Evidence for the Role of Ywha in Mouse Oocyte Maturation

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EVIDENCE FOR THE ROLE OF YWHA IN MOUSE OOCYTE

MATURATION

A thesis submitted

To Kent State University in partial

Fulfillment of the requirements for the

Degree of Master of Science

Ariana Claire Detwiler

August, 2015

Except for previously published materials

Thesis written by

Ariana Claire Detwiler

B.S., Pennsylvania State University, 2012

M.S., Kent State University, 2015

Approved by

______Douglas W. Kline, Professor, Ph.D., Department of Biological Sciences, Masters Advisor

______Laura G. Leff, Professor, PhD., Chair, Department of Biological Sciences

______James L. Blank, Professor, Dean, College of Arts and Sciences

TABLE OF CONTENTS

List of Figures ……………………………………………………………………………………v

List of Tables ……………………………………………………………………………………vii

Acknowledgements …………………………………………………………………………….viii

Abstract ……………………………………………………………………………………….....1

Chapter I

Introduction…………………………………………………………………………………..2

1.1 Introduction …………………………………………………………………………..2

1.2 Ovarian Function ……………………………………………………………………..2

1.3 Oogenesis and Folliculogenesis ………………………………………………………3

1.4 Oocyte Maturation ……………………………………………………………………5

1.5 Maternal to Embryonic Messenger RNA Transition …………………………………8

1.6 Meiotic Spindle Formation …………………………………………………………...9

1.7 YWHA Isoforms and Oocyte Maturation …………………………………………...10

Aim…………………………………………………………………………………………..15

Chapter II

Methods……………………………………………………………………………………...16

Collection of oocytes and eggs…………………………………………………………..16

Collection of morulae and blastocysts …………………………………………………..17

Messenger RNA isolation and cDNA synthesis for YWHA gene expression…………..18

Polymerase chain reaction and gel electrophoresis for YWHA gene expression………..19

Gene sequencing ………………………………………………………………………...20

Quantitative polymerase chain reaction …………………………………………………20

Protein isolation for pull-down ………………………………………………………….21

SDS-PAGE and Western blot for protein pull-down ……………………………………22

Chapter III

Results ………………………………………………………………………………………24

1. Do the levels of YWHA gene expression change during oocyte maturation? …….....24

Background and rationale ……………………………………………………………….24

1.1 Detecting YWHA isoform gene expression by RT-PCR ……………………….25

1.2 Sequencing YWHA isoform PCR products ……………………………………..26

1.3 Measuring levels of gene expression through oocyte maturation by quantitative

PCR………………………………………………………………………………34

2. Is YWHAH a binding partner for CDC25B in ovary, oocytes, and eggs? …………..45

Background and rationale ……………………………………………………………….45

2.1 Detecting the binding of CDC25B to YWHAH in oocytes and eggs through

pull-down experiments …………………………………………………………..46

Summary ………………………………………………………………………………………..48

iii

Chapter IV

Discussion …………………………………………………………………………………..49

References ……………………………………………………………………………………...55

Appendix of Abbreviations ……………………………………………………………………61

LIST OF FIGURES

CHAPTER I

Figure 1. Follicular development ………………………………………………………………...4

Figure 2. Mouse oocyte maturation ……………………………………………………………...5

Figure 3. Simplified diagram of proteins involved in oocyte maturation from arrest at

prophase (left) to release from meiotic arrest (right)……………………………………...7

Figure 4. Immunoprecipitation of CDC25B phosphatase and co-immunoprecipitation of

YWHA proteins with CDC25B ……………………………………………………………..12

Figure 5. Microinjection of antisense morpholino oligonucleotides ………………………...…14

CHAPTER III

Figure 6. Expression of YWHA isoform mRNA in oocytes and eggs of two mouse strains …..26

Figure 7. H2AfZ (NM_016750.3) mRNA Sequence …………………………………………...27

Figure 8. YWHAH (NM_011738.2) mRNA Sequence ………………………………………...28

Figure 9. YWHAB (NM_018753.6) mRNA Sequence ……………………………………..….29

Figure 10. YWHAE (NM_009536.4) mRNA Sequence ……………………………………….30

Figure 11. YWHAG (NM_018871.3) mRNA Sequence ……………………………………….31

Figure 12. YWHAQ (NM_011739.3) mRNA Sequence ……………………………………….32

Figure 13. YWHAZ (NM_011740.3) mRNA Sequence ……………………………………….33

Figure 14. Fold changes between oocyte and egg for the YWHAH isoform …………………..35

Figure 15. Fold changes between oocyte and egg for the YWHAE isoform …………………..36

Figure 16. Fold changes between oocyte and egg for the YWHAG isoform …………………..37

Figure 17. Fold changes between oocyte and egg for the YWHAQ isoform ...………………...38

Figure 18. Fold changes between oocyte and egg for the YWHAZ isoform …………………...39

Figure 19. Fold changes between morula and blastocyst for the YWHAH isoform ...…….…...43

Figure 20. Pull down of YWHAH and CDC25B proteins in oocyte and egg ………………….47

LIST OF TABLES

CHAPTER II

Table 1. Primers used to detect presence of each YWHA isoform in RT-PCR ………………19

Table 2. Primers used to detect each YWHA isoform in qPCR ………………………………21

CHAPTER III

Table 3. Quantitative PCR average CT values for each YWHA isoform and Luciferase,

ΔCT values, and ΔΔCT values ………………………………………………………………40

Table 4. Quantitative PCR Average CT values for YWHAH isoform and Luciferase,

ΔCT values, and ΔΔCT values calculated from morula and blastocyst ……………………..44

vii

ACKNOWLEDGEMENTS

I would like to express my sincere gratitude to all who have helped me to accomplish this goal, especially to my advisor, Dr. Kline. Without his patient guidance and encouragement, I would not have been able to complete this thesis. I have learned so much from his mentoring and

I will be forever grateful for the time that we spent working together. I would also like to extend my gratitude towards my lab mates, past and present, Santanu De and Alaa Eisa. I am so glad that we became such close friends and peers in the short time we had here at Kent State together.

I am also very grateful to Dr. Vijayaraghavan and his lab members, Suranjana Goswami,

Rahul Bhattacharya, Shawn Davis, Nidaa Awaja, Sabyasachi Sen, Tejasvi Dudiki, and Cameron

Brothag, as we worked closely as sister labs on our projects. I am also grateful and appreciative to the undergraduate students who helped me with a few projects, Patricia Marthey, Michele

Fabian, and Isabella Cruz. I must also express my great thanks to Dr. Jenny Marcinkievicz for serving on my committee and fostering my love of teaching. Many thanks to all of the other professors, graduate students, and staff here at Kent State who have assisted me, guided me, and encouraged me in my pursuit of this degree.

And last but not least, thank you to my parents, my brother, the rest of my extended family, and all of my close friends who have supported me and encouraged me during this process.

viii

ABSTRACT

Processes essential to the growth and differentiation of oocytes, such as meiosis and spindle formation, are dependent upon intricate interactions between key proteins. The Tyrosine

3-Monooxygenase/Tryptophan 5-Monooxygenase Activation (YWHA) family of proteins have been found to be essential to vertebrate cell cycle regulation and development. There are seven isoforms, each encoded by a different gene. Previous studies have shown that these proteins are involved in maintenance of prophase I arrest in the GV-intact oocyte, as well as proper spindle formation. Real-time and quantitative PCR methods were employed to study the relative levels of expression of these proteins’ transcripts in the mouse oocyte and egg. Protein pull-down experiments were also used to investigate the interaction of the key phosphatase in meiosis resumption, CDC25B, and the YWHAH protein in oocytes and eggs. PCR results confirm the presence of at least six of the seven isoforms in both oocytes and eggs of two strains of mice, and suggest a decrease in relative expression of the transcripts through oocyte maturation. The pull- down results confirm the binding and interaction of YWHAH with CDC25B. This study increases the knowledge available on the YWHA proteins, and provides a strong basis for further studies.

CHAPTER I

INTRODUCTION

1.1 Introduction

Processes essential to the growth and differentiation of germ cells, such as meiosis and spindle formation, are dependent upon intricate interactions between key proteins. The Tyrosine

3-Monooxygenase/Tryptophan 5-Monooxygenase Activation protein (YWHA) family of proteins has been found to be essential to vertebrate cell cycle regulation and development.

Recent studies have shown that these proteins also play vital roles in oocyte maturation, meiotic spindle formation, and early embryonic development. These processes are necessary and required to produce fertilizable eggs, and determining the function and expression of the YWHA proteins in immature oocytes, mature eggs, and the developing embryo is essential to understanding these pathways.

1.2 Ovarian Function

The mammalian ovary is a complex organ, characterized by continuous growth and differentiation throughout the animal’s maturation. The ovary is the primary reproductive organ for the female, and carries out two main functional roles. First, the ovary is the site of differentiation of the mature oocyte and after maturation, releases the fertilizable egg (McGee

2 and Hsueh, 2000). Second, the ovary synthesizes and secretes hormones essential for follicle development, maintenance of the reproductive tract, and regulating the menstrual/estrous cycle

(Hirshfield, 1991). In mice, the ovary is a small organ, filled with germ cells and stroma at birth.

