Article Figures & Data
Figures
- FIG. 1.
Generation of Tslc1−/− mice. (A) Wild-type allele, targeting construct, and targeted allele of the Tslc1/Igsf4 gene. An open box and solid lines indicate an exon and introns, respectively. IVS1B is a genomic fragment used as a probe for Southern blotting. WT2F and SA7R are the primers for PCR complementary to the wild-type genomic sequence of the Tslc1/Igsf4 gene, while N1F and SA5R are the PCR primers complementary to the targeted allele. P, restriction site of PvuII. (B) Southern blot analysis of the wild-type and targeted alleles of Tslc1/Igsf4. Genomic DNA was digested with a restriction enzyme, PvuII, blotted, and hybridized with a probe, IVS1B. Fragments of 10.9 kb and 2.9 kb were derived from the wild-type and targeted alleles, respectively. (C) PCR analysis for monitoring inheritance of the targeted allele of Tslc1/Igsf4 in the progeny of the Tslc1+/− intercross. W and T indicate the wild-type allele (1.9 kb) and the targeted allele (1.6 kb), respectively. M, molecular marker. (D) RT-PCR analysis of Tslc1/Igsf4 in the testes from Tslc1+/+ and Tslc1−/− mice. A fragment of 154 bp corresponds to exons 1 to 3 of the Tslc1/Igsf4 mRNA. A ribosomal protein gene, S16, served as a control endogenous gene. (E) Western blotting of testis lysates, with or without treatment for deglycosylation, from Tslc1+/+, Tslc1+/−, and Tslc1−/− mice. The filter was hybridized with the anti-TSLC1/IGSF4 antibody CC2 (top) or stained with Coomassie brilliant blue (CBB; bottom).
- FIG. 2.
Reproductive organs and semens from Tslc1+/+ and Tslc1−/− male mice. (A and B) Morphology of reproductive organs from Tslc1+/+ (A) and Tslc1−/− (B) mice. The bladder (open arrow in panel A), prostate (open arrowhead in panel A), seminal vesicles (closed arrowhead in panel A), testes (closed arrow in panels A and B), vasa deferentia (open arrow in B), caput epididymides (closed arrowhead in panel B), and cauda epididymides (open arrowhead in panel B) are demonstrated. Note that the testes from the Tslc1−/− mice are significantly smaller than those from the Tslc1+/+ mice. (C to H) Phase-contrast microscopy of semens from Tslc1+/+ (C and E) and Tslc1−/− (D and F to H) mice. (I and J) PAS staining of semens from Tslc1+/+ (I) and Tslc1−/− mice (J). The open arrowhead indicates a possible acrosome with PAS staining.
- FIG. 3.
Immunohistochemical and histological analyses of Tslc1+/+, Tslc1+/−, and Tslc1−/− mice. (A to H) Immunohistochemical analysis of TSLC1/IGSF4 protein in testes from Tslc1+/+ (A and D to G), Tslc1+/− (B), and Tslc1−/− (C and H) mice, using the anti-TSLC1/IGSF4 antibodies CC2 (A to F) and EC2 (G and H). (A and B) The TSLC1/IGSF4 protein was detected in the seminiferous tubules but not in the interstitial compartment, including the Leydig cells (open arrowhead in panel A). (C) The TSLC1/IGSF4 protein was not detected in a testis from a Tslc1−/− mouse. (D) No signals were detected by CC2 preincubated with an excess amount of antigenic polypeptides. (E) Seminiferous epithelium at stage I. The TSLC1/IGSF4 protein was localized along the membranes of step 13 spermatids (closed arrow) and early pachytene spermatocytes (open arrowhead) but was not detected in step 1 spermatids (open arrow) or the Sertoli cells (closed arrowhead). (F) Seminiferous epithelium at stage VII. The TSLC1/IGSF4 protein was localized along the membranes of step 7 spermatids (open arrow), step 16 residual bodies (closed arrow), and preleptotene spermatocytes (closed arrowhead) but was not detected in the late pachytene spermatocytes (open arrowhead). (I to T) Histological analyses of the testes (I to N), ductuli efferentes testis (O and P), epididymides (Q and R), and ovaries (S and T) from Tslc1+/+ (I, M, O, Q, and S), Tslc1+/− (J), and Tslc1−/− (K, L, N, P, R, and T) mice by HE staining (I to K and M to T) or PAS staining (L). (K) Degenerated round cells were accumulated in the lumen (closed arrowhead), and extensive vacuolization was observed at the basal side (open arrowheads). (L) A large number of round and degenerated cells were seen in the lumen (closed arrowhead). Note that some of the cells in the lumen were stained with PAS and appeared to be derived from round spermatids (open arrowhead), elongating spermatids (open arrow), or the pachytene spermatocytes (closed arrow). (M to R) The open arrows (M and N), open arrowheads (M and Q), and closed arrowheads (N and R) indicate the rete of the testis, the spermatozoa, and the degenerated round cells, respectively. (S and T) Closed arrows indicate the secondary follicle. Mice were examined at 25 weeks of age (A to R) and 40 weeks of age (S and T).
