Disruption of spermatogenesis in mice lacking A-type lamins

Nuclear lamins are members of the intermediate filament protein family. They form an extensive meshwork, the nuclear lamina, that underlies the inner nuclear membrane. Two types of lamins have been characterized: the A-type lamins that have developmentally regulated expression and the B-type lamins that are constitutively expressed (Goldman et al., 2002; Gruenbaum et al., 2000; Stuurman et al., 1998). Four A-type lamin isoforms have been identified in mammals, namely the lamins A, AΔ10, C and C2 (Benavente et al., 2004; Goldman et al., 2002; Gruenbaum et al., 2000). The first three are expressed in differentiated somatic cells. Lamin C2 has been detected in meiotic stages of spermatogenesis with no other A-type lamins being expressed during this process (Alsheimer and Benavente, 1996). The biological significance of the developmental regulation of the A-type lamins is largely unknown, although it has been suggested that they are involved in the terminal differentiation of cells (i.e. Collard et al., 1992). Despite a lack of evidence for such a role (Sullivan et al., 1999), the functional significance of A-type lamins has gained more relevance (Burke and Stewart, 2002; Cohen et al., 2001; Hutchison, 2002; Wilson et al., 2001) because of the recent discovery that certain human hereditary diseases, the laminopathies (Emery-Dreifuss muscular dystrophy, dilated cardiomyopathy and Dunningan-type familial partial lipodystrophy, mandibuloacral disease, Charcot-Marie-Tooth neuropathy type 2B1 and Hutchinson-Gilford progeria) are caused by mutations in LMNA, the gene coding for the A-type lamins (Bonne et al., 1999; Cao and Hegele, 2000; Cao and Hegele, 2003; De Sandre-Giovannoli et al., 2002; Fatkin et al., 1999; Eriksson et al., 2003). Surprisingly, although A-type lamins are expressed in the majority of adult cell types, pathological manifestations in individuals with LMNA mutations are largely restricted to muscle or fat tissue.

Similarities to the human conditions have been described in Lmna^–/– mice (Sullivan et al., 1999). These mice do not express the A-type lamins because of the targeted introduction of a deletion in the Lmna gene (Sullivan et al., 1999). At birth, Lmna^–/– mice are indistinguishable from their wild-type and heterozygous littermates. However, within 2 to 3 weeks after birth the Lmna^–/– mice show growth retardation and develop severe muscular dystrophy accompanied by the loss of white fat. Other tissues of the Lmna^–/– mice are overtly normal, apart from the thymus that atrophies (Sullivan et al., 1999). In our present study, we show that testes are also affected in Lmna^–/– mice. These mice display a dramatic increase in apoptosis during late pachytene stage and a breakdown of spermatogenesis. However, female Lmna^–/– gametogenesis and fertility remained unaffected, indicating that Lmna expression is required for normal spermatogenesis but not for oogenesis in the mouse. These data validate stringent synaptic checkpoint control in mammalian spermatogenesis (Hunt and Hassolt, 2002).

The wild-type, heterozygous and Lmna^–/– C57Bl/6×129/S1 mice used in this study have been described previously (Sullivan et al., 1999).

Testes were fixed overnight in 4% PBS-buffered formaldehyde (pH 7.4), dehydrated in an ethanol series and embedded in paraffin wax according to standard procedures. 5 μm sections were transferred to slides, paraffin wax was removed by dipping into xylene (three times 5 minutes each) and sections were rehydrated through an ethanol series. Afterwards, sections were stained with Hematoxylin and Eosin (HE) or Hematoxylin alone according to standard protocols. Alternatively, testis and ovary tissues were fixed in 2.5% cacodylate-buffered glutaraldehyde (1 hour, 4°C) and then postfixed with 1% osmium tetroxide (1 hour). After overnight staining with 0.5% uranyl acetate, testes were dehydrated in an ethanol series and embedded in Epon. Semi-thin sections were then prepared and stained with 1% Toluidine Blue.

TUNEL assay on paraffin wax sections of testes was performed according to the manufacturer's instructions (Qbiogene, Heidelberg, Germany). Methyl Green staining of the sections was done according to standard protocols.

Ultrathin sections of Epon-embedded testes (see above) were double stained with uranyl acetate and lead citrate according to standard procedures. Micrographs were obtained with a Zeiss EM-10 electron microscope. Partially overlapping micrographs of individual seminiferous tubules were put together using the Adobe Photoshop 6.0 program.