During the first week after birth, the ovary grows and differentiates exponentially, and by 7 days old, oocyte growth and follicle development has begun (Peters, 1969). Ovulation and thus, sexual maturity, of the female mouse occurs between 24 and 28 days after birth (Snell, 1956).

1.3 Oogenesis and Folliculogenesis

Oocytes, the female germ cells, start their development while the female is still developing in utero. Primordial oogonia undergo mitosis to form a primary oocyte surrounded by somatic cells called follicle cells. This follicle-enveloped oocyte is then arrested in prophase I of meiosis until the female becomes reproductively mature. This pool of about 8000 dictyate-stage oocytes per ovary is the sole source of fertilizable eggs for the female mouse’s lifetime. For selected oocytes, the follicle cells continue to grow around the arrested oocyte in preparation for ovulation. A single layer of epithelial cells expands to three layers of cuboidal granulosa cells.

Theca cells, which develop lutenizing hormone (LH) receptors, are then recruited to surround the granulosa cells. As seen in Figure 1, the follicle continues to grow, soon forming an antrum, and resulting in the Graafian follicle (Biggers and Schuetz, 1972; Wassarman, 1991; Wassarman and

Kinloch, 1992).

Figure 1. Follicular development. The female germ cell, the oocyte, begins in a primordial follicle, which surrounds the oocyte with a single layer of follicle cells. After it is selected for development and ultimately ovulation, the follicle begins to grow. More layers of follicle cells are recruited, called the cumulus cells. An antrum soon develops within the follicle. The oocyte is fully grown and meiotically competent when the follicle reaches this Graafian stage.

Resumption of meiosis begins just before the oocyte is expelled from the ovary.

As the antrum expands, the oocyte becomes surrounded by two layers of granulosa cells

(cumulus cells), the innermost layer becoming columnar. This layer of cells is called the corona radiata. The oocyte reaches its fully grown size in the Graafian follicle and becomes meiotically competent, as well. Resumption of meiosis takes place prior to ovulation and is triggered by the

LH surge in vivo or by manual release from the follicle in vitro (Biggers and Schuetz, 1972;

Zuckerman and Weir, 1977; Jones, 1978; Austin and Short, 1982; Knobil and Neill, 1988;

Wassarman, 1991; Wassarman and Kinloch, 1992). Resumption of meiosis begins with nuclear envelop breakdown (germinal vesicle breakdown), followed by the formation of the first polar body, and arrest at metaphase II (Figure 2) (Jones, 2004; Mehlmann, 2005). This mature,

4 fertilizable egg only completes meiosis after fertilization by sperm, resulting in the formation of the zygote.

Figure 2. Mouse oocyte maturation. The left bright field image shows a mouse oocyte still enveloped in some cumulus cells at the equatorial plane. The intact germinal vesicle and nucleolus are visible in the center of the cell. The right bright field image shows a mature, fertilizable mouse egg with its first polar body at the equatorial plane. This cell has undergone germinal vesicle breakdown.

1.4 Oocyte Maturation

The process of oocyte maturation produces a mature, fertilizable egg. Immature mammalian oocytes are held in prophase I arrest by an inhibitory phosphorylation of Cyclin-

Dependent Kinase I (CDK1) by the activity of WEE1/MYT family of kinases, including oocyte- specific WEE2 (Mitra and Schultz, 1996; Kanatsu-Shinohara et al., 2000; Han et al., 2005; Oh et al., 2010). CDK1 and the regulatory cyclin B1 (CCNB1) make up the Maturation Promoting

Factor (MPF), which has been well characterized in mammalian and amphibian oocytes and somatic cells, as it is the cell cycle regulatory factor for both meiotic and mitotic cells (Masui,

2001; Kishimoto, 2003; Brunet and Maro, 2005; Ma and Poon, 2011). Signaling molecules produced in the oocyte and the surrounding somatic cells control protein phosphatases and kinases that, in turn, regulate the phosphorylation status and activity of CDK1.

Dephosphorylation of CDK1 by M-phase inducer phosphatase 2 (CDC25B) causes resumption of meiosis I. It has been suggested that phosphorylated CDC25B is bound to YWHA (14-3-3) proteins in the oocyte cytoplasm, sequestering it there in an inactive state (Duckworth et al.,

2002).

Elevated levels of cyclic adenosine monophosphate (cAMP) within the oocyte maintain meiotic arrest. Oocytes spontaneously resume meiosis when cAMP levels fall, either by removal of the oocyte from the ovary or by luteinizing hormone (LH) which causes reduced cAMP levels and protein kinase A (PKA) activity within follicles preparing for ovulation (Pincus and

Enzmann, 1935; Cho et al., 1974; Masciarelli et al., 2004; Vaccari et al., 2008). High concentrations of both cAMP and PKA in immature oocytes are maintained by constitutively active heterotrimeric G protein receptors linked to stimulatory G proteins that activate adenylyl cyclase (Mehlmann et al., 2002; Kalinowski et al., 2004; Mehlmann et al., 2004; Diluigi et al.,

2006). The phosphorylation, and therefore inactivation, of CDC25B is accomplished by PKA and possibly protein phosphatase 1 (PP1) and/or PP2 (Solc et al., 2010). Evidence suggests that

PKA also phosphorylates WEE2 to maintain prophase I arrest (Han et al., 2005). MPF is activated by reduced phosphorylated WEE2 and the dephosphorylation of CDK1 by CDC25 proteins (Nurse, 1990; Morgan, 1995; Malumbres and Barbacid, 2005). Genetic studies performed in mouse oocytes have shown that CDC25B, not CDC25C, is the primary

6 phosphatase regulating the dephosphorylation of CDK1 (Chen et al., 2001; Lincoln et al., 2002).

Thus, resumption of meiosis and maturation of oocytes is dependent upon the activation of

CDC25B by dephosphorylation and its translocation to the nucleus where it dephosphorylates

CDK1, triggering the activation of MPF (Figure 3).

Figure 3. Simplified diagram of proteins involved in oocyte maturation from arrest at prophase (left) to release from meiotic arrest (right). The left pathway, the phosphorylated and inactive Maturation Promoting Factor (MPF) and thus, the prophase arrested oocyte, shows the CDC25B phosphorylated, inactive, and bound to a YWHA (14-3-3) protein. The right pathway shows the active form of MPF, which releases the oocyte from meiotic arrest and allows it to mature to an egg. The CDC25B and YWHA (14-3-3) proteins have dissociated, the

CDC25B dephosphorylated, and able to then dephosphorylate MPF. This process, as well as dephosphorylation by WEE1, activates MPF.

1.5 Maternal to Embryonic Messenger RNA Transition

The oocyte is a very unique and special cell type because of its ability to store and thrive off of a finite supply of RNA molecules and proteins until early embryonic development. During oogenesis, oocyte cytoplasm undergoes maturation as well as the nuclear material to form a competent oocyte. This cytoplasmic maturation includes the storage of maternal mRNAs, proteins, and nutrients to support oocyte and embryonic development. Transcription and storage of mRNAs continues during folliculogenesis until the oocyte is fully grown. At that point, transcription ceases and the oocyte is dependent upon the stored supply of mRNA transcripts and proteins during meiosis I and II, as well as during fertilization and formation of the zygote

(reviewed in Bell et al., 2008). Species of mammals vary in the time it takes for the maternal message to be replaced by the activation of the transcriptional activity of the embryonic genome, a point called the maternal-embryonic transition. Mice undergo the maternal-embryonic transition as early as the 2-cell stage embryo. Species such as pigs and humans undergo the transition between the 4- and 8-cell stage, and rabbits, sheep, and bovine switch during the 8- and 16-cell stage embryos. Non-mammalians, such as Xenopus and Drosophila, don’t undergo this transition until very late in early embryonic development, after 12 rapid cleavages of the blastula and after the fourteenth interphase, respectively (reviewed in Gandolfi, 2000).

Total RNA for the fully grown oocyte has been calculated to be between 0.43 and 0.6 ng, while it falls between 0.3 and 0.55 ng for the mature, fertilizable mouse egg (reviewed in Peaston et al., 2010). Polyadenylated RNA was found to be 0.5 - 0.9 pg and 0.25 - 0.35 pg, respectively

(Bachvarova et al., 1985; Bachvarova, 1985). Shortly after fertilization, the maternal mRNAs are quickly turned over by being degraded or deadenylated, and protein levels fall (Brinster, 1967;

Bachvarova and Deleon, 1980; Piko and Clegg, 1982). Total RNA content declines to 0.24 ng in

8 the 2-cell stage embryo, with a sharp decrease in mRNA from 0.83 pg to 0.26 pg between the 1- and 2-cell stage embryo. After the large decrease in stored maternal message, embryonic genomic transcription begins and levels of RNA and mRNA spike in the developing embryo.

