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The DNA damage checkpoint protein RAD9A isessential for male meiosis in the mouse

Ana Vasileva1,2, Kevin M. Hopkins1, Xiangyuan Wang2, Melissa M. Weisbach2, Richard A. Friedman3,Debra J. Wolgemuth2,* and Howard B. Lieberman1,4,*1Center for Radiological Research, College of Physicians and Surgeons, Columbia University Medical Center, 630 West 168th St., VC 11-219/220,New York, NY 10032, USA2Genetics & Development and Obstetrics and Gynecology, The Institute of Human Nutrition, Herbert Irving Comprehensive Cancer Center, ColumbiaUniversity Medical Center, Russ Berrie 608, 1150 St. Nicholas Avenue, New York, NY 10032, USA3Biomedical Informatics Shared Resource of the Herbert Irving Comprehensive Cancer Center and Department of Biomedical Informatics, ColumbiaUniversity Medical Center, 1130 St. Nicholas Avenue, Room 824, New York, NY 10032, USA4Department of Environmental Health Sciences, Mailman School of Public Health, Columbia University Medical Center, New York, NY 10032, USA

*Authors for correspondence (DJW3@columbia.edu; HBL1@columbia.edu)

Accepted 28 May 2013Journal of Cell Science 126, 3927–3938� 2013. Published by The Company of Biologists Ltddoi: 10.1242/jcs.126763

SummaryIn mitotic cells, RAD9A functions in repairing DNA double-strand breaks (DSBs) by homologous recombination and facilitates the

process by cell cycle checkpoint control in response to DNA damage. DSBs occur naturally in the germline during meiosis but whetherRAD9A participates in repairing such breaks is not known. In this study, we determined that RAD9A is indeed expressed in the malegerm line with a peak of expression in late pachytene and diplotene stages, and the protein was found associated with the XY body. As

complete loss of RAD9A is embryonic lethal, we constructed and characterized a mouse strain with Stra8-Cre driven germ cell-specificablation of Rad9a beginning in undifferentiated spermatogonia in order to assess its role in spermatogenesis. Adult mutant male micewere infertile or sub-fertile due to massive loss of spermatogenic cells. The onset of this loss occurs during meiotic prophase, and there

was an increase in the numbers of apoptotic spermatocytes as determined by TUNEL. Spermatocytes lacking RAD9A usually arrested inmeiotic prophase, specifically in pachytene. The incidence of unrepaired DNA breaks increased, as detected by accumulation of cH2AXand DMC1 foci on the axes of autosomal chromosomes in pachytene spermatocytes. The DNA topoisomerase IIb-binding protein 1(TOPBP1) was still localized to the sex body, albeit with lower intensity, suggesting that RAD9A may be dispensable for sex body

formation. We therefore show for the first time that RAD9A is essential for male fertility and for repair of DNA DSBs during meioticprophase I.

Key words: RAD9A, Double-strand breaks, Meiosis, Spermatogenesis

IntroductionMouse Rad9a is an evolutionarily conserved gene with a diverse

set of functions important primarily for promoting genomic

integrity (Lieberman, 2006). RAD9A protein is a member of the

heterotrimeric 9-1-1 complex, which consists of RAD9A, HUS1

and RAD1, and carries out many of its activities as part of this

complex. Deletion of Rad9a causes embryonic lethality (Hopkins

et al., 2004) and mice with a conditional knock out in

keratinocytes demonstrate enhanced susceptibility to the

development of carcinogen-induced skin tumors (Hu et al.,

2008). Mice heterozygous for Rad9a are prone to spontaneous as

well as radiation-induced cataractogenesis (Kleiman et al., 2007).

Conversely, aberrantly high levels of human RAD9A play a

functional role in prostate carcinogenesis (Broustas and

Lieberman, 2012; Zhu et al., 2008).

The RAD9A protein is intimately involved in the cellular

response to DNA damage. It regulates cell cycle checkpoints in

mitosis through participation in both the ATR as well as ATM

signaling pathways (Furuya et al., 2004; Lee et al., 2007; Niida

and Nakanishi, 2006). Human RAD9A colocalizes with the

phosphorylated form of histone H2A variant X (cH2AX) after

DNA damage, and this is independent of ATM function (Greer

et al., 2003). Furthermore, RAD9A inactivation delays the

appearance of ionizing radiation-induced cH2AX foci, which

together argue that RAD9A may modulate chromatin structure in

response to DNA damage (Pandita et al., 2006). RAD9A also

influences DNA repair directly. It can physically interact with

proteins involved in base excision repair, mismatch repair and

homologous recombination repair, and consequently, modify

their activity and modulate the respective DNA repair pathways

(Chang and Lu, 2005; Gembka et al., 2007; Guan et al., 2007; He

et al., 2008; Pandita et al., 2006; Park et al., 2009; Smirnova et al.,

2005; Toueille et al., 2004; Wang et al., 2004). There is also

evidence that RAD9A participates in nucleotide excision repair

(Li et al., 2013).

DNA repair is an important part of the normal progression of

meiosis and the function of the 9-1-1 complex for meiosis in vivo

has been demonstrated in several non-mammalian systems. The

S. cerevisiae homolog of RAD9A, Ddc1, is found at sites of

double-strand breaks (DSBs) in meiosis and is required for the

pachytene checkpoint (Hong and Roeder, 2002). It appears that in

this model, the 9-1-1 complex is bound to the synaptonemal

complex (SC) via Red1 and is essential for SC formation

(Eichinger and Jentsch, 2010). A possible role in DNA repair for

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one member of the complex, HUS1, had been described during

oogenesis in Drosophila melanogaster wherein females with a

null hus1 mutation were sterile, mainly due to oocyte nuclear

defects similar to those produced by mutations in the spindle

class of DNA DSB repair enzymes (Abdu et al., 2007).

Furthermore, the Drosophila hus1 gene is required for proper

disassembly of the SC for efficient processing of DNA DSBs

(Peretz et al., 2009).

The roles of mammalian RAD9A in cell cycle checkpoint

control, apoptosis and DNA repair are well established in

mitotically dividing cells. All of these activities are critical for

gametogenesis, but no prior studies have documented

involvement of the 9-1-1 complex and specifically RAD9A in

mammalian meiosis. The early embryonic lethality of Rad9a null

mice is accompanied by increased apoptotic activity and reduced

cellular proliferation (Hopkins et al., 2004). While these findings

clearly underscore that Rad9a is an essential gene in mammals,

the embryonic lethality obviated understanding its function in the

germ line by use of this null mutation. We initially determined

RAD9A’s pattern of expression in the male germ line and then

examined the effect of loss of its function on the progression of

cells through mitosis and meiosis. In order to investigate its

function during spermatogenesis, we generated mice in which a

conditional Rad9a allele was ablated specifically in

undifferentiated spermatogonia by Cre recombinase expressed

under the Stra8 promoter. While the mitotic divisions of

spermatogonia appeared to be unaffected, RAD9A-deficiency

had drastic effects on meiosis. We determined for the first time

that RAD9A is essential in males for progression through meiosis

and for the efficient repair of meiotic DNA DSBs.