- FIG. 4.
Staging analyses of the testes from 21-week-old Tslc1+/+ and Tslc1−/− mice by PAS staining.
- FIG. 5.
Flow cytometric analyses of cells isolated from the testes of Tslc1+/+ and Tslc1−/− mice. (A) The flow cytograms demonstrate four discrete peaks: an HN (haploid) peak representing elongated spermatids, a 1N (haploid) peak representing round spermatids, a 2N (diploid) peak representing G1-phase spermatogonia, and a 4N (tetraploid) peak representing pachytene spermatocytes and G2-phase spermatogonia. (B) Relative amounts of four cell populations in the testes. Open and closed boxes indicate cells from Tslc1+/+ and Tslc1−/− mice, respectively.
- FIG. 6.
Detection of apoptosis by TUNEL assays. (A and B) Histochemistry of the testes from Tslc1+/+ (A) and Tslc1−/− (B) mice by TUNEL assay. Cells stained brown are TUNEL-positive cells. Nuclei were counterstained with methyl green (green). Closed arrowheads and open arrowheads indicate spermatocytes and spermatids, respectively. The open arrow indicates the sloughed cell. (C) Ratios of TUNEL-positive tubules to total tubules. (D) Average numbers of TUNEL-positive cells in TUNEL-positive tubules.
- FIG. 7.
Morphological analysis of germ cells from Tslc1+/+ and Tslc1−/− mice during postnatal development. Testes (T) and epididymides (E) of juvenile mice from 2 to 11 weeks of age were examined by HE staining. Closed arrowheads, closed arrows, open arrowheads, and open arrows in black indicate spermatogonia, spermatocytes, round spermatids, and elongated spermatids, respectively. Closed arrowheads, closed arrows, open arrowheads, and open arrows in yellow indicate spermatozoa, sloughed cells, multinucleated giant cells, and vacuoles, respectively.
- FIG. 8.
Weights of testes and numbers of normal sperm during postnatal development of Tslc1+/+ and Tslc1−/− mice. (A) Weights of testes. (B) Numbers of normal sperm. *, P < 0.05; **, P < 0.005; ***, P < 0.0001.
- FIG. 9.
Electron microscopic analysis of spermatogenic cells and Sertoli cells from Tslc1+/+ and Tslc1−/− mice. (A to H) Spermatids from Tslc1−/− mice (A to C, G, and H) and Tslc1+/+ mice (D to F). Spermatids in step 5 (A and D), step 7 (B and E), step 8 (C and F), and step 10 or later (G and H) are demonstrated. (I) A degenerated cell in the epididymis from a Tslc1−/− mouse. Densely staining materials corresponding to the acrosome (open arrowhead), as well as numerous degenerated vacuoles, were observed. (J to L) Numerous figures of phagocytosis (open arrowhead in panel J) and vacuolization (closed arrowhead in panel J) were observed within the Sertoli cells. Note that the Sertoli cell-Sertoli cell junction (open arrows in panels J and K) and ectoplasmic specialization (closed arrow in panel L) were unaffected in the testes from Tslc1−/− mice. S, Sertoli cell; Sg, spermatogonium; Sc, spermatocyte; St, spermatid; ac, acrosome.
