Jan Nevoral

Bisphenol S negatively affects the meotic maturation of pig oocytes

Bisphenol A (BPA), a chemical component of plastics, is a widely distributed environmental pollutant and contaminant of water, air, and food that negatively impacts human health. Concerns regarding BPA have led to the use of BPA-free alternatives, one of which is bisphenol S (BPS). However, the effects of BPS are not well characterized, and its specific effects on reproduction and fertility remain unknown. It is therefore necessary to evaluate any effects of BPS on mammalian oocytes. The present study is the first to demonstrate the markedly negative effects of BPS on pig oocyte maturation in vitro, even at doses lower than those humans are exposed to in the environment. Our results demonstrate (1) an effect of BPS on the course of the meiotic cell cycle; (2) the failure of tubulin fibre formation, which controls proper chromosome movement; (3) changes in the supply of maternal mRNA; (4) changes in the protein amounts and distribution of oestrogen receptors α and β and of aromatase; and (5) disrupted cumulus cell expansion. Thus, these results confirm that BPS is an example of regrettable substitution because this substance exerts similar or even worse negative effects than those of the material it replaced.

Bisphenol S instead of bisphenol A: a story of reproductive disruption by regretable substitution – a review

A range of substances that are released into the environment, foodstuffs and drinking water as a result of human activity were originally considered relatively harmless, and it was only later that their adverse effects were discovered. In general the use of such substances is currently restricted, and they are often replaced by other substances. This applies also in the case of a range of endocrine disruptors. These substances have the capacity to disturb the balance of physiological functions of the organism on the level of hormonal regulation, and their pleiotropic spectrum of effects is very difficult to predict. Endocrine disruptors include the currently intensively studied bisphenol A (BPA), a prevalent environmental pollutant and contaminant of both water and foodstuffs. BPA has a significantly negative impact on human health, particularly on the regulation mechanisms of reproduction, and influences fertility. The ever increasingly stringent restriction of the industrial production of BPA is leading to its replacement with analogues, primarily with bisphenol S (BPS), which is not subject to these restrictions and whose impacts on the regulation of reproduction have not yet been exhaustively studied. However, the limited number of studies at disposal indicates that BPS may be at least as harmful as BPA. There is therefore a potential danger that the replacement of BPA with BPS will become one of the cases of regrettable substitution, in which the newly used substances manifest similar or even worse negative effects than the substances which they have replaced. The objective of this review is to draw attention to ill-advised replacements of endocrine disruptors with substances whose effects are not yet tested, and which may represent the same risks for the environment, for the reproduction of males and females, and for human health as have been demonstrated in the case of the originally used substances.

Hydrogen sulfide donor protects porcine oocytes against aging and improves the developmental potential of aged porcine oocytes.

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.

Dual effects of hydrogen sulfide donor on meiosis and cumulus expansion of porcine cumulus-oocyte complexes.

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 in vitro 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.

Endogenously produced hydrogen sulfide is involved in porcine oocyte maturation in vitro.

Hydrogen sulfide, one of three known gasotransmitters, is involved in physiological processes, including reproductive functions. Oocyte maturation and surrounding cumulus cell expansion play an essential role in female reproduction and subsequent embryonic development. Although the positive effects of exogenous hydrogen sulfide on maturing oocytes are well known, the role of endogenous hydrogen sulfide, which is physiologically released by enzymes, has not yet been described in oocytes. In this study, we observed the presence of Cystathionine β-Synthase (CBS), Cystathionine γ-Lyase (CTH) and 3-Mercaptopyruvate Sulfurtransferase (3-MPST), hydrogen sulfide-releasing enzymes, in porcine oocytes. Endogenous hydrogen sulfide production was detected in immature and matured oocytes as well as its requirement for meiotic maturation. Individual hydrogen sulfide-releasing enzymes seem to be capable of substituting for each other in hydrogen sulfide production. However, meiosis suppression by inhibition of all hydrogen sulfide-releasing enzymes is not irreversible and this effect is a result of M-Phase/Maturation Promoting Factor (MPF) and Mitogen-Activated Protein Kinase (MAPK) activity inhibition. Futhermore, cumulus expansion expressed by hyaluronic acid (HA) production is affected by the inhibition of hydrogen sulfide production. Moreover, quality changes of the expanded cumuli are indicated. These results demonstrate hydrogen sulfide involvement in oocyte maturation as well as cumulus expansion. As such, hydrogen sulfide appears to be an important cell messenger during mammalian oocyte meiosis and adequate cumulus expansion.

Nitric Oxide Donor s‐Nitroso‐n‐Acetyl Penicillamine (SNAP) Alters Meiotic Spindle Morphogenesis in Xenopus Oocytes

Nitric Oxide (NO) has been involved in both intra‐ and extra‐cellular signaling pathways in a wide range of organisms, and can be detected in some reproductive tissues. Based upon previous results reporting that NO‐donor SNAP (s‐nitroso‐n‐acetyl penicillamine) promoted the release from the metaphase II‐anaphase II block in amphibian eggs, the aim of the present study was to assess the influence of SNAP on the activation of the molecular mechanisms triggering meiotic resumption of Xenopus oocytes, analogous to G2/M transition of the cell cycle. A high concentration of SNAP (2.5 mM) was found to inhibit the appearance of the white spot (meiotic resumption) and promoted alteration of spindle morphogenesis leading to atypical structures lacking bipolarity and correct chromosomes equatorial alignment. The medium acidification (pH = 4) promoted by SNAP specifically impacted the white spot occurrence. However, even when pH was restored to 7.4 in SNAP medium, observed spindles remained atypical (microtubule disorganization), suggesting SNAP impacted spindle assembly regardless of the pH. n‐Acetyl‐d,l‐penicillamine disulfide, a degradation product of SNAP with the same molecular characteristics, albeit without release of NO, yielded spindle assemblies typical of metaphase II suggesting the specificity of NO action on meiotic spindle morphogenesis in Xenopus oocytes. J. Cell. Biochem. 116: 2445–2454, 2015.