ResultsThe distinct expression pattern of RAD9A in testicular

cells suggests a role in meiosis

It is well established that in somatic cells RAD9A has multiple

functions, including DNA repair and checkpoint control

(Lieberman, 2006). To determine whether RAD9A has similar

functions in male germ cells, we first characterized the pattern of

expression of its protein and mRNA in wild type (WT) mouse

testis. We examined the abundance of RAD9A protein by

immunoblotting of total testis extracts from mice of ascending

age (Fig. 1A). With the onset of meiosis at postnatal day (pnd)

,10, the level of RAD9A increased and peaked coincident with

the abundance of spermatocytes in late pachytene and diplotene

stage at pnd18 (Bellve et al., 1977). The appearance of haploid

germ cells at ,pnd20 and their relative prevalence in the testis

thereafter was accompanied by a drop in RAD9A levels at pnd28

and in adult testis. This most likely reflected low or no expression

in spermatids. To test this hypothesis and determine the cellular

localization of the protein, we detected RAD9A in testis by

immunohistochemistry and observed strong signal in the nuclei

of prophase spermatocytes (Fig. 1B, red arrows). Lower levels of

Fig. 1. Levels of RAD9A protein in mouse testis. (A) RAD9A protein abundance detected by immunoblotting of total testis extracts (50 mg/lane) isolated from

juvenile mice of ascending age, adult (Ad) wild-type testis and wild-type mouse ES cells. Beta-actin was a loading control. Mouse age (postnatal day, pnd) is

indicated under each lane. (B) Localization of RAD9A protein in adult mouse testis determined by immunohistochemistry in sections from adult Rad9af/+ testis at

406 (left panel or at 1006, right panel). RAD9A signal (brown) was detected in the nucleus of pachytene spermatocytes (red arrows) and Sertoli cells (green

arrowheads). Roman numerals indicate the stage of the mouse seminiferous epithelial cycle. Hematoxylin (blue) marks the nuclei. Spermatids were only weakly

labeled. (C) Immunostaining for RAD9A, SYCP3 and DMC1 in squash preparations of adult seminiferous tubules. Left panels show RAD9A (green) and DAPI

(blue; revealing nuclear localization); middle panels, SYCP3 (far red), RAD9A (green); right panels, DMC1 (red), RAD9A (green). Upper panels: leptotene/

zygotene stage; lower panels: pachytene stage. In leptotene/zygotene spermatocytes, there are bright DMC1 foci at DSBs and the RAD9A signal is diffuse

with some speckled aggregates. In pachytene, RAD9A localizes primarily to the XY body (arrowhead), with a few additional regions staining positive, in distinct

foci not associated with chromatin. (D) Colocalization of RAD9A (green) with SUMO1 (red). SUMO1 was not detected in zygotene spermatocytes (upper panel).

In pachytene spermatocytes (lower panel) RAD9A localized primarily to the XY body (arrowhead) with a few additional foci as in C. Colocalization of RAD9A

and SUMO1 in a pachytene spermatocyte confirms localization of RAD9A to the XY body (arrowhead).

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nonetheless consistent signal were also seen in Sertoli cells

(Fig. 1B, green arrowheads). Spermatids contained a

comparatively weak signal, if at all, and spermatogonia did not

seem to express RAD9A. It is possible, however, that basal

expression of RAD9A in spermatogonia is below the detection

limits of the assay.

These data are consistent with the profile of Rad9a RNA

expression measured by qRT-PCR in total RNA isolated from

testes of similarly aged juvenile mice (supplementary material

Fig. S1A). RNA levels reflected RAD9A protein expression, with

an increase at the entry of germ cells into meiosis and peaking

toward the end of the first meiotic prophase at pnd17. This trend

was supported by the higher levels of Rad9a RNA in isolated

primary spermatocytes as compared to round spermatids

(supplementary material Fig. S1A). The cellular distribution of

Rad9a RNA was also confirmed by in situ hybridization analysis

on adult testis, where Rad9a signal was detected in meiotic

prophase spermatocytes and also in Sertoli cells, but not in round

spermatids nor in spermatogonia (supplementary material Fig.

S1B).

To determine the distribution and precise sub-cellular

localization of RAD9A in spermatocytes, we carried out a

detailed immunofluorescence study on squash preparations of

testicular tubules. Staging of meiotic prophase I nuclei was based

on morphology of the SC (detected with mouse anti-SYCP3

antibody), as well as the presence of foci of DNA DSBs

(visualized with rabbit anti-DMC1 antibody). Leptotene-to-

zygotene spermatocytes are characterized by fragmentary

SYCP3 signal on homologous chromosomes and the presence

of multiple DMC1 foci (Fig. 1C, top row) (Moens et al., 2002).

At this stage, we found RAD9A clustered in multiple foci as well

as diffusely distributed throughout the nucleus. In contrast, by

mid-pachytene, when DSBs on autosomes are repaired and

chromosome synapsis is complete, RAD9A localizes primarily to

the XY body (Fig. 1C, bottom row). There were a few remaining

strong RAD9A foci throughout the nucleus, which apparently

were not colocalized with SYCP3 at axial elements (AE) on

chromosomes. In pachytene spermatocytes, SUMO1 appears

exclusively at the X and Y bivalent (Rogers et al., 2004).

Colocalization of RAD9A with SUMO1 confirmed its

association with the XY body at this stage (Fig. 1D). This

distinct nuclear distribution of RAD9A in spermatocytes suggests

at least two possible functions for RAD9A during male meiosis –

participation in the repair of DSBs and/or a role in the formation

and maintenance of the XY body.

Mice with conditional deletion of Rad9a in the testis are

infertile

Whole body Rad9a deletion in mice is embryonic lethal (Hopkins

et al., 2004), thus precluding analysis of RAD9A-deficient germ

cells under these conditions. Therefore, to study the role of the

protein in spermatogenesis it was necessary to generate animals

with a male germ cell-specific deletion of a floxed Rad9a allele.

As we reported previously (Hu et al., 2008), Rad9af/f mice are

viable and normal, consistent with our expectation that the

‘floxed’ Rad9a allele is functionally WT. We crossed

homozygous Rad9af/f mice with a ‘deleter’ strain, in which Cre

expression is driven by the Stra8 promoter, shown previously to

drive Cre expression in undifferentiated type A spermatogonia

(Sadate-Ngatchou et al., 2008). Excised floxed alleles are

designated as Rad9adel to distinguish them from the originally

obtained Rad9a2 alleles (Hopkins et al., 2004). We compared

expression of Rad9a in Rad9af/+, heterozygous-like Rad9af/del,

and mutant Rad9af/delCre+ testes, and found about half the levels

of Rad9af/+ expression in Rad9af/del testes and over a 3.5-fold

reduction of RNA levels in the mutant testes (Fig. 2A).

Next, we determined if loss of RAD9A function in the germ line

affects the reproductive potential of the mutant males. Three

rounds of mating of an initial cohort of Rad9af/delCre+ males

(n57) each with two wild-type CD1 females resulted in complete

infertility (Table 1; supplementary material Fig. S2A–C, group

Rad9af/delCre+-1). A second cohort of Rad9af/delCre+ males (n57)

was produced by further matings of the Rad9af/+Cre+ progeny,

which was maintained on a mixed 129SvEv, C57Bl/6 and

FVB background to Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J

originating from a cross between 129X1/SvJ and C57Bl/6J. Most

mutant mice from this cohort were initially sub-fertile rather than

completely infertile (Table 1; supplementary material Fig. S2A–C,

group Rad9af/delCre+-2). Overall, nine out of fourteen (64.3%)

males did not produce any pups over the course of three months.

The five remaining males exhibited declining fertility as they only

fathered pups during the first or the first and second rounds of

Fig. 2. Testis-specific deletion of Rad9a leads to reduced testis size

and loss of germ cells. (A) Quantitative RT-PCR for Rad9a mRNA in

whole testis from Rad9af/+, Rad9af/del and Rad9af/delCre+ mice. Arbp is

used as an internal control. The twofold reduction in Rad9a mRNA level

in the heterozygous-like Rad9af/del mouse testis is similar to that in

Rad9a+/2 ES cells (Hopkins et al., 2004). In the Rad9af/delCre+ mutant,

Rad9a message was reduced to less than a third of that in the Rad9af/+

control. Expression of Rad9a in Sertoli cells probably accounts for the

levels detected. (B) Testes from Rad9af/+, Rad9af/del and Rad9af/delCre+

mice. Rad9af/+ and Rad9af/del mouse testes were equivalent in size.

