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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2009 Oct 6;106(40):17055–17060. doi: 10.1073/pnas.0906414106

MEIG1 is essential for spermiogenesis in mice

Zhibing Zhang a,b,1, Xuening Shen a, David R Gude a,b, Bonney M Wilkinson c, Monica J Justice c, Charles J Flickinger d, John C Herr d, Edward M Eddy e, Jerome F Strauss III a,b
PMCID: PMC2746124  PMID: 19805151

Abstract

Spermatogenesis can be divided into three stages: spermatogonial mitosis, meiosis of spermatocytes, and spermiogenesis. During spermiogenesis, spermatids undergo dramatic morphological changes including formation of a flagellum and chromosomal packaging and condensation of the nucleus into the sperm head. The genes regulating the latter processes are largely unknown. We previously discovered that a bi-functional gene, Spag16, is essential for spermatogenesis. SPAG16S, the 35 kDa, testis-specific isoform derived from the Spag16 gene, was found to bind to meiosis expressed gene 1 product (MEIG1), a protein originally thought to play a role in meiosis. We inactivated the Meig1 gene and, unexpectedly, found that Meig1 mutant male mice had no obvious defect in meiosis, but were sterile as a result of impaired spermatogenesis at the stage of elongation and condensation. Transmission electron microscopy revealed that the manchette, a microtubular organelle essential for sperm head and flagellar formation was disrupted in spermatids of MEIG1-deficient mice. We also found that MEIG1 associates with the Parkin co-regulated gene (PACRG) protein, and that testicular PACRG protein is reduced in MEIG1-deficient mice. PACRG is thought to play a key role in assembly of the axonemes/flagella and the reproductive phenotype of Pacrg-deficient mice mirrors that of the Meig1 mutant mice. Our findings reveal a critical role for the MEIG1/PARCG partnership in manchette structure and function and the control of spermiogenesis.


Spermatogenesis can be divided into three stages: spermatogonial mitosis, meiosis of spermatocytes, and spermiogenesis, the final step of spermatogenesis. During this stage, the haploid round spermatids differentiate into species-specific shaped spermatozoon, with dramatic morphological changes, including elongation and condensation of the nucleus, and formation of the flagellum (1, 2). Even though some genes have been reported to be indispensable for this process (3, 4), the underlying mechanisms remains largely unknown and need to be elucidated.

Mouse meiosis expressed gene 1 (Meig1) was originally identified in a search for mammalian genes potentially involved in meiosis. Two Meig1 transcripts, 11a2 and 2a2, were identified previously, both containing three exons. The two transcripts share the same ORF and 3′ UTR, but differ in their 5′ UTRs. Each has a unique non-translated exon 1. The 11a2 message was expressed in somatic cells in the testis, including Leydig cells, whereas the predominant 2a2 isoform was reported to be germ cell-specific. The 2a2 transcript begins to accumulate in the testis at day (d)8–9 of postnatal (pn) development, coinciding with the entry of germ cells into meiosis, and is expressed most abundantly at pn d14 and subsequent stages, when spermatocytes enter the pachytene stage. In situ hybridization analysis showed that Meig1 expression level was low in leptotene cells and increased as the cells progressed through zygotene and pachytene stages. In addition, Meig1 message was also detected in embryonic ovary after d15 of gestation when the cells entered the pachytene stage of meiosis 1, but not in adult ovary, suggesting that Meig1 is a meiosis-associated gene (59). A recent transcriptional profile study revealed that the Meig1 message is also present in Sertoli cells in fetal gonads and a Sertoli cell line TTE3 (10, 11). Although the molecular weight is 10 kDa according to its amino acid composition, MEIG1 protein migrates as a 14-kDa band in Western blots because of its basic nature. MEIG1 protein contains multiple consensus sequences for serine and threonine phosphorylation. There is also evidence that MEIG1 protein is phosphorylated and forms a dimer in vivo (8). Furthermore, the phosphorylated dimer enters the nucleus during the first meiotic prophase and binds to meiotic chromatin (9).