Total RNA increases to 0.69 ng and 1.47 ng in the 8- to 16-cell stage embryo and in early blastocysts. Messenger RNA also increases during these stages, from 0.44 pg to 1.42 pg (Piko and Clegg, 1982).

Even before the reduction in RNA after fertilization, there is a significant selective reduction in maternal transcripts as the GV-arrested oocyte continues meiosis to form a mature egg (Su et al., 2007; Peaston et al., 2010). Given the potentially important role of the YWHA proteins in oocyte maturation (see section 1.7 below) and early development, it will be important to evaluate the expression of the seven isoforms of YWHA in oocytes, eggs, and early embryos.

Determining if any of the isoforms are more stable during oocyte maturation will be useful knowledge to gain. It has been suggested that a number of transcripts associated with prophase I arrest are thought to be degraded during oocyte maturation, while those that may be required in the metaphase II egg are stable (Su et al., 2007).

1.6 Meiotic Spindle Formation

Spindles are responsible for chromosome segregation and alignment during meiotic events. They are built of microtubules composed of α/β-tubulin dimers (Nigg and Raff, 2009).

Oocytes’ meiotic spindles are assembled without centrosomes, unlike male gametes (Szollosi et al., 1972; Dumont and Desai, 2012). These spindles assemble from microtubule organizing centers (MTOCs) present in the prophase-arrested oocyte and increase in number after germinal vesicle breakdown (GVBD) (Schuh and Ellenberg, 2007). Proper spindle formation is essential

9 to oocyte development and meiosis, as the chromosomes must separate completely and in a timely manner to form the mature egg and polar bodies (Wassarman and Fujiwara, 1978; Vogt et al., 2008; Yin et al., 2008; Schatten and Sun, 2011; Dumont and Desai, 2012; Jones and Lane,

2012). Inability to form and align proper spindles or to separate chromosomes can result in aneuploidy, which is common in meiosis I and increases with maternal age (Pan et al., 2008;

Grondahl et al., 2010). Studies have shown that the YWHA proteins interact with α- and β- tubulin in both interphase and mitotic HeLa cells (Meek et al., 2004; Rubio et al., 2004). Recent data has shown that a specific YWHA protein, YWHAH, interacts with α-tubulin in both the metaphase I and II spindles and in the first polar body, and may be required for normal meiotic spindle formation (De and Kline, 2013).

1.7 YWHA Isoforms and Oocyte Maturation

14-3-3 proteins of the Tyrosine 3-Monooxygenase/Tryptophan 5-Monooxygenase

Activation protein family (YWHA) are highly conserved, homologous proteins encoded by separate genes. Seven mammalian isoforms exist: 14-3-3β (YWHAB), 14-3-3γ (YWHAG), 14-

3-3ε (YWHAE), 14-3-3ζ (YWHAZ), 14-3-3η (YWHAH), 14-3-3τ (YWHAQ), and 14-3-3σ

(SFN) (reviewed in Aitken, 2006). First identified during a systematic classification of brain proteins (Moore & Perez, 1967), these proteins were given the name “14-3-3” to denote the elution fraction for these proteins after DEAE-cellulose chromatography and their migration position after starch gel electrophoresis (reviewed in Aitken, 2006). YWHA proteins have been found to be central mediators varying cell signaling processes, including cell cycle regulation and apoptosis (Rubio et al., 2004; Aitken, 2006; Morrison, 2009; Freeman and Morrison, 2011).

There is evidence that these proteins are involved in regulating cell division in somatic cells

(Hermeking and Benzinger, 2006; Gardino and Yaffe, 2011); coordinate interactions between mitotic spindles and cytokinesis (Zhou et al., 2010); and are involved with mitotic apparatus in mammalian cells (Pietromonaco et al., 1996).

All seven YWHA protein isoforms have been detected in both immature oocytes and mature eggs by immunofluorescence microscopy, immunohistochemistry, and Western blotting.

Though not yet fully quantified, their individual protein expression levels were varied, as well as their distribution subcellularly. YWHAH was found to co-localize in the meiotic spindle region with α-tubulin (De et al., 2012). In situ proximity ligation assays (PLA) showed a binding interaction between YWHAH and α-tubulin at the single molecule level within mouse eggs matured in vivo. These assays employ specific antibodies that bind to the target proteins. A pair of secondary oligonucleotide-conjugated antibodies binds to the primary antibodies and, when in close proximity to one another, the DNA strands join through enzymatic ligation. This then creates a circular DNA molecule, which is amplified through rolling circle amplification. The amplified DNA is visualized with a fluorescent probe, revealing the interaction sites of the two target protein. None of the other six isoforms were found to accumulate at the meiotic spindle through this method. To further understand the role of YWHAH in spindle formation, its expression was knocked down via injection of antisense morpholino oligonucleotide against

YWHAH. After being allowed to mature and undergo GVBD, the reduction of YWHAH protein caused a significant decrease in the number of cells that formed a normal bipolar spindle (De and

Kline, 2013).

Dephosphorylation of CDK1 within the MPF complex by dephosphorylated CDC25B causes meiosis to resume, leading to GVBD and oocyte maturation. Prior to the initiation of oocyte maturation, it is thought that CDC25 is held in check by binding to YWHA proteins. To

11 further illuminate the role of YWHA, the interactions between all seven isoforms and CDC25B were studied to determine which was involved in regulating CDC25B and meiotic arrest in mouse oocytes. In situ PLA and co-immunoprecipitation experiments were utilized to examine the molecular interactions between each of the YWHA isoforms and CDC25B. All seven

YWHA isoforms were found to interact with CDC25B in oocytes and eggs in the PLA experiment. The co-immunoprecipitation results indicate that six of the seven isoforms interact with CDC25B within mouse oocytes and eggs (Figure 4). (Interaction of CDC25B with SFN

(14-3-3σ) was indicated by the in situ PLA but could not be confirmed in Western blots as the antibody used may not recognize the denatured antigen).

Figure 4. Immunopreciptation of CDC25B phosphatase and co-immunoprecipitation of

YWHA proteins with CDC25B. (A) CDC25B is detected by the goat anti-CDC25B antibody in

12 the positive control (PC; ovary lysate). CDC25B was immunoprecipitated from extracts of ovaries (Ov), oocytes (O), and eggs (E) with goat anti-CDC25B. (B) The Western blot membrane in A was stripped and reprobed for each YWHA isoform co-immunoprecipitated with

CDC25B, omitting SFN. All six YWHA isoforms appear to co-immunoprecipitate with

CDC25B in ovaries and oocytes as detected by prominent bands. Reduced band intensities were seen in eggs. (C) The blot was stripped and reprobed to confirm minimal loss of protein by repeated membrane stripping (De, 2014).

These experiments suggest that CDC25B can interact with all seven YWHA isoforms.

However, any one of the isoforms may play a more central role in holding CDC25B and therefore, meiotic resumption in arrest. Translation-blocking morpholino oligonucleotides specific for each isoform were employed to reduce protein expression in order to determine with isoforms are central in regulating CDC25B. Microinjection of the antisense mopholino oligonucleotides (MO) for each YWHA isoform was used to study the maintenance of meiotic arrest in GV-intact oocytes. It was found that 70% of oocytes injected with the YWHAH MO underwent GVBD, compared to 7.5% for controls (Figure 5). This isoform was the only one to show significant meiosis resumption compared to the controls, indicating that YWHAH is functionally important for maintaining the prophase I arrest in mouse oocytes. The reduction of this protein likely provided fewer binding sites for CDC25B. This would have allowed enough active CDC25B to trigger the activation of MPF and resumption of meiosis. Thus, the YWHAH protein may be the isoform that interacts with CDC25B phosphatase to sequester it in the cytoplasm, therefore inactivating it and inhibiting the release of the oocyte from meiotic arrest.

Figure 5. Microinjection of antisense morpholino oligonucleotides. An antisense morpholino oligonucleotide for each YWHA isoform was injected into GV-intact oocytes, and the percent that underwent GVBD was scored. YWHAH injection had the greatest number of GVBD stage oocytes, as well as the only statistically significant variation from the controls. Numbers indicate the number of cells examined and the asterisk indicates a significant difference for YWHAH compared to controls (De, 2014).

AIM

The aim of this project was to quantify the expression levels of each specific isoform of

YWHA by quantitative real-time PCR in order to further elucidate their importance during oocyte maturation in oocytes, eggs, and even in embryos. Transcripts of each isoform were expected to be seen, as indicated by previous protein expression experiments and through microarray studies of both mouse and human oocytes and eggs (Pan et al., 2008, Grondahl et al.,

2010; Grondahl et al., 2013). Investigating the YWHAH transcript level change during embryonic development may further our understanding of this protein’s importance in the developing embryo. Protein pull-down experiments were also conducted in order to confirm the binding of CDC25B to YWHAH in oocytes and eggs, to further our knowledge of their interactions and roles in oocyte maturation.

