Are Full-Length mRNA In Bos taurus Spermatozoa Transferred to the Oocyte During Fertilization?

This thesis focuses on the discovery of full-length mRNA transcripts in Bos taurus spermatozoa. The primary aim of this study is to id entify and validate fulllength mRNA primarily from RNA-Sequencing of bovine spermatozoa. The secondary aim is to determine if full-length sperma tozoal transcripts are delivered to the oocyte at fertlilization, allowing for future s tudies to track their inheritance from paternal sources to the embryo. The main hypothesis of this thesis is that full-length mRNA transcripts exist within the spermatozoal tran script profile in Bos taurus. The secondary hypothesis is that if spermatozoal mRNA i s functional after fertilization, then full-length transcripts should be present in t he early stage embryo. To examine these hypotheses, this thesis is divided into three main chapters. The first is a literature review, discussing the pr ocess of spermatogenesis, the unique properties of spermatozoal mRNAs, including some hypothesized functions of spermatozoal mRNAs. A summary of a new technique, R NA-Sequencing, will be discussed in this review as well as comparisons to previous literature techniques for identifying mRNA transcripts of interest. The second chapter is the manuscript published in t he journal Biology of Reproduction in January 2013, co-first-authored by Christopher Card. This manuscript uses the technique RNA-Seq to examine the transcrip t profile of nine Bos taurus bulls, and highlights several transcripts of interest for further study. This study found 6,166 total transcripts, and performed Gene Ontology anal ysis of the transcripts to categorize them into functional categories for further examina tion, the top most category of interest being translation. The third chapter of this thesis is a manuscript in preparation, formatted for submission to the journal of Molecular Reproduction and Development. This manuscript evaluates twenty four target mRNA transc ripts to see if they are fulllength. These transcripts were identified through f our main methods: their location on the Y chromosome, their high expression in the RNASeq data set from chapter 2, their presence in Gene Ontology categories of inter es f om chapter 2, and their discovery from previous literature studies. Sixteen tra scripts are found to be fulllength, eight are degraded, and four have alternati ve polyadenylation ends. In conclusion, several full-length transcripts were found in this study, which have the potential to create functional proteins do wnstream in the fertilized oocyte. Several transcripts were also proved to be degraded in the mature spermatozoa. This has confirmed the need for this type of study, and elucidates new transcript targets for further research to pursue.

x LIST OF TABLES CHAPTER 2:     Table 3 Sequencing of bovine sperm transcripts where base pairs matched the predicted transcript using primers in Table 1 Table 4 Transcript presence in testis, sperm, oocyte and embryos from published microarray studies. Y=transcript present, N= transcript absent, M=results inconclusive.
Oocyte & Embryo microarray data are from Kocabas et al. 2006      spermatozoa is presented here, as well as several hypotheses as to the functions that these spermatozoal mRNAs might serve. Following discussion of the spermatozoal RNAs, this review will examine a new method for identifying RNA transcripts of interest, called RNA-Seq, which was used for the Chapter 2 Biology of Reproduction publication. This will include a comparsion with previous methods for identifying RNAs. This review will conclude with the hypotheses and aims of this study.

II. Gametogenesis
Both males and females create specialized reproductive cells through the process of gametogenesis. Many mechanisms are shared between spermatogenesis (the generation of male spermatozoa) and oogenesis (generation of female oocytes). Despite these basic similarities between spermatogenesis and oogenesis, the two processes achieve their reproductive goals through two very different methods.
The main difference is that only males have a self-renewing system, which enables them to create much greater numbers of gametes compared to females (Holstein et al. 2003), discussed below. Unlike spermatozoa, the stem cells that create oocytes are incapable of self-renewing post-natally (Kocabas et al. 2006). For an oocyte to mature, they have to halt cell death, activate maternal transcription, unpack paternal DNA, and then kick-start embryo development (Potireddy et al. 2006). Once the spermatozoa fuse with the oocyte, the spermatozoa may be able to assist the oocyte with some of these functions. The maternal contribution produces a limited number of oocytes, but invests more energy into the production of oocytes with large biomass (Hayward and Gillooly 2011). In contrast, the paternal strategy focuses on the production of massive quantities of spermatozoa.

