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. 2011 Feb 23;84(6):1235–1241. doi: 10.1095/biolreprod.110.085720

Lymphoid-Specific Helicase (HELLS) Is Essential for Meiotic Progression in Mouse Spermatocytes1

Wenxian Zeng 4, Claudia Baumann 4,8, Anja Schmidtmann 5, Ali Honaramooz 4,6, Lin Tang 7, Alla Bondareva 7, Camila Dores 7, Tao Fan 5, Sichuan Xi 5, Theresa Geiman 5, Rahul Rathi 4, Dirk de Rooij 9, Rabindranath De La Fuente 4,8, Kathrin Muegge 5, Ina Dobrinski 4,7,2
PMCID: PMC3099587  PMID: 21349825

Abstract

Lymphoid-specific helicase (HELLS; also known as LSH) is a member of the SNF2 family of chromatin remodeling proteins. Because Hells-null mice die at birth, a phenotype in male meiosis cannot be studied in these animals. Allografting of testis tissue from Hells−/− to wild-type mice was employed to study postnatal germ cell differentiation. Testes harvested at Day 18.5 of gestation from Hells−/−, Hells+/−, and Hells+/+ mice were grafted ectopically to immunodeficient mice. Bromodeoxyuridine incorporation at 1 wk postgrafting revealed fewer dividing germ cells in grafts from Hells−/− than from Hells+/+ mice. Whereas spermatogenesis proceeded through meiosis with round spermatids in grafts from Hells heterozygote and wild-type donor testes, spermatogenesis arrested at stage IV, and midpachytene spermatocytes were the most advanced germ cell type in grafts from Hells−/− mice at 4, 6, and 8 wk after grafting. Analysis of meiotic configurations at 22 days posttransplantation revealed an increase in Hells−/− spermatocytes with abnormal chromosome synapsis. These results indicate that in the absence of HELLS, proliferation of spermatogonia is reduced and germ cell differentiation arrested at the midpachytene stage, implicating an essential role for HELLS during male meiosis. This study highlights the utility of testis tissue grafting to study spermatogenesis in animal models that cannot reach sexual maturity.

Keywords: chromosomes, developmental biology, HELLS, LSH, meiosis, spermatogenesis, testis, transplantation

Lymphoid-specific helicase (HELLS) plays an essential role during meiotic chromosome synapsis in the mouse testis.

INTRODUCTION

The lymphoid-specific helicase (HELLS; also known as LSH) is a member of the SNF helicase family that is required for normal development and survival in the mouse [1, 2]. SNF2-like helicases play a role in chromatin remodeling, and the HELLS protein has been implicated in the control of genome-wide DNA methylation [3]. Mice homozygous for a targeted deletion of the Hells gene die within a few hours after birth, likely because of kidney failure [1]. Analysis of female germ cells obtained from Hells−/− fetuses on Day 18.5 of gestation revealed the presence of incomplete homologous chromosome synapsis associated with abnormal methylation of transposable elements and tandem repeats at pericentric heterochromatin. In addition, analysis of ovarian explants obtained from Hells mutant females at a stage equivalent to Day 7 of postnatal development indicates that lack of HELLS function is also associated with severe oocyte loss and lack of ovarian follicle formation [4].

Although HELLS is expressed in undifferentiated embryonic stem cells, it is significantly downregulated upon differentiation [5]. The adult animal shows barely detectable levels of HELLS with the exception of the thymus and the testis, suggesting an important biological role in these tissues [6]. However, the neonatal lethality of the Hells−/− mice precludes the direct analysis of the role of HELLS in male germ cell development and differentiation. Shortly after birth, germ cells (gonocytes) within seminiferous tubules resume proliferation to give rise to spermatogonia [7]. Spermatogonia proceed through mitotic and meiotic divisions and complete cytological transformations resulting in the production of virtually unlimited numbers of spermatozoa throughout the life of the male. The complete process of spermatogenesis takes 33 days in mice, with appearance of prophase I spermatocytes by 10 days after birth [8]. An efficient, long-term in vitro culture system has not been established for testis tissue, making it difficult to study a potential phenotype in male meiosis for Hells mutant animals.

