Calsperin is a testis-specific chaperone required for sperm fertility

Abstract

Calnexin (CANX) and calreticulin (CALR) are homologous lectin chaperones located in the endoplasmic reticulum and cooperate to mediate nascent glycoprotein folding. In the testis, calmegin (CLGN) and calsperin (CALR3) are expressed as germ cell-specific counterparts of CANX and CALR, respectively. Here, we show that Calr3(-/-) males produced apparently normal sperm but were infertile because of defective sperm migration from the uterus into the oviduct and defective binding to the zona pellucida. Whereas CLGN was required for ADAM1A/ADAM2 dimerization and subsequent maturation of ADAM3, a sperm membrane protein required for fertilization, we show that CALR3 is a lectin-deficient chaperone directly required for ADAM3 maturation. Our results establish the client specificity of CALR3 and demonstrate that the germ cell-specific CALR-like endoplasmic reticulum chaperones have contrasting functions in the development of male fertility. The identification and understanding of the maturation mechanisms of key sperm proteins will pave the way toward novel approaches for both contraception and treatment of unexplained male infertility.

FIGURE 1.

Haploid-specific expression of CALR3 and chaperone activity. A, CALR3 was detected only in the testis when various tissues were examined by Western blot analysis. B, testis lysates collected from mice of different ages were examined by Western blotting. CALR3 appeared as a faint signal at 3 weeks (wks) of age, whereas CLGN was detected at 2 weeks of age. It should be noted that ADAM1 is composed of two independent proteins, ADAM1A and ADAM1B (43). C, immunofluorescence staining of a testicular section with anti-CLGN (shown in red) and anti-CALR3 (shown in green) antibodies. Sequential sections were stained with hematoxylin and eosin (upper left panel). CALR3 was detected in elongating spermatids, whereas CLGN was detected in pachytene spermatocytes to elongating spermatids. D, co-localization of CLGN and CALR3 was observed in elongating spermatids. Green, CALR3; red, CLGN; blue, Hoechst 33258. Scale bars = 200 μm (C) and 4 μm (D).

FIGURE 2.

Impaired sperm migration from the uterus into the oviduct in Calr3^−/− mice. A, pregnancy rate (pregnancy/vaginal plug formation) presented as a percentage. Calr3^−/− males copulated normally but failed to induce pregnancy. Four males were examined in each group. The total number of plugs observed is indicated in parentheses. B–D, sperm migration assay. The oviduct with the uterus was removed 2 h after coitus, and frozen sections containing the uterotubal junction (red dashed line in B) were prepared. Although wild-type sperm were observed in the uterine part and the uterotubal junction, Calr3^−/− sperm were observed only in the uterine part and not in the uterotubal junction (C and D). The boxes in C are magnified in D. Arrows indicate sperm. The sections were stained with hematoxylin and eosin.

FIGURE 3.

Impaired sperm binding to the zona pellucida in Calr3^−/− mice. A, wild-type sperm successfully bound to the egg zona pellucida, but Calr3^−/− sperm failed to adhere despite frequent collisions (supplemental Movies S1 and S2). B, Calr3^−/− sperm were not able to fertilize eggs in vitro. The average fertilization rate from three independent experiments is presented. The total number of eggs examined is indicated in parentheses. IVF, in vitro fertilization. C and D, zona–free eggs preloaded with Hoechst 33342 were incubated with +/+ or Calr3^−/− sperm, and fused sperm were observed under a fluorescence microscope. Arrows indicate MII chromosomes, and arrowheads indicate fused sperm (C). The average fusion rate (fused eggs/examined eggs) from three independent experiments is presented in D. The number of eggs examined is indicated in parentheses. E–G, successful fertilization was observed with Calr3^−/− sperm after partial zona dissection and in vitro fertilization. Arrowheads indicate the slit made by partial zona dissection (E). The offspring of Calr3^−/− males obtained after partial zona dissection, in vitro fertilization, and embryo transfer are shown in F. The average birth rate (pups/transplanted eggs) after transferring embryos subjected to partial zona dissection and in vitro fertilization (n = 3) is shown in G.

FIGURE 4.

Interactions between ADAM proteins and ER chaperones. A, Western blot analysis of testis lysates separated by SDS-PAGE under reducing conditions (upper four panels) and nonreducing conditions (lower panel). Comparable amounts of ADAM1B, ADAM2, and ADAM3 were detected in +/+, Clgn^−/−, and Calr3^−/− testes under reducing conditions. When ADAM1A/ADAM2 and ADAM1B/ADAM2 heterodimers were probed by anti-ADAM2 antibody under nonreducing conditions (22–24), ADAM1/ADAM2 heterodimerization was absent in Clgn^−/− testis but was not impaired in Calr3^−/− testis. B, immunoprecipitates (IP) with anti-CANX, anti-CALR, anti-CLGN, and anti-CALR3 antibodies from testis lysates (100 μg) were probed with the indicated antibodies. Testis lysates (10 μg) were loaded as the input control. Whereas CLGN associated with ADAM1B, ADAM2, and ADAM3, CALR3 associated with only ADAM3. C, testis lysates digested with endoglycosidase H (EndoH) were immunoprecipitated with the indicated antibodies and then probed with anti-ADAM3 antibody. Endoglycosidase H treatment changed the molecular mass of ADAM3 but did not affect the association with CLGN and CALR3. Arrowheads indicate ADAM3. Asterisks indicate the nonspecific signal. D, testis lysates from Clgn^−/− and Calr3^−/− mice were immunoprecipitated with anti-CALR3 and anti-CLGN antibodies and then probed with anti-ADAM3 antibody.

FIGURE 5.

Trypsin sensitivity of ADAM2 and ADAM3 during spermatogenesis in +/+, Clgn^−/−, and Calr3^−/− mice. A, testis lysates (5 μg) were incubated with tosylphenylalanyl chloromethyl ketone-treated trypsin and then subjected to Western blot analysis with the indicated antibodies. Asterisks indicate the IgG that reacted with the secondary antibody. B, live germ cells collected from testes were trypsinized and then analyzed by Western blotting. The lysate prepared from 5 × 10⁵ cells was loaded in each lane. ADAM2 was not digested in the Clgn^−/− germ cells, whereas ADAM3 was not digested in the Calr3^−/− germ cells.

FIGURE 6.

Disappearance of ADAM3 from mature Calr3^−/− sperm. A, Western blot analysis of sperm lysates. ADAM3 was not detected in Calr3^−/− sperm, whereas other sperm fertilizing proteins remained. ACE, angiotensin-converting enzyme. B, immunofluorescence staining of sperm with antibodies against ADAM1B, ADAM2, and ADAM3. Fresh sperm collected from the epididymis of mutant mice were fixed in 4% paraformaldehyde and then immunostained with the following antibodies: KS107–57 (rat anti-mouse ADAM1B), KS107–190 (rat anti-mouse ADAM2), and rabbit anti-mouse ADAM3 (25). All ADAM proteins were detected in the head region of control +/+ sperm. Whereas Clgn^−/− sperm lack all ADAM proteins, Calr3^−/− sperm lack only ADAM3.