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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Dev Biol. Author manuscript; available in PMC Apr 15, 2012.
Published in final edited form as:
PMCID: PMC3065526
NIHMSID: NIHMS270580

Cul4A is Essential for Spermatogenesis and Male Fertility

Abstract

The mammalian Cul4 genes, Cul4A and Cul4B, encode the scaffold components of the cullin-based E3 ubiquitin ligases. The two Cul4 genes are functionally redundant. Recent study indicated that mice expressing a truncated CUL4A that fails to interact with its functional partner ROC1 exhibit no developmental phenotype. We generated a Cul4A-/- strain lacking exons 4-8 that does not express any detectable truncated protein. In this strain, the male mice are infertile and exhibit severe deficiencies in spermatogenesis. The primary spermatocytes are deficient in progression through late prophase I, a time point when expression of the X-linked Cul4B gene is silenced due to meiotic sex chromosome inactivation. Testes of the Cul4A-/- mice exhibit extensive apoptosis. Interestingly, the pachytene spermatocytes exhibit persistent double stranded breaks, suggesting a deficiency in homologous recombination. Also, we find that CUL4A localizes to the double stranded breaks generated in pre-pachytene spermatocytes. The observations identify a novel function of CUL4A in meiotic recombination and demonstrate an essential role of CUL4A in spermatogenesis.

Keywords: Cul4A, spermatogenesis, homologous recombination

INTRODUCTION

CUL4A, a member of cullin family of proteins, is a core component of the CUL4A-based E3 ubiquitin ligases known to regulate cell cycle, DNA replication, DNA repair and chromatin remodeling. CUL4A acts as the scaffold component of the complex by interacting with ROC1 with its C terminal region and with Damaged DNA Binding protein 1 (DDB1) with its N terminal region. Through its interaction with different WD40-containing proteins that act as substrate adaptors, DDB1 is responsible for recruitment of specific target proteins, while the RING finger domain containing ROC1 brings the ubiquitin conjugating enzyme E2 to the complex. All cullin-based ubiquitin ligases are positively regulated by attachment of ubiquitin-like molecule NEDD8 (neural precursor cell expressed, developmentally down-regulated 8) to the cullin component.

The Cul4 gene has been conserved during evolution from fission yeast to humans. While S. pombe and A. thaliana encode a single Cul4 gene, the mammalian cells express two Cul4 paralogs, Cul4A and Cul4B. In mice, the Cul4A gene is localized on chromosome number 8 and in humans on chromosome 13. The region 13q34 containing Cul4A is found to be amplified in breast cancers (Melchor et al., 2009), hepatocellular carcinomas (Yasui et al., 2002), squamous cell carcinomas (Shinomiya et al, 1999), adrenocortical carcinomas (Dohna et al, 2000) and childhood medulloblastoma (Michiels et al, 2002). On the other hand, the Cul4B is an X-linked gene in both mice and humans and its mutation correlates with X-linked mental retardation in humans (Zou et al, 2007).

Although encoded by two different genes, the mammalian CUL4A and CUL4B proteins share 80% identity (Jackson and Xiong, 2009). CUL4B has an extended N terminal region. Both of them bind and utilize DDB1 as a linker protein, suggesting redundancy of their functions with regards to substrate ubiquitination. Indeed, many substrates of CUL4-based ubiquitin ligases have been shown to be targeted by both CUL4A and CUL4B complexes, including chromatin licensing and DNA replication factor 1 (CDT1) (Higa et al, 2006), histones H3 and H4 (Wang et al, 2006) and p53 (unpublished data from our lab).

