Small RNAs in meiotic and postmeiotic mouse male germ cells

Small RNAs in meiotic and postmeiotic mouse male germ cells

Ram Prakash Yadav

Master thesis research project in Biology 45 hp, 2011 Department of Physiology, Institute of Biomedicine University of Turku, Finland

Supervisor: Dr. Noora Kotaja, PhD, Docent

LIST OF ABBREVIATIONS

Ago argonaute

bp base pairs

cDNA complementary DNA

DAPI 4'-6-Diamidino-2-phenylindole

DcrF dicer forward primer

DcrR dicer reverse primer

DcrNull dicer null primer

DNase deoxyribonuclease

DNA deoxyribonucleic acid

dpp days post partum

dsRNAs double-stranded RNAs

EDTA ethylene diaminetetraacetic acid

endo-siRNA endogenous siRNA

ESCs embryonic stem cells

IAP intracisternal A particle

IR immature rhesus monkey

KO knockout

LINE1 long interspersed nuclear element-1

miRNA microRNA

mRNA messenger RNA

MR mature rhesus monkey

MH mature human

Ngn3 neurogenin 3

NSCs neural stem cells

PBS phosphate buffered saline

PCR polymerase chain reaction

pre-miRNAs precursor miRNAs

PFA paraformaldehyde

piRNA piwi-interacting RNA

PGCs primordial germ cells

pri-miRNAs primary miRNAs transcript

Pspc pachytene spermatocytes

qPCR real-time quantitative-PCR

RISC RNA-induced silencing complex

RT-qPCR reverse transcription real-time quantitative-PCR

RT reverse transcription

RNA ribonucleic acid

rpm revolutions per minute

RPE retinal pigmented epithelium

RNAi RNA interference

SINEB1 short interspersed nuclear element B1

SINEB2 short interspersed nuclear element B2

siRNA small interfering RNA

Spc spermatocytes

SQ starting quantity

T

annealing temperature

TBE tris-borate-EDTA

UTR untranslated region

WT wild type

YFP yellow fluorescence protein

S. No. TABLE OF CONTENTS Page 1.) Introduction …...

1.1) Dicer : a molecular scissor ...

1.2) RNA-induced silencing complex (RISC) …...

1.3) Roles of Dicer …...

1.4) Spermatogenesis …...

1.5) Small RNAs in spermatogenesis …...

1.6) Conditional germ cell-specific Dicer1 knockout mouse model …...

1.7) Aim of current project …...

1 1 2 2 4 5 6 7 2.) Materials and methods …...

2.1) Tissue samples …...

2.2) Isolation of spermatocytes and round spermatids ...

2.3) Total RNA isolation …...

2.4) Deoxyribonuclease (DNase) treatment …...

2.5) cDNA synthesis …...

2.6) Quantitative PCR (qPCR) …...

3.7) microRNA RT-qPCR …...

3.8) Data analysis …...

8 8 8 8 9 9 9 10 11 3.) Results …...

3.1) Expression of Dicer and miRNAs during mouse spermatogenesis …...

3.2) Confirmation of the downregulation of Dicer and Dicer-dependent

miRNAs in germ cell-specific Dicer1 knockout mice …...

3.3) Expression of transposable elements and centromeric repeat

transcripts in Dicer knockout testes …...

11 11 14 15

4.) Discussion …... 19

5.) Acknowledgements …... 21

6.) References …... 22

7.) Appendix A …... 25

1. Introduction

1.1) Dicer: a molecular scissor

Dicer is a RNase III endoribonuclease that slices pre-miRNA and double stranded RNA (dsRNA) into small RNA fragments of 18-25 nucleotides in length to generate short interfering RNAs (siRNAs) and microRNAs (miRNAs). siRNAs and miRNAs belong to a group of small non- coding RNAs that are not translated into proteins but possess per se functional activities.

Figure: 1. Overview of miRNA and siRNA biosynthesis and than processing by Dicer into 18-25 nucleotides of dsRNA molecules. The dsRNA unwinds, and one strand associates with Argonaute and other proteins to form RISC complex which binds to 3' UTR of target mRNA and acts either by translational repression or mRNA cleavage depending upon the complementarity between the small RNA and its target (Adopted from He et al. 2004).

Dicer has two ribonuclease III (RNase III) domains and one PAZ domain connected by

flexible hinges that assist dsRNA binding and processing. The PAZ domain is responsible for

binding within a highly conserved binding pocket of 3' overhang of dsRNA in a sequence-

independent manner and consequently two catalytic RNA III domains cut each strand of the dsRNA

to 18-25 nucleotides length (1, 2). Then the dsRNA is unwounded to single strands. One of them

acts as guide strand, which associates with the Argonaute protein and other factors to form the

RNA-induced silencing complex (RISC). The other strand is called passenger strand and is believed

to be eliminated by degradation pathways. siRNA precursors can be processed by Dicer alone, but hairpin-looped primary miRNA transcripts (pri-miRNAs) have to be processed first into approximately 60-100 nucleotides of precursor miRNAs (pre-miRNAs) in the nucleus by another endonuclease: Drosha (3, 4). Then, pre-miRNAs are then transported to cytoplasm for further processing by Dicer (Fig 1).

1.2) RNA-induced silencing complex (RISC)

The RISC complex is a multiprotein complex which possesses catalytic activities and mediates the functions of miRNAs. Argonaute (Ago) protein is one of the important catalytic components of the RISC complex. Ago proteins belong to the Argonaute family, which is highly conserved between species and many organisms. There are twenty-seven Argonaute family members in Caenorhabditis elegans, one in Schizosaccharomyces pombe and eight in mammals. In mammals, Ago1 to Ago4 are expressed ubiquitously and function in RISC complexes. It is found that Ago1 mediates the gene silencing and Ago2 has sequence-specific endonuclease activity responsible for the cleavage of the target mRNAs. RISC uses a guide strand from Dicer-processed double-stranded small RNAs (dsRNAs) as a template for recognizing the 3'-untranslated region (3' UTR) of the target mRNA in animals. The detailed mechanisms of RISC loading are still unknown.

However, the incorporation of the guide strand into the RISC complex is usually regulated by thermodynamic stability of both ends of the RNA strand (2). The nature of the sequence complementarity of guide strand to 3' UTR decides whether the target messenger RNA (mRNA) is silenced or degraded. A perfect complementary binding to 3' UTR results in the degradation of mRNA. In contrast, imperfect complementary may lead to the translational silencing of the target mRNA (2, 3, 4). These processes are important for either defending us against viral infections or the gene regulation by miRNAs.

