaco et al., 2007). However, only a few reports have implemented the use of ooplasm transfer to investigate its effects on development of iSCNT embryos (Sansinena et al., 2010).

OT has recently been carried out on SCNT embryos to determine the effects of the technique on developmental potential and to replenish ooplasm losses as a result of the enucleation procedures. Removal of large amount of ooplasm (20-25%) has been associated with decreased embryonic development (Gopalakrishna et al., 2014). Therefore, it was hypothesized that an equivalent percentage of ooplasm transferred with the somatic cell might restore the original volume and positively influences embryo competence. Gopalakrishna et al. (2014) showed a slight improvement in blastocyst development and higher fusion rates in the replenished group. Although OT has been associated with similar implantation rates to the

control group in mice (Meirelles and Smith, 1997), the use of this technology has been linked to detrimental effects during embryonic development (Cheng et al., 2009). Levron et al. (1996) reported that when nuclear maturation status of the donor mouse oocyte and recipient oocyte differed, significantly lower implantation rates were obtained.

The results from the studies described in chapter 2 showed that wood bison iSCNT embryos accumulated significantly less ATP, were more likely to undergo nuclear condensation, and showed altered gene expression compared to cattle SCNT embryos. Access to wood bison oocytes to conduct OT experiments was not feasible. Consequently, oocytes from plains bison oocytes were used as ooplasm donor in plains bison iSCNT embryos. Here, we established an

OT approach to investigate whether incorporation of domestic cattle or plains bison ooplasm could influence energy reserves, gene expression, and development rates in cattle SCNT and iSCNT bison embryos. However, previous pilot studies conducted in our laboratory revealed that the additional step required to transfer the ooplasm limits the use of OT in conjunction with iSCNT due to increased oocyte lysis and elevated exposure time to environmental factors during micromanipulation. Therefore, the aims of this study were to: 1) establish a 2-step micromanipulation technique to transfer ooplasm into SCNT and iSCNT embryos, and to 2) determine the effects of ooplasm transfer on in vitro embryo development, mitochondrial function, and expression of nuclear and mitochondrial genes in cattle SCNT and plains bison iSCNT embryos.

MATERIALS AND METHODS

Experimental design

To determine the effects of OT on embryo development rates, experimental groups consisted of the following: 1) cattle SCNT (C-SCNT), 2) cattle SCNT supplemented with cattle ooplasm (C-SCNT+ C-OT), 3) plains bison iSCNT (PB-iSCNT), 4) plains bison iSCNT supplemented with plains bison ooplasm (PB-iSCNT+ PB-OT). Domestic cattle oocytes were used as cytoplasts in all groups. Plains bison subspecies was selected due to existence of some commercial herds in Ontario, Canada, and availability of oocytes. Cleavage divisions at 2, 4, and

8-16 cell stages were determined at 36, 50, and 80 hours post activation (hpa), respectively.

Reconstructed embryos at 80 hpa (day 4) were randomly assigned to ATP or PCR analyses. A fifth group consisting of domestic cattle embryos produced by in vitro fertilization techniques using cattle sperm (C-IVF) was included only when carrying out qPCR experiments as an additional control. Cleavage rates were estimated based on the total number of reconstructed embryos in culture.

Chemicals

All chemicals were obtained from Sigma-Aldrich Chemical Co. (St Louis, MO) unless otherwise stated.

Culture and preparation of donor somatic cells

Procedures for culture and preparation of the donor somatic cells were described in chapter 2. The same cattle and plains bison cell lines used in previous nuclear transfer experiments were used to conduct OT studies. Cells used as somatic cell donors were at passages

3-5 and confluent for 2-3 days. 82

Oocyte collection, IVM, and IVC of reconstructed embryos

Oocyte collection and IVM of cattle and plains bison oocytes were carried out as described in chapter 1. Embryos from all groups were cultured in 30 µl drops containing synthetic oviductal fluid (SOF) (Chemicon-Millipore, Billerica, MA, USA) in a humidified atmosphere of 5% CO2, 5%O2, and 90% N2 for 80 hpa.

In vitro fertilization (IVF) of cattle oocytes

Frozen-thawed Bos taurus semen (EastGen, Ontario, Canada) with known in vitro fertility was used for IVF. Semen was prepared by swim-up in HEPES/Sperm TALP for 45 minutes at 38.5°C in a humidified atmosphere of 5% CO2 in air. Cumulus-oocyte complexes

(COCs) were washed three times in HEPES/Sperm TALP at 22 hours post IVM, and transferred to an 80 μl droplet containing fertilization medium (IVF-TALP) supplemented with 20 μg/ml heparin under silicone oil (Paisley Products, Scarborough, ON, Canada). A final concentration of

1 x 106 motile sperm/ml was used to fertilize 25-30 COCs per droplet. Co-incubation with sperm was carried out for 16 hours in a humidified atmosphere of 5% CO2 in air at 38.5°C.

Subsequently, groups of 30 presumptive zygotes were placed in 30 μl drops containing SOF medium. Embryos were cultured for 80 hours post fertilization using the same conditions mentioned in the previous section.

Embryo reconstruction and ooplasm transfer

Micromanipulation techniques including enucleation and somatic cell nuclear transfer were carried out as described in chapter 2 with some modifications. Ooplasm-donor oocytes

(cattle and plains bison) were selected based on the presence of an extruded polar body and homogenous ooplasm under light microscopy 18-20 hours post IVM. Thereafter 5 somatic cells 83

were introduced at the end of the transfer pipette (15 μm inner diameter) previously coated with

0.01% PVA/PVP followed by aspiration of 10 to 15% of either cattle or plains bison ooplasm. A schematic representation of the steps involved in iSCNT + OT is shown in Figure 7. Both ooplasm and somatic cell were then transferred into the perivitelline space (Figure 8).

Reconstructed embryos and ooplasm-donor oocyte were micromanipulated in 40 μl microdroplets containing HEPES/Sperm TALP medium and cytochalasin B (5 μg/ml) under silicone oil on a stage warmer. After removal of more than 70% of ooplasm from the ooplasm- donor oocyte, a new oocyte was selected and maintained in a droplet used for SCNT and OT.

Fusion of reconstructed embryos

Fusion of the reconstructed couplets in the case of C-SCNT and PB-iSCNT groups were performed as described in chapter 2. However, a pilot study was carried out to determine the adequate parameters to electrofuse the somatic cell and the portion of ooplasm using oocytes and somatic cells from domestic cattle (APPENDIX I). Each reconstructed embryo from C-SCNT+

C-OT and PB-iSCNT+ PB-OT groups received two pulses applied at a 30 minute interval. The first DC pulse was set at 1.5 kV/cm for 20 μs in a 0.5 mm electrofusion chamber to fuse the transferred ooplasm to the reconstructed embryo. A second DC pulse employing higher voltage and longer exposure time was used to fuse the somatic cell into the reconstructed embryo, once the portion of transferred ooplasm was not longer visible in the perivitelline space. Values for the second pulse were 2.1 kV/cm for 32 μs. After each pulse, reconstructed oocytes were subsequently washed in 3 ml of HEPES/Sperm TALP and placed in IVM medium under an atmosphere of 5% CO2 in air with maximum humidity at 38.5°C. Fusion rates for ooplasm and somatic cell were estimated 30 and 60 minutes after completion of the first and second pulse, respectively. Chemical activation protocol was performed as described in chapter 1.

ATP determination

ATP quantification in single embryos from all experimental groups was assessed as described in chapter 2. A total of 10 embryos per group were included for this analysis except for

C-SCNT+ C-OT which included 13 samples.

RNA extraction, reverse transcription, and gene expression analysis

Embryos from all nuclear transfer and IVF groups were collected at 80 hpa in pools of 5, washed three times in PBS-PVA 0.01%, snap-frozen in liquid nitrogen and stored at -80°C until needed. RNA was isolated from pooled embryos using the miRNeasy Micro Kit (QIAGEN, Inc,

Burlington, ON), following the Manufacturer’s instructions. Procedures for reverse transcription were indicated in chapter 2. cDNA samples were stored at -20°C until needed. Quantitative real time PCR (qPCR) was used to measure mRNA expression profiles of genes involved in mitochondria respiration, replication, and transcription in all nuclear transfer groups and C-IVF embryos. Techniques used for this analysis have been previously described (chapter 2). Primer sequences of genes used in this objective are listed in Table 6.

Figure 7. Representation of iSCNT and OT procedures. A) Large blue and yellow circles represent plains bison and cattle oocytes, respectively. Red diamonds denote a nucleus. B) A portion of plains bison ooplasm (small blue circle) and plains bison somatic cell (small brown circle) are introduced in the perivitelline space. C) Reconstructed embryos + OT after first electropulse (note how the portion of plains bison ooplasm has been fused (scattered blue pattern). D) Reconstructed embryo followed by a second electro pulse showing successful fusion of the somatic cell.