However, the Rad9af/delCre+ mouse testis was one-third the size of the

other two. (C) Histological examination of testicular sections from adult

Rad9af/+ (left), Rad9af/del (middle) and Rad9af/delCre+ (right) littermates

stained with periodic acid-Schiff (PAS). The testicular section from a

sterile Rad9af/delCre+ male shows depletion of germ cell populations in

most tubules, vacuolization, agametic tubules (asterisks) and lack of

round or elongating spermatids. Tubules with one layer of cells were

found at stages where there should normally be three to four layers.

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mating. Statistical analysis of the three rounds of matings was

performed and mouse cohorts with respective genotypes were

grouped into classes (‘a’ and ‘b’) according to similarities in

statistically significant differences in litter size. These analyses

demonstrated that after the first round, the fertility of the first

Rad9af/delCre+ cohort (Table 1, mice 1–7; supplementary material

Fig. S2A, Rad9af/delCre+-1) formed a class of its own

(supplementary material Fig. S2A, class b) and was significantly

different from the three other groups (supplementary material

Fig. S2A, class a), while that of the second Rad9af/delCre+

cohort (Table 1, mice 8–14; supplementary material Fig. S2A,

Rad9af/delCre+-2) was not significantly different from that of

Rad9af/+ and Rad9af/del littermates (Table 1; supplementary

material Fig. S2A, class a). However, after the second and third

matings (supplementary material Fig. S2B,C), the fertility of

Rad9af/+ and the second Rad9af/delCre+ cohort was found to be

significantly different and they fell into two different classes, a and

b, respectively.

After completion of the study at 5.5 months of age, the

reproductive tracts of the mated males were excised and

examined at the gross morphological and histological levels.

Testes of the completely infertile Rad9af/delCre+ males were

about a third of the size of Rad9af/+ or Rad9af/delCre2 testes

(Fig. 2B), and testicular sections showed severe loss of germ

cells (Fig. 2C). The most severely affected tubules had gross

vacuolization (Fig. 2C, asterisks) and appeared almost empty,

containing mainly Sertoli cells, spermatogonia and an occasional

spermatocyte.

We next compared the morphology and the degree of

disruption of spermatogenesis in the sub-fertile cohort with that

of the completely infertile mutant males (supplementary material

Fig. S3). Histological analysis of epididymal sections showed

that at the end of the study, both the initially sub-fertile

(supplementary material Fig. S3B) and the completely infertile

(supplementary material Fig. S3C) mutants were virtually devoid

of sperm, consistent with the low sperm counts and lack of

progeny at the third mating (Table 1). Although many testicular

tubules from sub-fertile males contained elongating spermatids

and even sperm (supplementary material Fig. S3E), they also

displayed a range of abnormalities characteristic of the

completely infertile cohort (supplementary material Fig. S3F)

but not seen in controls (supplementary material Fig. S3D). In

testes of infertile mice, between 91.2 and 97.8% of tubules

exhibited gross vacuolization with a single exception of a sterile

male having only 12.5% of tubules containing more than two

large vacuoles. In contrast, only 2–10% of testicular tubules in

sub-fertile males were highly vacuolized. Note that such

vacuolization was not observed in the Rad9afl/+ testes.

Multinucleated giant cells were present in ,7% of testicular

tubules from infertile males (supplementary material Fig. S3F,

red arrows) and in ,2% of tubules in sub-fertile males

(supplementary material Fig. S4A,B, red arrows). Among the

multiple abnormalities found in both cohorts were condensed,

apoptotic nuclei of early prophase I spermatocytes

(supplementary material Fig. S3E,F and Fig. S4, black arrows)

and abnormal metaphase figures (supplementary material Fig.

S4F, arrowheads). Partial germ cell loss in sub-fertile testes

(supplementary material Fig. S3E) was typically characterized

by lack of second and/or third germ cell layers on half or more

of the tubule perimeter (supplementary material Fig. S3E,

asterisk and Fig. S4C, asterisks). Interestingly, spermatogonia

appeared normal (supplementary material Fig. S4, marked with

‘s’).

We next asked whether incomplete excision of the floxed

allele could contribute to the heterogeneity of the mutant

phenotypes. We first examined the efficiency of the Rad9a

‘flox’ allele excision by immunostaining for RAD9A protein in

Rad9af/+ and Rad9af/delCre+ testicular sections. Rad9af/+

pachytene spermatocytes stain positive for RAD9A at the XY

body and form a continuous layer in the tubule (Fig. 3A).

Table 1. Summary of Rad9af/+, Rad9af/del and Rad9af/delCre+ mouse fertility study

Number of pups and embryos sired

Male genotype Testis weight (mg) Sperm count (6104) First mating Second mating Third mating

Rad9a f/delCre+

Mouse 1a 27.50 0 0 0 0Mouse 2a 35.00 163 0 0 0Mouse 3a 24.90 16 0 0 0Mouse 4a 27.90 0 0 0 0Mouse 5a 19.20 0 0 0 0Mouse 6a 17.00 0 0 0 0Mouse 7a 18.25 1 0 0 0Mouse 8b 15.15 0 0 0 0Mouse 9b 42.95 48.5 13 0 0Mouse 10b 44.75 490 31 0 0Mouse 11b 54.95 94 7 31 0Mouse 12b 32.45 34.5 23 8 0Mouse 13b 28.6 30 9 4 0Mouse 14b 32.1 0 0 0 0Rad9a f/del 74.8 505 23 17 13Rad9a f/del 95.6 3230 39 35 19Rad9a f/del 90.8 4630 22 19 25Rad9a f/+ 113.05 3750 30 25 29Rad9a f/+ 118.6 2562 25 30 19Rad9a f/+ 98.8 3290 24 20 28Rad9a f/+ 101.0 4700 34 26 31

aData from the first cohort of Rad9a f/delCre+ males; bdata from the second cohort of Rad9a f/delCre+ males.

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However, there were RAD9A-positive spermatocytes along part

of the perimeter of the tubules in mutant testes (Fig. 3B, arrows),

suggesting that Cre excision had not occurred in these cells.

Immunohistochemical staining of testicular sections with our

anti-RAD9A antibody revealed that RAD9A was still expressed

in ,37% of the remaining spermatocytes in testes of sub-fertile

(supplementary material Fig. S5B) and infertile (supplementary

material Fig. S5C,D) Rad9af/delCre+ males. This was not

surprising in view of the previously reported lack of expression

of the Stra8-Cre transgene (Sadate-Ngatchou et al., 2008) in

some PLZF-positive spermatogonia, resulting in a failure to

excise a floxed allele (Hobbs et al., 2012). The RAD9A-deficient

spermatocytes detected appeared abnormal, as indicated by a

weak and fragmentary SYCP3 signal (Fig. 3B, right panel).

