Dual Effects of Hydrogen Sulfide Donor on Meiosis and Cumulus Expansion of Porcine Cumulus-Oocyte Complexes

<div><p>Hydrogen sulfide (H₂S) has been revealed to be a signal molecule with second messenger action in the somatic cells of many tissues, including the reproductive tract. The aim of this study was to address how exogenous H₂S acts on the meiotic maturation of porcine oocytes, including key maturation factors such as MPF and MAPK, and cumulus expansion intensity of cumulus-oocyte complexes. We observed that the H₂S donor, Na₂S, accelerated oocyte <i>in vitro</i> maturation in a dose-dependent manner, following an increase of MPF activity around germinal vesicle breakdown. Concurrently, the H₂S donor affected cumulus expansion, monitored by hyaluronic acid production. Our results suggest that the H₂S donor influences oocyte maturation and thus also participates in the regulation of cumulus expansion. The exogenous H₂S donor apparently affects key signal pathways of oocyte maturation and cumulus expansion, resulting in faster oocyte maturation with little need of cumulus expansion.</p></div

Effect of Na₂S on meiosis resumption and transition to meiosis II in DOs.

<p>Proportion of GVBD (A) and meiosis I to II transition (B) during <i>in vitro</i> cultivation after 20 and 30 h <i>in vitro</i> cultivation, respectively. H₂S: 300 µM Na₂S. ^a,b,cStatistically significant differences among experimental groups (P<0.05).</p

Effect of Na₂S on meiotic resumption and transition to meiosis II during oocyte cultivation.

<p>Proportion of GVBD (A) and meiosis I to II transition (B) in oocytes during <i>in vitro</i> cultivation over 2 h time scale. H₂S: 300 µM Na₂S. *Statistically significant differences between control and H₂S groups (P<0.05).</p

Effect of different Na₂S concentrations on meiosis resumption and transition to meiosis II in oocytes.

<p>Proportion of GVBD (A) and meiosis I to II transition (B) during <i>in vitro</i> cultivation after 20 and 30 h <i>in vitro</i> cultivation, respectively. ^a,b,cStatistically significant differences among experimental groups (P<0.05).</p

Effect of Na₂S on HA content in expanded cumulus.

<p>(A) Total and retained HA content in COCs cultivated with 150–900 µM Na₂S for 48 hs, total HA is related to the control group. (B) Total and retained HA content in COCs during <i>in vitro</i> cultivation with 300 µM Na₂S over 12 h time scale, total HA is related to the control group after 48 h cultivation. (C) Total and retained HA content in COCs and OOXs cultivated with or without H₂S donor, total HA is related to the control group of COCs. H₂S: 300 µM Na₂S. ^a,b,cStatistically significant differences among experimental groups in total HA, ^1,2statistically significant differences among experimental groups in retained HA, *statistically significant differences in total HA between control and H₂S groups (P<0.05).</p

Effect of Na₂S on partenogenetic development of porcine oocytes.

<p>Oocytes were matured with or without Na₂S and partenogenetically activated using calcium ionophore. Pronucleus formation after 24 h zygote culture, cleavage rate after 2 days and blastocyst achievement after 7 days presumptive embryos culture were evaluated (%±SE).</p><p>H₂S: 300 µM Na₂S during oocyte maturation.</p><p>*Statistically significant differences between control and H₂S group – in column (P<0.05).</p

Effect of Na₂S on MPF and MAPK activities during oocyte cultivation.

<p>Representative autoradiograms and signal quantifications of phosphorylated histone H1 (A) and MBP (B) reflecting MPF and MAPK activity, respectively. Kinase activity was measured in oocytes cultivated with or without Na₂S over 2 h time scale. The kinase activity was related to oocytes cultivated for 24 hs. C: control; H₂S: 300 µM Na₂S. *Statistically significant differences between control and H₂S groups (P<0.05).</p

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