In 2002, Cul4A knockout mice were reported to be embryonic lethal (Li et al, 2002). Deletion of exon 1 of the Cul4A gene (Cul4AΔ1/Δ1) resulted in no viable knockout embryo beyond 7.5 dpc, suggesting that Cul4B could not compensate for the loss of Cul4A during mouse embryonic development. Interestingly, in 2009, a different Cul4A mutant strain of mice was described (Liu et al, 2009). That study deleted exons 17-19 of the Cul4A gene and observed no apparent developmental phenotype. In that mutant strain a C-terminal truncated protein was detectable in multiple tissues and in the embryonic fibroblasts (MEFs). Since the deleted region between exons 17-19 encodes the ROC1 binding site and the NEDD8 modification site, it failed to recruit ROC1 (Liu et al, 2009), which presumably prevents recruitment of E2. However, the truncated protein retained DDB1 binding domain. The obvious discrepancies between the two Cul4A mutant strains of mice were explained by unintentional deletion of a 529 bp region upstream of the first exon of the essential Psid2 gene in Cul4AΔ1/Δ1 mice, localized on the complementary strand adjacent to the Cul4A exon 1 (Liu et al, 2009). Psid2 encodes a protein that contains PCI domain that is found in the essential subunits of translation initiation factor 3, 26S proteasome and COP9 signalosome (Hofmann and Bucher, 1998).

Independently of the above studies, we generated a Cul4A mutant strain of mice with deletion of exons 4-8, generated from a previously described Cul4A fl/fl mice (Kopanja et al, 2009). The region between exons 4 and 8 encodes partial DDB1 binding site. In various tissues examined, we did not observe expression of truncated product of the Cul4A gene, suggesting complete loss of Cul4A expression. The Cul4A -/- mice were born at the expected Mendelian ratio, further supporting the notion that Cul4A is dispensable for embryonic development in mice. Surprisingly, we observed that Cul4A -/- male mice are infertile. We show that Cul4A is essential for spermatogenesis and it is involved in the double stranded break repair by homologous recombination during male meiosis.

RESULTS

Cul4A is dispensable for mouse embryonic development

To delete the Cul4A alleles, Cul4A fl/fl mice that have floxed exons 4-8 (Kopanja et al, 2009) were crossed with transgenic mice expressing EIIA-Cre (Fig. 1A). To avoid mosaicism, the following strategy was used. Homozygous floxed male animals were crossed with females carrying the EIIA-Cre transgene to obtain mosaic animals with Cul4A +/fl/- EIIA-Cre genotype that were subsequently crossed again with Cul4A fl/fl. From the progeny, mice with the germline transmission of one deletion allele, Cul4A fl/-, without the EIIA-Cre transgene were obtained, which were used to obtain the homozygous deletion strain (Cul4A -/-). Genotyping was performed using the tail DNA. In Figure 1B, scheme of genotyping strategy is shown. The Cul4A knockout (exons 4-8) mice were born alive, at expected Mendelian ratio. From a total of 100 pups born from heterozygous mating, 21 were born with Cul4A -/- genotype. Those animals appeared phenotypically normal and healthy, suggesting that Cul4A is dispensable for embryonic development in mice. Histopathological analyses, performed at Veterinary Diagnostic Laboratory, University of Illinois, confirmed that there are no gross abnormalities in the Cul4A knockout mice. Western blot analysis, shown in Figure 1C, confirmed the absence of the CUL4A protein in different tissues harvested from Cul4A knockout mice, as well as the apparent absence of any truncated protein that could potentially result from exons four to eight deletion. Testis, heart, liver and brain were chosen because these tissues express CUL4A at high levels in the wild type genotype. The arrow in Figure 1C indicates the full length CUL4A protein.

Figure 1
Generation of the Cul4A knockout mice

Male Cul4A-/- mice are infertile

Surprisingly, when Cul4A knockout male mice were used for mating, no pups were born. To verify that those mice are indeed infertile, matings were set up so that female and male Cul4A knockout mice mate with control wt or Cul4A fl/fl mice. They were observed for five months. As shown in Table 1, the average litter size of the female Cul4A knockout mice is smaller than that of control mice, suggesting reduced fertility, but nevertheless those mice are still capable of bearing and delivering live pups. In contrast, from the 9 male Cul4A knockout mice that were observed for five months, none were able to generate any pup, nor were the females noticed to be pregnant. However, copulatory plugs were frequently observed in the female mice, suggesting that the infertility of the male Cul4A -/- mice did not result from abnormal sexual behavior. Because the phenotype in males was more pronounced compared to females, we focused on the role of CUL4A in spermatogenesis.