The function of MEIG1 remains unknown. Our previous investigation revealed that MEIG1 associates with SPAG16S, a 35-kDa nuclear protein essential for spermatogenesis (12). Recent studies by others also suggested that MEIG1 might be essential for spermatogenesis and related to ciliary function. Meig1 message is dramatically reduced in heat shock transcription factor 2 (Hsf2) mutant mice, and this may result in impaired spermatogenesis and reduced fertility of Hsf2 mutant mice (13). A bioinformatic analysis revealed that Meig1 is most abundantly expressed in tissues rich in ciliated cells, such as, testis, lung, olfactory sensory neurons, and is, therefore, predicted to be important for cilia function (14).

To investigate the function of the Meig1 gene, we generated a Meig1-conditional knockout mouse model, and crossed it to CMV-Cre transgenic mice so that the Meig1 gene was deleted globally in vivo. Our studies with the globally targeted Meig1-knockout mice indicate that homozygous mice are viable, but the males are sterile, producing only a few sperm that are morphologically abnormal. Spermatogenesis is dramatically impaired at the stage of elongation and condensation. These studies indicate that MEIG1 is a key protein in the regulation of spermiogenesis.

Results

To predict potential function of the MEIG1 protein, the mouse MEIG1 protein sequence was analyzed for conserved domains with NCBI conserved domain program (http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml). No known functional domains were identified. However, the MEIG1 protein sequences were found to be highly conserved among different species (Fig. S1).

To further characterize the Meig1 gene, 5′ RACE was conducted with a Meig1 specific reverse primer localized in exon 1. The PCR products were cloned into pCR2.1 TA vector, and 20 inserts were fully sequenced. Three isoforms were identified, Meig1_v1, Meig1_v2, and Meig1_v3. Meig1_v1 and Meig1_v2 correspond to 11a2 and 2a2, respectively, Meig1_v3 is a previously unidentified isoform. This isoform also shares the same coding sequences as Meig1_v1 and Meig1_v2, but it has a unique non-translated exon in front of the first coding exon (Fig. 1A). Thus, the three isoforms can be distinguished by their non-translated exons. To further investigate the tissue distribution and expression patterns during the first wave of spermatogenesis, primer sets were designed so that the three isoforms could be amplified separately. The reverse primer is located in exon 1, the forward primers are in individual non-translated exons (Fig. 1A, 1a for Meig1_v1, 1b for Meig1_v2, and 1c for Meig1_v3). PCR was performed using cDNAs reversely transcribed from total RNA isolated from different tissues. Meig1_v1 message, which was reported to be a somatic isoform, was present in several tissues tested, including lung, liver, testis, and oocytes; Meig1_v2, which was reported to be germ cell-specific isoform, was present in almost all of the tissues analyzed (Fig. 1B). The other isoform we identified, Meig1_v3 was only expressed in the testis (Fig. 1B). Expression patterns of the three isoforms were also investigated during the first wave of spermatogenesis (Fig. 1C). Meig1_v1 message was present from d20 after birth, Meig1_v2 was present throughout the whole process of spermatogenesis, Meig1_v3 was detectable from d12 after birth, and the message was abundant in a late stage of spermiogenesis.

Fig. 1.

Fig. 1.

Expression patterns of the three Meig1 isoforms. (A) Structure of the Meig1 gene. Solid boxes indicate translated exons (1 and 2), open, notched, and crossed boxes indicate nontranslated exons (1a, 1b, and 1c). The arrows indicate location of RT-PCR primers. The red solid boxes indicate 3′UTR of the Meig1 transcripts. I, II, III under 1b represent three consecutive sequences in the exon. (B) Tissue distribution of the three Meig1 transcripts. H: heart; B: brain; SP: spleen; Lu: lung; Li: liver; Sm: skeletal muscle; K: kidney; T: testis; Oc: oocyte; Ov: oviduct. (C) mRNA expression of the three Meig1 transcripts during the first wave of spermatogenesis (RT-PCR analysis).

