Mutations in the Pick1 gene cause globozoospermia, a male infertility disorder, in both mice and humans. PICK1 is crucial for vesicle trafficking, and its deficiency in sperm cells leads to abnormal vesicle trafficking from the Golgi to the acrosome. This eventually disrupts acrosome formation and leads to male infertility. Here, we identified ICA1L, which has sequence similarities to ICA69 (also known as ICA1), as a new BAR-domain binding partner of PICK1. ICA1L is expressed in testes and brain, and is the major binding partner for PICK1 in testes. ICA1L and PICK1 are highly expressed in spermatids and trafficked together at different stages of spermiogenesis. ICA1L-knockout mice were generated by CRISPR-Cas technology. PICK1 expression was reduced by 80% in the testes of male mice lacking ICA1L. Sperm from ICA1L-knockout mice had abnormalities in the acrosome, nucleus and mitochondrial sheath formation. Both total and mobile sperm numbers were reduced, and about half of the remaining sperm had the characteristics of globozoospermia. These defects ultimately resulted in reduced fertility of male ICA1L-knockout mice, and ICA69/ICA1L-double knockout male mice were sterile.

Infertility is a health problem affecting 10–15% of couples worldwide (Jarow et al., 1989; Dunson et al., 2004). Around half of these cases are caused by male infertility (Jarow et al., 2002; Turek, 2005; Ferlin et al., 2006). Globozoospermia is a rare and severe sperm morphology disorder characterized by round-headed, acrosomeless spermatozoa (Sen et al., 1971). The acrosome locates at the head of mammalian sperm and contains various hydrolytic enzymes. The acrosome plays a crucial role during fertilization. When the sperm comes into contact with the zona pellucida of an egg, the acrosomal hydrolytic enzymes are released to facilitate sperm penetration into the zona pellucida prior to fusion with the egg (Ikawa et al., 2010). Human patients having a complete form of globozoospermia, characterized by 100% acrosomeless sperm, termed type I globozoospermia or total globozoospermia, are infertile. There are also a larger group of people whose sperm are partially acrosomeless, which is termed partial globozoospermia or type II globozoospermia (Dam et al., 2011). Several genetic models phenocopy human globozoospermia, such as mice deficient in CK2α′, Hrb, GOPC, GBA2, ZPBP1 and ZPBP2, Hsp90b1, Vps54, SPACA1, Dpy19l2, Atg7 and PICK1 (Xu et al., 1999; Kang-Decker et al., 2001; Yao et al., 2002; Yildiz et al., 2006; Lin et al., 2007; Xiao et al., 2009; Audouard and Christians, 2011; Paiardi et al., 2011; Fujihara et al., 2012; Pierre et al., 2012; Wang et al., 2014). In addition, mutations in SPATA16, PICK1 and DPY19L2 have been identified in human globozoospermia patients, with deletions and point mutations of DPY19L2 being a major cause of globozoospermia (Dam et al., 2007; Liu et al., 2010; Harbuz et al., 2011; Koscinski et al., 2011; Coutton et al., 2012; ElInati et al., 2012; Zhu et al., 2013).

Protein interacting with C-kinase 1 (PICK1) is a peripheral membrane protein that is important for protein and vesicle trafficking. PICK1 contains a PDZ (PSD-95, Dlg and ZO1) domain and a BAR (Bin, amphiphysin and Rvs) domain. The BAR domain of PICK1, which forms banana-shaped dimers, is capable of binding to the curved vesicle membrane (Jin et al., 2006). The PDZ domain of PICK1 interacts with α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits GluA2, GluA3 and GluA4c (Dev et al., 1999; Xia et al., 1999). PICK1 is involved in long-term depression (LTD) and long-term potentiation (LTP) by regulating subcellular localization and surface expression of AMPA receptors (Chung et al., 2000; Xia et al., 2000; Perez et al., 2001; Steinberg et al., 2006; Terashima et al., 2008).

Islet cell autoantigen 69 kDa (ICA69, also known as ICA1) was first identified as an autoantigen from type 1 diabetes patients (Pietropaolo et al., 1993). ICA69 also contains an N-terminal BAR domain. ICA69 forms heteromeric BAR domain dimers with PICK1 and is the major binding partner for PICK1 in the brain and pancreas (Cao et al., 2007,, 2013). ICA69 inhibits synaptic targeting of AMPA receptors by restricting the localization of PICK1 to dendritic shafts (Cao et al., 2007; Xu et al., 2014). In addition, the BAR domain complex of PICK1 and ICA69 is associated with insulin granule trafficking in pancreatic β-cells. Deficiency of PICK1 leads to abnormal trafficking of insulin granules and glucose intolerance (Cao et al., 2013; Holst et al., 2013). Moreover, this complex has also been found to promote the budding of growth hormone granules from the Golgi apparatus in the pituitary gland (Holst et al., 2013). ICA69-knockout (KO) mice share similar insulin trafficking defects and glucose intolerance to PICK1-KO mice (Cao et al., 2013). Interestingly, PICK1 and ICA69 are stabilized by each other in the complex, such that absence of one leads to the disappearance of the other from pancreatic β-cells (Cao et al., 2013).

Our previous work has demonstrated that mutation of PICK1 in mice also leads to phenotypes resembling globozoospermia, with primary defects in acrosome formation (Xiao et al., 2009). The acrosome malformation in PICK1-KO mice was caused by the abnormal trafficking of vesicles from the Golgi to the acrosome. These defects could be rescued by testes-specific re-expression of PICK1 (Li et al., 2013). In addition, mutation of PICK1 was identified in one human globozoospermia patient, providing further support to the importance of PICK1 in acrosome formation and male infertility (Liu et al., 2010). Interestingly, despite the ICA69-KO mice having very similar insulin trafficking defects to PICK1-KO mice, the ICA69-KO mice were very different from PICK1-KO mice in terms of acrosome formation and fertility, as no obvious defects were observed. Here, we report that we have identified protein islet cell autoantigen 1-like (ICA1L), which contains a BAR domain with high similarity to the BAR domain of ICA69, as the major binding partner for PICK1 in testes. ICA1L and PICK1 were highly expressed in spermatids and trafficked together at different stages of spermiogenesis. Both the number of total and mobile sperm were reduced, and about half of the remaining sperm had characteristics of globozoospermia in ICA1L-KO mice. These defects ultimately resulted in reduced fertility of ICA1L-KO male mice, and ICA69/ICA1L-double knockout male mice were sterile.

ICA1L is abundant in testes

ICA69 is the major binding partner of PICK1 in brain and pancreas (Cao et al., 2007,, 2013). ICA69-KO mice have very similar phenotypes to PICK1-KO mice in terms of insulin trafficking and glucose intolerance (Cao et al., 2013). However, unlike PICK1-KO mice, ICA69-KO mice have no obvious defect in fertility. Using a BLAST search, we found a new protein ICA1L, which contains a BAR domain with high similarity to that of ICA69 and a unique C-terminal region (Fig. 1A,B). Interestingly, the BAR domain of ICA1L is highly conserved during evolution, implying that it has a conserved role in different species (Fig. S1). Therefore, we wondered whether ICA1L has a compensatory or different role to that of ICA69. To investigate their relationship, ICA69- and ICA1L-specific antibodies, targeting their C-terminal regions, were generated (Fig. 1B–D). The ICA1L antibody specifically recognized ICA1L and the ICA69 antibody specifically recognized ICA69 (Fig. 1C). A strip test for the ICA1L antibody demonstrated that it recognized a single band in testes homogenates (Fig. 1D). A peptide antibody previously generated against ICA69 recognized both proteins (Cao et al., 2007) (Fig. 1C). However, this peptide antibody could also be used for western blotting because the molecular mass of ICA69 is bigger than that of ICA1L (Fig. 1C) and this antibody could only weakly recognize endogenous ICA1L (data not shown).

Fig. 1.

ICA1L is abundant in testes. (A) Schematic representations of structures of ICA69 and ICA1L. Both ICA69 and ICA1L are composed of an N-terminal BAR domain and a C-terminal domain (ICAC and ICALC, respectively). (B) Similarity between rat ICA69 and rat ICA1L. Alignment was performed by Clustal Omega and presented by Boxshade. The red bar represents BAR domains of both ICA69 and ICA1L, the borders of which (amino acids 21–248 and 15–242, respectively) were determined by SMART. The upper and lower purple box shows the antigens for ICA69 (amino acids 275–459) and ICA1L (amino acids 266–411), respectively. The red box shows the peptide antigen (amino acids 468–480) for generation of ICA69 antibody. (C) Antibody specificity test. HEK293T cells were transfected with Myc–ICA69 or Myc–ICA1L and cell lysates were used for western blotting. (D) A strip test for anti-ICA1L antibody. Testes sample was immunoblotted with following antibodies: lane1, serum, 1:1000; lane 2, flow through, 1:1000; lane 3, anti-ICA1L antibody preincubated with antigen, 1:300; lane 4, anti-ICA1L antibody, 1:300. (E) Different tissues were dissected from mouse and homogenized to obtain the total proteins. Equal amounts of proteins were loaded onto SDS-PAGE gels and analyzed by western blotting.

Fig. 1.

ICA1L is abundant in testes. (A) Schematic representations of structures of ICA69 and ICA1L. Both ICA69 and ICA1L are composed of an N-terminal BAR domain and a C-terminal domain (ICAC and ICALC, respectively). (B) Similarity between rat ICA69 and rat ICA1L. Alignment was performed by Clustal Omega and presented by Boxshade. The red bar represents BAR domains of both ICA69 and ICA1L, the borders of which (amino acids 21–248 and 15–242, respectively) were determined by SMART. The upper and lower purple box shows the antigens for ICA69 (amino acids 275–459) and ICA1L (amino acids 266–411), respectively. The red box shows the peptide antigen (amino acids 468–480) for generation of ICA69 antibody. (C) Antibody specificity test. HEK293T cells were transfected with Myc–ICA69 or Myc–ICA1L and cell lysates were used for western blotting. (D) A strip test for anti-ICA1L antibody. Testes sample was immunoblotted with following antibodies: lane1, serum, 1:1000; lane 2, flow through, 1:1000; lane 3, anti-ICA1L antibody preincubated with antigen, 1:300; lane 4, anti-ICA1L antibody, 1:300. (E) Different tissues were dissected from mouse and homogenized to obtain the total proteins. Equal amounts of proteins were loaded onto SDS-PAGE gels and analyzed by western blotting.