Grafting of testis tissue from newborn males of multiple mammalian species into mouse hosts results in recapitulation of complete spermatogenesis with the production of fertilization-competent sperm [9–12]. Previous studies revealed that functional gametes can be obtained from fetal male gonadal tissue grafted into mouse hosts [13]. Allografting of testis tissue harvested from neonatal, nonviable cloned mice to wild-type recipient mice has also been employed successfully to rescue male gonadal function and generate offspring by subsequent intracytoplasmic injection of sperm recovered from testis grafts [14]. Naughton et al. [15] applied whole-testis transplantation to overcome the limitation of neonatal lethality of Gdnf-, Gfra1-, and Ret-deficient mice and found that each of these genes is required for postnatal spermatogenesis but not for embryologic development of the testes. In the present study, allografting of fetal testis tissue recovered from Hells−/− mice into adult host mice was used to monitor progression of spermatogenesis.

MATERIALS AND METHODS

Experimental Design

Hells-heterozygous animals [1] were mated, and fetuses were recovered at Day 18.5 of gestation. Gender and Hells genotype of the pups were determined by PCR. Male gonadal tissue from wild-type, heterozygous, and homozygous knockout mice was grafted into adult mouse hosts and allowed to develop for 4–8 wk. Grafts were recovered and histologically analyzed for progression of spermatogenesis. Additional grafts were recovered 1 wk after grafting following a 1-h exposure to bromodeoxyuridine (BrdU) to determine germ cell proliferation or 22 days after grafting to collect prophase I spermatocytes for chromosome analysis.

Animals and Tissue Transplantation

Hells-heterozygous animals were maintained at the National Cancer Institute on a mixed C57BL/6J × 129/SvJ background. Matings were monitored, and pups were collected from pregnant females at Day 18.5 of gestation. Tissue was collected for genotyping, and gonads were isolated and placed individually in cold PBS and shipped overnight to the University of Pennsylvania. Genotyping of donor animals was done by PCR analysis utilizing the common upstream sense primer 936 (5′-GAG CCT TGA GTG CAT TGG ATC-3′) and the antisense neo primer 219 (5′-GCT GCT AAA GCG CAT GCT CCA GAC-3′) or the antisense Hells primer 918 (5′-GCA CTG AAC AGT CTT TCC CA-3′). Reactions were run for one cycle at 94°C for 3 min, followed by 30 cycles of 94°C for 1 min, 60°C for 1 min, and 72°C for 1 min, and then one cycle of 72°C for 7 min using Platinum PCR Supermix (Invitrogen). PCR reactions were subjected to electrophoresis. For internal comparison of grafting efficiency, B6C3F1 (bred in-house) wild-type testes were also collected at Day 18.5 of gestation at the University of Pennsylvania. Testes were cut in half and grafted into 6- to 8-wk-old male ICR-SCID mice (IcrTac:ICR-Prkdc-scid; Taconic, Inc.) as described earlier [9]. Briefly, recipient mice were castrated under anesthesia, and during the same procedure, three to six incisions were made into the dorsal skin. Tissue grafts were placed subcutaneously through the skin incision, and the incision was closed with a wound clip. Fetal testes from 21 litters were grafted to 23 recipient mice. Depending on availability of pups on a given day, recipients received three to six grafts. Each recipient animal received at least one graft from each genotype. Each testis was cut in half and grafted to two different recipient animals.

To characterize the role of HELLS in meiotic progression, a subset of grafts (n = 4 mice; i.e., four replicates) was recovered 22 days after grafting to collect prophase I spermatocytes for chromosome analysis. All animal procedures were approved by and performed according to the Institutional Animal Care and Use Committees at the National Cancer Institute and the University of Pennsylvania in accordance with the Guide for Care and Use of Laboratory Animals.

Analysis of Testis Grafts

Recipient mice were euthanized at 4 wk (n = 6), 6 wk (n = 6), and 8 wk (n = 11) after grafting, and tissue grafts were recovered. Tissue was fixed in Bouin solution and processed for histology. Each seminiferous tubule in each graft was examined for progression of spermatogenic differentiation, and the most advanced germ cell type present was recorded.