Table 1
The Cul4A knockout male mice are infertile

To evaluate the sperm cells, epididymides were isolated from the Cul4A fl/fl and the Cul4A -/- mice, processed as described in Materials and Methods and subsequently stained with H&E. As expected for fertile animals, epididymal tubules of the control animals were full of sperm cells (Fig. 2A, 2C). In contrast, epididymal tubules of the knockout mice were almost completely empty (Fig. 2B, 2D). A few sperm cells that were found in Cul4A -/- epididymides displayed abnormal head morphology (Ab in Fig. 2D, inset). To examine sperm cells in more details, epididymides were harvested from the sacrificed animals, briefly washed in PBS and minced in M2 medium. After incubation for fifteen minutes at 32 degree Celsius, released spermatozoa were frozen and kept at -80 degree Celsius. We did not observe significant difference between the control and the knockout sperm cells with regards to their fragility upon freezing. Compared to controls, the number of spermatozoa in the Cul4A -/- mice was reduced dramatically. Estimated number of sperm cells in the knockout epididymides was less than 10% of the controls (Fig. 2E, left panel). Also, the daily sperm production (DSP) in the knockout testes was significantly reduced compared to the control testes (Fig. 2E, right panel).

Figure 2
Sperm defects in the Cul4A knockout epididymides

More than 1000 sperm cells per animal were examined in detail. Near hundred percent of the sperm with Cul4A fl/fl genotype exhibited normal, hooked shape heads (Fig. 2F). By contrast, from more than 1000 of Cul4A -/- genotype, only 1.06±0.6 percent exhibited normal morphology, but very few of them were motile (only 0.2±0.14 sperm cells exhibited twitching or progressive motility). As seen in Figure 2G, 2H, 2J, 2K, 2L, morphological defects of the Cul4A -/- sperm cells were diverse. Oval small heads, headless or decapitated sperm as well as cytoplasmic droplets on sperm tails (data not shown) were observed.

Testicular phenotype of the Cul4A knockout mice

To investigate the spermatogenic defect in Cul4A knockout mice, we analyzed the testes. Compared to all tissues examined, CUL4A expression is the most abundant in testis (Fig. 3A). Testes from the Cul4A knockout mice were 2-fold smaller (Fig. 3B) compared to testes from control littermates (calculated as a percentage of total body weight). Absolute testes weights were also 2-fold smaller in Cul4A knockout mice (0.15±0.018g) compared to controls (0.3±0.03g). The epididymides isolated from the same animals were approximately 20% smaller compared to controls (Table 2), while there was no difference in the seminal vesicle weights. The data presented in the Table 2 are means from five different animals per genotype.

Figure 3
Cul4A plays role in testis development
Table 2
Weight of male reproductive organs in Cul4A fl/fl and Cul4A -/- mice

A clear difference was observed between testicular sections of control (Fig. 4A, 4B) and knockout mice (Fig. 4C, 4D, 4E and 4F), with a significant decrease in the number of postmeiotic cells and the presence of vacuoles (V in Fig. 4C) in the Cul4A -/- testes. Tenfold increase in apoptotic cells/field (9-fold when calculated as apoptotic cell/tubule cross section) was observed in the knockout testes (Figure 5A). Seminiferous epithelium in the Cul4A -/- testes also exhibited premature sloughing of germ cells (Supplementary Fig. 1). There was a clear deficiency in postmeiotic cells (round, elongated spermatids and sperm cells) as well as appearance of multinucleated giant cells (data not shown), abnormal spermatocytes (Aspc in Fig. 4D) and abnormal spermatids (Ab in Fig. 4C, 4E and 4F). Sertoli cell nuclei appeared normal along the basement membrane (see Supplementary Figure 1). The Leydig cells also appeared normal, morphologically and in numbers per interstitial space (Supplementary Figure 1).

Figure 4
Testicular defects of the adult Cul4A knockout mice
Figure 5
Induction of apoptosis and meiotic delay in Cul4A knockout testis

Cul4A deficiency disrupts male meiosis

The events in postnatal testicular development and spermatogenesis in mice are well documented and understood (Bellve et al, 1977; Russell et al, 1990; Borg et al, 2010). The first postmeiotic cells, haploid round spermatids, appear by day 20 and differentiate into elongated spermatids. Sperm are released into the lumen on approximately day 35.

