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SEPT12 deficiency causes sperm nucleus damage and developmental arrest of preimplantation embryos

2010; Elsevier BV; Volume: 95; Issue: 1 Linguagem: Inglês

10.1016/j.fertnstert.2010.07.1064

1556-5653

Ying‐Hung Lin, Chuan-Kai Chou, Y. Elizabeth Hung, I‐Shing Yu, Hsien-An Pan, Shu‐Wha Lin, Pao‐Lin Kuo,

Renal and related cancers

Oocytes fertilized with spermatozoa obtained from Septin 12+/– chimeric mice failed to develop beyond the morula stage after IVF and intracytoplasmic sperm injection because of significant DNA defects in the spermatozoa. Given that SEPT12 is expressed at the edge of the sperm nucleus in both humans and mice, we hypothesized the vital roles of Septin 12 in sperm head shaping, nuclear DNA condensation, and early embryonic development. Oocytes fertilized with spermatozoa obtained from Septin 12+/– chimeric mice failed to develop beyond the morula stage after IVF and intracytoplasmic sperm injection because of significant DNA defects in the spermatozoa. Given that SEPT12 is expressed at the edge of the sperm nucleus in both humans and mice, we hypothesized the vital roles of Septin 12 in sperm head shaping, nuclear DNA condensation, and early embryonic development. The septin gene family is part of a highly conserved family of polymerizing guanosine triphosphate (GTP) binding proteins (1Hall P.A. Russell S.E. The pathobiology of the septin gene family.J Pathol. 2004; 204: 489-505Crossref PubMed Scopus (242) Google Scholar). The function of Septins, SEPTs, includes membrane compartmentalization, mitosis, cytoskeletal remodeling, and vesicle trafficking. Septins have been implicated in the pathology of many diseases, including Alzheimer's disease, hereditary neuralgic amyotrophy, leukemia, ovarian tumor, breast cancer, and male infertility (1Hall P.A. Russell S.E. The pathobiology of the septin gene family.J Pathol. 2004; 204: 489-505Crossref PubMed Scopus (242) Google Scholar). The roles of septins in mammalian spermatogenesis have recently begun to be revealed (2Ihara M. Kinoshita A. Yamada S. Tanaka H. Tanigaki A. Kitano A. et al.Cortical organization by the septin cytoskeleton is essential for structural and mechanical integrity of mammalian spermatozoa.Dev Cell. 2005; 8: 343-352Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 3Kissel H. Georgescu M.M. Larisch S. Manova K. Hunnicutt G.R. Steller H. The Sept4 septin locus is required for sperm terminal differentiation in mice.Dev Cell. 2005; 8: 353-364Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). During spermatogenesis, SEPT4 plays important roles in maintaining proper mitochondrial architecture and establishing the annulus (2Ihara M. Kinoshita A. Yamada S. Tanaka H. Tanigaki A. Kitano A. et al.Cortical organization by the septin cytoskeleton is essential for structural and mechanical integrity of mammalian spermatozoa.Dev Cell. 2005; 8: 343-352Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 3Kissel H. Georgescu M.M. Larisch S. Manova K. Hunnicutt G.R. Steller H. The Sept4 septin locus is required for sperm terminal differentiation in mice.Dev Cell. 2005; 8: 353-364Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). In humans, men with asthenozoospermia tend to lose annulus (SEPT1/4/6/7) complexes in mature spermatozoa (2Ihara M. Kinoshita A. Yamada S. Tanaka H. Tanigaki A. Kitano A. et al.Cortical organization by the septin cytoskeleton is essential for structural and mechanical integrity of mammalian spermatozoa.Dev Cell. 2005; 8: 343-352Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 4Sugino Y. Ichioka K. Soda T. Ihara M. Kinoshita M. Ogawa O. et al.Septins as diagnostic markers for a subset of human asthenozoospermia.J Urol. 2008; 180: 2706-2709Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Gene targeting experiments showed that most Septin 12+/– chimeric mice were sterile because of severe spermatogenic defects, including immotile sperm, sperm with a bent tail, round-headed sperm, and sperm with acrosomal defects (5Lin Y.H. Lin Y.M. Wang Y.Y. Yu I.S. Lin Y.W. Wang Y.H. et al.The expression level of septin12 is critical for spermiogenesis.Am J Pathol. 2009; 174: 1857-1868Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Considering that haploinsufficiency of Septin 12 in mice results in severe sperm defects and male sterility, we hypothesized that sperm deficient in SEPT12 would fail to fertilize the oocyte in vivo. To analyze nuclear integrity, sperm isolated from Septin12+/– chimeric mice were examined using acridine orange (AO), toluidine blue (TB), and aniline blue (AB). To determine whether spermatozoa with a defective Septin 12 allele isolated from Septin12+/– chimeric mice could fertilize the egg and support early embryonic development, IVF and intracytoplasmic sperm injection (ICSI) were preformed. To illustrate the expression patterns of SEPT12 during human spermiogenesis, a testicular sample was analyzed by immuno-fluorescence assay (IFA). AO, TB, and AB staining assays were used to determine sperm nuclear integrity in Septin 12+/– chimeric mice. The experiment was performed according to standard protocols (6Talebi A.R. Moein M.R. Tabibnejad N. Ghasemzadeh J. Effect of varicocele on chromatin condensation and DNA integrity of ejaculated spermatozoa using cytochemical tests.Andrologia. 