The effects of crocetin supplementation on the blastocyst outcome, transcriptomic and metabolic profile of in vitro produced bovine embryos.

The earliest stages of embryo development are deeply influenced by reactive oxygen species (ROS), byproducts of the mitochondrial oxygen metabolism that play a key role as messengers in normal cell signal transduction and cell cycling. Despite its positive roles, the imbalance caused by the excess of ROS and an inefficient antioxidant system leads to oxidative stress, with negative consequences to the cell such as DNA damage, metabolic changes, mitochondrial stress and cell death. In the present work, crocetin - a natural antioxidant - was added to the culture media of bovine embryos to evaluate the efficiency of its antioxidant capability during embryo culture. Oocytes were in vitro matured (IVM) and fertilized according to standard protocols. Embryos were cultured at 38.5 °C under humidified air with 5% CO2, 7% O2, and 90% N2 in Synthetic Oviduct Fluid (SOF) medium supplemented with amino acids and either 5% of FBS (SOFaa) (control group) or SOFaa supplemented with 1  µM crocetin (crocetin group). After 5 days from the beginning of in vitro culture (IVC) (day 5 - D5), embryos were transferred to individual drops of culture media. At day 7 (D7), embryos were assessed by means of blastocyst rates, morphophysiological analyzes (total cell number, ROS and mitochondrial activity levels), transcript quantitation of 47 genes and metabolomic evaluation of the culture media by Raman spectroscopy. In the crocetin group blastocyst rates were higher and embryos had increased total cell number and decreased intracellular levels of ROS. These embryos also had upregulation of genes related with response to stress and lipid metabolism (ATF4, BAX, FOXO3, GADD45A, GPX1, GPX4, HSF1, SOD2, ACACA, SREBF1 and SREBF2). Raman spectroscopy corroborated these results indicating more active lipid and amino acid production in this group. The absence of crocetin in the culture media resulted in higher ROS level, as well as up regulation of genes related to DNA damage, stress response and energy metabolism (MORF4L2, SOD1, TXN, PFKP, PGK1 and PPARGC1A). In conclusion, crocetin supplementation during culture protects embryos from oxidative stress and influences the adaptive response to stress conditions, leading to an increase in both blastocyst yield and quality, as well as changes in transcriptomic and metabolic profile of in vitro produced bovine embryos.

Effect of superstimulation on the expression of microRNAs and genes involved in steroidogenesis and ovulation in Nelore cows.

To better understand the impact of ovarian superstimulation on bovine follicular microenvironment, Nelore cows (Bos taurus indicus) were subjected to ovarian superstimulation with follicle stimulating hormone (FSH, n = 10; P-36 protocol) or FSH combined with eCG (n = 10; P-36/eCG protocol). Follicular fluid was analyzed for cholesterol concentration. Granulosa cells were analyzed by RT-qPCR to assess the expression of genes involved in steroidogenic and ovulatory and expression of microRNAs involved in final follicular development and luteinizing hormone/choriogonadotropin receptor (LHCGR) expression. Plasma concentration of estradiol was also measured. Follicular fluid from the P-36 group showed higher concentration of cholesterol than that of control (non-superstimulated) cows. Plasma concentration of estradiol was higher in the P-36/eCG group. Abundance of STAR and FSHR mRNAs were lower in granulosa cells from the P-36/eCG group. In contrast, LHCGR mRNA abundance was higher in superstimulated granulosa cells from the P-36 group and showed a pattern opposite to that of miR-222 expression. Ovarian superstimulation did not affect the expression of other markers (mmu-miR-202-5p, has-miR-873, has-miR-144, and their target genes, CREB, TGFBR2, and ATG7) of antral follicle development. However, the mRNA expression of VEGF pathway components was modulated by P-36 treatment. Taken together, these results demonstrate that superstimulatory protocols modify steroidogenic capacity, increase plasma estradiol, and regulate the abundance of VEGF system, LHCGR mRNA and suppress the expression of miR-222 in bovine granulosa cells.

Lipid profiles of follicular fluid from cows submitted to ovarian superstimulation.

Ovarian superstimulation with exogenous gonadotropins has been extensively used to produce in vivo-derived embryos for embryo transfer in cattle. This process modifies the antral follicle microenvironment and affects oocyte and embryo quality as well the differentiation of granulosa cells. Lipids play significant roles in the cell, such as energy storage, cell structure, and fine-tuning of the physical properties and functions of biological membranes. The phospholipid (PL) contents as well as the effects of superstimulatory treatments on the PL profile of follicular fluid from cows, however, remain unknown. Therefore, to gain insight into the effects of superstimulation with follicle-stimulating hormone (FSH; P-36 protocol) or FSH combined with equine chorionic gonadotropin (eCG; P-36/eCG protocol) on the profile and abundance of PL from cows submitted or not submitted to superstimulatory protocols, were treated with these two superstimulatory protocols. As a control, non-superstimulated cows were only submitted to estrous synchronization. The follicular fluid was aspirated, the remaining cells removed and the follicular fluid stored at -80 °C until extraction. The lipid screening was performed by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) and this technique allowed the identification of sphingomyelins (SM) and phosphatidylcholines (PC) and phosphoethanolamines (PE). The relative abundance of the ions observed in the three experimental groups was analyzed by multivariate and univariate statistical models. The phospholipid SM (16:0) and PC (36:4) and/or PC (34:1) were less (P < 0.05) abundant in the P-36 group compared to the control or P-36/eCG groups. However, the PC (34:2) was more (P < 0.05) abundant in both group of superstimulated cows compared to the control. In summary, ovarian superstimulation seems to modulate the PL content of bovine follicular fluid with a significant increase in PC (34:2), which jointly with others PC and SM, seems to offer a suitable biomarker involved with reproductive processes successful as ovary superstimulation response and embryo development.

