327THE EFFECTS OF GLUCOSE AND GLUCOSAMINE ON CUMULUS EXPANSION AND 
NUCLEAR MATURATION OF BOVINE CUMULUS-OOCYTE COMPLEXES

Glucose is the primary energy substrate consumed by bovine COCs during in vitro maturation (IVM), with most accounted for by glycolysis (L-lactate production). However, antral follicular fluid (FF) contains less than half the glucose of standard IVM media (TCM199=5.6mM, FF=2.3mM). We have previously demonstrated that from 20 to 24h of IVM, a significant proportion of the glucose utilized is directed into pathways other than lactate production (Sutton et al., 2003 Reproduction 126, 27–34). We hypothesize that glucose is utilized for cumulus matrix synthesis. The aim of this study was to determine the influence of glucosamine (an intermediate for matrix components) on FSH-stimulated glucose uptake and cumulus expansion. The influence of different glucose concentrations and glucosamine on nuclear maturation was also investigated. Bovine COCs were collected from abattoir-derived ovaries. In Exp. 1, individual COCs (n=60, 3 replicates) were cultured in 10-μL drops of TCM199 (plus pyruvate, hCG and BSA, containing 5.6mM glucose), ±FSH (0.1IUmL−1) and ±glucosamine (5mM). After 20h, COCs were transferred to fresh media and cultured a further 4h. Cumulus expansion and glucose/L-lactate levels in spent medium from 0–4-h and 20–24-h culture periods were measured. In Experiment 2, COCs (n=300, 6 replicates) were cultured in groups of 10 in 100μL of Bovine FF medium (a defined medium based on the composition of bovine antral FF, also containing amino acids, FSH, hCG and BSA) ±glucosamine (5mM) in 2.3 or 5.6mM glucose, or in conventional TCM199 IVM media (as above). Nuclear maturation was assessed at 24 and 30h using orcein staining. Treatment differences were determined using two-way ANOVA. The influence of FSH and glucosamine (Exp. 1) on the measured parameters was evident at 20–24h, with FSH increasing diameter, glucose uptake and L-lactate production (P<0.05). Although glucosamine alone did not influence diameter or glucose/L-lactate concentrations, glucosamine plus FSH led to a decrease in glucose uptake compared to FSH-stimulation alone (P<0.05). The proportion of oocytes at MII (Exp. 2) was significantly lower when COCs were cultured in low glucose (main effect, 24h: 2.3mM=38% v. 5.6mM=64%; P<0.005). The presence of glucosamine tended to stimulate meiotic maturation (main effect, 24h: 0mM=45% v. 5mM=59%; P=0.1). MII frequency in TCM199 controls at 24h was 68%. These experiments support the hypothesis that synthesis of cumulus matrix is a major pathway for glucose metabolism, especially in the absence of glucosamine. Furthermore, oocytes matured in media based on a physiological concentration of glucose (2.3mM), have delayed meiosis compared to oocytes cultured in higher glucose (5.6mM). Thus, glucose has multiple functions, involving matrix formation and meiosis regulation during bovine IVM. Supplementation of medium with glucosamine appears to partly reduce the dependency of COCs on glucose. Supported by Australian Research Council and COOK Australia.

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341 OXYGEN TENSION AND EGF AFFECT METABOLISM OF IN VITRO-MATURED CUMULUS-ENCLOSED MOUSE OOCYTES

Reproduction Fertility and Development ◽

10.1071/rdv18n2ab341 ◽

2006 ◽

Vol 18 (2) ◽

pp. 278

Author(s):

K. A. Preis ◽

G. E. Seidel Jr ◽

D. K. Gardner

Keyword(s):

