Butyrolactone I Research Articles

Utilization of Assisted Reproductive Technologies in Breeding Auliekol Cattle: A Comparative Study.

This study evaluates the utilization of in vitro embryo production (IVEP) technology for the conservation and breeding of the Auliekol cattle breed, a primary beef breed in Kazakhstan facing population decline due to the cessation of breeding programs and the incursion of transboundary diseases. We assessed the effect of consecutive ovum pick-up (OPU) procedures on oocyte yield and embryo production in Auliekol and Aberdeen Angus cows. A total of 2232 and 3659 oocytes were aspirated from Auliekol and Aberdeen Angus donors, respectively, with significantly higher yields and embryo production observed in Aberdeen Angus cows. The application of a meiotic block using Butyrolactone I (BLI) and subsequent in vitro fertilization (IVF) protocols was employed, with embryo development monitored up to the morula/blastocyst stage. Results indicated that Auliekol cows exhibited lower oocyte recovery, cleavage, and blastocyst rates compared to Aberdeen Angus cows, likely due to genetic characteristics. Despite the challenges, IVEP presents a valuable tool for the preservation and future propagation of the Auliekol breed, highlighting the need for further research to enhance reproductive outcomes and conservation strategies.

Butyrolactone I attenuates inflammation in murine NASH by inhibiting the NF-κB signaling pathway

Nonalcoholic steatohepatitis (NASH) is the development of non-alcoholic fatty liver disease (NAFLD) and a key element in the exacerbation of NAFLD. Since there are currently no drugs approved by the U.S. Food and Drug Administration to treat this disease, the search for treatments that can be translated into clinical use is urgent. Butyrolactone I (BLI), isolated from Aspergillus terreus, is an active compound possessing multiple biological activities. However, the effects of BLI on NASH have never been reported. In this study, RAW264.7 cells stimulated by lipopolysaccharide (LPS) were applied to study the anti-inflammatory effect and the underlying mechanisms of BLI in vitro. Following this, mice fed with high-fat and -fructose diet (HFFD) were used to explore the alleviation of NASH by BLIin vivo. We found that BLI attenuated inflammation in LPS-induced RAW264.7 cells by inhibiting the NF-κB signaling pathway and downregulating the expression of iNOS and COX-2. Moreover, results of experiments in vivo demonstrated that BLI reduced serum transaminase levels, decreased hepatic fat accumulation, inhibited inflammation, suppressed oxidative stress, and ameliorated liver fibrosis. For the first time, we investigated the role of BLI in the treatment of murine NASH. We found that BLI alleviates NASH partly by inhibiting the NF-κB pathway of signaling. Given its hepatoprotective effects and non-toxic properties, BLI can be a novel and effective drug for NASH patients.

Marine fungal metabolite butyrolactone I prevents cognitive deficits by relieving inflammation and intestinal microbiota imbalance on aluminum trichloride-injured zebrafish

BackgroundMounting evidences indicate that oxidative stress, neuroinflammation, and dysregulation of gut microbiota are related to neurodegenerative disorders (NDs). Butyrolactone I (BTL-I), a marine fungal metabolite, was previously reported as an in vitro neuroprotectant and inflammation inhibitor. However, little is known regarding its in vivo effects, whereas zebrafish (Danio rerio) could be used as a convenient in vivo model of toxicology and central nervous system (CNS) diseases.MethodsHere, we employed in vivo and in silico methods to investigate the anti-NDs potential of BTL-I. Specifically, we established a cognitive deficit model in zebrafish by intraperitoneal (i.p.) injection of aluminum trichloride (AlCl3) (21 μg) and assessed their behaviors in the T-maze test. The proinflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) as well as acetylcholinesterase (AChE) activity or glutathione (GSH) levels were assayed 24 h after AlCl3 injection. The intestinal flora variation of the zebrafish was investigated by 16S rDNA high-throughput analysis. The marine fungal metabolite, butyrolactone I (BTL-I), was used to modulate zebrafish cognitive deficits evoked by AlCl3 and evaluated about its effects on the above inflammatory, cholinergic, oxidative stress, and gut floral indicators. Furthermore, the absorption, distribution, metabolism, excretion, and toxicity (ADMET) and drug-likeness properties of BTL-I were studied by the in silico tool ADMETlab.ResultsBTL-I dose-dependently ameliorated AlCl3-induced cognitive deficits in zebrafish. While AlCl3 treatment elevated the levels of central and peripheral proinflammatory cytokines, increased AChE activity, and lowered GSH in the brains of zebrafish, these effects, except GSH reduction, were reversed by 25–100 mg/kg BTL-I administration. Besides, 16S rDNA high-throughput sequencing of the intestinal flora of zebrafish showed that AlCl3 decreased Gram-positive bacteria and increased proinflammatory Gram-negative bacteria, while BTL-I contributed to maintaining the predominance of beneficial Gram-positive bacteria. Moreover, the in silico analysis indicated that BTL-I exhibits acceptable drug-likeness and ADMET profiles.ConclusionsThe present findings suggest that BTL-I is a potential therapeutic agent for preventing CNS deficits caused by inflammation, neurotoxicity, and gut flora imbalance.

