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FEBS Letters 585 (2011) 2171–2176

journal homepage: www.FEBSLetters.org

p125/Sec23-interacting (Sec23ip) is required for spermiogenesis

Nagisa Arimitsu a,1, Takeshi Kogure a,1, Takashi Baba a, Kazuki Nakao b, Hiroshi Hamamoto c, Kazuhisa Sekimizu c, Akitsugu Yamamoto d, Hiroki Nakanishi e,f, Ryo Taguchi e,f, Mitsuo Tagaya a, ⇑ Katsuko Tani a,

a School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo 192-0392, Japan b RIKEN Center for Developmental Biology, 2-2-3 Minatojima-Minamimachi, Chuo-ku, Kobe 650-0047, Japan c Department of Developmental Biochemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan d Faculty of Bioscience, Nagahama Institute of Bio-Science and Technology, Nagahama, Shiga 526-0829, Japan e Department of Metabolome, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan f Core Research for Evolutional Science and Technology, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan

article info abstract

Article history: p125/Sec23ip is a -like protein that interacts with Sec23, a coat component of COPII Received 12 April 2011 vesicles that bud from endoplasmic reticulum exit sites. To understand its physiological function, Revised 18 May 2011 we produced p125 knockout mice. The p125 knockout mice grew normally, but males were subfer- Accepted 18 May 2011 tile. Sperm from p125-deficient mice had round heads and lacked the acrosome, an organelle con- Available online 30 May 2011 taining the enzymes responsible for fertilization. p125 was found to be expressed at stages I–XII of Edited by Wilhelm Just spermatogenesis, similar to the expression pattern of involved in acrosome biogenesis. These results suggest that p125 plays an important role in spermiogenesis.

Keywords: COPII Structured summary of protein interactions: Endoplasmic reticulum exit site p125 and Sec31A colocalize by fluorescence microscopy (View interaction) Sec23 Ó 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Spermiogenesis

1. Introduction (PA-PLA1/iPLA1-a, p125/iPLA1-b and KIAA0725p/iPLA1-c), which are uniquely localized in cells [7–10]. The physiological importance

The export of secretory and membrane proteins from the ER is of iPLA1 family proteins has been demonstrated in C. elegans [11] mediated by COPII-coated vesicles [1]. The COPII coat consists of and Arabidopsis [12,13]. Like mammals, Xenopus has three mem- the small GTPase Sar1 and two heterodimers, Sec23–Sec24 and bers, and very recent orthologous phenotype analysis combined Sec13–Sec31. COPII vesicles bud at specialized ER subdomains, with morpholino-mediated knockdown in Xenopus embryos termed ER exit sites or transitional ER [2]. p125/Sec23ip was dis- predicted that p125/sec23ip is a candidate gene responsible for covered as a mammalian protein that interacts with Sec23 [3,4], Waardenburg syndrome, a disorder characterized by craniofacial and a later study showed that it interacts not only with Sec23 defects and pigment abnormalities [14]. but also with Sec31 [5]. Knockdown of p125 by RNA interference To elucidate the physiological function of p125 in mammals, we causes the redistribution of ER exit sites in cultured cells, suggest- generated p125 KO mice. We found that loss of p125 causes a de- ing its role in their organization [6]. fect in acrosome formation, leading to male subfertility.

p125 is a member of an iPLA1 family that is conserved among eukaryotes. While there is only one member in yeast, Caenorhabdi- 2. Materials and methods tis elegans and Arabidopsis, mammals have three members 2.1. Conditional targeting vector

A p125 gene fragment was isolated from a mouse genomic

Abbreviations: ER, endoplasmic reticulum; iPLA1, intracellular phospholipase A1; bacterial artificial library derived from the KO, knockout; WT, wild-type; IVF, in vitro fertilization; HE, hematoxylin and eosin; C57BL/6 mouse strain (RP23-121G8) and subcloned into pBlue- MEF, mouse embryo fibroblast; PNA, peanut agglutinin script II SK. A conditional targeting vector was constructed ⇑ Corresponding author. Fax: +81 42 676 5468. using the highly efficient recombineering-based method of Liu E-mail addresses: [email protected], [email protected] (K. Tani). 1 These authors contributed equally to this work. et al. [15].

