The genomic response of human granulosa cells (KGN) to melatonin and specific agonists/antagonists to the melatonin receptors.

Melatonin is a known modulator of follicle development; it acts through several molecular cascades via binding to its two specific receptors MT1 and MT2. Even though it is believed that melatonin can modulate granulosa cell (GC) functions, there is still limited knowledge of how it can act in human GC through MT1 and MT2 and which one is more implicated in the effects of melatonin on the metabolic processes in the dominant follicle. To better characterize the roles of these receptors on the effects of melatonin on follicular development, human granulosa-like tumor cells (KGN) were treated with specific melatonin receptor agonists and antagonists, and gene expression was analyzed with RNA-seq technology. Following appropriate normalization and the application of a fold change cut-off of 1.5 (FC 1.5, p ≤ 0.05) for each treatment, lists of the principal differentially expressed genes (DEGs) are generated. Analysis of major upstream regulators suggested that the MT1 receptor may be involved in the melatonin antiproliferative effect by reprogramming the metabolism of human GC by activating the PKB signaling pathway. Our data suggest that melatonin may act complementary through both MT1 and MT2 receptors to modulate human GC steroidogenesis, proliferation, and differentiation. However, MT2 receptors may be the ones implicated in transducing the effects of melatonin on the prevention of GC luteinization and follicle atresia at the antral follicular stage through stimulating the PKA pathway.

Zinc supports transcription and improves meiotic competence of growing bovine oocytes.

In the last years, many studies focused on the understanding of the possible role of zinc in the control of mammalian oogenesis, mainly on oocyte maturation and fertilization. However, little is known about the role of zinc at earlier stages, when the growing oocyte is actively transcribing molecules that will regulate and sustain subsequent stages of oocyte and embryonic development. In this study, we used the bovine model to gain insights into the possible involvement of zinc in oocyte development. We first mined the EmbryoGENE transcriptomic dataset, which revealed that several zinc transporters and methallothionein are impacted by physiological conditions throughout the final phase of oocyte growth and differentiation. We then observed that zinc supplementation during in vitro culture of growing oocytes is beneficial to the acquisition of meiotic competence when subsequently subjected to standard in vitro maturation. Furthermore, we tested the hypothesis that zinc supplementation might support transcription in growing oocytes. This hypothesis was indirectly confirmed by the experimental evidence that the content of labile zinc in the oocyte decreases when a major drop in transcription occurs in vivo. Accordingly, we observed that zinc sequestration with a zinc chelator rapidly reduced global transcription in growing oocytes, which was reversed by zinc supplementation in the culture medium. Finally, zinc supplementation impacted the chromatin state by reducing the level of global DNA methylation, which is consistent with the increased transcription. In conclusion, our study suggests that altering zinc availability by culture-medium supplementation supports global transcription, ultimately enhancing meiotic competence.

Responses of bovine early embryos to S-adenosyl methionine supplementation in culture.

AIM: There is a growing concern about the potential adverse effects of high dose folic acid (FA) supplementation before and during pregnancy. FA metabolism generates S-adenosyl methionine (SAM) which is an important cofactor of epigenetic programming. We sought to assess the impact of a large dose of SAM on early embryo development. MATERIALS & METHODS: In vitro cultured bovine embryos were treated with SAM from the eight-cell stage to the blastocyst stage. In addition to the phenotype, the genome-wide epigenetic and transcription profiles were analyzed. RESULTS: Treatment significantly improved embryo hatching and caused a shift in sex ratio in favor of males. SAM caused genome-wide hypermethylation mainly in exonic regions and in CpG islands. Although differentially expressed genes were associated with response to nutrients and developmental processes, no correspondence was found with the differentially methylated regions, suggesting that cellular responses to SAM treatment during early embryo development may not require DNA methylation-driven changes. CONCLUSION: Since bovine embryos were not indifferent to SAM, effects of large-dose FA supplements on early embryonic development in humans cannot be ruled out.

The impact of exposure to serum lipids during in vitro culture on the transcriptome of bovine blastocysts.