Pharmacokinetics and Metabolism Study of Deep-Sea-Derived Butyrolactone I in Rats by UHPLC-MS/MS and UHPLC-Q-TOF-MS.

Butyrolactone I (BTL-I) is a butanolide isolated from the deep-sea-derived fungus, Aspergillus sp. It provides a potential new target for the prevention and treatment of food allergies. This study aimed to investigate the metabolic and pharmacokinetic profile of BTL-I in rats. The metabolic profiles were obtained by UHPLC–Q-TOF-MS. As a result, eleven metabolites were structurally identified, and the proposed metabolic pathways of BTL-I were characterized. The main metabolites were the oxidative and glucuronidative metabolites. In addition, a sensitive UHPLC–MS/MS method was established for the quantitation of BTL-I in rat plasma (LOQ = 2 ng/mL). The method was fully validated and successfully applied to the pharmacokinetic study of BTL-I in rats after oral administration or intravenous administration. The oral bioavailability was calculated as 6.29%, and the maximum plasma concentrations were 9.85 ± 1.54 ng/mL and 17.97 ± 1.36 ng/mL for intravenous and intragastric dosing groups, respectively.

Effects of the addition of oocyte meiosis-inhibiting drugs on the expression of maturation-promoting factor components and organization of cytoplasmic organelles

The present study evaluated the effects of the blockade of meiosis in bovine oocytes by the cyclin-dependent kinase inhibitors roscovitine (ROS) and butyrolactone-I (BL-I) on nuclear maturation and extracellular signal-regulated kinase 1/2 (ERK1/2), cyclin B1 and p34cdc2 protein expression and localization. We also evaluated ultrastructural changes in oocytes. Immature oocytes were obtained from slaughtered bovines and divided into: (1) control (oocytes for in vitro maturation only in tissue culture medium-199 for 24 h), (2) oocytes that were treated with 12.5μMROS for 6 h, (3) oocytes that were treated with 50μMBL-I for 6 h and (4) oocytes that were treated with 6.25 μMROS+25 μMBL-I for 6 h. Incubation with inhibitors was followed by the reversal of blockade for 18 h. Oocytes then underwent immunohistochemical analysis to visualize chromatin and assess ERK1/2, cyclin B1 and p34cdc2 localization/expression, followed by preparation of the cells for ultrastructure analysis by electron microscopy. The groups at 6 h of maturation and before IVM exhibited the lowest number of oocytes in metaphase I. ROS group had the highest number of degenerating oocytes (p < 0.05). After maturation, majority of oocytes were in metaphaseII with no differences among groups (p> 0.05). ERK1/2, cyclin B1 and p34cdc2 expression differed throughout inhibition and oocyte maturation (p < 0.05). No difference was observed in the localization of these proteins in the ooplasm. No ultrastructural changes in oocytes were observed between treatments, with the exception of treatment with drugs that augmented lipid metabolism (p < 0.05). Results indicate that the effects of CDK1 inhibitors are reversible in bovine oocytes, indicated by nuclear, cytoplasmic, and molecular maturation parameters.

Treatment with roscovitine and butyrolactone I prior to in vitro maturation alters blastocyst production.

This study evaluated the effects of oocyte meiosis inhibitors roscovitine (ROS) and butyrolactone I (BL-I) on in vitro production of bovine embryos. Bovine oocytes were maintained in pre in vitro maturation (pre-IVM) with 25 µM ROS or 100 µM BL-I for 24 h to delay meiosis and for 24 h in in vitro maturation (IVM). Following this treatment, the nuclear maturation index was evaluated. All embryos degenerated following this procedure. In the second set of experiments, oocytes were maintained for 6 or 12 h in pre-IVM with the following three treatments: ROS (25 µM or 12.5 µM), BL-I (100 µM or 50 µM) or a combination of both drugs (6.25 µM ROS and 12.5 µM BL-I). Oocytes were cultivated for 18 or 12 h in IVM. When a meiosis-inducing agent was used during pre-IVM for 24 h, more degenerated oocytes were observed at the end of the IVM period. This effect decreased when the meiotic blocking period was reduced to 6 or 12 h. No significant differences were observed in the blastocyst production rate of oocytes in pre-IVM for 6 h with ROS, BL-I, or ROS + BL-I compared with that of the control group (P > 0.05). However, inhibition of oocytes for 12 h resulted in decreased embryo production compared with that in the controls (P < 0.05). There was no difference in the post-vitrification embryo re-expansion rate between the study groups, showing that the meiotic inhibition for 6 or 12 h did not alter the embryo cryopreservation process.

Deep-Sea-Derived Butyrolactone I Suppresses Ovalbumin-Induced Anaphylaxis by Regulating Mast Cell Function in a Murine Model.

