Vad1.3 is an evolutionarily-conserved, testis-specific gene identified from a retinol-treated Vitamin A-deficiency (VAD) rat model. VAD1.3 is expressed throughout spermiogenesis at the acrosome of spermatids and epididymal spermatozoa, suggesting a role in acrosome biogenesis or acrosome reaction. The present study aimed to explore the functional role of VAD1.3 in spermatogenesis and sperm functions by the cellular and gene-knockout approaches.
Double immunofluorescent microscopy confirmed the co-localization of VAD1.3 and syntaxin 1 in mouse spermatids and spermatozoa. Deletion analysis of the Vad1.3 gene in transfected mouse spermatocyte GC2-spd and human cervical cancer HeLa cells revealed a polarized peri-nuclear/Golgi expression pattern for the N-terminal GFP-VAD fusion proteins which contain a bipartite nucleus localization (BNL) motif, but a nuclear expression pattern for the C-terminal GFP-VAD. The N-terminal sequences of VAD1.3 mediated its interaction with syntaxin 1, as demonstrated by both co-localization and co-immunoprecipitation studies. The full-length GFP-VAD co-localized with the Golgi markers and was redistributed into the endoplasmic reticulum after brefeldin A treatment, suggesting that VAD1.3 was recruited through the ER-Golgi-acrosome pathway.
Vad1.3+/- mice was previously generated by the conventional knockout approach. The heterozygous mice had normal spermatogenesis during postnatal days and adulthood (6-8 weeks). At the age of 8-19 months, 6 out of 17 heterozygous mice but no wild-type exhibited a decrease in the epididymal sperm count and testicular weight (p < 0.05). Histological analyses unveiled disarrangement of the seminiferous epithelium and sloughing of germ cells, predominantly spermatids, which was mediated partially by apoptosis as a higher percentage of TUNEL-positive cells were detected in these heterozygous mice (p < 0.05). This phenotype was associated with a decrease in the mRNA (p < 0.05) and protein levels of VAD1.3 in the testis.
Crossing of the Vad1.3+/- mice produced wild-type and heterozygous offspring in a ratio of 1:3, but no Vad1.3-/- mice were found. There was no significant difference between the heterozygous intercrosses and the wild-type intercrosses in the number of oocytes ovulated, the developmental rate of embryos from zygotes to blastocysts, the number of implantation site, resorption site or the offspring could result from defective fertilization between Vad1.3 null gametes rather than developmental lethality. The role of VAD1.3 in fertilization was supported by the inhibitory effects of the anti-VAD1.3 antibody on in vitro fertilization and progesterone-induced acrosome reaction. Immuno-staining revealed that VAD1.3 was present in the acrosome-intact spermatozoa but not in acrosome-reacted spermatozoa, indicating a role of VAD1.3 in ZP-binding or acrosome reaction rather than sperm-egg fusion. In oocytes VAD1.3 was distributed in the cytoplasm near the cortex. litter size. Only a few Vad1.3-/- embryos were found at the zygotic (3.7%) and 2-cell (3%) stages in the heterozygous intercrosses. These findings suggested that the absence of the Vad1.3-/-
In sum, VAD1.3 may play important roles in fertilization and spermatogenesis in mice. The BNL motif of VAD1.3 directs its Golgi expression and the N-terminal sequence of the protein mediates its interaction with syntaxin 1. The use of tissue-specific knockout approach may help to answer the functional role of VAD1.3 in future. / published_or_final_version / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy
| Identifer | oai:union.ndltd.org:HKU/oai:hub.hku.hk:10722/179979 |
| Date | January 2012 |
| Creators | Gao, Jing, 高晶 |
| Contributors | Lee, CKF, Yeung, WSB |
| Publisher | The University of Hong Kong (Pokfulam, Hong Kong) |
| Source Sets | Hong Kong University Theses |
| Language | English |
| Detected Language | English |
| Type | PG_Thesis |
| Source | http://hub.hku.hk/bib/B4852170X |
| Rights | The author retains all proprietary rights, (such as patent rights) and the right to use in future works., Creative Commons: Attribution 3.0 Hong Kong License |
| Relation | HKU Theses Online (HKUTO) |
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