Using the ICA1L-specific antibody the tissue distribution of ICA1L was determined. Interestingly, ICA1L was abundant in testes but only weakly expressed in brain (Fig. 1E). ICA1L was absent in other tissues. PICK1 was highly expressed in brain and testes, followed by pancreas. ICA69 was mainly expressed in brain and pancreas but only weakly expressed in testes.

ICA1L interacts with PICK1 in vitro and in vivo

ICA69 forms a heteromeric BAR domain complex with PICK1 (Cao et al., 2007). ICA1L contains a BAR domain with high similarity to that of ICA69. Next, we investigated whether ICA1L could interact with PICK1. We found that ICA1L strongly bound to PICK1 when co-expressed in a heterologous system (Fig. 2A). In addition, when using anti-PICK1 antibodies to immunoprecipitate endogenous PICK1, ICA1L was robustly co-immunoprecipitated with PICK1 from both brain and testes homogenates (Fig. 2B,C). In addition, anti-ICA1L antibodies were used to immunoprecipitate endogenous ICA1L in testes homogenates, and PICK1 was readily co-immunoprecipitated with ICA1L (Fig. 2D). These results suggest that PICK1 and ICA1L strongly interact with each other. Interestingly, an additional higher band of ICA1L was observed in brain and when it was overexpressed in HEK293T cells (Fig. 2A,B, indicated by asterisks).

Fig. 2.

ICA1L binds to PICK1 in vitro and in vivo. (A) HEK293T cells were transiently transfected with the indicated constructs. Anti-GFP antibodies were used for immunoprecipitation (IP). In vivo co-immunoprecipitation from mouse brain homogenates (B) and testes homogenates (C) using anti-PICK1 antibodies. The asterisk marks an additional band of ICA1L (see text). (D) In vivo co-immunoprecipitation from mouse testes homogenates using anti-ICA1L antibodies. (E) Schematic representation of structure of ICA1L and deletion mutants of ICA1L. (F) HEK293T cells were transiently transfected with cDNA as indicated. Anti-GFP antibodies were used for immunoprecipitation. PICK1 strongly bound to the BAR domain of ICA1L. (G) Schematic representation of structure of PICK1 and deletion mutants of PICK1. NT, N-terminus; CT, C-terminus. (H) HEK293T cells were transiently transfected with indicated constructs. Anti-GFP antibodies were used for immunoprecipitation. ICA1L potently bound to the BAR domain of PICK1. (I) HEK293T cells were transiently transfected with indicated constructs. Anti-GFP antibodies were used for immunoprecipitation. The BAR domain of PICK1 interacted with the BAR domain of ICA1L.

Fig. 2.

ICA1L binds to PICK1 in vitro and in vivo. (A) HEK293T cells were transiently transfected with the indicated constructs. Anti-GFP antibodies were used for immunoprecipitation (IP). In vivo co-immunoprecipitation from mouse brain homogenates (B) and testes homogenates (C) using anti-PICK1 antibodies. The asterisk marks an additional band of ICA1L (see text). (D) In vivo co-immunoprecipitation from mouse testes homogenates using anti-ICA1L antibodies. (E) Schematic representation of structure of ICA1L and deletion mutants of ICA1L. (F) HEK293T cells were transiently transfected with cDNA as indicated. Anti-GFP antibodies were used for immunoprecipitation. PICK1 strongly bound to the BAR domain of ICA1L. (G) Schematic representation of structure of PICK1 and deletion mutants of PICK1. NT, N-terminus; CT, C-terminus. (H) HEK293T cells were transiently transfected with indicated constructs. Anti-GFP antibodies were used for immunoprecipitation. ICA1L potently bound to the BAR domain of PICK1. (I) HEK293T cells were transiently transfected with indicated constructs. Anti-GFP antibodies were used for immunoprecipitation. The BAR domain of PICK1 interacted with the BAR domain of ICA1L.

To determine which domain of ICA1L interacted with PICK1, deletion mutants of ICA1L were generated (Fig. 2E). The ICA1L BAR domain strongly bound to PICK1, whereas the C-terminal region weakly interacted with PICK1 (Fig. 2F). To map which domain of PICK1 interacted with ICA1L, deletion mutants of PICK1 were generated (Fig. 2G). The BAR domain of PICK1 bound to ICA1L, whereas the PDZ domain and the C-terminal region did not (Fig. 2H). In addition, the BAR domain of PICK1 interacted with the BAR domain of ICA1L (Fig. 2I). Taken together, these results suggest ICA1L interacts with PICK1 mainly in a BAR-domain-dependent manner.

ICA1L is the major binding partner for PICK1 in testes

Interestingly, we also found that ICA1L was absent in PICK1-KO mice, similar to ICA69 (Fig. 3A). We did not observe any difference in the presence of ICA69 and ICA1L mRNAs in the brain and testes of PICK1-KO mice, indicating that the disappearance of ICA69 and ICA1L in PICK1-KO mice was not due to downregulated gene transcription (Fig. 3B,C). The facts that ICA1L bound tightly to PICK1 and ICA1L was highly expressed in testes suggest that ICA1L could be the major binding partner for PICK1 in testes. To estimate the percentages of ICA1L and PICK1 that were associated with each other in testes, we performed quantitative co-immunoprecipitation from testes homogenates. First, anti-PICK1 antibodies were used to immunoprecipitate endogenous PICK1 in testes twice consecutively and ∼86% of ICA69 and ∼85% of ICA1L co-immunoprecipitated with PICK1 (Fig. 3D,E). Similarly, anti-ICA1L antibodies were used to immunoprecipitate endogenous ICA1L in testes and ∼80% of PICK1 co-immunoprecipitated with ICA1L (Fig. 3F,G). No ICA69 co-immunoprecipitated with ICA1L, suggesting that the three proteins did not form a complex (Fig. 3F,G). It also further confirmed the specificity of the ICA1L antibody. After immunoprecipitation with anti-ICA1L antibodies, the ICA69 antibodies were used to immunoprecipitate endogenous ICA69 in testes and ∼11% of PICK1 co-immunoprecipitated with ICA69 (Fig. 3F,G). As control, GAPDH was immunoblotted and it was not significantly changed by immunoprecipitation with anti-PICK1, -ICA69 or -ICA1L antibodies, further supporting the specificities of these antibodies (Fig. 3D–G). Taken together, the above results demonstrate that ICA1L is abundant and the major binding partner for PICK1 in testes.

Fig. 3.

ICA1L is the major binding partner for PICK1 in testes. (A) Brains, pancreas and testes were dissected from WT and PICK1-KO mice and homogenized to obtain the total proteins. Equal amounts of proteins were loaded to SDS-PAGE gels and analyzed by western blotting. RNAs were extracted from brains (B) and testes (C) of WT and PICK1-KO mice and cDNAs were synthesized by reverse transcription and amplified by PCR. (D) PICK1 was immunoprecipitated (IP) with PICK1 antibodies consecutively twice from mouse testes homogenates. The testes homogenates after the first and second immunoprecipitation were designated as AIP1 and AIP2, respectively. (E) Quantification for D; n=4, error bar represents s.e.m. ***P<0.001; #, not significant (Student's t-test). (F) ICA1L and ICA69 were immunoprecipitated with anti-ICA1L and -ICA69 antibodies, respectively, from mouse testes homogenates. The testes homogenates after immunoprecipitation with anti-ICA1L and -ICA69 antibodies were designated as AIP1 and AIP2, respectively. (G) Quantification for F; n=4, error bar represents s.e.m. ***P<0.001; #, not significant (Student's t-test). M1–M4 in D and F represent samples from four different mice.

Fig. 3.

ICA1L is the major binding partner for PICK1 in testes. (A) Brains, pancreas and testes were dissected from WT and PICK1-KO mice and homogenized to obtain the total proteins. Equal amounts of proteins were loaded to SDS-PAGE gels and analyzed by western blotting. RNAs were extracted from brains (B) and testes (C) of WT and PICK1-KO mice and cDNAs were synthesized by reverse transcription and amplified by PCR. (D) PICK1 was immunoprecipitated (IP) with PICK1 antibodies consecutively twice from mouse testes homogenates. The testes homogenates after the first and second immunoprecipitation were designated as AIP1 and AIP2, respectively. (E) Quantification for D; n=4, error bar represents s.e.m. ***P<0.001; #, not significant (Student's t-test). (F) ICA1L and ICA69 were immunoprecipitated with anti-ICA1L and -ICA69 antibodies, respectively, from mouse testes homogenates. The testes homogenates after immunoprecipitation with anti-ICA1L and -ICA69 antibodies were designated as AIP1 and AIP2, respectively. (G) Quantification for F; n=4, error bar represents s.e.m. ***P<0.001; #, not significant (Student's t-test). M1–M4 in D and F represent samples from four different mice.

