Search

Lipid metabolism is essential for ensuring oocyte maturation and embryo development. β-Oxidized fatty acids (FA) are a potent source of energy for cells, particularly for bovine somatic follicular cells. Superstimulatory protocols using follicle stimulating hormone (FSH) or FSH combined with equine chorionic gonadotropin (eCG) are capable of stimulating the follicular microenvironment and drive the expression of biomarker genes associated with lipid metabolism in the cumulus-oocyte complex (COC) for better embryo development. In this study, we assesed the effects of FSH and FSH/eCG protocols on the expression of genes related to lipid metabolism in bovine granulosa cells (GCs). Further, we measured triglyceride levels in follicular fluid (FF) obtained from both superstimulatd and non-superstimulated cows (synchronized cows). In summary, superstimulation with gonadotropins maintained the TG levels in bovine FF and ensured GCs mRNA abundance of ACSL1, ACSL3, ACSL6, SCD, ELOVL5, ELOVL6, FASN, FADS2, and SREBP1. We, however, found the abundance of CPTIB mRNA to be lower in GCs obtained from cows subjected to FSH/eCG protocols than synchronized cows. In conclusion, the findings of this study showed that ovarian superstimulation around the preovulatory phase has a mild impact on the lipid metabolism in GCs., Competing Interests: Conflicts of interest: The authors have no conflict of interest to declare., (Copyright © The Author(s).)

The polyspermy occurrence is considerably lower under in vivo compared to in vitro embryo culture conditions, suggesting that the presence of some factors in the maternal environment is responsible for this. The α-L-fucosidase (FUCA) is a natural glycosidase present in the oviductal fluid, therefore, this study aimed at investigating the effect of adding FUCA to the hardening of the zona pellucida (ZP), polyspermy control, and embryonic yield and quality of bovine blastocysts produced in vitro. In the first experiment, the effect of FUCA (0.125 U/mL) was evaluated during the entire in vitro fertilization (IVF). However, it was demonstrated to be embryotoxic by completely inhibiting the blastocyst formation. In the second experiment, the FUCA (0.125 U/mL) was tested as short-term incubation before IVF (pre-fertilization step) for 30 min or 2 h, which demonstrated that FUCA treatment for 30 min resulted in ZP hardening. In the third experiment, a pre-fertilization FUCA treatment (1 h) at different concentrations (0, 0.0625, and 0.125 U/mL) showed that FUCA (0.0625 U/mL) improved pre-fertilization ZP hardening and tended to increase monospermic fertilization rates but did not improve embryo yield and quality. Together, it has been demonstrated that FUCA can induce oocyte pre-fertilization ZP hardening and might improve monospermic fertilization performance, and this effect is dependent on both variables (protein concentration and incubation time)., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

During embryo development, the endoplasmic reticulum (ER) acts as an important site for protein biosynthesis; however, in vitro culture (IVC) can negatively affect ER homeostasis. Therefore, the aim of our study was to evaluate the effects of the supplementation of tauroursodeoxycholic acid (TUDCA), an ER stress inhibitor, in the IVC of bovine embryos. Two experiments were carried out: Exp. 1: an evaluation of blastocyst rate, hatching kinetics, and gene expression of hatched embryos after being treated with different concentrations of TUDCA (50, 200, or 1000 μM) in the IVC; Exp. 2: an evaluation of the re-expansion, hatching, and gene expression of hatched embryos previously treated with 200 µM of TUDCA at IVC and submitted to vitrification. There was no increase in the blastocyst and hatched blastocyst rates treated with TUDCA in the IVC. However, embryos submitted to vitrification after treatment with 200 µM of TUDCA underwent an increased hatching rate post-warming together with a down-regulation in the expression of ER stress-related genes and the accumulation of lipids. In conclusion, this work showed that the addition of TUDCA during in vitro culture can improve the cryotolerance of the bovine blastocyst through the putative modulation of ER and oxidative stress.

