Hydrogen Sulfide Donor Protects Porcine Oocytes against Aging and Improves the Developmental Potential of Aged Porcine Oocytes

<div><p>Porcine oocytes that have matured in in vitro conditions undergo the process of aging during prolonged cultivation, which is manifested by spontaneous parthenogenetic activation, lysis or fragmentation of aged oocytes. This study focused on the role of hydrogen sulfide (H₂S) in the process of porcine oocyte aging. H₂S is a gaseous signaling molecule and is produced endogenously by the enzymes cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (MPST). We demonstrated that H₂S-producing enzymes are active in porcine oocytes and that a statistically significant decline in endogenous H₂S production occurs during the first day of aging. Inhibition of these enzymes accelerates signs of aging in oocytes and significantly increases the ratio of fragmented oocytes. The presence of exogenous H₂S from a donor (Na₂S.9H₂O) significantly suppressed the manifestations of aging, reversed the effects of inhibitors and resulted in the complete suppression of oocyte fragmentation. Cultivation of aging oocytes in the presence of H₂S donor positively affected their subsequent embryonic development following parthenogenetic activation. Although no unambiguous effects of exogenous H₂S on MPF and MAPK activities were detected and the intracellular mechanism underlying H₂S activity remains unclear, our study clearly demonstrates the role of H₂S in the regulation of porcine oocyte aging.</p></div

Parthenogenetic activation of oocytes aged under the effect of the H₂S donor.

<p>At Oocytes were cultivated 48 hours to the metaphase II and then divided into 4 groups (see table). Control group (MII) was parthenogenetically activated immediately (without any exposure to prolonged cultivation). Other groups were exposed to prolonged cultivation (aging) for 24 hours in modified M199 medium supplemented with a H2S donor (Na₂S.9H₂O; 0μM, 150μM, and 300μM) and then parthenogenetically activated with calcium ionophore (25μM, 5 min) combined with 6-dimethyl aminopurine (2mM, 2 h). Subsequently, oocytes were cultured in NCSU 23 medium for the following 24 hours.</p><p>^a,b,c Statistically significant differences in the ratio of activated oocytes between individual treatments (in columns) are indicated with different superscripts (P<0.05).</p><p>Parthenogenetic activation of oocytes aged under the effect of the H₂S donor.</p

Effect of H₂S donor on MPF and MAPK activity. 6A

<p>Histone H1 kinase assay was carried out to determine the activity of MPF by measurement of MPF capacity to phosphorylate its substrate (histone H1). <b>6B</b>: MBP kinase assay was carried out to determine the activity of MAPK by measurement of MAPK capacity to phosphorylate its substrate (MBP – Myelin basic protein). MPF and MAPK activities were determined in the MII oocytes (C – control, white column), the oocytes aged 12h and 24h in modified M199 medium, the oocytes aged 12h and 24h in modified M199 medium supplemented with a H₂S donor (Na₂S, black column), and the oocytes aged 12h and 24h in modified M199 medium supplemented with triple combination of inhibitors (3Ki, grey column). The results are presented as a ratio relative to the group of oocytes at metaphase II. <i>(GV – germinal vesicle stage; MII – oocytes at metaphase II; A12–12 hours of aging; A24–24 hours of aging; C – control, white column; Na₂S—Na₂S.9H₂O, 300 μM, black column; 3Ki - 1mM oxamic acid + 1mM beta-kyano-L-alanine + 5mM alpha-ketoglutaric acid disodium salt dihydrate). ^a,b, Statistically significant differences in activity (MPF or MAPK) between individual treatments at the same time are indicated with different superscripts (P<0.05)</i>.</p

Reversion of the effects of CBS, CSE and MPST inhibitors using a H₂S donor.

<p>Oocytes were cultivated to metaphase II and then exposed to prolonged cultivation (24 hours) in a modified M199 medium supplemented with a H₂S donor (Na₂S.9H₂O; 300 μM) and the following individual inhibitors: oxamic acid (1mM, OA), beta-kyano-L-alanine (1mM, KA), and alpha-ketoglutaric acid disodium salt dihydrate (5mM, KGA). <i>Na₂S (300 μM, Na₂S.9H₂O); KGA—alpha-ketoglutaric acid disodium salt dihydrate (5 mM); KA – beta-kyano-L-alanine (1mM); OA—oxamic acid (1 mM); MII—intact oocytes (oocytes at metaphase II, anaphase II or telophase II), A—activated oocytes (oocytes with pronuclei or embryos), F—fragmented oocytes, L—lysed oocytes; Different letters and numbers indicate significant differences between different treatments and hours of aging (P<0.05). A,B – statistically significant differences in portion of MII stage oocytes between individual treatments. a,b,c,d – statistically significant differences in portion of activated oocytes between individual treatments. 1,2,3 – statistically significant differences in portion of fragmented oocytes between individual treatments</i>.</p

Effects of an elevated H₂S level and inhibition of H₂S producing enzymes during oocyte aging.