ICA1L regulates PICK1-mediated AMPA receptor clustering in a similar manner to ICA69 and is upregulated in the brain of ICA69-KO mice

PICK1 regulates subcellular localization and surface expression of AMPA receptors and is consequently involved in LTD and LTP (Chung et al., 2000; Xia et al., 2000; Perez et al., 2001; Steinberg et al., 2006; Terashima et al., 2008). ICA69 regulates AMPA receptor trafficking by disrupting PICK1 homodimer formation (Cao et al., 2007). The fact that both ICA69 and ICA1L are expressed in brain made us wonder whether ICA1L has a compensatory role for ICA69 (Figs 1E and 3A). GluA2 was diffuse in the cytoplasm when expressed in HEK293T cells, but upon co-expression of PICK1 GluA2 became clustered in HEK293T cells (Fig. 4A). Additional expression of ICA1L disrupted the PICK1-mediated GluA2 clustering (Fig. 4A), similar to what has been observed for ICA69 (Cao et al., 2007). PICK1 formed homodimers, which is important for the synaptic targeting of AMPA receptors (Fig. 4B). Both ICA1L and ICA69 could compete for the PICK1 homodimer formation (Fig. 4B,C). In addition, increasing the relative ratio of ICA1L or ICA69 to PICK1 further reduced PICK1 homodimer formation (Fig. 4D–G). ICA1L, ICA69 and PICK1 were expressed in similar regions of the brain (Fig. 4H). More importantly, ICA1L expression was upregulated in the brains of ICA69-KO mice (see Fig. 7G,J). Taken together, these results suggest ICA1L could have a compensatory role for ICA69 in brain.

Fig. 4.

ICA1L compensates for ICA69 in brain. (A) HEK293T cells were transfected with the indicated constructs. When PICK1 (green) and GluA2 (red) were co-expressed in cells, they formed many co-clusters in the cytosol. Co-expression with ICA1L (blue) disrupts the PICK1–GluA2 clusters as does ICA69 (blue). When PICK1, GluA2, ICA69 and ICA1L were expressed alone, they were all diffusely localized in the cytosol (lower panels). Scale bars: 10 μm. (B) HEK293T cells were transfected with the indicated constructs. Anti-GFP antibodies were used for immunoprecipitation (IP). Both ICA69 and ICA1L could compete for PICK1 homodimer formation. (C) Quantification for B. The amount of co-immunoprecipitated Myc–PICK1 normalized to total immunoprecipitated YFP–PICK1 represents the relative level of PICK1 homodimers, n=5, error bar represents s.e.m. (D) HEK293T cells were transfected with the indicated constructs. Anti-GFP antibodies were used for immunoprecipitation. Increasing the relative transfection ratio of ICA1L to PICK1 (from 1:1 to 3:1) could further reduce PICK1 homodimer formation. (E) Quantification for D. The amount of co-immunoprecipitated Myc–PICK1 normalized to total immunoprecipitated YFP–PICK1 represents the relative level of PICK1 homodimers, n=4, error bar represents s.e.m. (F) HEK293T cells were transfected with the indicated constructs. Anti-GFP antibodies were used for immunoprecipitation. Increasing the relative transfection ratio of ICA69 to PICK1 (from 1:1 to 3:1) could further reduce PICK1 homodimer formation. (G) Quantification for F. The amount of co-immunoprecipitated Myc–PICK1 normalized to total immunoprecipitated YFP–PICK1 represents the relative level of PICK1 homodimers, n=5, error bar represents s.e.m. (H) The indicated brain parts were dissected and homogenized for western blotting. ICA1L, ICA69 and PICK1 were expressed in similar regions of the brain. *P<0.05, **P<0.01, ***P<0.001 (Student’s t-test).

Fig. 4.

ICA1L compensates for ICA69 in brain. (A) HEK293T cells were transfected with the indicated constructs. When PICK1 (green) and GluA2 (red) were co-expressed in cells, they formed many co-clusters in the cytosol. Co-expression with ICA1L (blue) disrupts the PICK1–GluA2 clusters as does ICA69 (blue). When PICK1, GluA2, ICA69 and ICA1L were expressed alone, they were all diffusely localized in the cytosol (lower panels). Scale bars: 10 μm. (B) HEK293T cells were transfected with the indicated constructs. Anti-GFP antibodies were used for immunoprecipitation (IP). Both ICA69 and ICA1L could compete for PICK1 homodimer formation. (C) Quantification for B. The amount of co-immunoprecipitated Myc–PICK1 normalized to total immunoprecipitated YFP–PICK1 represents the relative level of PICK1 homodimers, n=5, error bar represents s.e.m. (D) HEK293T cells were transfected with the indicated constructs. Anti-GFP antibodies were used for immunoprecipitation. Increasing the relative transfection ratio of ICA1L to PICK1 (from 1:1 to 3:1) could further reduce PICK1 homodimer formation. (E) Quantification for D. The amount of co-immunoprecipitated Myc–PICK1 normalized to total immunoprecipitated YFP–PICK1 represents the relative level of PICK1 homodimers, n=4, error bar represents s.e.m. (F) HEK293T cells were transfected with the indicated constructs. Anti-GFP antibodies were used for immunoprecipitation. Increasing the relative transfection ratio of ICA69 to PICK1 (from 1:1 to 3:1) could further reduce PICK1 homodimer formation. (G) Quantification for F. The amount of co-immunoprecipitated Myc–PICK1 normalized to total immunoprecipitated YFP–PICK1 represents the relative level of PICK1 homodimers, n=5, error bar represents s.e.m. (H) The indicated brain parts were dissected and homogenized for western blotting. ICA1L, ICA69 and PICK1 were expressed in similar regions of the brain. *P<0.05, **P<0.01, ***P<0.001 (Student’s t-test).

ICA1L traffics together with PICK1 during spermiogenesis

ICA1L is mainly expressed in testes, so we wanted to focus on studying the function of ICA1L in testes. PICK1 is mainly expressed in spermatids and is involved in acrosome trafficking (Xiao et al., 2009). Given that ICA1L was the major binding partner for PICK1 in testes, this made us ask whether ICA1L was also expressed in spermatids. Using immunohistochemistry, we found that both ICA1L and PICK1 were expressed in spermatids and they colocalized very well with each other (Fig. 5A). The spermatids undergo metamorphic and biochemical changes to produce mature sperm during spermiogenesis. To assess whether ICA1L and PICK1 traffic together during different stages of spermiogenesis (the Golgi, cap, acrosome and maturation phases) dynamic locations of PICK1 and ICA1L were characterized. Both PICK1 and ICA1L partially colocalized or were close to the acrosome during the Golgi phase and started to move to the opposite ends of the acrosome during the cap and acrosome phases (Fig. 5B,C). PICK1 and ICA1L then completely located at the opposite ends of acrosome during the maturation phase (Fig. 5B,C). These results suggest ICA1L shares similar trafficking dynamics to PICK1 during spermiogenesis. Notably, and consistent with western blotting data, the ICA1L signal was absent in PICK1-KO mice (Fig. 5C). The acrosome was fragmented and nucleus formation was impaired in PICK1-KO mice, as previously reported (Xiao et al., 2009) (Fig. 5B,C). Taken together, these results suggest that ICA1L and PICK1 traffic together during spermiogenesis.

Fig. 5.

ICA1L traffics together with PICK1 during spermiogenesis. (A) ICA1L colocalized with PICK1 in spermatids. Rabbit anti-ICA1L (green) and guinea pig anti-PICK1 (red) antibodies were used for immunolabeling. Lower panels show enlarged images of box areas in upper panels. Colocalization of ICA1L and PICK1 is marked by arrows. Dynamic locations of PICK1 (arrowheads) (B) and ICA1L (arrowheads) (C) during acrosome formation. Guinea pig anti-PICK1 antibodies were used to label endogenous PICK1 (red). Rabbit anti-ICA1L antibodies were used to label endogenous ICA1L (red). Mouse anti-sp56 antibodies were used to label acrosomes (green, arrows). DAPI was used to label the nucleus (blue). Scale bars: 10 μm.

Fig. 5.

ICA1L traffics together with PICK1 during spermiogenesis. (A) ICA1L colocalized with PICK1 in spermatids. Rabbit anti-ICA1L (green) and guinea pig anti-PICK1 (red) antibodies were used for immunolabeling. Lower panels show enlarged images of box areas in upper panels. Colocalization of ICA1L and PICK1 is marked by arrows. Dynamic locations of PICK1 (arrowheads) (B) and ICA1L (arrowheads) (C) during acrosome formation. Guinea pig anti-PICK1 antibodies were used to label endogenous PICK1 (red). Rabbit anti-ICA1L antibodies were used to label endogenous ICA1L (red). Mouse anti-sp56 antibodies were used to label acrosomes (green, arrows). DAPI was used to label the nucleus (blue). Scale bars: 10 μm.

Generation of ICA1L-KO mice by CRISPR-Cas

Given that ICA1L is the major binding partner for PICK1 in testes and they traffic together during spermiogenesis, we asked whether deletion of Ica1l would affect spermiogenesis. CRISPR-Cas is a fast, efficient and reliable technology that can be used for multiplex genome editing (Ran et al., 2013; Wang et al., 2013; Yang et al., 2013). ICA1L-KO mice were therefore generated by CRISPR-Cas technology. Exon 2 was targeted and a restriction enzyme site for NspI was chosen for restriction fragment length polymorphism (RFLP) analysis (Fig. 6A). The PCR product containing the target region was gel purified, and NspI digestion gave the two predicted bands (Fig. 6B). The mutation rate was about 28% in vitro when the construct pX330 expressing single-guided RNA (sgRNA) targeting Ica1l was expressed in N2a cells as demonstrated by RFLP analysis (Fig. 6C). After synthesizing Cas9 mRNA and sgRNA, microinjection, transferring embryos into foster mothers, in total 12 founders from three foster mothers were identified and verified by sequencing (Fig. 6D,E; Fig. S2). Two injection parameters were chosen – large volume (‘L’) and relatively small volume (‘S’) (Fig. 6E). Injection of a large volume gave a higher mutation rate, whereas injection of a low volume resulted in a lower mutation rate (Fig. 6E; Table S1). Interestingly, we found that most insertions happened at sites between the third and fourth base pair (bp) before the protospacer adjacent motif (PAM) (Fig. S2). No off-targeting effects were found in our case (Table S2) and the mosaic rate was about 50%, similar to the previous report (Yang et al., 2013) (Fig. S2). A founder with a mutant allele of a 23 bp deletion (Founder 2 in Fig. S2) was back-crossed to C57BL/6J for the following experiments. For genotyping, the PCR products for wild-type (WT) and ICA1L-KO mice are 112 bp and 89 bp, respectively, and heterozygous mice contain both fragments (Fig. 6F).