We therefore asked whether this inefficiency of excision was

due to low Cre activity or was dependent on locus context. To

assess Cre activity, we crossed Rad9af/delCre+ mice with a

second mouse line, Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J

(mT/mG), designed as a reporter for efficiency of excision

(Muzumdar et al., 2007). The efficiency of excision of

the tdTomato transgene was assessed by scoring the fraction

of cells expressing EGFP, i.e. fluorescence turned from red to

green, in single-cell suspensions from Rad9af/+Cre+Tom+ andRad9af/delCre+Tom+ testes using a previously reportedflow cytometric assay (Bastos et al., 2005; Vasileva et al.,

2009). In the Rad9af/+Cre+Tom+ adult testes, 91.4% ofthe prophase spermatocytes were EGFP positive, while in theRad9af/delCre+Tom+ male there were 96.5% EGFP-positivespermatocytes (these were the mice used in cohort 2 of the

fertility studies), which corroborates previously published .95%efficiency of excision for the Stra8-Cre transgene (Sadate-Ngatchou et al., 2008; Yang et al., 2012). Therefore, incomplete

excision of the Rad9a allele is not likely a consequence of lowCre activity or frequent occurrence of cells lacking Creexpression. Incomplete excision, as demonstrated by an

unrecombined floxed allele, was also observed in offspringborn to wild-type females mated to Rad9af/delCre+ males fromthe fertility study. Taken together, these data indicate that Rad9a

deletion in the Stra8-Cre model was incomplete and that theheterogeneity of the Rad9a mutant phenotype may be due toinefficient excision precluded by locus-specific genomiccontext of the Rad9a conditional allele within a subset of

spermatogonia.

RAD9A-deficient germ cells are eliminated by apoptosis

It is well established in cell lines that RAD9A has an anti-

apoptotic function (Kobayashi et al., 2004), and Rad9a2/2 mouseES cells demonstrate high spontaneous levels of apoptosis (Zhuet al., 2005). We therefore assessed the number of apoptotic cells

by TUNEL staining in histological testicular sections obtainedfrom the infertile mice used in the fertility study (Fig. 4A–C). Asis standard in such analysis of apoptosis in testicular tubules,

quantification of apoptosis is presented as the number ofapoptotic cells counted per 100 tubules and by calculating theapoptotic index (by multiplying the percentage of tubulescontaining apoptotic germ cells by the number of apoptotic

germ cells per tubule; Fig. 4D) (Salazar et al., 2005). While theRad9af/+ testes had the expected occasional apoptoticspermatocyte (6–9 TUNEL-positive cells/100 tubules), in the

mutant there were numerous positive spermatocytes (Fig. 4B,C).We determined the approximate stage of the seminiferous cycleof the mutant tubules and observed apoptotic spermatocytes

in tubules corresponding to stages beyond mid-pachytene(Fig. 4B,C, red arrowheads). Mutant males 1, 2, 3, 4 and 7 hadintermediate loss of germ cells (Fig. 4B) and showed an ,3-fold

increase in the number of apoptotic cells/100 tubules, and morethan 20-fold increase in apoptotic index, relative to controls. Asexpected, values for both the total apoptotic cells and theapoptotic index were downwardly skewed in mutants containing

mainly agametic tubules (males 5 and 6), due to massive overallgerm cell loss (Fig. 4C).

Spermatocytes lacking RAD9A arrest at the late zygoteneor early pachytene stage of meiotic prophase I

To identify the specific stage at which apoptosis was occurring,we quantified the proportion of each germ cell stage with specific

attention to the number of spermatocytes at different stages ofprophase I using flow cytometry as previously described (Bastoset al., 2005; Vasileva et al., 2009). In most mouse strains, the first

meiotic division in the first spermatogenic wave occurs aroundpnd18–19 and round spermatids appear at ,pnd20 (Bellve et al.,1977). We chose this age for our analysis as differences in

Fig. 3. Spermatocytes in Rad9af/delCre+ testis can escape floxed allele

excision. (A,B) Immunofluorescence was performed on testicular sections

from adult Rad9af/+ (A) and Rad9af/delCre+ (B) mice. Left panels: detection

of RAD9A (green), SYCP3 (red) and nuclei (by DAPI staining; blue). Right

panels: for clarity, only RAD9A and SYCP3 are shown. Pachytene

spermatocytes (red SYCP3 on chromosomes) in Rad9af/+ testis formed an

uninterrupted concentric circle with bright RAD9A signal (green) on the XY

body (A, right panel, white arrows). RAD9A-positive spermatocytes were

readily detected in Rad9af/delCre+ tubules indicating lack of ‘flox’ allele

excision (B, right panel, white arrows). A few spermatocytes with interrupted

chromosome cores and lower intensity of SYCP3 appear RAD9A-negative

(white arrowheads). Rad9af/+ tubules were filled with structured layers

including round and elongated haploid spermatids, and fewer ‘DAPI only’

haploid nuclei were detected in the lumen of Rad9af/delCre+ tubules,

confirming that these cells have escaped excision of the conditional

Rad9a allele.

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cellularity between mutant and Rad9af/+ testes would likely be

the direct result of loss of RAD9A function rather than a

secondary effect of massive germ cell loss due to improper cell–

cell contact and general degeneration of seminiferous tubules,

which is observed in adult mutant testes (Chung et al., 2004). As

assays performed on different days tend to have variable intensity

of the Hoechst staining, we adjusted the parameters in each assay

by matching a control Rad9af/+ littermate for every mutant

littermate. Testes of Rad9af/+ and of Rad9af/delCre+ littermates

contained similar numbers of spermatogonia and preleptotene

spermatocytes (Table 2; supplementary material Fig. S6).

Strikingly, a notable reduction in the number of late prophase

spermatocytes was detected in Rad9af/delCre+ as compared to

Rad9af/+ littermates. This was occasionally accompanied by an

increase in the numbers of early prophase I leptotene/zygotene

spermatocytes. We conclude that the majority of RAD9A-

deficient spermatocytes fail to progress through pachytene or

they arrest early in this stage. Spermatids were readily detected in

Rad9af/+ pnd20 littermates (,7.07%) but not in mutant testes cell

suspensions.

RAD9A is essential for efficient repair of DNA DSBs onautosomes during prophase I

We next asked what might trigger arrest in early pachytene and

ensuing apoptosis of RAD9A-deficient spermatocytes. We

speculated that, given the role of RAD9A in DNA repair,

spermatocytes lacking RAD9A may have defects in repairing the

DNA DSBs necessary for homologous recombination. The

pattern of distribution of cH2AX, a marker for DSBs, during

meiosis is well documented (Bellani et al., 2005; Mahadevaiah

et al., 2001). In leptotene and zygotene spermatocytes, it is

usually interspersed throughout the nucleus and disappears by

late zygotene, whereas in pachytene, strong cH2AX signal is

solely restricted to the sex body (Fig. 5A, left panel). However,

in some RAD9A-deficient pachytene spermatocytes, cH2AX

was still seen throughout the nuclei (Fig. 5A, right panel).

To investigate this characteristic at higher resolution, we

assessed cH2AX in spermatocyte spreads from Rad9af/+

(Fig. 5B, left panels) and Rad9af/delCre+ testes (Fig. 5B, right

panels). Both Rad9af/+ and Rad9af/delCre+ leptotene and

zygotene spermatocytes displayed cH2AX signal (red)

associated with SC at DSBs formed on autosomes (Fig. 5B, top

and middle, left and right panels, respectively). While pachytene

spermatocytes in the Rad9af/+ exhibited normal localization of

cH2AX to the sex body (Fig. 5B, lower left panel), mutant

spermatocytes exhibited various patterns of cH2AX distribution.

Approximately 29% of RAD9A-deficient mid-pachytene

spermatocytes and 21% of those late spermatocytes detected

displayed normal localization of cH2AX to the sex body

(Fig. 5B, lower right panel; supplementary material Fig. S7Ai

and Bi, green arrows and Table S1). However, we also detected

cH2AX associated with autosomes indicating unrepaired DNA

Fig. 4. Elevated levels of apoptosis in adult testes of infertile

Rad9af/delCre+ mice. (A–C) Testes from adult Rad9af/+ (A) and Rad9af/

delCre+ (B,C) littermates were stained using the TUNEL technique, and

sections were counterstained with Hematoxylin. Higher numbers of apoptotic

cells appear in Rad9af/delCre+ testes with intermediate (B) as opposed to

nearly complete germ cell loss (C). Tubules were staged according to Russell

et al. (Russell et al., 1990) and stages indicated with Roman numerals.