0014-5793/$36.00 Ó 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2011.05.050 2172 N. Arimitsu et al. / FEBS Letters 585 (2011) 2171–2176

2.2. Generation of p125 KO mice

The p125 targeting vector was electroporated into embryonic stem cells, i.e., TT2 cells [16]. Two targeted cell lines were injected into C57BL/6 ICR eight-cell stage embryos to produce chimeric mice. The male chimeric mice obtained were crossed with female WT C57BL/6 mice to obtain p125+/flox offspring. To generate p125/ mice, p125+/flox mice were mated with CAG-Cre transgenic mice (RIKEN BRC), which express Cre recombinase at early stages of development. For genotyping by PCR analysis, the sequences of the primers used were as follows: Primer 1 (50-CGTCCAGGACA TCTCAGAATCTGCGACTTCG-30) and Primer 2 (50-CGCAATGACAGGA GTTCCAAGCCTCATCACC-30). All animal procedures and experi- ments were approved by the Animal Care Committee of Tokyo University of Pharmacy and Life Sciences.

2.3. IVF

IVF was carried out as described [17]. Eggs were placed in a drop of human tubal fluid medium and incubated with control or Fig. 1. Generation of p125 KO mice. (A) Targeted disruption of the p125 gene. / p125 sperm for 24 h at 37 °C under 5% CO2. The percentage of Schematic representation of the WT allele with the conditional targeting vector, and 2-cell stage embryos was determined. the targeted (floxed) and KO alleles. Exons of the p125 gene and loxP sequences are indicated by black boxes and closed triangles, respectively. Neo, neomycin-resistant 2.4. Histology and immunohistochemistry gene flanked by the PGK promoter and the bovine growth hormone poly(A) tail; DT- A, the diphtheria toxin A gene flanked by the PGK promoter; K, KpnI; N, NcoI. (B) Southern blot analysis of KpnI-digested genomic DNA from WT (+/+) and hetero- For histological analysis, mouse testes were fixed in 10% forma- zygous floxed (+/F) alleles. 50 probe-1 in (A) recognized 18.8- and 7.7-kb fragments lin in PBS, dehydrated and then embedded in paraffin. Paraffin sec- of the WT and floxed alleles, respectively. (C) Southern blot analysis of NcoI- tions (6 lm) were stained with HE according to the standard digested genomic DNA from WT (+/+), heterozygous (+/) and KO (/) mice. 50 protocol. For immunohistochemistry, testes were fixed with 4% probe-2 in (A) recognized 2.4- and 8.9-kb fragments of the WT and KO alleles, respectively. (D) Western blot analysis of MEF lysates (10 lg). paraformaldehyde in PBS and frozen in O.C.T. compound (Tissue- Tek; Sakura). Cryosections (10 lm) were prepared, permeabilized in TBS-T (20 mM Tris–HCl (pH 7.6), 137 mM NaCl and 0.1% Table 1 Tween-20), blocked with 2% normal goat serum in PBS, and then Subfertility of p125 KO males. analyzed. Genotype Number of Number of Average litter matings pregnancies sizea Male Female 2.5. In situ hybridization +/+ +/+ 27 27 7.4 +/ 3 3 8.7 To prepare a cRNA probe, a p125 cDNA fragment (nucleotides / 4 4 9.0 344-850) was labeled using a DIG RNA labeling mix (Roche) +/ +/+ 19 19 6.8 according to the manufacturer’s protocol. cRNA probes against +/ 12 12 7.4 H1t and protamine 1 were prepared as described previously [18]. / 8 8 7.6 / +/+ 78 49 3.3 +/ 20 11 1.6 2.6. Preparation of MEFs / 8 4 1.3

p125/ mice generated through p125+/ p125+/ mice were Data were obtained for mating between animals younger than 8 and 6 months of age for males and females, respectively. dissected on embryonic day 13.5 (E13.5), and MEFs were recovered a Average litter sizes were calculated as the number of pups per number of by trypsinization. matings.