Figure 8. Phase contrast micrograph of A) cattle oocyte + cattle ooplasm and somatic donor cell nucleus, arrow represents the cattle somatic cell while the arrow head denotes the portion of ooplasm being transferred into the perivitelline space. B) Micrograph of a different oocyte taken 5 minutes after first DC pulse. Note fusion process between cattle oocyte and the portion of cattle ooplasm (arrow head) with presence of the somatic cell in the back (thin arrow).

Statistical analysis

Fusion, development at 2, 4, 8-16 cell stage, and blastocyst rates were compared using

Chi-square in all experimental groups. ATP quantification and gene expression profiles were analysed using One-way ANOVA. A probability of <0.05 was considered significant. The same statistical computer software mentioned in chapter 1 was also used here to analyse data retrieved from all experiments.

RESULTS

Fusion rates

Cattle oocytes (n=919) were reconstructed to produce iSCNT and SCNT couplets with or without ooplasm. The percentage of oocytes with fused ooplasm within 30 minutes in C-SCNT+

C-OT and PB-iSCNT + PB-OT groups was 93.0 and 92.4%, respectively. After the second pulse

(30 minutes interval), fusion rates for the somatic cell in the former group was 40.76% (n=117), while for the latter was 53. 9% (n=75). Fusion rates for C-SCNT and PB-iSCNT were 42.5

(n=91) and 41.1 % (n=111), respectively. There was a significant difference in the proportion of fused oocytes between PB-iSCNT and PB-iSCNT+ PB-OT (p=0.01), however no significant differences were found when C-SCNT and C-SCNT+ OT groups were compared (p=0.6).

In vitro development of reconstructed embryos

The first three cleavage divisions in all groups (C-SCNT, C-SCNT+ C-OT, PB-iSCNT,

PB-iSCNT + PB-OT) were monitored to determine differences in development. No significant differences in the proportion of embryos among groups at 2, 4, and 8-16 cell stages were found

(p>0.05). Developmental rates for 2, 4, and 8-16 cell stage ranged between 87.5- 91.8%

(p=0.81), 76-83.5% (p=0.76), and 57.6-61.3% (p=0.96), respectively (Table 9).

Table 9. Number of reconstructed embryos developed (%) to the 2, 4, and 8-16 cell stage.

Stage of C- SCNT C-SCNT + PB-iSCNT PB-iSCNT + p-value development C-OT PB-OT

[n=85] [n=80] [n=85] [n=50]

2-cell stage 78(91.7) 70(87.5) 75(88.2) 44(88.0) 0.81

4-cell stage 71(83.5) 64(80.0) 68(80.0) 38(76.0) 0.76

8-16 cell stage 49(57.6) 49(61.2) 51(60.0) 29(58.0) 0.96

ATP quantification

Mitochondrial function was assessed by estimating the amount of ATP accumulated in single embryos at the 8-16 cell stage. ATP content (pmol/embryo) did not differ among groups

(p>0.05). ATP values in C-SCNT, C-SCNT+ C-OT, PB-iSCNT, PB-iSCNT + PB-OT were

1.35± 0.23, 1.31± 0.34, 1.27± 0.18, and 1.26± 0.31 pmol/embryo, respectively. ATP accumulation in embryos from all groups is shown in Figure 9.

Expression profiles of nuclear and mitochondria-encoded genes

Analysis of expression profiles of nuclear and mitochondrial gene transcripts was determined by qPCR. Expression of mRNA in SCNT, iSCNT, and IVF embryos at the 8-16 cell stage was compared to the house-keeping gene GAPDH. The expression profiles for mt-COX2 showed no significant difference among groups (p=0.36). In the case of nuclear encoded genes,

similar expression levels were found for NRF2 (p=0.93), and TFAM (p=0.80) in all groups

Expression profiles for all genes are shown in Figure 10.

Figure 9. ATP quantification in reconstructed embryos at 8-16 cell stage with or without OT.

2.5

2 COX2 mRNA

- 1.5

abundance vs vs GAPDH abundance 0.5 Relative mt Relative

0 A C-IVF C-SCNT C-SCNT+ C-OT PB-iSCNT PB-iSCNT + PB-OT

2 1.8 1.6

1.4 1.2

2 mRNA 2 abundance - vs GAPDH vs 0.8 0.6 0.4

Relative NRFRelative 0.2 0 B C-IVF C-SCNT C-SCNT+ C-OT PB-iSCNT PB-iSCNT + PB-OT

8 7

5 4

vs GAPDH vs 3 2

1 Relative TFAM TFAM Relative mRNAabundance 0 C C-IVF C-SCNT C-SCNT+ C-OT PB-iSCNT PB-iSCNT + PB-OT

Figure 10. Gene expression profiles of mt-COX-2(A), NRF-2(B), and TFAM (C) in IVF and reconstructed embryos at the 8-16 cell stage.

DISCUSSION

Application of OT in animal models have revealed insights into cell biology concepts, such as transmission of mutated mtDNA (Pinkert et al., 1997) or epigenetic modifications of imprinted genes (Cheng et al., 2009). Furthermore, OT has also been applied to advanced reproductive biotechnologies, such as SCNT and ICSI. These experimental models provide a unique opportunity to assess deleterious or beneficial effects on embryo development (Sansinena et al., 2010). One of the main limitations of this technique is related with the difficulty in establishing direct effects of the ooplasmic component on embryo development since a portion of ooplasm might contain multiple cellular and subcellular structures (Levy et al., 2004).

Consequently, different techniques for isolation and segregation of specific components, such as mitochondria, have been reported (Takeda et al., 2005; Ferreira et al., 2010). Nevertheless optimization procedures should be performed prior to transfer of mitochondria extract to guarantee maintenance of mitochondrial biochemical and physical properties (Pinkert et al.,

1997). Conversely, OT only uses mechanical approaches to extract and incorporate a portion of ooplasm into another gamete or embryo (Levron et al., 1996).

Current knowledge on OT studies has revealed not only potential advantages but also problems leading to embryo demise. A comprehensive mouse study used OT in combination with ICSI to analyse the effects of OT on DNA methylation and post-natal growth parameters.

The study showed decreased survival and birth rates but absence of gross abnormalities at birth, however epigenetic modifications in imprinted loci of genes such as H19/Igf2, Snrpn, Peg3,

Igf2r, Rasgrf were not observed (Cheng et al., 2009). Differences in methodologies for OT and

SCNT techniques might explain the variations in developmental outcomes documented by

several studies. However, one of the main factors influencing this variability might be explained by other factors, organelles, and nucleic acids present in a small portion of ooplasm.

Understanding the interactions occurring between nuclear and ooplasmic compartments is essential to determine the potential causes leading to reduced development in iSCNT embryos. iSCNT studies have demonstrated the ability of enucleated oocytes to support early development with subsequent failure to activate embryonic genes resulting in embryo arrest (Dominko et al.,

1999; Wang et al., 2009). It has been reported that embryo demise in iSCNT might be linked to the lack of species-specific proteins, organelles, and several molecules in domestic oocytes used for iSCNT (Mastromonaco et al., 2007; Jing et al., 2011; Lagutina et al., 2011). Therefore, it has been hypothesized that ooplasm containing mitochondria matching the same taxonomic origin as the somatic cell in iSCNT embryos could influence nuclear-ooplasmic interactions and developmental outcomes (Mastromonaco et al., 2007). However, limited information is available regarding the effects of OT on embryo development of iSCNT embryos. Sansinena et al. (2010) showed that supplementation of caprine ooplasm in iSCNT embryos (domestic cattle cytoplast x caprine somatic cell) yielded low fusion and cleavage rates, and poor blastocyst rates (0%).

However, their results might be attributed to technical difficulties since the study lacked a control group (goat ooplasm transferred into caprine SCNT embryos). On the other hand, studies applying OT to in vitro produced embryos have revealed contradictory results since both detrimental and advantageous effects have been attributed to OT (Cohen et al., 1997; Goud et al.,

2004; Takeda et al., 2005). Our investigation showed that a portion of ooplasm transferred into

SCNT and iSCNT had no effects on embryo development rates, ATP content, or expression of mitochondrial and nuclear genes.

Previous reports have successfully transferred and fused ooplasm into oocytes recruited for parthenogenesis and iSCNT experiments (Takeda et al., 2005; Chiaratti et al., 2010a;

Sansinena et al., 2010). However, the combination of OT and somatic cell nuclear transfer requires excessive micromanipulation that may lead to oocyte damage. Implementation of OT and iSCNT procedures includes three basic steps: enucleation of the oocyte donor, transfer of the somatic cell, and incorporation of a portion of ooplasm either before enucleation or after placing the somatic cell in the perivitelline space. The technique applied in our experiments reduced the number of micromanipulation steps resulting in acceptable reconstruction rates evidenced by maintenance of normal ooplasm architecture and high ooplasm fusion rates. However, fusion assessment performed after the second pulse revealed inefficiencies during and after this critical process. Low fusion rates, both in control and treatment groups, were obtained repeatedly resulting in loss of 50% of reconstructed embryos. Interestingly, more couplets were fused in the

PB-iSCNT+ PB-OT group than PB-iSCNT group which might be related to increased proximity between the plains bison somatic cell and the oolemma caused by an increase in total volume.