Deep-sea-derived butyrolactone I (BTL-I), which was identified as a type of butanolide, was isolated from Aspergillus sp. Ovalbumin (OVA)-induced BALB/c anaphylaxis was established to explore the antifood allergic activity of BTL-I. As a result, BTL-I was able to alleviate OVA-induced allergy symptoms, reduce the levels of histamine and mouse mast cell proteinases, inhibit OVA-specific IgE, and decrease the population of mast cells in the spleen and mesenteric lymph nodes. BTL-I also significantly suppressed mast-dependent passive cutaneous anaphylaxis. Additionally, the maturation of bone marrow-derived mast cells (BMMCs) declined as BTL-I caused down-regulation of c-KIT receptors. Furthermore, molecular docking analyses revealed that BTL-I interacted with the inhibitory receptor, FcγRIIB. In conclusion, the reduction of mast cell function by deep-sea-derived BTL-I as well as its interactions with the inhibitory receptor, FcγRIIB, may contribute to BTL-I-related protection against food anaphylaxis.

Isolation and identification of phase I metabolites of butyrolactone I in rats

1. Butyrolactone I (BL-I), one of the major secondary metabolites of fungus Aspergillus terreus, is a selective cdc2 kinase inhibitor. In the present study, the metabolism of BL-I in male Wistar rats was investigated by characterizing metabolites excreted into feces.2. Following an oral dose of 40 mg/kg BL-I, 10 phase I metabolites were isolated from the feces of rats, and their structures were identified on the basis of a range of spectroscopic data and ICD analysis. These metabolites were fully characterized as butyrolactone VI (M1), aspernolide E (M2), 7′′S-hydroxy-9′′-ene-butyrolactone I (M3), 7′′R-hydroxy-9′′-ene-butyrolactone I (M4), 7″S, 8″R-dihydroxy-aspernolide E (M5), 7″R, 8″S-dihydroxy-aspernolide E (M6), 7″R-acetyl-8″S-hydroxy-aspernolide E (M7), 7″S-acetyl-8″R-hydroxy-aspernolide E (M8), 7″R-methoxy-8″S-hydroxy-aspernolide E (M9), butyrolactone V (M10), respectively. It is the first time to describe the metabolites of BL-I in vivo, and metabolites M3 to M9 are new compounds.3. BL-I and metabolites M2 to M10 were evaluated for their antimicrobial activity and in vitro antiproliferative activities. Only M-3 and M-4 showed inhibitory effect against staphylococcus aureus both with MIC of 125 μg/ml. BL-I and metabolites M-4 and M-5 exhibited potent cancer cell growth inhibitory activities against HL-60 (human leukemia) cell lines with the IC50 values of 13.2, 28.8 and 35.7 μM, respectively.4. On the basis of metabolites profile, a possible metabolism pathway for BL-I in rats has been proposed. This is the first systematic study on the phase I metabolites of BL-I.

Effect of Temporary Meiotic Attenuation of Oocytes with Butyrolactone I and Roscovitine in Resistance to Bovine Embryos on Vitrification.

This study aimed to produce in vitro bovine embryos by the addition of two drugs, which is responsible for oocyte meiosis inhibition: roscovitine (ROS) and butyrolactone I (BL-I). Oocytes were recovered from slaughtered cows and matured in a commercial medium and maintained in a 5% CO2 atmosphere. Oocytes were maintained for 6 h in an in vitro maturation (IVM) medium containing ROS (12.5 μm), BL-I (50 μm) and association of drugs (ROS 6.25 μm and BL-I 25 μm). Oocytes were cultured for 18 h in an agent-free medium for the resumption of meiosis. After 24 h of maturation, oocytes were inseminated in the commercial in vitro fertilization (IVF) medium. Presumptive zygotes were cultured in SOFaa medium in a 5% CO2 atmosphere. On day 3, rate of cleavage was evaluated and on days 6 and 7, rate of blastocyst formation. BL-I and its association with the ROS increased the rates of cleavage and blastocyst formation (p < 0.05). The ROS alone was inefficient, impairing embryonic development, with low rates of blastocyst formation when compared to the control group and other treatments (p < 0.05). The embryos from BL-I and ROS+BL-I groups presented higher number of cells and lower rates of cellular apoptosis compared to other groups, either for the fresh or for post-thawing embryos. Embryos from ROS+BL-I group showed to be more resistant to the vitrification process, presenting a higher rate of embryonic re-expansion (p < 0.05). In conclusion, block of meiosis using BL-I or its association with ROS increased the rate of blastocyst formation, and the association of ROS+BL-I resulted in a better resistance to the embryo cryopreservation process.