Asterisks after Roman numerals indicate that mutant tubules were staged with

the closest possible approximation to a normal seminiferous tubule cycle. In

the Rad9af/delCre+ testes prophase I spermatocytes were observed in tubules

of all stages (red arrowheads). (D) Quantification of TUNEL-stained

apoptotic cells was performed in round cross sections of tubules. Data were

collected from at least 100 tubules per section from three different sections

and average total apoptotic cells/100 tubules (gray columns) or apoptotic

index (black columns) are shown. Numbers for the Rad9af/+ genotype were

from two randomly picked males in this group (see Table 1; results from

Rad9af/delCre+ males are listed individually).

Table 2. Percentage of each germ cell population in testes of Rad9af/+ or Rad9af/delCre+ littermates at pnd20

Spermatocytes (%a)

Genotype of littermates Spermatogonia (2N; %a)Pre-leptotene

(S-phase)Leptotene and

zygotenePachytene and

diplotene Spermatids (%a)

Rad9a f/+ 3.17 2.91 7.12 15.3 6.77Rad9af/delCre+ 3.35 2.56 8.54 9.13 0.44Rad9a f/+ 2.91 3.89 8.34 17.5 2.33Rad9af/delCre+ 3.94 3.19 8.31 4.06 0.86Rad9a f/+ –b 3.72 7.33 14.7 8.45Rad9af/delCre+ –b 3.7 5.25 1.54 1.17

aCalculated as a fraction of the total live cell population, which includes somatic cells and 2N cells not shown from this analysis.bSuspensions prepared by mechanical maceration resulting in very low numbers of spermatogonia, as opposed to all other samples prepared by enzymatic

digestion.

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DSBs. The cH2AX distribution in the remaining RAD9A-

deficient spermatocytes appeared multilobular (supplementary

material Fig. S7Bi, asterisks; Table S1).

To further evaluate the efficiency of DNA DSB repair in the

Rad9a mutant and obtain data independent of cH2AX, which

also localizes to broken DNA in cells undergoing apoptosis, we

stained spread spermatocytes with antibodies against DMC1,

which specifically mark meiotic DSBs. Rad9af/+ mid-pachytene

spermatocytes had cleared DMC1 from autosomes completely

(Fig. 5C, top panel), while same stage spermatocytes from

mutant testes had retained numerous DMC1 foci on AEs (Fig. 5,

white arrowheads). As expected DMC1 foci were found on the X

Fig. 5. Rad9af/delCre+ spermatocytes contain residual, unrepaired DNA DSBs. (A) Co-immunofluorescence on paraffin-embedded testis tissue from pnd13

Rad9af/+ (left panel) and Rad9af/delCre+ (right panel) littermates. Pachytene spermatocytes have fully synapsed chromosomes marked by SYCP3 (green) and

correctly formed XY bivalents stained with cH2AX (red). Rad9af/delCre+ spermatocytes often have excessive cH2AX staining resembling an apoptotic response

due to DNA damage (arrowheads). DAPI was used to stain nuclei. (B) Examples of typical Rad9af/+ and Rad9af/delCre+ leptotene (top panels) and zygotene

(middle panels) spermatocytes, with partially synapsed chromosomes as indicated by interrupted SYCP3 signal and ‘clouds’ of cH2AX localized to sites of

obligatory DNA DSBs. Lower panels: normal localization of cH2AX at the XY body of a Rad9af/+ (left panel) and Rad9af/delCre+ (right panel) pachytene

spermatocytes. Autosomes in RAD9A-deficient pachytene spermatocytes retained cH2AX indicating persistent DNA damage (right panel). (C) Double

immunolabeling of DMC1 (red) and SYCP3 (green) was performed on spermatocyte spreads from Rad9af/+ (top panel) and two different Rad9af/delCre+ (middle

and bottom panel) testes. DAPI was used to stain nuclei. Mid-pachytene stage was determined by the extent of axial element formation and chromosome

compaction and lack of thickening at the ends. In Rad9af/+ pachytene spermatocytes, DMC1 is cleared from autosomes, indicating complete repair of DNA DSBs.

Multiple DMC1 foci were present on at least three autosomes in Rad9af/delCre+ spermatocytes at mid-pachytene (arrowheads). Dashed ovals indicate the location

of the sex body.

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chromosome (Moens et al., 2002), but appeared more prominent

compared to foci in Rad9af/+ spermatocytes (Fig. 5C, encircled

XY).

TOPBP1 localization in RAD9A-deficient spermatocytes

Persistence of cH2AX and DMC1 foci in RAD9A-deficient

pachytene spermatocytes indicates a requirement for RAD9A

protein in the repair of DNA DSBs. In S. pombe the Rad9

interacting protein TOPBP1 functions together with ATR in the

activation of the recombination checkpoint during late prophase

(Perera et al., 2004). A study in mitotic mammalian cells further

demonstrated that RAD9A has a role in homologous

recombination repair (Pandita et al., 2006). TOPBP1 is found

on asynapsed, but not on synapsed chromosome axes in zygotene

and also in the sex body in pachytene spermatocytes (Perera et al.,

2004).

We therefore examined the localization of TOPBP1 in

Rad9a conditional knockout spermatocytes. The distribution of

TOPBP1 was similar in leptotene and early zygotene Rad9af/+

and mutant spermatocytes (Fig. 6A,Bi for Rad9af/+ and

Fig. 6E,Fi for Rad9af/delCre+, respectively). In Rad9af/+ control

late zygotene nuclei, we observed multiple TOPBP1 foci aligned

with the SC only on asynapsed autosomes (Fig. 6Bii, blue

arrowheads). This is in contrast to the previously reported

localization of TOPBP1 on multiple asynapsed autosomes in

zygotene (Perera et al., 2004). However, in RAD9A-deficient late

zygotene spermatocytes strong TOPBP1 signal was present along

the axes of multiple synapsed autosomes (Fig. 6Fii, blue

arrowheads).

Rad9af/+ pachytene and diplotene spermatocytes exhibited the

characteristic intense cloud-like TOPBP1 signal encompassing

the sex body (Fig. 6Ci, Cii and D). Although other characteristics

such as cH2AX and SUMO1 localization in Rad9a mutant

pachytene spermatocytes appear normal, TOPBP1 signal had

lower intensity and was confined to the chromosome axes of the

XY chromosomes in the sex body. In addition, RAD9A-deficient

pachytene spermatocytes retained diffuse TOPBP1 signal on

autosomes (Fig. 6Gi and Gii). We did not observe TOPBP1 at

sites of fragmentary SC (Fig. 6Gi and Gii, white arrowheads,

magnified in insets), and aligned, but not fully synapsed bivalents

were typically observed in mid-pachytene nuclei (Fig. 6H,

yellow arrowheads, upper inset). Along with fragmentary SC,

RAD9A-deficient spermatocytes occasionally contained an

extended XY body domain encompassing autosomes (Fig. 6Gi

and H, green arrowheads, insets).

DiscussionStudies of meiosis in yeasts, fly and nematode indicate that the 9-

1-1 complex plays critical roles in several activities related to this

process, including DNA repair, partner choice for homologous

recombination and induction of the meiotic checkpoint

(Grushcow et al., 1999; Hofmann et al., 2002; Lieberman,

2006; Peretz et al., 2009). However, a meiosis-specific function

for mammalian 9-1-1, and in particular the RAD9A component,

has not yet been demonstrated, complicated in part by the fact

that the Rad9a null is embryonic lethal (Hopkins et al., 2004).