Glucose Uptake ◽

Oxygen Tension ◽

Embryo Development ◽

Metabolic Activity ◽

Lactate Production ◽

Defined Medium ◽

Blastocyst Development ◽

Developmental Potential ◽

Exact Test

In vitro maturation of immature oocytes results in limited success in both clinical and research laboratories. Although reduced oxygen concentration is beneficial to embryo development, the optimal concentration for oocyte maturation has yet to be determined. The objective of this study was to determine whether oxygen tension (20% or 5% O2) affects oocyte physiology. Additionally, the effect of epidermal growth factor (EGF) in maturation medium on oocyte metabolic activity and subsequent embryo development was determined. Cumulus–oocyte complexes (COCs; n = 231) were collected from 28-day-old unprimed F1 (C57BL/6 × CBA/ca) mice. COCs were individually matured in defined medium at 37°C in 6% CO2 in one of four groups (Table 1). For the metabolism study, COCs were further divided into two groups: individual maturation in a 2-µL drop of medium for 16 h (n = 131); or individual maturation in 5-μL for 12 h and then placed in a 0.5-μL drop of medium for 4 h (n = 100), the time of greatest metabolic activity of the COC. At 17 h of maturation, COCs were individually fertilized, and zygotes were individually cultured until 96 h, at which time blastocyst development was assessed. Metabolic profiles were analyzed by ANOVA, and blastocyst rates were analyzed by Fisher's exact test. Maturation rates and blastocyst development were not different between groups. However, at 12–16 h of maturation, metabolism of COCs was affected by both oxygen tension and EGF (Table 1). Concerning metabolism over the entire course of maturation, glucose uptake and lactate production were higher in COCs in 5% O2 + 100 ng EGF (P < 0.05) than in the remaining three groups. There was no difference between 5% O2 and 20% O2 + 100 ng EGF, but 20% O2 caused less glucose uptake and lactate production than did the other three treatment groups (P < 0.05). Results of this study are the first to show that oxygen tension alters COC metabolism: COCs matured under 5% O2 were more active metabolically than COCs matured under 20% O2. The effect of oxygen tension is to some extent moderated by the presence of EGF, as metabolic activity of COCs matured under 20% O2 + 100 ng EGF was closer to that of COCs matured under 5% O2 conditions. Although blastocyst rates were similar across the four groups, embryos derived from oocytes matured in different oxygen tensions may exhibit different developmental potential. In conclusion, results of this study have implications for the improvement of maturation conditions in both clinical and research laboratories. Table 1. Carbohydrate metabolism of individual COCs at 12–16 h of maturation

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296 IMPACT OF CO-CULTURING CUMULUS-ENCLOSED PORCINE OOCYTES WITH DENUDED OOCYTES DURING IN VITRO MATURATION IN A DEFINED MEDIUM ON CUMULUS EXPANSION AND OOCYTE MATURATION

Reproduction Fertility and Development ◽

10.1071/rdv27n1ab296 ◽

2015 ◽

Vol 27 (1) ◽

pp. 237

Author(s):

R. Appeltant ◽

T. Somfai ◽

M. Nakai ◽

S. Bodo ◽

D. Maes ◽

...

Keyword(s):

Oocyte Maturation ◽

In Vitro Maturation ◽

Binary Logistic Regression ◽

Defined Medium ◽

Positive Influence ◽

Developmental Competence ◽

Cumulus Expansion ◽

Nuclear Maturation ◽

Morphological Criteria

Recent research has revealed that oocyte-secreted factors (OSF) affect cumulus expansion and play important roles during maturation and embryo development of mammalian oocytes. The use of denuded oocytes (DO) as supplements during in vitro maturation (IVM) in a nondefined medium improved developmental competence of cumulus-enclosed porcine oocytes (COC; Gomez et al. 2012 Zygote 20, 135–145). We investigated the effect of DO on cumulus expansion and nuclear maturation of COC in pigs during IVM using a defined medium. If the DO exert a positive influence on IVM, the defined medium can then be analysed for the presence of OSF. Immature COC were collected in the slaughterhouse from prepubertal gilts. To obtain DO, some COC were completely denuded by pipetting through a narrow-bore glass pipette. The COC used as a source for DO fulfilled the same morphological criteria as the COC used for IVM. The IVM medium was porcine oocyte medium (POM; Yoshioka et al. 2008 J. Reprod. Dev. 54, 208–213) with hormone supplementations applied only during the first 20 h of the IVM period. The COC were fixed to the bottom of 35-mm plastic Petri dishes in 3 × 3 grids by Cell-Tak (BD Bioscience, Bedford, MA, USA) in 100-µL droplets POM covered by paraffin oil. Culture droplets (each including 1 COC grid) were supplemented with (DO+ group, n = 179) or without 16 DO (DO– group, n = 143). After 20 h of IVM, the medium was replaced with a preincubated hormone-free POM and oocytes were cultured for an additional 28 h. At 0, 20, and 48 h of IVM, images of each grid were taken at the same magnification. The size of each COC was measured as a 2-dimensional area in pixels by analysing images with ImageJ software. Relative cumulus expansion was calculated at 20 and 48 h of IVM on the basis of the initial COC size at 0 h, which was assigned as 1. At 48 h of IVM, the COC were denuded and examined for oocyte maturation by orcein staining. The experiment was replicated 5 times. Cumulus expansion ratios at 20 and 48 h of IVM were compared between the DO+ and DO– groups by ANOVA. Maturation rates were compared between the DO+ and DO– groups by binary logistic regression. No difference in cumulus expansion between DO– and DO+ could be observed at 20 h (1.83 ± 0.04 and 1.75 ± 0.03, respectively) and 48 h (1.41 ± 0.03 and 1.47 ± 0.02, respectively) of IVM. Nuclear maturation rates of COC in DO– and DO+ groups did not differ significantly (39.0 ± 5.4 and 32.9 ± 8.8%, respectively). In conclusion, addition of DO to the defined IVM medium did not affect the cumulus expansion and oocyte maturation of follicular porcine COC. Further research is needed to assess the effects of DO during IVM on subsequent fertilization. If DO prove to be beneficial for fertilization, the nature of the OSF will be investigated.This study was supported by FCWO of UGent and by FWO-Flanders (grant number FWO11/ASP/276).