CHAPTER II

METHODS

Collection of oocytes and eggs

All mice were housed and used at Kent State University under an approved Institutional

Animal Care and Use Committee protocol following the National Research Council’s publication Guide for the Care and Use of Laboratory Animals. To extract a large number of oocytes or eggs from adult CD1 mice (2-3 months old), the females underwent superovulation before sacrifice. For egg extraction, 5 IU pregnant mare’s serum gonadotropin (PMSG) (G4877,

Sigma) was injected into the females to stimulate the follicles, followed by 5 IU human chorionic gonadotropin (hCG) (CG10, Sigma) to induce ovulation 48 hours later. The females were then sacrificed and their ovaries removed. The hCG was omitted for oocyte collection, with an injection of PMSG 36-48 hours before collection. The cells were removed from the females either through a break in the ampulla as for eggs, or through ovary punctures with a 26-gauge needle to rupture follicles for the oocytes. The cells were collected in Minimal Essential Medium

(MEM), containing 120 U/mL penicillin G (P4687, Sigma), 50 µL/mL streptomycin sulfate

(S1277, Sigma), 0.24 mM sodium pyruvate (P-4562, Sigma), 0.1% polyvinyl alcohol (P-8136,

Sigma), and buffered with 20 mM HEPES to pH 7.2. Threshold concentration (0.1 mg/mL) of dibutyryl cAMP (D0627, Sigma) was added to the MEM for oocyte collection to prevent

16 spontaneous oocyte maturation. The eggs were dissociated from their cumulus cells with

0.3mg/mL hyaluronidase (H4272, Sigma), and the zona pellucidae were removed for both oocytes and eggs using acid Tyrode’s solution (T1788, Sigma).

Collection of morulae and blastocysts

The embryos for the YWHAH qPCR analysis were generated through in vitro fertilization. 8-12 week old C57BL/6J females were superovulated as described above, and the eggs were collected. Sperm was collected from 3-6 month old C57BL/6J males. The cauda epididymides with vas deferentia were removed and placed in human tubal fluid (HTF) medium

(MR-070-D, EMD Millipore) after sacrifice. The cauda epididymides were slashed with a 26- gauge needle and the vas deferentia were squeezed with forceps to release the sperm. The tissues were then incubated at 37°C with 5% CO2 for 10 minutes, allowing for the remaining sperm to swim out into the media. After which time, the tissues were removed and the sperm was left to incubate for another 1-1.5 hours to allow for capacitation. 15 µL of the capacitated sperm was combined with the cumulus oocyte complexes from one female in a dish containing HTF medium and left to incubate at 37°C with 5% CO2 for 4 hours. The excess sperm were removed from the dish through 3 washes of clean HTF medium and the fertilized eggs were allowed to incubate overnight. The following day, the two-cell stage embryos were transferred to potassium simplex optimized medium (KSOM) (MR-121-D, EMD Millipore) and cultured. Three days after fertilization, the morulae were collected. Blastocysts were collected four days after fertilization.

Messenger RNA Isolation and cDNA Synthesis for YWHA Gene Expression

The oocytes, eggs, morulae, and blastocysts designated for mRNA isolation were stored in Lysis/Binding Buffer (100 mM Tris-HCl pH 7.5, 500 mM LiCl, 10 mM EDTA pH 8.0, 1%

LiDS, 5 mM DTT) acquired from the Dynabeads mRNA DIRECT Kit (61011, Ambion Life

Technologies) in 50 µL stocks of 25 cells, and stored at -80°C until use. Oocytes and eggs were taken from CD1 females, while morulae and blastocysts were created from mating C57BL/6J mice. Messenger RNA isolation was carried out using the Dynabeads kit. One mg of these magnetic oligo (dT)25 beads can bind up to 2 µg of mRNA. The cell lysates were incubated at room temperature for 3-5 minutes with 10 uL beads, which only bind the poly-A tails of mRNA.

After two washes each with two wash buffers, A (10 mM Tris-HCl pH 7.5, 0.15 M LiCl, 1 mM

EDTA, 0.1% LiDS) and B (10 mM Tris-HCl pH 7.5, 0.15 M LiCl, 1 mM EDTA), the supernatant was removed by placing the beads on a neodymium magnet (NB014, Applied

Magnets), allowing for easy removal of supernatant without removing the beads. The mRNA was then eluted from the beads with 10 mM Tris-HCl, pH 7.5. For samples designated for qPCR,

100 ng Luciferase Control RNA (L4561, Promega) was added to each 10 µL mRNA extraction prior to reverse transcription to be used as an internal control.

First strand complementary DNA was synthesized using the QuantiTect Reverse

Transcription Kit (205311, Qiagen). A step to eliminate any residual genomic DNA from the mRNA extracts was included before the reverse transcription. The template mRNA was then mixed with Reverse Transcriptase, oligo-dT and random primers, and RT buffer, following the manufacturer’s instructions. The mixture was incubated at 42°C to activate the Reverse

Transcriptase for 15 minutes, followed by a 3 minute incubation at 95°C to inactivate it.

Complementary DNA stocks were stored at -20°C until use.

Polymerase chain reaction and gel electrophoresis for YWHA gene expression

To confirm the presence of each YWHA isoform in both CD1 and C57BL/6J oocytes and eggs, RT-PCR was utilized. Primer pairs for each isoform were created using the Primer-BLAST program, and were designed according to NCBI published mouse mRNA. All primers were designed to span exon-exon junctions. 10 µL of GoTaq Green Master Mix (M712, Promega) was added to each reaction tube, as well as 0.2 µM of each primer (Table 1), 0.1 µL template cDNA, and water for a 20 µL reaction volume. The PCR protocol for YWHAQ, SFN, YWHAG, and

H2AfZ was as follows: denaturation (95°C for 3 min.), amplification repeated for 35 cycles

(94°C for 30 sec., 57°C for 45 sec., 72°C for 45 sec.), followed by a cooling step to 10°C. For

YWHAB, YWHAH, YWHAE, and YWHAZ, a gradient annealing temperature was used

(53/58.2°C). The PCR products were then run on a 2% agarose gel stained with GelRed Nucleic

Acid Stain (RGB-4103, Phenix) and visualized under exposure to UV light.

Table 1: Primers used to detect presence of each YWHA isoform in RT-PCR

Product Gene Forward Primer Reverse Primer Size YWHAB 5’-AACGATGTGCTGGAGCTGT-3’ 5’-CGGATGCAACTTCAGAAAGA-3’ 121 bp

YWHAE 5’-CAGAACTGGACACGCTGAGT-3’ 5’-TTCTGCTCTTCACCATCACC-3’ 118 bp

YWHAH 5’-CATGAAGGCGGTGACAGA-3’ 5’-TAACCCTCCAAGAAGATCGC-3’ 110 bp

YWHAG 5’-TCCTTCTTTCCAGCCGATCC-3’ 5’-GTTCAGCTCGGTCACGTTCTT-3’ 139 bp

YWHAQ 5’-CGGTGGCCTACAAAAACGTG-3’ 5’-ACAATTCCAGGACCGTGGTG-3’ 168 bp

YWHAZ 5’-CCAGCGACCACCCATTGT-3’ 5’-ACGATGACGTCAAACGCTTC-3’ 139 bp

SFN 5’-TGTGGCGAAGACTAGGAGGA-3’ 5’-GTCTCGAGAGTAACGCTGGG-3’ 134 bp

H2AfZ 5’-TGCAGCTTGCTATACGTGGA-3’ 5’-TCCTTTCTTCCCGATCAGCG-3’ 110 bp

Gene sequencing

To confirm the isolation of each YWHA isoform, each PCR product was cleaned and sent out for sequencing. The products were created following the PCR protocol as stated above.

Then, the PCR products were isolated from the residual PCR buffers and primers by DNA Clean and Concentrator -5 (D4003, Zymo Research) spin columns. 10-20 µL of the purified PCR DNA template was combined with 0.4 µM forward and reverse primers in separate tubes, and then shipped to Eurofins MWG Operon LLC for sequencing.

Quantitative polymerase chain reaction

To quantify the level of gene expression for each YWHA isoform during oocyte maturation, quantitative PCR was employed. It was accomplished using the SYBR Green

Jumpstart Taq ReadyMix (S4438, Sigma-Aldrich). All samples were run in triplicate.

Complementary DNA stocks created as described above with the Luciferase Control RNA were utilized. A baseline control is mandatory for qPCR and is usually an unchanging endogenous gene. However, because significant fluctuations in mRNA levels through oocyte maturation occur, we chose not to rely on an internal housekeeping gene. As stated previously, the

Luciferase control RNA spike-in was instead used as an internal control. Because the spike-in occurred before the reverse transcription reaction, the Luciferase data also confirms that the cDNA reactions, as well as the qPCR, were successful and consistent.

Primers for each isoform were designed using Primer-BLAST software, were designed to span exon-exon junctions, and to be between 100 and 200 bp. Primer pairs for YWHAB,

YWHAE, YWHAH, and YWHAZ were redesigned for the qPCR experiment to maintain consistent annealing temperatures for all isoforms, as well as for the Luciferase control RNA

(Table 2). The primer pairs for the other isoforms already had an annealing temperature of 57°C.