Insulin-like growth factor-1 (IGF-1) regulates cellular lipid content, whereas pregnancy-associated plasma protein-A (PAPP-A) increases IGF-1 bioavailability. Using in vitro-matured cumulus-oocyte complexes, we aimed to evaluate the impact of PAPP-A on the blastocyst lipid content, embryo cryotolerance and embryonic transcriptional profile. We determined that PAPP-A did not affect the lipid content of oocytes, blastocysts, or blastocyst yield (P > 0.05). However, PAPP-A modulated the embryo transcriptional profiles by downregulating PPARGC1A and AKR1B1, which are related to lipid metabolism; CASP9, a pro-apoptotic gene; and IFN-τ, a marker of embryo quality (P < 0.05). Furthermore, the use of PAPP-A improved blastocyst re-expansion in the first 3 h of culture after vitrification (P < 0.05). Although PAPP-A did not affect the blastocyst lipid content or embryo production, we suggest that embryonic transcriptional modulation could contribute to maintain the balance in embryo lipid metabolism. Furthermore, PAPP-A's approach seems to control key intracellular pathways that improve post-cryopreservation development of blastocysts., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

Final antral follicle development and future ovulation are mediated by gonadotropin-induced changes with spatio-temporally regulated expression of genes. Here, we aimed to quantify the relative mRNA abundance of bta-miR-222 and its predicted target, LHCGR, in granulosa cells (GCs) from follicles, after follicle deviation, as well as from GCs cultured in vitro with follicle stimulating hormone (FSH) and/or insulin. Thus, to study the impact of follicle deviation, Nelore heifers (n = 10; Bos taurus indicus) were hormonally synchronized and slaughtered 3 days after ovulation. Then, GCs from the dominant follicle (DF) and its respective subordinate follicle (SF) were recovered for RT-qPCR. For in vitro analysis, small follicles (2-5 mm) were dissected from bovine ovaries collected from a local abattoir. The GCs were isolated and cultured in serum-free medium, or treated with insulin (1 ng/mL or 10 ng/mL) alone or in combination with human recombinant FSH (1 ng/mL), for 6 days. Our findings showed that the relative mRNA abundance of LHCGR in GCs was higher in the DF compared to the SF (p = 0.01). Inversely, bta-miR-222 expression was lower in the DF compared to the SF (p = 0.01). Furthermore, GCs cultured with FSH and insulin together resulted in a higher abundance of LHCGR and a lower abundance of bta-miR-222 (p ≤ 0.05) when compared to GCs cultured with insulin alone. In conclusion, we found that the LHCGR upregulation in GCs from the DF is inversely related to bta-miR-222 expression. We also suggest the involvement of FSH in bta-miR-222 suppression in healthy bovine GCs., Competing Interests: Declaration of interest We declare that there is no conflict of present work., (Copyright © 2022 Elsevier Inc. All rights reserved.)

In several species, oocyte and embryo competence are improved by the addition of endoplasmic reticulum (ER) stress inhibitors to in vitro maturation (IVM) medium and/or in vitro culture (IVC) medium. This study aimed to evaluate the effects of three concentrations of tauroursodeoxycholic acid (TUDCA; 50, 200, and 1,000 μM), a chemical chaperone for relieving ER stress, during IVM of bovine cumulus-oocyte complexes (COCs) for 24 h. Treated oocytes were analyzed for nuclear maturation, reactive oxygen species (ROS) production, mitochondrial activity, and abundance of target transcripts. In addition, the number of pronuclei in oocytes was evaluated after 18-20 h of insemination, and the rates of blastocyst and hatched blastocyst formation were evaluated after 7 and 8/9 days of culture, respectively. We further evaluated the transcript abundance of embryonic quality markers. Our findings showed that supplementation of IVM medium with 200 μM of TUDCA decreased ROS production and increased abundance of transcripts related to antioxidant activity in oocytes ( CAT , GPX1 , and HMOX1 ) and embryos ( GPX1 and PRDX3 ). Interestingly, high concentration of TUDCA (1,000 μM) was toxic to oocytes, reducing the nuclear maturation rate, decreasing mitochondrial activity, and increasing the abundance of ER stress ( HSPA5 ) and cellular apoptosis ( CASP3 and CD40 ) related transcripts. The results of this study suggest that treatment with 200 μM of TUDCA is associated with a greater resistance to oxidative stress and indirectly with ER stress relief in bovine oocytes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Pioltine, Costa, Barbosa Latorraca, Franchi, dos Santos, Mingoti, Paula-Lopes and Nogueira.)

