Human exhaled air can efficiently support in vitro maturation of porcine oocytes and subsequent early embryonic development

Abstract

Air phase is an indispensable environmental factor affecting oocyte maturation and early embryo development. Human exhaled air was previously proved to be a reliable and inexpensive atmosphere that sustains normal early development of mouse and bovine embryos. However, whether human exhaled air can support in vitro maturation (IVM) of porcine oocytes is not yet known. To evaluate the feasibility of maturing oocytes in human exhaled air, we examined oocyte morphology, BMP15 expression, nuclear and cytoplasmic maturation. We found that cumulus expansion status, expression levels of BMP15 important for cumulus expansion and the rate of first polar body emission were similar among human exhaled air, 5% O₂ or 20% O₂ in air after IVM of 44 h. Furthermore, the percentage of metaphase II (MII) oocytes showing normal cortical and sub-membranous localization of cortical granules and diffused mitochondrial distribution patterns is comparable among groups. The cleavage, blastocyst rate and total cell number were not apparently different for parthenogenetic activated and somatic cloned embryos derived from MII oocytes matured in three air phases, suggesting oocytes matured in human exhaled air obtain normal developmental competence. Taken together, human exhaled air can efficiently support in vitro maturation of porcine oocytes and subsequent early embryonic development.

Keywords: early embryo; human exhaled air; oocyte maturation; pig; somatic cell nuclear transfer.

Figure 1. Human exhaled air maintains normal meiotic maturation of porcine oocytes. (A) The procedures of human exhaled air preparation. a. Porcine cumulus-oocyte complexes (COCs) at germinal vesicle (GV) stage were cultured in four-well plates containing in vitro maturation (IVM) medium covered with mineral oil. Four-well plates were transferred into the sterilized airtight aluminium bag and then experimenters exhaled air into the bag. b. Outside edge of bag should be sealed immediately when bag was inflated with appropriate human exhaled air. c. Shown was an aluminium bag inflated with human exhaled air. (B) Representative images of COCs with expanded cumulus cells matured in vitro in different air phases for 44 h. Scale bars: 100 µm. (C) Quantitative analysis of polar body extrusion (PBE) rate for COCs matured in vitro in different air phases for 44 h. The experiment was conducted four times with 240 GV oocytes per group. All the percentage data are expressed relative to the number of GV oocytes and shown as mean ± S.E.M. Values with different superscripts across groups indicate significant differences (P < 0.05). MII denotes metaphase stage of meiosis II.

Figure 2. Human exhaled air sustains robust distribution of cortical granules and mitochondria during porcine oocyte maturation. (A) Immunofluorescence analysis of cortical granules (CG) with cortical area and peripheral sub-membranous distribution in denuded metaphase-II (MII) oocytes cultured in different air phases. Cortical granules and DNA in denuded MII oocytes were labeled with FITC-conjugated peanut agglutinin (green) and propidium iodide (red). Bottom panels showed the merged images (yellow) between cortical granules signals and DNA staining. Shown are representative Z-stacks obtained by epifluorescence microscopy from one experiment. Arrow indicates the first polar body, arrowhead denotes sub-membranous region. Scale bars: 20 µm. (B) Quantification of the percentage of MII oocytes with GC displaying cortical area and peripheral sub-membranous distribution in A. Data are shown as mean ± S.E.M. The experiment was repeated three times with 60 oocytes (human exhaled air), 64 oocytes (5% O₂), 48 oocytes (20% O ₂), respectively. Values with different superscripts across groups indicate significant differences (P < 0.05). (C) Immunofluorescence analysis of active mitochondria with diffused distribution in denuded metaphase-II (MII) oocytes cultured in different air phases. Mitochondria and DNA were labeled with MitoTracker Red CMXRos (red) and Hoechst33342 (blue). Bottom panels showed the merged images between cortical granules signals and DNA staining. Shown are Z-stacks obtained by epifluorescence microscopy from a representative experiment. Scale bars: 20 µm. (D) Quantification of the percentage of MII oocytes exhibiting diffused mitochondrial distribution in C. Data are shown as mean ± S.E.M. The experiment was performed three times with 44 oocytes (human exhaled air), 56 oocytes (5% O₂), 52 oocytes (20% O₂), respectively. Values with different superscripts across groups indicate significant differences (P < 0.05).

Figure 3. Human exhaled air maintains correct expression of BMP15 essential for porcine oocyte maturation. (A) Representative images of COCs matured in vitro in different air phases for 18 h. Scale bars: 100 µm. (B) Real-time qPCR analysis of BMP15 transcripts in denuded oocytes derived from COCs of A. Expression levels were normalized against endogenous housekeeping gene EF1α1 and human exhaled air group was set to 1. A total of three biological replicates were performed. Data are shown as mean ± S.E.M. Values with different superscripts across groups indicate significant differences (P < 0.05). IVM denotes in vitro maturation.

Figure 4. Oocytes matured in human exhaled air support the early development of parthenogenetic activated embryos. Metaphase II (MII) oocytes matured in vitro in different air phases were parthenogenetically activated (PA) by electric pulse. PA embryos were then cultured for 7 days in fresh PZM-3 medium at 38.5°C and 5% CO ₂ in humidified air. The cleavage rate (48 h) (A), blastocyst rate (day 7) (B) and total cell number per blastocyst (C) were statistically analyzed. Data are shown as mean ± S.E.M. The experiment was repeated four times with 240 embryos per group. Values with different superscripts across groups indicate significant differences (P < 0.05). (D) Representative images of PA blastocysts derived from MII oocytes matured in vitro in different air phases. DNA was labeled with Hoechst33342 (blue). Scale bars: 100 µm.

Figure 5. Oocytes matured in human exhaled air support the early development of somatic cloned embryos. Somatic cell nuclear transfer (SCNT) embryos were generated though transplanting donor cells into enucleated MII oocytes matured in vitro in different air phases. SCNT embryos were then cultured in fresh PZM-3 medium at 38.5°C and 5% CO₂ in humidified air for 7 days. The cleavage rate (48 h) (A), blastocyst rate (day 7) (B) and total cell number per blastocyst (C) were statistically analyzed. Data are shown as mean ± S.E.M. The experiment was repeated four times with 120 embryos per group. Values with different superscripts across groups indicate significant differences (P < 0.05). (D) Representative images of SCNT blastocysts derived from MII oocytes matured in vitro in different air phases. DNA was labeled with Hoechst33342 (blue). Scale bars: 100 µm.