A time course histopathological study of testis development in Cul4A fl/fl and Cul4A -/- mice was performed, at postnatal days 10, 14, 18, 27 and 35. At days 10 and 14, no difference was observed between Cul4A -/- and controls, suggesting that in the knockout testes germ cells enter meiosis and progress normally to the beginning of prophase I as in control testes (Supplementary Fig. 2). However, at day 18 disparities between the two genotypes were detected. Seminiferous tubules from fl/fl mice were normal, with evidence of sustained spermatogenesis from spermatogonia to spermatocytes, and some spermatocytes reaching late stage pachytene (P in Figure 4G). In contrast, the Cul4A -/- testes contained fewer pachytene spermatocytes and cells with pycnotic nuclei were more prominent (arrowheads in Figure 4H). Preleptotene and zygotene spermatocytes appeared normal in Cul4A -/- testes.

At day 27, the differences between testes of Cul4A fl/fl and Cul4A -/- were even more pronounced. Significant hypocellularity was seen in knockout testes (Fig. 4J) compared to the control. In Cul4A proficient testes, most of the tubules had postmeiotic, round spermatids (Rs in Fig. 4I), symbolizing the end of meiosis in the first wave of spermatogenesis. Metaphase, anaphase or telophase spermatocytes in Stage XII tubules also were observed (data not shown). On the contrary, the Cul4A -/- testes had almost no postmeiotic cells (Fig. 4J). The most matured meiotic spermatocytes were those in prophase I, in late pachytene or diplotene (P in Fig. 4J). To quantify the difference in tubules with postmeiotic cells, a total of 500 tubules of each genotype were examined. In the control testes, 388 out of 500 tubules had round spermatids present, compared to only 18 out of 500 tubules in the knockout testes (Fig. 5C). The Cul4A-/- tubules with post-meiotic cells had only up to 7 round spermatids per tubule section, while 70 percent of control tubules had more than 70 round spermatids per tubule section.

In 35 days old Cul4A fl/fl testes, all stages of elongated sperm cells were seen (Es in Fig. 4K), but very few were present in Cul4A -/- testes (Fig. 4L). In Cul4A -/- males, only 143 of 500 seminiferous tubules had elongated sperm cells compared to 459 of 500 in Cul4A fl/fl testes (Fig. 5D). Multinucleated giant cells, dying cells, abnormally shaped elongated spermatids, and prematurely released cells into the lumen of tubules were often seen in the Cul4A-/- testes. To examine if increased cell death during the first wave of spermatogenesis could contribute to the deficiency in postmeiotic cells in the Cul4A knockout testes, TUNEL assay was performed. Compared to the age-matched controls there was nearly threefold more apoptotic spermatocytes in the knockout 18 days old testes (Figure 5B), fourfold more in the 27 day old and fivefold more in the 35 days old knockout testes (Figure 5D and 5E respectively), suggesting that increased cell death could contribute to the deficiency in postmeiotic cells in the developing Cul4A knockout testes.

Cul4A is not critical for synapsis of homologous chromosomes and formation of XY-body

During meiotic prophase I the homologous chromosomes pair, synapse and recombine. The most apparent element associated with these processes is the synaptonemal complex (SC). Synapsing can be studied by analyzing surface spermatocyte spreads stained for components of synaptonemal complex (Heyting et al, 1987; Kidane et al, 2010) including SCP3, SCP2 and SCP1. As seen in the Figure 6A, both, control and knockout pachytene spermatocytes exhibited complete axial element formation with sporadic fragmentation of autosomal axial elements in the Cul4A -/- testes. Co-staining with SCP1 and SCP3 antibodies revealed complete synaptonemal complex formation (Figure 6B) in most of pachytene spermatocytes of both genotypes. From 590 knockout bivalents only 30 exhibited synaptic abnormalities, and in the controls 8 out of 349 were aberrant (data not shown), indicating that Cul4A is not critical for synapsis of the homologous chromosomes.