Evaluation of Culture Media for Butyrolactone I Production by Aspergillus terreus LS07 as Antidiabetic

Butyrolactone I (BL-I) has been reported as inhibitor of α-glucosidase and antioxidant compound isolated from culture broth of Aspergillus terreus. The purpose of this study was to determine the effect of various liquid media compositions on production of BL-I from A. terreus LS07. Fermentation was conducted in four liquid medium: potato dextrose broth (PDB), potato malt peptone (PMP), malt extract (ME), and Czapek-Dox broth (CDB), which were incubated for seven days at room temperature, with agitation of 70rpm. Ethyl acetate (EtOAc) was used to extract the active metabolite from each broth. Glucosidase inhibitory activity was measured by the inhibition of yeast α-glucosidase, while antioxidants measured by testing the total phenol and DPPH free radical scavenger activity. The results showed that the EtOAc extract on CDB had strong inhibition activity against the enzyme α-glucosidase and strong antioxidant with IC50 values 6.45 and 24.05μg/mL, respectively. The CDB extract also showed the highest content of total phenols (349.66mg Gallic Acid Equivalent (GAE)/g extract) and BL-I (58.5% w/w). Based on these results, among the four kinds of medium used, Czapek-Dox broth is an ideal culture medium of A. terreus LS07 that could be applied for increase production of BL-I as natural antidiabetic source from terestrial fungi

162 EFFECT OF MEIOTIC ARREST USING BUTYROLACTONE I AND ROSCOVITINE IN RESISTANCE TO EMBRYO CRYOPRESERVATION

In vitro embryo production by maturation, fertilization, and culture has become a valuable assisted reproductive technology in cattle breeding systems. However, even with the notable innovations in this system, the greatest obstacles for the complete success of this biotechnology are the low pregnancy rates after transfer and the greater sensitivity to cryopreservation. Thus, this study evaluated the effect of the addition of a meiotic arrest using butyrolactone I and roscovitine on in vitro production of bovine embryos. Nelore oocytes were matured in TCM-199 with Earle's salt + 10% FCS, FSH, and LH, in 5% CO2 atmosphere. To delay meiosis, the oocytes were maintained for 6 h in medium in presence of butyrolactone I (BL-I) 25 μM + roscovitine (ROS) 6.25 μM. Then, the oocytes (Control n = 405; BL-I + ROS n = 386) were cultured for 18 h in agent-free medium to resume meiosis, completing 24 h of maturation. After 24 h of maturation (Day 0), oocytes were fertilized in human tubal fluid (HTF, Irvine, New Zealand) under the conditions above. Semen was selected through Percoll gradient and the concentration adjusted to 2 × 106 sperm mL–1. The presumed zygotes were culture in 90-μL droplets of SOFaa + 0.6% BSA + 2.5% FCS in 5% CO2 until Day 7, when the blastocyst rate was evaluated. We performed 5 replicates. Data were analysed with ANOVA, followed by Tukey test using the general linear model (PROC GLM) of SAS 9.2 (SAS Institute Inc., Cary, NC, USA). The level of significance adopted was 5%. Statistical differences were observed in blastocyst production rate: Control: 42.64% and BL-I + ROS: 55.96% (P &lt; 0.05). The remaining embryos (Control n = 163; BL-I + ROS n = 182) were vitrified, warmed, and recultured in SOFaa with 10% FCS for 12 h. After this period, re-expansion rate was determined. We found difference in re-expansion rates of groups (Control: 84.66% and BL-I + ROS: 93.40%; P &lt; 0.05). Additionally, differences were found among total number cell groups: Control: 54.33 and BL-I + ROS: 97.5; P &lt; 0.05). The use of BL-I + ROS is able to better a producing rate of in vitro embryos and improve the cryotolerance. The authors acknowledge FAPESP 2010/18994-4 and 2011/08926-4 for financial support.

232 EFFECT OF MEIOTIC ARREST USING BUTYROLACTONE I AND ROSCOVITINE ON IN VITRO PRODUCTION OF BOVINE EMBRYOS

Butyrolactone I (BL-I) and roscovitine (ROS) are selective inhibitors of the cyclin-dependent kinases, and both have been shown to reversibly inhibit meiotic resumption in cattle oocytes without having a negative effect on subsequent development to the blastocyst stage. The aim of this study was to evaluate the in vitro production of bovine embryos in the presence of BL-I (Enzo Life Sciences International Inc., Plymouth Meeting, PA, USA) and ROS (Sigma-Aldrich, St. Louis, MO, USA). For that, Nellore oocytes were matured in TCM-199 with Earle’s salts + 10% FCS, FSH, and LH, in a 5% CO2 atmosphere. To delay meiosis, the oocytes were maintained for 6 h in medium in the presence of 25 µM BL-I + 6.25 µM ROS. The oocytes were then cultured for 18 h in agent-free medium to resume meiosis, completing 24 h of maturation. After 24 h of maturation (Day 0), oocytes were fertilized in human tubal fluid (HTF, Irvine, New Zealand), in a 5% CO2 atmosphere. Semen was selected through Percoll gradient and the concentration was adjusted to 2 × 106 sperm mL–1. The presumed zygotes were cultured inside a bag in 90-µL droplets of SOFaa + 0.6% BSA + 2.5% FCS in a 5% CO2, 5% O2, and 90% N2 atmosphere until Day 7, when blastocyst rate was evaluated. Five replicates were done. Data were analysed with ANOVA, followed by Tukey’s test using the general linear models procedure (PROC GLM) of SAS, version 9.2 (SAS Institute Inc., Cary, NC, USA). The level of significance adopted was 5%. No statistical differences were observed in blastocyst production rate: control, 33.3 ± 3.74%; BL-I + ROS, 38.0 ± 4.5%. Thus, the presence of BL-I + ROS in the prematuration medium of bovine oocytes did not compromise their subsequent developmental competence. The next steps of the present study are evaluating the kinetics of blastocyst formation and detecting apoptotic cells in situ by TUNEL, which will show that these substances do not compromise embryonic quality.