1.3) Roles of Dicer

Dicer serves as a molecular ruler by chopping dsRNA substrates into smaller pieces to generate miRNAs and exogenous or endogenous siRNAs. The best-studied Dicer-dependent small RNAs are miRNAs, which function mainly at posttranscriptional level to control the expression of their target mRNAs (5). Mammalian genomes comprise many miRNA genes. miRNA processing is globally affected by Dicer and regulates a variety of biological processes (6). A miRNA can downregulate the expression level of hundreds of genes and acts as a regulatory factor to control various physiological processes and diseases. Furthermore, the important components of RNA interference (RNAi) pathways, Dicer and Drosha are inter-linked with poor clinical outcome in the epithelial ovarian cancers patients (7), suggesting the association of RNAi machinery with tumorigenesis and cancer biology.

The importance of Dicer in mammalian development is clearly demonstrated by deleting

Dicer1 gene in mice. The deletion of Dicer1 gene results in early developmental arrest and the mice

die at embryonic day 7.5. Knockout mice were deficient of stem cells, suggesting the role of Dicer

in maintaining stem cell population in the early development (9). Similar type of consequences were

also observed when conditionally inactivating the Dicer1 gene in the zebrafish (10). Mouse

embryonic stem cells (ESCs) lacking functional Dicer show several defects in differentiation and

the phenotype can be rescued by re-expressing Dicer in the mutant ESCs (11). Dicer1 knockout ESCs show induction in mammalian centromeric transcripts without affecting histone modifications at pericentric repeats or expression of ESCs markers (12). These studies suggest the involvement of Dicer in stem cell differentiation and maintenance and silencing of centromeric heterochromatin.

The data from high-throughput pyrosequencing of small RNAs also revealed the roles of Dicer in mouse ESCs (13).

The role of Dicer in specific tissues has begun to be uncovered through various conditional knockout mouse models, in which the Dicer1 deletion takes place only in desired tissue.

Conditional removal of Dicer from the limb mesoderm using Cre lines Prx1Cre and ShhgfpCre in which Cre expression is under the control of promoters of Prx1 and Shh genes respectively, leads to lack of processed miRNAs, delay in development of limb, massive cell death, disregulation of specific gene expression and development of smaller limb size. Surprisingly, no defects in tissue- specific differentiation or basic patterning in the Dicer-deficient limb buds were found (8). This studies implies that Dicer is not required for the patterning but for the morphogenesis of the vertebrate limb. Dicer-deficient neural stem cells (NSCs) display normal karyotype, self-renewal capacity and heterochromatin protein expression levels. However, the cells show enlarged nuclei, abnormal differentiation, increase in pro-apoptosis proteins, and decrease in anti-apoptosis proteins.

In depth protein analysis by mass spectrometry revealed changes in the expressions of many proteins (14). In this study the authors illustrated the function of Dicer in the NSC development, cell survival and differentiation.

The important role of Dicer in female reproduction has been demonstrated by several studies (15). The deletion of Dicer in mouse oocytes resulted in the blockage of meiosis I, appearance of disorganized spindles, severe chromosomal defects and induction of transposable elements (16), signifying the critical roles of Dicer during meiosis in oocytes and protection of female germline from transposable elements. In the Dicer1 knockout mouse model that uses Cre recombinase under the control of anti-Mullerian hormone receptor type 2 promoter, Dicer1 is inactivated in mesenchyme-derived cells of the oviducts, uterus, mullerian ducts and ovarian somatic granulosa cells. Several reproductive defects such as decreased ovulation rates, oocyte and embryo integrity, prominent bilateral oviductal cysts, and shorter uterine horns were observed. The deep sequencing data of small RNAs from the oviduct showed downregulation of specific sets of miRNAs in Dicer1 knockout as compared to wild type oviduct. Interestingly, it was found that these differentially expressed miRNAs regulate important genes involved in differentiation of mullerian duct and mesenchyme-derived structures (17). These studies illustrate the involvement of somatic Dicer in the function of female reproductive tract.

In addition to the posttranscriptional regulation of protein-coding genes, Dicer-dependent

pathways have been implicated in a variety of other cellular and molecular processes such as

regulation of heterochromatin domain organization, gene silencing, recombination suppression and

chromosome segregation in fission yeast (18). The conditional ablation of Dicer1 induces

accumulation of Alu RNA in human retinal pigmented epithelium (RPE) cells and causes

cytotoxicity and macular degeneration in the geographic atrophy (19). This clearly illustrates the

roles of Dicer in the degradation of retrotransposon transcripts. The human Alu RNA shows

similarity with Alu-like B1 (SineB1) and B2 (SineB2) RNAs in the mouse. The conditional deletion

of Dicer in a chicken–human hybrid DT40 cell line showed cell death with abnormal mitotic cells

and induction of high levels of α-satellite transcripts of human centromeric repeats. The abnormal localization of heterochromatin proteins, cohesin protein (Rad21) and checkpoint protein (BubR1) were observed by using immunocytochemical analysis (20), suggesting the involvement of Dicer in the formation of heterochromatin structure. The deletion of RNAi machinery in the fission yeast leads to loss of cohesin at centromeres which affects the chromosome segregation during mitosis (21). The deletion also resulted in slow growth rate, chromosomes lagging during anaphase and disturbed silencing of centromeric repeats (22). These studies clearly indicate the role of RNAi machinery in the regulation and maintenance of chromosome architecture, and in the chromosome segregation during mitosis and meiosis in fission yeast.

1.4) Spermatogenesis

Spermatogenesis is the process of forming mature haploid spermatozoa or sperm from diploid spermatogonial stem cells. It is a highly complex, precisely organized and timely regulated developmental process and it occurs throughout most of the mammalian adult life. Spermatogenesis takes place inside the seminiferous tubules of the testis. Primordial germ cells (PGCs) are the main precursors for spermatogenic cells in the prenatal testis. After birth, PGCs give rise to spermatogonial stem cells that provides the pool of undifferentiated cells for the adult spermatogenesis. Along with germ cells, adult mouse testis contain many somatic cells such as Sertoli cells, testosterone-producing Leydig cells, and peritubular myoid cells. Sertoli cells and their tight junction dynamics play a vital role in nurturing and supporting the events of spermatogenesis (23, 24).