II. A. Spermatogenesis
Spermatogenesis is the process that spermatogonial stem cells undergo in the testis to mature into functional spermatozoa which is supported by Sertoli nurse cells 3 make large quantities of spermatozoa, males use a self-renewing stem cell system to allow for production of spermatozoa from sexual maturity up until death. To this end, spermatozoa develop from a group of stem cells in the testis, which are capable of self-renewal through continuous mitosis (Holstein et al. 2003). The developing germ cells then enter meiosis I and II, now called spermatocytes, and, after a final homologous recombination event, become haploid round spermatids (Iguchi et al. 2006).
During the early round spermatid stage of the second meiosis, a significant increase of transcription and translation also occurs, depositing all the mRNAs the mature spermatozoa will maintain (Braun 2000;Eddy 2002;Holstein et al. 2003).
From this point forward in spermatogenesis, the spermatids become transcriptionally silent and cease making mRNAs, although new proteins are later produced for the morphological changes necessary for mature spermatozoa formation. Several transcripts that remain in round spermatid are modified through post-transcriptional mechanisms in the 5' and 3' untranslated regions to hold them in an inactive state for later translation (Braun 1998). This is regulated by accessory proteins such as Tarbp2 (Braun 2000), and is known to be used on the transcripts PRM1 and PRM2 (Mali et al. Throughout the process of spermatogenesis, several unique mechanisms are utilized to regulate temporal and quality control of the spermatozoal mRNAs. Transcriptional silencing, discussed above, is one of the standard mechanisms used to regulate timing of mRNA transcript use during spermatogenesis (Braun 2000). Around the same time that selective mRNAs are being silence, the developing spermatozoa also performs DNA-repair and apoptosis to ensure quality of the mature spermatozoa (Smirnova et al. 2006). Prior to nuclear condensation ubiquitin-mediated proteolysis removes selective proteins from the cell, is responsible for replacing histones with sperm-specific protamines (Sutovsky 2003), and degrades selective mRNAs in the developing embryo (Thompson et al. 2003). The exact percentage of spermatozoa mRNAs that are degraded is unknown. When these regulatory mechanisms malfunction, the spermatozoal quality and fertility decrease (Foote 2003).

III. A. Composition
The focus of this thesis is investigating the existence of full-length transcripts in bovine spermatozoa thus providing more evidence that spermatozoa mRNAs may be functional. Spermatozoa carry not only paternal DNA, but also paternal RNAs to 6 the oocyte at fertilization, but no previous studies have examined whether these mRNAs are full-length, a prerequisite for functionality as a protein. Finding the functionality of these transcripts is particularly interesting since spermatozoa are translationally silent at maturity, indicating that they are incapable of utilizing these mRNAs themselves (Miller and Ostermeier 2006

III. B. 2. c. Oocyte meiotic division
One way that spermatozoal mRNAs may impact embryogenesis is through control of the cell cycle, such as preventing or promoting the timing of cell divisions.
For example, the microRNA-34c has been demonstrated to be responsible for the first cleavage of the embryo in mice. MicroRNA-34c is also known to be carried by the spermatozoa rather than the oocyte ). This demonstrates that at a basic level, spermatozoa determine the timing of development.
Other transcripts directly regulate the cell cycle, such as CKS2 that is involved in the MI anaphase transition in the cell cycle. Mutations present in this gene are responsible for sterility in both men and women (Donovan and Reed 2003). Cell cycle regulation is very important in early embryo development because it helps to determine when the embryo will overtake its own gene expression and cease using paternal or maternal sources of mRNAs and proteins (Hecht et al. 2009).

III. B. 2. d. Embryo imprinting
In reproduction, an eternal arms race exists between which copy of a gene will be used by the embryo: the maternal copy or the paternal copy? A large portion of this is controlled by a process called imprinting, which marks which copy of the allele to use by methylating the unused gene copy . This process is thought to be partially controlled by spermatozoal antisense RNAs, which act to maintain and protect the paternal copies of genes from degradation mechanisms as they enter the oocyte. The RNAs are hypothesized to work through the formation of a transcriptional silencing complex, which tags paternal DNA for imprinting in the oocyte (Miller and Ostermeier 2006).

III. B. 2. e. Epigenetic influences
There are many different ways that spermatozoal mRNAs might help the earliest stages of fertilization to establish and maintain the paternal genome . This touches on the idea of selfish genes: that the mRNAs of the father may be acting to further paternal interests, while maternal mRNAs compete against them.

