Omega-3 α-linolenic acid and omega-6 linoleic acid are essential fatty acids for health maintenance of human and animals because they are not synthesized in vivo. The purpose of this study was to evaluate the effect of α-linolenic acid and linoleic acid supplementation on in vitro maturation and developmental potential of porcine oocytes. Various concentrations of α-linolenic acid and linoleic acid were added into in vitro maturation medium, and we evaluated the degree of cumulus expansion, oocyte nuclear-maturation rate, blastocyst rate, blastocyst quality, and levels of prostaglandin E2, 17b-estradiol, and progesterone in the spent medium. High doses (100 μM) of α-linolenic acid and linoleic acid supplementation significantly inhibited cumulus expansion and oocyte nuclear maturation, and prostaglandin E2 synthesis also significantly decreased compared with other groups (p < 0.05). Supplementation of 50 μM α-linolenic acid and 10 μM linoleic acid showed higher quality blastocysts in terms of high cell numbers and low apoptosis when compared with other groups (p < 0.05), and synthesis ratio of 17b-estradiol / progesterone also significantly increased compared with control group (3.59 ± 0.22 vs. 2.97 ± 0.22, 3.4 ± 0.28 vs. 2.81 ± 0.19, respectively; p < 0.05). Our results indicated that supplementation with appropriate levels of α-linolenic acid and linoleic acid beneficially affects the change of hormone synthesis (in particular, an appropriate increase in the 17b-estradiol / progesterone synthesis ratio) for controlling oocyte maturation, leading to improved embryo quality. However, high doses of α-linolenic acid and linoleic acid treatment results in detrimental effects.

This study aimed to recover the ovarian function through in vitro culture of preantral follicles from aged mice. First, we isolated the preantral follicles from ovaries of sixty-seven-week old B6D2F1 mice with decreased fecundity to know how many follicles were present in them, which was 6 preantral follicles including 2 primary, 2 early secondary and late secondary follicles from 8 aged mice. It was confirmed that a few follicles (~2) were present in aged mice through histological analysis compared to adult mice as control. The 9 days of in vitro culture of preantal follicles showed in vitro growth and induced maturation after treatment with hCG (2.5 IU/mL) and EGF (5 ng/mL). Cumulus cells in the cumulus-oocyte complexes (COCs) were removed using hyaluronidase and oocytes at the germinal vesicle (GV) and GV breakdown (GVBD) were obtained from preantral follicle culture of aged mice in vitro. In conclusion, these observations demonstrated that there still were a few preantral follicles in the ovaries of 67 week-old mice, which we were able to culture in vitro and oocytes were obtained from them. This study proposed an in vitro culture system using preantral follicle as a therapeutic strategy for fertility preservation in humans for assisted reproductive medicine.

Morphology of cumulus-oocyte-complexes (COCs) at germinal vesicle (GV) stage as one of the evaluation criteria for oocyte maturation quality after in vitro maturation (IVM) plays important roles on the meiotic maturation, fertilization and early embryonic development in pigs. When cumulus cells of COCs are insufficient, which is induced the low oocyte maturation rate by the increasing of reactive oxygen species (ROS) in porcine oocyte during IVM. The ROS are known to generate including superoxide and hydrogen peroxide from electron transport system of mitochondria during oocyte maturation in pigs. To regulate the ROS production, the cumulus cells is secreted the various antioxidant enzymes during IVM of porcine oocyte. Our previous study showed that Mito-TEMPO, superoxide specific scavenger, improves the embryonic developmental competence and blastocyst formation rate by regulating of mitochondria functions in pigs. However, the effects of Mito-TEMPO as a superoxide scavenger to help the anti-oxidant functions from cumulus cells of COCs on meiotic maturation during porcine oocyte IVM has not been reported. Here, we categorized experimental groups into two groups (Grade 1: G1; high cumulus cells and Grade 2: G2; low cumulus cells) by using hemocytometer. The meiotic maturation rate from G2 was significantly (p < 0.05) decreased (G1: 79.9 ± 3.8% vs G2: 57.5 ± 4.6%) compared to G1. To investigate the production of mitochondria derived superoxide, we used the mitochondrial superoxide dye, Mito-SOX. Red fluorescence of Mito-SOX detected superoxide was significantly (p < 0.05) increased in COCs of G2 compared with G1. And, we examined expression levels of genes associated with mitochondrial antioxidant such as SOD1, SOD2 and PRDX3 using a RT-PCR in porcine COCs at 44 h of IVM. The mRNA levels of three antioxidant enzymes expression in COCs from G2 were significantly (p < 0.05) lower than COCs of G1. In addition, we investigated the anti-oxidative effects of Mito-TEMPO on meiotic maturation of porcine oocyte from G1 and G2. Meiotic maturation and mRNA levels of antioxidant enzymes were significantly (p < 0.05) recovered in G2 by Mito-TEMPO (0.1 μM, MT) treatment (G2: 68.4 ± 3.2% vs G2 + MT: 73.9 ± 1.4%). Therefore, our results suggest that reduction of mitochondria derived superoxide by Mito-TEMPO may improves the meiotic maturation in IVM of porcine oocyte.