Therefore, we constructed and characterized a mouse strain

bearing a conditional Rad9a knockout in undifferentiated

spermatogonia to assess its role in spermatogenesis.

RAD9A is expressed in a distinct pattern in meiotic

prophase spermatocytes

We demonstrated that RAD9A was readily detected in

spermatocytes but surprisingly not in spermatogonia, implying

Fig. 6. Localization of TOPBP1 in Rad9af/+ and Rad9af/delCre+ spermatocytes. Rad9af/+ (top row) and confirmed RAD9-deficient Rad9af/delCre+ (bottom

row) nuclei labeled with anti-TOPBP1 (green), anti-SYCP3 (red), anti-RAD9A (far red, not shown) and DAPI (blue) are shown. Both Rad9af/+ (A) and

Rad9af/delCre+ (E) leptotene spermatocytes contain multiple TOPBP1 foci throughout the nucleus. In Rad9af/+ (Bi,Bii) and Rad9af/delCre+ (Fi,Fii) zygotene

spermatocytes, TOPBP1 is found along the unsynapsed regions of chromosome axes (blue arrowheads in Bi, Bii and Fii) and presumably on the X and Y (dashed

oval in Bii). TOPBP1 signal in Rad9af/+ (Ci,Cii) and RAD9A-deficient (Gi,Gii) pachytene nuclei was observed at the unsynapsed regions of the X and Y (dashed

ovals, X and Y label); however, it was quite weak in Rad9af/delCre+ pachytene-like spermatocytes (Gi,Gii,H) as opposed to a strong signal encompassing the

entire sex body in Rad9af/+ spermatocytes (Ci,Cii). RAD9A-deficient nuclei retain diffuse TOPBP1 staining along the SC of autosomes; white arrowheads

indicate interruptions in the SC in RAD9A-deficient pachytene spermatocytes (also insets in Gi,Gii); yellow arrowheads in H indicate paired homologs with

partial asynapsis (top inset); green arrowhead in Gi shows TOPBP1 retained on autosomes in pachytene stage; green arrowhead in H indicates a mixed

XY+autosomal domain (also H, bottom inset). Diplotene spermatocytes appeared normal in spreads from Rad9af/+ testis (D) and were not found in RAD9A-

deficient spermatocytes from Rad9af/delCre+ testes.

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a specific requirement during the meiotic phase of male germ cell

differentiation. Early in meiosis, during the leptotene stage,RAD9A protein was present throughout the nucleus.Subsequently, in zygotene, this diffuse nuclear staining was

accompanied by foci of RAD9A presumably associated with thechromosome axes, similar to the distribution of DMC1 foci. Thisobservation, together with the previously reported physicalassociation of RAD9A with RAD51 in mitotic cells (Pandita

et al., 2006), suggested a similar interaction between RAD9A andthe recombination related protein DMC1 during homology-directed repair of DSBs in meiosis. However, RAD9A foci did

not fully overlap with DMC1 foci (Fig. 1C), which suggests thatrather than a direct role in DNA end binding and homologysearch, RAD9A might be affecting DNA DSB repair indirectly,

via its association with the checkpoint apparatus. Similarobservations were made for RAD1, which did not colocalizecompletely with DMC1 in early meiotic prophase (Freire et al.,

1998). The precise spatial and functional relationship betweenearly recombination nodules containing DMC1 and mouseRAD9A foci needs further resolution.

It was previously shown that TOPBP1, a well-known binding

partner of RAD9A (Makiniemi et al., 2001), shares a similarlocalization pattern at DNA repair foci together with ATR(Perera et al., 2004). Human CHK1, which requires

phosphorylation by TOPBP1-bound ATR for full activation,was also found on meiotic chromosomes and has been proposedto function in DNA damage-induced checkpoint responses(Flaggs et al., 1997). The association of RAD9A with the sex

body during pachytene and diplotene is similar to the patternreported for RAD1, TOPBP1 and ATR, all strongly localizing tothe XY pair (Freire et al., 1998; Perera et al., 2004). This

overlapping localization pattern indicates that, although the sexbody is formed, TOPBP1-related checkpoint signaling during lateprophase I may be RAD9A-dependent. Therefore, it is possible

that RAD9A may play multiple functions in meiosis, during bothearly and later stages.

Rad9af/delCre+ male mice display variable fertility

Male mice deficient in RAD9A function from theundifferentiated spermatogonial stage developed normally andshowed no unusual reproductive behavior. However, theyexhibited reduced testis size and low sperm counts that led to

infertility or sub-fertility, underscoring the essential role ofRAD9A in male germ cell differentiation. While there washeterogeneity in the severity of the infertility, all males examined

eventually became sterile. A failure to fully excise the floxedRad9a allele in some spermatogonia due to the lack of expressionof Stra8-Cre in some spermatogonia (Hobbs et al., 2012; Sadate-

Ngatchou et al., 2008) may have contributed in part to thisheterogeneity. Nonetheless, the loss of RAD9A results in astriking and surprising meiotic phenotype.

Histological analysis of the testis of Rad9af/delCre+ mutant

males indicated the presence of the various types ofspermatogonia and the cells appear to enter into meioticprophase normally. However, few of the spermatocytes

progressed beyond zygotene into pachytene and virtually noRAD9A-deficient diplotene spermatocytes are seen. Our TUNELassay revealed that RAD9A-deficient testis demonstrated higher

levels of apoptosis, predominantly in spermatocytes as judged bypositive signal in the first and second layers of cells from thebasal membrane of the seminiferous tubules.

A prominent meiotic defect in RAD9A-deficientspermatocytes is unrepaired DNA DSBs

In mammals, spermatocyte apoptosis can be activated in mutants

that fail to complete meiotic DNA repair and recombination (e.g.

Dmc12/2, Msh42/2 or Msh52/2), and both male and female micewith such mutations are sterile (Edelmann et al., 1999; Pittman

et al., 1998). Also, spermatocytes with defective SC formation orDSB repair are eliminated at mid-pachytene, demonstrating thatcoordination between SC dynamics and DSB repair is necessary

to permit meiotic progression (Bolcun-Filas et al., 2009; Hameret al., 2008a; Hamer et al., 2008b). Synapsis and DNA repair areinterrelated and interdependent events, making it difficult to

discriminate which process, either one or the other or both, isaffected by RAD9A deficiency. Human RAD9A is retained atsites of damaged DNA and colocalizes with cH2AX. Most of thecH2AX foci on autosomes of RAD9A-deficient spermatocytes

were presumably at sites of DNA DSBs generated in leptoteneand not at sites of asynapsed chromatin. Unlike in SPO11-deficient spermatocytes (Barchi et al., 2005), in those cells

lacking RAD9A, DNA DSBs were normally produced and sexbodies seem to form. We found RAD9A-deficient spermatocyteswith significant retention of cH2AX throughout the nucleus up to

early pachytene stage, which pointed to unrepaired DNA damage.This was accompanied by a significant increase in the number ofleptotene/zygotene spermatocytes in the mutant concomitant with

a decrease of cells in pachytene. Persistent DMC1 foci onautosomes in mid-pachytene further support this idea andindicate that the abnormal cH2AX presence in pachytene was adirect result of compromised DNA DSB repair in the absence of

RAD9A. Together, these data strongly indicate that aberrantlypersistent cH2AX foci in mutant spermatocytes were at sites ofunrepaired DNA damage and that early apoptosis has been

triggered either due to a failure of RAD9A to aid homologousrepair directly or because of failed activation of the DNA DSBcheckpoint, normally controlled by the 9-1-1 clamp.