3. Results

3.1. Generation of p125 KO mice

Given that many related to membrane trafficking are essential in yeast, we decided to construct a conditional target- ing vector to generate p125 KO mice. A p125-targeting vector was designed to contain exon 1 comprising the initiation codon flanked by two loxP sites (Fig. 1A). Embryonic stem cells with the targeted allele were screened with G418, and p125+/flox mice were generated according to a conventional procedure (Fig. 1B). p125+/flox mice were crossed with transgenic mice expressing Cre recombinase to generate p125+/ mice. Homozygous p125/ mice were obtained by intercrossing the heterozygous mice. Loss Fig. 2. IVF experiments with zona pellucida-intact eggs from WT females, and of exon 1 and p125 protein expression were confirmed by sperm from control and KO males. Fertilization was determined by microscopy at Southern blotting (Fig. 1C) and Western blotting (Fig. 1D), 24 h post-insemination. Error bars represent the S.D. for each mouse genotype. respectively. ⁄⁄P < 0.005 by Student’s t-test. N. Arimitsu et al. / FEBS Letters 585 (2011) 2171–2176 2173

3.2. Male p125 KO mice are subfertile results suggest that the major cause of the subfertility is a male problem. Contrary to our expectation, p125 KO mice were born at Grossly, the testes of p125/ mice were normal and their sizes Mendelian frequency, and grew indistinguishably from WT were not much different from those of control mice at any age. In and heterozygous mice. However, male p125 KO mice appeared addition, the total number of sperm from a single cauda epididy- to be subfertile. Thus, we carefully examined the pregnancy and mis of a p125-deficient mouse was only slightly smaller than that litter sizes in females with vaginal plugs, evidence of mating. from a control (WT and heterozygous) mouse (data not shown). IVF When WT (p125+/+) males were crossed with p125+/+, p125+/ assays showed that sperm obtained from p125-deficient mice and p125/ females, pregnancy almost always occurred in fe- exhibited a markedly reduced fertilization rate (31%) compared males with vaginal plugs (Table 1). The litter sizes ranged be- with in control mice (84%) (Fig. 2), suggesting that the subfertility tween 7.4 and 9.0. By contrast, when p125/ males were in p125-deficient mice is due to a sperm dysfunction. mated, failure of conception was frequently observed. The aver- age litter sizes on mating with p125+/+, p125+/ and p125/ fe- 3.3. Abnormal spermatogenesis in p125 KO mice males were 3.3, 1.6 and 1.3, respectively. Notably, no marked difference in pregnancy was observed between p125/ and To determine why sperm from p125-deficient mice are p125+/+ females on crossing with WT males (9.0 vs 7.4). These dysfunctional, we examined the morphology of the seminiferous