Conversely, this was not evident in C-SCNT+ C-OT group. Thus, in order to provide some explanations regarding the low fusion rates obtained in this study, a small experiment was conducted to investigate the causes of low fusion rates by modifying voltage, time of exposure, molar concentration of ionic compounds, and number of pulses (Appendix II). Altering original parameters resulted in no improvement in fusion rates of cattle SCNT embryos. Therefore, it was not clear if higher ooplasm volumes caused by OT enhances fusion in embryos reconstructed by

SCNT.

Animal and human OT studies have recommended the transfer of approximately 10 to

15% of ooplasm volume but there is no strong evidence indicating that higher volumes

negatively affect embryo development (Cohen et al., 1997; Chiaratti et al., 2010a). Therefore, a similar approach was used in this study to assess the effects of OT on plains bison iSCNT embryos. Interestingly, domestic cattle SCNT embryos reconstructed by alternative protocols, such as hand-made cloning (HMC), contained volumes surpassing 50 or 100% of their original ooplasm due to hemi-cytoplast aggregation. It has been reported that HMC embryos showed similar or higher developmental competence than smaller cattle embryos reconstructed by micromanipulation procedures (Vajta et al., 2004; Ambrosio et al., 2009). In HMC water buffalo embryos, a positive association between HCM procedures and improved developmental outcomes has not been established yet (Chauhan et al., 2011). However, findings from HMC studies are difficult to relate to SCNT or conventional OT experiments because the methodology required for embryo reconstruction differs significantly in the two approaches.

Optimal ATP content in oocytes and embryos has been associated with embryo competence (Stojkovic et al., 2001; Van Blerkom et al., 2004). Thus, energy production is fundamental for resumption of meiosis, fertilization and cleavage, activation of the embryonic genome, and several cellular processes during the preimplantation stages (reviewed by Van

Blerkom, 2008). It has been hypothesized that transfer of ooplasm or mitochondria could enhance energy levels in the newly formed embryo resulting in improved development (Malter and Cohen, 2002; Yi et al., 2007). A study conducted by Van Blerkom et al (1998) showed that fertilized mouse oocytes microinjected with somatic mitochondria accumulated 30% more ATP

36 hours post injection. Additionally, the authors confirmed the presence of viable somatic mitochondria 80 hours post transfer. The results from our study showed that ATP values were similar among groups at the 8-16 cell stage (80 hpa). Furthermore, absence of significant differences in ATP content among groups correlates with embryo development rates at the 2, 4

and 8-16 cell stages. Van Blerkom et al. (1998) demonstrated gradual increases in ATP content reported in fmol/embryo throughout development although assessment at the 8-16 cell stage was not performed. A single ATP determination was carried out in our study four days after ooplasm supplementation. Therefore, an increase in ATP content during the first cleavage divisions followed by subsequent reduction at later stages could explain the similarities found in ATP content in cattle and plains bison groups supplemented with or without ooplasm. Serial ATP determinations may be necessary to determine metabolic changes during the first cleavage divisions. Similar ATP content among groups can also be explained by the ability of the bovine oocyte to target and eliminate foreign mitochondria at the 8-16 cell stage resulting in regulation of ATP production (Sutovsky et al., 2004). Moreover, many reports have also documented the presence and replication of the foreign mtDNA in SCNT and iSCNT embryos during the preimplantation and post implantation stages (John et al., 2005; Hua et al., 2012;

Imsoonthornruksa et al., 2012), which suggests that a portion of these embryos escape mitochondrial degradation events favouring heteroplasmy.

Several investigations of SCNT embryos have provided valuable information regarding the percentage of somatic mitochondria present in embryos and offspring but have failed to identify the mechanisms leading to transmission of two or more mitochondrial genomes although some theories have been proposed. Therefore, replication or elimination of somatic mitochondria occurring during the preimplantation stages is very difficult to predict (reviewed by St John et al., 2005). Similarly, OT experiments have also failed to determine distinct mitochondrial inheritance patterns. Chiaratti et al., (2010a) showed that Bos indicus zygotes transferred with water buffalo ooplasm (~10-15%) had similar developmental potential to that of the control group. Furthermore, water buffalo mitochondrial copy number remained stable from the zygote

to the blastocyst stage. Interestingly, Bos indicus adult females (n=2) resulting from the zygotes with mixed cytoplasm had only cattle mtDNA in germ line and numerous other tissues examined suggesting that elimination of water buffalo mtDNA could also take place at post-implantation stages. However, Chiaratti et al. (2010)’s findings are difficult to extrapolate to iSCNT studies because the proportion of mitochondrial and nuclear genomes differ significantly between embryos generated by OT and iSCNT techniques. Their study showed that the cattle nuclear genome interacts with more than 90% of the corresponding cattle mitochondrial genome. In contrast, the nuclear genome in iSCNT embryos must cross-talk with 99% of mtDNA that belongs to another species which might result in incompatibilities between genomes (Wen et al.,

2005; Bowles et al., 2007; Lagutina et al., 2010).

Improvement in developmental outcomes by implementation of OT studies has been linked to abundance of mRNAs, functional mitochondria, and presence of other molecules (Levy et al., 2004). Mitochondrial transfer into oocytes and embryos has been used to determine the effects of mitochondrial heteroplasmy on subsequent developmental (Van Blerkom et al., 1998).

Evidence has suggested that addition of mitochondria sharing the same taxonomic origin as the somatic cell might lead to disturbances in iSCNT embryos. This concept was illustrated by Hua et al. (2012) who reported impaired development in ovine iSCNT embryos (i.e., ovine somatic cell and domestic cattle cytoplast) microinjected with mitochondria isolated from ovine granulosa cells. Significantly fewer embryos were able to progress through the 8-cell stage in comparison with iSCNT embryos injected with mitochondria derived from domestic cattle granulosa cells. Their findings demonstrated that heteroplasmy (close to 0.29%) contributed to poor development in ovine iSCNT embryos injected with ovine somatic mitochondria.

Furthermore, the same authors reported downregulation of pluripotency genes, such as SOX-2

and OCT4, in conjunction with upregulation of TFAM and POLRMT, which are genes involved in mitochondrial replication and transcription (Asin-Cayuela and Gustafsson, 2007; Ringel et al.,

2011).

In the last decade, revolutionary disciplines such as transcriptomics have allowed comprehension of complex cellular processes occurring during early development (Uyar et al.,

2013). Quantification of transcripts in embryos has been used to assess embryo developmental competence in vitro and in vivo (de Oliveira et al., 2005; Cánepa et al., 2014; Sudano et al.,

2014). However, very few OT reports have included PCR studies to understand the interactions taking place between ooplasmic and nuclear components. Thus, we studied expression profiles of genes involved in mitochondrial events such as respiration, transcription and replication (e.g. mt-

COX2, TFAM, and NRF-2). The results from our study showed similar gene expression profiles among groups. Moreover cattle IVF embryos were included in this study as an additional control group to investigate expression of these genes at the 8-16 cell stage. Findings from the IVF group were in accordance with previous investigations, which documented low expression levels of mitochondrial replication and transcription factors (Bowles et al., 2007; May-Panloup et al.,

2005). Interestingly, our study revealed that plains bison ooplasm transferred into bison iSCNT embryos did not alter gene expression of mitochondrial and nuclear encoded genes. The lack of differences in gene expression between PB-iSCNT and PB-iSCNT +PB-OT groups might have resulted from proper degradation of maternal transcripts at the time of genome activation, limited involvement of bison mtDNA in mitochondrial events such as replication and transcription due to dilution and segregation of the bison mitochondria, and active elimination of bison mtDNA by unknown mechanisms. Our results suggest that mitochondrial events depending on nuclear and ooplasmic communication are not negatively affected by supplementation of either cattle or

plains bison ooplasm. However, further experiments are required to address these questions and determine if the embryo arrest in bison iSCNT embryos could be ameliorated by implementation of OT.

Application of OT in iSCNT experiments can provide valuable information to understand complex interactions occurring between nuclear and ooplasmic components in reconstructed embryos. However, confounding factors in the ooplasm may mask the beneficial or deleterious effects of supplementing mitochondria in a portion of ooplasm. Although our study found no differences at the 8-16 cell stage, further studies are required to understand whether the potential effects associated with OT can be observed at later cleavage stages. Microinjection of mitochondria extract isolated from bison reproductive somatic cells, easily available by cell culture, might provide additional information regarding the role of this organelle in nuclear- ooplasmic incompatibilities.