Initial RT-PCR products were examined via gel electrophoresis to confirm correct product size.

Each qPCR reaction was analyzed on the Qiagen Rotor-Gene Q.

Table 2 : Primers used to detect each YWHA isoform in qPCR

Product Gene Forward Primer Reverse Primer Size YWHAB 5’-AGTCCTCCGCGAAAATGAC-3’ 5’-CACGTTCTTGTAGGCAACAG-3’ 173 bp

YWHAE 5’ -GCCATTTTTCCTGCTCGGAC-3’ 5’-CCACCATTTCGTCGTATCGC-3’ 172 bp

YWHAH 5’-GAAGGCGGTGACAGAGCTGAA-3’ 5’-CGCCTGGCACCAACTACATT-3’ 90 bp

YWHAG 5’- TCCTTCTTTCCAGCCGATCC-3’ 5’-GTTCAGCTCGGTCACGTTCTT-3’ 139 bp

YWHAQ 5’- CGGTGGCCTACAAAAACGTG-3’ 5’-ACAATTCCAGGACCGTGGTG-3’ 168 bp

YWHAZ 5’- TTCTACGATCACGTCCAACC-3’ 5’-CGATGACGTCAAACGCTTC-3’ 196 bp

SFN 5’- TGTGGCGAAGACTAGGAGGA-3’ 5’-GTCTCGAGAGTAACGCTGGG-3’ 134 bp

Luciferase 5’- AGCGAAGGTTGTGGATCTGG-3’ 5’-GTGTTCGTCTTCGTCCCAGT-3’ 184 bp

The qPCR thermal cycling program was as follows for each isoform: denaturation (95°C for 5 min), amplification repeated for 35-45 cycles, depending upon the isoform, (57°C for 20 sec), and extension (72°C for 20 sec). During the extension step, fluorescence level was acquired. Melting points were also measured between 65° and 94°C, rising by 1°C each step.

Protein isolation for pull-down

To create the full ovary lysate, CD1 females were sacrificed, their ovaries removed and cleaned. The organs were then homogenized in lysis buffer, centrifuged, and the supernatant was separated from the pellet. The samples were then stored at -80°C until use. The oocytes and eggs

21 to be used for protein pull-down experiments were placed in lysis buffer (10 mM Tris-HCl pH

7.0, 1 mM EGTA, 1 mM EDTA pH 8.0, 0.1% β-mercaptoethanol, 1% Triton X-100, 1x MS-

SAFE Protease and Phosphatase Inhibitor (MSSAFE, Sigma)), centrifuged, and stored at -80°C in 50 µL stocks of 50 oocytes and 50 eggs. MagneGST glutathione particles (V8611, Promega) were incubated with Glutathione S-transferase (GST) – tagged human, recombinant YWHAH protein (BML-SE315, Enzo) for 1-3 hours at room temperature with continuous mixing. After washing with Binding and Wash Buffer (50 mM NaH2PO4, 140 mM NaCl), the particles with the bound YWHA proteins were then incubated overnight with the ovary, oocyte, or egg lysates.

Like the Dynabeads, these particles are also magnetic, so separation of supernatant from the beads was easily accomplished by placing the beads on a neodymium magnet and pipetting off the liquid. The bound YWHA and their prey proteins were eluted from the GST beads by

Laemmli buffer (300 mM Tris-HCl pH 7.0, 50% glycerol, 5% SDS, 0.05% bromophenol blue,

250 mM DTT). The samples were incubated in 95°C for 5 minutes to denature the proteins in preparation for SDS-PAGE.

SDS-PAGE and Western blot for protein pull-down

Mini PROTEAN TGX Precast 12% gels (456-1043, BioRad) were loaded with 10 µL of protein eluates in Laemmli buffer and electophoresed for 30 minutes at 200V. After a rinse in dH2O, the gel was placed in the transfer sandwich and the Western blot transfer was carried out at 100V for 30 minutes. After blocking non-specific binding sites with 5% nonfat dry milk in

Tris-buffered saline and Tween 20 (1x TTBS: 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 01%

Tween 20), the PVDF-Plus transfer membrane (GE Water and Process Technologies) was then incubated with rotation overnight at 4°C with rabbit anti-CDC25B primary antibody (10644,

Proteintech), diluted 1:200. After washing with TTBS, blots were incubated for one hour at room temperature with the goat anti-rabbit secondary antibody conjugated to horseradish peroxidase

(SC-2004, Santa Cruz), diluted 1:2000. The blots were washed with 1x TTBS twice for 15 minutes each, followed by 2 more washes for 5 minutes each. They were then developed with homemade ECL chemiluminescence preparation (20 µL H2O2 and 2 mL Luminol) using the

Fujifilm LAS-3000 luminescent image analyzer.

CHAPTER III

RESULTS

AIM

1. Do the levels of YWHA gene expression change during oocyte maturation?

Background and Rationale:

Previously, each of the seven YWHA isoform proteins have been found to be present in both immature oocytes and mature eggs by immunofluorescence microscopy, immunohistochemistry, and Western blotting. Though not yet fully quantified, their individual protein expression levels were varied, as well as their distribution subcellularly (De et al., 2012).

Messenger RNA microarray data indicate that at least six isoforms of YWHA are expressed in mouse oocytes (Pan et al., 2008), as well as in human oocytes (Grondahl et al., 2010; Grondahl et al., 2013). However, at least one contradictory paper has been published (Meng et al., 2013) stating that only YWHAB and YWHAE are present in GV-intact oocytes. To resolve this discrepancy, RT-PCR and qPCR were employed in order to confirm the presence of mRNA for each protein and to quantify the levels of gene expression for each of the seven YWHA isoforms in oocytes and eggs. To investigate how YWHAH protein levels change throughout embryonic

24 development, qPCR was also carried out on morulae and blastocysts. RT-PCR products were sequenced in order to confirm the amplification of the correct mRNA product.

1.1 Detecting YWHA isoform gene expression by RT-PCR

In order to support the previous findings that each isoform of YWHA is present in oocytes and eggs, RT-PCR was utilized. Each primer set was designed to span exon-exon junctions, to confirm that only cDNA created from mRNA was being detected. These primers were tested on brain and skin mRNA extracts to confirm competency. Messenger RNA was isolated from oocytes and eggs, and complementary DNA was created from oocytes and eggs from both CD1 and C57BL/6J mice. Using these strains, outbred and inbred, respectively, allowed us to compare and demonstrate the consistency of the YWHA proteins across two different mouse strains. These samples were analyzed via RT-PCR, the products electrophoresed, and imaged. As seen in Figure 6, each isoform is present in both oocytes and eggs for both strains except for SFN, which is only slightly detectable in the CD1 oocytes. H2A Histone

Family, Member Z (H2AfZ) was used as an endogenous positive control, and can be seen in each of the cell types.

Figure 6. Expression of YWHA isoform mRNA in oocytes and eggs of two mouse strains.

Messenger RNA, isolated from oocytes and eggs, was transcribed into first strand complementary DNA, and prepared for RT-PCR. The PCR reaction using isoform-specific

YWHA primers was repeated twice for each isoform and the products were loaded in adjacent lanes. The upper panel displays PCR products of oocyte and egg from outbred CD1 mice. The lower panel displays those same products for C57BL/6J mice. H2AfZ served as the internal positive control for this PCR. In all cases, no bands were seen in the negative control lanes consisting of all the PCR reactants save the template. Each isoform was analyzed at least twice by this method.

1.2 Sequencing YWHA isoform PCR products

To confirm that the PCR products were indeed the seven isoforms of YWHA genes, the samples were sent out to undergo Sanger sequencing. Each PCR product was cleaned of any

26 residual PCR reagents and primers, and then combined with the forward or the reverse isoform- specific primers. Eurofins MWG Operon carried out the sequencing. Each of the seven isoforms were sequenced and aligned to published mouse mRNA sequences (Figures 7 - 13), except for

SFN as the product yield was too low for accurate sequencing. Due to common sequencing inaccuracies at the beginning and end of sequences, 50-60% of the expected PCR sequence was seen for each isoform. For some of the isoforms, only one of the primers gave a reliable sequence, such as YWHAH, YWHAG, and YWHAQ (Figures 10, 13, 14). Even in the cases of the other sequences with sequences from both forward and reverse primers, only a partial sequence from the predicted product sequence was reported. It is common in routine sequencing that the first twenty or so bases of a sequence do not usually resolve due to chemical stabilization and sometimes primer dimers, and no clean-up protocol will result in a 100% yield. However, the sequence data base calls for each sample were matched with 100% accuracy to published

NCBI mouse mRNA RefSeq sequences.

Figure 7. H2AfZ (NM_016750.3) mRNA Sequence. The internal positive control for the PCR, the protein-coding H2A Histone Family, Member Z (H2AfZ), was sequenced along with the

27 seven YWHA isoforms. The forward and reverse primers are underlined. The highlighted PCR sequence was generated from both the forward and reverse primers. Of the 110 bp of the predicted PCR product, 56 bp were detected, with 100% base call accuracy to published mouse mRNA sequence.