The Ensemble Genome Browser shows five distinct Meig1 transcripts in the mouse: Meig1-001, 002, 003, 004, and 005. Sequences of these five transcripts were compared with the three Meig1 transcripts we identified. Meig1-002 is identical to Meig1-v1; Meig1-001 is identical to Meig1-v2, except that it lacks sequence I; and Meig1-005 contains an extra sequence (IV) compared to Meig1-001. Meig1-003 and Meig1-v3 share the same coding exons and the nontranslated exon 1c, but Meig1-003 contains an extra nontranslated exon, sequence III in 1b. Meig1-004 contains the same coding sequence as the other Meig1 transcripts, but a shorter 3′UTR, in addition, its nontranslated exon is located between 1b and 1c (Fig. S2). Specific primers were designed for PCR amplification of Meig1-004 and Meig1-005. Meig1-004 is only present in the testis. Besides being highly expressed in the testis, Meig1-005 was present in most of the tissues tested, including brain, lung, and oviduct (Fig. S2Ba). During the first wave of spermatogenesis, Meig1-004 is detectable from d8 after birth, Meig1-005 is expressed as early as d6 after birth, and the message is up-regulated during spermatogenesis (Fig. S2Bb).

To investigate the role of the Meig1 gene in vivo, we generated a Meig1-conditional knockout mouse model using the LoxP system. The targeting strategy is shown in Fig. S3A. LoxP sites were inserted upstream and downstream of the first coding exon, exon 1. The targeting construct was linearized by ClaI digestion at the plasmid/gene insert junction and transfected into the XY ES cells by electroporation. The ES clones were screened by PCR with a forward primer located in the Neo cassette of the target construct, and a reverse primer located downstream of the right arm; or a forward primer located upstream of the left arm, and a reverse primer located in the Neo cassette (as shown in Fig. S3A). Five positive ES cell clones were identified by PCR and were further confirmed by Southern blotting using a probe as shown in Fig. S3A corresponding to an upstream region flanking the mutation site. The probe recognized a 10-kb DNA fragment in the wild-type allele and a 7-kb DNA in the targeted allele when DNA was digested by BamH I (Fig. S3B). ES cells from 2 different clones verified to contain the properly Meig1-mutated gene were injected into blastocysts to produce chimeric mice. The chimeric male mice were bred to wild-type female mice to create heterozygous mice without deletion of any genomic sequence (Fig. S3C). These heterozygous mice were crossed to FLP recombinase transgenic mice to delete the Neo cassette, and the resulting mice (Meig1flox mice) were crossed with CMV-Cre transgenic mice expressing Cre recombinase and thus deleting exon 1 of the Meig1 gene during embryogenesis.

When Meig1flox mice were crossed to CMV-Cre transgenic mice, it was found that the mutant allele could be transmitted from both Meig1 heterozygous males and females, and that homozygous mice could be obtained from interbreeding of the heterozygous male and female mice (Fig. 2A). Both male and female null mice were viable and showed no gross abnormalities associated with cilia dysfunction in mice, including failure to thrive, hydrocephalus, situs inversus, sinus, and pulmonary infection. Northern blot and RT-PCR analyses revealed that the Meig1 messages were undetectable in the testis, and all other somatic tissues evaluated (Fig. 2 B and C and Fig. S2Bc). Western blot analysis using testis extracts demonstrated that MEIG1 protein was absent in the Meig1-null mice (Fig. 2D).

Fig. 2.

Fig. 2.