CHAPTER FOUR

Quality assessment of in vivo-derived and interspecies somatic cell nuclear transfer

wood bison embryos

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INTRODUCTION

Every year, conservation organizations show alarming statistics regarding the number of species listed as threatened or endangered. The wood bison population, an essential herbivore in

Canadian ecosystems, has been reduced to approximately 7000 individuals (COSEWIC, 2013).

Initial efforts to increase the number of individuals through the use of assisted reproductive technologies (ARTs) have resulted in suboptimal outcomes (Thundathil et al., 2007; Palomino et al., 2013). One of the main obstacles to successfully apply these technologies is related to the lack of information regarding normal reproductive events taking place from the early stages of gametogenesis to acquisition of gamete competence. Thus, investigation of embryonic processes becomes a fundamental step before embarking into application of ARTs in wood bison.

In vitro embryo production (IVP) procedures used to culture oocytes and embryos from wood bison species have been previously documented (Thundathil et al., 2007; Seaby et al.,

2012, Cervantes et al., 2013). However, the limited number of studies, small sample sizes, and low efficiency of IVP reported in most studies clearly demonstrate an urgent need to further accelerate research regarding the factors negatively influencing in vitro bison development.

Although the studies mentioned previously have showed some potential to support early development, information regarding embryo quality and/or development from in vivo produced embryos is lacking in wood bison (Seaby et al., 2013). Therefore, when determining factors associated with embryo demise, careful interpretation should be performed since proper comparison using in vivo embryos has not yet been established. However, accessing early in vivo bison embryos before the morula stage would require the use of invasive techniques including surgical approaches. At later stages of development, the embryo enters the uterus which facilitates its recovery by non-surgical protocols requiring chemical/physical restraint, placing

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the animal at considerable risk for injury. Thus, techniques originally implemented in domestic species are more difficult to apply in bison (Dorn, 1995).

In large domestic species, in vivo embryos are subjected to morphological assessment to subjectively predict their ability to implant and successfully culminate in healthy offspring.

Commonly, this evaluation uses stereoscopic illumination to assess embryo viability based on a number of characteristics, such as degree of fragmentation, number and shape of blastomeres, integrity of the zona pellucida, and stage of development at specific time points, among other features (Gardner et al., 2000; Mori et al., 2002; Meseguer et al., 2011). Although morphological assessment is one of the most common tests implemented, some limitations have been linked to this technique regarding subjectivity among technicians and the limited amount of information obtained by visual evaluation (Farin et al., 2009). There are other essential parameters that can be used to determine embryo quality, including the time required to advance from one embryonic stage to the next (Lechniak et al., 2008), quantification of total cell number, and allocation of trophectoderm cells (TE) and inner cell mass (ICM), which are cells responsible to give rise the placenta and embryo proper, respectively (De la Fuente and King, 1997). In the last decade, noninvasive techniques have been developed to predict embryonic developmental ability, such as quantification of viability markers present in the culture medium (Muñoz et al., 2014).

Therefore, using a combination of these techniques might lead to objective interpretation of embryo viability. However, fundamental parameters in bison embryos such as normal morphology, length of embryo preimplantation stages, and total cell number are unknown. This study aimed to: 1) describe morphological features and time of development of vitrified-thawed in vivo-derived wood bison embryos, and 2) to compare the total cell number and incidence of apoptosis between wood bison embryos produced in vivo and in vitro by superovulation and

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artificial insemination techniques and interspecies somatic cell nuclear transfer (iSCNT), respectively.

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MATERIALS AND METHODS

Experimental design

Groups consisted of in vivo-derived wood bison morulae and blastocysts, and in vitro wood bison expanded blastocysts produced by iSCNT using cattle oocytes as cytoplasts. In vivo embryos were thawed and cultured for 48 hours. Development was assessed at 4 hours, 24, and

48 post thawing. Morphological embryo evaluation was determined by visual inspection on a stereoscope Leica MZ125 using the scoring system implemented by the International Embryo

Transfer Society. In the case of wood bison embryos produced by iSCNT techniques, cleavage rates were determined 36 hours post chemical activation (hpa) whilst blastocyst rates were determined every 24 hours starting on day 7 (~172 hpa) until day 9 (~220 hpa). Cleavage and blastocyst rates were estimated based on the total number of reconstructed iSCNT embryos in culture. In vivo and in vitro blastocysts were assessed for cell number and apoptosis index. Cell allocation was determined only in domestic cattle IVF blastocysts and wood bison iSCNT blastocysts on day 9 of development.

Chemicals

All chemicals were obtained from Sigma-Aldrich Chemical Co. (St Louis, MO) unless otherwise stated.

Sample collection

In vivo wood bison embryos (n=24) were obtained from the University of Saskatchewan

(Dr. Gregg Adams, Department of Veterinary Biomedical Sciences). Embryos were collected from superovulated and artificially inseminated wood bison females (n=10) following protocols

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described by Palomino et al., (2013). Embryos were retrieved on day 6 post ovulation by transcervical uterine lavage during the non-breeding season of 2011 and the breeding season of

2013. Collected embryos were vitrified and placed in a Cryotop® as described by Dr.

Muhammad Anzar (University of Saskatchewan, unpublished) and stored in liquid nitrogen until needed.

In vitro fertilization (IVF) of cattle oocytes

Procedures for in vitro fertilization of mature domestic cattle oocytes were carried out as described in chapter 3.

Thawing procedure

Embryos were thawed by placing each cryotop into 2.5 ml TCM-199® supplemented with

20% steer serum (Cansera; Rexdale, ON, Canada) + 17% sucrose at 37°C for 5 minutes.

Embryos were then washed three times in synthetic oviductal fluid (SOF) (Chemicon-Millipore,

Billerica, MA, USA) supplemented with 2% Cansera at 37°C and cultured in SOF in 5 μl droplets in a humidified atmosphere of 5% CO2, 5%O2, and 90% N2 for 2 days (48 hours).

Wood bison iSCNT embryo production

Procedures for oocyte collection, in vitro maturation (IVM), reconstruction of embryos by iSCNT techniques, and chemical activation of couplets were performed as described in chapter 1 and 2. Wood bison iSCNT embryos were cultured in 30 µl drops containing SOF for 9 days maximum (~220 hpa) using the same conditions as previously mentioned.

TUNEL staining

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The terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling (TUNEL) assay (Roche Diagnostics, Indianapolis, IN, USA) was used to determine apoptosis in wood bison embryos produced in vivo and in vitro by iSCNT. Blastocysts on day 8 (in vivo-derived wood bison) and day 9 (wood bison iSCNT) were stored and treated as described in chapter 1.

Differential staining for TE and ICM cells

Wood bison iSCNT blastocysts and domestic cattle IVF blastocysts were collected on day

9 of culture (~220 hpa). Only embryos displaying normal morphological features were included.

Procedures for staining TE and ICM were performed according to the protocol by Thouas et al.,

(2001). Briefly, embryos were placed in 500 μl of a solution containing 100 μg/ml propidium iodine in 1% PBS-Triton X-100 for 30 seconds at room temperature. Subsequently, they were moved to 500 μl of 25 μg/ml Hoechst in ethanol and incubated for 12 hours at 4°C in the dark.

Embryos were then placed onto a slide containing 13 μl of anti-fade solution (VECTASHIELD®,

Vector Laboratories, California) diluted in 0.1 % PBS-PVA solution (1:1 ratio). Analysis of differential staining was performed using a Leica DM5500 B fluorescence microscope at 20X magnification.

Statistical analysis

Descriptive statistics were used to determine the proportion of embryos progressing until day 8 (in vivo-derived) or day 9 (iSCNT) of development. The proportion of TUNEL positive

(apoptotic) nuclei and total cell number and allocation of TE and ICM cells were assessed by

Wilcoxon rank-sum test. A probability of <0.05 was considered significant. Data were analysed using the computer program RSTUDIO Version 0.97.551/IC 10.1.

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RESULTS

In vitro development of wood bison embryos

Thawed embryos were placed in culture and monitored for development for 2 days. At 4 hours post-warming, only a few embryos (n=5) showed morphological characteristics associated with embryo viability while a large proportion of embryos (n=19, 79.16%) showed features commonly linked to degeneration and/or fragmentation (Table 10). During the 48 hours monitoring, progression from the morula to the hatching stage was observed (n=3). However, complete hatching out of the zona pellucida was not documented in this experiment. Illustrations describing the stage of development and quality of embryos from in vivo-derived embryos are shown in Figures 11 and 12. In the case of wood bison embryos produced by iSCNT, first cleavage division and blastocyst rates at day 7, 8, and 9 of development were 81.9 ± 1.79, 0, 4.8

± 0.43, and 12.4% ± 3.27, respectively.