Figure 2. Male germ cells differentiation in mouse (Modified from Meikar et al. 2011). A schematic representation of spermatogenesis process in the mouse where germ cells undergo mitotic, meiotic and postmeiotic events. The time course of appearance of germ-specific cell types during first wave of spermatogenesis in juvenile mice is indicated as dpp.

The process of postnatal spermatogenesis involves three main stages: mitotic, meiotic and

postmeiotic. During mitotic phase, spermatogonia undergo several successive mitotic divisions. The

spermatogonia resides in the basal compartment of the seminiferous epithelium. In contrast meiotic

and postmeiotic germ cells locate in the luminal compartment. Meiotic germ cells are known as

spermatocytes (Spc). The important events that occur during meiosis are synaptonemal complex

formation, crossing over and homologous recombination. Each meiotic process produces haploid

round spermatids (RS), which further undergo haploid differentiation, or spermiogenesis. Late

spermatocytes and round spermatids are transcriptionally highly active. During postmeiotic spermiogenesis, chromatin is reorganized and condensed by replacing histones first by transition proteins and then by protamines in the elongating spermatids (ES). Protamine-incorporated genes are transcriptionally silent. Thus, this process allows only limited histone-bound genes to be expressed after chromatin compaction, which highlights the importance of mRNA storing and translational regulation in late phases of spermatogenesis (Fig 2). The postmeiotic events also include acrosome, flagellum formation and cytoplasm ejection. In general, the whole spermatogenesis process completes within 34-35 days in the mouse (23, 25).

1.5) Small RNAs in spermatogenesis

The Dicer1 knockout mouse model, in which Dicer1 is deleted in embryonic PGCs, showed defects in PGC proliferation and germ cell differentiation (26, 27). These studies suggest that Dicer is important for the proliferation of PGCs and spermatogonia. The miRNA microarray expression data showed 19 differentially expressed miRNAs between immature and mature mouse testes which was further validated by a quantitative real-time RT-PCR assay (28). Another miRNA microarray expression studies on immature rhesus monkey (IR), mature rhesus monkey (MR), and mature human (MH) demonstrated 26 differentially expressed miRNAs in the samples IR/MR and IR/MH (29). All these studies suggest roles of miRNAs in the developmental control of mammalian male germ cell differentiation.

Postnatal male germ cells express several classes of small RNAs, including miRNAs and endogenous siRNAs, as well as germline-predominant piwi-interacting RNAs (piRNAs), which are processed by Dicer-independent mechanisms. Several miRNAs are demonstrated to be specifically expressed in certain types of differentiating male germ cells. miR-34c and miR-184 which were also used in this study, are two examples of germ cell-specific miRNAs. miR-34c is highly expressed during late steps of spermatogenesis and was also shown to be involved in tumor suppressive functions in human prostate (30, 31). miR-184 has been depicted to be round spermatid-specific in testis (unpublished). It is also found to be involved in the germline stem cell differentiation and development of the female germline in Drosophila and it is also implicated in epigenetic regulations during neural stem cell proliferation and differentiation (32, 33). Recently, endo-siRNAs which are Dicer-dependent but Drosha-independent, were shown to be highly expressed in male germ cells (34). Similar to miRNAs, they were shown to act at the posttranscriptional level to control mRNA expression, but other functions were also predicted. However, the specific processes controlled by miRNAs and endo-siRNAs during spermatogenesis and the mechanisms of their actions in male germline remain to be characterized.

The spermatogenesis requires not only germ cells but also other associated cells such as

Sertoli cells. The selective deletion of Dicer in Sertoli cells resulted in the significant

downregulation of essential genes for Sertoli cell function in the neonatal testes and morphological

alterations started after the postnatal day 5. Due to defects in Sertoli cell maturation, germ cells lost

the ability to undergo meiosis and spermiogenesis. Consequently, low number or absence of

spermatozoa and progressive testicular degeneration were observed (35). Another study (36) reveals

that deletion of Dicer in Sertoli cells resulted in the massive upregulation of testicular proteins

which was quantified by mass spectrometry analysis. It clearly indicates the involvement of Dicer-

dependent pathways in Sertoli cell-mediated male reproductive functions.

1.6) Conditional germ cell-specific Dicer1 knockout mouse model

Recently, mouse model systems have been exploited to answer many fundamental and experimental questions in mammalian research that can not be addressed by other model systems.

Besides the difference between mice and humans, genetically modified mice provide a good system to test hypotheses and to identify novel mechanisms both in biology and diseases. The knockout strategy delineates the cause and effect relationships of expressed genes. Knockout refers to the deletion of a specific gene and subsequent monitoring of its loss-of-function to understand the biological functions of that gene. This strategy may create problems if the gene deletion affects whole biological systems for example causing embryonic lethality or severe developmental abnormalities that prevents assessment of target gene functions in a specific time during developmental processes (37). To address this problem, it is necessary to conditionally delete the target gene. Cre/Lox system has been shown as an elegant tool for dissecting the spatial and temporal deletion of genes in a specific cell type and at correct time (38).

Figure 3. Our Dicer1 knockout mouse model (Modified from Korhonen et al. 2011). On the top general floxed (fx) Dicer1 allele construct showing Dicer forward (DcrF), reverse (DcrR), Dicer null (DcrNull) primers and possible bands size of different mouse genotypes {fx/fx (420 bp), wt/wt (351 bp) and fx/wt (420 and 351 bp)} in the gel. The Dicer1 deletion was verified by genomic PCR revealing the band (600 bp) specific for deleted allele of the Dicer1 knockout testis.{KO: Dcr(fx/fx);Ngn3Cre, WT: Dcr(fx/fx)}. On the bottom, transgenic mice expressing Cre recombinase under the control of Neurogenin 3 (Ngn3) promoter mice were crossed with reporter mice expressing yellow fluorescence protein (YFP), only upon Cre-mediated recombination to analyze the efficiency and penetrance of Ngn3Cre transgene. High level fluorescence signal was detected in postnatal male germ cells of Ngn3Cre;ROSA26YFP testes as compared to control littermates.