Expression of mRNA Encoding the LH Receptor (LHR) and LHR Binding Protein in Granulosa Cells from Nelore (Bos indicus) Heifers Around Follicle Deviation.

The time at which follicles acquire LHR in bovine granulosa cells is the subject of some controversy among researchers. The main objective of the present study was to assess the mRNA expression of LHR and LRBP (mRNA protein binding), a post-transcriptional suppressor of LHR mRNA expression, in granulosa cells from the two largest follicles around the expected time of follicle deviation in Nelore heifers. First, the interval between ovulation and follicle deviation in 20 Nelore heifers was determined (2.3 ± 0.2 days after ovulation). Ovulation was hormonally synchronized, and then, heifers were slaughtered on days 2, 2.5 and 3 after ovulation (before, during and after, respectively, the expected time of follicle deviation), and granulosa cells from the two largest follicles were collected. The mRNA abundance of an LHR fragment common to all isoforms (total LHR) and LRBP was assessed by real-time RT-PCR, and LHR alternative transcripts were assessed by semiquantitative RT-PCR followed by electrophoresis. LHR mRNA expression was not detected before the expected time of deviation. Total LHR mRNA abundance was greater in the largest follicle and increased from day 2.5 to 3. In contrast, LRBP mRNA was detected starting on day 2 and was more expressed in the second largest follicle on days 2.5 and 3. The present data suggest that the expression of LHR mRNA in bovine granulosa cells is established after follicle deviation and that the lower abundance of LRBP mRNA after the expected time of deviation may contribute to greater expression of LHR in the bovine dominant follicle.

Ovarian superstimulation using FSH combined with equine chorionic gonadotropin (eCG) upregulates mRNA-encoding proteins involved with LH receptor intracellular signaling in granulosa cells from Nelore cows.

The LH plays a key role in controlling physiological processes in the ovary acting via LH receptor (LHR). In general, the effects of LHR on the regulation of granulosa cell differentiation are mediated mainly via the Gs-protein/adenylyl cyclase/cAMP system; however, the LHR activation could also induce phospholipase C (PLC)/inositol trisphosphate (IP3) via Gq/11 system. Additionally, the expression of G-proteins (GNAS, GNAQ, and GNA11) and PLC ß has been showed in bovine antral follicle, concomitant with an increase in LHR expression. To gain insight into the effects of superstimulation with FSH (P-36 protocol) or FSH combined with equine chorionic gonadotropin (eCG; P-36/eCG protocol) on the mRNA expression of proteins involved in LHR signaling in bovine granulosa cells, Nelore cows (Bos indicus) were treated with two superstimulatory protocols: P-36 protocol or P-36/eCG protocol (replacement of the FSH by eCG administration on the last day of treatment). Nonsuperstimulated cows were only submitted to estrous synchronization without ovarian superstimulation. The granulosa cells were harvested from follicles and mRNA abundance of GNAS, GNAQ, GNA11, PLCB1, PLCB, PLCB4, and adenylyl cyclase isoforms (ADCY3, ADCY4, ADCY6, ADCY8, and ADCY9) was measured by real-time reserve transcription followed by polymerase chain reaction. No differences on mRNA abundance of target genes were observed in granulosa cells of cows submitted to P-36 protocol compared with control group. However, the cows submitted to P-36/eCG protocol showed upregulation on the mRNA abundance of target genes (except ADCY8) in granulosa cells. Although the P-36 protocol did not regulate mRNA expression of the proteins involved in the signaling mechanisms of the cAMP and IP3 systems, the constant presence of GNAS, GNAQ, GNA11, PLCB1, PLCB3, PLCB4, and adenylyl cyclase isoforms (ADCY3, ADCY4, ADCY6, and ADCY9) mRNA and the upregulation of these genes in granulosa cells from cows submitted to P-36/eCG protocol reinforce the participation of Gq/11/PLC/IP3 signaling as well as Gs-protein/adenylyl cyclase/cAMP system on LHR pathways during bovine granulosa cell differentiation submitted to superstimulatory treatments.

Expression of LH receptor mRNA splice variants in bovine granulosa cells: changes with follicle size and regulation by FSH in vitro.

In cattle, most evidence suggests that granulosa cells express LH receptors (LHR) after (or as) the follicle becomes dominant, however there is some suggestion that granulosa cells from smaller pre-dominant follicles may express several LHR mRNA splice variants. The objective of this study was to measure LHR expression in bovine follicles of defined size and steroidogenic ability, and in granulosa cells from small follicles (<6 mm diameter) undergoing differentiation in vitro. Semiquantitative RT-PCR demonstrated that LHR mRNA was undetectable in granulosa cells of follicles <7 mm diameter (nondominant follicles), and increased with follicle diameter in follicles >7 mm diameter. Splice variants with deletions of exon 10 and part of exon 11 were detected as previously described, and we detected a novel splice variant with a deletion of exon 3. Cultured granulosa cells contained LHR mRNA, but with significantly greater amounts of variants with deletions of exon 10 and/or exon 11 compared with cells from dominant follicles. FSH increased the abundance of some but not all LHR mRNA splice variants in cultured granulosa cells. The addition of LH to cultured cells did not increase progesterone secretion, despite the presence of LHR mRNA. Collectively, these data suggest that granulosa cells do not acquire functional LHR until follicle dominance occurs.