Gene targeting to study a novel testis-specific gene Vad1.2 in spermatogenesis

Abstract

Spermatogenesis is regulated by steroid hormones which induce expression of various genes responsible for the growth, proliferation and differentiation of spermatogonia to form mature haploid spermatozoa. The surrounding somatic cells including Leydig and Sertoli cells support the whole process in vivo. Previously, we used the post-vitamin A treated vitamin-A-deficient (PVA-VAD) rat model to study spermatogenesis, and identified 24 genes that are differentially up-regulated after retinol treatment. Vad1.2 is one of the up-regulated transcripts expressed in the rat testis from postnatal day 25. Vad1.2 transcript is localized to the round and elongating spermatids in the adult mouse testis. In silico analyses showed that Vad1.2 transcript is down-regulated in patients with teratozoospermia and non-obstructive azoospermia, suggesting that Vad1.2 may have important roles in spermatogenesis. However, how Vad1.2 affects spermatogenesis remains unclear. Therefore, the present study was designed to study the functional roles of Vad1.2 protein in mice using gene targeting approach and investigate the molecular changes in mice with Vad1.2 deficiency. Vad1.2 polyclonal antibody was raised against the full-length mouse Vad1.2 recombination protein and affinity purified. Vad1.2 protein was localized to the cytoplasm and flagellum of condensing spermatids, specifically to the fibrous sheath (FS) in cauda epididymal spermatozoa. Vad1.2 conditional knockout vector was constructed and used to generate Vad1.2 null mice. Vad1.2-/- male mice developed normally but were subfertile with reduced sperm count and motility. Vad1.2-/- male mice had smaller testis and higher incident of sloughing of immature germ cells into the seminiferous lumens when compared to the wild-type. Yet, the rates of germ cell proliferation and apoptosis were similar between the wild-type and the mutant testis. Interestingly approximately 50% of the mutant cauda epididymal spermatozoa showed deformed flagella and demonstrated structural defects typically associated with bending of flagellum at the principal piece or at the midpiece/principal piece junctions. The acrosome, nucleus and mitochondrial sheath of these spermatozoa appeared normal, while the flagellum displayed structural abnormalities including deformation of the two longitudinal columns of the FS and disruption of a portion of FS, suggesting that Vad1.2 might be involved in the biogenesis of FS in spermatogenesis. Furthermore, Vad1.2 interacted with Akap4 in vivo, and the two proteins were co-immunoprecipitated from the testis or cauda epididymal spermatozoa lysates. Akap4 and Vad1.2 were localized to the tail region of the testicular spermatids and cauda epididymal spermatozoa. The expression levels of pro- and mature Akap4 in Vad1.2-/- testes were markedly increased when compared with the wild-type mice. However, a significant decrease of Akap4 was found in the mutant cauda epididymal spermatozoa, suggesting that most of the mature Akap4 failed to incorporate into the FS. Taken together, Vad1.2 plays an important role in spermatogenesis and Vad1.2 deficiency leads to subfertility in mice with the deformed flagella in mature spermatozoa. Further studies on the regulation of FS formation may uncover the underlying molecular changes associated with Vad1.2 deficiency, and may provide fundamental information for treatment of infertile patients with FS defect in the spermatozoa.