Usually, Cre recombinase is expressed under the control of a tissue-specific promoter to remove a target gene in a desired tissue. Cre-expressing mice are crossed with mice carrying the target gene within flanking loxP sites. The loxP site is Cre recombinase protein specific binding site where Cre protein dependent recombination occurs. Multiple Cre delivery systems has been developed and their utility depend upon specific research interest. Here, we used Neurogenin 3-Cre system. Neurogenin 3 (Ngn3) is a transcription factor that is required for differentiation of endocrine cells and development of pancreatic islets. It is expressed in undifferentiated spermatogonia in testis, but also during development of spinal cord and hypothalamus and in the progenitors of pancreatic islet (39). Exon of Dicer1 gene which encodes for the second RNaseIII domain of Dicer, is flanked by two loxP sites. Ngn3Cre transgene is activated postnatally around day 5 in spermatogonia. It produces recombinase enzyme, which drives the recombination within two flanking loxP sites resulting in conditional removal of exon of Dicer1 in germ cells. To evaluate the male germ cell-specific Cre activity, transgenic Cre mice were crossed with reporter mice expressing transgenic ROSA26YFP gene having a stop codon flanked with LoxP sites before the YFP coding region. Cre recombinase excises the stop codon, which results in the expression of YFP in differentiating spermatogonia, spermatocytes and spermatids of Ngn3Cre;ROSA26YFP seminiferous epithelium in a highly penetrant manner (40). Furthermore, the deletion of Dicer in the knockout testis was checked by genomic PCR using DcrF primer (upstream of the deleted exon) and DcrNull primer designed in the region downstream of the deleted exon. The expected deletion specific band (600 bp) is only amplified from the knockout testis samples collected at postnatal day 10 (Fig 3), which clearly indicates that Dicer1 deletion has occurred in the germline. Dicer1 knockout males are infertile and spermatogenesis is affected which indicates a crucial role of Dicer in male germ cell differentiation.

1.7) Aim of current project

Dicer is constitutively expressed in a variety of cell types and regulates many biological processes. Understanding and elucidating the functions of Dicer with respect to specific cell types is crucial for deciphering cell-specific molecular and physiological events. We have generated a germ cell-specific Dicer1 knockout model and analyzed the phenotype to understand the role of Dicer- dependent pathways during postnatal spermatogenesis. My project was a part of this analysis. The specific aims of my project were:

1. To analyze the expression of Dicer and germ cell-specific miRNAs in meiotic and postmeiotic male mouse germ cells

2. To validate the absence of full-length Dicer1 mRNA and Dicer-dependent miRNAs in the Dicer1 knockout testes.

3. To study the involvement of Dicer in the expression of transposable elements and pericentric

and centromeric repeat transcripts in male mouse germ cells.

2. Materials and Methods

2.1) Tissue samples

Testis and liver from 8 dpp, 14 dpp, 20 dpp, 28 dpp and 34 dpp wild type male mice, and also testis from 28 days and adult wild type and Dicer1 knockout mice were collected. Tissue samples were frozen in liquid nitrogen and stored at -70 ˚C until further processed.

2.2) Isolation of spermatocytes and round spermatids

Late pachytene spermatocytes and round spermatids were isolated from adult testes by using centrifugal elutriation (Beckman JE-6B Rotor Elutriator). Testes were decapsulated, fragmented into small pieces and then transferred to collagenase (0.5 mg/ml) solution prepared in PBS containing 0.1 % glucose. After digestion, the cell suspension was filtered (100 μm pore size) and centrifuged at 1500 rpm for 5 minutes to remove collagenase solution. The pellet was resuspended in ice cold PBS containing 0.1 % glucose and the cell suspension was filtered twice through glass wool and once through 50 μm filter to remove elongated spermatids. After centrifugation, the pellet was resuspended in small volume of PBS containing 0.1% glucose and the suspension was injected through the injection port in the Beckman elutriator. Cells were collected at 2800 and 2000 rpm by adjusting different flow rates as shown in Appendix A.

To confirm the success of the isolation, cell slides were prepared from different fractions.

Cells were fixed in 1% PFA (paraformaldehyde) in PBS containing 0.15% Triton-X 100. Slides were incubated overnight in a humidified chamber. After drying the slides, they were post-fixed in 4% PFA in PBS for 15 minutes. Slides were washed three times in PBS and mounted in mounting medium containing DAPI (Santa Cruz Biotechnology, Santa Cruz, USA). Finally, visualized with the Leica DFC320 digital color camera mounted onto a Leica DMRB microscope (Leica Microsystems, Wetzlar, Germany) using either a PL FLUOTAR 40X/0.70 or PL FLUOTAR 100X/1.30 OIL PH3 objective and Leica IM 500 version 4.0 software. Isolated cells were stored as pellets at -70˚C until further processed.

2.3) Total RNA isolation

Tissue samples were homogenized in TRI Reagent solution (Cat. No. TR118, Molecular

Research Center, Inc.). TRI reagent was added 1 ml per 50 to 100 mg of tissue, and the

homogenates were incubated for 5 minutes at room temperature allowing nucleoprotein complexes

to completely dissociate. Chloroform (0.2 ml) was added per 1 ml of TRI Reagent. Samples were

mixed vigorously and then centrifuged at 12,000 x g for 15 minutes at 4 °C. The upper colorless

aqueous phase was mixed with equal volume of isopropanol followed by addition of 15 mg of

GlycoBlue (Cat. No. AM 9515, Ambion). The mixture was vortexed and incubated at room

temperature for 5 min and centrifuged at 12,000 x g for 10 minutes at 4 °C. Supernatant was

removed without disturbing the pellet and washed with 75 % ethanol. Finally, the pellet was air

dried and dissolved in nuclease free water. RNA concentration was measured by using NanoDrop

ND-1000 Spectrophotometer (Thermo Fisher Scientific,Wilmington, USA).

2.4) Deoxyribonuclease (DNase) treatment

Total RNA was treated with Deoxyribonuclease (Turbofree DNA kit, Ambion) to digest the genomic DNA contamination prior to RT-qPCR. The DNase treatment was done in a mixture containing 1 μg of total RNA, 0.1 volume of 10X buffer and 0.1 volume of DNase. The reaction volume was adjusted to 10 µl by adding nuclease free water. The reaction mixture was incubated for 15 minutes at room temperature, followed by addition of 1 μl of 25 mM EDTA solution and treated for 10 minutes at 65 °C to inactivate the DNase.

2.5) cDNA synthesis

The cDNA synthesis was performed by using DyNAmo™ cDNA synthesis kit (Finnzymes).

The total reaction mixture (20 μl) contained 0.5 μg of total RNA, 0.3 μg of random hexamer primers, 0.1 volume of M-MuLV RNase H

reverse transcriptase, and 0.5 volume of 2X RT buffer.