2008; 40: 245-251Crossref PubMed Scopus (76) Google Scholar). These three assays unanimously showed increased DNA damage in the sperm isolated from Septin 12+/– chimeric mice, compared with that from the wild type mice. The rate of abnormal staining of spermatozoa isolated from Septin 12-deficient mice was significantly higher than that of the wild type mice (AO, wild-type, 7.33 ± 2.35%; Septin 12-deficient, 58.23 ± 11.06%, P < 0.05; TB, wild type, 6.57 ± 1.54%; Septin 12-deficient, 90.44 ± 2.77%, P < 0.0001; AB, wild type, 4.13 ± 1.58%; Septin 12-deficient, 58.33 ± 13.22%, P < 0.05; by Student's t test; Fig. 1A ). These findings suggest that SEPT12 deficiency during spermatogenesis results in mouse sperm nuclear defects. We went on to test if ICSI could be used to circumvent the sperm defect of the Septin 12+/– chimeric mice. For the IVF and ICSI experiments, mouse sperm were isolated from the vas deferens of Septin 12+/– chimeric and wide-type (C57BL/6) mice. The IVF and ICSI procedures were performed according to the methods described in our previous publication, with minor modifications (7Chu P.C. Wang T.Y. Lu Y.T. Chou C.K. Yang Y.C. Chang M.S. Involvement of p29 in DNA damage responses and Fanconi anemia pathway.Carcinogenesis. 2009; 30: 1710-1716Crossref PubMed Scopus (16) Google Scholar). For the ICSI procedure, spermatozoa with abnormal morphology (e.g., bent tail, abnormal head) were selected for microinjection into the metaphase II–arrested eggs from the C57BL/6 mice. After 3–5 days, the cultured embryos were observed to determine their developmental stages. Strain-specific microsatellite markers were used to determine the genetic backgrounds (C57BL/6 and/or 129) (8Cho C. Willis W.D. Goulding E.H. Jung-Ha H. Choi Y.C. Hecht N.B. et al.Haploinsufficiency of protamine-1 or -2 causes infertility in mice.Nat Genet. 2001; 28: 82-86Crossref PubMed Google Scholar). For the IVF and ET experiment, a total of 642 oocytes were inseminated with spermatozoa isolated from the Septin 12+/– chimeric mice. The fertilization rate was 55.5% (365/642), a figure comparable to routine mouse IVF outcome (40%–60%). Embryos (n = 151) developing to the blastocyst or morula stage were transferred to the pseudopregnant female mice. After transfer, a total of 43 pups were generated, and all were of the 129 genetic backgrounds (28.4% vs. routing assay 52.4%). There are two explanations for this result. First, spermatozoa with poor motility (caused by the tail or mitochondrial defects) or an acrosome defect have difficulty fertilizing the oocyte. Second, after fertilization, the nuclear defects cause developmental arrest of the preimplantation embryos. Of 51 oocytes injected, only seven were fertilized and developed to the blastocyst stage. In addition, all embryos were found to contain only the wild type allele without the mutant Septin 12 allele. This finding suggests that embryos fertilized by sperm with a SEPT12 deficiency underwent developmental arrest and failed to develop beyond the morula stage. IFA was performed to observe the SEPT12 expression pattern during human spermiogenesis. The human experiment was approved by the Institutional Review Board of National Cheng Kung University Medical Centre, and informed consent was obtained from the enrollee. Human testicular tissue was retrieved from an infertile man with congenital bilateral absence of vas deferens, separated by the density gradients and subjected to IFA (5Lin Y.H. Lin Y.M. Wang Y.Y. Yu I.S. Lin Y.W. Wang Y.H. et al.The expression level of septin12 is critical for spermiogenesis.Am J Pathol. 2009; 174: 1857-1868Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). The following observations on the human testicular material could be made. The SEPT12 signal started to appear at the round spermatid stage (Fig. 1B, a and a′). At the first step of spermiogenesis, SEPT12 concentrated around the acrosome (Fig. 1B, b and b′). Afterward, SEPT12 was expressed between the edge of the acrosome and the perinuclear mantle of the manchette (Fig. 1B, c and c′). Next, SEPT12 encircled the upper site of the acrosome (Fig. 1B, d and d′) and formed the rim of the sperm nucleus (Fig. 1B, e and e′). With the formation of mitochondria and mature spermatozoa, SEPT12 was localized at the neck and annulus regions (Fig. 1B, f–h′). Given that SEPT12 was localized at the edge of the sperm nucleus during human spermiogenesis, we hypothesize that SEPT12 forms a barrier to facilitate sperm head shaping and nuclear compaction. Loss of SEPT12 results in loss of nuclear integrity. In conclusion, sperm isolated from Septin 12+/– chimeric mice have nuclear defects. Oocytes fertilized with these spermatozoa have poor developmental potential. This finding (haploinsufficiency of Septin 12 results in sperm nuclear DNA damage) is reminiscent of the protamine knockout mice (8Cho C. Willis W.D. Goulding E.H. Jung-Ha H. Choi Y.C. Hecht N.B. et al.Haploinsufficiency of protamine-1 or -2 causes infertility in mice.Nat Genet. 2001; 28: 82-86Crossref PubMed Google Scholar). In humans, SEPT12 is expressed at the edge of the sperm nucleus, an expression pattern similar to that of the mouse (5Lin Y.H. Lin Y.M. Wang Y.Y. Yu I.S. Lin Y.W. Wang Y.H. et al.The expression level of septin12 is critical for spermiogenesis.Am J Pathol. 2009; 174: 1857-1868Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). These findings suggest vital roles for Septin 12 in sperm head shaping, nuclear DNA condensation, and subsequent embryonic development. The authors thank the Transgenic Mouse Models Core of the National Research Program for Genome Medicine for generating the knockout mice and the National Laboratory Animal Center for handling the assisted reproduction technology.