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. 2016 Jun;33(6):757-63.
doi: 10.1007/s10815-016-0709-1. Epub 2016 Apr 8.

Plasma membrane and acrosome loss before ICSI is required for sheep embryonic development

Debora A Anzalone  1 , Domenico Iuso  1 , Marta Czernik  1 , Grazyna Ptak  1   2 , Pasqualino Loi  3
Affiliations

Plasma membrane and acrosome loss before ICSI is required for sheep embryonic development

Debora A Anzalone et al. J Assist Reprod Genet. 2016 Jun.

Abstract

Purpose: This study aims to determine if the integrity of the sperm plasma membrane and acrosome vesicle could be limiting factors in sheep intracytoplasmic sperm injection (ICSI).

Methods: Prior to in vitro fertilization (IVF) or ICSI, the oocytes were subjected to in vitro maturation (IVM) for 24 h. First, to evaluate the need of artificial activation for ovine ICSI, 226 oocytes were injected with intact spermatozoa (IS), from which 125 were activated by incubation in ionomycin and 101 were cultured without activation. Next, spermatozoa were mechanically (by piezo-electrical pulses) and/or chemically (by ionomycin/Triton X-100) treated to break membranes and acrosomes and were injected into oocytes, grouped as follows: (i) piezo-pulsed spermatozoa (PPS), (ii) PPS pre-treated with ionomycin (PPS-I), (iii) PPS pre-treated with Triton X-100 (PPS-T), and (iv) intact and untreated spermatozoa as a control (CTR-IS).

Results: No differences were observed in the zygote/cleavage/blastocyst rate between chemically activated and non-activated oocytes (50 vs. 45 %, 11.6 vs. 10.1 %; 1.8 vs. 1.1 %, respectively), after ICSI with CTR-IS. Injection of PPS compared to CTR-IS increased the proportion of zygotes and blastocysts (84.6 vs. 45 %, p < 0.01; 15.5 vs. 1.1 %, p < 0.0001, respectively). Moreover, the percentage of PPS-derived blastocysts was not significantly different from that obtained by conventional IVF (15.5 vs. 20.2 %). The ICSI blastocysts' development was also improved with PPS pre-treated with ionomycin (15.6 %), but was completely impeded with PPS pre-treated with Triton X-100 (0 %).

Conclusion: Our findings confirm that ICSI with spermatozoa whose plasma membrane and acrosome have been mechanically damaged substantially improves embryonic development until the blastocyst stage.

Keywords: Acrosome; ICSI; Ovine sperm; Plasma membrane.

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Figures

Fig. 1
Fig. 1
Acrosomal integrity after PSA-FITC staining. Spermatozoa with acrosome show brilliant green signal on the acrosomal cap; spermatozoa without acrosome show green signal only in the equatorial region of the head. All heads (nuclei) were counterstained with DAPI (blue). Merge means DAPI + PSA-FITC. Scale bar = 5 μm
Fig. 2
Fig. 2
Pronuclear formation after ICSI. The presence of pronuclei (PN) was analyzed 14–16 h post sperm injection. a Activated oocytes showed two clearly distinguishable pronuclei; b non-activated oocytes showed chromosomal metaphase (MII) and a non-decondensed sperm head (SPZ head) closer to it. All nuclei were counterstained with propidium iodide. Scale bar = 10 μm
Fig. 3
Fig. 3
Integrity of sperm plasma membrane evaluation. The integrity of sperm plasma membrane was evaluated by Transmission Electron Microscopy (TEM) in intact (IS) and piezo-pulsed spermatozoa (PPS). Abbreviations: pm plasma membrane, dpm damaged plasma membrane, a acrosome, n nucleus, t tail, pnm perinuclear material
Fig. 4
Fig. 4
Sheep blastocysts at 8th day of culture. Image compares blastocysts derived from IVF (left) and ICSI with PPS (right). IVF in vitro fertilized blastocyst, PPS-ICSI blastocysts obtained from ICSI with piezo-pulsed spermatozoa. Scale bar = 100 μm
See this image and copyright information in PMC

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