Phenotype of Meig1/CMV-Cre mutant mice. (A) Representative PCR results using a primer set (P3 and P4 in Fig. S3A, primer sequences in Table S1) showing heterozygous (+/−), wild-type (+/+), and homozygous (−/−) genotypes. (B) Northern blot analysis of testicular Meig1 mRNA expression in wild-type, heterozygous and homozygous mice with Meig1 full-length cDNA as the probe. (C) All of the three Meig1 transcripts are disrupted in all of the tissues in Meig1/CMV-Cre mice as revealed by PCR using isoform specific primers sets. B: brain; Li: liver; Lu: lung; Sp: spleen; K: kidney; H: heart; T: testis. (D) Western blot analysis of testicular MEIG1 protein expression in wild-type, heterozygous and homozygous mice. (E) Representative testicular H&E staining images from wild-type and Meig1 homozygous mutant mice. (F) Analysis of haploid (D), diploid (2D), and tetraploid cells (4D) cell populations in wild-type and knockout mice by flow cytometry. (G) Representative images of epididymides from a wild-type mouse (+/+) and a Meig1 homozygous mutant mouse (−/−). (H) Representative sperm collected in 2 mL PBS from cauda epididymides from a wild-type (+/+) and a Meig1 homozygous (−/−) mice. The arrow points to a sperm with a round head, the arrow head points to a sperm with a detached head. (I) Sperm count of wild-type (+/+), heterozygous (+/−), and homozygous (−/−) mice. n = 8 for each group. *: P < 0.001.

To test fertility of the Meig1 mutant mice, mature nullizygous mice (>6 weeks of age) were mated with wild-type mice. All of the wild-type (Meig1flox), heterozygous and homozygous female mice tested delivered pups with normal litter sizes. Wild-type and heterozygous males were also fertile. However, all nine homozygous mutant male mice tested were sterile through 4 months of breeding (Table 1).

Table 1.

Fertility, fecundity, and testis weight of wild-type and Meig1 mutant mice

Genotype Male fertility Litter size (n = 8) Testis/body weight (n = 8, mg/g) Female fertility Litter size (n = 8)
+/+ 8/8 8.6 ± 0.6 8.15 ± 0.41 8/8 9.4 ± 0.5
+/− 8/8 8.4 ± 0.4 8.05 ± 0.35 8/8 9.6 ± 0.4
−/− 9/0 0 7.95 ± 0.41 8/8 10.1 ± 0.6

To test fertility, sexually mature mice were bred to wild-type animals for four months. Litter size was recorded for each mating. Testis weight and body weight were measured from adult mice.

The testis weight of the nullizygous mice euthanized at 6 months of age was comparable to age-matched wild-type mice (Table 1). Light microscopy revealed that in wild-type and heterozygous adult mice, all stages of spermatogenesis were present (Fig. 2E and Fig. S4). However, in Meig1 homozygous mutant mice, microscopy revealed that most spermatids developed only up to steps 12–13, a few to steps 14–15, and beyond that most had been shed (Fig. 2E and Fig. S4). Even though spermiogenesis is arrested before complete spermatid elongation, there was no significant differences in the DNA content of cell populations between wild-type and Meig1 mutant mice as analyzed by flow cytometry using adult mice, indicating no defects in meiosis (Fig. 2F). In epididymides from wild-type adult mice, typical adult sperm concentrations were found (Fig. 2G), but epididymides from Meig1 homozygous mutant mice only contained debris and degenerating sperm, none of which were motile as examined by visual inspection with a light microscope (Fig. 2 G–I). Histological evaluation of the lung, brain, heart, kidney and liver revealed normal tissue architecture. Transmission electron microscopy demonstrated that spermatids in wild-type mice undergo the normal developmental process, including flagella formation and chromatin condensation and have a normal manchette structure (Fig. 3 A–D and Fig. S5). However, in Meig1-deficient mice, there was failure of flagellar formation, disorganization of sperm axonemes, and deformed sperm heads, no obvious or disrupted manchette structures were seen in spermatids (Fig. 3 E–L and Fig. S6). We also observed condensed Sertoli cell cytoplasm. A normal “9 + 2” axoneme arrangement was not present in the flagellum of MEIG1-deficient spermatids, and some flagella contained multiple axoneme structures. Interestingly, flagellar components such as microtubules and outer dense fibers could be detected but were not assembled correctly. This is consistent with the normal expression of genes encoding axoneme and fibrous sheath proteins in the MEIG1-deficient mice (Fig. S7) and the observation that TNP2 failed to translocate into nuclei of spermatids in Meig1-deficient mice (Fig. S8).