These data demonstrate a function for RAD9A during earlyprophase I. However, the distinct pattern of RAD9A localizationto the sex body during late prophase I may indicate an additionalrole at this stage. Spermatocyte apoptosis at mid-pachytene can

also result from abnormal sex body formation and defectivemeiotic sex chromosome inactivation (MSCI) (Mahadevaiahet al., 2008; Royo et al., 2010). Definitive analysis of such a role

in meiosis was precluded in RAD9A-deficient spermatocytes dueto induction of apoptosis in early pachytene spermatocytes, andlate-pachytene spermatocytes were rare. However, sex bodies

formed and key events of MSCI such as localization of cH2AXand SUMO1 at the XY pair appeared normal in RAD9A-deficientspermatocytes that did reach mid-pachytene. Therefore, while

RAD9A is not likely required for the formation of the sex body, aweak TOPBP1 signal and its restriction to the axes of the X andY suggest that RAD9A may be important for the functioning ofthe sex body and could play a role in MSCI.

There is also the interesting possibility that, at different stages,RAD9A physically interacts with different protein complexes tomediate distinct activities. For example, it is known that in

somatic cells RAD51 binds to RAD9A, which in our systemcould be involved in the repair of obligatory DSBs. Studies in S.

pombe have also shown that the Rad9 interacting protein TopBP1

functions in the meiotic recombination checkpoint (Perera et al.,2004). Since RAD9A and TOPBP1 are found to interact inmitotic mammalian cells, it is likely that RAD9A and TOPBP1

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are in complexes functioning in mammalian meioticrecombination as well as in late prophase I.

In summary, our findings demonstrate that RAD9A is required

for progression through prophase I of mammalian male meiosis.RAD9A deficiency causes distinct abnormalities in prophase Ispermatocytes, mainly the accumulation of DNA DSBs andincreased apoptosis. This in turn results in infertility or sub-

fertilility. RAD9A is a multifunctional protein with activities inDNA repair, cell cycle checkpoint control and apoptosis, studiedprimarily in the context of the mitotic cell cycle. Investigations

reported herein reveal a role for RAD9A in events in the meioticcell cycle and thus importantly provide an avenue to evaluate thesignificance of specific RAD9A-related functions in mitotic

versus meiotic processes, as well as impact on male fertility.

Materials and MethodsmRNA expression analyses

Q-RT-PCRTotal testis RNA was isolated from mice of the indicated genotype and age usingTrizol reagent (Invitrogen) according to the manufacturer’s recommendations,treated with DNase I (Invitrogen) and reverse-transcribed using random primers(Invitrogen). In each real-time PCR 100 ng cDNA was combined with qPCRMastermix Plus for SYBR Green I (Applied Biosystems) containing 300 nM ofspecific primers: Rad9a-q forward: 59-GGCTGTCCATTCGCTATCCC-39 Rad9a-q reverse: 59-GTGGGGCAAAAAGGAAGCAG-39 designed to anneal to exon twoand three. Intervening intron precludes the amplification of a larger fragment underthese conditions.

Arbp was amplified with primers: Arbp-q forward: 59-CAAAGCTGAA-GCAAAGGAAGAG-39 Arbp-q reverse: 59-AATTAAGCAGGCTGACTTGGTT-G-39 as previously reported (Yabuta et al., 2011). Reactions were performed intriplicate in an ABI 7300 Real-time PCR System (Applied Biosystems, FosterCity, CA) and RNA load normalized with Arbp.

In situ hybridizationRad9a mRNA was detected in situ in 5 mm paraffin sections of adult wild-typemice using digoxigenin-labeled riboprobes as described previously (Batourina,et al., 2001; Chung, et al., 1998b). A cDNA fragment spanning nucleotides 1–180of the Rad9a mRNA was cut with NdeI (Invitrogen, Carlsbad, CA) from full ORFcloned in pGEMT and T7 polymerase used to generate the sense probe. Antisenseprobe was prepared by AatII (Invitrogen, Carlsbad, CA) digest followed by SP6polymerase conversion.

Protein extracts and immunoblotting

Cytoplasmic and nuclear extracts from fresh testis were prepared as describedpreviously (Andrews and Faller, 1991; Revenkova et al., 2001). Immunoblot analysiswas performed according to standard protocols with the following antibodies: mousemonoclonal against RAD9A (cat. no. 200784, Zen Biosciences, CA) and goatpolyclonal against beta-actin (cat. no. sc-163637, Santa Cruz Biotechnology, CA).

Generation of Rad9af/f or Rad9af/del and Stra8-Cre Rad9a male mice

All mice were housed in a pathogen-free facility, and manipulations performed instrict adherence to IACUC guidelines. The generation of the conditional Rad9a floxallele has been described previously (Hu et al., 2008). Rad9af/f mice were interbredwith Stra8-Cre mice (Sadate-Ngatchou et al., 2008) obtained from The Jackson Labsin order to generate Rad9af/+Cre+ mice. In a second round of mating, Rad9af/+Cre+

males were crossed with Rad9af/f females to generate Rad9af/delCre+ males. Forassessment of male fertility, female mice of the inbred CD1 line purchased fromCharles River Labs (Wilmington, MA) were mated with adult Rad9af/delCre+ males.

Rad9af/delCre+ mice were crossed with Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J (mT/mG) (Muzumdar et al., 2007) to assess efficiency of excision by the Stra8-Credriver. The mouse colony was maintained in a mixed genetic background of 129SvEv, C57BL/6 and FVB/NJ.

Fertility studies

The fertility of males with Rad9af/delCre+ and Rad9af/fCre+ genotypes wasassessed, beginning at 3 months of age, by mating to two, 8-week-old CD1females. At day 30 females were sacrificed and the number of live pups anddeveloping embryos recorded. At the end of each mating period two new 8-week-old CD1 females were added for another month, for a total of three rounds.Statistical analysis was performed for litter size using a negative binomial modelwith correction for multiple comparisons. Data were plotted at the 95% confidencelimit. At the completion of the three mating periods, males were sacrificed andtheir testis weight and sperm counts from cauda epididymes determined as

described (Wang, 2003). Progeny of mutant males were genotyped to determinethe efficiency of conditional allele excision.

Tissue preparation and histology

Testes of mice at indicated ages were dissected from anesthetized animals, weighed,then fixed in either Bouin’s solution (Sigma) or 3.7% paraformaldehyde overnight at4 C. Fixed tissues were embedded in paraffin, sectioned at 5-mm thick, and mounted onslides. Periodic acid-Schiff (PAS) staining was used for histological analysis, accordingto standard procedures (Sotomayor and Handel, 1986). Staging of spermatogenesis wasperformed according to a published method (Russell et al., 1990).

TUNEL staining of apoptotic cells and calculation of the AI

TUNEL staining was performed on tissue sections using the in situ cell deathdetection kit (Roche Diagnostics, Indianapolis, IN), according to themanufacturer’s instructions. Briefly, histological sections were treated as forimmunohistochemistry without subsequent antigen retrieval. The tailing labelingreaction was carried out in the terminal deoxynucleotidyl transferase (TdT) bufferwith HRP-labeled UTP. TUNEL-positive cells were visualized with DAB/H2O2

and sections counterstained with Hematoxylin. Only clearly stained cells werescored as apoptotic, and only tubules cut perpendicular to the length of the tubule(yielding round tubules in section) were evaluated. At least 100 tubules per sectionfrom six different testicular sections (more than 60 mm apart) of the same animalwere counted. Apoptosis level quantified by the apoptotic index (AI), wasdetermined as described (Woolveridge et al., 1999; Yu et al., 2001). Tubulescontaining three or more TUNEL-positive cells were considered apoptotic. The AIwas assessed by multiplying the percentage of apoptotic tubules by the meannumber of TUNEL-positive cells per tubule. Significant differences (P,0.01)between groups were assessed by statistical analysis.