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272 INTRACELLULAR NITRIC OXIDE LEVEL OF PORCINE OOCYTES IS NEGATIVELY CORRELATED WITH OOCYTE MATURATION RATE AND CUMULUS EXPANSION INDEX IN A CHEMICALLY DEFINED MEDIUM

Reproduction Fertility and Development ◽

10.1071/rdv23n1ab272 ◽

2011 ◽

Vol 23 (1) ◽

pp. 234

Author(s):

T. Uozumi ◽

H. Funahashi

Keyword(s):

Nitric Oxide ◽

Oocyte Maturation ◽

Defined Medium ◽

Chemically Defined Medium ◽

Dibutyryl Camp ◽

Cumulus Expansion ◽

Nuclear Maturation ◽

Porcine Oocyte ◽

Maturation Rate

Nitric oxide (NO) has been known to inhibit nuclear maturation in cumulus–enclosed oocytes in rodents. The objective of this study was to examine if meiotic stimulators, such as dibutyryl cAMP and epidermal growth factor (EGF), influence intracellular NO level of oocytes and if the level is correlated with oocyte maturation rate and cumulus expansion in a chemically defined medium. Oocyte–cumulus complexes (OCC) were aspirated from mid-size follicles (3–6 mm in diameter) of prepuberal porcine ovaries. The OCC were cultured in modified porcine oocyte medium with various supplements – gonadotropins plus dibutyryl cAMP (Gn + cAMP), EGF plus dibutyryl cAMP (EGF + cAMP), dibutyryl cAMP alone (cAMP), EGF alone (EGF), and non-supplements (none) – for a first 20-h period and then in fresh porcine oocyte medium (without those supplements) for another 24 h in an atmosphere of 5% CO2 in air at 39°C. Following in vitro maturation culture, OCC were assessed for the degree of cumulus expansion (scored from 0 as cumulus free to 5 as full expansion) and then additionally cultured with DAF2-DA, an indicator of NO, for an additional 1-h period in the same condition. The oocytes were denuded with 0.1% hyaluronidase, and the intensity of fluorescence was measured. The oocytes were also fixed, stained with acetic orcein, and observed for meiotic stage. Statistical analysis was performed by ANOVA with a Bonferroni-Dunn post hoc test (significance, P < 0.05). Maturation rates and cumulus expansion indexes were significantly affected by various supplement conditions (Table 1). The intensity of fluorescence showing intracellular NO level was also different among experimental groups (Table 1). A negative correlation was found between intracellular NO intensity and maturation rate (r2 = 0.71) or cumulus expansion index (r2 = 0.70). From these results, we conclude that there is a synergistic effect of cAMP and EGF on cumulus expansion and oocyte maturation and the reduction of oocyte NO levels in a chemically defined medium. Furthermore, a reduction of oocyte NO level seems to be included in the induction of cumulus expansion and oocyte maturation. Table 1.Effects of supplements on nuclear maturation, cumulus expansion, and intracellular NO level of porcine oocytes1

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Human TNF-α Affects the Egg-laying Dynamics and Glucose Metabolism of Schistosoma Mansoni Adult Worms in Vitro

10.21203/rs.3.rs-853245/v1 ◽

2021 ◽

Author(s):

Ednilson Hilário Lopes-Junior ◽

Gilbert de Oliveira Silveira ◽

Camila Banca Guedes ◽

Gratchela Dutra Rodrigues ◽

Viviane Sousa Ribeiro ◽

...