A simple method for assessing hyaluronic acid production by cumulus-oocyte complexes

The cumulus expansion of cumulus-oocyte complex (COC) is an essential regulating process of oocyte maturation and as such it is a possible biomarker of the in vitro maturing oocytes quality. Cumulus expansion is usually assessed by non-invasive methods based on visual evaluation with many inaccuracies. On the other hand, analytical measurement of the quantity of hyaluronic acid (HA), the most abundant compound of expanded cumuli, is one of possible methods to evaluate cumulus expansion precisely. Therefore, this study aimed to verify the applicability of HA analysis for evaluating the cumulus expansion and testing oocyte maturation. The COCs were cultured in modified M199 medium for 8–48 h. The samples for the HA analysis were prepared on an 8-hour time scale, and HA retained in COCs was measured using a spectrophotometric method adapted for this purpose. We observed an increasing quantity of HA during the in vitro cultivation. A comparison with expanded COCs’ classification or expansion area proved the proposed method of HA analysis suitable for the evaluation of cumulus expansion in vitro. Our findings consider the quantity of HA-expressed cumulus expansion to be a valuable marker of COC quality enabling an adequate oocyte meiotic stage estimation.

Cumulus Cell Expansion, Its Role in Oocyte Biology and Perspectives of Measurement: A Review

Abstract Cumulus expansion of the cumulus-oocyte complex is necessary for meiotic maturation and acquiring developmental competence. Cumulus expansion is based on extracellular matrix synthesis by cumulus cells. Hyaluronic acid is the most abundant component of this extracellular matrix. Cumulus expansion takes place during meiotic oocyte maturation under in vivo and in vitro conditions. Quantification and measurement of cumulus expansion intensity is one possible method of determining oocyte quality and optimizing conditions for in vitro cultivation. Currently, subjective methods of expanded area and more exact cumulus expansion measurement by hyaluronic acid assessment are available. Among the methods of hyaluronic acid measurement is the use of radioactively labelled synthesis precursors. Alternatively, immunological and analytical methods, including enzyme-linked immunosorbent assay (ELISA), spectrophotometry, and high-performance liquid chromatography (HPLC) in UV light, could be utilized. The high sensitivity of these methods could provide a precise analysis of cumulus expansion without the use of radioisotopes. Therefore, the aim of this review is to summarize and compare available approaches of cumulus expansion measurement, respecting special biological features of expanded cumuli, and to suggest possible solutions for exact cumulus expansion analysis.

Prostaglandin E2 stimulates the expression of cumulus expansion-related genes in pigs: the role of protein kinase B

The production of prostaglandin E2 (PGE2) seems to play an important role in the ovulation process. PGE2 was found to induce cumulus expansion and meiosis resumption in mice, but little is known about its role in pigs. The goals of this study were (a) to assess the effect of PGE2 on the expression levels of cumulus expansion-related genes, (b) to define the signaling pathways that drive the PGE2-stimulated expression of cumulus expansion-related genes, (c) to measure the effect of PGE2 on the activation of key signaling molecules (MAPK3/1, PKB) and on hyaluronan production in cumulus cells, and (d) to assess the effect of PGE2 on meiosis resumption. We documented that PGE2 is able to induce the expression of cumulus expansion-related genes (HAS2, TNFAIP6) as well as genes involved in steroidogenesis (CYP11A1) or prostaglandin production (PTGS2). PGE2 is able to activate PKB and MAPK3/1 and induce mild cumulus expansion and meiosis resumption, but less efficiently than FSH.

Gasotransmitters in Gametogenesis and Early Development: Holy Trinity for Assisted Reproductive Technology—A Review

Creation of both gametes, sperm and oocyte, and their fusion during fertilization are essential step for beginning of life. Although molecular mechanisms regulating gametogenesis, fertilization, and early embryonic development are still subjected to intensive study, a lot of phenomena remain unclear. Based on our best knowledge and own results, we consider gasotransmitters to be essential for various signalisation in oocytes and embryos. In accordance with nitric oxide (NO) and hydrogen sulfide (H2S) physiological necessity, their involvement during oocyte maturation and regulative role in fertilization followed by embryonic development have been described. During these processes, NO- and H2S-derived posttranslational modifications represent the main mode of their regulative effect. While NO represent the most understood gasotransmitter and H2S is still intensively studied gasotransmitter, appreciation of carbon monoxide (CO) role in reproduction is still missing. Overall understanding of gasotransmitters including their interaction is promising for reproductive medicine and assisted reproductive technologies (ART), because these approaches contend with failure of in vitro assisted reproduction.