The lumicrine system is a postulated signaling system in which testis-derived (upstream) secreted factors enter the male reproductive tract to regulate epididymal (downstream) pathways required for sperm maturation. Until now, no lumicrine factors have been identified. We demonstrate that a testicular germ-cell–secreted epidermal growth factor–like protein, neural epidermal growth factor–like–like 2 (NELL2), specifically binds to an orphan receptor tyrosine kinase, c-ros oncogene 1 (ROS1), and mediates the differentiation of the initial segment (IS) of the caput epididymis. Male mice in which Nell2 had been knocked out were infertile. The IS-specific secreted proteases, ovochymase 2 (OVCH2) and A disintegrin and metallopeptidase 28 (ADAM28), were expressed upon IS maturation, and OVCH2 was required for processing of the sperm surface protein ADAM3, which is required for sperm fertilizing ability. This work identifies a lumicrine system essential for testis-epididymis-spermatozoa (NELL2-ROS1-OVCH2-ADAM3) signaling and male fertility.

More than 1000 genes are predicted to be predominantly expressed in mouse testis, yet many of them remain unstudied in terms of their roles in spermatogenesis and sperm function and their essentiality in male reproduction. Since individually indispensable factors can provide important implications for the diagnosis of genetically related idiopathic male infertility and may serve as candidate targets for the development of nonhormonal male contraceptives, our laboratories continuously analyze the functions of testis-enriched genes in vivo by generating knockout mouse lines using the CRISPR/Cas9 system. The dispensability of genes in male reproduction is easily determined by examining the fecundity of knockout males. During our large-scale screening of essential factors, we knocked out 30 genes that have a strong bias of expression in the testis and are mostly conserved in mammalian species including human. Fertility tests reveal that the mutant males exhibited normal fecundity, suggesting these genes are individually dispensable for male reproduction. Since such functionally redundant genes are of diminished biological and clinical significance, we believe that it is crucial to disseminate this list of genes, along with their phenotypic information, to the scientific community to avoid unnecessary expenditure of time and research funds and duplication of efforts by other laboratories.

Several hundred fertilized pig eggs were prepared by an in vitro fertilization (IVF) technique in which follicular phase ovarian eggs were matured in vitro to metaphase II before incubation with capacitated epididymal spermatozoa for 12 h at 39 degrees C. Parthenogenetic eggs were also prepared by stimulation of the mature eggs with an electric pulse. The zonae were solubilized with 0.2% pronase/phosphate-buffered saline (PBS) or lactic acid/PBS. The time taken for solubilization was 30-40% shorter than for unfertilized eggs, indicating that zona hardening was induced during fertilization. At the same time, the sperm receptor activity of the zona was reduced. Electrophoretic analyses of zona glycoproteins from the ovarian, mature and fertilized eggs revealed that the amount of 90 kDa proteins decreased substantially during fertilization. This fraction could barely be detected in the zonae from parthenogenetic eggs. However, modification with a fluorescent probe showed that the general architecture of the zona remained unchanged during fertilization. These results suggest that the minor 90 kDa proteins are specifically degraded by the protease(s) released from the oocyte at fertilization, thereby leading to the block to polyspermy.

Spermatozoa are produced in the testis but gain their fertilizing ability during epididymal migration. This necessary step in sperm maturation includes posttranslational modification of sperm membrane proteins that includes protein processing by proteases. However, the molecular mechanism underpinning this epididymal sperm maturation remains unknown. In this study, we focused on transmembrane serine protease 12 (Tmprss12). Based on multi-tissue expression analysis by PCR, Tmprss12 was specifically expressed in the testis, and its expression started on day 10 postpartum, corresponding to the stage of zygotene spermatocytes. TMPRSS12 was detected in the acrosomal region of spermatozoa by immunostaining. To reveal the physiological function of TMPRSS12, we generated two knockout (KO) mouse lines using the CRISPR/Cas9 system. Both indel and large deletion lines were male sterile showing that TMPRSS12 is essential for male fertility. Although KO males exhibited normal spermatogenesis and sperm morphology, ejaculated spermatozoa failed to migrate from the uterus to the oviduct. Further analysis revealed that a disintegrin and metalloprotease 3 (ADAM3), an essential protein on the sperm membrane surface that is required for sperm migration, was disrupted in KO spermatozoa. Moreover, we found that KO spermatozoa showed reduced sperm motility via computer-assisted sperm analysis, resulting in a low fertilization rate in vitro. Taken together, these data indicate that TMPRSS12 has dual functions in regulating sperm motility and ADAM3-related sperm migration to the oviduct. Because Tmprss12 is conserved among mammals, including humans, our results may explain some genetic cases of idiopathic male infertility, and TMPRSS12 and its downstream cascade may be novel targets for contraception.

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