Acentriolar microtubule organization centers and Ran‐mediated microtubule formation pathways are both required in porcine oocytes

Abstract

Mammalian oocytes lack centrioles but can generate bipolar spindles using several different mechanisms. For example, mouse oocytes have acentriolar microtubule organization centers (MTOCs) that contain many components of the centrosome, and which initiate microtubule polymerization. On the contrary, human oocytes lack MTOCs and the Ran‐mediated mechanisms may be responsible for spindle assembly. Complete knowledge of the different mechanisms of spindle assembly is lacking in various mammalian oocytes. In this study, we demonstrate that both MTOC‐ and Ran‐mediated microtubule nucleation are required for functional meiotic metaphase I spindle generation in porcine oocytes. Acentriolar MTOC components, including Cep192 and pericentrin, were absent in the germinal vesicle and germinal vesicle breakdown stages. However, they start to colocalize to the spindle microtubules, but are absent in the meiotic spindle poles. Knockdown of Cep192 or inhibition of Polo‐like kinase 1 activity impaired the recruitment of Cep192 and pericentrin to the spindles, impaired microtubule assembly, and decreased the polar body extrusion rate. When the RanGTP gradient was perturbed by the expression of dominant negative or constitutively active Ran mutants, severe defects in microtubule nucleation and cytokinesis were observed, and the localization of MTOC materials in the spindles was abolished. These results demonstrate that the stepwise involvement of MTOC‐ and Ran‐mediated microtubule assembly is crucial for the formation of meiotic spindles in porcine oocytes, indicating the diversity of spindle formation mechanisms among mammalian oocytes.

Phase Separation during Germline Development

“…Centrioles degenerate during early oogenesis, and the pericentriolar material is lost in most species (except for rodents) [91]. Whereas rodent oocytes nucleate spindle microtubules using both acentriolar MTOCs (aMTOCs) and the RanGTP pathway [92][93][94], non-rodent oocytes seem to require the RanGTP pathway only [95,96]. Apart from nucleation factors, spindle assembly involves motor proteins as well as proteins that regulate the growth and stability of microtubules.…”

PAK1 Is Involved in the Spindle Assembly during the First Meiotic Division in Porcine Oocytes

“…In mouse oocytes, the nucleation of microtubules (MTs) relies on the function of acentrioloar microtubule-organizing centers (aMTOCs) [ 15 ], and the depletion of pericentrin (PCNT) from the germinal vesicle breakdown (GVBD) stage, an aMTOCs material, results in disorganized spindle [ 16 ]. However, in porcine oocytes, the aMTOCs are absent before meiosis I, and the nucleation of microtubules in the oocytes is initially mediated by chromatin and Ran GTP gradients [ 17 ]. It is unclear whether PAK1 is required for the assembly of microtubules in porcine oocytes, and its function during the meiotic maturation of porcine oocytes remain unknown.…”

Mechanism of spindle pole organization and instability in human oocytes

“…In addition to anchoring microtubules, aMTOCs promote microtubule nucleation (30,32,35). However, NUMA neither nucleates microtubules nor interacts with microtubule nucleators, in line with the crucial requirement of the Ran pathway for microtubule nucleation in nonrodent mammalian oocytes (2,36).…”

“…The mechanism of spindle pole organization in nonrodent mammalian oocytes, including humans, cows, and pigs, remains unclear. Unlike mouse oocytes, oocytes from these species lack distinct aMTOC foci at their spindle poles (2,36). Although studies of these oocytes have reported localization of a few proteins such as g-tubulin and NUMA (nuclear mitotic apparatus protein) at the spindle poles (2,(37)(38)(39)(40), how these oocytes assemble their spindle poles and control their spindle polarity without centrosomes and aMTOCs remains poorly understood.…”