Protein Disulfide Isomerase Homolog PDILT Is Required for Quality Control

Correction DEVELOPMENTAL BIOLOGY Correction for “Protein disulfide isomerase homolog PDILT is required for quality control of sperm membrane protein ADAM3 and male infertility,” by Keizo Tokuhiro, Masahito Ikawa, Adam M. Benham, and Masaru Okabe, which appeared in issue 10, March 6, 2012, of Proc Natl Acad Sci USA (109: 3850–3855; first published February 22, 2012; 10.1073/pnas. 1117963109). The authors note that the title appeared incorrectly. The title should instead appear as “Protein disulfide isomerase homolog PDILT is required for quality control of sperm membrane protein ADAM3 and male fertility.” The online version has been corrected. www.pnas.org/cgi/doi/10.1073/pnas.1204275109 CORRECTION www.pnas.org PNAS | April 10, 2012 | vol. 109 | no. 15 | 5905 Downloaded by guest on September 24, 2021 Protein disulfide isomerase homolog PDILT is required for quality control of sperm membrane protein ADAM3 and male fertility Keizo Tokuhiroa, Masahito Ikawaa,1, Adam M. Benhama,b, and Masaru Okabea,c,d aResearch Institute for Microbial Diseases, cGraduate School of Pharmaceutical Sciences, and dImmunology Frontier Research Center, Osaka University, Suita, Osaka 565-0871, Japan; and bSchool of Biological and Biomedical Sciences, Durham University, Durham DH1 3LE, United Kingdom Edited by Ryuzo Yanagimachi, Institute for Biogenesis Research, University of Hawaii, Honolulu, HI, and approved January 31, 2012 (received for review November 1, 2011) A disintegrin and metalloproteinase 3 (ADAM3) is a sperm mem- misfolded proteins. Whereas homologous lectin chaperones, brane protein critical for both sperm migration from the uterus calnexin/calreticulin (CANX/CALR), chiefly mediate nascent into the oviduct and sperm primary binding to the zona pellucida glycoprotein folding in the somatic cells (14), testicular germ (ZP). Here we show that the testis-specific protein disulfide isom- cells contain additional homologs, called calmegin (CLGN) and erase homolog (PDILT) cooperates with the testis-specific calreti- calsperin (CALR3), respectively (15, 16). We have previously culin-like chaperone, calsperin (CALR3), in the endoplasmic demonstrated that the disruption of either Clgn or Calr3 genes reticulum and plays an indispensable role in the disulfide-bond resulted in male infertility concurrent with the absence of −/− formation and folding of ADAM3. Pdilt mice were male infertile ADAM3 from mature spermatozoa. Whereas CLGN mediates because ADAM3 could not be folded properly and transported to ADAM1A/ADAM2 heterodimerization that is required for the sperm surface without the PDILT/CALR3 complex. Peculiarly −/− −/− subsequent ADAM3 maturation (6, 17), CALR3 directly binds we find that not only Pdilt , but also Adam3 , spermatozoa to and regulates ADAM3 maturation (11). effectively fertilize eggs when the eggs are surrounded in cumulus Appropriate intra- and intermolecular disulfide bond formation oophorus. These findings reveal that ADAM3 requires testis-spe- also promotes protein folding in the ER. For disulfide bond for- cific private chaperones to be folded properly and that the princi- mation in the ER, more than 20 protein disulfide isomerase (PDI) ple role of ADAM3 is for sperm migration into the oviduct but not family proteins have been implicated in the process of oxidation, for the fertilization event. Moreover, the importance of primary reduction, and isomerization (18, 19). Among the family members, sperm ZP binding, which has been thought to be a critical step in PDIA3 (ERP57) is notable because it associates with CANX/CALR mammalian fertilization, should be reconsidered. and contributes to the quality control cycle of newly synthesized glycoproteins in the ER (20, 21). In addition to ubiquitous PDIs calmegin | cyritestin | gamete | uterotubal | disease including PDIA3, male germ cells also express a testis specificPDI- like protein, PDILT (22). Although PDILT does not have consensus n mammals, ejaculated spermatozoa are not able to fertilize eggs CXXC motifs and is not a classical oxidoreductase, it does interact Iimmediately, but acquire their fertilizing ability after spending with CLGN and with model proteins in vitro, suggesting that it may some time in the female reproductive tract. The phenomenon un- have a role in the maturation of spermatid proteins and conse- derlying this acquisition of fertilizing ability is called sperm capaci- quently, male infertility in vivo (23). tation, and the discovery made it possible to perform mammalian In the present study, we showed that PDILT interacts with in vitro fertilization (IVF) by mixing capacitated sperm with ovu- CALR3 in the testicular germ cells and plays an indispensable role lated eggs (1, 2). Although various sperm factors have been reported in the disulfide formation in the ADAM3. The PDILT knockout to be important using the IVF system and inhibitory assays, recent mice were male infertile, consistent with the previous observations gene-knockout studies have revealed that many of the factors are that spermatozoa lacking ADAM3 cannot migrate through the not essential for the