Heat stress impairs gap junction communication and cumulus function of bovine oocytes.

The intimate association of cumulus cells with one another and with the oocyte is important for regulating oocyte meiotic arrest and resumption. The objective of this study was to determine the effects of heat stress on cumulus cell communication and functions that may be related to accelerated oocyte meiosis during early maturation. Bovine cumulus-oocyte complexes underwent in vitro maturation for up to 6 h at thermoneutral control (38.5°C) or elevated (40.0, 41.0 or 42.0°C) temperatures. Gap junction communication between the cumulus cells and the oocyte was assessed using the fluorescent dye calcein after 4 h of in vitro maturation. Dye transfer was reduced in cumulus-oocyte complexes matured at 41.0°C or 42.0°C; transfer at 40.0°C was similar to control (P < 0.0001). Subsequent staining of oocytes with Hoechst revealed that oocytes matured at 41.0 or 42.0°C contained chromatin at more advanced stages of condensation. Maturation of cumulus-oocyte complexes at elevated temperatures reduced levels of active 5' adenosine monophosphate activated kinase (P = 0.03). Heat stress exposure had no effect on active extracellular-regulated kinase 1/2 in oocytes (P = 0.67), associated cumulus cells (P = 0.60) or intact cumulus-oocyte complexes (P = 0.44). Heat-induced increases in progesterone production by cumulus-oocyte complexes were detected during the first 6 h of maturation (P = 0.001). Heat-induced alterations in gap junction communication and other cumulus-cell functions likely cooperate to accelerate bovine oocyte meiotic progression.

Spindle abnormalities and chromosome misalignment in bovine oocytes after exposure to low doses of bisphenol A or bisphenol S.

STUDY QUESTION: What are the effects of exposure to bisphenol A (BPA) or bisphenol S (BPS) during IVM on bovine oocyte maturation, spindle morphology and chromosome alignment? SUMMARY ANSWER: Exposure to BPA or BPS during IVM resulted in increased spindle abnormalities and chromosome misalignment, even at very low concentrations. WHAT IS KNOWN ALREADY: BPA is an endocrine disrupting chemical that alters oocyte maturation, spindle morphology and chromosome alignment in a range of species. The use of BPA substitutes, such as BPS, is increasing and these substitutes often display different potencies and mechanisms of action compared with BPA. STUDY DESIGN, SIZE, DURATION: Bovine cumulus-oocyte complexes (COCs) underwent IVM with BPA or BPS for 24 h, together with vehicle-only controls. Overall, 10 different concentrations of BPA or BPS were used ranging from 1 fM to 50 µM in order to detect low dose or non-monotonic effects. An incomplete block design was utilized for the study, with at least three replicates per block. A total of 939 oocytes (250 of which were controls) were used for the BPA experiments, and 432 (110 controls) for the BPS experiments. Following the IVM period, the oocytes were denuded and fixed for immunocytochemistry. PARTICIPANTS/MATERIALS, SETTING, METHODS: Immunocytochemistry was used to label the chromatin, actin, and microtubules in the fixed oocytes. The meiotic stage was assessed using immunofluorescence, and the metaphase-II (MII) oocytes were further assessed for spindle morphology and chromosome alignment (in all MII oocytes regardless of spindle morphology) using immunofluorescence and confocal microscopy. Significant differences between the treatment and control groups were determined using chi-square and Fisher's exact tests. MAIN RESULTS AND THE ROLE OF CHANCE: There was no effect of BPA or BPS on the proportion of bovine oocytes that reached MII (P > 0.05). BPA and BPS increased spindle abnormalities in MII oocytes at almost all concentrations tested, including those as low as 1 fM (P = 0.013) or 10 fM (P < 0.0001), respectively, compared to control. Oocytes with flattened spindles with broad poles were observed at a higher frequency at some concentrations of BPA (P = 0.0002 and P = 0.002 for 10 nM and 50 µM, respectively) or BPS (P = 0.01 for 100 nM BPS), while this spindle phenotype was absent in the controls. BPA increased chromosome misalignment at concentrations of 10 fM, 10 nM and 50 µM (P < 0.0001 to P = 0.043 depending on the dose). BPS increased chromosome misalignment at concentrations of 10 fM, 100 pM, 10 nM, 100 nM and 50 µM (P < 0.0001 to P = 0.013 depending on the dose). LIMITATIONS REASONS FOR CAUTION: Exposures to BPA or BPS were performed during the IVM of COCs to allow for determination of direct effects of these chemicals on oocyte maturation. Whole follicle culture or in vivo studies will confirm whether follicular cell interactions modify the effects of BPA or BPS on oocyte meiotic maturation. Investigation into the effects of BPA or BPS on other oocyte functions will determine whether these chemicals alter oocyte quality via mechanisms independent of the meiotic endpoints characterized here. WIDER IMPLICATIONS OF THE FINDINGS: The findings of this study show that both BPA and BPS induce spindle abnormalities and chromosome misalignment in bovine in a non-monotonic manner, and at concentrations that are orders of magnitude below those measured in humans. Taken in context with previous studies on the effects of BPA in a range of species, our data support the literature that BPA may reduce oocyte quality and lead to subsequent infertility. Additionally, these results contribute to the burgeoning field of research on BPS and suggest that BPS may indeed be a 'regrettable substitution' for BPA. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by funding from the National Institutes of Health (NIH) (Grant 1R15ES024520-01). The authors declare no conflict of interest.