Ganglioside GM3 is known as an inhibition factor of cell differentiation and proliferation via inhibition of epidermal growth factor receptor (EGFR) phosphorylation. Our previous study showed that the exogenous ganglioside GM3 reduced the meiotic maturation of porcine oocytes and induced apoptosis at 44 h of in vitro maturation (IVM). However, the role of ganglioside GM3 in the relationship between EGFR signaling and apoptosis during porcine oocyte maturation has not yet been studied. First, porcine cumulus-oocyte complexes (COCs) were cultured in the NCSU-23 medium with exogenous ganglioside GM3 according to maturation periods (non-treated, only IVM I: 0 - 22 h, only IVM II: 22 - 44 h and IVM I & II: 0 - 44 h). We confirmed that the proportion of germinal vesicle breakdown (GVBD) increased significantly in the IVM I treated group than in the control group. We also confirmed that the meiotic maturation until M II stage and polar body formation decreased significantly in the only IVM I treated group. Cumulus cell expansion and mRNA levels of the expansion-related factors (HAS2, TNFAIP6 and PTX3) decreased significantly in the IVM I treated group than in the control group. Protein levels of EGFR, p-EGFR, ERK1/2, and p-ERK1/2 decreased significantly in the GM3-treated groups, during the IVM I period. In addition, cellular apoptosis, determined using TUNEL assay, and protein levels of Cleaved caspase 3, were increased significantly in the GM3-treated COCs during the IVM I period. Based on these results, ganglioside GM3 exposure of porcine COCs during the IVM I period reduced meiotic maturation and cumulus cell expansion via inhibition of EGFR activity in pigs.

Here, we evaluated the mode of programmed cell death during porcine oocyte maturation by comparing the two major pathways associated with programmed cell death, apoptosis (type I), and autophagy (type II). We investigated the expression and localization of major genes involved in autophagy and apoptosis at mRNA and protein levels. Furthermore, the effect of hormonal stimulation on autophagy and apoptosis was analyzed. We found that the activity of autophagy-associated genes was increased in the cumulus-oocyte complexes (COCs) following follicle-stimulating hormone (FSH) treatment, while the addition of luteinizing hormone (LH) reversed this effect. The expression of proteins associated with autophagy was the highest in FSH-treated COCs. On the other hand, caspase-3 protein level was maximum in COCs cultured with LH. The treatment with rapamycin resulted in the effect similar to that observed with FSH treatment and increased autophagy activity. Thus, hormonal stimulation of pig oocytes resulted in distinct patterns of maturation. The high-quality oocytes majorly relied on the type II pathway (autophagy), while the type I pathway (apoptosis) was more prominent among poor-quality oocytes. Further investigation of this distinction may allow the development of techniques to produce high-quality oocytes in porcine in vitro fertilization.