Tables
- TABLE 1.
Weights of organs, sperm parameters, and serum testosterone levels in Tslc1−/− and Tslc1+/+ mice
Parameter Value for Tslc1−/− mice No. of mice analyzed Value for Tslc1+/+ mice No. of mice analyzed P valueb Total body wt (g) (25 wks) 38.7 ± 2.8 4 37.8 ± 1.6 4 NS Wet wt of organs (mg)a Brain 425 ± 10 3 442 ± 7.9 4 NS Eye 25.5 ± 0.4 5 24.4 ± 2.1 3 NS Thymus 38.7 ± 0.9 3 42.4 ± 2.2 3 NS Lung 174 ± 28 3 181 ± 7.2 4 NS Heart 222 ± 23 5 189 ± 19 4 NS Spleen 85.5 ± 19 5 94 ± 5.0 4 NS Kidney 318 ± 22 5 309 ± 22 4 NS Liver 1,900 ± 80 5 1,770 ± 260 4 NS Stomach 420 ± 54 4 405 ± 54 3 NS Intestinum tenue, pancreas, and mesenterium 2,310 ± 130 5 2,470 ± 280 4 NS Intestinum crassum 584 ± 78 3 651 ± 47 3 NS Seminal vesicle 163 ± 14 4 153 ± 4.2 4 NS Bladder and prostate 133 ± 18 4 143 ± 10 4 NS Epididymis and vas deferens 59.4 ± 9.5 5 85.1 ± 13 4 NS Testis 94.7 ± 3.0 5 134 ± 8.7 4 0.003 Sperm parameters (11 wks) Total no. of cells (106) 7.3 ± 1.0 3 20 ± 2.4 3 0.008 No. of normal sperm (103) 1.2 ± 0.8 3 15,000 ± 2,200 3 0.002 Motile sperm/normal sperm (%) 1.1 ± 0.4 3 83.3 ± 3.3 3 <0.0001 Serum testosterone level (ng/ml) 16 wks 4.5 ± 4.0 3 2.5 ± 2.0 3 NS 25 wks 1.7 ± 0.5 4 3.2 ± 3.0 4 NS - TABLE 2.
Sloughed cells from seminiferous epithelium in testes from Tslc1−/− micea
Type of cells No. of cells (%) Spermatogonia 0 (0.0) Spermatocytes 12 (2.5) Spermatids 474 (97.5) Steps 1-3 16 (3.3) Steps 4-6 84 (17.3) Steps 7-9 298 (61.3) Steps 10-13 38 (7.8) Steps 14-16 38 (7.8) Total 486 (100) ↵ a Twelve testicular sections from three 25-week-old Tslc1−/− mice were examined.
- TABLE 3.