Figure 8. YWHAH (NM_011738.2) mRNA Sequence. The data for the YWHAH isoform resulted in a 58 bp sequence out of the 110 bp-long predicted product with 100% base call accuracy matched to the published mouse mRNA data. This data was generated from the forward primer only; the reverse primer did not produce a clear sequence. The forward and reverse

28 primers are underlined, and the sequence results are highlighted. The exon-exon junction is indicated by the arrowed line.

Figure 9. YWHAB (NM_018753.6) mRNA Sequence. The results for YWHAB mRNA show a

70 bp sequence out of the 121 bp-long predicted product, with a 100% accurate base call match to published mouse mRNA data. The resulting sequence was generated from both the forward and reverse primers. The forward and reverse primers are underlined, and the sequence results are highlighted. The exon-exon junction is indicated by the arrowed line.

Figure 10. YWHAE (NM_009536.4) mRNA Sequence. The results for YWHAE mRNA show a 67 bp sequence out of the 118 bp-long predicted product, with 100% base call match to published mouse mRNA data. The resulting sequence was generated from both the forward and

30 reverse primers. The forward and reverse primers are underlined, and the sequence results are highlighted. The exon-exon junction is indicated by the arrowed line.

Figure 11. YWHAG (NM_018871.3) mRNA Sequence. The results for YWHAG mRNA displays an 82 bp sequence detected out of the 139 bp-long predicted product with a 100% base call match to published mouse mRNA data. The sequence was generated from the forward primer only; the reverse primer did not produce a clean sequence. The forward and reverse primers are underlined, and the sequence results are highlighted.

Figure 12. YWHAQ (NM_011739.3) mRNA Sequence. The results for YWHAQ mRNA show a 114 bp sequence detected out of the 168 bp-long predicted product with a 100% base call match to published mouse mRNA data. The sequence was generated from the forward primer only; the reverse primer did not produce a clean sequence. The forward and reverse primers are underlined, and the sequence results are highlighted. The exon-exon junction is indicated by the arrowed line.

Figure 13. YWHAZ (NM_011740.3) mRNA Sequence. The results for YWHAZ mRNA show an 80 bp sequence detected out of the 139 bp-long predicted product with a 100% base call match to published mouse mRNA data. The sequence was generated from both the forward and

33 reverse primers. The forward and reverse primers are underlined, and the sequence results are highlighted. The exon-exon junction is indicated by the arrowed line.

1.3 Measuring levels of gene expression through oocyte maturation by quantitative PCR

To determine the how the levels of mRNA change through oocyte maturation for each

YWHA isoform, qPCR was carried out on both oocytes and eggs. As described in the Methods, mRNA was isolated from pools of 25 oocytes and eggs and cDNA stocks were created from these. Luciferase spike-in RNA was used as an internal control, as a consistent, unchanging endogenous gene was not found through this maturation process (Salisbury et al., 2009). Primers were designed to span exon-exon junctions for each isoform to produce a product of 100-200 base pairs in length. Each isoform was examined thrice independently, with three technical replicates per cell type. Technical replicates outside of the accepted CT value difference were omitted. Such outlying values may be due to errors such as small pipetting errors or unaccounted for technical errors in the procedure. Each analysis was run with at least two accepted technical

-ΔΔ C replicates. Fold changes were calculated using the 2 T method (Figures 14 – 18). Student’s t – test was used to evaluate difference between the expression levels of oocytes and eggs (Table 3).

14-3-3 eta 1.6 1.4 1.2 1 0.8 Oocyte 0.6 Egg 0.4

FoldChange (RQ) 0.2 0 -0.2 1 2 3 -0.4

Figure 14. Fold changes between oocyte and egg for the YWHAH isoform. The fold changes in gene expression from oocyte to egg were calculated for each qPCR trial (1, 2, 3) for the

-ΔΔ C YWHAH isoform using the 2 T method. All CT values for eta were less than 33 and were less than 14 for Luciferase. Accepted technical replicates were within 1.02 CT difference. Error bars indicate standard error for each trial, calculated from the ΔCT averages of both the oocyte and egg samples. Amplification was carried out in 35 cycles for each trial. The oocyte values were set to 1.

14-3-3 epsilon 1.2

0.8

0.6 Oocyte Egg

0.4 FoldChange (RQ) 0.2

0 1 2 3

Figure 15. Fold changes between oocyte and egg for the YWHAE isoform. The fold changes in gene expression from oocyte to egg were calculated for each qPCR trial (1, 2, 3) for the

-ΔΔ C YWHAE isoform using the 2 T method. All CT values for epsilon were less than 30 and were less than 12 for Luciferase. Accepted technical replicates were within 0.51 CT difference. Error bars indicate standard error for each trial, calculated from the ΔCT averages of both the oocyte and egg samples. Amplification was carried out in 35 cycles for each trial. The oocyte values were set to 1.

14-3-3 gamma 1.4

1.2

0.8 Oocyte 0.6 Egg

FoldChange (RQ) 0.4

0.2

0 1 2 3

Figure 16. Fold changes between oocyte and egg for the YWHAG isoform. The fold changes in gene expression from oocyte to egg were calculated for each qPCR trial (1, 2, 3) for the

-ΔΔ C YWHAG isoform using the 2 T method. All CT values for gamma were less than 31 and were less than 14 for Luciferase. Accepted technical replicates were within 0.73 CT difference. Error bars indicate standard error for each trial, calculated from the ΔCT averages of both the oocyte and egg samples. Amplification was carried out in 35 cycles for each trial. The oocyte values were all set to 1.

14-3-3 theta 1.2

0.8

0.6 Oocyte

0.4 Egg

FoldChange (RQ) 0.2

0 1 2 3 -0.2

Figure 17. Fold changes between oocyte and egg for the YWHAQ isoform. The fold changes in gene expression from oocyte to egg were calculated for each qPCR trial (1, 2, 3) for the

-ΔΔ C YWHAQ isoform 2 T method. All CT values for theta were less than 30 and were less than 15 for Luciferase. Accepted technical replicates were within 0.67 CT difference. Error bars indicate standard error for each trial, calculated from the ΔCT averages of both the oocyte and egg samples. Amplification was carried out in 35 cycles for each trial. The oocyte values were set to

14-3-3 zeta 4.5 4 3.5 3 2.5 Oocyte 2 Egg 1.5

FoldChange (RQ) 1 0.5 0 -0.5 1 2 3

Figure 18. Fold changes between oocyte and egg for the YWHAZ isoform. The fold changes in gene expression from oocyte to egg were calculated for each qPCR trial (1, 2, 3) for the

-ΔΔ C YWHAZ isoform using the 2 T method. All CT values for zeta were less than 40 and were less than 12 for Luciferase. Accepted technical replicates were within 0.67 CT difference. Error bars indicate standard error for each trial, calculated from the ΔCT averages of both the oocyte and egg samples. Amplification was carried out in 45 cycles for each trial. The oocyte values were set to 1.

Table 3. Quantitative PCR average CT values for each YWHA isoform and Luciferase, ΔCT values, and ΔΔCT values. The table below displays average CT values for each YWHA isoform run for both cell types, as well as those values for the positive exogenous Luciferase control. ΔCT values were calculated from the difference between each isoform and Luciferase average CT values. The standard error of the mean was calculated for each of these, as well as the P-value using a Student’s t-test. ΔΔCT values were calculated from the difference between the isoform and Luciferase ΔCT values.

Isoform Cell Type Isoform Avg CT Luciferase Avg CT ΔCT SEM P-value ΔΔCT YWHAH Oocyte 24.66 10.31 14.35

24.52 12.02 12.50

27.44 9.90 17.54

Oocyte Average 25.54 10.74 14.80 1.47 0.00

Egg 27.75 12.06 15.69

29.95 13.37 16.58

32.90 10.48 22.42

Egg Average 30.20 11.97 18.23 2.11 0.253 3.428

YWHAE Oocyte 23.50 10.23 13.27

25.30 10.14 15.16

28.08 10.30 17.78

Oocyte Average 25.62 10.22 15.40 1.31 0.00

Egg 26.02 10.43 15.59

28.77 11.32 17.45

29.06 10.95 18.11

Egg Average 27.95 10.90 17.05 0.755 0.3367 1.64

YWHAG Oocyte 30.83 13.22 17.61

27.14 12.39 14.75

26.20 10.60 15.60

Oocyte Average 28.05 12.07 15.98 0.85 0.00

Egg 30.83 12.63 18.20

28.69 11.34 17.35

28.66 11.30 17.36

Egg Average 29.39 11.76 17.64 0.28 0.1385 1.65

YWHAQ Oocyte 22.63 9.34 13.29

22.22 9.64 12.58

24.86 8.97 15.90

Oocyte Average 23.24 9.31 13.92 1.01 0.00

Egg 28.09 14.55 13.54

28.36 10.52 17.84

29.74 10.51 19.23

Egg Average 28.73 11.86 16.87 1.71 0.2124 2.95

YWHAZ Oocyte 31.44 10.55 20.89

39.56 11.77 27.80

35.33 10.57 24.76

Oocyte Average 35.44 10.96 24.48 2.00 0.00

Egg 32.69 10.00 22.69

37.49 11.71 25.78

38.96 9.82 29.14

Egg Average 36.38 10.51 25.87 1.86 0.6385 1.39

SFN could not be analyzed perhaps due to low copy number. One run of 45 cycles produced CT values between 42 and 44. However, subsequent runs did not have any samples that crossed the threshold. YWHAB was also unable to be quantified using this method. CT values between technical replicates varied greatly, sometimes climbing above a difference of 8. The addition of 2% dimethyl sulfoxide (DMSO) added to the stability of the CT values and promoted primer binding to cDNA, but enough replicates were not able to be calculated for statistical analysis. Luciferase values remained consistent, as with other isoform assays, even with the addition of the DMSO.