Fig. 6.

Generation of ICA1L-KO mice by CRISPR-Cas. (A) Targeting region for Ica1l. The red sequence is the PAM motif. The uppercase letters are the NspI restriction site. The targeting sequence is underlined. (B) A fragment containing the targeting region was PCR amplified from mouse genomic DNA and gel purified. The PCR product was left untreated or incubated with NspI. The fragment (703 bp) could be cut into two bands (308 bp and 395 bp). (C) Testing CRISPR-Cas efficiency in vitro. N2a cells were transfected with empty vector (EV) or pX330 expressing the sgRNA targeting Ica1l. Gel purified PCR products were digested by NspI. Fragments carrying mutations could not be digested by NspI. (D) Work flow of microinjection. (E) Screening of founders. PCR products from newborns were gel purified and digested by NspI. Three littermates from three foster mothers were screened. Embryos in recipient 1 were injected at a relatively higher volume, whereas embryos in recipient 2 and 3 were injected at a lower volume. Injection of a higher volume resulted in a higher mutation rate. (F) Genotyping of ICA1L-KO mice. The PCR product for WT mice is 112 bp. The PCR product for KO mice is 89 bp. Heterozygous mice contain both products.

Fig. 6.

Generation of ICA1L-KO mice by CRISPR-Cas. (A) Targeting region for Ica1l. The red sequence is the PAM motif. The uppercase letters are the NspI restriction site. The targeting sequence is underlined. (B) A fragment containing the targeting region was PCR amplified from mouse genomic DNA and gel purified. The PCR product was left untreated or incubated with NspI. The fragment (703 bp) could be cut into two bands (308 bp and 395 bp). (C) Testing CRISPR-Cas efficiency in vitro. N2a cells were transfected with empty vector (EV) or pX330 expressing the sgRNA targeting Ica1l. Gel purified PCR products were digested by NspI. Fragments carrying mutations could not be digested by NspI. (D) Work flow of microinjection. (E) Screening of founders. PCR products from newborns were gel purified and digested by NspI. Three littermates from three foster mothers were screened. Embryos in recipient 1 were injected at a relatively higher volume, whereas embryos in recipient 2 and 3 were injected at a lower volume. Injection of a higher volume resulted in a higher mutation rate. (F) Genotyping of ICA1L-KO mice. The PCR product for WT mice is 112 bp. The PCR product for KO mice is 89 bp. Heterozygous mice contain both products.

PICK1 is reduced by 80% in the testes of ICA1L-KO mice

After ICA1L-KO mice were generated, protein expression levels were investigated in testes, brain and pancreas. Interestingly, we found that PICK1 was reduced by ∼80% in the testes of ICA1L-KO mice (Fig. 7A,B), which correlates to the earlier finding that 80% of PICK1 was associated with ICA1L in testes (Fig. 3F,G). This suggests that PICK1 was degraded without binding to ICA1L in testes. However, the levels of PICK1 were not significantly changed in the testes of ICA69-KO mice, implying that ICA69 does not play an important role in testes (Fig. 7A,B). The levels of ICA69 were not significantly altered in the testes of ICA1L-KO mice (Fig. 7A,C) and, similarly, the levels of ICA1L were also not significantly changed in the testes of ICA69-KO mice (Fig. 7A,D). In addition, we mated ICA69/ICA1L double-knockout (DKO) mice and found that PICK1 was reduced by ∼82% in the testes of ICA69/ICA1L DKO mice, which is similar to the 80% reduction of PICK1 found in ICA1L-KO mice, again confirming that the loss of ICA69 did not contribute much to the loss of PICK1 in testes (Fig. 7E,F).

Fig. 7.

PICK1 is reduced by 80% in the testes of ICA1L-KO mice. The testes (A), brain (G) and pancreas (K) from WT, ICA69-KO and ICA1L-KO mice were dissected and homogenized for western blotting. Quantification of relative expression levels of PICK1 (B), ICA69 (C), ICA1L (D) normalized to GAPDH in A; WT, n=3; ICA69-KO, n=3; ICA1L-KO, n=3, error bar represents s.e.m. The testes (E) from WT and ICA69/ICA1L DKO mice were dissected and homogenized for western blotting. Quantification of relative expression levels of PICK1 (F) normalized to GAPDH in E; WT, n=4; ICA69/ICA1L DKO, n=3, error bar represents s.e.m. Quantification of relative expression levels of PICK1 (H), ICA69 (I), ICA1L (J) normalized to GAPDH in G; WT, n=6; ICA69-KO, n=8; ICA1L-KO, n=5, error bar represents s.e.m. Quantification of relative expression levels of PICK1 (L) and ICA69 (M) normalized to GAPDH in (K), WT, n=3; ICA69-KO, n=3; ICA1L-KO, n=3, error bar represents s.e.m. *P<0.05; **P<0.01; ***P<0.001; #, not significant (Student’s t-test).

Fig. 7.

PICK1 is reduced by 80% in the testes of ICA1L-KO mice. The testes (A), brain (G) and pancreas (K) from WT, ICA69-KO and ICA1L-KO mice were dissected and homogenized for western blotting. Quantification of relative expression levels of PICK1 (B), ICA69 (C), ICA1L (D) normalized to GAPDH in A; WT, n=3; ICA69-KO, n=3; ICA1L-KO, n=3, error bar represents s.e.m. The testes (E) from WT and ICA69/ICA1L DKO mice were dissected and homogenized for western blotting. Quantification of relative expression levels of PICK1 (F) normalized to GAPDH in E; WT, n=4; ICA69/ICA1L DKO, n=3, error bar represents s.e.m. Quantification of relative expression levels of PICK1 (H), ICA69 (I), ICA1L (J) normalized to GAPDH in G; WT, n=6; ICA69-KO, n=8; ICA1L-KO, n=5, error bar represents s.e.m. Quantification of relative expression levels of PICK1 (L) and ICA69 (M) normalized to GAPDH in (K), WT, n=3; ICA69-KO, n=3; ICA1L-KO, n=3, error bar represents s.e.m. *P<0.05; **P<0.01; ***P<0.001; #, not significant (Student’s t-test).

PICK1 was reduced by ∼73% in the brains of ICA69-KO mice, whereas PICK1 was slightly reduced in the brains of ICA1L-KO mice (Fig. 7G,H). This result suggests that ICA69 is the major binding partner for PICK1 in brain whereas ICA1L is a minor binding partner. ICA69 was not changed in the brains of ICA1L-KO mice (Fig. 7G,I). PICK1 was reduced to almost undetectable levels in the pancreas of ICA69-KO mice (Fig. 7K,L). ICA1L was absent from the pancreas (Fig. 7K). The levels of ICA69 and PICK1 were not significantly altered in the pancreas of ICA1L-KO mice (Fig. 7K–M).

Defective sperm and fertility in ICA1L-KO and ICA69/ICA1L DKO mice

Although 80% of PICK1 was lost in the testes of ICA1L-KO mice, ICA1L-KO male mice were still fertile (Fig. 8A). However, the fertility of ICA1L-KO male mice was significantly reduced. The litter size of ICA1L-KO male mice was reduced by ∼36% compared to WT mice, whereas no significant change was found in ICA69-KO male mice (average litter size, mean±s.e.m., male WT, 7.4±0.4, n=8; male ICA69-KO, 6.0±0.5, n=8; male ICA1L-KO, 4.7±0.7, n=16) (Fig. 8A). More importantly, male ICA69/ICA1L DKO mice were sterile (Fig. 8A). To investigate how deficiency of ICA1L led to reduced fertility and why ICA69/ICA1L DKO male mice were sterile, sperm from the cauda epididymis were examined. The total sperm number from the cauda epididymis of ICA1L-KO mice was reduced by ∼46% (Fig. 8B). The numbers of mobile and linear mobile sperm from the cauda epididymis were also reduced by ∼54% and ∼60%, respectively (Fig. 8C,D). Although there were trends of reduction in the total, mobile and linear mobile sperm numbers for ICA69-KO mice, they did not reach significance compared to WT controls (Fig. 8B–D). By contrast, ICA69/ICA1L DKO mice had severe defects. Total sperm number was reduced by ∼98% (Fig. 8B). The mobile sperm number was reduced by ∼99% compared to WT mice, and no linear mobile sperm were observed in ICA69/ICA1L DKO mice (Fig. 8C,D). Hematoxylin and Eosin (H&E) staining of the cauda epididymis confirmed that the numbers of mature sperm in ICA1L-KO and ICA69/ICA1L DKO mice were reduced (Fig. 8E). From the H&E staining of seminiferous tubules of testes, it could be seen that the layers of cells (spermatogonia, spermatocytes and round spermatids) were preserved in ICA69-KO, ICA1L-KO and ICA69/ICA1L DKO mice (Fig. 8F). However, sperm with round nuclei could be detected in ICA1L-KO and ICA69/ICA1L DKO mice. This suggests that an abnormal transition from round spermatids to mature sperm during spermiogenesis could result in defective sperm in ICA1L-KO and ICA69/ICA1L DKO mice.

Fig. 8.