fertilization process in vivo [including acrosin UTJ and cannot bind to the ZP. However, we found that the for zona pellucida (ZP) binding and penetration, hyaluronidase for spermatozoa lacking ADAM3 do fertilize eggs effectively in vivo cumulus oophorus penetration, zonadhesin for ZP binding, and when capacitated spermatozoa were directly deposited into the – fertilin for sperm egg fusion] (ref. 3 and refs. therein). oviduct. Our data demonstrate the importance of a testis-specific, In contrast, gene knockout studies unexpectedly discovered ER-quality control system for sperm fertilizing ability and call into the fertilization-related molecules (3). ADAM3, one of these question the importance of primary sperm ZP binding that has fi – essential factors, was rst reported as a sperm egg fusion protein been thought to be a critical step in mammalian fertilization. candidate but later was proven to be dispensable in fusion re- action (4, 5). However, Adam3 knockout male mice are infertile Results because the mutant spermatozoa cannot migrate through the PDILT Forms a Chaperone Complex and Interacts with ADAM3. We uterotubal junction (UTJ) in vivo and are unable to bind to ZP first examined the expression of PDILT in adult mouse organs and fi in vitro (6). It was also reported that ADAM3 has af nity to the in developing testes by Western blotting analysis (Fig. S1 A and B). ZP (7). There are at least seven lines of knockout mice showing PDILT was specifically detected in testis, consistent with previous similar male infertility phenotypes: [Ace (8), Adam1a (9), Adam2 (10), Adam3 (5), Calr3 (11), Clgn (12), and Tpst2 (13)]. ADAM3 was lost from the sperm or was defective in all these mutant Author contributions: K.T., M.I., and A.M.B. designed research; K.T. and M.I. performed mice. Because ADAM3 is involved in so many infertility phe- research; K.T., M.I., A.M.B., and M.O. analyzed data; and K.T., M.I., A.M.B., and M.O. notypes, elucidating its maturation and function is important in wrote the paper. understanding the process of mammalian fertilization. The authors declare no conflict of interest. ADAM3 is a cysteine-rich, glycosylated membrane protein that This article is a PNAS Direct Submission. is cotranslationally translocated into the endoplasmic reticulum 1To whom correspondence should be addressed. E-mail: [email protected]. (ER) of spermatids, where numerous molecular chaperones and This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. catalysts promote glycoprotein folding as well as the disposal of 1073/pnas.1117963109/-/DCSupplemental. 3850–3855 | PNAS | March 6, 2012 | vol. 109 | no. 10 www.pnas.org/cgi/doi/10.1073/pnas.1117963109 observations in rat and human (23, 24). In developing mouse tes- CALR3, PDI, and PDIA3 (Fig. S2E). When we looked at testic- tes, whereas PDIA3 (ERP57) was continuously detected through- ular sections by optical microscopy, disruption of PDILT caused out development, PDILT was not detectable until the mice be- no deleterious effect on spermatogenesis and sperm produced by came 3 wk old, indicating postmeiotic expression. As we have the mutants were morphologically normal (Fig. S2F). shown previously (11), CALR3 and ADAM3 were also expressed −/− from 3 wk old, whereas CLGN expression starts from 2 wk after Disappearance of ADAM3 from Pdilt Spermatozoa. The CLGN birth. When we immunostained testicular germ cells (Fig. S1C), protein is involved in the heterodimerization of ADAM1A/ CLGN was detected from spermatocytes to spermatids, whereas ADAM2 (only found in the ER) and ADAM1B/ADAM2 (fer- CALR3 and PDILT were only expressed in haploid spermatids. tilin, found in both ER and at the cell surface) (17, 26). Although These data confirmed that mouse PDILT is a male haploid germ- PDILT interacts with CLGN, normal ADAM1/2 hetero- − − cell–specific protein coexpressed with CLGN and CALR3. dimerization was observed in Pdilt / testis (Fig. 1C) and the We next examined the interaction of PDILT with ER chaper- ADAM1B/ADAM2 protein was successfully presented on ma- ones and ADAM proteins by coimmunoprecipitation analysis ture spermatozoa (Fig. 1 D and E). However, ADAM3 dis- − − (Fig. 1 A and B), because CLGN- and CALR3-deficient mice had appeared selectively from mature Pdilt / spermatozoa, whereas infertile phenotypes attributed to failures in ADAM1/ADAM2 various sperm-fertilizing proteins were normally presented, as − − heterodimerization and ADAM3 maturation, respectively (11). observed in Calr3 / mice (11). − − PDILT interacted with neither CANX nor CALR, but did bind to To trace the fate of ADAM3 in Pdilt / mice, we analyzed CLGN in wild-type mice, consistent with the data from rat (23). spermatozoa from testis and epididymis by Western blotting. We further showed that PDILT interacted with CALR3 and that ADAM3 is known to be processed from an uncleaved ∼90-kDa the signal was stronger than with CLGN (Fig. 1A). PDILT inter- form to a functional ∼30-kDa form in the epididymis (27). In − − acted with neither ADAM1B nor ADAM2, but did interact with Pdilt / mice, ADAM3 was expressed normally in testis, but was ADAM3 (Fig. 1B).

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