Total volume was adjusted to 20 µl by adding nuclease free water. The negative RT (reverse transcription) reactions for respective samples were prepared without adding reverse transcriptase.

The parameters of PCR were adjusted as: primer extension at 25 °C for 10 minutes, cDNA synthesis at 37 °C for 30 minutes, reaction termination at 85 °C for 5 minutes and cooling at 10 °C for ∞.

2.6) Quantitative PCR (qPCR)

The qPCR was performed with DyNAmo™ Flash SYBR® Green qPCR Kit (Finnzymes) by using CFX 96 Real Time System (Bio-Rad). The reaction was prepared by mixing 0.5 volume of master mix, 0.1 volume of primers (forward and reverse) and 0.4 volume of respective cDNA (1:20 dilution) in the 20 μl of total reaction volume in each well of 96 well PCR plate.

Standard dilutions (1:20, 1:40, 1:80 and 1:160) were prepared from adult wild type mouse testis cDNA to plot a standard curve. qPCR was performed with triplicates for each sample (both wild type and Dicer1 knockout) and corresponding negative RT reactions, and duplicates for each standard dilution. In the whole set of experiment, L19 gene expression was used as reference gene to normalize the data. Primers for L19 were forward : 5´-GGA CAG AGT CTT GAT GAT CTC- 3´

and reverse : 5´-CTG AAG GTC AAA GGG AAT GTG- 3´. Primers used in qPCR are shown in Table 1.

Table 1. Primer pair sequences and their annealing temperature.

S. No. Name Primer sequences T

1.) Dicer Dicer F1: 5´-CTT GAC TGA CTT GCG CTC TG- 3´

Dicer R1: 5´-AAT GGC ACC AGC AAG AGA CT- 3´

60

2.) Major satellite Major F1: 5´- GAC GAC TTG AAA AAT GAC GAA ATC- 3´

Major R1: 5´-CAT ATT CCA GGT CCT TCA GTG TGC- 3´

57

3.) Minor satellite MINF1 : 5´-CAT GGA AAA TGA TAA AAA CC- 3´

MINR1 : 5´-CAT CTA ATA TGT TCT ACA GTG TGG- 3´

57

4.) Line1 Line1 sense : 5´-TTT GGG ACA CAA TGA AAG CA- 3´

Line1 antisense : 5´- CTG CCG TCT ACT CCT CTT GG- 3´ 60 5.) SineB1 SineB1 sense : 5´-GTG GCG CAC GCC TTT AAT C- 3´

SineB1 antisense : 5´-GAC AGG GTT TCT CTG TGT AG-3´

60

6.) IAP IAP gag sense : 5´-AGC AGG TGA AGC CAC TG- 3´

IAP gag antisense : 5´-CTT GCC ACA CTT AGA GC- 3´

62

IAP, intracisternal A particle. T

annealing temperature.

The qPCR parameters were set as: initial denaturation at 95 °C for 7 minutes, then in cycles, denaturation 95 °C for 10 seconds, annealing at 60 °C for 15 seconds and extension at 72 °C for 5 minutes. PCR product was run in the 3 % agarose gel which was prepared in 1X TBE buffer.

2.7) microRNA RT-qPCR

All the total RNA samples were treated with Deoxyribonuclease I (Cat. No. 18068-015, Invitrogen) to digest the genomic DNA contamination prior to RT-qPCR. In contrast to previous DNase treatment, here total reaction mixture of 10 μl was prepared which contains only 50 ng of total RNA. TaqMan miRNA kit (Applied Biosystems) was used for quantification of miRNAs according to the manufacturer’s instructions using primers for miR-34c and miR-184. U6 snRNA (RNU6B) was used as reference to normalize the miRNA expression. The cDNA was synthesized separately for each samples by using U6 (5X) and specific miRNA (5X) primers, and corresponding negative RT reactions. The cDNA synthesis was performed with DyNAmo™ cDNA synthesis kit (Finnzymes). The total reaction mixture (15 μl) was made with 10 ng of total RNA, 0.3 μg of random hexamer primers, 0.1 volume of M-MuLV RNase H

reverse transcriptase, 0.5 volume of 2X RT buffer and remaining volume adjusted by nuclease free water. Negative RT reactions for respective samples were prepared without adding reverse transcriptase. PCR parameters for cDNA synthesis were adjusted as: primer extension at 16 °C for 30 minutes, cDNA synthesis at 42 °C for 30 minutes, reaction termination at 85 °C for 5 minutes and cooling at 10 °C for ∞. Standard dilutions (1:1, 1:2, 1:4, 1:8 and 1:16) of cDNA were prepared from adult wild type testis cDNA to plot the standard curve. The miRNA qPCR was performed by preparing total mix of 10 μl containing 0.5 volume of TaqMan Universal PCR master mix II, 0.05 volume of TaqMan primers (20X) for U6 or miR-184 or miR-34c, 0.07 volumes of respective cDNA and remaining volume adjusted with nuclease free water. miRNA qPCR parameters were adjusted as: initial denaturation at 95 °C for 10 minutes, then in cycles, 95 °C for 15 seconds and extension at 60 °C for 60 seconds.

The qPCR was performed in triplicates for each sample and corresponding negative RT reactions,

and duplicates for each standard dilution.

2.8) Data analysis

Standard curve is a simple and reliable method for the analysis of qPCR data. A standard plot was generated from mRNA expression profiles based on the range of standard dilutions of known concentration of cDNA. The standard starting quantity (SQ) mean value was calculated for each sample triplicates from the standard curve. The relative gene (Dicer1 or miRNAs) expression was calculated by dividing each SQ mean value of gene to the respective sample SQ mean value of reference gene (L19 or U6). A graph was plotted by taking samples in X-axis and relative gene expression values in Y-axis.

3. Results

3.1) Expression of Dicer and miRNAs during mouse spermatogenesis

Dicer has a central role in the processing of double-stranded RNA precursors into small RNA effector molecules in the miRNA and RNAi pathways (3, 5). It is ubiquitously expressed in almost all cells and regulates various processes. On the other hand, the exact expression pattern of Dicer is unclear during the meiotic and postmeiotic maturation of male mouse germ cells. Since we are interested in characterizing the role of Dicer-mediated pathways in male germ cell differentiation, RT-qPCR was carried out to detect Dicer mRNA expression in juvenile mouse testes along with specific type of germ cells such as pachytene spermatocytes and round spermatids.