Figure 6
Cul4A is not critical for the synapsis

Furthermore, we analyzed meiotic sex chromosome inactivation (MSCI) using γH2AX and uH2A as markers of the sex body domain. MSCI involves transcriptional silencing of the X and Y chromosomes in pachytene, when the autosomes are fully synapsed and sex chromosomes are compartmentalized into a peripheral nuclear domain called XY- or sex-body (Solari, 1974; McKee and Handel, 1993; Turner, 2007). MSCI persists throughout pachytene and diplotene and it is mediated by extensive chromatin remodeling of the sex chromosomes. The central chromatin modification in MSCI is γH2AX phosphorylation. In spermatocytes H2AX gets phosphorylated in two waves; first, throughout chromatin in leptotene as a cellular response to DSBs induced by SPO11 (necessary for initiation of homologous recombination) and second at the zygotene-pachytene transition only on the chromatins of the X and Y chromosomes. The second wave of H2AX phosphorylation initiates MSCI (Turner et al, 2004; Bellani et al, 2005). It persists on XY-body until the diplotene-metaphase transition. To investigate if initiation of MSCI takes place in Cul4A -/- spermatocytes, spreads were probed with antibody against γH2AX. Interestingly, both, CUL4A proficient and deficient pachytene spermatocytes had prominent γH2AX positive sex-body (Fig. 8A). From 150 pachytene spreads from a total of three mice per genotype, 144 exhibited γH2AX signal over X and Y chromatin.

Figure 8
Persistance of DSBs in the Cul4A knockout pachytene spermatocytes

Shortly after initiation of MSCI with γH2AX phosphorylation, X and Y chromosomes undergo further chromatin modification (rewieved in Handel, 2004) including H2A ubiquitination (Baarends et al, 1999), deacetylation of histones H3 and H4 (Khalil et al, 2004), dimethylation of H3 forming H3K9me2 (Khalil et al, 2004) and sumoylation (Rogers et al, 2004; Vigodner and Morris, 2005). Because CUL4A has been shown to ubiquitinate H2A in vitro (Kapetanaki et al, 2006), we sought to investigate whether histone H2A on X and Y chromosomes is ubiquitinated in the Cul4A knockout spermatocytes following MSCI initiation. Interestingly, both Cul4A fl/fl and Cul4A -/- pachytene and diplotene spermatocytes exhibited uH2A positive sex-body (Fig. 7), suggesting that there is no deficiency in uH2A formation on sex body chromatin during MSCI in the CUL4A deficient spermatocytes.

Figure 7
Cul4A is not critical for H2A ubiquitination on XY body

CUL4A is required for the repair of DSBs in the pachytene spermatocytes

Next, we investigated whether homologous recombination is defective in the Cul4A knockout testis. Recombination starts with SPO11 topoisomerase – dependent double strand DNA breaks (DSBs) in one sister chromatid in one homolog (Keeney et al., 1997; Longhese et al., 2009). It's been shown that often more than two hundred DSBs per genome occur in leptotene. Once formed, DSBs are substrates for homologous recombination (HR). They are recognized by HR repair machinery and marked by ATM-depended γH2AX phosphorylation (Barchi et al., 2005; Bellani et al., 2005). By pachytene, most of the DSBs are repaired, and the γH2AX foci disappear from the autosomal chromatin (Li et al., 2007). To investigate whether the Cul4A knockout spermatocytes are proficient in homologous recombination, γH2AX phosphorylation staining of surface spermatocyte spreads was used.

As shown in Figure 8A, spermatocytes of both genotype were equally susceptible to SPO11 induced DSBs in leptotene of prophase I. By pachytene, in most of the Cul4A fl/fl spermatocytes, positive signal for γH2AX was detected only on the XY-body. Interestingly, positive γH2AX foci persisted in pachytene knockout spreads, mostly along chromosomal axes, suggesting that repair of DSBs is defective and that homologous recombination is impaired in the Cul4A -/- spermatocytes. To verify that in the knockout spermatocytes incompletely repaired breaks remain longer than those in the control spermatocytes, we staged pachytene spreads following previously described criteria of X and Y chromosomes pairing (Tres, 1977). While most of the mid- and late knockout pachytene spermatocytes exhibited persistent DSBs, the majority of controls did not (Fig. 8A). Fifty pachytene spreads from three different pairs of mice were analyzed for γH2AX foci and quantification is shown in Figure 8B. While 24±4 out of 50 Cul4A fl/fl pachytene spreads had no autosomal foci, only 8.75±1.9 out of 50 Cul4A -/- spreads belong to the same group. The majority of the knockout pachytene spermatocytes had more than five foci per spread; with 11±3.5 having 6 to 20 foci and 23.25±1.25 having more than 20 autosomal γH2AX foci, whereas only 9±1 and 8±1 of control spreads exhibited 6-20 and more than 20 foci respectively. Persistence of γH2AX foci in pachytene Cul4A -/- spermatocytes suggests that CUL4A plays role in homologous recombination in male meiosis.

