Toxicological investigation of lilial.

Lilial (also called lysmeral) is a fragrance ingredient presented in many everyday cosmetics and household products. The concentrations of lilial in the final products is rather low. Its maximum concentration in cosmetics was limited and recently, its use in cosmetics products was prohibited in the EU due to the classification as reproductive toxicant. Additionally, according to the European Chemicals Agency, it was under assessment as one of the potential endocrine disruptors, i.e. a substance that may alter the function of the endocrine system and, as a result, cause health problems. Its ability to act as an androgen receptor agonist and the estrogenic and androgenic activity of its metabolites, to the best of our knowledge, have not yet been tested. The aim of this work was to determine the intestinal absorption, cytotoxicity, nephrotoxicity, mutagenicity, activation of cellular stress-related signal pathways and, most importantly, to test the ability to disrupt the endocrine system of lilial and its Phase I metabolites. This was tested using set of in vitro assays including resazurin assay, the CHO/HPRT mutation assay, γH2AX biomarker-based genotoxicity assay, qPCR and in vitro reporter assays based on luminescence of luciferase for estrogen, androgen, NF-κB and NRF2 signalling pathway. It was determined that neither lilial nor its metabolites have a negative effect on cell viability in the concentration range from 1 nM to 100 µM. Using human cell lines HeLa9903 and MDA-kb2, it was verified that this substance did not have agonistic activity towards estrogen or androgen receptor, respectively. Lilial metabolites, generated by incubation with the rat liver S9 fraction, did not show the ability to bind to estrogen or androgen receptors. Neither lilial nor its metabolites showed a nephrotoxic effect on human renal tubular cells (RPTEC/TERT1 line) and at the same time they were unable to activate the NF-κB and NRF2 signalling pathway at a concentration of 50 µM (HEK 293/pGL4.32 or pGL4.37). Neither lilial nor its metabolites showed mutagenic activity in the HPRT gene mutation test in CHO-K1 cells, nor were they able to cause double-strand breaks in DNA (γH2AX biomarker) in CHO-K1 and HeLa cells. In our study, no negative effects of lilial or its in vitro metabolites were observed up to 100 µM using different in vitro tests.

The bisphenol S contamination level observed in human follicular fluid affects the development of porcine oocytes.

Bisphenol S (BPS), the main replacement for bisphenol A (BPA), is thought to be toxic, but limited information is available on the effects of Bisphenol S on ovarian follicles. In our study, we demonstrated the presence of Bisphenol S in the follicular fluid of women at a concentration of 22.4 nM. The effect of such concentrations of Bisphenol S on oocyte maturation and subsequent embryo development is still unknown. Therefore, we focused on the effect of Bisphenol S on in vitro oocyte maturation, fertilization, and embryo development. As a model, we used porcine oocytes, which show many physiological similarities to human oocytes. Oocytes were exposed to Bisphenol S concentrations similar to those detected in female patients in the ART clinic. We found a decreased ability of oocytes to successfully complete meiotic maturation. Mature oocytes showed an increased frequency of meiotic spindle abnormalities and chromosome misalignment. Alarming associations of oocyte Bisphenol S exposure with the occurrence of aneuploidy and changes in the distribution of mitochondria and mitochondrial proteins were demonstrated for the first time. However, the number and quality of blastocysts derived from oocytes that successfully completed meiotic maturation under the influence of Bisphenol S was not affected.

Evidence of endogenously produced hydrogen sulfide (H2S) and persulfidation in male reproduction.

Persulfidation contributes to a group of redox post-translational modifications (PTMs), which arise exclusively on the sulfhydryl group of cysteine as a result of hydrogen sulfide (H2S) action. Redox-active molecules, including H2S, contribute to sperm development; therefore, redox PTMs represent an extremely important signalling pathway in sperm life. In this path, persulfidation prevents protein damage caused by irreversible cysteine hyperoxidation and thus maintains this signalling pathway. In our study, we detected both H2S and its production by all H2S-releasing enzymes (cystathionine γ-lyase (CTH), cystathionine ß-synthase (CBS), and 3-mercaptopyruvate sulfurtransferase (MPST)) in male reproduction, including spermatozoa. We provided evidence that sperm H2S leads to persulfidation of proteins, such as glyceraldehyde-3-phosphate dehydrogenase, tubulin, and anchor protein A-kinase. Overall, this study suggests that persulfidation, as a part of the redox signalling pathway, is tightly regulated by enzymatic H2S production and is required for sperm viability.