199 PREMATURATION OF BOVINE OOCYTES WITH BUTYROLACTONE I AND/OR CILOSTAMIDE: EFFECTS ON MEIOSIS PROGRESSION, CYTOPLASMIC MATURATION AND GENE EXPRESSION

Meiotic block during a prematuration culture (pre-IVM) before in vitro maturation (IVM) is suggested as a way to provide additional time to synchronize oocyte-somatic cell communication, leading to improved cytoplasmic maturation and nuclear meiotic competence and the acquisition of critical cellular functions necessary for developmental competence. This study was designed to evaluate the effects of the inhibitors butyrolactone I (Bl-I) and cilostamide on oocyte nuclear and cytoplasmic maturation. For that, 2 well-established methods of pre-IVM and IVM for cilostamide (Albuz et al. 2010 Hum. Reprod. 25, 2999–3011) or Bl-I (Hashimoto et al. 2002 Biol Reprod. 66, 1696–1701) were used. Abattoir-collected oocytes were IVM in maturation medium (MM: mSOF with 0.8% BSA and hormones) for 24 h, without a previous pre-IVM culture (control). Cilostamide-group oocytes were treated for the first 2 h in vitro (pre-IVM) with 100 μM of the adenylate cyclase activator forskolin and 500 μM IBMX (nonspecific phosphodiesterase inhibitor) and then oocytes underwent extended IVM for 30 h in the presence of 20 μM cilostamide (type 3 phosphodiesterase inhibitor; pre-IVM 2 h + IVM 30 h). The Bl-I-group oocytes were pre-IVM for 24 h with 100 μM Bl-I diluted in TCM-199 supplemented with 0.2 mM pyruvate and then were IVM in MM for 20 h (pre-IVM 24 h + IVM 20 h). The Bl-I + Cilost group was a combination of both procedures: oocytes were first pre-IVM with Bl-I for 24 h and then cultured as described for the cilostamide group (pre-IVM 24 h + IVM 30 h). Cultures were carried out at 38.5°C in 5% CO2 in humidified air. After IVM, oocytes were stained with 500 nM mitotracker red to assess the mitochondrial membrane potential (Δψm) and with 10 μg mL–1 of Hoescht 33342 to evaluate the nuclear maturation (n = 207). Images were captured by an Olympus confocal microscope and analyzed with Fluoview software. The TUNEL assay was used to detect oocyte DNA fragmentation (n = 74). Relative amounts of mRNA for apoptotic-related genes were quantified after IVM in individual oocytes using real-time PCR (Kameyama et al. 2007 Reproduction 133, 423–32). Means were compared by ANOVA and Tukey's test or by chi-square (P ≤ 0.05). The percentage of oocytes reaching metaphase II after IVM did not differ among groups (73.8 to 90.4%; P ≥ 0.05), indicating that in vitro meiotic resumption was normal. The Δψm, expressed in arbitrary units of fluorescence, was 1.0 ± 0.1a (control), 2.7 ± 0.4b (Bl-I), 3.2 ± 0.5b (Cilost) and 2.1 ± 0.3ab (Bl-I + Cilost). The percentage of TUNEL-positive oocytes (18.8–41.3%) did not differ among groups (P ≥ 0.05). The relative abundance of BAX (1.0 ± 0.4 to 2.3 ± 0.4) and BCL-XL (1.0 ± 0.3 to 0.3 ± 0.1) transcripts was unaffected by pre-IVM and IVM (P ≥ 0.05). In conclusion, except for an increase in mitochondrial activity, pre-IVM with cilostamide and/or Bl-I did not affect cytoplasmic and nuclear oocyte maturation. However, oocyte developmental potential needs to be better evaluated in a future study through assessment of embryonic development. We acknowledge FAPESP and NSERC.