Immunocytochemistry for HELLS

To evaluate the expression pattern of the HELLS protein in wild-type mouse testes, mouse testis specimens were fixed in 4% paraformaldehyde (PFA) at 4°C overnight, dehydrated through a series of ethanol and xylenes, and embedded into paraffin. Sections (thickness 5 μm) were rehydrated sequentially, then boiled for 10 min in a 10 mM citrate buffer (pH 6.0) in a microwave oven, cooled, washed in PBS (1.5 mM KH2PO4, 8.3 mM Na2HPO4, 137 mM NaCl, and 2.7 mM KCl; pH 7.4), and permeabilized two times for 5 min each with 0.1% Triton X-100 in PBS. The samples were blocked with 5% normal donkey serum in PBS for 30 min at room temperature before incubation overnight at 4°C with a custom polyclonal rabbit anti-HELLS antibody raised against full-length recombinant HELLS (1:400 dilution). The sections were washed with PBST (0.025% Triton X-100 in PBS) and incubated for 10 min with 3% H2O2 to quench the endogenous peroxidase activity. Samples were washed again with PBST and then incubated with diluted (1:500 in PBS) peroxidase-conjugated donkey anti-rabbit immunoglobulin (Ig) G (Jackson ImmunoResearch Laboratories) for 1 h at room temperature, washed with PBST, briefly rinsed with PBS, and then exposed to NovaRed substrate (Vector Laboratories) for 2 min, rinsed with water, and counterstained in Gill hematoxylin for 1 min. The sections were subsequently dehydrated through a series of ethanol and xylenes and mounted in Permount (Fisher Scientific). Sections not exposed to the anti-HELLS antibody were used as a negative control.

Germ Cell Counting

To compare the number of germ cells in donor testes of all three genotypes, testes from Fetal Day 18.5 Hells+/+, Hells+/−, and Hells−/− pups were fixed in 2% PFA overnight and embedded in paraffin for sectioning. Sections (thickness 5 μm) were cut and processed for standard hematoxylin-and-eosin staining. For each genotype, two or three pups were used for evaluation. For each sample, germ cells and Sertoli cells were counted based on nuclear morphology (Fig. 1A) in 20–30 cross sections of seminiferous tubules. The ratio of Sertoli cells to germ cells was calculated and used for comparison between genotypes.

FIG. 1.

FIG. 1.

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Histological appearance of donor testis, detection of germ cell proliferation based on BrdU incorporation, and immunocytochemical detection of HELLS in adult mouse testis. A) Gestational Day 18.5 Hells+/+ donor testis. Germ cells (arrow) and Sertoli cells (arrowhead) can be clearly identified based on nuclear morphology. Hematoxylin-and-eosin staining. B) Testis tissue allograft (Hells+/+) exposed to BrdU and recovered 1 wk after grafting. Proliferating germ cells are indicated by arrows and Sertoli cells by arrowheads. C and D) Immunocytochemical detection of HELLS in adult mouse testis. HELLS protein is present in premeiotic (spermatogonia; arrowheads) and meiotic germ cells up to zygotene at stage X (arrows). Bars = 20 μm (A, B, and D) and 40 μm (C).

Immunostaining for BrdU

To detect cell proliferation in the transplanted testis tissue, a subset of recipient mice (n = 3) received an intraperitoneal injection of BrdU (100 mg/kg) at 1 wk after grafting, and mice were euthanized by CO2 inhalation 1 h after BrdU injection. Immunohistochemical staining of cells in the S phase was performed as previously described [11, 16]. Briefly, after deparaffinizing and rehydrating, the slides were incubated in 1 M HCl for 8 min at 70°C in a hybridization oven. After washing in distilled water and Tris-buffered saline (TBS; 10 mM Tris and 150 mM NaCl; pH 7.6), the slides were incubated in 0.1% trypsin (Type II; Sigma Chemical Co.) in TBS for 15 min at room temperature. Nonspecific staining was blocked by incubation with 5% donkey serum in TBS for 20 min at room temperature. A monoclonal mouse anti-BrdU antibody (1:30 dilution with TBS plus 0.1% bovine serum albumin [BSA]; DakoCytomation) was added for 1 h at 37°C. The slides were incubated with secondary donkey anti-mouse IgG linked to horseradish peroxidase (1:100 diluted with PBS plus 1% BSA; Jackson ImmunoResearch Laboratories, Inc.) for 60 min at room temperature. The label was visualized using a Vector DAB kit (Vector Laboratories, Inc.). The reaction was stopped by washing in distilled water. Slides were counterstained with hematoxylin, dehydrated, and mounted.