Exposure to alternative bisphenols BPS and BPF through breast milk: Noxious heritage effect during nursing associated with idiopathic infertility.

There is increasing evidence that bisphenols BPS and BPF, which are analogues of BPA, have deleterious effects on reproduction even at extremely low doses. Indirect exposure via the maternal route (i.e. across the placenta and/or by breastfeeding) is underestimated, although it can be assumed to be a cause of idiopathic female infertility. Therefore, we hypothesised the deleterious effects of exposure to BPA analogues during breastfeeding on the ovarian and oocyte quality of offspring. A 15-day exposure period of pups was designed, whilst nursing dams (N ≥ 6 per experimental group) were treated via drinking water with a low (0.2 ng/g body weight/day) or moderate (20 ng/g body weight/day) dose of bisphenol, mimicking real exposure in humans. Thereafter, female pups were bred to 60 days and oocytes were collected. Immature oocytes were used in the in-vitro maturation assay; alternatively, in-vivo-matured oocytes were isolated and used for parthenogenetic activation. Both in-vitro- and in-vivo-matured oocytes were subjected to immunostaining of spindle microtubules (α-tubulin) and demethylation of histone H3 on the lysine K27 (H3K27me2) residue. Although very low doses of both BPS and BPF did not affect the quality of ovarian histology, spindle formation and epigenetic signs were affected. Notably, in-vitro-matured oocytes were significantly sensitive to both doses of BPS and BPF. Although no significant differences in spindle-chromatin quality were identified in ovulated and in-vivo-matured oocytes, developmental competence was significantly damaged. Taken together, our mouse model provides evidence that bisphenol analogues represent a risk to human reproduction, possibly leading to idiopathic infertility in women.

Acute low-dose bisphenol S exposure affects mouse oocyte quality.

Bisphenol S (BPS) is widely used to replace the known endocrine disruptor BPA in various products. We evaluated the effect of acute in vivo BPS exposure on oocyte quality, simulating the oral route of exposure via oral gavage. Eight-week-old ICR female mice (N = 15 per experimental group) were exposed to vehicle or BPS1-BPS4 (0.001, 0.1, 10, and 100 ng BPS x g bw-1 day-1, respectively) for seven days. Oocytes were isolated and matured in vitro. We observed that BPS exposure increased aberrant spindle formation in mature oocytes and induced DNA damage. Moreover, BPS3 significantly increased the chromatin repressive marks 5-methyl cytosine (5meC) and H3K27me2 in immature oocytes. In the BPS2 group, the increase in 5meC occurred during oocyte maturation. Transcriptome analysis revealed differential expression of early embryonic development transcripts in BPS2-exposed oocytes. These findings indicate that the biological effect of BPS is non-monotonic, affecting oocyte quality even at concentrations that are orders of magnitude below those measured in humans.

Epigenetic and non-epigenetic mode of SIRT1 action during oocyte meiosis progression.

BACKGROUND: SIRT1 histone deacetylase acts on many epigenetic and non-epigenetic targets. It is thought that SIRT1 is involved in oocyte maturation; therefore, the importance of the ooplasmic SIRT1 pool for the further fate of mature oocytes has been strongly suggested. We hypothesised that SIRT1 plays the role of a signalling molecule in mature oocytes through selected epigenetic and non-epigenetic regulation. RESULTS: We observed SIRT1 re-localisation in mature oocytes and its association with spindle microtubules. In mature oocytes, SIRT1 distribution shows a spindle-like pattern, and spindle-specific SIRT1 action decreases α-tubulin acetylation. Based on the observation of the histone code in immature and mature oocytes, we suggest that SIRT1 is mostly predestined for an epigenetic mode of action in the germinal vesicles (GVs) of immature oocytes. Accordingly, BML-278-driven trimethylation of lysine K9 in histone H3 in mature oocytes is considered to be a result of GV epigenetic transformation. CONCLUSIONS: Taken together, our observations point out the dual spatiotemporal SIRT1 action in oocytes, which can be readily switched from the epigenetic to non-epigenetic mode of action depending on the progress of meiosis.

