Mammalian fertilization comprises sperm migration through the female reproductive tract, biochemical and morphological changes to sperm, and sperm-egg interaction in the oviduct. Recent gene knockout approaches in mice have revealed that many factors previously considered important for fertilization are largely dispensable, or if they are essential, they have an unexpected function. These results indicate that what has been observed in in vitro fertilization (IVF) differs significantly from what occurs during “physiological” fertilization. This Review focuses on the advantages of studying fertilization using gene-manipulated animals and highlights an emerging molecular mechanism of mammalian fertilization.

Proteasomes are proteolytic complexes involved in non‐lysosomal degradation which are localized in both the cytoplasm and the nucleus. The dynamics of proteasomes in living cells is unclear, as is their targeting to proteins destined for degradation. To investigate the intracellular distribution and mobility of proteasomes in vivo, we generated a fusion protein of the proteasome subunit LMP2 and the green fluorescent protein (GFP). The LMP2–GFP chimera was quantitatively incorporated into catalytically active proteasomes. The GFP‐tagged proteasomes were located within both the cytoplasm and the nucleus. Within these two compartments, proteasomes diffused rapidly, and bleaching experiments demonstrated that proteasomes were transported slowly and unidirectionally from the cytoplasm into the nucleus. During mitosis, when the nuclear envelope has disintegrated, proteasomes diffused rapidly throughout the dividing cell without encountering a selective barrier. Immediately after cell division, the restored nuclear envelope formed a new barrier for the diffusing proteasomes. Thus, proteasomes can be transported unidirectionally over the nuclear membrane, but can also enter the nucleus upon reassembly during cell division. Since proteasomes diffuse rapidly in the cytoplasm and nucleus, they may perform quality control by continuous collision with intracellular proteins, and degrading those proteins that are properly tagged or misfolded.

In the endoplasmic reticulum (ER), disulfide bonds are simultaneously formed in nascent proteins and removed from incorrectly folded or assembled molecules. In this compartment, the redox state must be, therefore, precisely regulated. Here we show that both human Ero1‐Lα and Ero1‐Lβ (hEROs) facilitate disulfide bond formation in immunoglobulin subunits by selectively oxidizing PDI. Disulfide bond formation is controlled by hEROs, which stand at a crucial point of an electron‐flow starting from nascent secretory proteins and passing through PDI. The redox state of ERp57, another ER‐resident oxidoreductase, is not affected by over‐expression of Ero1‐Lα, suggesting that parallel and specific pathways control oxidative protein folding in the ER. Mutants in the Ero1‐Lα CXXCXXC motif act as dominant negatives by limiting immunoglobulin oxidation. PDI‐dependent oxidative folding in living cells can thus be manipulated by using hERO variants.

Significance: Protein disulfide isomerase (PDI) and its homologs have essential roles in the oxidative folding and chaperone-mediated quality control of proteins in the secretory pathway. In this review, the importance of PDI in health and disease will be examined, using examples from the fields of lipid homeostasis, hemostasis, infectious disease, cancer, neurodegeneration, and infertility. Recent Advances: Recent structural studies, coupled with cell biological, biochemical, and clinical approaches, have demonstrated that PDI family proteins are involved in a wide range of physiological and disease processes. Critical Issues: Critical issues in the field include understanding how and why a PDI family member is involved in a given disease, and defining the physiological client specificity of the various PDI proteins when they are expressed in different tissues. Future Directions: Future directions are likely to include the development of new and more specific reagents to study and manipulate PDI family function. The development of conditional mouse models in concert with clinical data will help us to understand the in vivo function of the different PDIs at the organism level. Taken together with advances in structural biology and biochemical studies, this should help us to further understand and modify PDIs' functional interactions. Antioxid. Redox Signal. 16, 781–789.

A disintegrin and metalloproteinase 3 (ADAM3) is a sperm membrane protein critical for both sperm migration from the uterus into the oviduct and sperm primary binding to the zona pellucida (ZP). Here we show that the testis-specific protein disulfide isomerase homolog (PDILT) cooperates with the testis-specific calreticulin-like chaperone, calsperin (CALR3), in the endoplasmic reticulum and plays an indispensable role in the disulfide-bond formation and folding of ADAM3. Pdilt ^−/− mice were male infertile because ADAM3 could not be folded properly and transported to the sperm surface without the PDILT/CALR3 complex. Peculiarly we find that not only Pdilt ^−/− , but also Adam3 ^−/− , spermatozoa effectively fertilize eggs when the eggs are surrounded in cumulus oophorus. These findings reveal that ADAM3 requires testis-specific private chaperones to be folded properly and that the principle role of ADAM3 is for sperm migration into the oviduct but not for the fertilization event. Moreover, the importance of primary sperm ZP binding, which has been thought to be a critical step in mammalian fertilization, should be reconsidered.

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.