Sperm in ICA1L-KO mice have globozoospermia-like phenotypes. (A) Average pup numbers from WT, ICA69-KO, ICA1L-KO and ICA69/ICA1L DKO male mice mated with female mice. WT, n=8; ICA69-KO, n=8; ICA1L-KO, n=16; ICA69/ICA1L DKO, n=6, error bar represents s.e.m. Total sperm numbers (B), mobile sperm numbers (C) and linear mobile sperm numbers (D) of WT, ICA69-KO, ICA1L-KO and ICA69/ICA1L DKO mice. WT, n=8; ICA69-KO, n=11; ICA1L-KO, n=5; ICA69/ICA1L DKO, n=3, error bar represents s.e.m. (E) H&E staining of the cauda epididymis of WT, ICA69-KO, ICA1L-KO and ICA69/ICA1L DKO mice. The numbers of mature sperm in ICA1L-KO and ICA69/ICA1L DKO mice were reduced. Insets, enlarged views of the heads of the sperm. (F) H&E staining of the testes of WT, ICA69-KO, ICA1L-KO and ICA69/ICA1L DKO mice. The morphology of spermatogonia, spermatocytes, round spermatids and sperm is labeled by white arrows, white arrowheads, black arrows and black arrowheads, respectively. The lower panel shows higher magnification views of the boxed regions in the upper panels. (G) Bright field images of sperm from the cauda epididymis of WT, ICA69-KO, ICA1L-KO and ICA69/ICA1L DKO mice. Sperm from the cauda epididymis of WT, ICA69-KO, ICA1L-KO and ICA69/ICA1L DKO mice were labeled for the acrosome marker sp56 (green) (H) and the mitochondrial marker Tomm20 (red) (I); nuclei (blue) were labeled by DAPI. Scale bars: 5 μm. (J) The percentage of globozoospermia-like sperm in WT, ICA69-KO, ICA1L-KO and ICA69/ICA1L DKO mice. WT, n=8; ICA69-KO, n=11; ICA1L-KO, n=4; ICA69/ICA1L DKO, n=3, error bar represents s.e.m. nu, nucleus; ms, mitochondrial sheath. *P<0.05; **P<0.01; ***P<0.001; #, not significant (Student’s t-test).

Fig. 8.

Sperm in ICA1L-KO mice have globozoospermia-like phenotypes. (A) Average pup numbers from WT, ICA69-KO, ICA1L-KO and ICA69/ICA1L DKO male mice mated with female mice. WT, n=8; ICA69-KO, n=8; ICA1L-KO, n=16; ICA69/ICA1L DKO, n=6, error bar represents s.e.m. Total sperm numbers (B), mobile sperm numbers (C) and linear mobile sperm numbers (D) of WT, ICA69-KO, ICA1L-KO and ICA69/ICA1L DKO mice. WT, n=8; ICA69-KO, n=11; ICA1L-KO, n=5; ICA69/ICA1L DKO, n=3, error bar represents s.e.m. (E) H&E staining of the cauda epididymis of WT, ICA69-KO, ICA1L-KO and ICA69/ICA1L DKO mice. The numbers of mature sperm in ICA1L-KO and ICA69/ICA1L DKO mice were reduced. Insets, enlarged views of the heads of the sperm. (F) H&E staining of the testes of WT, ICA69-KO, ICA1L-KO and ICA69/ICA1L DKO mice. The morphology of spermatogonia, spermatocytes, round spermatids and sperm is labeled by white arrows, white arrowheads, black arrows and black arrowheads, respectively. The lower panel shows higher magnification views of the boxed regions in the upper panels. (G) Bright field images of sperm from the cauda epididymis of WT, ICA69-KO, ICA1L-KO and ICA69/ICA1L DKO mice. Sperm from the cauda epididymis of WT, ICA69-KO, ICA1L-KO and ICA69/ICA1L DKO mice were labeled for the acrosome marker sp56 (green) (H) and the mitochondrial marker Tomm20 (red) (I); nuclei (blue) were labeled by DAPI. Scale bars: 5 μm. (J) The percentage of globozoospermia-like sperm in WT, ICA69-KO, ICA1L-KO and ICA69/ICA1L DKO mice. WT, n=8; ICA69-KO, n=11; ICA1L-KO, n=4; ICA69/ICA1L DKO, n=3, error bar represents s.e.m. nu, nucleus; ms, mitochondrial sheath. *P<0.05; **P<0.01; ***P<0.001; #, not significant (Student’s t-test).

When carefully examining the morphology of the sperm from the cauda epididymis, we observed that a large number of sperm from ICA1L-KO and ICA69/ICA1L DKO mice did not have the typical hook-like morphology, but had abnormal heads resembling irregularly shaped balls (Fig. 8G). Next, we examined the sperm from the cauda epididymis by labeling nuclei with DAPI and the acrosomes with sp56 (also known as ZP3R). The sperm from WT and ICA69-KO mice have typical crescent moon shape acrosomes marked by sp56 whereas the acrosomes of some sperm in ICA1L-KO mice have defects including fragmentation, mislocalization and deformation (Fig. 8H). The acrosomes in ICA69/ICA1L DKO mice were either fragmented or completely lost (Fig. 8H). Smaller and irregular nuclei could also be observed in ICA1L-KO and ICA69/ICA1L DKO mice (Fig. 8H). Immunostaining with Tomm20, which labels mitochondria, revealed that the mitochondrial sheaths, which are responsible for sperm movement, had a variety of defects in the sperm of ICA1L-KO and ICA69/ICA1L DKO mice, including wrapping around the deformed nucleus, aggregating near the deformed nucleus and splitting into separate aggregates (Fig. 8I). Quantification of the numbers of sperm from the cauda epididymis of WT and ICA69-KO mice showed that the percentages of abnormal sperm with defects in the nucleus, acrosome and mitochondrial sheath were only 4% and 9% in WT and ICA69-KO mice, respectively (Fig. 8J). However, for ICA1L-KO and ICA69/ICA1L DKO mice, the percentages of abnormal sperm with defects in the nucleus, acrosome and mitochondrial sheath were ∼51% and ∼95% in ICA1L-KO and ICA69/ICA1L DKO mice, respectively (Fig. 8J). These abnormalities are reminiscent of the defects seen in globozoospermia, a human infertility disorder characterized by round-headed sperm with deformed nuclei, abnormal acrosomes and malformed mitochondrial sheaths. Taken together, our results demonstrate that deficiency of ICA1L leads to reduced male fertility in a mouse model with characteristics of type II globozoospermia, whereas deficiency of both ICA1L and ICA69 lead to complete infertility in male mice.

In this study, we identified ICA1L as a new binding partner of PICK1. ICA1L is homologous to ICA69, another BAR-domain-containing protein that we have previously reported as the major binding partner for PICK1 in brain and pancreas (Cao et al., 2007,, 2013). Our previous results suggested that ICA69 associates with 76% of PICK1 in brain by forming a tight BAR-domain complex. Around 73% of PICK1 is lost in the brains of ICA69-KO mice. In this study, we found that ICA1L also forms BAR domain complexes with PICK1. ICA1L interacts with PICK1 in brain and ICA1L disrupts PICK1-mediated GluA2 clustering and competes with PICK1 for homodimer formation, similar to ICA69. In addition, the expression levels of ICA1L is similar to that of ICA69 and PICK1 in different brain regions and ICA1L is increased in the brains of ICA69-KO mice. Taken together, ICA1L has a compensatory role for ICA69 in the brain.

Despite the similarity, the tissue distribution of ICA1L is quite different from that of ICA69. Expression of ICA69 is high in brain where ICA1L is low. Expression of ICA69 is low in testes where ICA1L is high. In addition, ICA1L is completely absent in pancreas, where ICA69 expression is high. ICA1L accounts for binding to 80% of PICK1 whereas ICA69 accounts for only 11% in testes. PICK1 was reduced by 80% in the testes of ICA1L-KO mice. ICA69 and ICA1L are completely absent in PICK1-KO mice. We did not observe any difference in the presence of ICA69 and ICA1L mRNAs in the brain and testes of PICK1-KO mice. One possible explanation is that ICA69 and ICA1L form complexes with PICK1 and stabilize each other in the complexes. Loss of one protein would lead to the degradation of the other partner in the complexes.

In western blotting, although the ICA69 band of the expected full-length size was gone in ICA69-KO mice, a smaller band was detected by our ICA69 antibodies in the testes of ICA69-KO mice but not in the brains and pancreas. The antibodies we used were C-terminal antibodies. The smaller size of this additional band suggests that it could be a truncated form or abnormal spliced form of ICA69. The ICA69-KO mice used here were generated by deleting exon 2 of Ica1 gene (Winer et al., 2002). It is possible that abnormal splicing isoform could be generated in ICA69-KO mice by joining the exons before and after exon 2. Alternatively, a truncated form of ICA69 could be produced by using alternative start codon downstream of the original start codon. The fact that this additional band was only found in testes, but not in brain or pancreas suggests that this abnormal splicing or alternative start codon was only utilized in testes. This truncated protein, however, could not compensate for the loss of ICA69 in the double knockout mice, suggesting that it is not functional.

Although PICK1 was reduced by 80% in the testes of ICA1L-KO mice, male mice deficient in ICA1L were still fertile. The mice became sterile only after both ICA1L and ICA69 were knocked out. This suggests that there might be two independent pathways or types of vesicles that contribute to the biogenesis of acrosomes. The major one is mediated by ICA1L and the minor one depends on ICA69. Despite the minor role of ICA69 in testes, it is still able to form heteromeric complex with PICK1 and compensate for the loss of ICA1L to a certain degree in acrosome biogenesis. In the absence of both ICA69 and ICA1L, PICK1 could only form homodimers, which is not sufficient to support the normal vesicle trafficking needed for acrosome formation. As a result, acrosome formation was severely impaired in ICA69/ICA1L DKO mice.

Mice deficient in PICK1 are sterile and have a globozoospermia-like phenotype (Xiao et al., 2009). In addition, mutation of PICK1 causes globozoospermia in humans, emphasizing the importance of PICK1 in acrosomal trafficking (Liu et al., 2010). The mutation of PICK1 (G393R), which causes globozoospermia in human, is in the C-terminal acidic region of PICK1. PICK1 G393R does not affect the interaction with ICA1L (our unpublished data). The acidic region of PICK1 negatively regulates the lipid-binding ability of the BAR domain of PICK1, which is also important for protein trafficking (Jin et al., 2006). Hence, it is possible that the lipid-binding ability of the BAR domain is affected by the mutation of the acidic region, which would influence normal acrosome trafficking (Liu et al., 2010).