In vitro culture has a detrimental impact on early embryonic development, and serum addition to IVC is recognized to compromise blastocyst quality. Particularly, serum fatty acids affect embryonic lipid composition and reduce cryopreservation survival. To understand the molecular pathways of serum-induced embryonic stress, this study examined the early development of bovine embryos produced in different protein- or lipid-supplemented culture media: BSA alone (control), BSA + serum lipid fraction (SELF), delipidated serum and total serum. These protein-lipid treatments were applied from the eight to 16 cell stages to the blastocyst stage. As planned, SELF treatment increased the fatty acid concentration in the medium compared with control medium but did not induce embryo toxicity. However, microarray comparison between blastocysts cultured in BSA without or with SELF revealed differential transcriptomic profile associated with ceramide-induced oxidative stress and inflammation. Moreover, the SELF treatment had a significant impact on genes involved in cholesterol metabolism (LDLR, HMGCS1), with the potential upstream control of the transcription factors SREBP and PPARA, two major regulators of cholesterol metabolism. In addition, the expression of pluripotence-related genes (APEX, CLDN6) was downregulated in blastocysts subjected to either SELF or total serum. Taken together, these results illustrate how the early embryonic transcriptome responds to increased lipid exposure through an inflammatory and metabolic signature.

Identification of differentially expressed markers in human follicular cells associated with competent oocytes.

BACKGROUND: The development of an accurate method for selection of high-quality embryos is essential to achieve high pregnancy rates with single embryo transfer in human IVF. The developmental competence of the oocyte is acquired during follicle maturation and strong communication also exists between the follicular cells (FCs) and the oocytes; thus oocyte developmental competence may be determined by markers expressed in the surrounding FCs. METHODS: From consenting patients (n = 40), FCs were recovered on a per follicle basis by individual follicle puncture. Hybridization analyses using a custom-made complementary DNA microarray containing granulosa/cumulus expressed sequence tags (ESTs) from subtracted libraries and an Affymetrix GeneChip were performed to identify specific genes expressed in follicles leading to a pregnancy. The selected candidate genes were validated by quantitative-PCR (Q-PCR). RESULTS: Subtractive libraries prepared from pooled samples representing pregnant versus non-pregnant patients produced 1694 ESTs. Hybridization data analysis discriminated 115 genes associated with competent follicles. Selected candidates were confirmed by Q-PCR: 3-beta-hydroxysteroid dehydrogenase 1 (P = 0.0078), Ferredoxin 1 (P = 0.0203), Serine (or cysteine) proteinase inhibitor clade E member 2 (P = 0.0499), Cytochrome P450 aromatase (P = 0.0359) and Cell division cycle 42 (P = 0.0396). CONCLUSIONS: Microarray technologies are useful to mine the transcriptome of FCs expressed in follicles associated with competent oocytes and could be used to improve embryo selection with the objective of successful single embryo transfer.

Effect of serum and cumulus cell expansion on marker gene transcripts in bovine cumulus-oocyte complexes during maturation in vitro.

OBJECTIVE: To determine the distribution of transcripts encoding the FSH receptor (FSHr), LH receptor (LHr), connexin 43 (Cx43), cyclooxygenase-2 (COX-2), and prostaglandin E(2) receptors 2 and 3 (EP2 and EP3) within bovine cumulus-oocyte complexes (COCs) and denuded oocytes and investigate the influence of gonadotropins, serum, and cumulus cell expansion on the abundance of transcripts encoding these genes. DESIGN: Prospective controlled animal study. SETTING: University research laboratory. PATIENT(S): Animal models for human studies. INTERVENTION(S): Cumulus-oocyte complexes were treated in culture with serum and gonadotropin-supplemented media to examine the effects to mRNA transcript levels. MAIN OUTCOME MEASURE(S): Variation in mRNA transcript levels. RESULT(S): Luteinizing hormone receptor, FSHr, and EP3 mRNAs were detected in intact COCs and not in cumulus cell-denuded oocytes, whereas Cx43, COX-2, and EP2 mRNAs were found in both COCs and oocytes. The relative abundance of marker gene mRNAs did not vary in media containing no additives or FSH alone, independent of whether the media induced cumulus cell expansion. However, the presence of serum in maturation media significantly decreased expression of all mRNAs except LHr. CONCLUSION(S): The relative abundance of COC mRNAs is altered by serum in the maturation medium, which may signify long-term consequences for embryonic development.