Fig. 3. Abnormal acrosome biogenesis in p125 KO mice. (A) HE staining of seminiferous tubules of testes from WT and p125 KO mice. The right images show higher magnification views of the boxed regions in the left images. In WT mice, there were many elongated spermatids (blue arrows), whereas only a few were observed in p125 KO mice (light blue box). Bars, 50 lm. (B) Sperm from the cauda epididymis was stained with Coomassie Brilliant Blue (CBB, left) (Bar, 10 lm) or viewed by electron microscopy (EM, right) (Bar, 1 lm). (C and E) Sperm from the cauda epididymis was triple-stained with FITC-PNA, Hoechst 33342, and Mitotracker (C) or sp56 (E). Bar, 10 lm. (D) Quantitation of PNA-positive sperm in control and p125 KO mice. Error bars represent the S.D. 2174 N. Arimitsu et al. / FEBS Letters 585 (2011) 2171–2176 tubules and sperm in p125-deficient mice. The seminiferous tu- deficiency in acrosomes, whose formation is highly dependent on bules contain germ cells at all stages of maturation; stem cells the Golgi function [20]. and spermatogonia on one side of the junction, and spermatocytes, and round, elongating and elongated spermatids organized in strict 3.4. p125 is expressed in spermatocytes and round spermatids order of maturation toward the lumen [19]. HE staining showed that the layers of cells in the seminiferous tubules were largely Next, we examined the expression pattern of p125 in the testis. preserved in p125-deficient mice (Fig. 3A, KO), implying no As shown in Fig. 4A, p125 expression was detected in testicular remarkable aberrancy of spermatogonia, spermatocytes or round cells, but not in sperm. Immunostaining of the seminiferous tu- spermatids. However, spermatids near the lumen of the tubules bules showed that p125 is expressed in spermatocytes and round were not elongated in p125-deficient mice (Fig. 3A, KO, light blue spermatids, but not in spermatogonia (Fig. 4B, top row). p125 box). Consistent with this finding, many sperm in the cauda epidid- immunoreactivity was not detected in whole cross-sections of ymis of p125-deficient mice had round heads (Fig. 3B, KO). To seminiferous tubules prepared from p125 KO mice (top row, right), examine the morphology of organelles including the acrosome, a indicating that the anti-p125 antibody can specifically recognize specialized secretory organelle localized in the head of mammalian p125. The expression of p125 mRNA in spermatocytes and round sperm [20], we stained sperm with Hoechst 33342, Mitotracker spermatids was confirmed by in situ hybridization (Fig. 4B, bottom and an acrosome marker, PNA [21]. Fig. 3C shows that sperm from row). Detailed in situ hybridization analysis demonstrated that p125-deficient mice had a round-shaped nucleus (blue) and an p125 mRNA is expressed at stages I-XII of the 12-stage growth cy- abnormal mitochondrial sheath (red). Notably, while the cres- cle of mouse spermatogenesis (Fig. 4C) [23]. cent-shaped acrosome in WT sperm was unequivocally stained Subcellular localization of p125 in spermatocytes was further with FITC-PNA and an antibody against an acrosome marker, examined by immunofluorescence microscopy (Fig. 5). In spermat- sp56, few PNA-, sp56-positive structures were observed in sperm ocytes, p125 was localized in a ring-shaped structure with some from p125-deficient mice (Fig. 3C–E). This sperm phenotype is large dots in the cytoplasm (middle column). Its distribution mark- quite similar to that observed in males with globozoospermia edly overlapped that of Sec31A (an ER exit site marker) in the per- [22], which is characterized by round-headed sperm with a inuclear ring-like structure and large dot-like structures, whereas

Fig. 4. Stage-specific expression of p125 during spermatogenesis. (A) Expression of p125. Total protein lysates (20 lg) of testicular cells and sperm were analyzed by Western blotting. (B) For immunohistochemistry (top row; bars, 5 lm), testis sections were double-stained with the anti-p125 antibody and Hoechst 33342. In situ hybridization (bottom row; bar, 10 lm). H1t, testicular H1 histone; Pro1, Protamine 1. SC, spermatocyte; RS, round spermatid; ES, elongate spermatid. (C) In situ hybridization of serial sections of seminiferous tubules. H1t and protamine 1 cRNA probes were used to determine the stage of spermatogenesis; H1t (stages VI–XII) and protamine 1 (stages I–III and IX–XII of the 12-stage growth cycle of mouse spermatogenesis [18]. p125 is expressed in stages I–XII. N. Arimitsu et al. / FEBS Letters 585 (2011) 2171–2176 2175

Fig. 5. Localization of p125 in spermatocytes. Cell smears were prepared as described in [27] and double-stained with antibodies against p125 and Sec31A (ER exit site), KDEL (ER) or GM130 (Golgi). Arrowheads indicate p125, Sec31A-positive large dots. Bar, 5 lm.