13
This may also explain why certain transcripts are expressed highly and selectively in spermatozoa versus oocytes (Kleene 2005).
An expanding field of interest as to the mechanism of these changes is the field of epigenetics. Epigenetic changes are changes in the genetics or phenotype resulting from modifications other than to the underlying DNA. This is to say that epigenetics are post-processing modifications made to DNA, mRNAs, and proteins that affect areas other than the DNA coding (see Figure 2 below Another spermatozoal transcript that is delivered to the oocyte at fertilization is DDX3Y. DDX3Y is a DEAD-box RNA helicase, and is one of 33 total genes found on the Y chromosome (Marshall Graves 2000), which also has an X chromosome homolog (Vong et al. 2006). The X homolog of DDX3Y also shares in similar functions, but it has been demonstrated that when the X-encoded isoform is mutated, that DDX3Y is capable of rescuing some of the functionality for the embryo (Sekiguchi et al. 2004). DDX3Y is located in a known azoospermia region on spermatozoa, a region known for causing infertility in the spermatozoa when damaged

III. B. 3. Spermatozoal mRNA use as a fertility assay
Assessing fertility of spermatozoa has been limited to tests of morphology, As previously discussed, many of the mRNAs in spermatozoa are degraded or will be degraded rapidly in the oocyte. However, even incomplete mRNA transcripts may be used as a snapshot of fertility, and also as a predictor of infertility ( knockouts fail to express any morphological or motility abnormalities, so they would not be detected using standard fertility assays (Yamagata et al. 2002).

IV. Full-Length mRNA Transcripts
While some specific spermatozoal transcripts have been identified, functionally depends on the presence of intact, full-length transcripts that can be translated into proteins either in the spermatozoa or in the early stage embryo.
Spermatozoal transcript profile methods, primarily microarray studies, have so far only identified the presence of transcripts but have not designated between whether the mRNA transcripts are full-length or degraded remnants of spermatogenesis. If spermatozoal mRNA is functional after fertilization, then full-length transcripts should exist within the spermatozoa transcript profile.

VI. Hypotheses
1. Full-length transcripts exist within the spermatozoal transcript profile.
2. If spermatozoal mRNA is functional after fertilization, then full-length transcripts will be found in the early embryo.

VII. Aims
The primary aim of this study is to identify and validate full-length mRNA primarily from RNA-Sequencing of bovine spermatozoa. The secondary aim is to determine if full-length spermatozoal transcripts are delivered to the oocyte at fertlilization, allowing for future studies to track their inheritance from paternal sources to the embryo.

VIII. References
Ameur represented. This is the first report of the spermatozoal transcript profile in any species 31 using high-throughput sequencing, supporting the presence of mRNA in spermatozoa for further functional and fertility studies.

Introduction
In addition to delivering the paternal genome to the oocyte at fertilization, ejaculated spermatozoa retain a pool of RNAs, containing mRNAs, rRNAs and short non-coding RNAs [1][2][3][4]. Spermatozoal antisense RNAs epigenetically regulate early embryonic development and have a structural role in maintaining histone-bound spermatozoa chromosomal regions [3][4][5][6]. Although the complete spermatozoal mRNA profile is not known, spermatozoa contain at least 3,000-7,000 mRNAs with predominantly short fragments, probably indicative of a predominance of degraded RNA [7][8][9]. Individual spermatozoal transcripts that have been identified include mRNAs for ribosomal proteins, mitochondrial proteins, protamines, and proteins involved in signal transduction and cell proliferation [7][8][9][10][11][12]. The hypothesized function of the spermatozoal transcripts in transcriptionally-silent spermatozoa is currently unknown although spermatozoa-derived mRNAs, including PRM1, PRM2, PSG-1, CLU, HLA-E, DBY and PLCZ1, can be detected in embryos post-fertilization suggesting a role for spermatozoal mRNAs in the zygote [13][14][15][16][17][18]. However, only translation of PLCZ1 has been demonstrated in embryos and many of these spermatozoal transcripts are rapidly degraded in the embryo rendering them nonfunctional [15][16][17][18]. Some spermatozoal transcripts may be translated in the mitochondria during capacitation [19]. Additionally, the diagnostic potential of the total spermatozoal RNA population as a snapshot of spermatogenic gene expression is 32 emerging. Individual transcripts are stably regulated within and between individual males and perturbation of the ubiquitin-proteosome pathway during spermatogenesis can be detected in spermatozoal RNA making this a promising area for male fertility assay development [20][21][22][23].
The bovine spermatozoal transcript profile remains incomplete because previous studies have relied on hybridization-based techniques, which evaluate a limited pool of transcripts and do not provide information about full-length transcripts [7,9,10,24,25]. In contrast, RNA-Sequencing (RNA-Seq), based on high-throughput sequencing technology, is revolutionizing our understanding of transcriptomics by enabling sequencing of complete transcript profiles, including full-length mRNAs and identifying novel splicing junctions and exons [26,27]. Also unique to this direct sequencing, absolute quantification of a broad range of expression levels across transcripts can be obtained. High-throughput sequencing of the total RNA in human spermatozoa has focused on rRNA and small non-coding RNA populations but the complete mRNA profile has not been reported [2,4].
We hypothesize that the transcript profile of cryopreserved bovine spermatozoa can be directly sequenced using RNA-Seq. Over 6000 spermatozoal transcripts were sequenced with this approach and a heterogeneous population of degraded and fulllength mRNAs was identified. Previously reported spermatozoal transcripts were confirmed while a number of transcripts not previously found in spermatozoa of any species have also been identified including HMGB4, GTSF1, and CKS2. This is the first study to date to utilize RNA-Seq to sequence the spermatozoal mRNA population and report full-length transcripts for any species. 33