The following affinity purified antibodies were used in the immunostaining experiments. TRF1: rabbit anti-TRF1 antibodies, #647 (Zhu et al., 2000). RAP1: rabbit anti-human-RAP1 antiserum, #765 (Li et al., 2000). SCP3: guinea pig anti-SCP3 antiserum (Alsheimer and Benavente, 1996). Anti-histone H1t: rabbit-anti-H1t (Moens, 1995). All antisera were diluted in PBS containing 0.05% Tween 20, 0.2% BSA and 0.1% gelatin (PBTG). All antibodies were tested in individual staining reactions for their specificity and performance. Controls without primary antibodies were all negative (not shown).

Mouse testes preparations were obtained from wild-type, Lmna^+/– and Lmna^–/– mice (Sullivan et al., 1999). Animals were killed and testes immediately resected and shock frozen for 5 minutes in isopentane on dry ice and stored at -80°C until further use. Preparation of nuclear suspensions, centromere/telomere fluorescence in situ hybridization (FISH) procedure and detergent spreading of spermatocytes was performed as described previously (Scherthan et al., 2000b) with the following modifications. About 10 μl of a testicular suspension was placed on a glass slide and mixed gently with 80 μl of 1% Lipsol ionic detergent solution (LIP equipment, UK). After about 10 minutes 90 μl of freshly made fixative solution (1% formaldehyde, 10% 50 mM NaBH₃, 0.15% Triton X-100) was added to the slide and gently mixed by tilting. The solution was then dried down, and the slides washed three times with 0.1% agepon (Agfa) and stored at -70°C until further use.

Examination of the Lmna^–/– males revealed that testes development is affected in these mice. We found that the testes in Lmna^–/– males were smaller than in their wild-type and heterozygous littermates (Fig. 1A-C). Subsequent histological analysis revealed a smaller diameter of seminiferous tubules with spermatogenesis also being profoundly abnormal (Fig. 1A-C). The periphery of the seminiferous tubules of Lmna^–/– animals, where spermatogonia and Sertoli cells are located, was apparently normal. In contrast, spermatocytes I were reduced in number, while postmeiotic stages (spermatids and spermatozoa) were largely absent in mutant testes (Fig. 1C) and epididymis (Fig. 2B).

Next, we searched for defects in the progression through first meiotic prophase in spread nuclei of Lmna^–/– spermatocytes by immunostaining of the axial element (AE) protein SCP3 of the synaptonemal complex (SC) (Lammers et al., 1994; Alsheimer and Benavente, 1996). In the Lmna^–/– testes preparations we noted a significantly increased frequency of mid-preleptotene [χ², P<0.001; as determined by cen/tel FISH (Scherthan et al., 1996)] leptotene and zygotene spermatocytes in the knockout as compared to wild type (χ², P<0.01 for leptotene and P<0.001 for zygotene) as well as a significant reduction in pachytene and diplotene spermatocytes in the knockout as compared to wild type or heterozygote (χ², P<0.01) (Fig. 3). The reduction in late spermatocyte stages was also evident when we compared the frequencies of H1t-expressing spermatocytes (Fig. 3) (see Scherthan et al., 2000a). H1t expression becomes detectable at mid pachytene and is seen up to late sperm development (Moens, 1995). In Lmna^–/– testis preparations 28% spermatocytes expressed H1t, while in wild-type and Lmna^+/– spermatocytes there were 52% and 42%, respectively. The differences between wild type and Lmna^+/– were significant (χ², P<0.01) and between the knockout testis preparations and the wild type or heterozygote being highly significant (χ², P<0.0001), which is consistent with elimination of spermatocytes during the pachytene stage (Fig. 3).

