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. 2021 Nov 19;11(1):22629.
doi: 10.1038/s41598-021-02000-z.

Controlled spermatozoa-oocyte interaction improves embryo quality in sheep

Debora Agata Anzalone #  1 , Luca Palazzese #  2 , Marta Czernik  1   2 , Annalaura Sabatucci  3 , Luca Valbonetti  3 , Emanuele Capra  4 , Pasqualino Loi  5
Affiliations

Controlled spermatozoa-oocyte interaction improves embryo quality in sheep

Debora Agata Anzalone et al. Sci Rep. .

Abstract

The current protocols of in vitro fertilization and culture in sheep rely on paradigms established more than 25 years ago, where Metaphase II oocytes are co-incubated with capacitated spermatozoa overnight. While this approach maximizes the number of fertilized oocytes, on the other side it exposes them to high concentration of reactive oxygen species (ROS) generated by active and degenerating spermatozoa, and positively correlates with polyspermy. Here we set up to precisely define the time frame during which spermatozoa effectively penetrates and fertilizes the oocyte, in order to drastically reduce spermatozoa-oocyte interaction. To do that, in vitro matured sheep oocytes co-incubated with spermatozoa in IVF medium were sampled every 30 min (start of incubation time 0) to verify the presence of a fertilizing spermatozoon. Having defined the fertilization time frame (4 h, data from 105 oocytes), we next compared the standard IVF procedures overnight (about 16 h spermatozoa/oocyte exposure, group o/nIVF) with a short one (4 h, group shIVF). A lower polyspermic fertilization (> 2PN) was detected in shIVF (6.5%) compared to o/nIVF (17.8%), P < 0.05. The o/nIVF group resulted in a significantly lower 2-cell stage embryos, than shIVF [34.6% (81/234) vs 50.6% (122/241) respectively, P < 0.001]. Likewise, the development to blastocyst stage confirmed a better quality [29% (70/241) vs 23.5% (55/234), shIVF vs o/nIVF respectively] and an increased Total Cell Number (TCN) in shIVF embryos, compared with o/n ones. The data on ROS have confirmed that its generation is IVF time-dependent, with high levels in the o/nIVF group. Overall, the data suggest that a shorter oocyte-spermatozoa incubation results in an improved embryo production and a better embryo quality, very likely as a consequence of a shorter exposure to the free oxygen radicals and the ensuing oxidative stress imposed by overnight culture.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Experimental design. Fertilization window has been detected by localization of sperm within 4 h from sperm-oocyte coincubation. Then, polyspermy, embryo development and blastocysts quality were compared between short and overnight IVF. ROS production from medium only and spermatozoa has been evaluated at t0, after short and overnight incubation. Further details are in the text.
Figure 2
Figure 2
Controlled time-IVF. (a) spermatozoon is passing through the zona pellucida (arrow head) 90 min post the beginning of IVF. (b) a spermatozoon (arrow head) position at 120 min post IVF. (c) Spermatozoon (arrow head) at the perivitelline space at 180 min post-IVF. (d) early sperm nucleus decondensation at 240 min after-IVF. (e,f) Pronuclear evaluation at 14 h post-IVF. (e) o/nIVF embryos, on the left normal 2 Pronuclear (2PN) fertilization, on the right polyspermic fertilization (> 2PN). (f) shIVF group, on the left normal 2 Pronuclear (2PN) fertilization, on the right polyspermic fertilization (> 2PN). Red signal: Propidium Iodide (P.I.); green signal: Pisum Sativum Agglutinin (PSA). Scale bar in (a,d,e,f): 50 μm. Scale bar in (b,c right): 10 μm.
Figure 3
Figure 3
Embryonic development and blastocyst’s cells counting. (a) Expanded blastocyst at day 7 of embryo culture produced with convention overnight IVF (on the left) and short IVF (on the right). Scale bar represents: 150 µm. (b) Differential staining for blastocyst’s cells counting. On the left, o/nIVF blastocyst, on the right shIVF blastocyst. Chromatin was stained with Hoechst 33,342 (in blue) and Propidium Iodide (in red). (c) Graphic representation of blastocyst total cells number. For each comparison a total of 16 blastocysts for o/nIVF and 14 blastocysts for shIVF were evaluated. ** means P = 0.0062.
Figure 4
Figure 4
Analysis of molecular markers. The relative expression of the Proliferating Cells Nuclear Antigen (PCNA) and the antioxidant gene (SOD1) were investigated in shIVF and o/nIVF groups (n = 3 each) by RT-qPCR. ACTB were used as reference genes. Gene relative expressions and P-Value (t-test) between shIVF and o/nIVF groups were calculated by the PCR R package.
Figure 5
Figure 5
Spermatozoa ROS production. Reactive oxygen species (ROS) generation measured as 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) oxidation rate, calculated as mean and standard deviation of fluorescence intensity slope as a function of time (DF/min) in the linear region (corresponding to the first 10 min of the kinetic measurement. (a) ROS level detected in PBS and IVF medium with and without spermatozoa in time points t = 0, t = sh (shIVF), t = o/n (o/nIVF). (b) ROS level produced by spermatozoa in PBS and IVF medium in time points t = 0, t = sh (shIVF), t = o/n (o/nIVF). * means P < 0.005.
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