(A) (B)

Figure 4. Germ specific cell isolation from male mouse testis by centrifugal elutriation. (A) Pachytene spermatocytes (B) and round spermatids, both account up to 80 % of the total cells. Slides were fixed in a fixation solution, mounted with DAPI and visualized in the Leica DFC320 digital colour camera using 40X objective. All captured images were processed equally to reduce the background noise.

Testis samples isolated from mice sacrificed at different time points during the synchronized first wave of spermatogenesis represent different phases of germ cell differentiation within the seminiferous epithelium. Testis collected at 8 dpp contains spermatogenic cells just prior to or at the onset of meiosis; at 14 dpp pachytene spermatocytes appear, at 20 dpp round spermatids have already been generated and at 28 dpp the elongation of spermatids has started. Specific types of germ cells were isolated by centrifugal elutriation (41). From our optimized protocol of centrifugal elutriation, pachytene spermatocytes were enriched up to 80-90 % (Fig. 4A), whereas round spermatids were refined up to 70-80 % of total number of cells, with the predominant contaminating cell population being elongated spermatids (Fig. 4B). The total RNA was extracted from total testis and isolated germ cells and quantified using NanoDrop spectrophotometer (Table 2). Table 2 includes RNA concentrations of the samples from wild type (WT) and knockout (KO) testes and also from isolated pachytene spermatocytes and round spermatids.

Table 2. Quantification of total RNA extracted from wild (WT) and knockout (KO) testes, or from isolated pachytene spermatocytes (Pspc) and round spermatids (RS).

Sample ID 1 WT Testis

2 WT Testis

3 WT Testis

4 WT Testis

5 WT Testis

6 WT RS - 18/04

7 WT Pspc - 18/04

30 (45) WT Testis

13 (44) KO Testis

Matteo's WT Testis

Matteo's KO Testis

Age/

days post partum (dpp)

8 dpp 14 dpp 20 dpp 28 dpp 34 dpp Adult Adult 28 dpp 28 dpp Adult Adult

RNA Conc.

(ng/µl)

1292.9 1313.9 1554.5 2239.5 1994.8 329.4 415.6 749.3 885.5 1426.3 915.3 260/280 2.02 2.03 1.99 2.01 2.01 1.93 1.92 2.00 2.00 2.01 2.01

Dicer mRNA expression during the first wave of spermatogenesis was monitored by RT- qPCR. Higher levels of Dicer mRNA was detected at 8 dpp and 14 dpp than at 20 dpp, 28 dpp and 34 dpp (Fig. 5). These results suggest that Dicer expression is relatively higher in spermatogonia and early spermatocytes and the expression is decreased in late spermatocytes and round spermatids; appearance of these later cell types dilutes the Dicer signal, which results in the decrease in the relative Dicer expression detected from total testis RNA. This is further supported by low levels of Dicer mRNA in late pachytene spermatocytes (Pspc) and round spermatids (RS). It has to be kept in mind that as RNA was extracted from the total testis tissue, a part of the signal represents the Dicer expression in the somatic cells of testis such as Sertoli cells, Leydig cells, interstitial cells and peritubular myoid cells.

Next we verified the expression patterns of two selected Dicer-dependent miRNAs, miR-184

and miR-34c that we wanted to use in our studies. miR184 has been suggested to be specific for

round spermatids (unpublished) and has been demonstrated to be associated with germline cell

proliferation, patterning and embryonic development in Drosophila (32). miR-34c is specifically

expressed in the late stages of spermatogenesis (30) and has reported roles in the cellular

senescence, apoptosis and control of the cell cycle (42, 43). miRNA RT-qPCR was performed with

similar sets of samples as used for Dicer mRNA detection.

Figure 5. Expression of Dicer during spermatogenesis. Dicer expression in postnatal testis samples collected at 8 dpp, 14 dpp, 20 dpp, 28 dpp and 34 dpp, and in late pachytene spermatocytes (Pspc) and round spermatids (RS). The RT-qPCR data was normalized with mRNA expression of the reference gene L19. The mean values and standard error of at least two independent experiments are shown.

Interestingly, It was shown that both miR-184 and miR-34c expressions were gradually increased from 8 dpp to 34 dpp (Fig. 6). miR-184 expression appeared to peak in round spermatids (Fig. 6A) whereas miR-34c seemed to be more widely expressed (Fig. 6B). miR-34c signal appeared at 14 dpp corresponding to the appearance of pachytene spermatocytes and its expression was further increased at 20, 28 and 34 dpp, indicating that it is also present in the round and elongating spermatids.

(A)

8 dpp 14 dpp 20 dpp 28 dpp 34 dpp Pspc RS

0.00 0.20 0.40 0.60 0.80 1.00 1.20

Relative Dicer expression

8 dpp 14 dpp 20 dpp 28 dpp 34 dpp Pspc RS

0.00 0.20 0.40 0.60 0.80 1.00 1.20

Relative miR-184 expression

(B)

Figure 6. Expression of miRNAs during spermatogenesis. Expression of (A) miR-184 and (B) miR-34c in postnatal testis samples (8 dpp, 14 dpp, 20 dpp, 28 dpp and 34 dpp) and also in isolated pachytene spermatocytes (Pspc) and round spermatids (RS). miRNA RT-qPCR data was normalized with the U6 mRNA expression (reference gene). Each RT reaction was carried out at least two times and used to calculate the mean and standard error.

3.2) Confirmation of the downregulation of Dicer and Dicer-dependent miRNAs in germ cell–specific Dicer1 knockout mice

To validate the deletion of Dicer1 gene in our Dicer1 knockout mouse model, we performed a RT-qPCR analysis from the WT and KO testis RNA by using primers amplifying Dicer mRNA.

One primer was designed to hybridize within the deleted area, and thus no product is expected when Dicer1 has undergone Cre-loxP-mediated deletion. Dicer mRNA expression showed a sharp drop in the Dicer1 knockout testes (Fig. 7A). Furthermore, we were interested in confirming the absence of Dicer-dependent miRNAs in our knockout model. The expressions of both miR-184 and miR-34c were dramatically reduced in KO compared to WT 28 days old adult testes (Fig. 7B and C).