A protective role of cumulus cells after short-term exposure of rat cumulus cell-oocyte complexes to lifestyle or environmental contaminants.

Ovarian follicular fluid provides a potential reservoir for exogenous compounds that may adversely affect oocyte quality. This study examined the effects of common lifestyle and environmental contaminants, namely bisphenol-A (BPA), caffeine, 3,4-methylenedioxymethamphetamine (MDMA), nicotine and Δ9-tetrahydrocannabinol (THC) on gap junction genes (Gja1, Gja4) and proteins (GJA1), glucose metabolism genes (Gfpt1, Pfkp) and oocyte growth factor genes (Bmp15, Gdf9), as well as gap junction transfer rate, in rat cumulus-oocyte complexes (COCs). In vitro exposure to MDMA and THC accelerated the timing of meiotic resumption and all contaminants altered either gap junction gene expression (BPA, caffeine, MDMA and THC) or transfer rate (BPA and nicotine). In vitro exposure of COCs to MDMA also altered glucose metabolism genes. Overall, oocyte-derived genes were largely unaffected following exposure to any contaminant. In summary, the impact of short-term exposure to lifestyle and environmental contaminants on oocyte function may be diminished due to protective properties of cumulus cells.

The in-vitro effects of cAMP and cGMP modulators on inter-cellular dye transfer and gene expression levels in rat cumulus cell--oocyte complexes.

Supplementation of in-vitro maturation medium with reagents that inhibit meiotic resumption whilst supporting normal function of cumulus cell-oocyte complexes (COC) is challenging. This study compared the in-vitro effects of synthetic and physiologically-relevant reagents on meiotic resumption, gap junction activity and gene expression of rat COC. Higher doses of forskolin reduced gap junction activity. Whilst addition of phosphodiesterase inhibitors initially promoted gap junction activity, this decreased with time in-vitro. Moreover despite oocytes remaining in meiotic arrest, there was a concomitant decline in expression of genes critical for oocyte maturation, and evidence of a reduction in overall transcription rate. Similarly, supplementing media with C-type natriuretic peptide and/or oestradiol delayed meiotic resumption and only initially maintained gap junction activity. In contrast, several key genes were stimulated and overall transcription rates remained constant with time in-vitro. In summary, supplementation of media with physiologically-relevant reagents may better enable normal functions of the COC.