The regions in the Lmna^–/– seminiferous tubules where prophase I stages predominated were characterized by a high frequency of cell death (Fig. 1C, Fig. 4E). Dead or dying cells were distinctly stained by Hematoxylin and showed conspicuous morphological alterations (see Russell et al., 1990). In contrast, cell death was rare in heterozygous and wild-type testes (see below). Transmission electron microscopy (TEM) was used to further characterize the defective cells in the mutant testes according to established criteria (Russell et al., 1990). In agreement with the results obtained by SCP3 staining in spread preparations (Fig. 3), apparently normal leptotene/zygotene spermatocytes as well as early-pachytene spermatocytes were observed in mutant seminiferous tubules (Fig. 4G). TEM provided additional evidence that cell death in the Lmna^–/– testes was primarily occurring in the pachytene spermatocytes. Dead cells were found in groups within regions of the seminiferous tubules containing normally appearing pachytene spermatocytes (Fig. 4G) with SC-like structures in the residual nuclei (Fig. 4H). Using the TUNEL assay we established that apoptosis was the most likely cause of cell death (Fig. 1D-F, Fig. 4F). As expected, TUNEL-positive cells were very frequent and present in most of the sectioned seminiferous tubules in the mutant animals (Fig. 1F, Fig. 4F). In contrast, TUNEL-positive cells were rarely found in the seminiferous tubules of heterozygous and wild-type animals (Fig. 1D,E). Further analysis revealed that Lmna^–/– testis tubules up to stage V of the seminiferous cycle (Oakberg, 1956) generally failed to show TUNEL-positive cells (Fig. 4B,C). However, tubules at later stages of the epithelial cycle, i.e. stage VII (Fig. 4E,F), had dying cells. This indicates that cell death is initiated in mid-pachytene.

In summary, the histological, ultrastructural and quantitative analysis indicates that spermatogenesis in Lmna^–/– mice is disrupted by apoptosis of pachytene stage spermatocytes. It is noteworthy that testis size reduction and failure of prophase I progression were also observed in young Lmna^–/– mice (e.g. Fig. 4G), when these animals were still hardly distinguishable from their wild-type and heterozygous littermates.

We then looked for possible defects in Lmna^–/– spermatocytes that could explain spermatogenesis disruption. Since the nuclear lamina has been suspected to play a role in telomere attachment in meiosis (Alsheimer et al., 1999), we performed TEM (Fig. 5) and 3D telomere/centromere FISH analyses [not shown (Scherthan et al., 2000a)]. It was found that meiotic telomere attachment to the nuclear envelope (NE) and 3D distribution of telomeres were morphologically indistinguishable in wild-type and Lmna^–/– spermatocytes (Fig. 5). Likewise, axial elements and SCs of Lmna^–/– spermatocytes were found to contain TRF1, TRF2 and mRAP1 telomere proteins at their ends, which mirrors the situation in the wild type (Fig. 6, and not shown). Furthermore, bouquet analysis by TRF1 immunofluorescence (Scherthan et al., 2000b) as well as telomere/centromere FISH to suspension nuclei (see Scherthan et al., 2000a) revealed similar low frequencies of bouquet spermatocytes (Fig. 3; 0.3% in Lmna^–/–, 0.2% in Lmna^+/– and 0.36% in Lmna^+/+ meiosis; 2535, 2029 and 1956 nuclei investigated, respectively). This indicates that the bouquet stage and meiotic telomere movements are virtually unaffected in Lmna^–/– mice.

Since the telomere complex and its interaction with the NE were overtly normal, and since the presence of a synaptic checkpoint has been postulated to be operational at male prophase I (Odorisio et al., 1998), we performed analysis of homologue pairing and synapsis progression by SCP3-staining of detergent-spread spermatocytes I (Fig. 6). This revealed an abundance of aberrant zygotene-like nuclei in Lmna^–/– spermatogenesis: 87% of abnormal zygotene nuclei (n=24) had 5-15 complete SCs in combination with long unpaired axial elements (Fig. 6A). In such nuclei several short univalents were noted (Fig. 6B). Moreover, almost every second Lmna^–/– pachytene spermatocyte (45%; n=27) had univalent sex chromosomes (Fig. 6C), a condition rarely seen in heterozygous (5%; n=39; Fig. 6D) and wild-type C57Bl/6 spermatocytes (0.7%; n=149). Additionally, 15% of Lmna^–/– nuclei showed terminal X/autosome associations, while this was only observed in 5% of heterozygous cells (n=26) and 0.5% of wild-type spermatocytes (n=200).