Our objectives were to investigate potential changes in the size of steroidogenic large luteal cells (LLC) during partial luteolysis induced by a sub-dose of cloprostenol in early diestrus and to determine transcriptional variations in genes involved in corpus luteum (CL) functions. Cows were subjected to an Ovsynch protocol, with the time of the second GnRH treatment defined as Day 0 (D0). On D6, cows were randomly allocated into three treatments: Control (2 mL saline, im; n = 10), 2XPGF (two doses of 500 μg of cloprostenol, im, 2 h apart; n = 8) or 1/6PGF (single dose of 83.3 μg of cloprostenol, im; n = 10). Before treatments and every 8 h during the 48-h experimental period, blood samples were collected and CL volumes measured. Furthermore, two CL biopsies were obtained at 24 and 40 h post-treatment. The 1/6PGF treatment caused partial luteolysis, characterized by sudden decreases in plasma progesterone (P 4 ) concentrations, luteal volume and LLC size, followed by increases (to pretreatment values) in P 4 and luteal volume at 24 and 40 h post-treatment, respectively. However, at the end of the study, P4, luteal volume and LLC size were all significantly smaller than in Control cows. Temporally associated with these phenotypes, there was a lower mRNA abundance of VEGFA at 24 and 40 h, and ABCA1 at 24 h (P < 0.05). In conclusion, a sudden reduction in CL size during partial luteolysis induced by a sub-dose of PGF 2α analog on day 6 of the estrous cycle was attributed to a reduction in LLC size, although these changes did not account for the entire phenomenon. In addition to its involvement in reducing CL size, decreased VEGFA mRNA abundance impaired CL development, resulting in a smaller luteal gland and lower plasma P 4 concentrations compared to Control cows., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Although studies have focused on extracellular matrix (ECM) remodeling during the formation and functioning of adult ovaries, there is no comprehensive understanding of the mechanisms controlling preantral follicle development in fetal bovine ovaries during gestation. Thus, to gain insights into ECM remodeling during initial ovarian development, we used fetal ovaries to quantify the fractal dimension (FD), total collagen, and relative mRNA abundance of genes related to ECM remodeling (COL1A1, COL1A2, COL4A1, MMP2, MMP9, MMP14, TIMP1, and TIMP2). For this, pairs of fetal ovaries were obtained from cows in a local abattoir at days 60, 90, 120, and 150 of gestation; one of each pair was submitted to RNA extraction for target transcript analysis, and the other was used for total collagen and FD evaluation. From day 120 total collagen appeared to occupy a greater area in the fetal ovary. The fractal analysis with picrosirius red staining shows higher at day 150 when compared with that on day 60. On the contrary, we found an inverse pattern when we used the hematoxylin and eosin staining approach. Concerning target gene expression, the relative abundances of COL1A1, COL4A1, MMP2, MMP14, TIMP1, and TIMP2 mRNA were higher on day 150 when compared with that on day 60. We conclude that fractal analysis reflects the morphological changes occurring during structural organization of the fetal ovary and that the expression of genes related to ECM remodeling is modulated throughout gestation in bovine fetal ovaries., Competing Interests: Declaration of competing interest We declare that there is no conflict of interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Insulin-like growth factor 1 (IGF-1) activity is established by the regulation of IGF binding protein activity, which blocks IGF-1 functions, whereas pregnancy-associated plasma protein-A (PAPP-A) improves IGF-1 bioavailability and facilitates binding to IGF receptors. To further extend our understanding of the effect of exogenous PAPP-A on bovine embryo production, we added this protein during in vitro maturation of cumulus-oocyte complexes (COCs); moreover, we assessed its effects on IGF-1 quantity in the maturation medium, embryonic yield and postwarming survival, blastocyst quality, and transcript abundance. Bovine COCs were matured in a serum-free medium, either with PAPP-A supplementation (100 ng/ml) or without (control). The treatment group produced higher IGF-1 concentrations in the maturation medium; however, showed no difference on cleavage, blastocysts rates, and embryonic survival 3 and 24 hr postcryopreservation. Regarding gene expression, VNN1 was upregulated, whereas AGPAT9, FASN, EGFR, HAS2, and IMPDH1 were downregulated in PAPP-A treated. PAPP-A treated, CPT2, DNMT3A, and TFAM were upregulated, whereas ATF4 and IFITM3 were downregulated. We concluded that although the addition of PAPP-A did not affect embryo yield and blastocyst survival, higher IGF-1 levels may affect embryo competence through differential expression of genes involved in lipid metabolism, oocyte competence, and mitochondrial function., (© 2019 Wiley Periodicals, Inc.)