(A)

WT Adult Testis KO Adult Testis 0.0

0.2 0.4 0.6 0.8 1.0 1.2

Relative Dicer mRNA expression

8 dpp 14 dpp 20 dpp 28 dpp 34 dpp Pspc RS

0.000 0.200 0.400 0.600 0.800 1.000 1.200

Relative miR-34c expression

(B) (C)

Figure 7. Validation of the Cre-mediated deletion of Dicer1 gene in Dcr(fx/fx);Ngn3Cre knockout testes.

Expression of (A) Dicer mRNA (B) miR-184 and (C) miR-34c in the Dicer1 KO versus WT testes. The RT-qPCR was performed with adult or 28 days old testes RNA samples. The data was normalized with L19 (for Dicer mRNA) or U6 mRNA (for miRNAs) expressions. Each reaction was performed at least two times and used to calculate the mean and standard error.

Nevertheless, some expression of Dicer or miRNAs in the knockout mouse may come from the somatic cells present in the testis. These results confirm that the Cre-mediated deletion of Dicer1 gene is efficient in the testes of Dicer1 knockout mice.

3.3) Expression of transposable elements and centromeric repeat transcripts in Dicer1 knockout testes

Transposons are the mobile genetic elements that can move from one location to another in the genome (44). In recent years, the regulatory role of transposons have been implicated in many biological processes such as heterochromatin and epigenetic control and regulatory roles in human embryonic stem cells (45, 46). Expression of transposable elements is induced in mouse oocytes lacking Dicer (16).

(A) (B)

28 days Testis Adult Testis 0.00

0.50 1.00 1.50 2.00 2.50

Relative Line1 expression

28 days Testis Adult Testis 0.00

0.50 1.00 1.50 2.00 2.50

Relative SineB1 expression

28 days Testis Adult Testis 0.000

0.200 0.400 0.600 0.800 1.000 1.200

Relative miR-34c expression

28 days Testis Adult Testis 0.000

0.200 0.400 0.600 0.800 1.000 1.200

Relative miR-184 expression

(C)

Figure 8. Expression of transposable elements in the Dicer1 knockout testes. (A) Line1 (B) SineB1 and (C) IAP in 28 days old and in adult WT versus KO testes. The RT-qPCR data was normalized with L19 mRNA expression.

Reactions were repeated at least twice for each transposable elements and calculated mean value and standard error.

In addition, human Dicer mediates the degradation of Alu/SINE transposable elements in retinal pigmented epithelium, and Dicer deficit induces Alu RNA accumulation in the age-related muscular degeneration (19). So we attempted to check the expression of transposable elements (Line1, SineB1 and IAP) in our Dicer1 knockout mouse model. We found no significant change in the expression of transposable elements in 28 days old or adult Dicer1 knockout testes as compared to wild type (Fig. 8).

Centric and pericentric heterochromatin domains consist of minor and major satellites respectively (47). It is found that active protein coding genes also reside within centromeric chromatin (48). They are silenced through RNA interference pathways in fission yeast (49). Dicer1 deficient mouse embryonic stem cells displayed severe defects in differentiation and maintenance of centromeric heterochromatin structure and centromeric silencing (12). Therefore, we prompted to analyze major and minor satellite expression in our Dicer1 knockout model using RT-qPCR. We demonstrated induction in major satellite expression in 28 days KO testes as compared to WT testes (Fig. 9B) whereas no product was detected in the negative control (Fig. 9C). The primers were designed so that different sized bands should be detected depending on whether the PCR product consists of one, two or three repeats (Fig. 9A). In our optimized conditions of 29 cycles for RT- qPCR, only the shortest 308 bp product was detected. We further optimized the protocol by using standard non-quantitative PCR and were able to detect the 542 band in addition to 308 band (Fig.

9D). Interestingly, in contrast to 28 days old mice, testes of adult wild type mice showed expression of major satellite repeats, suggesting that Dicer may be involved in the temporal regulation of major satellite expression and by silencing prevents their premature induction. We did not find any significant differences in the expression of minor satellite in Dicer1 knockout testes (data not shown).

28 days Testis Adult Testis 0.00

0.50 1.00 1.50 2.00 2.50

Relative IAP expression

(A)

(B)

(C)

(D)

Figure 9. Induction of pericentric repeat (major satellite) transcripts in the Dicer1 knockout mouse testis.

(A) A diagram of mouse major satellite repeats showing the organization and primers used to detect transcripts. (B) RT positive reactions for L19 and major satellite were performed with triplicates for each sample. (C) RT negative reactions for L19 and major satellite. RT-qPCR reactions were run for 29 cycles. (D) Verification of major satellite by using normal PCR in an optimized conditions for 27 cycles. All the PCR products were run in 3% agarose gel to visualize the bands of transcripts, and pictures were inverted and processed equally to reduced the background noise.

4. Discussion

Spermatogenesis is a complex and precisely regulated dynamic process, which occurs throughout the adult life of mammals. The exact regulatory mechanisms of meiotic and postmeiotic events in the spermatogenesis are still unclear. Thus, the current project has been focused on Dicer- dependent small RNA pathways and their involvement in the spermatogenesis process.

Dicer is an endoribonuclease which acts as central processing enzyme for siRNA, endo- siRNA and miRNA pathways and it globally affects the processing of these small RNAs (6). The conditional Dicer1 knockout mouse represents an efficient model system to study tissue or cell- specific Dicer-mediated regulations in the mammals (37, 38). The importance of Dicer-dependent pathways are demonstrated in the various biological processes such as neural stem cell development and differentiation (14), differentiation of the male mouse germ cells (26), stem cell differentiation, maintenance of centromeric heterochromatin and silencing in the mouse (12), morphogenesis (8), degradation of retrotransposon (19), maintaining stem cells population in early mouse and zebrafish development (9, 10), formation of heterochromatin structure (20), maintenance and regulation of chromosome architecture (21), meiosis in the female mouse germline (16) and chromosome segregation (22).

In our conditional Dicer1 knockout mouse, Dicer1 deletion takes place around postnatal day 5 in spermatogonia (40). On microscopic level, Dicer1 knockout testes show disorganized stages of seminiferous epithelial cycle, increase in the number of apoptotic spermatocytes and decreased number of haploid cells (40). The main problems are found during haploid differentiation as defects in nuclear shaping and chromatin condensation of developing spermatozoa. miR-184 and miR-34c were shown to be highly expressed in male mouse germ cells. In this project, these miRNAs were chosen to check the functionality of Dicer-dependent pathways in male germ cells.

