Abstract
As a major protein of the polyhedral coat of coated pits and vesicles, clathrin molecules have been shown to play a stabilization role for kinetochore fibers of the mitotic spindle by acting as inter-microtubule bridges. Clathrin heavy chain 1 (CLTC), the basic subunit of the clathrin coat, plays vital roles in both spindle assembly and chromosome congression during somatic-cell mitosis. However, its function in oocyte meiotic maturation and early embryo development in mammals, especially in domesticated animals, has not been fully investigated. In this study, the expression profiles and functional roles of CLTC in sheep oocytes were investigated. Our results showed that the expression of CLTC was maintained at a high level from the germinal vesicle (GV) stage to metaphase II stage and that CLTC was distributed diffusely in the cytoplasm of cells at interphase, from the GV stage to the blastocyst stage. After GV breakdown (GVBD), CLTC co-localized with beta-tubulin during metaphase. Oocyte treatments with taxol, nocodazole, or cold did not affect CLTC expression levels but led to disorders of its distribution. Functional impairment of CLTC by specific morpholino injections in GV-stage oocytes led to disruptions in spindle assembly and chromosomal alignment, accompanied by impaired first polar body (PB1) emissions. In addition, knockdown of CLTC before parthenogenetic activation disrupted spindle formation and impaired early embryo development. Taken together, the results demonstrate that CLTC plays a vital role in sheep oocyte maturation via the regulation of spindle dynamics and an essential role during early embryo development.
Highlights
Successful oocyte maturation and early embryo development depend on correct spindle formation and chromosome separation during cell division (Duan and Sun, 2019; Radonova et al, 2019)
Clathrin heavy chain 1 protein expression and subcellular localization during sheep oocyte meiotic maturation were examined by immunoblotting and immunofluorescence staining, respectively
Combined with the immunoblotting results that Clathrin heavy chain 1 (CLTC) was expressed in germinal vesicle (GV)-stage oocytes (Figure 1A), this suggests that CLTC was distributed evenly throughout the GV stage cytoplasm
Summary
Successful oocyte maturation and early embryo development depend on correct spindle formation and chromosome separation during cell division (Duan and Sun, 2019; Radonova et al, 2019). The oocyte meiotic spindle is mainly composed of microtubules (MTs), and spindle organization depends on the activities of numerous MT-associated proteins (Xu et al, 2012; Petry, 2016; Namgoong and Kim, 2018). Clathrin molecules stabilize kinetochore fibers of the mitotic spindle by acting as inter-MT bridges and promote chromosome congression in somatic cells (Booth et al, 2011; Cheeseman et al, 2011; Rao et al, 2016). Centrosome positioning in Caenorhabditis elegans embryos has been shown to be regulated by CLTC (Spiro et al, 2014)
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