Studies have shown that the use of equine chorionic gonadotropin (eCG), which binds both follicle stimulating hormone (FSH) and luteinizing hormone (LH) receptors, could modify the female reproductive tract. We, thus, aimed to quantify the messenger RNA (mRNA) abundance of genes related to cumulus-oocyte complexes (COCs) and embryo quality in Nelore cows (Bos taurus indicus) submitted to ovarian superstimulation using only FSH (FSH group; n = 10) or replacement of the last two doses of FSH by eCG (FSH/eCG group; n = 10). All animals were slaughtered and the ovarian antral follicles from both groups (10-14 mm in diameter) were aspirated for cumulus, oocyte and in vitro embryo production gene expression analysis. The relative mRNA abundance of 96 genes related to COCs development and embryo quality was measured by RT-qPCR. We found that oocytes are more affected by eCG use and that 35 genes involved in lipid metabolism, oxidative stress, transcriptional control, and cellular development were upregulated in the FSH/eCG group. In blastocysts, lipid metabolism seems to be the main pathway regulated by eCG use. We suggest that these multiple effects could be due to the ability of eCG to bind LHR and FSHR, which could activate multiple signal transduction pathways in the superstimulated ovary, further impacting the transcriptional profile of COCs and blastocysts., (© 2019 Wiley Periodicals, Inc.)

The aim of this study was to determine the expression of fibroblast growth factor 22 (FGF22) in the bovine corpus luteum (CL) and to investigate the effects of in vivo total or partial cloprostenol-induced luteolysis on the mRNA abundance of FGF22 and its receptor, FGFR1B. Corpora lutea at different stages of development were then dissected from abattoir ovaries (n = 10/stage); a portion of the tissue samples was fixed in paraformaldehyde and the remaining samples were homogenized and subjected to total RNA extraction. To assess mRNA abundance of target genes during induced luteolysis, nineteen cows were synchronized and then randomly assigned to a Latin square design as follows: Control; 2 administrations of prostaglandin F 2α (PGF 2α , total luteolysis; 2 × 250 μg of cloprostenol sodium) and 1/6PGF 2α (partial luteolysis; 83.33 μg of cloprostenol sodium). FGF22 and FGFR1B expression levels were measured by RT-qPCR, and FGF22 protein expression was detected by immunohistochemistry. In summary, FGF22 mRNA was detected at all stages of CL development, and FGF22 protein was also detected in luteal tissue. FGF22 mRNA expression was lower at stage IV than at stage III (P < 0.05), and the same pattern was observed in luteal immunoreactivity. Furthermore, cloprostenol-induced luteolysis, both total and partial, increased FGFR1B mRNA abundance in luteal tissue (P < 0.05), but did not affect FGF22 mRNA abundance. In conclusion, these data suggest a potential role for the FGF22-FGFR1B system during development and regression of bovine CL., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Mammalian oocytes resume meiosis spontaneously after removal from the ovarian follicle. We tested the effects of a 2-h prematuration treatment (Pre-IVM) with forskolin (FSK) and 3-isobutyl-1-methylxanthine (IBMX) in bovine cumulus-oocyte complexes (COCs) on the lipid content of oocytes and blastocysts, on the membrane lipid composition of blastocysts and on the transcriptional profiling of cumulus cells and blastocysts in a high-throughput platform. Embryonic development rates to the morula (mean 56.1%) or blastocyst (mean 26.3%) stages were unaffected by treatment. Lipid content was not affected after Pre-IVM, but was increased after IVM in treated oocytes. Conversely, the lipid content was reduced in Pre-IVM blastocysts. Pre-IVM COCs generated blastocysts containing blastomeres with more unsaturated lipids in their membranes. Pre-IVM also altered the relative abundance of 31 gene transcripts after 2h and 16 transcripts after 24h in cumulus cells, while seven transcripts were altered in blastocysts. Our results suggest that the Pre-IVM treatment affected the lipid composition and transcriptional profiles of COCs and blastocysts. Therefore, Pre-IVM with FSK and IBMX could be used either to prevent spontaneous meiotic resumption during IVM or to modulate lipid composition in the membrane and cytoplasm of blastocysts, potentially improving bovine embryos.

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., (Copyright © 2017. Published by Elsevier Inc.)

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., (Copyright © 2017. Published by Elsevier Inc.)