There are two different types of globozoospermia in human. Type I globozoospermia (total globozoospermia) is characterized by 100% acrosomeless sperm. Type II globozoospermia (partial globozoospermia) presents an increased proportion of round-headed sperm and acrosome malformations compared to normozoospermia (Dam et al., 2011). The sperm in ICA1L-KO mice are not completely acrosomeless. Some of the sperm still have some acrosomes and mitochondria in their heads, characteristics of type II globozoospermia, suggesting ICA1L could be involved in the pathogenesis of type II globozoospermia. It would be interesting to search for potential mutations of ICA1L in human patients with type II globozoospermia.

In some animal models of globozoospermia, such as mice deficient in GOPC, or ZPBP1 and ZPBP2, the sperm concentrations are normal (Yao et al., 2002; Lin et al., 2007). In other animal models, such as mice deficient in CK2α′, Hrb, GBA2, Vps54, Dpy19l2 or Atg7, sperm concentrations are reduced (Xu et al., 1999; Kang-Decker et al., 2001; Yildiz et al., 2006; Paiardi et al., 2011; Pierre et al., 2012; Wang et al., 2014). Interestingly, the sperm concentrations of total globozoospermia patients are normal, but the sperm concentrations of partial globozoospermia patients are reduced (Dam et al., 2011). Sperm numbers are reduced in ICA1L-KO and ICA69/ICA1L DKO mice as well as in PICK1-KO mice. PICK1, ICA69 and ICA1L are expressed in hypothalamus (Fig. 4H). This raises the possibility that the reduction in sperm concentration could be a consequence of impaired hypothalamic–pituitary–gonadal axis function. However, the defects in spermiogenesis are the primary effect of PICK1 deficiency in testes and are not caused by altered hypothalamic–pituitary–gonadal axis function because re-expression of PICK1 by lentiviral infection into the seminiferous tubules can rescue the abnormal spermiogenesis and male infertility of PICK1-KO mice (Li et al., 2013). The functions of ICA1L, ICA69 and PICK1 depend on each other, so it is unlikely that defective spermiogenesis is caused by impaired neuroendocrine function in ICA1L-KO or ICA69/ICA1L DKO mice.

Antibodies

ICA1L-specific antibody was generated against amino acid residues 266–411 of rat ICA1L. ICA69-specific antibody used in immunoprecipitation was generated against amino acid residues 275–459 of rat ICA69. A peptide antibody against ICA69 which could recognize both ICA69 and ICA1L was previously generated against amino acid residues 468–480 (IGKTDKEHELLNA) of rat ICA69 (Cao et al., 2007). The guinea pig anti-PICK1 antibody used in immunostaining was previously generated against the C-terminal 100 amino acid residues of mouse PICK1 (Cao et al., 2007). Rabbit anti-PICK1 antibody (PC100) used in immunoblotting was previously generated against the C-terminal 100 amino acid residues of mouse PICK1 (Cao et al., 2007). Rabbit anti-PICK1 antibody previously generated against the N-terminal 29 amino acid residues of mouse PICK1 was used in immunoprecipitation (Cao et al., 2007). Rabbit anti-GFP antibody was generated using GFP fusion protein as the antigen. Primary antibodies used include the following: mouse anti-PICK1 antibody (antigen from amino acid residues 10–130) was purchased from NeuroMab. Mouse anti-Myc antibody was purchased from the Developmental Studies Hybridoma Bank (9E10). Mouse anti-GAPDH antibody was purchased from Abcam. Mouse anti-GluA2 antibody was purchased from Millipore. Mouse anti-sp56 antibody was purchased from QED Bioscience. Rabbit anti-Tomm20 antibody was purchased from Santa Cruz Biotechnology.

cDNA cloning

Rat PICK1, Rat ICA69 and mouse ICA1L cDNA were subcloned into corresponding expression vectors in frames by restriction enzyme SalI/NotI. All constructs were verified by sequencing.

Preparation of tissue samples for western blotting

Tissues were dissected and homogenized using homogenate buffer [10 mM Tris-HCl and 320 mM sucrose supplemented with protease inhibitor cocktail tablet (Roche), pH 7.4] to obtain the total proteins. Homogenate was centrifuged at 700 g for 10 min at 4°C and the supernatant was retained. Protein concentration was determined by a Bradford protein assay (Pierce, Rockford, IL). Equal amounts of proteins were loaded to SDS-PAGE gels and analyzed by western blotting.

Genotyping PCR and RT-PCR

PICK1 mice were genotyped by PCR using the following three primers: 5′-TCACTTGCCAGAGGAGAAAACTG-3′, 5′-AAAAATAGGCGTATCACGAGGC-3′ and 5′-CACTCGCAGCTTGTTCTGATCTG-3′. The WT PCR product is a 400-bp band whereas the mutant band is a 200-bp band. The brains and testes from WT and PICK1-KO mice were used for RT-PCR analysis. Total RNAs were extracted with TRIzol reagent (Invitrogen) according to manufacturer's instructions, and cDNAs were synthesized by reverse transcription using a first-strand cDNA Synthesis kit (Fermentas). PCR was performed with Q5 High-Fidelity DNA Polymerase (New England Biolabs). The primers used for PCR amplification of ICA69 were: forward 5′-AAGGATGACCTCTTGCTGTTGAATG-3′ and reverse 5′-ATAGCGATAGAAACAGGGCCTTGAC-3′. The primers used for PCR amplification of ICA1L were: forward 5′-GCATCCGATGCAGAACTGGACGCTAAGTTGGA-3′ and reverse 5′-TCCATCATTTCGCCAGCTTGAGTCGAATCCCGCT-3′. The primers used for PCR amplification of PICK1 were: forward 5′-GTCACCCTACAGAAGGATGCCCAGAACCTGATTG-3′ and reverse 5′-GTCCGCCTGCAGCTTGTTGTAATGGATGGTC-3′. The primers used for PCR amplification of β-actin were: forward 5′-TGAGAGGGAAATCGTGCGTG-3′ and reverse 5′-TGCTTGCTGATCCACATCTGC-3′.

HEK293T cell culture and transfection

Human embryonic kidney 293T cells (HEK 293T) were cultured in a humidified atmosphere containing 5% CO2 in MEM (Gibco) supplemented with 10% fetal bovine serum, 1 mM sodium pyruvate, 100 U/ml penicillin, and 100 µg/ml streptomycin and passaged every 2–3 days when the cell confluence reached 80–90%. Calcium phosphate co-precipitation method was used for transient transfection and medium was changed 9 h after transfection.

Immunocytochemistry

Cells were fixed 48 h after transfection by 4% paraformaldehyde plus 4% sucrose in PBS for 20 min at room temperature. The cells were then permeabilized by 0.2% Triton X-100 in PBS for 10 min at room temperature. After blocking with 10% normal donkey serum (NDS) in PBS for 1 h, the cells were incubated with primary antibody in 3% NDS for 1 h at room temperature, followed by a 1-h incubation with fluorescent-dye-conjugated secondary antibodies (Jackson Immuno). After washing with PBS three times at 10-min interval, the coverslips were mounted with Permafluor (Immunon).

Immunohistochemistry

Testes were fixed by cardiac perfusion through the left ventricle with 4% paraformaldehyde and 4% sucrose in PBS followed by post fixation with 4% paraformaldehyde and 4% sucrose in PBS for 4 h at 4°C. Cryo-protection was performed by incubating testes in gradients of sucrose in PBS solution (10% sucrose, 20% sucrose and 30% sucrose in PBS) at 4°C. Cryosections of 5 µm thickness were used for immunohistochemistry. All animal procedures were approved by the Animal Ethic Committee of the Hong Kong University of Science and Technology.

CRISPR-Cas-mediated gene targeting

ICA1L-KO mice were generated as previously reported (Wang et al., 2013). Briefly, a DNA template for sgRNA was cloned into pX330 by BbsI (New England Biolabs). The sgRNA oligonucleotides used for generating sgRNA expression vector are: forward 5′-CACCGGGAAAGAAGGAGGACGAGCATG-3′ and reverse 5′-AAACCATGCTCGTCCTCCTTCTTTCCC-3′. Empty vector and the construct expressing the sgRNA were transfected into N2a cells respectively and RFLP analysis was used for the efficiency testing. Primers used for PCR amplification of fragment containing the targeting region are: forward 5′-AGCAGGCTGAGCAAGCCACTAAGCAGTACTTCCTATG-3′ and reverse 5′-TAGAATTATTTCTAGCCATAATTATAAAATCTTAACAAT-3′. The PCR products were digested with NspI (New England Biolabs) and separated on a GelRed-stained agarose gel (1%). Fragments carrying mutations could not be cut by NspI. After efficiency testing in vitro, T7 promoter was added to Cas9 template by PCR amplification using the following primers: forward 5′-TAATACGACTCACTATAGGGAGAATGGACTATAAGGACCACGAC-3′ and reverse 5′-GCGAGCTCTAGGAATTCTTAC-3′. The PCR product was gel purified and used as the template for in vitro transcription using the mMESSAGE mMACHINE T7 Ultra kit (Life Technologies). T7 promoter was added into sgRNA template by PCR amplification using the following primers: forward 5′-TTAATACGACTCACTATAGGGAAAGAAGGAGGACGAGCATG-3′ and reverse 5′-AAAAGCACCGACTCGGTGCC-3′. The PCR product was gel purified and used as the template for in vitro transcription using MEGAshortscript T7 kit (Life Technologies). Both the Cas9 mRNA and the sgRNA were purified using MEGAclear kit (Life Technologies). Cas9 mRNA (100 ng/μl) and sgRNA (20 ng/μl) were microinjected into the cytoplasm of B6CBAF2 zygotes in M2 medium (Millipore). Thereafter, 20 embryos were transferred into the oviducts of each pseudopregnant B6CBAF1 female. In total 60 embryos were transferred into three foster mothers.

Off-targeting analysis

Potential off-targets were predicted by searching the mouse genome (mm10) for matches to the 23-nucleotide sgRNA sequence containing up to three mismatches followed by the NGG PAM motif. Fragments containing the potential off-targeting sites were PCR amplified from genomic DNA of founders and cloned into pEGFP-C3 vector and sequenced. More than 12 clones were sequenced for each site. Primers used for cloning are listed in Table S3.