The expression of Dicer mRNA and miRNAs were found to be dramatically decreased in either 28 days or adult knockout testes (Fig. 7), signifying that our knockout mouse model is good enough to proceed further in the analysis.

Dicer mRNA and germ cell-specific miRNA expressions at different time points during

postnatal testis development were quantified using RT-qPCR. It has to be kept in mind that the total

testis RNA samples contain RNA also from the somatic cells of the testis (Sertoli cells, Leydig cells

and myoid cells), thus some of the Dicer mRNA signal may be derived from somatically expressed

Dicer which may interfere with the interpretation of the results. The higher expression of Dicer in

earlier time points as compared to later time points suggest either that (a) Dicer mRNA level is

higher in spermatogonia and early spermatocytes than in late spermatocytes and round spermatids,

or (b) Dicer is expressed at high levels in Sertoli cells and when Sertoli cells get diluted in the

epithelium upon appearance of full spermatogenesis, the level of Dicer expression as detected from

the total testis RNA decreases (Fig. 5). Importantly, RT-qPCR from purified pachytene

spermatocytes and round spermatids confirmed the expression of Dicer in meiotic and postmeiotic

male germ cells. In addition, miR184 and miR34c expressions were shown to increase gradually in

the course of the progress of first wave of spermatogenesis (Fig 6), thus demonstrating that Dicer is

active in later germ cell types despite the relatively low expression of Dicer mRNA. Dicer is known

to be involved in the regulation of somatic cells of gonads, as demonstrated by the knockout mouse models with selective ablation of Dicer in Sertoli cells of testis (35) or mesenchyme-derived cells of female reproductive organs (17). These studies delineate the complexity of the regulation of germ cell differentiation and the significance of other germ cell-associated surrounding cells for the normal progress of spermatogenesis.

Dicer-dependent small RNAs may function by several different mechanisms in male germ cells. miRNAs and endo-siRNAs are known to control gene expression by targeting protein-coding mRNAs. This is why it is important to check the changes in the Dicer1 knockout transcriptome, and this work is in progress in our laboratory. Dicer is also involved in the regulation of transposon and centromeric repeat expression. Centromeric transcripts are found to be induced in the mouse embryonic stem cell lines and chicken-human hybrid DT40 cell lines (12, 20). Interestingly, the induction of major satellite repeat expression at 28 day old knockout as compared to wild type testes (Fig. 9B) indicates that pericentric major satellite in differentiating male germ cells are controlled by Dicer-dependent mechanisms. I did not find any significant changes in the expression levels of transposable elements in our knockout mice (Fig. 8), even though transposons were found to be aberrantly expressed in the mouse oocytes lacking Dicer1 (16), and increased transposon expression was detected in Ddx4Cre Dicer1 knockout mice, in which Dicer is deleted in prospermatogonia (50).

In conclusion, the Dicer deletion affects small RNA processing that is required for normal

spermatogenesis. In our mouse model, Dicer is deleted already in spermatogonia. The lack of gross

abnormalities in meiosis suggests that Dicer-dependent pathways are not essential for meiosis to

complete. The defects appeared only in postmeiotic cells and the phenotype points towards the

possible role of Dicer in the chromatin organization prior to and during the histone-protamine

transition. Whether these defects derive from earlier stages, for example from the disorganization of

pericentric heterochromatin in spermatogonia, will be characterized in the future. The discovery of

endo-siRNAs in male germline (34) opens interesting potential mechanisms to explain the

phenotype of male germ cell-specific Dicer1 knockout mice. Endo-siRNAs could be, for example,

involved in heterochromatin regulation and in the control of the major satellite via yet

uncharacterized mechanisms. The future research in our laboratory will aim for elucidating and

addressing these unknown molecular mechanisms hidden behind the postmeiotic defects. Revealing

the molecular pathways and components involved in the complex process of sperm production will

provide further understanding in the factors behind male infertility and may reveal potential targets

for developing novel male contraceptives in future.

5. Acknowledgements

Department of Physiology, Institute of Biomedicine University of Turku, Turku, Finland

I feel highly privileged to express my deep sense of gratitude to Noora Kotaja (Principal Investigator) for giving me an opportunity to conduct my thesis project. It has been a wonderful experience to work under her excellent supervision. I really appreciate her timely motivation and invaluable advices. A profound thanks to Oliver Meikar (PhD student) for his marvelous guidance throughout this research project work.

I would like to thank all other members of research group: Matteo Da Ros (PhD student), Hanna Korhonen (PhD student) for their continuous support and insightful comments while preparing this report, and also Ida Björkgren (PhD student) and Juho-Antti Mäkelä (PhD student) for their valuable suggestions and discussions during lab work. Special thanks to Tuula Hämäläinen for providing me good lab instructions and helping me at various circumstances, and also to other members of the department for their kind co-operation and help to complete this project work successfully.

Biology Education Centre (IBG) Uppsala University, Uppsala, Sweden

I would like to express my deepest and sincere gratitude to Prof. Håkan Rydin (programme director) and Prof. Lars Liljas (project coordinator) for their timely advice, and helping with issues related to master programme and master thesis research work. I would also like to thank Dr.

Katariina Kiviniemi Birgersson (international coordinator) for her suggestions, instructions and guidelines and Mrs Eva Damm (counselor and international coordinator) for her admirable counselling, help with administrative issues and arranging my health insurance coverage during project work in Finland.

Others

Last but not least, I would also like to thanks my parents, sister, brother Bhagwan Yadav

and friends for their endless and countless love and support.

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7. Appendix A

Optimized protocol for germ cells separation using centrifugal elutriation. Round spermatids (RS) are eluted in fractions 10-15 and pachytene spermatocytes (Pspc) are eluted in fractions 27-32.

S. No. Steps Flow rate (ml/min) Speed (rpm)

Load 15 2800

Wash I

1 21.12

2 20.48 2800

3 21.07

Wash II

4 25.09

5 25.07

6 25.21 2800

7 25.03

8 25.15

9 25.32

RS

10 31.57

11 31.45

12 31.19 2800

13 31.33

14 31.44

15 31.29

Wash I

16 14.06 2000

17 14.27

18 14.57

19 14.15

20 14.22

Wash II

21 25.15

22 25.45

23 25.33 2000

24 25.55

25 25.44

26 25.13

Pspc

27 39.47

28 39.46

29 39.47 2000

30 39.48

31 39.47

32 39.47

33 Final wash Switch off the centrifugation