Nitric oxide (NO) has an important role in oocyte maturation and embryonic development in mammals. This study examined the effect of exogenous NO donor S-nitroso-N-acetylpenicillamine (SNAP) in a maturation medium on meiotic progression and embryonic development after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT) in pigs. When oocytes were exposed to 0.1 μM SNAP for first 22 h of in vitro maturation (IVM) in Experiment 1, SNAP significantly improved blastocyst development in both defined and standard follicular fluid-supplemented media compared to untreated control (48.4 vs. 31.7-42.5%). SNAP treatment significantly arrested meiotic progression of oocytes at the germinal vesicle stage at 11 h of IVM (61.2 vs. 38.7%). However, there was no effect on meiotic progression at 22 h of IVM (Experiment 2). In Experiment 3, when oocytes were treated with SNAP at 0.001, 0.1 and 10 μM during the first 22 h of IVM to determine a suitable concentration, 0.1 μM SNAP (54.2%) exhibited a higher blastocyst formation than 0 and 10 μM SNAP (36.6 and 36.6%, respectively). Time-dependent effect of SNAP treatment was evaluated in Experiment 4. It was observed that SNAP treatment for the first 22 h of IVM significantly increased blastocyst formation compared to no treatment (57.1% vs. 46.2%). Antioxidant effect of SNAP was compared with that of cysteine. SNAP treatment significantly improved embryonic development to the blastocyst stage (49.1-51.5% vs. 34.4-37.5%) irrespective of the presence or absence of cysteine (Experiment 5). Moreover, SNAP significantly increased glutathione (GSH) content and inversely decreased the reactive oxygen species (ROS) level and mitochondrial oxidative activity in IVM oocytes. SNAP treatment during IVM showed a stimulating effect on in vitro development of SCNT embryos (Experiment 7). These results demonstrates that SNAP improves developmental competence of PA and SCNT embryos probably by maintaining the redox homeostasis through increasing GSH content and mitochondrial quality and decreasing ROS in IVM oocytes.

본 연구에서는 다양한 온도 (5, 10, 15, 20, 25℃), 조도 (10, 20, 40, 60, 80 μmol photons m-2 s-1) 및 광주기 (10:14, 12:12, 14:10 h L:D) 조건에서 개다시마 유리배우체 단편의 생장 및 성숙 유도조건을 파악하였다. 암배우체 단편의 생장은 20℃, 20 μmol photons m-2 s-1, 10:14 h (L:D) 조건에서 빠른 생장을 보인 반면 수배우체는 15℃, 20 μmol photons m-2 s-1, 14:10 h (L:D) 조건에서 빠른 생장을 보여 차이를 나타내었다. 암배우체의 최적 성숙조건은 15℃, 40 μmol photons m-2 s-1, 14:10 h (L:D) 조건에서 가장 빠른 성숙을 보인 반면 수배우체는 10℃, 20 μmol photons m-2 s-1, 10:14 h (L:D) 조건에서 빠른 성숙을 보여 차이를 나타내었다. 본 연구 결과 확인된 개다시마 유리배우체 단편의 생장 및 성숙조건은 개다시마의 자원조성 및 대량양식 기반조성의 기초자료로 활용될 수 있다.

This study investigated the use of bovine serum albumin (BSA) as alternatives to fetal bovine serum (FBS) in in vitro maturation medium. The oocyte maturation, cumulus cell-oocyte gap junctional communication, and development of bovine embryos were determined by assessing their cell number, lipid content, mitochondrial activity, gene expression and cryo-tolerance. Oocytes were cultured in TCM-199 supplemented with 1 μg/ml estradiol-17ß, 10 μg/ml FSH, 10 ng/ml EGF, 0.6 mM cysteine, 0.2 mM sodium pyruvate and either 8% BSA (BSA group), 10% FBS (FBS group), or neither BSA nor FBS (TCM group), and followed by in vitro fertilization and the zygotes were cultured in SOF-BE1 medium. The differences in embryo development between experimental groups were analyzed by one-way ANOVA. We have shown that the percentages of embryos that underwent cleavage and formed a blastocyst were non significantly different among all experimental groups (37.4 ± 1.5% for FBS group vs. 31.1 ± 3.9% for BSA group and 34.5 ± 1.6% for TCM group, six replicates were performed). Furthermore, there was no significant difference between the percentage of MII oocyte between FBS (71.8 ± 1.9%) and BSA groups (69.3 ± 2.3%). However, culture of oocytes with FBS increased (P < 0.05) the cumulus cell expansion as well as expression of gape junction proteins, CX37 and CX43, at both transcriptional and translation levels. We also found that FBS significantly increased total cell number and decreased the apoptotic index in day-8 blastocyst comparing to BSA group. The beneficial effects of BSA on embryos were associated with significantly reduced intracellular lipid content and increased mitochondrial activity in both oocytes and blastocyst. Taken together, these data suggest that supplementation of maturation medium with BSA, as alternatives to FBS, can be used as defined medium that support consistently the development of IVP bovine embryos.