Fig. 3.

Fig. 3.

Testicular ultrastructure in adult wild-type and Meig1 mutant mice. Representative transmission electronic microscopy images from an adult wild-type mouse (A–D) and a Meig1 homozygous mutant mouse (E–L). A and B show normal spermatogenesis and axoneme structure in wild-type testes, C and D shows normal condensing spermatids heads and manchette structure (arrow heads) in wild-type testes. E shows failure of spermatogenesis as evaluated by absence of sperm in the lumen of seminiferous tubule. F represents highly condensed Sertoli cell cytoplasm (arrow). G and H represent some condensing spermatids that lack manchette structure as seen in the wild-type testes (C and D). Inserts in F and G represent two deformed sperm heads. I–L and inserts show disorganized flagella. Note that the flagella components, such as microtubules and outer dense fibers seem to be made normally, but are not assembled correctly into flagella. Some flagella contain multiple axonemal structure (J). Arrows point to disorganized flagella.

To explore possible mechanisms of MEIG1 action, we carried out a yeast two-hybrid screen to identify interacting partners. Among the 70 positive clones sequenced, 18 represented PACRG. This interaction was confirmed by co-immunoprecipitation from lysates of MEIG1 and PACRG co-transfected COS-1 cells (Fig. 4 A and B), and testicular extracts (Fig. S9). Interestingly, testicular PACRG protein, but not mRNA expression, was reduced in Meig1 mutant mice. However, in quaking mutant mice in which the Pacrg gene is deleted, testicular MEIG1 protein was unchanged (Fig. 4 C–E).

Fig. 4.

Fig. 4.

MEIG1 associates with PACRG. (A) Expression of PACRG in COS-1 cells. COS-1 cells were transfected with the PACRG/pEGFP-C1 or empty pEGFP-C1 vector. Forty-eight hours after transfection, total cell lysates were prepared and Western blots were performed with an anti-GFP antibody (i) and an anti-PACRG antibody (ii). (B) Co-immuniprecipitation of MEIG1 and PACRG. COS-1 cells were co-transfected with PACRG/pEGFP-C1 and MEIG1/pTarget plasmids. Forty-eight hours after transfection, total cell lysates were pulled down with a preimmune serum or an anti-MEIG1 antibody. Western blots were conducted with an anti-GFP antibody (i) and an anti-MEIG1 antibody (ii). (C) Representative Western blot analysis of testicular PACRG protein expression in Meig1 mutant mice. v/v: quakingviable mutant mice; v/v TG: Pacrg-rescued qkv mice. (D) Real-time PCR analysis of Pacrg mRNA expression in Meig1 mutant mice. (E) Representative Western blot analysis of testicular MEIG1 protein expression in Pacrg mutant mice.

Discussion

The tissue distribution of the three Meig1 tanscripts we identified is different. Meig1_v1 is expressed in several tissues, including testis, lung, liver, and oocytes; it is the most abundant in the testis. Meig1_v2 is expressed in almost all of the tissues tested, and the abundance is comparable in these tissues, suggesting that Meig1_v2 is not a germ cell-specific transcript. Meig1_v3 is only present in the testis, indicating that this is a testis-specific transcript.