Apoptosis assessment

The proportion of total cell number and TUNEL positive labeling was determined to assess embryo quality in in vivo-derived and iSCNT wood bison embryos. There were no significant differences in the proportion of TUNEL positive nuclei between groups (p=0.18).

Although more TUNEL positive nuclei per blastocyst were found in in vivo-derived embryos while iSCNT had less apoptotic cells. Total cell number per blastocyst did not differ between groups (p=0.19), however there was a numerical difference between groups. In vivo derived embryos had more cells than embryos produced by iSCNT (Table 11). Fluorescence micrographs highlighting these findings are shown in Figure 13.

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Table 10. Embryo assessment of in vivo wood bison embryos based on recommendations by the International Embryo Transfer Society.

Day of assessment

Day 6 Day 7 Day 8

Donor Embryo ID Development Embryo Development Embryo Development Embryo animal stage (+) quality(*) Stage quality stage quality 707 1 2 4 2 4 2 4 55 2 3 4 3 4 3 4 65 3 4 4 4 4 4 4 54 4 4 4 4 4 4 4 64 5 Unknown 4 unknown 4 unknown 4 700 6 Unknown 4 unknown 4 unknown 4 255 7a 5 2 6 2 7 2 255 7b 6 2 8 1 8 1 255 8a 4 1 6 1 8 1 255 8b 4 2 6 2 8 1 255 8c 4 4 4 4 4 4 69 9a 4 4 4 4 4 4 69 9b Unknown 4 unknown 4 unknown 4 69 9c Unknown 4 unknown 4 unknown 4 69 9d Unknown 4 unknown 4 unknown 4 65 10a 5 3 5 4 5 4 65 10b 4 4 4 4 4 4 65 10c 4 4 4 4 4 4 65 10d 4 4 4 4 4 4 2013-J 11a 4 2 6 2 7 3 2013-J 11b unknown 4 unknown 4 unknown 4 2013-J 11c unknown 4 unknown 4 unknown 4 2013-J 12a 4 4 4 4 4 4 2013-J 12b 4 4 4 4 4 4

(+) Abbreviated descriptions for development stages are as follows: 2= 2- to 12- cell, 3= early morula, 4= morula, 5= early blastocyst, 6= blastocyst, 7= expanded blastocyst, 8= hatched blastocyst, 9= expanded hatched blastocyst.

(*) The codes assigned for embryo quality ranged from “1” to “4”. Values represent a visual evaluation of morphological features. Code 1, and 2 indicates excellent, and fair quality embryos, respectively, while code 3 and 4 describes poor morphological integrity, and degenerating embryos, respectively.

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DAY 7 DAY 8 Fluorescent micrograph

Figure 11. Phase contrast micrographs (day 7 and 8) from in vivo-derived wood bison embryos. Fluorescent micrographs denote total cell number 48 hours after thawing. Expanded blastocyst are observed in A) and B). Hatching blastocyst is indicated in C. Embryo identification in A, B, and C corresponds to 7a, 11a, and 7b, respectively.

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Phase contrast micrograph Fluorescent micrograph

13-cell embryo

Morula

Early blastocyst

Figure 12. Phase contrast and fluorescent micrographs of grade 4 embryos at different stages of development. Note abnormalities in shape of embryonic cells (morula and early blastocyst), and nuclear condensation (13- cell embryo).

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Table 11. Median [25%; 75%] total cell number and proportion of TUNEL labeling (apoptotic) nuclei in wood bison blastocsyts.

Parameter In vivo iSCNT P-value

[n=5] [n=15]

Total cell 98 [90; 99] 63 [56; 105] 0.19 number/embryo

Proportion of 11 [3; 11] 3 [2; 5.5] 0.18

TUNEL positive nuclei/embryo

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Red Channel Green Channel Merge

Figure 13. A) Wood bison in vivo-derived blastocyst displaying numerous cells TUNEL positive labeling (filled arrow). B) Wood bison iSCNT blastocyst with isolated apoptotic cells (thin arrow).

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Differential staining

The proportion of cells between TE and ICM was analyzed in this study as a measure of embryo viability. Wood bison iSCNT embryos were compared to domestic cattle IVF embryos since few in vivo-derived wood bison embryos survived the vitrification protocol (n=5). Total cell number from blastocysts in both groups ranged between 77- 165 cells. iSCNT embryos had significantly less cells compared to IVF cattle embryos. Additionally, significant differences were found both in TE and ICM population. However, the ratio of ICM cells to total cell number did not differ between groups (Table 12). Fluorescent micrographs representing the two cell types are indicated in Figure 14.

Table 12. Cell number and allocation in cattle IVF and wood bison iSCNT embryos.

Cattle IVF Wood bison iSCNT P value

[n=8] [n=6]

Total cell number 130[123; 144] 98[86; 107] <0.01

TE 93[90; 106] 68[58; 76] <0.01

ICM 37[35; 40] 30[28; 30] <0.01

Ratio (ICM/total cell) 27[27; 28] 30[29; 31] 0.15

TE= trophectoderm, ICM= inner cell mass

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Cattle IVF

Wood bison iSCNT

Figure 14. Differential staining of IVF cattle and iSCNT wood bison embryos. The two cell lineages are stained in blue (ICM) and pink (TE).

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DISCUSSION

The ability of mammalian embryos to successfully progress through the different preimplantation stages depends on complex interactions in response to intrinsic and extrinsic stimuli (McGeady et al., 2006). Insults occurring during this period will result in embryo demise or pathological alterations at subsequent stages (Penaloza et al., 2006). Numerous approaches have been recommended to determine the quality of embryos produced by in vitro or in vivo techniques (Maddox-Hyttel et al., 2003). Non-invasive techniques have been proposed as a novel model to assess viability by isolation and quantification of markers present in the culture medium

(Stamatkin et al., 2011; Muñoz et al., 2014). Other techniques, such as embryonic cell biopsy might provide useful information to detect chromosomal aberrations and differences in expression profile of genes linked to embryo competence. Although this technique is a powerful diagnostic technique, it requires expensive equipment and micromanipulation techniques which could negatively affect development (Ghanem et al., 2011; Ponsart et al., 2013). Morphological assessment by standard microscopy techniques has been considered an essential criterion to estimate embryo viability. However, its use in commercial and research programs has received criticism since subjective grading scores provided by trained technicians might lead to inaccurate evaluation (Farin et al., 2009).

Numerous embryo scoring quality systems have been described for cattle embryos

(Lindner and Wright, 1983; Mrkun and Kosec, 2000). However, a standardized system was developed by The International Embryo Transfer Society which has been universally used to certify and identify cattle embryos for commercial purposes. The system classifies each developmental stage and assigns a code to summarize phenotypic attributes compatible with normal morphology, moderate irregularities, or degeneration. It has been recommended that

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international commercialization of bovine embryos should only include code 1 (excellent or good). Although this evaluation system has been implemented for more than three decades in domestic species, in other non-domestic bovids, such as the wood bison, no classification criterion for embryos or oocytes has been described. Furthermore, parameters associated with embryo viability, such as total cell number, allocation of cell lineages, and incidence of apoptosis of in vivo-derived wood bison blastocysts have not yet been investigated. Consequently, previous bison studies have extrapolated data from domestic cattle studies to hybrid and iSCNT wood bison embryos (Seaby et al., 2012; Seaby et al., 2013). Thus, we aimed to characterize embryo development from in vivo-derived wood bison blastocysts during 48 hours of in vitro culture and, to compare embryo quality between in vivo and in vitro wood bison embryos produced by iSCNT. The results from our study showed that only 20% of the embryos survived after thawing

(n=5). Due to the high percentage of embryos displaying major alterations, developmental stage was not determined in 30% of in vivo-derived wood bison embryos (n=6). The remaining embryos showed morphological characteristics commonly associated with degeneration or loss of cellular shape. Therefore, adequate comparisons between in vivo and in vitro-derived wood bison embryos could not be established due to small sample size. No significant differences were found in total cell number or incidence of apoptosis in both groups (in vivo wood bison vs iSCNT wood bison). However, when comparing cattle domestic IVF and wood bison iSCNT blastocysts, there was a significant difference in total cell number, TE, and ICM but no significant differences were found when assessing the ratio between ICM to total cell number.