Involvement of K+ATP and Ca2+ channels in hydrogen sulfide-suppressed ageing of porcine oocytes.

BACKGROUND: Hydrogen sulfide has been shown to improve the quality of oocytes destined for in vitro fertilization. Although hydrogen sulfide is capable of modulating ion channel activity in somatic cells, the role of hydrogen sulfide in gametes and embryos remains unknown. Our observations confirmed the hypothesis that the KATP and L-type Ca2+ ion channels play roles in porcine oocyte ageing and revealed a plausible contribution of hydrogen sulfide to the modulation of ion channel activity. RESULTS: We confirmed the benefits of the activation and suppression of the KATP and L-type Ca2+ ion channels, respectively, for the preservation of oocyte quality. CONCLUSIONS: Our experiments identified hydrogen sulfide as promoting the desired ion channel activity, with the capacity to protect porcine oocytes against cell death. Further experiments are needed to determine the exact mechanism of hydrogen sulfide in gametes and embryos.

Long-term exposure to very low doses of bisphenol S affects female reproduction.

Bisphenols belong to the endocrine disruptors, affecting reproduction even in extremely low doses. Bisphenol S (BPS) has become widely used as a substitute for the earlier-used bisphenol A; however, its harmlessness is questionable. The aim of this study was to evaluate the effect of BPS on folliculogenesis and oocyte quality after in vivo exposure to low doses of BPS. Four-week-old ICR females (n = 16 in each experimental group) were exposed to vehicle control (VC), BPS1 (0.001 ng BPS.g/bw/day), BPS2 (0.1 ng.g/bw/day), BPS3 (10 ng.g/bw/day) and BPS4 (100 ng.g/bw/day) for 4 weeks. Ovaries were subjected to stereology and nano liquid chromatography-mass spectrometry (LC/MS). Simultaneously, metaphase II oocytes were obtained after pregnant mare serum gonadotrophin and human chorionic gonadotrophin administration, followed by immunostaining. In particular, mating and two-cell embryo flushing were performed. We observed that BPS decreases the amount of ovarian follicles and BPS2 (0.1 ng.g/bw/day) affects the volume of antral follicles. Accordingly, ovarian proteome is affected after BPS2 treatment. While BPS2 dosing results mainly in cytoskeletal damage in matured oocytes, the effects of BPS3 and BPS4 seem to be due instead to epigenetic alterations in oocytes. Arguably, these changes lead to observed affection of in vivo fertilization rate after BPS3 and BPS4 treatment. BPS significantly affects female reproduction astoundingly in extremely low doses. These findings underline the necessity to assess the risk of ongoing BPS exposure for public health.

Involvement of K+ATP and Ca2+ channels in hydrogen sulfide-suppressed ageing of porcine oocytes

BACKGROUND: Hydrogen sulfide has been shown to improve the quality of oocytes destined for in vitro fertilization. Although hydrogen sulfide is capable of modulating ion channel activity in somatic cells, the role of hydrogen sulfide in gametes and embryos remains unknown. Our observations confirmed the hypothesis that the KATP and L-type Ca2+ ion channels play roles in porcine oocyte ageing and revealed a plausible contribution of hydrogen sulfide to the modulation of ion channel activity. RESULTS: We confirmed the benefits of the activation and suppression of the KATP and L-type Ca2+ ion channels, respectively, for the preservation of oocyte quality. CONCLUSIONS: Our experiments identified hydrogen sulfide as promoting the desired ion channel activity, with the capacity to protect porcine oocytes against cell death. Further experiments are needed to determine the exact mechanism of hydrogen sulfide in gametes and embryos.

SIRT1-dependent modulation of methylation and acetylation of histone H3 on lysine 9 (H3K9) in the zygotic pronuclei improves porcine embryo development.

