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

p125 and Sec31A colocalize by fluorescence microscopy (View interaction) The export of secretory and membrane proteins from the ER is mediated by COPII-coated vesicles [1]. The COPII coat consists of the small GTPase Sar1 and two heterodimers, Sec23–Sec24 and Sec13–Sec31. COPII vesicles bud at specialized ER subdomains, termed ER exit sites or transitional ER [2]. p125/Sec23ip was discovered as a mammalian protein that interacts with Sec23 [3, 4], and a later study showed that it interacts not only with Sec23 but also with Sec31 [5]. Knockdown of p125 by RNA interference causes the redistribution of ER exit sites in cultured cells, suggesting its role in their organization [6]. p125 is a member of an iPLA1 family that is conserved among eukaryotes. While there is only one member in yeast, Caenorhabditis elegans and Arabidopsis, mammals have three members (PA-PLA1/iPLA1-α, p125/iPLA1-β and KIAA0725p/iPLA1-γ), which are uniquely localized in cells [7-10]. The physiological importance of iPLA1 family proteins has been demonstrated in C. elegans [11] and Arabidopsis [12, 13]. Like mammals, Xenopus has three members, and very recent orthologous phenotype analysis combined with morpholino-mediated gene knockdown in Xenopus embryos predicted that p125/sec23ip is a candidate gene responsible for Waardenburg syndrome, a disorder characterized by craniofacial defects and pigment abnormalities [14]. To elucidate the physiological function of p125 in mammals, we generated p125 KO mice. We found that loss of p125 causes a defect in acrosome formation, leading to male subfertility. A p125 gene fragment was isolated from a mouse genomic bacterial artificial chromosome library derived from the C57BL/6 mouse strain (RP23-121G8) and subcloned into pBluescript II SK. A conditional targeting vector was constructed using the highly efficient recombineering-based method of Liu et al. [15]. 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 (5′-CGTCCAGGACATCTCAGAATCTGCGACTTCG-3′) and Primer 2 (5′-CGCAATGACAGGAGTTCCAAGCCTCATCACC-3′). All animal procedures and experiments were approved by the Animal Care Committee of Tokyo University of Pharmacy and Life Sciences. IVF was carried out as described [17]. Eggs were placed in a drop of human tubal fluid medium and incubated with control or p125−/− sperm for 24 h at 37 °C under 5% CO2. The percentage of 2-cell stage embryos was determined. For histological analysis, mouse testes were fixed in 10% formalin in PBS, dehydrated and then embedded in paraffin. Paraffin sections (6 μm) were stained with HE according to the standard protocol. For immunohistochemistry, testes were fixed with 4% paraformaldehyde in PBS and frozen in O.C.T. compound (Tissue-Tek; Sakura). Cryosections (10 μm) were prepared, permeabilized in TBS-T (20 mM Tris–HCl (pH 7.6), 137 mM NaCl and 0.1% Tween-20), blocked with 2% normal goat serum in PBS, and then analyzed. To prepare a cRNA probe, a p125 cDNA fragment (nucleotides 344-850) was labeled using a DIG RNA labeling mix (Roche) according to the manufacturer's protocol. cRNA probes against H1t and protamine 1 were prepared as described previously [18]. p125−/− mice generated through p125+/− × p125+/− mice were dissected on embryonic day 13.5 (E13.5), and MEFs were recovered by trypsinization. Given that many genes related to membrane trafficking are essential in yeast, we decided to construct a conditional targeting 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. 1 A). 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 of exon 1 and p125 protein expression were confirmed by Southern blotting (Fig. 1C) and Western blotting (Fig. 1D), respectively. Contrary to our expectation, p125 KO mice were born at Mendelian frequency, and grew indistinguishably from WT and heterozygous mice. However, male p125 KO mice appeared to be subfertile. Thus, we carefully examined the pregnancy and litter sizes in females with vaginal plugs, evidence of mating. When WT (p125 +/+) males were crossed with p125 +/+, p125 +/− and p125 −/− females, pregnancy almost always occurred in females with vaginal plugs (Table 1 ). The litter sizes ranged between 7.4 and 9.0. By contrast, when p125 −/− males were mated, failure of conception was frequently observed. The average litter sizes on mating with p125 +/+, p125 +/− and p125 −/− females were 3.3, 1.6 and 1.3, respectively. Notably, no marked difference in pregnancy was observed between p125 −/− and p125 +/+ females on crossing with WT males (9.0 vs 7.4). These results suggest that the major cause of the subfertility is a male problem. Data were obtained for mating between animals younger than 8 and 6 months of age for males and females, respectively. Grossly, the testes of p125 −/− mice were normal and their sizes were not much different from those of control mice at any age. In addition, the total number of sperm from a single cauda epididymis of a p125-deficient mouse was only slightly smaller than that from a control (WT and heterozygous) mouse (data not shown). IVF assays showed that sperm obtained from p125-deficient mice exhibited a markedly reduced fertilization rate (31%) compared with in control mice (84%) (Fig. 2 ), suggesting that the subfertility in p125-deficient mice is due to a sperm dysfunction. To determine why sperm from p125-deficient