Gene Expression in Bovine Oocytes and Cumulus Cells After Meiotic Inhibition with the Cyclin‐Dependent Kinase Inhibitor Butyrolactone I

The aim of this study was to determine the effect of temporary inhibition of meiosis using the cyclin-dependent kinase inhibitor butyrolactone I (BLI) on gene expression in bovine oocytes and cumulus cells. Immature bovine cumulus-oocyte complexes (COCs) were assigned to groups: (i) Control COCs collected immediately after recovery from the ovary or (ii) after in vitro maturation (IVM) for 24 h, (iii) Inhibited COCs collected 24 h after incubation with 100 μm BLI or (iv) after meiotic inhibition for 24 h followed by IVM for a further 22 h. For mRNA relative abundance analysis, pools of 10 denuded oocytes and respective cumulus cells were collected. Transcripts related to cell cycle regulation and oocyte competence were evaluated in oocytes and cumulus cells by quantitative real-time PCR (qPCR). Most of the examined transcripts were downregulated (p < 0.05) after IVM in control and inhibited oocytes (19 of 35). Nine transcripts remained stable (p > 0.05) after IVM in control oocytes; only INHBA did not show this pattern in inhibited oocytes. Seven genes were upregulated after IVM in control oocytes (p < 0.05), and only PLAT, RBP1 and INHBB were not upregulated in inhibited oocytes after IVM. In cumulus cells, six genes were upregulated (p < 0.05) after IVM and eight were downregulated (p < 0.05). Cells from inhibited oocytes showed the same pattern of expression regarding maturation profile, but were affected by the temporary meiosis inhibition of the oocyte when the same maturation stages were compared between inhibited and control groups. In conclusion, changes in transcript abundance in oocytes and cumulus cells during maturation in vitro were mostly mirrored after meiotic inhibition followed by maturation.

Effect of Cyclin‐dependent Kinase (CDK) Inhibition on Expression, Localization and Activity of Maturation Promoting Factor (MPF) and Mitogen Activated Protein Kinase (MAPK) in Bovine Oocytes

This study aimed to assess the effects of cyclin-dependent kinase (CDK) inhibition on factors involved in the control of meiosis in bovine oocytes: maturation promoting factor (MPF) (p34(cdc2) and cyclin B1) and mitogen activated protein kinase (MAPK). Oocytes were maintained at germinal vesicle (GV) stage in vitro with 10 μM of the CDK inhibitor butyrolactone I (BLI) for 24 h (inhibited). After this period, some of the oocytes were transferred to in vitro maturation (IVM) culture for 24 h (inhibited and matured). Control oocytes were assessed immediately after follicle aspiration (immature) or after in vitro maturation for 24 h (matured). Real-time PCR analyses showed that transcripts for p34(cdc2) and MAPK were detected in immature and inhibited oocytes and decreased after maturation, irrespective of CDK inhibition with BLI. Cyclin B1 was detected at similar levels in all oocyte groups. The p34(cdc2) and MAPK proteins were detected by Western blotting at similar levels in all oocyte groups, and cyclin B1 protein was detected only after maturation. Immunofluorescence detection showed that p34(cdc2) was localized in the cytoplasm and GV of immature oocytes, and then throughout the cytoplasm after maturation. Cyclin B1 and MAPK were detected in the cytoplasm in all oocyte groups. Maturation promoting factor and MAPK activities were similar throughout most of maturation for oocytes treated with or without BLI. In conclusion, CDK inhibition did not affect the expression (mRNA and protein levels) and localization of MPF and MAPK, and had nearly no effect on kinase activities during maturation.