When sperm penetrates into the ovum, hyaluronidase plays a role of hydrolyzing the hyaluronic acid present in the membrane surrounding the oocytes. The zona pelucida of the ovum is hydrolysed to facilitate sperm entry. Therefore, the aim of this study was to investigate the effects of hyaluronidase during the in vitro maturation in porcine oocytes. The cumulus-oocyte complexes (COCs) were cultured during in vitro maturation (IVM) medium containing 0 and 0.1mg/ml hyaluronidase for 44 h. Representative images of oocytes were captured after cultured for 0 h and 22 h by using a microscope. The area was quantified using a image J software. After 44 h of IVM, nuclear maturation stage was assessed by the aceto-orcein method. In results, cumulus cells expansion was no significant difference between control and hyaluronidase treatment groups in 0 h. However, after 22 h of IVM, in 0.1mg/ml hyaluronidase group, cumulus cells diffusion was significantly reduced than control group (p<0.05). After 22 h matured COCs, the cumulus cells were normally expanded in the control group, but there was a significantly lower 0.1mg/ml hyaluronidase group than control group (p<0.05). The nuclear maturation rate was treated with 0.1mg/ml hyaluronidase, it was significantly decrease than control group (p<0.05). In conclusion, our study indicated that hyaluronidase exposure could reduce nuclear maturation in vitro by reducing the expansion of cumulus cells. According to the results, we conjectured that hyaluronidase treatment disrupted the oocyte maturation by hydrolyzing the hyaluronic acid around the oocytes and it reduces the activity of the intercellular gap junction because it weakens cumulus cell bonds and interferes with communication. However, additional studies on hyaluronidase are needed. This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (Ministry of Education) (2016R1D1A1B03931746).

Generally, in vivo, primary oocytes are grown and matured into secondary oocytes in the ovarian follicles. Quality of the oocytes matured in vivo is higher than that of oocytes matured in vitro, indicating the importance of materializing the microenvironment of ovarian follicles for production of high quality oocyte. Therefore, we tried to mimic the stiffness of ovarian follicles using an agarose as a biocompatible natural polymer. Unfortunately, to date, there are no many reports on whether the quality of porcine oocytes can be increased effectively under the soft matrix. Accordingly, we tried to evaluate the effects of IVM using different mechanical properties of agarose substrate on developmental competence of porcine oocytes. Agarose substrate was constructed and cumulus-oocyte-complexes (COCs) retrieved from porcine medium antral follicles were matured on non-coated (control) culture dish or dishes coated with 1% and 2% (w/v) agarose substrate. Then, cumulus expansion, embryonic development after parthenogenetic activation, and gene expression level were analyzed and compared. As the results, significant increase in blastocyst formation and cumulus expansion were detected in COCs matured on 1% (w/v) agarose substrate compared with control. Moreover, oocytes of COCs matured on 1% (w/v) agarose substrate showed significantly higher BMP15 expression level compared with control. Pro-apoptotic gene BAX expression was significantly increased in oocytes of COCs matured on 2% (w/v) agarose substrate compared with control. In the glycolytic enzyme phosphofructokinase (PFKP) gene expression, cumulus cells of COCs matured on agarose substrate showed significantly higher PFKP expression than control while they showed significantly lower BAX expression than control. These results demonstrated that quality of porcine oocytes could be increased efficiently by the IVM of immature oocytes on the soft culture matrix using agarose.

Sex hormones including progesterons, androgens, and estrogens are influential in differentiation of ovarian tissues and competence of fertility. These steroid hormones derived from cholesterol are required for cumulus-oocyte complexes(COCs) during oocyte maturation. COCs is a total functional and active entity playing a central role in oocyte. Lipid metabolism in the mammalian COCs is controlled by environmental factors. The intracellular cholesterol contents go through remarkable changes. It plays an important part of oocyte developmental competence. However, heat stress affects steroid hormone by decreasing progesterone, estrogen concentrations, and resumption of meiosis in COCs maturation. Reduction of the hormone and meiotic resumption might lead to the decline of ovarian function, follicle maturation, and subsequent embryogenesis. In the same vein, heat stress also influence on germinal vesicle breakdown, lipolytic variations, and loss of the nuclear envelope in the course of maturation of oocytes. In summary, we examined the effects of thermal stress on oocyte maturation through steroid hormone contents of change identifying the molecular mechanisms of lipids metabolism. It may have the solution to further the therapy methods for disorders regarding sterility.