To investigate whether CUL4A directly or indirectly participates in HR, its presence on the sites of DSBs was studied. To perform the analysis, spermatocyte spreads prepared from 14 days old testes were co-stained with CUL4A and γH2AX antibodies. Spermatocytes with fewer γH2AX foci were analyzed to facilitate the co-localization analyses. As seen in Figure 8C, in about 68% (75 out of 111 examined) of the pre-pachytene spermatocytes, CUL4A co-localized with the γH2AX foci, while an additional 10% (11 out of 111) of the spermatocytes exhibited partial co-localization. Co-localization of CUL4A with γH2AX foci suggests physical presence of CUL4A on the sites of DSBs and its direct role in homologous recombination.

DISCUSSION

Work presented here provides strong genetic evidence that Cul4A is essential for male fertility and spermatogenesis. The Cul4A-/- males produced drastically reduced number of sperm. Moreover, the sperm cells found in the Cul4A-/- epididymides exhibited severe morphological defects and reduced motility. We found increase in apoptotic cells in knockout testes. Moreover, Cul4A -/- spermatocytes were deficient in progression through late prophase I.

Key events in prophase I are generation of double stranded breaks, synapsing of the homologous chromosomes, meiotic sex chromosome inactivation (MSCI), and repair of double stranded DNA breaks (DSBs). Generation of the double stranded breaks (DSBs) at leptotene stage of meiotic prophase I appeared to be normal in Cul4A -/- background. Moreover, in our analysis, significant defects in synapsis were not detected in the knockout spermatocytes (only 30 out of 590 bivalents displayed synaptic abnormalities). However, there was a deficiency in repair of the DSBs in knockout spermatocytes, suggesting impairment of homologous recombination. It has been shown that defective homologous recombination can cause failure of MSCI, most likely by sequestering proteins needed for MSCI induction at the sites of persistent DSBs (Mahadevaiah et al., 2008). Failure of MSCI is believed to be sufficient to cause apoptosis of the pachytene cells (Turner et al., 2005; Turner et al., 2006). Because MSCI is a male specific event in mammals, it can explain also the bias for impaired fertility in males compared to females. Still, we did not detect any significant deficiency in MSCI in the Cul4A-/- spermatocytes based on_γH2AX positive sex-body. However, we cannot exclude the possibility that other chromatin modification is missing that induces MSCI failure.

We propose that the persistent DSBs in the presence of normal synapsing and MSCI induce pachytene spermatocyte apoptosis in the Cul4A -/- spermatocytes. The normal mid-pachytene spermatocytes X chromosome carries approximately nine unrepaired DSBs that are tolerated in the presence of normal synapsis and efficient MSCI (Mohadevaiah et al., 2008). Additionally, studies with the Down syndrome mouse model, which carries extra chromosome that encompass most of the human chromosome 21 (h21), demonstrated that approximately four unrepaired DSBs on asynapsed h21 chromosome were not sufficient to trigger pachytene apoptosis (Mohadevaiah et al., 2008). In both cases, DSBs were persistent into mid-pachytene, but were repaired by the end of pachytene, allowing cells to precede to metaphase I. It is possible that a certain threshold number of DSBs is required to induce apoptosis. Recently, it was suggested that mitotic DSB checkpoint required a threshold number (around 20) of DSBs to induce arrest (Deckbar et al., 2007). In that regard it is interesting that around 50% of Cul4A -/- pachytene spermatocytes exhibited more than twenty autosomal γH2AX foci.