Epididymal sperm count and morphology classification

The number of epididymal sperm was determined as previously described (Cooper and Castilla, 2009). Briefly, the cauda epididymis was dissected and incised. Sperm were exuded into 5 ml sperm extract medium for 30 min at 37°C under 5% CO2 and counted by using a hemocytometer or spread on slides for morphological observation.

Histology

The cauda epididymis and testes were fixed by cardiac perfusion through the left ventricle with 4% paraformaldehyde and 4% sucrose in PBS followed by post fixation with 4% paraformaldehyde and 4% sucrose in PBS for overnight at 4°C. The cauda epididymis and testes were dehydrated through a graded ethanol series, and then embedded in paraffin. Sections (5 μm) were cut on a microtome (Shandon Finesse; Thermo Fisher Scientific). H&E staining was performed to stain the sections.

Quantitative co-immunoprecipitation

Testes from WT mice were homogenized using homogenate buffer [10 mM Tris-HCl and 320 mM sucrose supplemented with protease inhibitor cocktail tablet (Roche), pH 7.4]. Homogenates were centrifuged at 700 g for 10 min at 4°C, and the supernatant was kept. 2% Triton X-100 was added to the supernatant to solubilize proteins for 2 h at 4°C. The solution was centrifuged at the maximum speed (16,873 g) for 20 min. The supernatant was kept, and protein concentrations were determined by performing a Bradford protein assay (Pierce, Rockford, IL). The final concentration was adjusted to 1 mg/ml. To 0.5 ml of the testes samples, immunoprecipitation was performed by using protein A beads (GE Healthcare) previously incubated with antibodies. The testes homogenates before immunoprecipitation was designated as ‘input’. The testes homogenates after the first and second immunoprecipitation were designated as AIP1 and AIP2, respectively. The immunoprecipitation and co-immunoprecipitation efficiencies were calculated by determining the amount of proteins as a AIP:input ratio by densitometry analysis.

Confocal microscopy

Fixed cells or testes slices were imaged using inverted microscopes LSM510 or LSM710 Meta (Carl Zeiss, Inc.) with a 63×1.4 NA oil DIC Plan Apo objective. Images were acquired by using the software LSM or ZEN 2009. Images were processed by Adobe Photoshop to adjust intensity and contrast, to select a region of interest and to overlay images. All images were taken in grayscale and artificially colored for presentation.

Statistical analyses

Statistical analyses of group data were performed using unpaired, two-tailed Student's t-tests. Asterisks indicate a significant difference: *P<0.05, **P<0.01, ***P<0.001.

We thank Dr Chung Nga Tam (Transgenic Service, HKUST) for assistant in microinjection of Cas9 mRNA and sgRNA and Mr Ho-chun Lai for drawing the mouse cartoon. Special thanks are given to Miss Shui Wa Yun for assistance in taking care of mice and genotyping.

Author contributions

J.X. conceived, directed this project and wrote the manuscript. M.X. purified the ICA1L-specific antibody. W.H.T. performed the microinjection of Cas9 mRNA and sgRNA into one-cell embryos. K.L.C. revised this manuscript. J.H. conducted the remaining experiments and wrote the manuscript.

Funding

This work was supported in part by the Research Grants Council of the Hong Kong SAR, China [grant numbers 16102914, 663613, HKUST10/CRF/12R, C4011-14R, T13-607/12R and AoE/M-05/12].