Cells within seminiferous tubules were counted based on BrdU staining for nuclei in the S phase and hematoxylin counterstaining to identify germ cells or Sertoli cells based on nuclear morphology (Fig. 1B). In three replicates, 17 seminiferous tubules from Hells+/+ samples and 7 seminiferous tubules from Hells−/− samples were counted, for a total of 248 of germ cells and 528 Sertoli cells from Hells+/+ samples and 56 germ cells and 148 Sertoli cells from Hells−/− samples.

Analysis of Meiotic Configuration in Hells−/− Spermatocytes

Testis grafts of different genotypes were recovered on Day 22 postgrafting and immediately processed for the analysis of meiotic configuration on surface spread preparations as described previously [4]. Briefly, tissue grafts were exposed for 20 min to a hypotonic solution of sodium citrate. Following gentle tissue dissociation, a drop of cell suspension was subsequently fixed on wet slides containing 1% PFA and 0.15% Triton X 100 (Bio-Rad). The stage of prophase I of meiosis in control wild-type and heterozygous spermatocytes was compared with that of Hells mutant spermatocytes following staining of the synaptonemal complex protein SYCP3 using a 1:1000 dilution of a rabbit anti-SYCP3 antibody [17] followed by a 2-h incubation with a 1:500 dilution of Alexa Fluor 488 goat anti-rabbit secondary antibody (Molecular Probes, Inc.). In addition, homologous chromosome synapsis, resolution of DNA double-strand breaks at the pachytene stage, and formation of an XY body were assessed by immunochemical detection of the phosphorylated (Ser-139) form of histone H2A (H2AFX) using a mouse monoclonal antibody (Abcam) at a 1:500 dilution followed by a 2-h incubation with a 1:1000 dilution of Alexa Fluor 555 goat anti-mouse secondary antibody (Molecular Probes, Inc.) Slides were counterstained with 4′,6′-diamidino-2-phenylindole and meiotic configurations analyzed using a DMRX/E microscope (Leica Microsystems) under a 100× objective as described previously [4].

Statistical Analysis

Data were analyzed using SigmaStat 3.0 (SPSS. Inc.). A Student t-test was performed to compare two groups. Data were expressed as the mean ± SEM, and P < 0.05 was considered to be significant. The percentage of control and Hells mutant spermatocytes that exhibited asynapsed chromosomes in three independent experimental replicates was analyzed using arcsine-transformed data and compared by one-way ANOVA. Groups were compared using the Fisher protected least-significant difference post-hoc test using JMP Start Statistics (SAS Institute, Inc.). Differences were considered to be significant when P < 0.05. Variation among replicates is presented as the SD.

RESULTS

Expression of HELLS and Germ Cell Proliferation

Immunocytochemistry for HELLS on wild-type adult mouse testis revealed the presence of HELLS protein in premeiotic and meiotic germ cells up to stage X (zygotene stage) (Fig. 1, C and D). To determine if germ cell proliferation was affected in the absence of HELLS, the number of germ cells per 100 Sertoli cells was counted in Day 18.5 fetal donor testes of all three genotypes, and the number of proliferating germ cells was counted based on cell morphology and BrdU incorporation after a 1-h exposure in the host mouse 1 wk after grafting. No difference was found between testes of all three genotypes in the number of germ cells/100 Sertoli cells at Fetal Day 18.5 (mean ± SEM, 17.2 ± 0.38, 17.5 ± 0.31, and 17.2 ± 0.38 germ cells/100 Sertoli cells for Hells+/+, Hells+/−, and Hells−/− testes, respectively; P > 0.05). However, the percentage of BrdU-positive germ cells was significantly higher in Hells+/+ grafts than in Hells−/− grafts (39% ± 11.8% vs. 22% ± 3.8%; n = 3 mice, P < 0.05). These results indicate that fewer germ cells proliferated in Hells−/− tissue. Hells+/+ grafts tended to have more BrdU-positive Sertoli cells than Hells−/− grafts, but the difference was not significant (33.4% ± 3.7% vs. 22% ± 1.4%).