The expression pattern of the three transcripts during the first wave of spermatogenesis is also different. Meig1_v2 is stably expressed during the whole process of spermatogenesis, indicating it is a house-keeping isoform. This result contrasts with the results of previous studies, which suggested that 2a2 (Meig1_v2) was a germ cell-specific isoform (59). Meig1_v1 and Meig1_v3 are expressed from d20 and d12 after birth, respectively, indicating that that they are late-meiosis and postmeiosis-expressed isoforms, suggesting a special role at this stage. The presence of multiple Meig1 transcripts in vivo suggests that the protein might have multiple functions depending upon cell type and timing of expression.

The phenotype of the Meig1-deficient mice generated using CMV-Cre excision was surprising, since previous studies suggested that MEIG1 might play a role in meiosis (59). However, we found that spermatocytes completed meiosis and spermatogenesis was arrested in the elongation and condensation stage, revealing that the MEIG1 is not necessary for meiosis, but is required for the completion of spermiogenesis.

With the exception of male infertility, no other phenotype related to immotile cilia syndrome was observed (15, 16), and the major feature of infertility is failure of spermiogenesis rather than sperm motility. Thus, MEIG1 is not a major candidate for immotile cilia syndrome.

To further investigate the mechanisms of MEIG1 in the regulation of spermatogenesis, a yeast two-hybrid screen was conducted, and PACRG was identified to be a major partner of MEIG1 in the testis. The Pacrg gene is co-regulated with the Parkin gene, a gene involved in Parkinson's disease, and it is a reverse strand gene located upstream of the Parkin gene (17). In the quaking mouse, there is a deletion of approximately 1.17 Mb of mouse chromosome 17, referred to as the quakingviable (qkv) deletion, including the entire promoter region, the first five coding exons of Parkin, and the complete coding sequence of Pacrg. The deletion results not only in a neurological phenotype, including a severe lack of myelin and the tremor of voluntary movements from which quaking mice get their name, but also in male infertility (18, 19), a phenotype that mirrors that of the Meig1 mutant mice described here. A subsequent study demonstrated that the infertility phenotype of the chromosome 17 deletion is due to deletion of the Pacrg gene (20).

Although studies have linked PACRG to certain genetic diseases, such as parkinsonism (21, 22), leprosy (23, 24), and even cancer (25, 26), further investigation conclusively established its role in ciliogenesis. Proteomics and biochemical studies revealed that it is a component of Chlamydomonas reinhardtii centriole/basal bodies (27, 28). In Trypanosoma brucei, two PACRG proteins localize along the full length of the axoneme. Ablation of both proteins by RNA interference knockdown experiments produced slow growth and paralysis of the flagellum due to disorganized axoneme structure (29). Of particular interest, variation in the human PACRG promoter was demonstrated to be a risk factor associated with azoospermia (30). A recent study demonstrated that PACRG associates with tubulin and microtubules (31).