Spermatozoa Samples
Cryopreserved spermatozoa from Holstein bulls with conception rate (CR) scores ranging from -2.9 to 3.5 were obtained from Genex Cooperative Inc. (Shawano, WI).
Spermatozoa from nine bulls (-2.9 to 3.5 CR) was used to generate the amplified cDNA pool for RNA-Seq, for qPCR validation and PCR amplification of the 5' and 3' exons. Spermatozoal RNA from nine additional bulls was also converted to cDNA   Transcripts were analyzed in two different populations: FPKM>0 and FPKM>100.

Bovine spermatozoal RNA purity
Using the Trizol method, the total amount of RNA isolated from two spermatozoa straws from an individual bull resulted in an average of 31 fg RNA per spermatozoa.
Bioanalyzer analysis of the spermatozoa RNA population shows a peak of smaller RNAs and a lack of 18S and 28S ribosomal RNA peaks present in testis RNA ( Figure   1A). The spermatozoal RNA was free of leukocytes, testicular germ cells and  Table   2.
A heterogeneous population of degraded and full-length transcripts was identified.
Degraded transcripts (lacking reads mapping to all exons) were more prevalent below  Table 1). Some of these full-length transcripts also included intronic reads that potentially represent novel exons. Retention of the 5'and 3' exons for PLCZ1, CRISP2, and GSTM3 were validated while many transcripts with FPKM<100 did not retain the 5' exon, including DDX3Y ( Figure 3A). The presence of full-length transcripts for GSTM3 and GSTF1 was confirmed by PCR amplification of the intact transcript from the first to last exon in unamplified cDNA ( Figure 3B). A preliminary survey of the bovine spermatozoal transcript profile for previously reported spermatozoal RNA candidates identified several transcripts in bovine, human, porcine and mouse (Table 3). These transcripts represented a wide range of FPKM levels, and nine of these transcripts retained the 5' and 3' exons, potentially indicating that these transcripts are also full-length (Table 3).