Progression of Spermatogenesis In Vivo

Before grafting, gonocytes and Sertoli cells were present in the seminiferous cords in Day 18.5 fetal testis tissue from all genotypes (Fig. 2A). At 4 wk after grafting, testis tissue from wild-type and heterozygous pups had progressed through meiosis with round spermatids present as the most advanced germ cell stage (Fig. 2B), whereas spermatogenesis arrested at stage IV and midpachytene spermatocytes were the most advanced germ cell stage observed in Hells−/− tissue (Fig. 2D and Table 1). In the control B6C3F1 wild-type tissue at 6 wk after grafting, round spermatids and elongated spermatids were observed (Fig. 2C). Similar to previous observations [11], the majority of tubules had an expanded lumen compared to normal wild-type mouse spermatogenesis at all time points and in grafts from all genotypes.

FIG. 2.

FIG. 2.

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Histological appearance of testis tissue before and after allografting. A) Before grafting, gonocytes (arrow) and Sertoli cells are present in the seminiferous tubules in Day 18.5 embryonic testis tissue from all genotypes (Hells−/− shown). B) At 6 wk after grafting, round spermatids (arrow) were found in Hells+/+ (shown) and Hells+/− tissue. C) In the control B6C3F1 wild-type tissue at 6 wk after grafting, round spermatids and elongated spermatids (arrow) were observed. D) In Hells−/− tissue, spermatogenesis arrested at stage IV (asterisk indicates tubule after stage IV, and arrow indicates type B spermatogonia) and germ cells did not differentiate beyond the midpachytene stage (tubule before stage IV; arrowheads). Bar = 50 μm.

TABLE 1.

Most advanced germ cell stage present in testis grafts.

graphic file with name bire-84-06-21-t01.jpg

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Taken together, Hells−/− testis grafts display a complete stage IV arrest at the midpachytene stage, whereas in Hells+/+ and Hells+/− testis grafts, germ cells differentiated into spermatids, suggesting a severe maturation defect in the absence of HELLS.

Analysis of Chromosome Configuration During Meiotic Prophase I

Testis tissue allografting proved to be an efficient approach to obtain Hells mutant spermatocytes at different stages of meiotic prophase I. Analysis of meiotic configuration in wild-type (n = 250) and heterozygous (n = 354) spermatocytes revealed the presence of 19 autosomal chromosome bivalents in addition to the partially synapsed sex chromosomes, indicating that complete homologous chromosome synapsis occurred in the majority (>80%) of control cells analyzed (Fig. 3A). However, tissue allografting induced a slight increase in the proportion of wild-type (18.9%) and heterozygous (19.1%) pachytene-stage spermatocytes that exhibit one or more asynapsed chromosomes (Fig. 3C). In contrast, lack of HELLS function induced a significant increase (P < 0.0001) in the proportion (97.8%) of mutant spermatocytes (n = 670) that exhibited abnormal synapsis of homologous chromosomes (Fig. 3, B and C) as identified by the presence of asynapsed univalents (Fig. 3B, arrows) as well as the formation of axial gaps in the synaptonemal complex of one or more chromosomes per cell (Fig. 3B, arrowhead).

FIG. 3.

FIG. 3.

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Impaired progression of prophase I in Hells−/− spermatocytes. A) Pachytene-stage spermatocyte obtained from wild-type testis grafts exhibiting 19 fully synapsed autosomal bivalents. The position of the partially synapsed sex chromosome bivalent is indicated (arrow). B) Lack of HELLS function is associated with incomplete synapsis of homologous chromosomes (arrows) as indicated by the presence of asynapsed univalents and axial gaps at the synaptonemal complex (arrowhead) in a high proportion of mutant spermatocytes. C) Proportion of pachytene-stage spermatocytes that exhibit asynapsed univalents. Data are presented as the mean ± SD of three independent experimental replicates. Different superscripts indicate significant differences (P < 0.0001).