<p>Oocytes were cultivated to metaphase II and then exposed to prolonged cultivation in a modified M199 medium for 24, 48 and 72 hours in the presence of a H₂S donor or H₂S producing enzymes inhibitors. <b>3A</b>. Na₂S (Na₂S.9H₂O; 300 μM) was used as the H₂S donor. <b>3B</b>. Oxamic acid (1mM, OA) was used as a CBS inhibitor, beta-kyano-L-alanine (1mM, KA) was used as a CSE inhibitor and alpha-ketoglutaric acid disodium salt dihydrate (5mM, KGA) was used as a MPST inhibitor. <i>C- control; Na₂S (300 μM, Na₂S.9H₂O); KGA—alpha-ketoglutaric acid disodium salt dihydrate (5 mM); KA – beta-kyano-L-alanine (1mM); OA—oxamic acid (1 mM); MII—intact oocytes (oocytes at metaphase II, anaphase II or telophase II), A—activated oocytes (oocytes with pronuclei or embryos), F—fragmented oocytes, L—lysed oocytes. Different letters and numbers indicate significant differences between different treatments and hours of aging (P<0.05). A,B,C – statistically significant differences in portion of MII stage oocytes between individual treatments. a,b,c,d – statistically significant differences in portion of activated oocytes between individual treatments. 1,2,3 – statistically significant differences in portion of fragmented oocytes between individual treatments</i>.</p

Effect of aging on morphology of porcine oocyte.

<p>MII—intact oocyte at metaphase II, A—activated oocyte, F—fragmented oocyte, L—lysed oocyte.</p

Determination of endogenous H₂S production during porcine oocyte aging.

<p>Oocytes were cultivated to metaphase II (MII). Hydrogen sulfide production was carried out using the spectrophotometric method. MII oocytes, as well as oocytes exposed to prolonged cultivation for 24, 48 and 72 hours, were examined. The results of the measurement are presented as a ratio relative to the MII oocyte group. ^{<i>a, b</i>}<i>Statistically significant differences in spontaneous hydrogen sulfide production are indicated by different superscripts (P<0.05). Each experiment was repeated four times. The total number of oocytes in each sample was 100</i>.</p

Effects of concurrent CBS, CSE and MPST inhibition and its reversion using a H₂S donor.

<p>Oocytes were cultivated to metaphase II and then exposed to prolonged cultivation in a modified M199 medium supplemented with a H₂S donor (Na₂S.9H₂O; 300 μM) and the inhibitors for 24, 48 and 72 hours. Various combinations of oxamic acid (1mM, OA) which was used as a CBS inhibitor, beta-kyano-L-alanine (1mM, KA) which was used as a CSE inhibitor and alpha-ketoglutaric acid disodium salt dihydrate (5mM, KGA) which was used as a MPST inhibitor were used in this experiment. To reverse effects of inhibitors, a H₂S donor (300 μM, Na₂S.9H₂O) was added to each experimental group. <i>C- control; Na₂S (300 μM, Na₂S.9H₂O); KGA—alpha-ketoglutaric acid disodium salt dihydrate (5 mM); OA—oxamic acid (1 mM); KA – beta-kyano-L-alanine (1mM); MII—intact oocytes (oocytes at metaphase II, anaphase II or telophase II), A—activated oocytes (oocytes with pronuclei or embryos), F—fragmented oocytes, L—lysed oocytes; Different letters and numbers indicate significant differences between different treatments and hours of aging (P<0.05). A,B,C,D – statistically significant differences in portion of MII stage oocytes between individual treatments. a,b,c,d,e – statistically significant differences in portion of activated oocytes between individual treatments. 1,2,3,4 – statistically significant differences in portion of fragmented oocytes between individual treatments</i>.</p

Early embryonic development of parthenogenetically activated oocytes aged under the effect of the H₂S donor.

<p>Oocytes were cultivated 48 hours to the metaphase II and then divided into 3 groups (see table). Control group (MII) was parthenogenetically activated immediately (without any exposure to prolonged cultivation). Other groups were exposed to prolonged cultivation (aging) for 24 hours in modified M199 medium supplemented with a H2S donor (Na₂S.9H₂O; 0μM, 150μM, and 300μM) and then parthenogenetically activated with calcium ionophore (25 μM, 5 min) combined with 6-dimethyl aminopurine (2 mM, 2 h). Subsequently, oocytes were cultured in NCSU 23 medium for 168 hours (7 days). The ratio of cleaved embryos was evaluated after the first 48 hours of culture.</p><p>^a,b,c,d Statistically significant differences in type of embryo stage between individual treatments (in columns) are indicated with different superscripts (P<0.05).</p><p>Early embryonic development of parthenogenetically activated oocytes aged under the effect of the H₂S donor.</p