In general, the shape of cumulus-oocyte-complexes (COCs) at germinal vesicle (GV) stage is important roles on meiotic maturation of porcine oocyte during in vitro maturation (IVM). Then, mitochondria produce reactive oxygen species (ROS) such as superoxide from electron transport system during oocyte maturation. ROS levels on oocytes are regulated by various antioxidant enzymes in cumulus cells (CCs). However, the effect of mitochondria derived superoxide production from CCs during IVM of porcine oocyte has not been reported. Firstly, we divided groups according to large number of CCs (Grade 1: G1) and small number of CCs (Grade 2: G2). Then, we counted cumulus cells of G1 and G2 oocyte by using haemocytometer. The oocyte maturation rate was significant decreased (p < 0.05) in G2 oocytes than that of G1 oocytes. We measured mitochondria derived superoxide in G1 and G2 COCs by using Mito-SOX staining. Mitochondrial superoxide was higher in G2 COCs than G1 COCs. Then, the mRNA expression levels of antioxidant enzymes (SOD1, SOD2 and PRDX3) in G2 COCs were decreased compared to G1 COCs. To reduce mitochondria derived superoxide, we used Mito-TEMPO as mitochondrial superoxide scavenger. Oocyte maturation rates in both G1 and G2 groups treated with Mito-TEMPO were increased than that of non-treated groups. Mitochondrial superoxide was lower in G1 and G2 groups treated with Mito-TEMPO than that of non-treatment groups. The mRNA expression levels of antioxidant enzymes in G1 and G2 COCs treated with Mito-TEMPO were increased compared to non-treated groups. Based on these findings, we suggest that reduction of mitochondria derived superoxide by Mito-TEMPO assists maturation competence in porcine oocytes.

U0126 is a highly selective inhibitor of both MEK1 and MEK2, a type of MAPK/ERK kinase. This study was conducted to evaluate the effect of U0126 treatment during in vitro maturation (IVM) on nuclear maturation, intra-oocyte glutathione content, and embryonic development after parthenogenesis (PA). U0126 (5 μM) was supplemented to IVM medium during the first 0 (control), 2, and 4 h. The basic medium used for IVM was medium-199 supplemented with 10% (v/v) porcine follicular fluid (standard), 0.6 mM cysteine, 0.91 mM pyruvate, 75 μg/ml kanamycin, and 1 μg/ml insulin. Immature pig oocytes were matured for 44 h and then oocytes reached metaphase II stage were electrically activated to induce PA. The in vitro culture medium for embryonic development was porcine zygote medium-3 containing 0.3% (w/v) fatty acid-free BSA. When immature oocytes were treated with U0126 during the first 0, 2, 4 h of IVM culture, nuclear maturation was significantly (P < 0.05) increased by the U0126 treatment for 4 h (96.2 ± 1.3%) compared to standard IVM (90.6 ± 2.1%). Cleavage of PA embryos was significantly increased by 4 h- treatment (90.6 ± 2.2%) compared to standard medium (83.9 ± 1.8%). In addition, blastocyst formation of PA embryos was significantly (P < 0.05) increased by the treatment for 4 h (55.8 ± 5.7%) compared to 2 h (38.1 ± 6.1%). The glutathione contents in IVM oocytes were not altered by the U0126 treatments for 0, 2, and 4 h (1.28 ± 0.10, 1.16 ± 0.09, and 1.10 ± 0.09, respectively). Our results demonstrated that 5 μM U0126 treatment during the first 4 h of IVM showed positive effects on nuclear maturation, cleavage, and embryonic development in pigs.