During the leptotene and zygotene stages of meiosis, H2AFX is a bona fide marker for DNA double-strand breaks. At these stages of prophase I, H2AFX immunostaining reveals a diffuse signal encompassing the entire nucleus [18, 19]. However, as meiosis proceeds and homologous chromosomes synapse during the transition from the zygotene to the pachytene stage, H2AFX signals become fragmented and progressively resolved at autosomal bivalents. In striking contrast, H2AFX accumulates at the partially asynapsed sex chromosomes in male spermatocytes as a component of the macrochromatin domain at the XY body, consistent with the process of meiotic sex chromatin inactivation [18]. Analysis of H2AFX phosphorylation in wild-type (n = 237) and Hells mutant spermatocytes (n = 241) after 22 days of allografting revealed comparable proportions of spermatocytes at the leptotene stage (19% and 15%, respectively) and the zygotene stage (34% and 34%, respectively) of meiosis as assessed by the patterns of H2AFX nuclear staining (red; Fig. 4, A and B) and the degree of homologous chromosome alignment (SYCP3, green; Fig. 4, A and B). The pachytene stage was determined by the resolution of double-strand breaks at completely synapsed chromosome bivalents and the exclusive accumulation of H2AFX at the XY body [18, 20, 21]. Typical pachytene-stage chromosome configurations were found in 47% of wild-type prophase I spermatocytes, of which a large majority (97%) showed characteristic H2AFX labeling only at the XY body (Fig. 4 C, arrow, and E), whereas H2AFX was not detectable at autosomal bivalents, indicating resolution of double-strand DNA breaks and complete homologous chromosome synapsis. In striking contrast, chromosome synapsis in mutant spermatocytes was evident only in a subset of bivalents (Fig. 4D, asterisk), whereas widespread asynapsis of univalents was accompanied by persistence of H2AFX signals (Fig. 4D, arrowhead). Importantly, a typical H2AFX-labeled XY body was not detected in Hells−/− spermatocytes (Fig. 4, D and E). These results indicate that lack of HELLS function interferes with homologous chromosome synapsis and formation of an XY body, consistent with meiotic arrest at the midpachytene stage.

FIG. 4.

FIG. 4.

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Persistence of H2AFX foci associated with double-strand DNA breaks at asynapsed chromosomes in Hells−/− spermatocytes. A and B) Hells+/+ (A) and Hells−/− (B) spermatocytes showing diffuse H2AFX staining throughout the zygotene stage of meiosis. C) Hells+/+ pachytene-stage spermatocyte with 19 fully synapsed bivalents and exclusive association of H2AFX with the partially asynapsed sex chromosome bivalent at the XY body (arrow). D) Hells−/− spermatocytes lacking HELLS protein reveal persistence of H2AFX foci at asynapsed chromosomes (arrowhead) as well as failure to form a sex body in all (n = 122) early pachytene spermatocytes analyzed. E) Proportion of spermatocytes at indicated stages of meiotic prophase I. The proportion of pachynema demonstrating formation of a characteristic XY body is also displayed. Data are presented as the mean ± SD of two independent experimental replicates. Different superscripts indicate significant differences (P < 0.05).

DISCUSSION

Meiotic prophase I in the mouse male germline begins shortly before the onset of puberty [22]. However, the perinatal mortality observed in Hells mutant males precludes the analysis of HELLS function during spermatogenesis. Transplantation of male germ cells and testis tissue has been established in the mouse to induce germ cell differentiation [9, 23]. Spermatogonia transplanted into the seminiferous tubules of a recipient mouse can proliferate and differentiate, and production of functional spermatozoa can be achieved in host testis. However, efficiency of recipient testis colonization and induction of donor-derived spermatogenesis is very low when germ cells are recovered from late-gestation fetal testes [24], and it was considered to be impractical to obtain sufficient numbers of spermatogonia from Hells−/− fetuses for transplantation. On the other hand, transplantation of testis tissue can lead to functional spermatogenesis in recipient mice [9, 13, 14]. In the present study, we applied this model of testis tissue grafting to overcome the limitation imposed by Hells−/− neonatal lethality for the study of HELLS function in male spermatogenesis.

