MODIFICACIONES ACORDE CON LAS SENTENCIAS DE LA CORTE

Testis cells from mouse pups (aged 6-14 days) that were frozen for ~14 years, were thawed and transplanted into recipient testes of mice in which endogenous

spermatogenesis had been destroyed by busulfan treatment. Sperm from these recipient males were isolated and used for ICSI. A total of 5 pups were born from the ICSI procedure that appeared grossly normal (Wu et al., 2012) (figure 5.5). To assess the possibility that epigenetic defects occurred as a result of these procedures, we analyzed DNA methylation in these pups.

LUMA was performed on genomic liver DNA (as described in chapter 4.3) to assess genome-wide DNA methylation. Methylation levels of 5 control livers (sired from natural matings) and 4 ICSI derived livers were assessed. Genomic methylation levels in the ICSI derived mice were indistinguishable from control mice at ~72% CpGs

methylated (figure 5.6). Therefore, repetitive elements appeared to be properly methylated in mice derived from ICSI using sperm from cryopreserved SSCs.

Next, we asked if imprinted DMRs have abnormal levels of methylation.

Figure 5.5 Experimental design to analyze offspring derived from SSCs cryopreserved ~14 years

frozen for ~14 years, were thawed and transplanted into rec

which endogenous spermatogenesis had been destroyed. Sperm from these recipient males were used for ICSI. A total of 5 pups were born from the ICSI procedure

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Figure 5.5 Experimental design to analyze offspring derived from SSCs

cryopreserved ~14 years. Testis cells from mouse pups (aged 6-14 days) that were

frozen for ~14 years, were thawed and transplanted into recipient testes of mice in which endogenous spermatogenesis had been destroyed. Sperm from these recipient

for ICSI. A total of 5 pups were born from the ICSI procedure

Figure 5.5 Experimental design to analyze offspring derived from SSCs

14 days) that were ipient testes of mice in which endogenous spermatogenesis had been destroyed. Sperm from these recipient

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Figure 5.6 Normal genome-wide DNA methylation detected in offspring derived from ICSI using sperm from cryopreserved SSCs. Methylation levels of liver DNA

were determined by the LUminometric Methylation Assay (LUMA) for naturally sired mice (control, closed circles) and ICSI-derived mice using sperm from cryopreserved SSCs (ICSI, open circle). Each circle represents an individual sample with the mean methylation for each group indicated by a horizontal bar.

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can induce abnormal methylation and expression of imprinted genes (de Waal et al., 2012a). We analyzed two paternally-methylated DMRs; H19 ICR and IG-DMR. Pyrosequencing of bisulfite treated liver DNA indicated normal levels of methylation at the H19 ICR in ICSI derived as compared to control mice (figure 5.7A). COBRA was performed to analyze methylation at the IG-DMR. Using this assay, levels of methylation detected in the ICSI mice were indistinguishable from controls (figure 5.7B). Therefore, methylation at these paternally-methylated DMRs was maintained in SSCs after long- term cryopreservation, and properly inherited in the next generation despite the use of ICSI.

Additionally, we analyzed methylation at maternally-methylated DMRs; Snrpn and Peg3. Pyrosequencing analysis of bisulfite treated liver DNA did not uncover any significant difference between methylation levels at the Snrpn DMR in ICSI derived mice and control mice (figure 5.8A). COBRA analysis of the Peg3 DMR also indicated

normal levels of methylation in the ICSI derived mice when compared to control mice (figure 5.8B). We therefore conclude that mice derived by ICSI using sperm from SSCs cryopreserved for ~14 years had normal levels of methylation both genome-wide and at imprinted loci. These data confirms the prospect of using these procedures to restore fertility in males who had undergone gonadotoxic treatment as boys.

Figure 5.7 Normal methylation at

from ICSI using sperm from cryopreserved SSCs

performed on bisulfite mutagenized liver DN circles) mice and ICSI- derived

circles) to determine DNA methylation levels at the

each experimental group is represented with a black horizontal line. represents an individual sample.

DNA to analyze methylation at the IG

Percent methylation as quantified by comparing band intensities of methylated to unmethylated fragments (indicated to the right) is written below panel.

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Figure 5.7 Normal methylation at paternally-methylated DMRs in mice derived from ICSI using sperm from cryopreserved SSCs. (A) Pyrosequencing was

performed on bisulfite mutagenized liver DNA from naturally sired (control, derived mice using sperm from cryopreserved SSCs

to determine DNA methylation levels at the H19 ICR. Mean methylation for each experimental group is represented with a black horizontal line. Each circle represents an individual sample. (B) COBRA was performed on bisulfite mutagenized DNA to analyze methylation at the IG-DMR and run out on a 12% acrylamide gel Percent methylation as quantified by comparing band intensities of methylated to unmethylated fragments (indicated to the right) is written below panel.

methylated DMRs in mice derived

(A) Pyrosequencing was A from naturally sired (control, closed

reserved SSCs (ICSI, open . Mean methylation for

Each circle fite mutagenized and run out on a 12% acrylamide gel. Percent methylation as quantified by comparing band intensities of methylated to

Figure 5.8 Normal methylation at maternally from ICSI using sperm from cryopreserved SSCs

performed on bisulfite mutagenized circles) mice and mice derived from

open circles) to determine DNA methylation levels at the methylation for each experimental group is repres

Each circle represents an individual sample. mutagenized DNA to analyze methylation at the

acrylamide gel. Percent methylation as quantified by compari

methylated to unmethylated fragments (indicated to the right) is written below panel.