Cattle embryos enter the uterus at the 8-16 cell stage, four to five days after ovulation and start blastocoele formation around day 7 (reviewed by Vejlstead, 2010). In other bovid species, such as the water buffalo, these embryonic events occur faster than in domestic cattle (Drost and

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Elsderv, 1987). In bison species, embryos and unfertilized oocytes can be found in the uterus 7 days after induction of ovulation (Dorn et al., 1990, Palomino et al., 2013). However, the exact embryo transport times in the bison’s oviduct have not yet investigated. The developmental stage of embryos used in this study demonstrated that at day 6 after ovulation, morulae (n=13) and blastocysts (n=2) are already present in the uterus. Morphological evaluation at the time of embryo recovery or before vitrification was not available. Therefore, conclusive statements about the impact of the vitrification process on embryo quality cannot be drawn. Visual evaluation of embryo developmental stages and determination of quality codes were performed daily following the criteria recommended by the International Embryo Transfer Society which provides fundamental information about gross morphology (Chapter 9, IETS Manual, 3rd edition). The results from this evaluation indicated that wood bison embryos with codes 1 and 2 progressed from the morula to the hatching stage. The zona pellucida began thinning with subsequent hatching at 24 (n=1), and 48 (n=2) hours after in vitro culture. Complete hatching out the zona pellucida was not observed after 24 hours in culture (n=1) which could be an indication of impaired embryo viability. Niimura et al., (2010) showed that the length of hatching in both fresh-cultured and frozen-thawed cattle embryos ranged from 19 to 39 and 21 to 40 hours depending on location of cell protrusion in the TE cells. Since wood bison embryos were only cultured for 48 hours and there is no information available on in vitro development to the hatching stage in wood bison, further studies are necessary to adequately determine these parameters in bison embryos. However, it has been suggested that a significant proportion of cattle frozen-thawed embryos are not able to complete the hatching process (Massip et al., 1982).

Another indicator of developmental potential is related with the proportion of cells positive for apoptosis (Matwee et al., 2000). The proportion of positive TUNEL cells found in our study was

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in accordance with the values reported for wood bison iSCNT embryos (Seaby et al., 2013).

Gjørret et al., (2003) reported higher incidence of apoptosis in cattle blastocysts produced by in vitro techniques compared to in vivo-derived blastocysts. However, we observed similar apoptosis incidence between groups but more apoptotic cells were found in in vivo-derived embryos near the ICM. Thus, further investigation is required to determine if this apparent increase in apoptosis affecting ICM is attributed to suboptimal culture conditions, detrimental effects due to the vitrification process, or intrinsic factors affecting ICM. Although this is the first report documenting total cell number and incidence of apoptosis in in vivo-derived wood bison embryos, careful interpretation of these findings is recommended due to the limitations previously mentioned.

Embryo quality can also be estimated by determining the proportion of the two cell lines

(ICM and TE). It has been estimated that a positive relationship between embryo quality and number of ICM compared to total cell number (~30-34%) exists (van Soom et al., 1997).

Evidence has suggested that low number of these population lineages might negatively affect processes such as implantation and placenta attachment (reviewed by Bavister, 1996). However, embryo biopsy at the blastocyst stage, effectively reducing the number of cells by 50% does not prevent development to term (Picard et al., 1986). The median number of TE and ICM was significantly lower in wood bison iSCNT than cattle IVF embryos, which could impair post implantation events. However, further studies including proper control (in vivo derived wood bison embryos) are necessary to determine if the low TE and ICM cell number observed in iSCNT embryos is attributed to the use of cattle oocytes as cytoplasts or intrinsic to the wood bison species. Nevertheless, the total cell number obtained from in vivo-derived embryos correlates with the total cell count from wood bison iSCNT, however the similarities might be

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attributed to differences in development at which embryos were analysed (i.e. wood bison in vivo-derived embryos were assessed at day 8 compared to day 9 in iSCNT wood bison embryos).

Therefore, fluctuations in the proportion of ICM and TE to total cell number between in vivo- derived wood bison and iSCNT embryos might be expected.

Factors such as small sample size, low survival rate post warming, and individual variability are the main limitations of this study. For instance, 80% of the embryos that survived the vitrification procedure, confirmed by morphological assessment and progression through development during 48 hours of in vitro culture, were obtained from the same female which could lead to misinterpretation due to lack of individual variation. Therefore, it is not clear if deleterious processes associated with cryopreservation of cells including physical and chemical damage could result in decreased survival rates in wood bison embryos. Vitrification or ultra- rapid freezing of gametes and embryos has gained significant preference over slow conventional cryopreservation techniques in the field of human and animal reproduction (Kuwayama, 2007).

Although both techniques have successfully been applied to cryopreserve embryos from many species (Massip, 2001), the success of these techniques has not been assessed in bison embryos.

It has been reported that the success rate of vitrified domestic cattle embryos from Bos taurus donors ranges between 40 to 50% (Hasler, 2010). Although, there are no reports regarding pregnancy rates of vitrified bison embryos, the suboptimal survival rates obtained in this study require further investigation.

It has been documented that embryos with low developmental competence are less likely to survive the vitrification process (Ren et al., 2012). Embryos obtained from this study were collected during the breeding (n=5) and the non breeding season (n=19). Evidence has suggested that embryos retrieved from seasonal breeders such as sheep and cats during the non-breeding

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season show reduced developmental ability in comparison with embryos obtained during the non-breeding season (Freistedt et al., 2001; Zheng et al., 2001). Although the effect of seasonality on embryo competence has not been fully investigated in wood bison, Palomino et al., (2013) documented better responses to estrus synchronization and superovulation protocols during the months where bison females start to show estrous behavior which suggests a positive association between the breeding season and embryo quality in wood bison species.

The results from this study showed low developmental competence of in vivo-derived wood bison embryos. Although, the sample size of this study was a main limitation to determine significant differences between groups, the reduced survival rates post thawing associated with poor quality embryo scores are highly suggestive of compromised development prior to in vitro culture. However, this hypothesis requires further investigation. Thus, the use of iSCNT techniques represent an efficacious model to study the preimplantation stages in species where embryos are difficult to obtain due to a limited number of individuals and/or unknown normal reproductive and embryological parameters.

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GENERAL DISCUSSION

The main objective of this thesis was to investigate the developmental potential of early embryos produced by interspecies somatic cell nuclear transfer (iSCNT) in the North American bison (Bison bison bison and Bison bison athabascae), using domestic cattle oocytes (Bos taurus) as cytoplasts. Although numerous studies on iSCNT have reported reduced developmental rates coincidentally with the time of embryonic genome activation (EGA), there is limited data regarding the factors associated with embryo demise in bison embryos generated by iSCNT. Thus, identification of potential factors linked to embryo failure during early development might provide valuable information about the feasibility of iSCNT as a potential aid in preservation and/or propagation of endangered genotypes. There is a negative association between developmental rates and species divergence between the genetic donor and oocyte donor (reviewed by Mastromonaco et al., 2014). Altered communication between ooplasmic and nuclear components has been recognized as a key factor affecting development in iSCNT embryos (Lagutina et al., 2010, Amarnath et al., 2011; Mastromonaco et al., 2010). Thus, assessment of organelle function, quantification of nucleic acids, and embryo quality using invasive techniques was investigated in this thesis to provide information about potential causes of embryo incompetence in bison embryos.

This thesis was based on observations performed on 2405 samples including cattle and plains bison oocytes for parthenogenesis studies, cattle SCNT, plains bison and wood bison iSCNT, ooplasm transfer (OT) experiments in cattle and plains bison reconstructed embryos, and characterization of in vivo-derived wood bison blastocysts. The experiments described in chapter

1 were carried out to determine the feasibility of cattle in vitro production systems (IVP) to mature and culture plains bison cumulus-oocyte complexes (COCs). Development of techniques

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to fertilize cattle gametes in vitro originated almost four decades ago (Iritani and Niwa, 1977). In domestic cattle not only the success rate has been improved but also a large repertoire of events leading to acquisition of nuclear and cytoplasmic oocyte maturation have been elucidated

(reviewed by Wrenzycki and Stinshoff, 2013). Conversely, in bison species the efficiency of these protocols has not been properly addressed. Studies regarding in vitro culture conditions of bison gametes suggest that a suboptimal in vitro environment might be associated with low developmental rates (Thundathill et al., 2007, Seaby et al., 2012). Thus, the progress in this field has been significantly slower when compared with domestic cattle. IVP implementation using more advanced techniques, such as somatic cell nuclear transfer and spindle transfer after cryopreservation of immature oocytes would be almost impossible to apply to bison species if proper conditions for IVM and IVC are not fully investigated. However, identification of optimal parameters, including adequate hormone concentration, selection of growth factors and numerous components present in the culture medium involves extensive research and multiple sample collections. Unfortunately, these experiments are impractical in threatened species due to the reduced number of individuals allowed for research. Thus, current knowledge of in vitro production of bison embryos has extrapolated information from cattle models often leading to poor success rates or misinterpretation of findings. Given this adverse scenario, we aimed to test protocols initially developed for cattle which used co-culture for IVM in conjunction with IVC to study the resumption of meiosis (Sirard and Bilodeau, 1990) and the role of somatic cells

(granulosa and cumulus cells) on oocyte competence (Dey et al., 2012). Numerous studies have shown advantageous effects on development when reproductive somatic cells have been incorporated into the in vitro system (Orsi and Reischl, 2007; Gilchrist et al., 2008). It has been reported that the co-culture system might be responsible for secretion of factors promoting

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embryo competence, removal of toxic components, and facilitating better interactions between cumulus cells and oocytes through gap junctions (Nagai et al., 2013; Hashimoto et al., 1998).