Genes up- and down-regulated in testes from Tslc1−/− mice
Gene product Accession no. Microarray analysis results Quantitative RT-PCR analysis resultsb P valuec Signal intensitya Ratio of Tslc1−/− value/Tslc1+/+ value Tslc1−/− value Tslc1+/+ value Tslc1−/− Tslc1+/+ Up-regulated genes in Tslc1−/− testes Phospholipase A2, group XIIA (Pla2g12a) AI845798 13,102 771 17.0 11 ± 2.1 4.1 ± 0.8 0.03 Purine-nucleoside phosphorylase (Pnp) U35374 1,629 410 4.0 6.1 ± 0.7 2.5 ± 0.6 0.02 Mus musculus cDNA 5′ end clone AA874329 706 272 2.6 FUS interacting protein 1 (Fusip1) AF060490 695 282 2.5 Hemoglobin, beta adult major chain (Hbb-b1) J00413 1,446 636 2.3 DnaJ homolog, subfamily A, member 2 (Dnaja2) AA763945 1,833 860 2.1 470 ± 160 300 ± 120 NS Vascular cell adhesion molecule 1 (Vcam1) U12884 2,321 1,546 1.5 110 ± 5.8 38 ± 2.5 0.0002 Guanylate kinase 1 (Guk1) U53514 3,225 2,294 1.4 23 ± 12 4.4 ± 0.1 NS Angiopoietin-like 4 (Angpt14) AF110520 6,143 4,823 1.3 3.1 ± 0.2 1.2 ± 0.2 0.002 Down-regulated genes in Tslc1−/− testes Immunoglobulin superfamily 4 (Igsf4/Tslc1) AF061260 444 3,943 0.11 0 ± 0 100 ± 18 0.001 Immunoglobulin superfamily 4 (Igsf4/Tslc1) AB021966 690 3,739 0.18 Platelet/endothelial cell adhesion molecule 1 (Pecam1) L06039 181 799 0.23 1.6 ± 0.2 1.2 ± 0.3 NS Mus musculus cDNA 3′ end clone AW047207 241 895 0.27 Peroxiredoxin 2 (Prdx2) U20611 281 892 0.31 16 ± 3.0 11 ± 0.6 NS Dehydrogenase/reductase X chromosome (Dhrsx) AI846822 4,509 11,760 0.38 11 ± 1.6 12 ± 2.1 NS Ornithine decarboxylase antizyme 3 (Oaz3) AB016275 17,735 44,962 0.39 94 ± 6.8 150 ± 9.2 0.007 Splicing factor, arginine/serine-rich 16 (Sfrs16) AF042799 430 1,030 0.42 Protein phosphatase 2, regulatory subunit B, beta isoform (Ppp2r2b) AW048155 6,134 14,358 0.43 9.2 ± 1.9 23 ± 1.5 0.004 Mouse endogenous murine leukemia virus modified polytopic provirus DNA M17327 789 1,769 0.45 Deleted in polyposis 1-like 1 (Dplll) AA755260 14,224 29,836 0.48 340 ± 30 970 ± 130 0.008 Lysophospholipase 1 (Lypla1) U89352 6,971 14,600 0.48 7.8 ± 0.4 5.6 ± 1.6 NS Suppressor of K+ transport defect 3 (Skd3) U09874 7,709 15,612 0.49 S100 calcium binding protein A13 (S100a13) X99921 773 1,430 0.54 Protamine 1 (Prm1) Z47352 18,600 31,700 0.58 12,000 ± 2,200 16,000 ± 1,700 NS RAN GTPase activating protein 1 (Rangap1) U20857 4,960 8,480 0.58 72 ± 33 110 ± 47 NS Growth arrest-specific protein 6 (Gas6) X59846 1,120 1,820 0.61 80 ± 8.2 170 ± 35 0.04 Bcl2-associated athanogene 1 (Bag1) AF022223 3,412 5,546 0.61 0.1 ± 0.1 1.1 ± 0.4 NS Cyclin-dependent kinase inhibitor 1C (Cdkn1c) U22399 1,090 1,650 0.66 2.3 ± 0.2 1.8 ± 0.1 NS Mothers against decapentaplegic homolog 6 (Smad6) AF010133 2,070 2,930 0.70 79 ± 11 94 ± 8.4 NS Sperm mitochondrion-associated cysteine-rich protein (Smcp) M88463 29,700 38,900 0.76 430 ± 43 930 ± 81 0.002
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Files in this Data Supplement:
- Supplemental file 1
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Fig. S1 (Differential expression of alpha-tubulin in the testes from Tslc1+/+ and Tslc1-/- mice)
Zipped PDF document, 7.6MB. - Supplemental file 2
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Legend for Fig. S1
MS Word document, 25K. - Supplemental file 3
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Table S1 (Primer sequences examined)
Zipped PDF document, 83K.
- Supplemental file 1
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Fig. S1 (Differential expression of alpha-tubulin in the testes from Tslc1+/+ and Tslc1-/- mice)