276 CORTICAL GRANULE MIGRATION AND PRONUCLEAR FORMATION IN BOVINE OOCYTES TREATED WITH BUTYROLACTONE I DURING TRANSPORT

Butyrolactone I (BLI) can be used to maintain meiosis blocked during transport of oocytes to the laboratory, aiming to maintain their quality and competence. This work evaluated the effect of BLI during transport of bovine oocytes on the cortical granules (CG) distribution, and fertilization. Meiotic block culture was in basic medium (TCM-199 with 0.2 mM pyruvate, 25 mM sodium bicarbonate, and 75 mg mL-1 gentamicin) with 10 μLM BLI (B10) or 100 (AM BLI + 0.3% BSA (B100). During meiotic block, oocytes were cultured in cryogenic vials (15/vial) containing 60 μLL of medium recovered with mineral oil, packed in a portable incubator (Thawing Unit MT 35/42, Minitub) at 38.5°C during the first 5 h, and then transferred to a incubator at 38.5°C and 5% CO2 in air for the remaining 19 h. Then, they were matured at 38.5°C and 5% CO2 in basic medium supplemented with 0.6% BSA and hormones (maturation medium, MM) for 20 h. As controls, a group of oocytes was matured in MM during 24 h, without prior meiotic block. Control oocytes were cultured at 38.5°C and 5% CO2 (control 1, C1) or in the same portable incubator conditions in which the blocked oocytes were cultured (C2). A group of oocytes was fixed immediately after follicular aspiration (C0). At the end of cultures, a group of oocytes (n = 853) were stained with 1 μg mL-1 Lens culinaris (FITC-LCA) and assessed for the distribution of CG (arrangement in clusters = immature; peripheral = mature). The remaining oocytes (n = 563) were fertilized for 18 h, and zygotes were stained with 10 μg mL-1 Hoechst 33342 and assessed for sperm penetration and pronuclear formation. Oocytes with 2 pronuclei (2PN) were considered to present normal fertilization, and oocytes with &gt;2PN were considered to be polyspermic. The data were analyzed by ANOVA and Tukey’s test, and correlation between CG positioning and pronuclear formation was analyzed with the Pearson correlation test. After meiotic block, the proportion of immature CG were higher (P &lt; 0.05) in B100 (90.7%) and C0 (88.2%) than in B10 (69.0%). After maturation, mature CG rates in C2 (61.7%) were similar to B10 (52.3%) and B100 (72.9%), but only B100 was similar to C1 (83.6%). Irrespective of the presence or absence of BLI, culture of oocytes in the portable incubator adversely affected the normal pronuclear formation rates, which were higher (P &lt; 0.05) in C1 (78.9%) than in C2 (56.8%), B10 (56.0%), and B100 (51.8%). The incidence of &gt;2PN was higher (P &lt; 0.05) in B10 (5.9%) than in C1, C2, and B100 (0.0, 1.4, and 0.0%, respectively). There was a fine correlation between mature CG and normal pronuclear formation (R = 0.5; P &lt; 0.05). We conclude that (1) CG migration is reversibly blocked by BLI at a concentration of 100 μLM during transport; (2) oocyte transport in the portable incubator, but not BLI itself, adversely affects the normal pronuclear formation; and (3) CG maturation is essential for normal pronuclear formation. Financial support: FAPESP.

345 GENE EXPRESSION IN OOCYTES AFTER MEIOTIC INHIBITION WITH THE CYCLIN-DEPENDENT KINASE INHIBITOR BUTYROLACTONE I

Once removed from the follicle, mammalian oocytes resume meiosis spontaneously and progress through breakdown of the germinal vesicle to the matured state at metaphase II. The ability to reversibly inhibit such meiotic resumption has been reported and is a potentially useful method for studying developmental competence acquisition in oocytes as well as in some cases allowing flexibility in an IVF system where oocytes are collected from distant locations or on different days. The aim of the present study was to determine the effect of temporary inhibition of meiotic resumption using the cyclin-dependent kinase inhibitor butyrolactone I (BLI) on gene expression in bovine oocytes. Immature bovine oocytes were recovered from the ovaries of slaughtered heifers at a commercial abattoir and assigned to 1 of 4 groups: (1) Control: immature oocytes were collected either immediately or (2) after IVM for 24 h in TCM-199 containing 10 ng mL-1 EGF and 10% (v/v) FCS, (3) Inhibited oocytes collected either 24 h after incubation in the presence of 100 μM BLI in TCM-199 with 3 mg mL-1 BSA or (4) after meiotic inhibition for 24 h followed by in vitro maturation. All cultures were carried out at 38.5°C under 5% CO2 in air and maximum humidity. For mRNA relative abundance analysis, cumulus cells were removed and pools of 10 denuded oocytes were snap frozen in liquid nitrogen and stored at -80°C until use. A total of 42 transcripts, previously reported to be related to cell cycle regulation and/or oocyte competence were evaluated by quantitative real time PCR. Differences in relative abundance were analyzed by ANOVA and Student’s t-test. The majority of transcripts were downregulated (P &lt; 0.05) after IVM in control oocytes (23 out of 42) and the same pattern was observed in inhibited oocytes that were allowed to mature. Twelve transcripts remained stable (P &gt; 0.05) after IVM in control oocytes; of these, only two (PTTG1 and INHBA) did not show the same pattern in inhibited and matured oocytes. Few genes (7) were upregulated after IVM in control oocytes (P &lt; 0.05) and of these, three (PLAT1, RBP1, and INHBB) were not upregulated in inhibited oocytes after IVM. Inhibited oocytes showed similar levels of expression (P &gt; 0.05) as immature control oocytes, except for two genes (LUM and INHBB), which were increased in these oocytes (P &lt; 0.05). The expression profiles of cell cycle genes were mostly unaffected by the BLI treatment. The few genes affected were previously reported as competence-related and could be useful markers of oocyte competence following pretreatment. In conclusion, the changes occurring in transcript abundance during oocyte maturation in vitro were to a large extent mirrored following inhibition of meiotic resumption prior to IVM and subsequent release from inhibition and maturation. CLV Leal was supported by CNPq, Brazil (PDE 201487/2007-1); Supported by Science Foundation Ireland (07/SRC/B1156).