Although the majority of these studies reported positive effects on in vitro micro-environment, few studies have fully investigated the mechanisms leading to improved developmental rates

(Orsi and Reischl, 2007). Thus, we implemented a system for culture of plains bison oocytes and parthenogenic embryos using granulosa and cumulus cells derived from domestic cattle. Due to the small sample size and difficulty in accessing plains bison oocytes, the maturation system applied in chapter 1 did not include oocytes cultured without somatic cells which could have provided definite answers regarding the effects of somatic cells on plains bison oocyte/embryo competence. Nevertheless, the encouraging results obtained from these experiments suggest a beneficial effect of cattle somatic cells on maturation and development of plains bison parthenogenic embryos since this is the first study showing developmental rates similar to domestic cattle. However, delayed development in plains bison was observed at the blastocyst stage which might indicate suboptimal in vitro conditions with potential deleterious effects in plains bison parthenogenic embryos. By implementing and testing the granulosa co-culture maturation protocol, we also ensured the selection of an adequate maturation system to provide competent cytoplasts for ooplasm transfer experiments (chapter 3). Furthermore, these protocols can create the foundation for future applications of IVP systems in bison species.

In an age governed by human development, biodiversity conservation has become prioritized due to habitat loss leading to accelerated extinction rates, globally. Modern biotechnologies including iSCNT have received considerable attention due to its potential roles in species preservation (Monfort, 2014). Somatic cells from rare or scarce individuals are obtained and cryopreserved to ultimately reconstruct embryos using cytoplast from domestic

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species (Loi et al., 2001; Srirratana et al., 2012; Hwang et al., 2013). Since iSCNT implementation in 1999, a large number of studies have focussed on investigating the feasibility of iSCNT to produce embryos from 32 mammals included in threatened or endangered species lists (reviewed by Mastromonaco et al., 2014). On the other hand, limited knowledge is available regarding the causes of early embryo failure when iSCNT procedures are used. Multiple alterations involving reprogramming errors, epigenetic modifications, and aberrant gene expression have been reported in embryos produced by iSCNT (Wang et al., 2009, Amarnath et al., 2011). However, it has been suggested that other factors regarding incompatibilities between nuclear and ooplasmic components might negatively impact development resulting in high embryonic arrest rates commonly observed at the time of EGA in iSCNT embryos (Yin et al.,

2006; Song et al., 2006).

One of the main organelles in the eukaryotic cell is the mitochondria. Embryo development is highly dependent on normal mitochondrial function even before fertilization takes place (Van Blerkom, 2004). It has been documented that mitochondrial function can be negatively influenced by species divergence in iSCNT embryos, which in turn could affect interactions between nuclear and mitochondrial genomes, such as respiration, mitochondrial replication, and energy production. The results obtained in chapter 2 suggested that bison embryo development might be impaired due to abnormal function of ooplasmic components in both plains and wood bison iSCNT embryos. It has been proposed that a foreign oocyte, selected as cytoplast, has the potential to drive early development until EGA (Dominko et al., 1999).

Interestingly, many iSCNT embryos fail to progress in vitro resulting in low blastocyst rates if the species are taxonomically related (Gómez et al., 2008; Seaby et al., 2013) or complete embryo arrest when the reconstructed embryos are derived from very distant species (Tecirliogu

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et al., 2006; Lagutina et al., 2010). Thus, our results showed that impaired mitochondrial function as indicated by significantly lower ATP content in bison embryos might compromise multiple cellular functions. Furthermore, it has been shown that apoptosis in early embryos confers protective mechanisms by removal of abnormal blastomeres but also it can lead to embryo death by deleterious stimuli during in vitro culture (Hardy, 1997). Mitochondria, on the other hand, play important roles by regulating a cascade of events leading to apoptosis (Maloyan et al., 2005). Therefore, it is plausible to assume that altered mitochondrial function led to increased apoptosis incidence in early plains bison embryos. However, establishing a direct relationship between low ATP content and development of apoptosis requires additional studies since other conditions might also be triggering apoptosis, such as excessive accumulation of free- radicals during in vitro culture (Kamjoo et al., 2002). In addition to the factors potentially causing deleterious effects in 8-16 cell bison embryos, changes in gene expression could also provide some insight into pathological processes occurring during the early pre-implantation stages. It is well recognized that embryos produced by SCNT display differences in gene expression (Zheng et al., 2012). For instance, downregulation of pluripotent genes or upregulation of somatic transcripts during the early stages of development have been described

(Chung et al., 2009; Beyhan et al., 2012). Moreover, aberrant expression of genes involved in mitochondrial function has also been associated with developmental problems (Bowles et al.,

2007). Evidence has suggested that normal development favours the transmission of only maternal mtDNA during embryogenesis. Thus, sperm-derived mitochondria are eliminated to guarantee inheritance of one mtDNA genome (Sutovsky et al., 2004). The use of reproductive biotechnologies, such as iSCNT, OT, spindle and pronuclear transfer introduce a portion of mitochondria that belong to a different individual from the same or a different species resulting

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in replication of foreign mtDNA or elimination of the newly introduced organelle (Cibelli et al.,

2002; Hua et al., 2012; Amato et al., 2014). This situation has generated two decades of debate.

To date, the mechanisms leading to heteroplasmy are difficult to predict. Studies have shown differences in heteroplasmy rates in OT and SCNT studies during the pre and post implantation stages (Barrit et al., 2001; John et al., 2005; Burgstaller et al., 2007). Therefore, it is difficult to determine an association between pathological heteroplasmy and compromised development.

Conversely, heteroplasmy has been reported in embryos and offspring from human and mammalian species lacking physiological alterations (St John et al., 2010).The qPCR experiments from our second chapter showed no differences in the level of expression for NRF-2 and mt-COX2 but higher levels of expression of TFAM in wood bison iSCNT embryos which might suggest the occurrence of both premature mitochondrial replication and transcriptional events. Interestingly, cattle and plains bison showed similar levels of expression and this similarity could be associated with presence of cattle genes in the plains bison genome (Ward et al., 200; Hedrick, 2010). However, further nuclear and mtDNA comparison between the cell lines used in this study should confirm this hypothesis.

Evidence has suggested that absence of compatibility among cellular and/or subcellular compartments in iSCNT embryo leads to embryo demise (Lagutina et al., 2011, Mastromonaco et al., 2007). Therefore, it has been hypothesized that incorporation of ooplasm matching the genetic origin of the somatic cell might positively influence development (Sansinena et al.,

2010). However, very little information has confirmed this hypothesis. The model established in chapter 3 resulted in the reduction of manipulation steps necessary to perform iSCNT + OT and study the effects of plains bison supplementation on early development. Our model used two species that share the same subfamily (Bovinae) but belong to different genus (Bison vs. Bos).

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No detrimental or beneficial effects on early development of cattle SCNT or bison iSCNT embryos as a result of OT were observed. Developmental rates, ATP accumulation, or gene expression were similar among groups. Thus, two scenarios can be envisioned from our results:

1) the amount of ooplasm transferred into the reconstructed embryos was not sufficient to produce quantifiable differences related to energy metabolism and gene expression, 2) the evolutionary relationship between domestic and plains bison species might be more permissive to cytoplasmic and nuclear discrepancies, a phenomenon observed in hybrid offspring (domestic cattle x bison) when the two species are bred (Basrur and Moon, 1967).

Significant and rapid advances in the field of assisted reproductive techniques have been documented in the last two decades. Soon after SCNT was successfully applied to mammalian species, it was suggested that iSCNT would facilitate the propagation of genotypes from endangered species. However, extensive research conducted on iSCNT has shown poorer results when compared to SCNT. While many studies have linked SCNT failure with incomplete reprogramming as well as aberrant expression of key genes, the role of nuclear-cytoplasmic interactions on embryonic development in iSCNT embryos has been addressed by few researchers. The results from our studies showed compromised embryo development in early bison iSCNT potentially linked to alterations in mitochondrial function affecting several cellular processes. We modified available cattle in vitro embryo production protocols to successfully mature and culture plains bison gametes. Application of these techniques might increase current

IVP efficiency and provide valuable tools to understand the causes of embryo incompetence in bison species. On the other hand, implementation of cattle protocols in bison species allowed us to produce competent plains bison oocytes used as cytoplast donors. Differences in development, mitochondrial function, or gene expression were not observed in 8-16 cell stage iSCNT embryos

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supplemented with our without plains bison ooplasm. This last experiment was fundamental to determine the effects of ooplasm transfer on embryo development. However, future research should explore different stages of development, variations in the amount of ooplasm, and optimization of procedures used for isolation of the cellular compartment to determine the role of specific ooplasmic components on nuclear-ooplasmic interactions and their impact on subsequent development.