mice are dysfunctional, we examined the morphology of the seminiferous tubules and sperm in p125-deficient mice. The seminiferous tubules contain germ cells at all stages of maturation; stem cells and spermatogonia on one side of the junction, and spermatocytes, and round, elongating and elongated spermatids organized in strict order of maturation toward the lumen [19]. HE staining showed that the layers of cells in the seminiferous tubules were largely preserved in p125-deficient mice (Fig. 3 A, KO), implying no remarkable aberrancy of spermatogonia, spermatocytes or round spermatids. However, spermatids near the lumen of the tubules were not elongated in p125-deficient mice (Fig. 3A, KO, light blue box). Consistent with this finding, many sperm in the cauda epididymis of p125-deficient mice had round heads (Fig. 3B, KO). To examine the morphology of organelles including the acrosome, a specialized secretory organelle localized in the head of mammalian sperm [20], we stained sperm with Hoechst 33342, Mitotracker and an acrosome marker, PNA [21]. Fig. 3C shows that sperm from p125-deficient mice had a round-shaped nucleus (blue) and an abnormal mitochondrial sheath (red). Notably, while the crescent-shaped acrosome in WT sperm was unequivocally stained with FITC-PNA and an antibody against an acrosome marker, sp56, few PNA-, sp56-positive structures were observed in sperm from p125-deficient mice (Fig. 3C–E). This sperm phenotype is quite similar to that observed in males with globozoospermia [22], which is characterized by round-headed sperm with a deficiency in acrosomes, whose formation is highly dependent on the Golgi function [20]. Next, we examined the expression pattern of p125 in the testis. As shown in Fig. 4 A, p125 expression was detected in testicular cells, but not in sperm. Immunostaining of the seminiferous tubules showed that p125 is expressed in spermatocytes and round spermatids, but not in spermatogonia (Fig. 4B, top row). p125 immunoreactivity was not detected in whole cross-sections of seminiferous tubules prepared from p125 KO mice (top row, right), indicating that the anti-p125 antibody can specifically recognize p125. The expression of p125 mRNA in spermatocytes and round spermatids was confirmed by in situ hybridization (Fig. 4B, bottom row). Detailed in situ hybridization analysis demonstrated that p125 mRNA is expressed at stages I-XII of the 12-stage growth cycle of mouse spermatogenesis (Fig. 4C) [23]. Subcellular localization of p125 in spermatocytes was further examined by immunofluorescence microscopy (Fig. 5 ). In spermatocytes, p125 was localized in a ring-shaped structure with some large dots in the cytoplasm (middle column). Its distribution markedly overlapped that of Sec31A (an ER exit site marker) in the perinuclear ring-like structure and large dot-like structures, whereas numerous small Sec31A-positive puncta throughout the cytoplasm were negative for p125 (top row). Staining for KDEL (an ER marker) did not show colocalization with p125 (middle row). It is of note that the ring-like structure positive for GM130 (a cis-Golgi marker) was more closely connected (bottom row) compared with that visualized by p125 or Sec31A. These results together suggest that p125 is localized in a certain population of ER exit sites in spermatocytes. Spermiogenesis is the last phase of spermatogenesis and characterized by a complex process in which round spermatids differentiate into elongated spermatids and finally spermatozoa [23]. During spermiogenesis, the acrosome, a secretory organelle that contains the proteolytic enzymes required to dissolve zona pellucida proteins during fertilization, is formed in close vicinity to the nucleus. The acrosome is formed by the fusion of pro-acrosomal vesicles derived from the trans-Golgi network [20]. p125 is expressed at stages I-XII of spermatogenesis, similar to the expression pattern of proteins involved in acrosome biogenesis [24]. Indeed, acrosome formation is impaired in p125 KO mice. The abnormal sperm observed in p125 KO mice are reminiscent of those observed in males with globozoospermia [22]. A globozoospermia-like phenotype has been observed in mice lacking Hrb [25], GOPC [26] and PICK1 [27]. These proteins are expressed at stages I-XII of spermatogenesis and are supposed to function at the Golgi-acrosome interface. To our knowledge, p125 is the first example of a COPII-interacting protein that is required for acrosome biogenesis. Our results suggest the importance of the ER transport system in spermiogenesis. Mutation in Sec23A, one of the two Sec23 paralogues in vertebrates, has been shown to cause craniofacial defects [28, 29], and a very recent study predicted that Sec23-interacting protein, p125, is also involved in craniofacial development in Xenopus [14]. However, we did not observe apparent craniofacial symptoms in p125 KO mice. Xenopus and mammalian p125 may have somewhat different physiological functions. We thank Dr. S. Aizawa of the Riken Center for Developmental Biology for the donation of the TT2 cells and Dr. K. Touhara of the University of Tokyo for providing the plasmids for in situ hybridization. We are also grateful to Ms. K. Shimada for her technical assistance. This work was supported in part by Grants-in-Aid for Scientific Research, #20570190 (to K.T.) and #20790078 (to N.A.), from the .