The similar phenotypes of Meig1 and Pacrg mutant mice, the direct interaction of the MEIG1 and PACRG proteins, and the fact that PACRG protein is markedly reduced in MEIG1-deficient mice, suggests that the two proteins regulate spermiogenesis through a common mechanism, and MEIG1 may stabilize PACRG and/or serve as a co-factor for PACRG action. Notably, the impact of the absence of these two proteins is on formation of the sperm flagella and not more broadly on cilia, as might have been anticipated from the studies on Chlamydomonas and trypanosomes. The severe defects in sperm head shaping and flagella formation suggest a failure of intramanchette transport, which plays a key role in shaping the spermatid head, centrosome, and tail (32), and it has been shown that centrosome and associated microtubules play an essential during spermiogenesis (3335). The manchette is a transient microtubular structure assembled concurrently with the elongation and condensation of the spermatid nucleus and growth of the centrosome-derived axoneme (36, 37). Several findings suggest that the manchette can sort structural proteins to the centrosome and the developing sperm tail through a mechanism of intramanchette transport (IMT) (32). In addition, IMT can also transport cargo proteins necessary for spermatid nuclear condensation (38). Disruption of manchette structure in Meig1 deficient mice may impair the intramanchette transport system, resulting in the failure of spermatid head shaping and the formation of flagella. Indeed, our ultrastructural analysis of testis of MEIG1-deficient mice revealed that normal manchette structures were not present or were severely disrupted, whereas they were observed in wild-type testis. The defect in formation of normal flagella does not appear to be caused by a failure to produce components of the axoneme and fibrous sheath, since the expression of flagellar genes, including Spag6, Spag16L, Spag17, and Akap4 was not affected in MEIG1-deficient mice (Fig. S7). Moreover, genes involved in the remodeling that forms the sperm head or that encode proteins that localize to the sperm head such as transition protein 2 (Tnp2), testis-specific histone H1 (Histone H1t) and Spag16S were not altered in Meig1 mutant mice. However, we cannot rule out the possibility that the expression of other genes playing important roles in flagellar formation and remodeling of the sperm head are altered in the absence of MEIG1. This possibility must be entertained since MEIG1 has been reported to bind chromatin, and because MEIG1 is also present in somatic cells of the testis, where it might affect spermatogenesis indirectly via interactions in the supporting Sertoli cells and/or Leydig cells. In the same yeast two hybrid screen, a protein that is present in the Chromatoid body was identified seven times, a RNA splicing factor was also identified five time, suggesting that MEIG1 might, in addition, play a role in RNA processing, which is required for spermiogenesis (39, 40).

In summary, we discovered that MEIG1, possibly through an interaction with PACRG, is essential for spermiogenesis, rather than its originally proposed role in meiosis. The profound sperm head and tail defects associated with the absence or disruption of normal manchettes, implicates MEIG1 as a critical gene for manchette structure and function and the control of spermiogenesis.

Methods

Rapid Amplification of cDNA Ends (RACE).

5′- and 3′- RACE were carried out to define the 5′- and 3′-untranslated region sequence of the mouse Meig1 using mouse testis poly (A)+ RNA and the Marathon cDNA amplification kit (Clontech) according to the manufacturer's instructions.

Generation of an Anti-MEIG1 Antibody.

A cDNA encoding full-length MEIG1 was inserted into pET28a vector (Novagen). The resulting fusion protein was induced and subsequently purified as reported previously (41). The purified recombinant protein was used to generate polyclonal antisera in rabbits by a commercial organization (Rockland).

Targeted Disruption of Meig1.

The 1.1-kb deletion region (exon 1 and flanking region) was amplified by PCR, and BamHI and LoxP sites were added to the left end and a NotI site to the right end. The PCR product was digested with BamHI/NotI, and cloned into the pBluescript plasmid (this construct was named M2/pBlue). A 5-kb left arm (red line in Fig. S3A) was amplified and cloned into ClaI/BamH I sites of M2/pBlue to create M1, 2/pBlue construct. The 3-kb right arm (green line) was amplified by PCR and KpnI sites were included on both ends. The PCR product was cloned into KpnI site of the plasmid backbone for the knockout construct pND1(M3/pND1). The M1, 2/pBlue plasmid was digested with ClaI/NotI and cloned into M3/pND1 to finish the final construct. The resulting targeting construct was linearized by ClaI digestion at the plasmid/gene insert junction and transfected into an XY ES line (TL) by electroporation. Positive ES cells were screened by PCR (primer sequences as shown in Table S1) and Southern blotting. Two positive ES cell clones were injected to generate chimeric mice.

Southern Blot Analysis.

Fifteen micrograms of DNA from ES cells was digested with BamHI and separated by 0.8% agarose gel electrophoresis. The DNA was transferred to nylon membranes and probed with a Meig1 gene-specific probe upstream of the homologous recombination region.

Northern Blot Analysis.

Northern blots containing total testicular RNA (30 μg per lane) were probed with a full-length coding region of mouse Meig1 cDNA or other cDNAs for genes involved in sperm function.