BACKGROUND: The histone code is an established epigenetic regulator of early embryonic development in mammals. The lysine residue K9 of histone H3 (H3K9) is a prime target of SIRT1, a member of NAD+-dependent histone deacetylase family of enzymes targeting both histone and non-histone substrates. At present, little is known about SIRT1-modulation of H3K9 in zygotic pronuclei and its association with the success of preimplantation embryo development. Therefore, we evaluated the effect of SIRT1 activity on H3K9 methylation and acetylation in porcine zygotes and the significance of H3K9 modifications for early embryonic development. RESULTS: Our results show that SIRT1 activators resveratrol and BML-278 increased H3K9 methylation and suppressed H3K9 acetylation in both the paternal and maternal pronucleus. Inversely, SIRT1 inhibitors nicotinamide and sirtinol suppressed methylation and increased acetylation of pronuclear H3K9. Evaluation of early embryonic development confirmed positive effect of selective SIRT1 activation on blastocyst formation rate (5.2 ± 2.9% versus 32.9 ± 8.1% in vehicle control and BML-278 group, respectively; P ≤ 0.05). Stimulation of SIRT1 activity coincided with fluorometric signal intensity of ooplasmic ubiquitin ligase MDM2, a known substrate of SIRT1 and known limiting factor of epigenome remodeling. CONCLUSIONS: We conclude that SIRT1 modulates zygotic histone code, obviously through direct deacetylation and via non-histone targets resulting in increased H3K9me3. These changes in zygotes lead to more successful pre-implantation embryonic development and, indeed, the specific SIRT1 activation due to BML-278 is beneficial for in vitro embryo production and blastocyst achievement.

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 ß 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.

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 ß-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.

Src family kinases are involved in the meiotic maturation of porcine oocytes.

Mammalian meiotic maturation is regulated by changes in the phosphorylation state of proteins involved in signalling pathways. The regulatory proteins include the family of Src tyrosine kinases. Src family kinases (SFKs) are required for meiotic maturation of mouse oocytes, and it remains to be elucidated whether they play the same role in porcine oocytes. To clarify the role of SFKs in the meiotic maturation of porcine oocytes we used inhibition of SFKs, western blotting and immunolocalisation to determine the presence of SFKs and localisation in the oocytes and assays to determine the activity of maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK). Inhibition of SFKs resulted in the disruption of oocyte maturation and led to a decline in MPF and MAPK activity. The fluorescence intensity of SFKs in the cytoplasm and membrane of MI oocytes decreased significantly compared with germinal vesicle oocytes. The highest fluorescence intensity for SFKs was detected on the membrane of MII oocytes. Only weak fluorescence was detected in the perichromosomal area of MI and MII oocytes. These results prove that SFKs play an active role in the meiotic maturation of porcine oocytes by regulating MPF and MAPK activity.

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.

Calcineurin expression and localisation during porcine oocyte growth and meiotic maturation.

The processes of oocyte growth, acquisition of meiotic competence and meiotic maturation are regulated by a large number of molecules. One of them could be calcineurin consisting of catalytic subunit A (Aα, Aß, Aγ isoforms) and regulatory subunit B (B1, B2 isoforms). Calcineurin is involved in the meiotic maturation of oocytes in invertebrates or in lower vertebrates. In the mammalian oocytes, the possible role of calcineurin in the regulation of oocyte meiosis has not been clarified to date. In this study, to investigate the role of calcineurin during porcine oocyte growth, acquisition of meiotic competence and meiotic maturation, we analysed the expression and localisation of calcineurin subunits and the mRNA expression of calcineurin isoforms. Calcineurin was expressed in growing porcine oocytes, in fully grown oocytes and during their in vitro meiotic maturation. We found both subunits of calcineurin. Calcineurin A and calcineurin B were localised mainly in the cortex in all porcine oocytes. The changes in the intracellular localisation of separate calcineurin subunits during meiotic maturation were determined. We detected mRNA for calcineurin isoforms Aß, Aγ, B2 in oocytes and mRNA for calcineurin isoforms Aß, Aγ, B1, and B2 in cumular cells. To our knowledge, this is the first confirmation of calcineurin presence in porcine oocytes.