Effects of activation on functional aster formation, microtubule assembly, and blastocyst development of goat oocytes injected with round spermatids

Abstract

A systematic study was conducted on round spermatids (ROS) injection (ROSI) using the goat model. After ROSI, the oocytes were treated or not with ionomycin (ROSI+I and ROSI-I, respectively) and compared with intracytoplasmic sperm injection (ICSI). After ROSI-I, most oocytes were arrested with premature chromatin condensation and few oocytes formed pronuclei. In contrast, most oocytes formed pronuclei after ROSI+I. Some ROS were observed to form asters that organized a dense microtubule network after ROSI+I, but after ROSI-I, no ROS asters were observed. Whereas most of the oocytes showed Ca(2+) rises and a significant decline in maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK) activities after ROSI+I, no such changes were observed after ROSI-I. Due to the lack of Ca(2+) oscillations after ROSI-I, oocytes were injected with more ROS. Interestingly, different from the results observed in a single ROS injection, injection with four ROS effectively activated oocytes by inducing typical Ca(2+) oscillations. Whereas ROSI+I oocytes and ICSI oocytes both showed extensive microtubule networks, no such a network was observed in parthenogenetic oocytes. Together, the results suggest that goat ROS is not able to trigger intracellular Ca(2+) rises and thus to inhibit MPF and MAPK activities, but artificial activation improved fertilization and development of ROSI goat oocytes. Goat ROS can organize functional microtubular asters in activated oocytes. A ROS-derived factor(s) may be essential for organization of a functional microtubule network to unite pronuclei. Goat centrosome is of paternal origin because both ROS and sperm asters organized an extensive microtubule network after intra-oocyte injection.

FIG. 1.

Phase-contrast micrographs of goat oocytes with different nuclear configurations after ROSI or ROSI followed by ionomycin activation (ROSI+I). (A) ROS (arrows) selected for use for injection. (B–D) Oocytes after ROSI alone (ROSI−I) with ROSN (arrows) showing intact nuclear envelope, NEBD, and PCC, respectively. The insets in B, C, and D represent enlarged views of ROSN. (E) A ROSI+I oocyte with PCC chromosomes separated by the anaphase spindle into two groups that move to the opposite poles of the oocyte. (F) A ROSI+I oocyte with two well-developed pronuclei. Magnification, 400×.

FIG. 2.

Confocal micrographs of goat oocytes after ICSI, ROSI, or ROSI followed by ionomycin activation (ROSI+I). The α-tubulin and chromatin are pseudo-colored green and blue, respectively. (A–C) Oocytes observed at 2, 3, and 6 h after ICSI, respectively. (A) Oocyte anaphase II spindle (F) and the decondensing sperm head (M). (B) Aster formation around the decondensed sperm chromatin (M) and the oocyte spindle in telophase II (F). (C) Microtubule network surrounding the apposed male and female pronuclei (PN). (D–F) Oocytes observed at 1, 1.5, and 6 h after ROSI, respectively, with D showing the prematurely condensed spermatid chromatin surrounded with microtubules (M) and the oocyte metaphase II spindle (F), and E and F showing oocyte metaphase II spindle (F) and the spindle-like structure around the spermatid condensed chromatin (M). (Insets, E and F) Two times amplifications of the spermatid spindle-like structures. (G–J) Oocytes observed at 2, 4, and 6 h after ROSI+I, with G showing oocyte spindle in telophase II (F) and intact ROSN with an aster-like structure (M), H and I showing spermatid (M) and oocyte (F) pronuclei and the microtubule network emerging from the spermatid pronucleus, and J showing well-developed ooplasmic microtubule network surrounding the apposed pronuclei (PN). (K and L) Oocytes observed at 2 and 6 h, respectively, after ionomycin treatment for parthenogenetic activation, with K showing oocyte anaphase II spindle (F) and L showing microtubules observed only in the pronucleus (PN) area. (Inset, L) Image before merging to show only the α-tubulin (pseudo-colored green). First (P1) and second (P2) polar bodies are often observed. Scale bar, 15 μm.

FIG. 3.

Relative MPF (histone H1 kinase) and MAPK (MBP kinase) activity of goat oocytes at different times after ICSI, ROSI, or ROSI+I. Values without a common letter above their bars differ significantly (P<0.05).

FIG. 4.

Dynamics of intracellular concentration of free calcium ion in goat oocytes. Ca²⁺ rises were observed after ICSI or ROSI+I, as shown in the top panel and the bottom panel, respectively. No Ca²⁺ rises were observed after ROSI, as shown in the middle panel.

FIG. 5.

Dynamics of intracellular concentration of free calcium ion in mouse and goat oocytes. (Upper panel) Pattern of Ca²⁺ oscillations in mouse oocytes after ROSI with one ROS. (Lower panel) Ca²⁺ rises in goat oocytes were observed only after ROSI with four ROS as demonstrated here.