Discussion
Here, we report the first cryopreserved bovine spermatozoal transcript profile using RNA-Seq, which includes degraded and full-length nuclear-encoded transcripts and mitochondrial-encoded RNA. The dynamic range of RNA-Seq allows for accurate identification and quantification of transcripts present at very low and high levels as well as the discovery of more transcripts, novel splicing junctions and novel exons than reported in previous microarray studies [7,9,10]. In addition to the identification of transcripts not previously reported in spermatozoal RNA, several known spermatozoal transcripts from a number of different species were also found. Gene ontology analysis of the highly abundant spermatozoal transcripts (FPKM>100) revealed that translation was the most predominant biological process represented.
The presence of full-length transcripts in transcriptionally-silent spermatozoa suggests that these transcripts could be translated after spermatogenesis is complete, potentially contributing to capacitation and early embryogenesis [1,3]. In this study, RNA was isolated from the whole cryopreserved semen straw, after a wash to remove the cryoprotectant, without the removal of non-motile spermatozoa.
Using the entire spermatozoa population is representative of the natural transcript variation present across a range of fertility scores for bulls used in artificial insemination and is consistent with the approach used in other studies [12,21,24,34].
The focus of this study was to enrich for and sequence the polyA + transcripts present in transcriptionally-silent spermatozoa. The mitochondrial-encoded rRNAs and mRNAs sequenced in this population were some of the most abundant transcripts although these mitochondrial RNAs represented only 0.5% of the total transcripts.
Mitochondrial rRNAs and mRNAs have been previously amplified in spermatozoa [10,19] and the presence of these transcripts is likely due to intact mitochondria present during the RNA isolation procedure and the high mitochondrial activity of spermatozoa. Poly(A-) transcripts and microRNAs were not evaluated in this study but probably present in the total bovine spermatozoa RNA population [4].
Using RNA-Seq, we identified several full-length transcripts in the bovine cryopreserved spermatozoal transcript profile. While some of these transcripts were previously reported in spermatozoa, the presence of full-length transcripts could not be determined from the microarray studies. The most abundant full-length transcript, PRM1, has been reported in spermatozoa from other species as well, including humans 43 and porcine [7,13,20,35]. The high level of PRM1 is probably due to retention of this transcript in elongating spermatids during the later stages of spermatogenesis. A function for PRM1 after spermatozoa leave the testis is doubtful as Prm1 transcripts are rapidly degraded in the mouse embryo [15,16]. Other transcripts are delivered to the oocyte after fertilization, including the Y chromosome-linked DBY and RPS4Y, were not identified as full-length transcripts in this study, therefore, a functional role in embryogenesis for these transcripts is also unlikely [17].
Polyubiquitin is also an abundant full-length transcript in bovine spermatozoa. The ubiquitin system has several functions during spermiogenesis and fertilization, including: histone removal, removal of damaged epididymal spermatozoa, and aiding in zona penetration [36,37]. Disruption of the ubiquitin-proteosome pathway during spermatogenesis is characteristic of teratozoospermic males and can be detected in human spermatozoal RNA [22]. Spermatozoa-derived ubiquitin RNAs may also have a role in directing the degradation of paternal mitochondrial RNAs, ensuring exclusive maternal mitochondrial DNA inheritance [36]. Further investigation of a role for spermatozoal-derived polyubiqutin mRNA pre-and post-fertilization is warranted.
Previously reported spermatozoal transcripts involved in capacitation and fertilization were also identified as full-length, including: PLCZ1, CRISP2 and CLGN1. PLCΖ1, a well-characterized activator of the calcium wave after fertilization, is translated in the oocyte and injections of PLCZ1 RNA into the oocyte are also sufficient for function [18]. PLCZ1 is present at lower amounts (FPKM= 41.3) in the bovine spermatozoa transcript profile demonstrating that functional transcripts may not be the most abundant transcripts in this population. The presence of full-length CRISP2 could be indicative of potential translation at fertilization as CRISP2 is one of the spermatozoal proteins involved in oocyte binding [38]. The CLGN1 protein is necessary for heterodimerization of fertilization proteins [39,40]. full-length in this study therefore a role for these transcripts in the early stage embryo is an interesting area for further investigation.
One-third of the transcripts with FPKM>100 were degraded (all exons were not mapped). A predominance of degraded transcripts was also found in the FPKM<100 transcript population although this was not quantified. A degraded RNA population is characteristic of the spermatozoal RNA populations isolated in previous studies [7,8] and The spermatozoal transcript profile reported here was sequenced from a pool of bulls that represent a normal range of fertility scores. While the presence of specific transcripts did vary in an independent population of bulls, further quantitative analysis in a much larger population will better assess the level of individual bull variation and a correlation of transcript levels with fertility scores. This is the first report of the spermatozoal transcript profile in any species using high-throughput sequencing, supporting the presence of mRNA in spermatozoa.
Further studies of the spermatozoal mRNA candidates identified will contribute to our 47 knowledge of the function of spermatozoal mRNA and expand our approaches to assay male fertility.