To investigate the changing levels of transcript during embryonic genome activation, the

YWHAH isoform was also analyzed via qPCR using morulae and blastocysts. One trial was performed, with three technical replicates per cell type. Technical replicates outside of the accepted CT value difference were omitted. The Luciferase control RNA was used as the exogenous positive control. The fold change from morula to blastocyst was calculated using the same method as the other qPCR analyses and was compared to the average of the oocyte and egg

YWHAH trials (Figure 19). Average CT values, ΔCT values, and ΔΔCT values are displayed in

Table 4.

14-3-3 eta morula & blastocyst 1.2

1.15

1.1

1.05 Morula Blastocyst

1 FoldChnage (RQ) 0.95

0.9 1

Figure 19. Fold changes between morula and blastocyst for the YWHAH isoform. The fold changes in gene expression for each cell type were calculated for the YWHAH isoform using the

-ΔΔ C 2 T method. The morula value was set to 1. All accepted CT values for YWHAH were less than 33 and were less than 12 for Luciferase. Fold change values for oocyte and egg came from the average of all three trials. Accepted technical replicates were within 0.74 CT difference.

Amplification was carried out in 35 cycles.

Table 4. Quantitative PCR Average CT values for YWHAH isoform and Luciferase, ΔCT values, and ΔΔCT values calculated from morula and blastocyst. The table below displays average CT values for the YWHAH isoform run for morulae and blastocysts, as well as those values for the positive exogenous Luciferase control. ΔCT values were calculated from the difference between YWHAH and Luciferase average CT values. ΔΔCT values were calculated from the difference between the isoform and Luciferase ΔCT values.

Isoform Cell Type Isoform Avg CT Luciferase Avg CT ΔCT ΔΔCT YWHAH Morula 31.07 11.66 20.04 0.00

Blastocyst 30.84 11.04 19.80 -0.24

AIM

2. Is YWHAH a binding partner for CDC25B in ovary, oocytes, and eggs?

Background and Rationale:

Previous studies have shown that the YWHA proteins, specifically YWHAH, are key in maintaining meiotic arrest in the immature oocyte, as well as regulating meiotic spindle formation (De et al., 2012; De and Kline, 2013). Six of the seven YWHA isoforms have been found to co-localize with CDC25B, which dephosphorylates MPF, triggering meiotic resumption and oocyte maturation, through PLA and co-immunoprecipitation experiments. Additionally, germinal vesicle breakdown (GVBD) was seen at 70% for GV-intact oocytes injected with antisense morpholino oligonucleotides against YWHAH, compared to only 7.5% GVBD in controls. This isoform was the only one to show significant meiosis resumption compared to controls and other isoforms. The reduction of the YWHAH protein likely provided fewer binding sites for CDC25B, thus providing enough active CDC25B to trigger the activation of MPF and resumption of meiosis. These studies indicate that YWHAH is functionally important for meiotic spindle formation, as well as maintaining the prophase I arrest in mouse oocytes (De, 2014).

The goal of this experiment was to study YWHAH’s possible role in maintaining prophase I arrest in the immature oocyte by sequestering CDC25B and keeping it inactive by using protein pull-down experiments conducted on oocyte and egg lysates. This process isolates

45 any YWHAH binding partners and by applying antibodies against CDC25B, its presence on the blot can be confirmed.

2.1 Detecting the binding of CDC25B to YWHAH in oocytes and eggs through pull-down experiments

To conduct this experiment, first protein was isolated from ovary, oocyte, and egg lysates by use of glutathione particles with bound GST-tagged YWHAH protein. The lysates were then incubated with the particles and any prey proteins captured by the YWHAH protein were also bound to the beads until elution. The samples were denatured, separated via SDS-PAGE, and transferred through Western blotting. The membrane was then incubated with rabbit anti-

CDC25B primary antibody, followed by goat anti-rabbit HRP conjugated secondary antibody.

The blot was imaged on the Fujifilm LAS-3000 luminescent image analyzer. Oocyte and egg lysates incubated with YWHAH are shown in Figure 20.

Figure 20. Pull down of YWHAH and CDC25B proteins in oocyte and egg. Two bands were detected by the rabbit anti-CDC25B antibody in the positive control lane consisting of full ovary lysate (Ov). The higher molecular weight band (~73 kDa) may be the phosphorylated form of

CDC25B, the lower molecular weight band (~63 kDa) may be a dephosphorylated form. A nonspecific band at a higher molecular weight is also seen in this preparation. Potential YWHAH binding partners were pulled from the 100-cell oocyte and egg lysates by GST-tagged YWHAH protein bound to glutathione particles. CDC25B was detected on the blot by use of rabbit anti-

CDC25B antibody. The lanes which contain the proteins eluted from the glutathione beads from oocytes (Oo) and eggs (Egg) show two bands each, one at ~73 kDa and the other at ~63 kDa, suggesting that YWHAH can bind both a phosphorylated and dephosphorylated form of

CDC25B. No CDC25B was detected in the recombinant GST protein negative control lanes.

This experiment was repeated a second time with similar results.

SUMMARY

The main objective of this project was to confirm the presence of the seven YWHA isoform proteins in oocytes and eggs. The transcripts of each isoform were found to be present in oocytes and eggs from both CD1 and C57BL/6J mice, except for SFN. This was confirmed through RT-PCR as well as through Sanger DNA sequencing. Quantitative PCR also confirmed the presence of the mRNA in oocytes and eggs for six of the seven isoforms. YWHAH was also detected in morulae and blastocysts. As predicted, the relative levels of expression of these six proteins generally appeared to drop during oocyte maturation and increased during embryonic development for YWHAH. More definitive studies using total RNA isolations are needed to determine levels of these transcripts through oocyte maturation, as two of the seven isoforms were unable to be analyzed with confidence.

A second objective of this project was to confirm the interaction of YWHAH with

CDC25B through a protein pull-down experiment. Human YWHAE GST-tagged recombinant protein was pulled down with its binding partners from oocytes and eggs. Anti-CDC25B antibodies were then added to visualize the binding of this protein with YWHAH. Two bands, one at ~73 kDa and one at ~63 kDa were seen from both cell types. This suggests that YWHAH is capable of binding both the phosphorylated and dephosphorylated forms of CDC25B in mouse oocytes and eggs.

CHAPTER IV

DISCUSSION

The seven isoforms of the YWHA proteins have long been known to control various cellular processes, such as cell cycle control, apoptosis, and coordinating mitotic spindles

(Mackintosh, 2004; Aitken, 2006; Morrison, 2009; Zhou et al., 201b; Freeman and Morrison,

2011). Recent work has suggested that these proteins also play a major role in meiotic events, such as meiotic spindle formation and controlling meiotic arrest and release. Previously, all isoforms of the YWHA family have been found to be present in immature oocytes and mature eggs through Western blot, immunofluorescence microscopy, and immunohistochemistry (De and Kline, 2013). To investigate the potential role of the YWHA proteins in meiotic spindle formation, interactions were studied between each YWHA isoform and CDC25B, the phosphatase responsible for dephosphorylating CDK1, thus releasing the oocyte from prophase I arrest and activating the resumption of meiosis. All seven isoforms were found to interact with

CDC25B. Co-immunoprecipitation experiments also suggest that six of the seven isoforms bind and interact with CDC25B in oocytes and eggs. Injection of translation-blocking morpholino oligonucleotides into GV-intact oocytes found that a significant number of oocytes underwent

GVBD and resumption of meiosis when the YWHAH protein was knocked down (De, 2014).

Experiments to study the role of YWHA proteins in meiotic spindle formation have shown an interaction between YWHAH and α-tubulin through PLA assays, as well as a

49 localization of YWHAH at the MI spindle by immunofluorescence. None of the other isoforms were found to localize in this region. Interruption of YWHAH translation by antisense morpholino injection into GV-intact oocytes also caused deformed or missing spindles (De and

Kline, 2013). These experiments, as well as the MO injections, suggest that the YWHAH isoform protein is key in controlling meiotic spindle formation in oocytes and in controlling meiotic arrest. To further understand the importance of the YWHA isoforms, and specifically the

YWHAH isoform, we proposed an investigation of the levels of expression of each isoform during oocyte maturation and into embryonic development.