Audouard
,
C.
and
Christians
,
E.
(
2011
).
Hsp90b1 knockout targeted to male germline: a mouse model for globozoospermia
.
Fertil. Steril.
95
,
1475
-
U1326.e4
.
Cao
,
M.
,
Xu
,
J.
,
Shen
,
C.
,
Kam
,
C.
,
Huganir
,
R. L.
and
Xia
,
J.
(
2007
).
PICK1-ICA69 heteromeric BAR domain complex regulates synaptic targeting and surface expression of AMPA receptors
.
J. Neurosci.
27
,
12945
-
12956
.
Cao
,
M.
,
Mao
,
Z.
,
Kam
,
C.
,
Xiao
,
N.
,
Cao
,
X.
,
Shen
,
C.
,
Cheng
,
K. K. Y.
,
Xu
,
A.
,
Lee
,
K.-M.
,
Jiang
,
L.
, et al. 
(
2013
).
PICK1 and ICA69 control insulin granule trafficking and their deficiencies lead to impaired glucose tolerance
.
PLoS Biol.
11
,
e1001541
.
Chung
,
H. J.
,
Xia
,
J.
,
Scannevin
,
R. H.
,
Zhang
,
X. Q.
and
Huganir
,
R. L.
(
2000
).
Phosphorylation of the AMPA receptor subunit GluR2 differentially regulates its interaction with PDZ domain-containing proteins
.
J. Neurosci.
20
,
7258
-
7267
.
Cooper
,
T. G.
and
Castilla
,
J. A.
(
2009
).
WHO laboratory manual for the examination and processing of human semen
.
J. Androl.
30
,
9
.
Coutton
,
C.
,
Zouari
,
R.
,
Abada
,
F.
,
Ben Khelifa
,
M.
,
Merdassi
,
G.
,
Triki
,
C.
,
Escalier
,
D.
,
Hesters
,
L.
,
Mitchell
,
V.
,
Levy
,
R.
, et al. 
(
2012
).
MLPA and sequence analysis of DPY19L2 reveals point mutations causing globozoospermia
.
Hum. Reprod.
27
,
2549
-
2558
.
Dam
,
A. H. D. M.
,
Koscinski
,
I.
,
Kremer
,
J. A. M.
,
Moutou
,
C.
,
Jaeger
,
A.-S.
,
Oudakker
,
A. R.
,
Tournaye
,
H.
,
Charlet
,
N.
,
Lagier-Tourenne
,
C.
,
van Bokhoven
,
H.
, et al. 
(
2007
).
Homozygous mutation in SPATA16 is associated with male infertility in human globozoospermia
.
Am. J. Hum. Genet.
81
,
813
-
820
.
Dam
,
A. H.
,
Ramos
,
L.
,
Dijkman
,
H. B.
,
Woestenenk
,
R.
,
Robben
,
H.
,
van den Hoven
,
L.
and
Kremer
,
J. A.
(
2011
).
Morphology of partial globozoospermia
.
J. Androl.
32
,
199
-
206
.
Dev
,
K. K.
,
Nishimune
,
A.
,
Henley
,
J. M.
and
Nakanishi
,
S.
(
1999
).
The protein kinase C alpha binding protein PICK1 interacts with short but not long form alternative splice variants of AMPA receptor subunits
.
Neuropharmacology
38
,
635
-
644
.
Dunson
,
D. B.
,
Baird
,
D. D.
and
Colombo
,
B.
(
2004
).
Increased infertility with age in men and women
.
Obstet. Gynecol.
103
,
51
-
56
.
ElInati
,
E.
,
Kuentz
,
P.
,
Redin
,
C.
,
Jaber
,
S.
,
Vanden Meerschaut
,
F.
,
Makarian
,
J.
,
Koscinski
,
I.
,
Nasr-Esfahani
,
M. H.
,
Demirol
,
A.
,
Gurgan
,
T.
, et al. 
(
2012
).
Globozoospermia is mainly due to DPY19L2 deletion via non-allelic homologous recombination involving two recombination hotspots
.
Hum. Mol. Genet.
21
,
3695
-
3702
.
Ferlin
,
A.
,
Arredi
,
B.
and
Foresta
,
C.
(
2006
).
Genetic causes of male infertility
.
Reprod. Toxicol.
22
,
133
-
141
.
Fujihara
,
Y.
,
Satouh
,
Y.
,
Inoue
,
N.
,
Isotani
,
A.
,
Ikawa
,
M.
and
Okabe
,
M.
(
2012
).
SPACA1-deficient male mice are infertile with abnormally shaped sperm heads reminiscent of globozoospermia
.
Development
139
,
3583
-
3589
.
Harbuz
,
R.
,
Zouari
,
R.
,
Pierre
,
V.
,
Ben Khelifa
,
M.
,
Kharouf
,
M.
,
Coutton
,
C.
,
Merdassi
,
G.
,
Abada
,
F.
,
Escoffier
,
J.
,
Nikas
,
Y.
, et al. 
(
2011
).
A recurrent deletion of DPY19L2 causes infertility in man by blocking sperm head elongation and acrosome formation
.
Am. J. Hum. Genet.
88
,
351
-
361
.
Holst
,
B.
,
Madsen
,
K. L.
,
Jansen
,
A. M.
,
Jin
,
C.
,
Rickhag
,
M.
,
Lund
,
V. K.
,
Jensen
,
M.
,
Bhatia
,
V.
,
Sorensen
,
G.
,
Madsen
,
A. N.
, et al. 
(
2013
).
PICK1 deficiency impairs secretory vesicle biogenesis and leads to growth retardation and decreased glucose tolerance
.
PLoS Biol.
11
,
e1001542
.
Ikawa
,
M.
,
Inoue
,
N.
,
Benham
,
A. M.
and
Okabe
,
M.
(
2010
).
Fertilization: a sperm's journey to and interaction with the oocyte
.
J. Clin. Invest.
120
,
984
-
994
.
Jarow
,
J. P.
,
Espeland
,
M. A.
and
Lipshultz
,
L. I.
(
1989
).
Evaluation of the azoospermic patient
.
J. Urol.
142
,
62
-
65
.
Jarow
,
J. P.
,
Sharlip
,
I. D.
,
Belker
,
A. M.
,
Lipshultz
,
L. I.
,
Sigman
,
M.
,
Thomas
,
A. J.
,
Schlegel
,
P. N.
,
Howards
,
S. S.
,
Nehra
,
A.
,
Damewood
,
M. D.
, et al. 
(
2002
).
Best practice policies for male infertility
.
J. Urol.
167
,
2138
-
2144
.
Jin
,
W.
,
Ge
,
W.-P.
,
Xu
,
J.
,
Cao
,
M.
,
Peng
,
L.
,
Yung
,
W.
,
Liao
,
D.
,
Duan
,
S.
,
Zhang
,
M.
and
Xia
,
J.
(
2006
).
Lipid binding regulates synaptic targeting of PICK1, AMPA receptor trafficking, and synaptic plasticity
.
J. Neurosci.
26
,
2380
-
2390
.
Kang-Decker
,
N.
,
Mantchev
,
G. T.
,
Juneja
,
S. C.
,
McNiven
,
M. A.
and
van Deursen
,
J. M. A.
(
2001
).
Lack of acrosome formation in Hrb-deficient mice
.
Science
294
,
1531
-
1533
.
Koscinski
,
I.
,
ElInati
,
E.
,
Fossard
,
C.
,
Redin
,
C.
,
Muller
,
J.
,
de la Calle
,
J. V.
,
Schmitt
,
F.
,
Ben Khelifa
,
M.
,
Ray
,
P.
,
Kilani
,
Z.
, et al. 
(
2011
).
DPY19L2 deletion as a major cause of globozoospermia
.
Am. J. Hum. Genet.
88
,
344
-
350
.
Li
,
X. M.
,
Mao
,
Z.
,
Wu
,
M.
and
Xia
,
J.
(
2013
).
Rescuing infertility of Pick1 knockout mice by generating testis-specific transgenic mice via testicular infection
.
Sci. Rep.UK
3
,
2842
.
Lin
,
Y.-N.
,
Roy
,
A.
,
Yan
,
W.
,
Burns
,
K. H.
and
Matzuk
,
M. M.
(
2007
).
Loss of zona pellucida binding proteins in the acrosomal matrix disrupts acrosome biogenesis and sperm morphogenesis
.
Mol. Cell. Biol.
27
,
6794
-
6805
.
Liu
,
G.
,
Shi
,
Q.-W.
and
Lu
,
G.-X.
(
2010
).
A newly discovered mutation in PICK1 in a human with globozoospermia
.
Asian J. Androl.
12
,
556
-
560
.
Paiardi
,
C.
,
Pasini
,
M. E.
,
Gioria
,
M.
and
Berruti
,
G.
(
2011
).
Failure of acrosome formation and globozoospermia in the wobbler mouse, a Vps54 spontaneous recessive mutant
.
Spermatogenesis
1
,
52
-
62
.
Perez
,
J. L.
,
Khatri
,
L.
,
Chang
,
C.
,
Srivastava
,
S.
,
Osten
,
P.
and
Ziff
,
E. B.
(
2001
).
PICK1 targets activated protein kinase C alpha to AMPA receptor clusters in spines of hippocampal neurons and reduces surface levels of the AMPA-type glutamate receptor subunit 2
.
J. Neurosci.
21
,
5417
-
5428
.
Pierre
,
V.
,
Martinez
,
G.
,
Coutton
,
C.
,
Delaroche
,
J.
,
Yassine
,
S.
,
Novella
,
C.
,
Pernet-Gallay
,
K.
,
Hennebicq
,
S.
,
Ray
,
P. F.
and
Arnoult
,
C.
(
2012
).
Absence of Dpy19l2, a new inner nuclear membrane protein, causes globozoospermia in mice by preventing the anchoring of the acrosome to the nucleus
.
Development
139
,
2955
-
2965
.
Pietropaolo
,
M.
,
Castaño
,
L.
,
Babu
,
S.
,
Buelow
,
R.
,
Kuo
,
Y. L.
,
Martin
,
S.
,
Martin
,
A.
,
Powers
,
A. C.
,
Prochazka
,
M.
,
Naggert
,
J.
, et al. 
(
1993
).
Islet-cell autoantigen 69-Kd (Ica69) - molecular cloning and characterization of a novel diabetes-associated autoantigen
.
J. Clin. Invest.
92
,
359
-
371
.
Ran
,
F. A.
,
Hsu
,
P. D.
,
Lin
,
C.-Y.
,
Gootenberg
,
J. S.
,
Konermann
,
S.
,
Trevino
,
A. E.
,
Scott
,
D. A.
,
Inoue
,
A.
,
Matoba
,
S.
,
Zhang
,
Y.
, et al. 
(
2013
).
Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity
.
Cell
154
,
1380
-
1389
.
Sen
,
C. G. S.
,
Holstein
,
A. F.
and
Schirren
,
C.
(
1971
).
über die Morphogenese rundköpfiger Spermatozoen des Menschen
.
Andrologia
3
,
117
-
125
.
Steinberg
,
J. P.
,
Takamiya
,
K.
,
Shen
,
Y.
,
Xia
,
J.
,
Rubio
,
M. E.
,
Yu
,
S.
,
Jin
,
W.
,
Thomas
,
G. M.
,
Linden
,
D. J.
and
Huganir
,
R. L.
(
2006
).
Targeted in vivo mutations of the AMPA receptor subunit GluR2 and its interacting protein PICK1 eliminate cerebellar long-term depression
.
Neuron
49
,
845
-
860
.
Terashima
,
A.
,
Pelkey
,
K. A.
,
Rah
,
J.-C.
,
Suh
,
Y. H.
,
Roche
,
K. W.
,
Collingridge
,
G. L.
,
McBain
,
C. J.
and
Isaac
,
J. T. R.
(
2008
).
An essential role for PICK1 in NMDA receptor-dependent bidirectional synaptic plasticity
.
Neuron
57
,
872
-
882
.
Turek
,
P. J.
(
2005
).
Practical approaches to the diagnosis and management of male infertility
.
Nat. Clin. Pract. Urol.
2
,
226
-
238
.
Wang
,
H.
,
Yang
,
H.
,
Shivalila
,
C. S.
,
Dawlaty
,
M. M.
,
Cheng
,
A. W.
,
Zhang
,
F.
and
Jaenisch
,
R.
(
2013
).
One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering
.
Cell
153
,
910
-
918
.
Wang
,
H.
,
Wan
,
H.
,
Li
,
X.
,
Liu
,
W.
,
Chen
,
Q.
,
Wang
,
Y.
,
Yang
,
L.
,
Tang
,
H.
,
Zhang
,
X.
,
Duan
,
E.
, et al. 
(
2014
).
Atg7 is required for acrosome biogenesis during spermatogenesis in mice
.
Cell Res.
24
,
852
-
869
.
Winer
,
S.
,
Astsaturov
,
I.
,
Gaedigk
,
R.
,
Hammond-McKibben
,
D.
,
Pilon
,
M.
,
Song
,
A.
,
Kubiak
,
V.
,
Karges
,
W.
,
Arpaia
,
E.
,
McKerlie
,
C.
, et al. 
(
2002
).
ICA69(null) nonobese diabetic mice develop diabetes, but resist disease acceleration by cyclophosphamide
.
J. Immunol.
168
,
475
-
482
.
Xia
,
J.
,
Zhang
,
X.
,
Staudinger
,
J.
and
Huganir
,
R. L.
(
1999
).
Clustering of AMPA receptors by the synaptic PDZ domain-containing protein PICK1
.
Neuron
22
,
179
-
187
.
Xia
,
J.
,
Chung
,
H. J.
,
Wihler
,
C.
,
Huganir
,
R. L.
and
Linden
,
D. J.
(
2000
).
Cerebellar long-term depression requires PKC-regulated interactions between GluR2/3 and PDZ domain-containing proteins
.
Neuron
28
,
499
-
510
.
Xiao
,
N.
,
Kam
,
C.
,
Shen
,
C.
,
Jin
,
W.
,
Wang
,
J.
,
Lee
,
K. M.
,
Jiang
,
L.
and
Xia
,
J.
(
2009
).
PICK1 deficiency causes male infertility in mice by disrupting acrosome formation
.
J. Clin. Invest.
119
,
802
-
812
.
Xu
,
X.
,
Toselli
,
P. A.
,
Russell
,
L. D.
and
Seldin
,
D. C.
(
1999
).
Globozoospermia in mice lacking the casein kinase II alpha′ catalytic subunit
.
Nat. Genet.
23
,
118
-
121
.
Xu
,
J.
,
Kam
,
C.
,
Luo
,
J.-H.
and
Xia
,
J.
(
2014
).
PICK1 mediates synaptic recruitment of AMPA receptors at neurexin-induced postsynaptic sites
.
J. Neurosci.
34
,
15415
-
15424
.
Yang
,
H.
,
Wang
,
H.
,
Shivalila
,
C. S.
,
Cheng
,
A. W.
,
Shi
,
L.
and
Jaenisch
,
R.
(
2013
).
One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering
.
Cell
154
,
1370
-
1379
.
Yao
,
R.
,
Ito
,
C.
,
Natsume
,
Y.
,
Sugitani
,
Y.
,
Yamanaka
,
H.
,
Kuretake
,
S.
,
Yanagida
,
K.
,
Sato
,
A.
,
Toshimori
,
K.
and
Noda
,
T.
(
2002
).
Lack of acrosome formation in mice lacking a Golgi protein, GOPC
.
Proc. Natl. Acad. Sci. USA
99
,
11211
-
11216
.
Yildiz
,
Y.
,
Matern
,
H.
,
Thompson
,
B.
,
Allegood
,
J. C.
,
Warren
,
R. L.
,
Ramirez
,
D. M. O.
,
Hammer
,
R. E.
,
Hamra
,
F. K.
,
Matern
,
S.
and
Russell
,
D. W.
(
2006
).
Mutation of beta-glucosidase 2 causes glycolipid storage disease and impaired male fertility
.
J. Clin. Invest.
116
,
2985
-
2994
.
Zhu
,
F.
,
Gong
,
F.
,
Lin
,
G.
and
Lu
,
G.
(
2013
).
DPY19L2 gene mutations are a major cause of globozoospermia: identification of three novel point mutations
.
Mol. Hum. Reprod.
19
,
395
-
404
.

Competing interests

The authors declare no competing or financial interests.

Supplementary information