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Figure 5.8 Normal methylation at maternally-methylated DMRs in mice derived from ICSI using sperm from cryopreserved SSCs. (A) Pyrosequencing was

performed on bisulfite mutagenized liver DNA from naturally sired (control,

mice derived from ICSI using sperm from cryopreserved SSCs (ICSI, to determine DNA methylation levels at the Snrpn DMR. Mean

methylation for each experimental group is represented with a black horizontal line. Each circle represents an individual sample. (B) COBRA was performed on bisulfite mutagenized DNA to analyze methylation at the Peg3 DMR and run out on a 12%

. Percent methylation as quantified by comparing band intensities of methylated to unmethylated fragments (indicated to the right) is written below panel.

methylated DMRs in mice derived

(A) Pyrosequencing was from naturally sired (control, closed

ved SSCs (ICSI, . Mean

ented with a black horizontal line. (B) COBRA was performed on bisulfite

DMR and run out on a 12% ng band intensities of methylated to unmethylated fragments (indicated to the right) is written below panel.

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CHAPTER SIX

DISCUSSION AND FUTURE DIRECTIONS

Genomic imprinting is a complex epigenetic phenomenon required for normal mammalian development. Imprinted expression requires marking the parental origin of the chromosome so that a specific parental allele is stably repressed or stably expressed. DNA methylation is essential for marking and silencing imprinted genes. These DNA methylation imprints and allele-specific expression patterns must be maintained throughout early development and in differentiated somatic tissues. Additionally, marking of the alleles must be reset in the germline to allow for establishment of sex- specific marks in the mature gamete, which will be transmitted to the next generation. These processes are critical for normal imprinting, and can be disrupted by environmental stress. This dissertation focused on identifying both cis and trans mechanisms by which DNA methylation confers imprints and how environmental stresses can disrupt imprinted regulation. We have defined a novel regulatory role for CpG content at the H19 ICR in silencing paternal H19. We have also identified TET1 and TET2 as trans factors

involved in resetting imprints in the germline and have provided evidence that individual MBD proteins are dispensable for normal imprinting. Moreover, we have demonstrated that methylation imprints are in fact stable in spermatogonial stem cells that have undergone aging and cryopreservation, suggesting that these techniques can be valuable sources for male infertility treatment.

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6. 1 Non-promoter methylation in gene silencing

Whereas DNA methylation at the H19 ICR is established in sperm and maintained in the zygote, methylation at the paternal H19 promoter region is not detected until midgestation (Tremblay et al., 1997; Tremblay et al., 1995). Intriguingly, mice that paternally inherited an allele in which the H19 ICR had been deleted did not gain methylation at the H19 promoter region (Srivastava et al., 2000; Thorvaldsen et al., 2006). It was proposed that the paternal ICR acts as a center for spreading of methylation to the paternal H19 promoter, indicating a cis-regulatory role for the ICR in paternal repression. This had been the prevailing model for how the ICR silences paternal H19 in

cis. Our data indicates a novel regulatory role for DNA methylation at the H19 ICR in

repression of H19 directly. In mice that inherited a mutant paternal allele in which 8 CpGs within the ICR but outside of CTCF binding sites had been deleted, H19ICR-8nrCG allele, significant levels of paternal H19 expression were detected despite maintaining a hypermethylated ICR and promoter (figures 2.5, 2.7-2.9). Therefore, we conclude that while H19 promoter methylation is necessary for repression, it is clearly not sufficient. This is particularly the case when total H19 is expressed very highly. In these tissues, the silent H19 allele is in an environment that is conducive to expression, with all the proper transcriptional machinery actively transcribing the normally active allele. Therefore, multiple levels of repression may be necessary to ensure silencing. Our data highlights the importance of non-promoter methylation as a means of repression when promoter methylation alone is insufficient.

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One remaining question is how does methylation at the H19 ICR act from a distance to silence H19? The repressive NuRD complex has been implicated in regulation of paternal H19 repression. Depletion of NuRD components MTA-2 or MBD3 resulted in biallelic H19 expression in blastocysts (Ma et al., 2010; Reese et al., 2007). It is possible that DNA methylation at the H19 ICR recruits the NuRD complex, resulting in a repressive chromatin environment and H19 silencing. With depletion of methylation at the ICR, there might be a decrease in NuRD recruitment resulting in a more permissive chromatin environment. Of note, MBD2 also is a NuRD complex component, although MBD2 and MBD3 form mutually exclusive complexes (Baubec et al., 2013; Gunther et al., 2013; Le Guezennec et al., 2006). Because we did not detect any loss of imprinting in Mbd2 null mice, it is likely that MBD3-NuRD complexes exclusively act at H19 in preimplantation embryos and cannot be compensated for by MBD2-NuRD complexes. However, this hypothesis is difficult to confirm biochemically due to the low amounts of material that can be collected from preimplantation embryos.