Consistent with previous studies [9, 11, 12], the efficiency of spermatogenesis in the grafted Hells−/− and B6C3F1 fetal testis was low, with some degree of damage to the seminiferous epithelium. Although the underlying causes are not yet known, this possibly resulted from an imbalance in fluid production and resorption [11], as was also observed in bovine and equine testis tissue grafts [25, 26].

Notwithstanding these limitations, Day 18.5 embryonic mouse testis tissue can survive and differentiate after transplanting into immunodeficient mice. Germ cells in Hells+/+ and Hells+/− testis tissue differentiated through meiosis to form haploid cells. In contrast, zygotene/early pachytene spermatocytes were the most advanced germ cell type present in Hells−/− tissue at all analyzed time points. In addition, whereas germ cell numbers were not different between Hells+/+ and Hells−/− donor testes at Day 18.5 of gestation, proliferation of germ cells was reduced in Hells−/− grafts.

Previous studies indicate that HELLS plays a critical role in homologous chromosome synapsis and maintenance of genome stability in mammalian oocytes [4]. To our knowledge, however, whether HELLS plays a role during male meiosis has remained unexplored. Results in the present study indicate that testis tissue allografting is an efficient system to overcome the limitations imposed by the perinatal mortality phenotype observed in Hells mutant pups. Analysis of meiotic configurations revealed that the absence of HELLS function in the male germline is associated with abnormal synapsis of homologous chromosomes in a high proportion of mutant spermatocytes. HELLS-deficient spermatocytes exhibit a range of meiotic abnormalities, including persistence of H2AFX foci at one or more asynapsed univalents indicative of the presence of unrepaired DNA double-strand breaks and axial gaps at the synaptonemal complex. Consistent with the phenotype observed in Hells−/− mutant spermatocytes, extensive asynapsis of the autosomal chromatin compartment might interfere with meiotic progression and, hence, sex body formation, resulting in midpachytene arrest and tubule stage IV apoptosis [20, 21, 27, 28]. These results provide evidence that HELLS plays a key role during male meiosis and, together with our previous observations [4], indicate that HELLS is required for the progression of meiosis in both the male and the female germline.

The mechanisms through which HELLS mediates homologous chromosome synapsis are not known. However, our previous studies indicate that HELLS is also involved in mediating the epigenetic silencing of tandem repeats at centromeric heterochromation as well as transposable elements through its primary role in the establishment of DNA methylation patterns at repetitive elements in mammalian oocytes [4, 29]. Thus, it is conceivable that in the absence of HELLS function, transcriptional reactivation of transposons of the intracisternal-A particle class during meiosis may interfere with homologous chromosome search and synapsis [4]. Hence, the results of the meiotic phenotype, in particular failure to form a characteristic XY body, are consistent with the observation that midpachytene-stage spermatocytes are the most advanced germ cell type observed in Hells-null testis tissue allografts. Our results indicate that testis tissue allografting supports the progression through different stages of the meiotic prophase I in control and heterozygous testis samples. However, this approach induced a slight increase in the proportion of spermatocytes exhibiting asynapsed chromosomes compared with that observed in vivo [22]. The mechanisms involved in this process are not fully understood. However, this methodology provides a unique system to further characterize both environmental and hormonal factors that might adversely affect meiotic progression and warrants further investigation.

In conclusion, allografting of testis tissue can overcome the limitations on studying male germ cell differentiation imposed by neonatal lethality. In the present study, allografting revealed that proliferation of spermatogonia is reduced and that germ cell differentiation is arrested in the absence of HELLS, suggesting an essential role for HELLS during male meiosis, as was reported previously for female meiosis [4]. Meiotic germ cells harvested from testis tissue allografting can now be studied to define the mechanism underlying the observed arrest of spermatogenesis and to further characterize the role of HELLS in meiotic chromatin remodeling.

ACKNOWLEDGMENTS

We thank C. Heyting for the generous gift of antibodies. We also thank Drs. Martin Dym and Richard Oko for critical analysis of spermatogenic progression in grafted testis tissue.

Footnotes

1

Supported by National Institutes of Health (NCRR 2R01RR17359-08 to I.D. and NICHD 2RO1HD042740-04 to R.D.L.F.) and National Cancer Institute (contract N01-C0-12400 to K.M.).

3

These authors contributed equally to this work.

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