Abstract:
Spermatozoa are now known to contain a limited number of mRNAs and the contribution of these mRNAs to early embryonic development has been controversial.
Although the spermatozoal transcript profile contains a predominance of degraded transcripts, only full-length mRNA transcripts will be capable of producing To determine if any of the selected transcripts were potentially transferred to the oocyte by the spermatozoa, previous microarray studies were used to find transcripts that were previously present in spermatozoa ) but absent in the oocyte (Kocabas et al. 2006).

Tissue Samples
Bovine testes were collected from a local abatoir for RNA isolation. Bovine cumulus-free oocytes and two-cell stage embryos were obtained from Sexing Technologies (Navasota, TX). Cryopreserved bovine spermatozoa straws were obtained from Genex Cooperative Inc., (Shawano, WI). Spermatozoa from nine individual bulls was pooled after RNA isolation (see below) for cDNA amplification.
These individuals had Conception Rate (CR) scores ranging from -2.9 to 3.5.
Spermatozoa from an additional three bulls were used for mRNA Reverse  (Table 3A).
Eight additional transcripts could not be sequenced in entirety, most likely because they are degraded in spermatozoa ( Figure 1B). These eight transcripts found in the bovine spermatozoal transcript RNA-Seq data had similar missing regions in PCR amplifications (Figure 3; Table 3). An example of this is the transcript PEBP1, 87 which had read mappings missing from the 5' end exon entirely; corresponding to missing reads found using PCR amplification (Figure 1 (Figure 4).

Discussion:
Visual inspection of RNA-Seq reads mapped to the bovine genome from the bovine spermatozoa transcript profile was used to identify sixteen full-length transcripts and these were validated by PCR amplification and subsequent sequencing, While other studies have looked at the mRNAs contained in spermatozoa, this is the first study to identify full-length transcripts. The importance of this is noted by the fact that this study found transcripts of interest from previous literature ( Table 1) that are not full-length (Table 3), and therefore likely non-functional. An example of this is DDX3Y, which has been examined in previous studies. One study found that reduction in sperm-derived DDX3Y mRNA has a role in spermatogenesis (Abdelhaleem 2005 are full-length in spermatozoa, this study demonstrates the potential for spermatozoa to have an epigenetic effect on embryonic development.
However, it is important to highlight the inherent bias to these selection methods, with a clear focus on isolating genes with a high probability of being fulllength. This study cannot be used to comment on the percentage of transcripts that may be full-length in the spermatozoa, only to prove that some do exist.
This study is incapable of determining if full-length spermatozoal transcripts are transferred to the oocyte at fertilization. The attempted oocyte and 2-cell embryo PCRs yielded inconclusive results. Several suspect amplifications indicated that the oocytes and 2-cell embryos were likely contaminated in both the cDNA amplified and unamplified RNA experiments ( Figure 4). The presence of known sperm-specific transcripts and Y chromosome transcripts in these populations, as well as varying results from gel to gel (Figure 4), make it impossible to conclude more about the spermatozoa to oocyte inheritance of these transcripts at this time.
In conclusion, full-length transcripts have been definitively proven to exist within bull spermatozoa, lending credence to the potential function of these mRNAs within the oocyte and embryo. Global comparisons of oocyte and embryo transcripts to spermatozoal transcripts are also needed to further identify targets that may be translated into proteins and impact embryogenesis.    Tables   Table 1: Transcript identification methods. Lit=Literature Searches, Y Csome= Y chromosome located, GO=found in gene ontology translation category     6. Cap the tube, and place it in the preheated thermal cycler. If you are NOT using a hot-lid thermal cycler, overlay the reaction mixture with two drops of mineral oil. divide the PCR reaction mix between the "Experimental" and "Optimization" tubes, using the Optimization tube for each reaction to determine the optimal number of PCR cycles, as described in Step 8.
b) For applications requiring longer cDNA transcripts, increase to 6 min.
8. Subject each reaction tube to 15 cycles, then pause the program. Transfer 30 µl from each tube to a second reaction tube labeled "Optimization". Store the "Experimental" tubes at 4°C. Using the Tester PCR tube, determine the optimal number of PCR cycles (see Figure 3 11. When the cycling 11. is completed, analyze a 5 µl sample of each PCR product alongside 0.1 µg of 1 kb DNA size markers on a 1.2% agarose/EtBr gel in 1X TAE buffer. Compare your results to Figure 4 to confirm that your reactions were successful.
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