A contradictory paper from Meng et al., stated that only two isoforms, YWHAE and

YWHAB, were present in GV-intact oocytes, although mRNA microarray data indicate that at least 6 isoforms of YWHA are expressed in mouse oocytes (Pan et al., 2008). SFN was not reported in this article. This current project was designed to resolve this discrepancy using RT-

PCR and quantitative PCR. Expression of the seven YWHA isoform proteins was determined through RT-PCR in both oocytes and eggs, for two strains of mice. By using an inbred and an outbred strain, C57BL/6J and CD1 respectively, the conservation of the genes was also confirmed. Each isoform was detected in each cell type for both mouse strains. However, SFN was only detected in a very faint band in oocytes from the outbred strain. Previously, SFN proved difficult to identify in mouse oocytes and eggs by Western blot techniques and co- immunoprecipitation. Uncertainty in levels of protein and of mRNA for SFN, as well as its role in meiotic cell has made it difficult to work with.

SFN, primarily found in epithelial cells, is unique compared to the other YWHA isoforms. It varies in structure, with unique amino acids (Met202, Asp204, and His206) that are not found in the others. SFN is able to regulate cell cycle in somatic cells. After DNA damage

50 occurs, SFN is induced by the p53 tumor suppressor protein, resulting in a G2 block. SFN accomplishes this negative regulation by binging and sequestering CDC2 and CDK2 complexes in the cytoplasm (Hermeking et al., 1997; Lee and Lozano, 2006). In CD1 oocytes, it is possible that SFN also binds cyclin-CDK complexes to maintain meiotic arrest. However, this may be secondary to the major component of prophase I arrest, YWHAH, which would explain its very low expression levels.

To confirm the presence of each YWHA isoform within oocytes and eggs, PCR products were cleaned and sent out for sequencing. Six of the seven isoforms’ identities were confirmed when compared to published NCBI mouse mRNA data. SFN was not able to be sequenced as the level of PCR product was too low to be detected in the sequencing assay. These findings support the microarray data from Pan et al., as well as previous findings on expression of the YWHA proteins (De et al., 2012; De and Kline, 2013). Each reported YWHA isoform sequence showed a product of about 50-60% of the predicted product. However, each PCR product base call was matched to published mRNA sequences with 100% accuracy, indicating that the products were sequenced with high accuracy. We can say with certainty that these products are indeed the

YWHA isoforms described.

During oogenesis, oocytes stockpile and store large amounts of maternal RNA that is its sole source of template during the interval of transcriptional silence beginning after growth completion until the maternal-embryonic transition. During this period, much of the maternal

RNA is degraded or deadenylated. Large-scale destruction of mRNA also occurs during oocyte maturation. Levels of polyadenylated mRNA decrease from about 85 pg to 35 pg., while total

RNA levels decrease by about 30% (reviewed in Bell et al., 2008 & Gandolfi, 2000). A study by

Su et al., in 2006 concluded through microarray assays that, in general, transcripts involved in

51 processes associated with prophase I arrest and oocyte maturation, such as protein synthesis, oxidative phosphorylation, and energy production were largely degraded. Transcripts involved in pathways essential to maintaining meiotic arrest at MII, such as the ERK/MAPK pathway, were found to be stable during oocyte maturation.

In order to determine if any of the YWHA isoform transcripts were stable during oocyte maturation, quantitative RT-PCR was employed to determine the relative levels of gene expression. Each isoform has been detected in both oocytes and eggs through various methods; however their mRNA levels were never measured. Following qPCR, the fold changes for each

-ΔΔ C isoform were calculated using the 2 T method (Livak and Schmittgen, 2001). Each isoform exhibited a general drop in average mRNA levels from oocyte to egg over three trials. YWHAH mRNA from C57BL/6J mice was also detected in morulae and blastocysts, increasing in expression through embryonic development. These findings support the previous studies that once the embryonic genome begins transcription after 2-cell stage, levels of mRNA increase dramatically in the embryo. SFN and YWHAB were unable to be analyzed, however. SFN mRNA levels were already shown to be too low to be detected in oocytes and eggs of C57BL/6J mice, and in eggs of CD1, with very low levels in oocytes (Figure 6). This was also the case when sequencing was attempted on the PCR products from the oocytes of CD1 mice. Definitive data for YWHAB proved difficult to acquire because of very inconsistent CT values, though transcript levels were detected for both oocytes and eggs.

This data supports the previous data that at least six of the seven isoforms are present in both mouse oocytes and eggs, as well as in human (Pan et al., 2008; Grondahl et al., 2010;

Grondahl et al., 2013). These findings provide a basis for further experimentation to study the roles of YWHA proteins in oocyte maturation. Because of the major degradation and

52 deadenylation events occurring during this period, poly-A mRNA isolation may not be the best method of mRNA extraction. The Dynabead Oligo (dT)25 beads that were used in this study only captured mRNA via its poly-A tail. Complementary DNA stocks could be better created from total RNA isolations to include truncated poly-A mRNA, for example.

Previous studies have suggested that YWHA proteins, specifically YWHAH, bind and sequester CDC25B in the cytoplasm of GV-intact oocytes. This maintains prophase I arrest, as

CDC25B cannot dephosphorylate CDK1 and activate MPF to resume meiosis. Co- immunoprecipitation experiments have shown that six of the seven YWHA isoforms interact with CDC25B. To confirm that YWHAH does indeed bind and interact with CDC25B in oocytes and eggs, protein pull-down experiments were conducted using human YWHAH recombinant

GST-tagged protein. The YWHAH protein is very conserved between human and mouse, with only two different amino acids at position 164 and 102, Gln to His and Ser to Ala in human to mouse, respectively (Toyooka et al., 2002). Two bands were seen for CDC25B in ovary, oocyte, and egg lysates, detected by rabbit anti-CDC25B antibody (Figure 20). These two bands, one at

~73 kDa and one at ~63 kDa, suggest that YWHAH is capable of binding a phosphorylated and a dephosphorylated form of CDC25B. However, for both cell types, the ~63 kDa band was much stronger. This may mean that YWHAH is better able to bind the dephosphorylated form of

CDC25B, but this finding requires additional examination. These findings confirm the previous findings that YWHAH does indeed bind to and interact with CDC25B in immature oocytes and mature eggs. Further studies pulling down the other isoforms of YWHA with CDC25B may also further elucidate the interactions between these proteins and the phosphorylation status of

CDC25B during binding.

The goal of this project was to further our understanding of the YWHA proteins through mouse oocyte maturation. These results confirm the presence of these isoforms in immature oocytes and mature eggs for two different strains of mice. A greater understanding of the relative expression of the transcripts for each isoform has also been gained. Binding and interaction of

CDC25B and YWHAH has also been confirmed through this study. Overall, this project has increased the bank of knowledge for these proteins and has set a greater base for further studies.

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APPENDIX OF ABBREVIATIONS

Ala: Alanine Asp: Aspartic Acid cAMP: Cyclic Adenosine Monophosphate CCNB1: Cyclin B1 CDC25B/C: Cell Division Cycle 25 homolog B/C CDK1: Cyclin-Dependent Kinase 1 DEAE: Diethylaminoethanol DMSO: Dimethyl sulfoxide DTT: Dithiothreitol ECL: Enhanced Chemiluminescence EDTA: Ethylenediaminetetraacetic acid EGTA: Ethylene glycol tetraacetic acid ERK/MAPK: Extracellular Signal-Regulated Kinases/Mitogen-Activated Protein Kinases Gln: Glutamine GST: Glutathion S-transferase GV: Germinal Vesicle GVBD: Germinal Vesicle Breakdown H2AfZ: H2A Histone Family, Member Z hCG: Human Chorionic Gonadotropin HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid His: Histidine HRP: Horseradish Peroxidase HTF: Human Tubal Fluid KSOM: Potassium Simplex Optimized Medium LH: Lutenizing Hormone LiCl: Lithium Chloride LiDS:

MEM: Minimal Essential Medium Met: Methionine MI: Meiosis I MII: Meiosis II MO: Morpholino Oligonucleotide MPF: Maturation Promoting Factor MTOC: Microtubule Organizing Complex NaCl: Sodium chloride

NaH2PO4: Monosodium phosphate NCBI: National Center for Biotechnology Information PKA: Protein Kinase A PLA: Proximity Ligation Assay PMSG: Pregnant Mare Serum Gonadotropin PP1/2: Protein Phosphatase 1/2 PRKACA: cAMP-Dependent Protein Kinase Catalytic Subunit Alpha PVDF: Polyvinylidene Fluoride qPCR: Quantitative Polymerase Chain Reaction RT-PCR: Real-time Polymerase Chain Reaction SDS: Sodium Dodecyl Sulfate SDS-PAGE: Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis Ser: Serine SFN: Stratifin (14-3-3σ) TBS: Tris Buffered Saline Tris-HCl: Tris-hydrochloride TTBS: Tween Tris Buffered Saline YWHA: Tyrosine 3-Monooxygenase/Tryptophan 5-Monooxygenase Activation Protein