The expression of MMPs in the development of the fertilized egg has a very important role in cell configuration. Objective To evaluate the clinical, the effect of differentially expressed MMPs on serum and serum - free medium on the maturation of blastocysts. The expression patterns of MMPs in serum and serum-free medium were compared at 6 h, 18 h and at the blastocyst stage using real-time PCR, ELISA and immunofluorescence. The results showed that the expression of MMPs was increased in the embryos of the serum medium, as a result of analysis of MMPs and TIMPs, MMP-2 was expressed in the cytoplasm of embryos in the serum-free medium, And it was found to be higher in expression than MMP-9. The serum medium was different from the bloodless badge: overall, TIMPs showed a higher expression in the ovarian cells than cyanosis, and TIMP-3 was more pronounced. Development rate of blastocyst according to in vitro culture method was higher than that of serum - free medium (61.22% 60/98) and serum - free medium (48.28% 28/58). Analysis of the protein release locations of MMPs and TIMPs showed that MMPs and TIMPs are highly expressed in serum mediums, focusing on the inner cell mass. However, very low expression appeared in the tropoblast. On the other hand, serum - free medium showed different expression from serum medium and TIMPs expression was generally low. Therefore, in the case of serum media, the expression of MMPs is highly expressed in the cytoplasm of the fertilized egg, increasing the reconstruction of cells.

Alpha-linolenic acid (ALA; n-3 18:3), a one of omega-3 fatty acid, is mainly contained in chloroplast of plant and ALA is an essential fatty acid, not synthesized in mammalian body, it must be supplied from foods. Polyspermy is especially high on in vitro fertilization (IVF) in pigs, which is a major obstacle to in vitro embryo production systems. In our previous study, when ALA was supplemented during in vitro maturation (IVM), the methaphase-II rate and gluthathione level was increased. The objective of this study was to evaluate the effects of alpha-linolenic acid (ALA) supplementation during IVM and subsequent of IVF in pigs. The cumulus-oocyte complexes (COCs) were submitted to IVM medium containing 0, 25, 50, and 100 μM ALA for 44 h. After 44 h of IVM, denuded oocytes were co-cultured with spermatozoa during 18 h. After 18 h of in vitro fertilization, oocyte were using aceto-orcein method, to evaluated penetration rate, monospermy (number of monospermy oocytes/total oocytes), and the IVF efficiency (number of monospermy/total penetrated oocytes). In results, 25 and 50 μM ALA groups were significantly increased on penetration rate compared with 100 μM ALA group (p<0.05). Similarly, monospermy rate were significantly increased 25 and 50 μM ALA groups than control group (p<0.05). IVF efficiency was no significant difference between control and ALA treatment groups. Our findings suggested that treatment of ALA supplementation during in vitro maturation (IVM) and subsequent of in vitro fertilization in pigs, ALA can increase IVF efficiency by effectively blocking polyspermy and increasing monospermy some mechanism in porcine oocytes. However, the study of mechanism by which ALA blocks polyspermy are needed, and this study suggests that ALA has a positive effect on in vitro production of porcine oocytes by decreasing polyspermy. This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (Ministry of Education) (2016R1D1A1B03931746).

In the present study of this experiment was to understand the expression of apoptotic gene expression in the ovary of miniature pigs and pigs on the 15th day of estrus. Also the compare and analyze of programmed cell death type(Apoptosis and autophagy) expression pattern during mature oocyte on the miniature and normal pig cells. Analysis of mRNA gene expression of ovary in miniature and normal pigs on the 15th day of estrus showed that the expression of genes related to Autophagy (ATG13, MAP1LC3, Beclin1) was high in normal pigs but the expression of ATG1 and ATG5 genes was low. In addition, the expression of genes related to apoptosis (Casp-3, BAX) was high in the mini pigs, and the gene related to the LH hormone was high in the miniature pigs, whereas the expression of the gene related to the FSH hormone was high in the normal pigs. On the other hand, the result of muture oocyte on the miniature and normal pig cells is the expression of Casp-3 protein was moust high from treatment of FL+rapa (FSH+LH and Rapamycin) of the oocyte on the miniature pig cell. However, MAP1LC3 expression was higher in the oocytes of treatment of rapanycine treatment on the nomal pig cells. There was no gene expression in cumulus cells of matured oocytes in mini pig cells, whereas MAP1LC3 expression was higher in oocyte cumulus cells matured in normal pig cells. It was confirmed that the miniature and normal pigs showed different programmed cell death patterns, In the case of oocytes matured in miniature pig cells, MAP1LC3 gene expression was found to be low in spite of treatment with Autophagy regulator.