Preparation of chromosome spreads and tubule squashes

Testis cell preparations were generated from juvenile or adult (2- to 4-months old)mice. Usually, one testis was freshly frozen in OCT or fixed in 4% PFA to be laterembedded in paraffin and used for immunohistochemistry, while the other wasprocessed for surface spreading according to established techniques (Peters et al.,1997). For long-term storage, slides were kept at 280 C.

For squashes, tubules were dispersed and fixed for 10 minutes in 2%formaldehyde in PBS with 0.05% Triton X-100, placed on slides, pressed with acoverslip, frozen in liquid nitrogen and kept at 280 C prior to processing. Washeswere carried out in 0.1% Triton X-100 and 10% antibody dilution buffer (ADB: 10%donkey serum, 3% BSA, 0.05% Triton X-100) in PBS. Then primary antibodies atdilutions of 1:50–1:100 in ADB in PBS were incubated overnight at 4 C. Secondaryantibodies diluted 1:300–1:500 were added to the tubule preparations and incubatedat room temperature. After washing, slides were mounted in ProLong Gold antifadereagent (Molecular Probes, Eugene, OR, USA), with 5 mg/ml DAPI.

Antibody generation and immunohistochemistry

The full-length Rad9a cDNA fragment (encoding amino acid residues 1–390) wassubcloned into the Escherichia coli expression vector pGEX-4T-3 (Invitrogen),expressed and purified on glutathione agarose (Sigma). A mixture of native anddenatured protein was used for immunization of rabbits and polyclonal antibodieswere generated (Cocalico Biologicals, Reamstown, PA). Specific antibodies werepurified on Protein A–Sepharose Fast-flow (Invitrogen), followed by passagethrough GST–Sepharose to reduce background. The resulting antibodies weredesignated as a-RAD9A. The specificity of the antibodies was confirmed byimmunoblotting and used to detect RAD9A protein in paraffin embedded testiculartissue sections by immunohistochemistry.

Immunohistochemical analyses were performed using a Vectastain ABC kit(Vector Laboratories, Burlingame, CA), as previously described (Liu et al., 1998).Antigen unmasking was performed by boiling slides in a microwave twice for5 minutes in 0.01 M citrate buffer, pH 6.0 (Shi et al., 1991). Endogenousperoxidase activity was saturated in 0.3% H2O2 for 30 minutes. Antibody stainingwith RAD9A primary antibodies at 1:100 dilution was performed according to theVectastain ABC kit manufacturer’s instructions. RAD9A protein was visualizedwith DAB and nuclei counterstained with Hematoxylin. Slides were viewed on aNikon photomicroscope under brightfield optics and images were captured with aSpot digital camera using SPOT software.

Immunofluorescence

Labeling of paraffin and frozen sections of mouse testis was performedas described previously (Revenkova et al., 2004). The following antibodieswere used: goat polyconal anti-RAD9A (sc-10465, Santa Cruz Biotechnology),mouse monoclonal anti-SYCP3 (ab-97672, Abcam), rabbit polyclonal anti-SYCP3(ab-15093, Abcam), rabbit polyclonal anti-DMC1 (sc-22768, Santa CruzBiotechnology), rabbit polyclonal anti-TOPBP1 (ab-105109, Abcam), mousemonoclonal anti-cH2AX Ser-139 (cat. no. 05-636, Millipore) and anti-SUMO1(sc-5508, Santa Cruz Biotechnology). For double or triple immunostaining withprimary antibodies from the same species, appropriate monovalent Fab fragments

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were employed. Secondary antibodies used were DyLight-647-labeled donkeyanti-mouse IgG (1:300; Jackson Immunoresearch), DyLight-488-labeled donkeyanti-goat (Jackson Immunoresearch, PA), DyLight-594-labeled donkey anti-rabbitIgG (Jackson Immunoresearch, PA) and Alexa-Fluor-647-labeled donkey anti-goatIgG (Invitrogen). Nuclei of testis sections were visualized by staining with DAPI.In addition to the antibodies listed above, on squash preparations the followingpairs of secondary antibodies were applied at 1:500 dilution: anti-rabbit-549 (cat.no. 111-605-144, Jackson Immunoresearch, PA) and anti-mouse Alexa-647(SUMO1 and SYCP3; cat no. A-31571, Molecular Probes, Life Technologies,CA) and anti-goat Alexa-488 (RAD9A; cat. no. A-11055, Molecular Probes, LifeTechnologies, CA). Both PBT and ADB buffers were made using immunoglobulinG-free protease-free BSA (cat. no. 001-000-161, Jackson ImmunoResearch, PA).

Analysis of excision efficiency by Stra8-Cre and quantification of germ cellpopulations by flow cytometry

For staining of individual germ cell types, single-cell suspensions were prepared fromtestes as described previously (Bastos et al., 2005; Vasileva et al., 2009) with minormodifications. When the same testes were used for spermatocyte spreads, suspensionswere prepared by mechanical maceration as opposed to enzymatic digestion, amodification, which resulted in very low numbers of spermatogonia (Table 2). Todiscriminate between cell types based on their DNA content and the progressive lossof activity of a nuclear ATP pump, which excludes dye from the nucleus as cellsdifferentiate, we stained cells with Hoechst 33342 and performed flow cytometry.Single-cell suspensions from Rad9af/+Cre+Tom+ and Rad9af/delCre+Tom+ testeswere used to determine the efficiency of excision of the tdTomato allele as thepercentage of EGFP-positive germ cells from the total germ cell pool. Suspensionswere analyzed on BD LSRII (BD Biosciences, Seattle, WA), with UV laserexcitation wavelength 355 nm and emission filters UV-Blue 460/50 and UV-Red692/40. Data were acquired using FACSDiva software (BD Biosciences, CA) andanalyzed with FlowJo 8.7 (Tree Star, Inc., OR) as described (Bastos et al., 2005;Vasileva et al., 2009). Cells with 2N DNA content and inactive ATP pump includesomatic cells and germ cells (secondary spermatocytes) are not shown. Thesubpopulation of 4N primary spermatocytes was subdivided into three regions, Ho-redlow, Ho-redmed and Ho-redhi, which contained, respectively, leptotene andzygotene, pachytene and diplotene spermatocytes. The identity of each populationwas confirmed by assessing the type of SCP3 immunofluorescence on chromosomesof sorted cells from each subset. To confirm that cell populations, which by Hoechststaining appeared as 4N are indeed spermatocytes, we crossed the Rad9af/delCre+

line to transgenic mice expressing EGFP under the meiosis-specific Smc1b promoterdescribed previously (Adelfalk et al., 2009). In this mouse line EGFP expressionstarts at the onset of prophase I.

AcknowledgementsWe thank Alastair Tulloch for help with quantification of the varioustesticular abnormalities, Dr Marcia Manterola for expert advice onpachytene stage matching and Dr Sanny S.W. Chung for help withstaging of mutant tubules, and comments on the manuscript.

Author contributionsA.V., D.J.W. and H.B.L. contributed equally to interpretation of dataand writing of the manuscript. A.V. helped formulate the studydesign and performed all experiments apart from setting up fertilitystudies. K.M.H. and X.W. provided technical assistance withgenotyping and histology. M.M.W. organized and conducted thefertility experiments. R.A.F. organized the fertility data andperformed statistical analyses. D.J.W. and H.B.L. initiallyconceived and designed the study and provided oversight.

FundingThis work was supported by the National Institutes of Health [grantnumbers R01CA130536 to H.B.L., R01GM079107 to H.B.L. andD.J.W.]. Deposited in PMC for release after 12 months.

Supplementary material available online at

http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.126763/-/DC1

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