A hypomorphic Trip 13 mutation causes meiotic failure in mice of both sexes (Li and Schimenti, 2007) without obvious defects in synapsis or failure in MSCI in spermatocytes, but with defects in DSB repair. However, a later study (Roig et al., 2010) found subtle synaptic defects in the same mutant mice (the length of autosomal sinaptonemal complex was around 11% shorter than expected). The phenotype in Cul4A knockout mice differ from the Trip 13 mutant strain because the female Cul4A knockout mice are capable of bearing and delivering live pups, although with slightly decreased fertility potential than the control littermates. It is possible that in the Cul4A -/- females, expression of CUL4B compensates for the loss of CUL4A. Cul4B is an X-linked gene, and thus its expression is diminished in male spermatocytes during MSCI. We propose that because of silencing, CUL4B could not compensate for the loss of CUL4A in spermatocytes resulting in defective homologous recombination and subsequent apoptosis.

How exactly the E3 ubiquitin ligase component CUL4A participates in HR is unclear, however, mounting evidence indicates a role of ubiquitination in the assembly of the proteins involved in DSB repair. For example, localization of the Fanconi syndrome protein FANCD2 and FANCI to the DSBs involves monoubiquitination by the FANCL E3 ubiquitin ligase (Meetei et al, 2004; Wang et al, 2004; Sims et al, 2007; Smogorzewska et al, 2007). Similarly, localization of the DSB repair protein CtIP involves ubiquitination by BRCA1 (Yu et al, 2006). The tumor suppressor BRCA1 associates with the RING domain protein BARD1 to form an active E3 ligase that is critical for DSB repair (Messick and Greenberg, 2010 and references therein). Interestingly, the recruitment of the BRCA1 protein to the DSBs was also shown to be dependent upon ubiquitination. BRCA1 is recruited by a complex containing the RAP80 protein, a ubiquitin interacting motif-containing protein that localizes to DSBs through an interaction with an unknown ubiquitinated protein, which is ubiquitinated by RNF8 (Kim et al, 2007; Sobhian et al, 2007; Wang et al, 2007; Yan et al, 2007). In addition, H2A and H2AX have been shown also to be ubiquitinated by RNF8 and RNF168, and the ubiquitination of these histones is important for the recruitment of DSB repair proteins (Huen et al, 2007; Kolas et al, 2007; Mailand et al, 2007). In that regard it is noteworthy that CUL4A has been shown to ubiquitinate histones (Kapetanaki et al, 2006; Wang et al, 2006). It is possible that the histone-ubiquitinating activity of CUL4A plays a role in the repair of DSB by homologous recombination.

MATERIALS AND METHODS

Generation of the Cul4A knockout mice

Mice harboring floxed alleles of Cul4A were described previously (Kopanja et al, 2009). To obtain Cul4A -/- mice, homozygous floxed male animals were crossed with females carrying the EIIACre transgene to obtain mosaic animals with the Cul4A +/fl/- EIIACre genotype that were subsequently crossed again with the Cul4A fl/fl. From the progeny, mice with germline transmission of heterozygous knockout allele (Cul4A fl/-) without the EIIACre transgene were obtained, which were used to obtain knockout mice (Cul4A -/-). Genomic DNA isolated from tails was used for genotyping with following primers: 5’-gtgtttaaacGACCACAGCACACAGTAAGTAAGCCCT-3’ and 5’-gtctcgagATGAAGACATGGGTGGACAGTGGC-3’ and 5’-GTGTTTAAACTTAAGGCCAGTCTTGGGCAGAGTGACA-3’ at 58°C as annealing temperature.

In the studies of postnatal testicular development and spermatogenesis, the day of the birth of mice was considered day 0. If otherwise not noted, three mice per genotype were used for experiments.

Tissue protein extracts preparation

Various tissues were harvested, homogenized and lysed in protein extraction buffer containing 50mM Hepes-KOH (ph 7.5), 300mM NaCl, 1mM EDTA, 1 mM EGTA, 1 mM DTT, 0.1% Tween-20, 10% glycerol, 10mM β-glycerolphosphate, 1 mM NaF, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 0.2 mM PMSF. Protein extracts were pre-cleared for IgG with 50% slurry of Protein A-Agarose and Protein G-Sepharose for 4hrs at 4°C, and 0.2mg of total cell protein was subjected to polyacrylamide-SDS gel electrophoresis followed by blotting to nitrocellulose. Cul4A antibody used in Fig 1C and in immunolocalization studies has been previously described (Li et al 2002), as well as the Cul4A antibody used in Fig 3A (Shiyanov et al 1999).

