From Nitric Oxide Toward S-Nitrosylation: Expanding Roles in Gametes and Embryos

Nitric oxide (NO) is a gasotransmitter involved in various aspects of reproduction. The observational data from different species, such as sea urchin, ascidians, amphibians, rodents, porcine, bovine, and human, suggest that NO might have a significant role in reproduction through several mechanisms. This proposed role might appear preserved through evolution; however, the effects of NO also depend on the species or stages considered. There has been debate over the physiological relevance of NO, though the benefits of its use in assisted reproduction are now widely recognized. Over the past years, S-nitrosylation has provided a new angle to decipher the mechanisms through which NO exerts its actions. This chapter summarizes, in a nonexhaustive manner, research that explores the role of NO in gametes and embryos

Ligands and Receptors Involved in the Sperm-Zona Pellucida Interactions in Mammals

Sperm-zona pellucida (ZP) interaction, involving the binding of sperm surface ligands to complementary carbohydrates of ZP, is the first direct gamete contact event crucial for subsequent gamete fusion and successful fertilization in mammals. It is a complex process mediated by the coordinated engagement of multiple ZP receptors forming high-molecular-weight (HMW) protein complexes at the acrosomal region of the sperm surface. The present article aims to review the current understanding of sperm-ZP binding in the four most studied mammalian models, i.e., murine, porcine, bovine, and human, and summarizes the candidate ZP receptors with established ZP affinity, including their origins and the mechanisms of ZP binding. Further, it compares and contrasts the ZP structure and carbohydrate composition in the aforementioned model organisms. The comprehensive understanding of sperm-ZP interaction mechanisms is critical for the diagnosis of infertility and thus becomes an integral part of assisted reproductive therapies/technologies

Nitric oxide-donor SNAP induces Xenopus eggs activation.

Nitric oxide (NO) is identified as a signaling molecule involved in many cellular or physiological functions including meiotic maturation and parthenogenetic activation of mammalian oocytes. We observed that nitric oxide donor SNAP was potent to induce parthenogenetic activation in Xenopus eggs. NO-scavenger CPTIO impaired the effects of SNAP, providing evidence for the effects of the latter to be specific upon NO release. In Xenopus eggs, SNAP treatment induced pigment rearrangement, pronucleus formation and exocytosis of cortical granules. At a biochemical level, SNAP exposure lead to MAPK and Rsk inactivation within 30 minutes whereas MPF remained active, in contrast to calcium ionophore control where MPF activity dropped rapidly. MAPK inactivation could be correlated to pronuclear envelope reformation observed. In SNAP-treated eggs, a strong increase in intracellular calcium level was observed. NO effects were impaired in calcium-free or calcium limited medium, suggesting that that parthenogenetic activation of Xenopus oocytes with a NO donor was mainly calcium-dependent

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

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

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

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

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