Prematuration of bovine oocytes with butyrolactone I reversibly arrests meiosis without increasing meiotic abnormalities after in vitro maturation

Objectives Asynchrony between nuclear and cytoplasmic maturation, and possibly damage to the oocyte meiotic spindle, limits the application of in vitro maturation (IVM) in assisted reproduction. Several studies have suggested that Prematuration with meiosis blockers may improve oocyte quality after IVM, favoring early embryogenesis. Thus, we investigated the effect of Prematuration with the nuclear maturation inhibitor butyrolactone I (BLI) on the meiotic spindle and chromosomal configuration of bovine oocytes. Study design Immature oocytes obtained from cows slaughtered in a slaughterhouse ( n = 840) were divided into the following groups: (1) control ( n = 325), submitted only to IVM in TCM199 for 24 h; (2) BLI 18 h ( n = 208) submitted to meiotic blockage with 100 μM BLI for 24 h (Prematuration) and then induction of IVM in TCM199 for 18 h; and (3) BLI 24 h ( n = 307), pre-matured with 100 μM BLI for 24 h followed by 24 h of IVM in TCM199. The oocytes were then fixed, stained by immunofluorescence for morphological visualization of both microtubules and chromatin, and evaluated. Results Meiotic arrest occurred in 90.2% of the oocytes cultured with BLI. Maturation rates were similar for all groups (80.3%, 73.6% and 82.7% for the control, BLI 18 h and BLI 24 h groups, respectively). We observed 81.3% normal oocytes in metaphase II in the control group, and 80.0% and 81.2% in the BLI 18 h and BLI 24 h groups, respectively. The incidence of meiotic anomalies did not differ between groups (18.7%, 20.0% and 18.8% for the control, BLI 18 h and BLI 24 h, respectively). Conclusion Prematuration with butyrolactone I reversibly arrests meiosis without damaging the meiotic spindle or the chromosome distribution of bovine oocytes after in vitro maturation.

Regulation of the meiotic prophase I to metaphase I transition in mouse spermatocytes

The meiotic prophase I to metaphase I transition (G2/MI) involves disassembly of synaptonemal complex (SC), chromatin condensation, and final compaction of morphologically distinct MI bivalent chromosomes. Control of these processes is poorly understood. The G2/MI transition was experimentally induced in mouse pachytene spermatocytes by okadaic acid (OA), and kinetic analysis revealed that disassembly of the central element of the SC occurred very rapidly after OA treatment, before histone H3 phosphorylation on Ser10. These events were followed by relocalization of SYCP3 and final condensation of bivalents. Enzymatic control of these G2/MI transition events was studied using small molecule inhibitors: butyrolactone I (BLI), an inhibitor of cyclin-dependent kinases (CDKs) and ZM447439 (ZM), an inhibitor of aurora kinases (AURKs). The formation of highly condensed MI bivalents and disassembly of the SC are regulated by both CDKs and AURKs. AURKs also mediate phosphorylation of histone H3 in meiosis. However, neither BLI nor ZM inhibited disassembly of the central element of the SC. Thus, despite evidence that the metaphase promoting factor is a universal regulator of the onset of cell division, desynapsis, the first and key step of the G2/MI transition, occurs independently of BLI-sensitive CDKs and ZM-sensitive AURKs.

Prematuration of bovine oocytes with butyrolactone I: Effects on meiosis progression, cytoskeleton, organelle distribution and embryo development

The effects of prematuration (PM) of bovine oocytes with butyrolactone I (BLI) for 24 h on meiosis progression, cell structures and embryo development were assessed. Germinal vesicle (GV) rates decreased (97.4–65.1%, P < 0.05) with decreasing BLI concentrations (100–25 μM). Without BSA in PM medium, GV rates were similar (98.7–97.2, P > 0.05) with low BLI (10–25 μM). After in vitro maturation (IVM) for 24 h, metaphase II (MII) rates for controls (IVM only) were similar (91.1%, P > 0.05) to PM with 10 μM BLI in BSA-free medium (B10 = 91.5%) and 100 μM BLI in medium with BSA (B100 = 92.4%). Meiosis resumption occurred earlier in treated oocytes (71.4–74.3% in GV for B10 and B100, respectively, after 6 h IVM compared with 97.3% in controls, P < 0.05). By 18 h of IVM, most oocytes reached MII (72.0–78.9%, P > 0.05). Microtubules and microfilaments were unaffected by BLI. Cortical granules (CG) migration was reversibly blocked by BLI. Mitochondria translocation was partially blocked by PM culture and after IVM more oocytes in B10 and B100 (95.2 and 98.2%, respectively) had mitochondria translocated to a mature pattern (all cytoplasm) than controls (81.5%, P < 0.05). Cleavage rates were similar (81–87%, P > 0.05), but blastocysts (day 7) decreased in B100 (33.0%, P < 0.05) compared with controls and B10 (38.3 and 41.6%, respectively). Day 8 hatching rates (11.0–19.2%) and mean total cell numbers (136–150) were similar ( P > 0.05). PM did not improve oocyte competence but also did not cause major structural alterations, suggesting that PM may be improved and used to study the mechanisms involved in oocyte differentiation.