TUNEL assay

TUNEL assay was performed on slides of paraffin embedded tissues using ApopTag Fluorescein In Situ Apoptosis Detection Kit (S7110) (Chemicon), following manufacturer's protocol.

Histochemistry

After isolation testes and epididymides were briefly washed with PBS and fixed in modified Davidson's fixative (30% of 37% formaldehyde, 15% ethanol, 5% glacial acetic acid, 50% distilled water) for 48hrs rotating at 4C. After fixation, they were briefly rinsed with tap water and kept in 10% neutral buffered formalin till processing. Approximately 10mm wide sections were taken through grades of alcohols and finally embedded in paraffin. 5 μm sections were prepared, baked over night at 55C and stain with H&E the next day following standard procedure.

Epididymal sperm count and the daily sperm production (DSP)

Epididymides were dissected, briefly washed with PBS and placed in M2 medium (Sigma), previously warmed to 32C. After mincing, the epididymides were incubated at 32C for 30 minutes. Debris was removed and volume was made up to 1 ml with M2 medium. An aliquot of the sample was diluted in 4% paraformaldehyde and counted using the hemacytometer. The daily sperm production (DSP) was estimated using a previously described method (Robb et al., 1978; Cooke, 1991; Joyce et al., 1993).

Identification of the pachytene spermatocyte spreads

To discriminate between zygotene and pachytene spermatocytes with asynapsis, we used the following criteria: (1) Thickness and length of axial elements: in pachytene cells axial elements are thicker and shorter compared to zygotene cells (Ashley et al., 2004). (2) Occurrence of asynapsed bivalents in the presence of multiple fully synapsed bivalents (Mahadevaiah et al., 2008). (3) DAPI staining pattern: pachytene nuclei have more heterogeneous DAPI staining, with centromeres brightly stained and separated into multiple subdomains in contrast to leptotene/zygotene nuclei where centromeres are clustered in a few domains and DAPI is bright throughout the nucleus (Turner et al., 2001).

Surface spermatocyte spreads

Surface spermatocytes spreads were prepared as previously described in Barlow et al., 1997, and Mahadevaiah et al., 2008.

Antibodies used for staining were rabbit SCP3 (AbCam), goat SCP1 (Santa Cruiz), mouse γH2AX (Upstate) and mouse uH2A (Upstate). Briefly, the slides were defrosted for 5 minutes in PBS, air dried and blocked with 5% goat serum, 0.15%BSA, 0.1% TritonX-100 in PBS for 1hr at room temperature, followed by incubation with primary antibody diluted in the blocking buffer over night at 4C. Next day, slides were washed 3X with PBS and incubated with secondary antibodies (FITC-conjugated polyclonal rabbit anti-mouse antibody (Dako; 1:75) and/or Alexa Fluor 594 goat anti-mouse (Invitrogen) at dilution 1:500) at room temperature for 1hr. The slides were washed 3X with PBS, stained with DAPI, mounted using Vectashield (Vector) and viewed using Nikon microscope. When SCP1 antibody was used, slides were blocked in 5% horse serum in PBS and antibodies were diluted in the same buffer. Rhodamine (TRITC)-conjugated AffiniPure Mouse Anti-Goat (Jackson ImmunoResearch) secondary antibody was used.

For co-localization studies, separate images of the same field of gamma H2AX (TRITC filter), Cul4A (FITC filter) and chromatin (DAPI) were obtained. The images were subsequently merged using SPOT Advance software. The nuclei with co-localization, as well as nuclei without co-localization were quantified.

Supplementary Material

01

ACKNOWLEDGMENT

We are thankful to Dr. Kristin Chun, Indiana University School of Medicine, for providing Cul4A antibody. This work was supported by a grant CA77637 to PR and a grant CA156164 to SB and PR.

Footnotes

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