Taken together, these results reveal a failure of prophase I progression and defective sex chromosome pairing in Lmna^–/– spermatogenesis and indicate that there is an effect on homologue pairing even in the heterozygous state, albeit at reduced levels. The molecular basis for this failure is not clear at present. One hypothesis would predict that the observed pairing defects are a consequence of the lack of meiosis-specific lamin C2 that may alter the properties of the NE and consequently homologue pairing. However, no support for this view has been obtained from chimeric mice. Recently, three chimeric knockout mice were obtained from wild-type blastocysts injected with Lmna^–/– ES cells. When mated to wild-type females two of these three chimeras produced wild-type and Lmna^+/– offspring (L. Mounkes and C.L.S., unpublished observations). Most probably, the presence of wild-type somatic and spermatogenic cells may result in a more robust physiology in the chimeric animals, which would facilitate mutant sperm generation. However, these observations in chimeric mice indicate that the absence of lamin C2 in Lmna^–/– mice does not directly disrupt male meiosis; rather they favor the notion that spermatogenesis disruption in these animals is a consequence of a more general absence of A-type lamins. The pathophysiological mechanisms leading to the spermatogenic failure in the Lmna^–/– mice, as well as those involved in somatic tissue defects [e.g. muscle dystrophies (Sullivan et al., 1999)], remain to be elucidated. Further insights into this matter have to await data on the fidelity of spermatogenesis in chimeric Lmna knockouts that are not, for obvious reasons, currently available.

In any case, the high frequency of spermatocytes I with unsynapsed chromosome segments and/or sex chromosomes (Fig. 6) may explain the massive cell death during late pachytene (Figs 1, 4), since these defects are probably detected by a so-called pachytene checkpoint, particularly in the male, that monitors timely completion of synapsis and recombination and triggers apoptosis if these events fail within a given time frame (Hunt and Hassolt, 2002; Odorisio et al., 1998; Roeder and Bailis, 2000; Yuan et al., 2002).

In agreement with an assumed lax synaptic checkpoint control in female meiosis (Hunt and Hassolt, 2002) and in contrast to spermatogenesis, histological analysis of the ovaries of adult wild-type Lmna^+/– and Lmna^–/– females revealed no overt differences (Fig. 7). The course of oogenesis appeared to be normal, because dictyotene oocytes were identified in the Lmna^–/– ovaries. In the wild type, no A-type lamins are detectable by immunofluoresence microscopy in early stages of female rodent meiosis (data not shown). However, A-type lamins have been detected in ovulated and fertilized oocytes (Stewart and Burke, 1987). Thereafter, lamin A levels decline and are absent by the blastocyst stage only to first reappear in the trophoblast of the post-implantation embryo (Stewart and Burke, 1987). To determine whether loss of A-type lamins compromises the development of the female germline or early embryogenesis we surgically transferred one Lmna^–/– ovary to each of six ovariectomized wild-type females. The transferred Lmna^–/– ovaries readily engrafted and the recipient females were mated with wild-type males 4-6 weeks after the operation. Four of the recipients became pregnant and delivered a total of 15 offspring. Genotyping the offspring revealed all F1 animals (15/15) to be heterozygous for the mutant Lmna allele. Our analysis of oogenesis in Lmna^–/– mice provides evidence for the operation of a stringent synaptic checkpoint in male meiosis and stress important differences in the requirements and regulation of mammalian meiosis in both sexes (see Hunt and Hassolt, 2002; Yuan et al., 2002).

Finally, it is also noteworthy that to date, the vast majority of the amino acid substitutions in LMNA that are associated with the development of a pathological condition in humans follow autosomal dominant inheritance. Recently, recessive inheritance of a mutation in LMNA has been reported to be the underlying defect of Charcot-Marie-Tooth type 2 (i.e. AR-CMT2, or CMT2) axonal neuropathies (De Sandre-Giovannoli et al., 2002). Interestingly, the AR-CMT2 underlying R298C amino acid substitution localizes in the lamin A/C rod domain and affects all four expressed A-type lamin (A, AΔ10, C and C2) isoforms. Furthermore, sex-specific expression of a heterozygous LMNA mutation has been observed in Dunningan-type familial partial lipodystrophy [FPLD (Vigouroux et al., 2000)]. The spermatogenesis defects observed in the mouse model also follow recessive inheritance and relate to the inactivation of both Lmna alleles. It will thus be interesting to learn more about the fertility of affected AR-CMT2 individuals.

We thank Alexandra Klaus for excellent technical support. H.S. thanks T. de Lange (New York) for mRap1 and Trf1 antibodies and Peter Moens (York, Canada) for H1t antibodies. The work was supported by DFG grants to R.B. (Be 1168/4-4) and H.S. (Sche 350/8-4).