COP9 constitutive photomorphogenic homolog subunit 5 (COPS5), also known as Jab1 or CSN5, has been implicated in a wide variety of cellular and developmental processes. By analyzing male germ cell-specific COPS5 deficient mice, we demonstrated previously that COPS5 is essential to maintain male germ survival and acrosome biogenesis. To further determine the role of Cops5 in the peritubular myoid (PTM) cells, a smooth muscle lineage surrounding seminiferous tubules, we herein derived mice conditionally deficient for the Cops5 gene in smooth muscle cells (SMC) using transgenic Myh11-Cre mice. Although these conditional Cops5-deficient mice were born at the expected Mendelian ratio and appeared to be normal within the first week after birth, the homozygous mice started to show growth retardation after one week. These mice also exhibited a variety of developmental and reproductive disorders, including failure of development of reproductive organs in both males and females, spermatogenesis defects, impaired skeletal development, and immune functions. Furthermore, conditional Cops5-deficient mice revealed dramatic impairment of the endocrine system associated with testicular functions, including a marked reduction in serum levels of gonadotropins (FSH, LH), testosterone, insulin-like growth factor 1 (IGF1), and glucose, but not vasopressin. All homozygous mice died before 67 days old in the study. Collectively, our results provide novel evidence that Cops5 in smooth muscle lineage plays an essential role in postnatal development and reproductive functions.

The manchette is a transient and unique structure present in elongating spermatids and required for proper differentiation of the germ cells during spermatogenesis. Previous work indicated that the MEIG1/PACRG complex locates in the manchette and is involved in the transport of cargos, such as SPAG16L, to build the sperm flagellum. Here using co-immunoprecipitation and pull-down approaches in various cell systems, we established that DNALI1, an axonemal component originally cloned from Chlamydomonas reinhardtii, recruits and stabilizes PACRG and we confirm in vivo, the co-localization of DNALI1 and PACRG in the manchette by immunofluorescence of elongating murine spermatids. We next generated mice with a specific deficiency of DNALI1 in male germ cells, and observed a dramatic reduction of the sperm cells, which results in male infertility. In addition, we observed that the majority of the sperm cells exhibited abnormal morphology including misshapen heads, bent tails, enlarged midpiece, discontinuous accessory structure, emphasizing the importance of DNALI1 in sperm differentiation. Examination of testis histology confirmed impaired spermiogenesis in the mutant mice. Importantly, while testicular levels of MEIG1, PACRG and SPAG16L proteins were unchanged in the Dnali1 mutant mice, their localization within the manchette was greatly affected, indicating that DNALI1 is required for the formation of the MEIG1/PACRG complex within the manchette. Interestingly, in contrast to MEIG1 and PACRG-deficient mice, the DNALI1-deficient mice also showed impaired sperm spermiation/individualization, suggesting additional functions beyond its involvement in the manchette structure. Overall, our work identifies DNALI1 as a protein required for sperm development.

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