Nuclear Configuration, Spindle Morphology and Cytoskeletal Organization of In Vivo Maturing Horse Oocytes

Abstract

Horse oocytes (n = 37) were recovered in vivo from pre-ovulatory follicles 30 h after an ovulation-inducing hCG injection and were examined by fluorescent staining and confocal microscopy. Percentages of metaphase-I (MI), metaphase-II (MII) and atypical oocytes were 11%, 78% and 11% respectively. Microtubules were concentrated in the meiotic spindle in both MI and MII oocytes. Chromosomes in the metaphase plate were anchored at the equatorial region of the spindle. Spindle orientation was perpendicular to the oolema in all MI oocytes, whereas in MII oocytes, 66% were parallel and 34% were perpendicular. In MII oocytes, the nuclear material in the polar body had no specific organization and was intertwined with microtubules. Discrete foci of microfilaments at the sub-cortical region of the ooplasm formed an F-actin band, as seen in the inner confocal sections. The percentage area of oocyte image with discrete foci and/or the thickness of F-actin band was used to indicate microfilament content. Microfilament content was greater (p < 0.006) in MII oocytes than in MI oocytes and greater (p < 0.03) in MII oocytes with a perpendicular spindle than with a parallel spindle. The perpendicular spindle orientation in MII oocytes may have represented a later stage of maturation. Atypical oocytes were based on microtubules that were detached from the kinetochores and spread in the ooplasm or by microtubules that accumulated as an amorphous mass near the condensed chromatin. This is the first description of the nuclear configuration, spindle morphology and cytoskeletal organization of in vivo maturing horse oocytes.