The ultimate goal of any embryo IVP system is the generation of competent embryos

(Galli et al., 2004). Intrinsic and extrinsic factors can potentially alter the ability of an embryo to progress during development. In our study a significant proportion of bison iSCNT embryos arrested at the time of EGA coincidentally with alterations described in chapter 2. Consequently, fewer wood bison iSCNT embryos reached the blastocyst stage (chapter 4). Embryo viability assessment in iSCNT embryos from endangered or threatened species commonly requires the use of inadequate control groups due to the paucity of data from valuable in vivo-derived embryos.

The studies conducted in chapter 4 characterized in vivo-derived wood bison embryos for the first time, in terms of morphology assessment, total cell number, and apoptosis incidence.

Although a representative sample size was obtained to conduct comparative studies, few data were generated due to the low survival rates post embryo thawing. Differences in total cell number and TUNEL positive nuclei were not found. A larger sample size will be necessary to clarify whether differences actually occur between iSCNT and in-vivo derived wood bison embryos. The findings from our studies have provided substantial new information regarding the developmental potential of bison embryos as well as potential factors influencing early development. The results reported in this thesis provide a crucial platform for future implementation of ARTs in bison species. However, it is imperative to further increase our

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knowledge about embryo development in bison species during the post implantation stages to enable proper assessment of the ability of in vitro produced embryos to attach, grow, and adapt to the extra-uterine life. Therefore, iSCNT is a good model for studying bison embryos to gain information about embryo development which can then be used in field applications.

Conservation measures in wood bison were implemented at the beginning of the 20th century. However, the number of herds never rebounded to their original size. The wood bison conservation status was, in fact described by Frank Gilbert Roe (1970) as “a tragic example of abuse towards wildlife in the North American continent”. Thus, it seems almost impossible to believe that innumerable bison herds once existed across the continent. Habitat preservation has been highlighted as a powerful aid to perpetuate the number of wood bison. However, propagation of their genetic material through the use of assisted reproductive technologies should be regularly included and recommended in most programs aiming at preservation of threatened and endangered species.

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FUTURE DIRECTIONS

In the field of species conservation, ARTs have played an important role to propagate endangered genotypes. However, access to gametes and embryos is generally limited.

Implementation of in vitro techniques requires extensive investigation of factors influencing development. Therefore, domestic cattle IVP protocols have been used in a large repertoire of endangered species. Although cattle IVP protocols have shown modest success when applied to wood bison, experimental models including taxonomically less divergent species, such as plains bison might be more advantageous since there are more physiological similarities between subspecies. Therefore, obtaining gametes from plains bison females located not only in the province of Ontario but also within Canada would accelerate the current knowledge on in-vitro embryo production systems.

Incorporation of only mitochondria derived from somatic cells or bison oocytes has been successfully implemented in various species. The use of this technique has allowed the study of nuclear-mitochondrial interactions during early development. Although these techniques are complex, they provide valuable information since the effects of only one component of the ooplasm can be assessed. However, protocols for isolation, characterization, and storage of bison mitochondria should be fully investigated.

Assessment of iSCNT efficacy as a potential tool to propagate endangered species requires evaluation not only during the preimplantation stages but also at the post implantation stages. Therefore, the transfer of iSCNT embryos to bison females will provide answers regarding the use of this technique to produce competent embryos and healthy offspring.

Concepts regarding the transmission of foreign mitochondria into the offspring would reveal potential detrimental effects at subsequent extra-uterine stages.

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SUMMARY AND CONCLUSIONS

 Implementation of domestic cattle IVP protocols in plains bison oocytes to produce parthenogenic embryos resulted in similar maturation and developmental rates between cattle and plains bison but delayed development at the blastocyst stage was observed in the latter group. Thus, further investigation is necessary to determine the causes of suboptimal in vitro culture conditions from day 7 to day 9 in plains bison parthenogenic embryos.

 Assessment of development in bison iSCNT embryos was significantly different from cattle SCNT. Low ATP content, higher incidence of nuclear condensation and apoptosis, and higher expression of TFAM was found in plains and wood bison embryos at the 8-16 cell stage.

Therefore, these alterations could lead to embryonic arrest or subsequent detrimental effects at the blastocyst stage.

 Applying a modified ooplasm transfer technique to reconstruct embryos allowed the study of nuclear-ooplasmic interactions in iSCNT plains bison embryos. Early cleavage divisions, ATP content, and expression of mitochondrial and nuclear genes were not negatively or positively affected by supplementation of plains bison ooplasm into plains bison iSCNT.

Counfounding factors in the ooplasm might explain the lack of differences observed in OT experiments requiring further studies.

 Wood bison in vivo-derived embryos exhibited poor survival rates after vitrification and thawing. Although similar cell number and apoptosis incidence were found in wood bison embryos produced either in vivo or in vitro, higher cell number would be expected in in vivo- derived embryos since analyses were performed a day earlier in development when compared to in vitro iSCNT bison embryos. However, these studies provided basic evidence about wood bison embryo development which can then be used for further research.

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 The results obtained from the various experiments support our hypothesis that cattle ooplasmic components influence mitochondrial function and related gene expression in iSCNT bison embryos, thereby affecting pre-implantation embryonic stages. The experiments conducted in this thesis revealed fundamental data concerning culture and chemical activation of plains bison oocytes. In addition, bison embryo (both subspecies) produced by iSCNT exhibited developmental abnormalities. Advantageous or detrimental effects were not observed by ooplasm supplementation in reconstructed cattle SCNT or plains bison iSCNT embryos. The use of iSCNT is a valuable mechanism to study embryo development from endangered or threatened species such as wood bison.

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APPENDIX

APPENDIX I Parameters used to electrofuse a portion of cattle ooplasm and the somatic cell in cattle SCNT embryos

Trial Voltage Duration Number Sample Lysis Ooplasm Somatic (kV/cm ) of pulse of pulses size n (%) fusion cell fusion (μs) n (%) n (%) 1a 1.74 23 2 40 0(0) 40(100) - 2b 1.74 21 2 22 3(13.6) 20(91) 3(13.6) 3 1.74 21 2 17 6(35.3) 15(88.2) 0(0) 4c 1.54 18 1 40 0(0) 35(95) - 2.34 23 1 0(0) - 0(0) 5c 1.38 19 1 9 0(0) 9(100) - 2.16 30 1 2(22.2) - 4(44.4) 6c 1.38 19 1 9 0(0) 9(100) - 2.10 35 1 2(22.2) - 4(44.4) 7c 1.38 19 1 9 0(0) 9(100) - 2.10 40 1 0(0) - 4(44.4) 8c 1.38 19 1 10 0(0) 9(90) - 2.10 45 1 1(10) - 3(30) 9c 1.50 20 1 28 0(0) 22(78.5) - 2.10 40 1 4(14.2) - 13(46.4) 10c 1.5 20 1 38 0(0) 38(100) - 2.10 38 1 73.6(28) - 5(13.1) 11c 1.50 20 1 42 2(4.7) 38(90.4) - 2.04 35 1 9(21.4) - 20(47.6) athis trial was only intended to fuse ooplasm b ooplasm was placed opposite to the somatic cell c, two separate electrical stimuli were used at different parameters settings (voltage and duration) at 30 minutes interval

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APPENDIX II Preliminary parameters for determination of adequate settings for fusion of cattle somatic cell into cattle oocytes (2013-2014)

Ionic compound Parameter Fusion rate Lysis rate (mM) (%) (%)

Ca2Cl Mg2Cl kv/cm μs number of pulses 100 100 1.22 37 1 10 57 100 50 1.80 30 2 18 64 50 100 1.76 40 1 25 18 50 100 1.80 20 2 31 43 50 100 1.80 35 1 28 65 50 100 1.26 47 1 8 23 50 100 1.86 12 2 48 37 50 100 1.20 47 1 14 2 50 100 1.70 10 2 38 3 50 100 1.80 15 2 34 3 100 100 1.40 42 1 5 16 100 100 0.90 37 2 5 20 100 100 1.32 42 2 0 50 100 100 1.56 42 1 30 10 100 100 1.74 42 1 0 22 100 100 1.92 42 1 20 20 100 100 2.10 42 1 55 22 100 100 1.56 32 1 22 22 100 100 1.74 32 1 33 0 100 100 1.92 32 1 44 11 100 100 2.34 42 1 22 44 100 100 2.10 32 1 30 10 100 100 2.52 22 1 30 40 100 100 2.34 32 1 22 11 100 100 1.56 22 2 70 10 100 100 1.74 22 2 80 10 100 100 1.92 22 2 60 10 100 100 2.10 22 2 22 66 100 100 2.34 22 2 30 30 100 100 1.74 32 2 40 0 100 100 1.60 20 2 60 9 100 100 1.80 20 2 63 10 100 100 1.80 30 1 75 0

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