Western Blot Analysis.

An equal amount of indicated protein (50 μg per lane) was subjected to Western blot analysis by using indicated antibodies.

Histology and Transmission Electron Microscopy.

Testes were prepared for light and electron microscopy by using standard methods (42).

Flow Cytometry.

Fresh isolated mixed germ cells were washed twice in PBS and stained with a solution containing 50 μg/mL propidium iodide, 7k U/mL RNase B, and 0.1% Triton X-100 in Na Citrate (3.8 mM) at room temperature for 20 min. The stained cells were analyzed by using a FACScan flow cytometer.

Real-Time PCR.

Total RNA was isolated from whole testes using TRIzol reagent (Invitrogen). RT and QPCR were carried out as previously described (43). The relative abundance of the targets were divided by the relative abundance of 18S rRNA in each sample to generate a normalized abundance for each transcript in each sample.

Yeast Two-Hybrid Screen.

A pretransformed mouse testis cDNA library (Clontech) was screened with the full-length Meig1 coding region as bait following the protocol provided in the kit.

Expression Vector Constructs.

cDNAs containing the full length Meig1 and Pacrg- coding sequences were generated by RT-PCR. After sequencing, Meig1 cDNA was cloned into the BamH1/XhoI site of the pTarget vector, Pacrg cDNA was cloned into cloned into the EcoRI/BamHI sites of the pEGFP-C1 vector, creating Meig1/pTarget and Pacrg/pEGFP-C1 plasmids.

Co-immunoprecipitation.

COS-1 cells were transfected with Meig1/pTarget and Pacrg/pEGFP-C1 plasmids. Forty-eight hours later, the cells were harvested into immunoprecipitation buffer (150 mM NaCl/50 mM Tris·HCl, pH 8.0/5 mM EDTA/1% Triton X-100/1 mM PMSF/proteinase inhibitor mixture), and the lysates were passed through a 20-gauge needle. After centrifugation at 11,600 × g for 5 min, the supernatants were precleared with protein A beads at 4 °C for 30 min. The supernatants were then incubated with preimmune serum (negative control) or an anti-MEIG1 polyclonal antibody at 4 °C for 2 h, and protein A beads were added with a further incubation at 4 °C overnight. The beads were washed with immunoprecipitation buffer three or four times, and loading buffer was then added to the beads, which were boiled at 100 °C for 10 min; the samples were then processed for Western blotting with monoclonal anti-GFP antibody and anti-MEIG1 antibody. Co-immunoprecipitation was also performed using testicular extracts of wild-type mice with the same procedure as described above except that 1 mg testicular protein was incubated with anti-PACRG polyclonal antibody or preimmune rabbit serum, and Western blot was conducted using an anti-MEIG1 antibody.

Supplementary Material

Supporting Information

Acknowledgments.

We thank Glenn L. Radice and Igor Kostetskii for their suggestions of making Meig1 conditional knockout construct; Dr. Rita Shiang (Virginia Commonwealth University) for providing CMV-Cre transgenic mice; and Emily Jumet and Sonya Washington for assistance in photographing light microscopic histology preparations. This work was supported by National Institutes of Health Grants HD37416 (to J.F.S.), HD 045783 (to J.C.H.), CA115503 (to M.J.J.), and in part by the Intramural Research Program of the National Institutes of Health, National Institute of Environmental Health Sciences Grant ZO1-ES070076 (to E.M.E.) and National Institute of Child Health and Human Development F31 Predoctoral Fellowship IF3IHD0G2314-01 (to D.R.G.). Tissue processing and staining were performed in Histology Core Facility of Virginia Commonwealth University. Transmission electron microscopy was performed in the Imaging Core of Virginia Commonwealth University (5P30NS047463).

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission. A.K. is a guest editor invited by the Editorial Board.

This article contains supporting information online at www.pnas.org/cgi/content/full/0906414106/DCSupplemental.

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