Defective sperm head decondensation undermines the success of ICSI in the bovine

Abstract

The efficiency of intracytoplasmic sperm injection (ICSI) in the bovine is low compared to other species. It is unknown whether defective oocyte activation and/or sperm head decondensation limit the success of this technique in this species. To elucidate where the main obstacle lies, we used homologous and heterologous ICSI and parthenogenetic activation procedures. We also evaluated whether in vitro maturation negatively impacted the early stages of activation after ICSI. Here we showed that injected bovine sperm are resistant to nuclear decondensation by bovine oocytes and this is only partly overcome by exogenous activation. Remarkably, when we used heterologous ICSI, in vivo-matured mouse eggs were capable of mounting calcium oscillations and displaying normal PN formation following injection of bovine sperm, although in vitro-matured mouse oocytes were unable to do so. Together, our data demonstrate that bovine sperm are especially resistant to nuclear decondensation by in vitro-matured oocytes and this deficiency cannot be simply overcome by exogenous activation protocols, even by inducing physiological calcium oscillations. Therefore, the inability of a suboptimal ooplasmic environment to induce sperm head decondensation limits the success of ICSI in the bovine. Studies aimed to improve the cytoplasmic milieu of in vitro-matured oocytes and to replicate the molecular changes associated with in vivo capacitation and acrosome reaction will deepen our understanding of the mechanism of fertilization and improve the success of ICSI in this species.

Figure 1

Representative images of PN formation in bovine oocytes fertilized by conventional in vitro fertilization (IVF), chemically activated oocytes without and with sham ICSI injection, or fertilized by ICSI. Upper images were captured after Hoechst staining, whereas the corresponding phase-contrast images are at the bottom. A and B: In vitro fertilization: zygotes examined 14 h post insemination. C and D: Parthenogenetically activated group with Io + CHX (14 h post activation). E and F: Sham-injected group, where injection of comparable volume of PVP (5%) was performed followed by chemical activation with Io + CHX (14 h post activation). G and H: ICSI, bovine zygote generated by ICSI without exogenous activation. White circle in G denotes injected, intact sperm head. I and J: ICSI – Io + CHX 14 hpa, ICSI bovine zygote 14 h post-chemical activation. White circle in I surrounds a partially decondensed sperm head. K and L: ICSI – Io + CHX 18 hpa, ICSI bovine zygote 18 h post-chemical activation. M and N: ICSI + bPLCζ: ICSI bovine zygote activated with bovine PLCζ 18 hpa. Arrows indicate PBs and arrowheads PNs. Magnification: 200×, scale bar in microns.

Figure 2

[Ca²⁺]_i responses induced by injection of bovine sperm with and without injection of PLCζ cRNA (0.5 μg/μL) or by the addition of ionomycin. (A) Injection of sperm-induced [Ca²⁺]_i responses in the minority of injected oocytes. (B) Co-injection of bull sperm with bovine PLCzeta1 cRNA (0.5 μg/μL) caused [Ca²⁺]_i responses in all injected oocytes. (C) Addition of Io caused a single [Ca²⁺]_i rise in all exposed oocytes and caused the baseline remain high as long as Io was in the media.

Figure 3

Representative fluorescent (upper) and phase-contrast (bottom) images of mouse oocytes following IVF or ICSI using mouse or a bovine sperm. A and B: IVF. C and D: Homologous ICSI using ovulated oocytes. E and F: Homologous ICSI using IVM oocytes. G and H: Heterologous ICSI using ovulated oocytes. I and J: Heterologous ICSI using IVM oocytes. M: Male pronuclei. FM: Female pronuclei. MP: Metaphase plate. Arrows indicate PBs and red circles denote PNs. Magnification 200×. Scale bar in microns.

Figure 4

Source of sperm and oocytes affect the size of PNs following homologous and heterologous ICSI using ovulated and IVM mouse oocytes. (A) Female PN. (B) Male PN. Values represent the mean + s.d. Bars with different letters are significantly different (P < 0.05).

Figure 5

[Ca²⁺]_i responses after homologous and heterologous ICSI in mouse oocytes. (A and B) Injection of mouse sperm into MII_OV induced consistent and expected [Ca²⁺]_i responses, although injection into IVM oocytes induced variable responses [Ca²⁺]_i (B). (C) Injection of bull sperm into MII_OV induced mostly high-frequency oscillations, although a few oocytes showed low-frequency oscillations (inset). (D) Injection of bull sperm into IVM induced high-frequency oscillations in half of the oocytes, although in the other half, the oscillations were less frequent or absent (inset).