numerous small Sec31A-positive puncta throughout the cytoplasm a very recent study predicted that Sec23-interacting protein, were negative for p125 (top row). Staining for KDEL (an ER marker) p125, is also involved in craniofacial development in Xenopus did not show colocalization with p125 (middle row). It is of note [14]. However, we did not observe apparent craniofacial symptoms that the ring-like structure positive for GM130 (a cis-Golgi marker) in p125 KO mice. Xenopus and mammalian p125 may have some- was more closely connected (bottom row) compared with that what different physiological functions. visualized by p125 or Sec31A. These results together suggest that p125 is localized in a certain population of ER exit sites in Acknowledgements spermatocytes. We thank Dr. S. Aizawa of the Riken Center for Developmental 4. Discussion Biology for the donation of the TT2 cells and Dr. K. Touhara of the University of Tokyo for providing the plasmids for in situ hybrid- Spermiogenesis is the last phase of spermatogenesis and char- ization. We are also grateful to Ms. K. Shimada for her technical acterized by a complex process in which round spermatids differ- assistance. entiate into elongated spermatids and finally spermatozoa [23]. This work was supported in part by Grants-in-Aid for Scientific During spermiogenesis, the acrosome, a secretory organelle that Research, #20570190 (to K.T.) and #20790078 (to N.A.), from the contains the proteolytic enzymes required to dissolve zona pellu- Ministry of Education, Culture, Sports, Science and Technology of cida proteins during fertilization, is formed in close vicinity to Japan. the nucleus. The acrosome is formed by the fusion of pro-acroso- mal vesicles derived from the trans-Golgi network [20]. References p125 is expressed at stages I-XII of spermatogenesis, similar to the expression pattern of proteins involved in acrosome biogenesis [1] Jensen, D. and Schekman, R. (2011) COPII-mediated vesicle formation at a glance. J. Cell Sci. 124, 1–4. [24]. Indeed, acrosome formation is impaired in p125 KO mice. The [2] Bannykh, S.I., Rowe, T. and Balch, W.E. (1996) The organization of endoplasmic abnormal sperm observed in p125 KO mice are reminiscent of reticulum export complexes. J. Cell Biol. 135, 19–35. those observed in males with globozoospermia [22]. A globozoo- [3] Tani, K., Mizoguchi, T., Iwamatsu, A., Hatsuzawa, K. and Tagaya, M. (1999) spermia-like phenotype has been observed in mice lacking Hrb P125 is a novel mammalian Sec23p-interacting protein with structural similarity to phospholipid-modifying proteins. J. Biol. Chem. 274, 20505– [25], GOPC [26] and PICK1 [27]. These proteins are expressed at 20512. stages I-XII of spermatogenesis and are supposed to function at [4] Mizoguchi, T., Nakajima, K., Hatsuzawa, K., Nagahama, M., Hauri, H.P., Tagaya, the Golgi-acrosome interface. To our knowledge, p125 is the first M. and Tani, K. (2000) Determination of functional regions of p125, a novel mammalian Sec23p-interacting protein. Biochem. Biophys. Res. Commun. 279, example of a COPII-interacting protein that is required for acro- 144–149. some biogenesis. Our results suggest the importance of the ER [5] Ong, Y.S., Tang, B.L., Loo, L.S. and Hong, W. (2010) 125A exists as part of the transport system in spermiogenesis. mammalian Sec13/Sec31 COPII subcomplex to facilitate ER-Golgi transport. J. Cell Biol. 190, 331–345. Mutation in Sec23A, one of the two Sec23 paralogues in verte- [6] Shimoi, W., Ezawa, I., Nakamoto, K., Uesaki, S., Gabreski, G., Aridor, M., brates, has been shown to cause craniofacial defects [28,29], and Yamamoto, A., Nagahama, M., Tagaya, M. and Tani, K. (2005) P125 is localized 2176 N. Arimitsu et al. / FEBS Letters 585 (2011) 2171–2176

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