Keyword(s):

Schistosoma Mansoni ◽

Mrna Expression ◽

Glucose Uptake ◽

Lactate Production ◽

Egg Laying ◽

Dependent Manner ◽

Tnf Α ◽

Atp Metabolism ◽

Fine Regulation

Abstract Several studies described the effect of human TNF-α on Schistosoma mansoni. It affects the worm’s development, metabolism, egg-laying, changes in the parasite´s gene expression and protein phosphorylation. Data available concerning the influence of hTNF-α on egg-laying are controversial and understanding the mechanism of egg-laying regulation is essential in combating schistosomiasis. We characterized the effects of in vitro treatment of S. mansoni adult worms with different doses of hTNF-α (5, 20 and 40ng/mL) for five days. We explored the effects on the egg-laying rate, glucose, ATP metabolism, mRNA expression levels of lactate dehydrogenase, of glucose transporters and of SmTNFR, the parasite gene for hTNF-α receptor. hTNF-α influenced egg-laying in a time and dose dependent manner: with 40ng/mL, egg-laying increased on day 2 and decreased on days 3 and 4; 20 ng/mL dose, egg-laying decreased on day 3, while at 5ng/mL dose, egg-laying decreased on day 4. The total number of eggs produced was not affected, but the egg-laying dynamic was altered; the median egg-laying time decreased significantly due to treatment. At 5 and 20ng/mL hTNF-alpha, lactate production diminished on days 3 up to 5, while glucose uptake increased on day 5. At 40ng/mL, glucose uptake diminished on days 1 up to 3, while ATP accumulation was detected on day 5. No significant changes in the mRNA expression were detected in all treatments. Crosstalk involving the hTNF-alpha and the parasite signaling play a role in the fine regulation of the worm´s metabolism and physiology and points to new strategies for disease control.

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319IN VITRO MATURATION OF EQUINE OOCYTES IN A COMPLETELY DEFINED MEDIUM SUPPLEMENTED WITH PROGESTERONE

Reproduction Fertility and Development ◽

10.1071/rdv16n1ab319 ◽

2004 ◽

Vol 16 (2) ◽

pp. 279

Author(s):

B. Merlo ◽

E. Iacono ◽

F. Prati ◽

G. Mari

Keyword(s):

Polar Body ◽

Basal Medium ◽

Defined Medium ◽

Chi Square ◽

Nuclear Maturation ◽

Maturation Rate ◽

Chi Square Test ◽

The Media ◽

Cytoplasmic Maturation

A completely defined medium for in vitro maturation (IVM) of equine oocytes has not yet been developed, since most of the media used for IVM are supplemented with serum or BSA. Furthermore, in this species there is no report about the influence of progesterone on maturation, although it has already been used as supplement (500ngmL−1) in EMMI (Maclellan LJ et al., 2001, Theriogenolgy 55, 310 abst). The aims of this study were to develop a completely defined medium for equine oocyte maturation and to investigate the effect of progesterone on nuclear maturation. Equine oocytes were collected by follicular scraping of abattoir-derived ovaries between April and June. The basal medium for maturation was SOFaa supplemented with pFSH-LH 0.1IUmL−1 (Pluset, Laboratorios Calier, Barcelona, Spain), EGF* 50ngmL−1, ITS (Insulin, Transferrin, Sodium selenite), L-cysteine 1.2mM, Maturation SOF (MSOF). Compact cumulus-oocyte complexes were selected, washed three times in H-SOF and matured in one of the following media (15–20 oocytesmL−1): (1) MSOF+FCS 10% (MSOF-FCS), (2) MSOF+progesterone 100ngmL−1 (MSOF-P4), (3) MSOF. After 24h of culture in 5% CO2 in air at 38.5°C, the oocytes were denuded by gently pipetting in a 0.25% trypsin solution, washed and stained with Hoechst 33258 (10μgmL−1 in PBS) for 30min at room temperature. Oocytes were examined under a fluorescent microscope to assess nuclear maturation. Only oocytes with an evident polar body and metaphase II plate (MII) were considered mature. The experiment was done in 6 replicates. Chi Square test was used for statistical analysis (Statistica for Windows – Stat Soft Inc., Tusla, OK, USA). Significance was assessed for P<0.05. The results of this study show that MSOF can be considered a suitable completely defined medium for IVM of equine oocytes. Adding progesterone significantly (P<0.05) increases the nuclear maturation rate at 24h of culture. It can be speculated that although cumuls cells produce this hormone, supplementation is useful to reach progesterone concentrations similar to those present in follicular fluid (early dominant 63.4±19.3ngmL−1, healthy preovulatory follicle 1094.3±170.9ngmL−1; Gerard N et al., 2002, Reproduction 124, 241–248). Further studies are needed to investigate the influence of progesterone on cytoplasmic maturation and to test the effect of different progesterone concentrations and time of maturation in a completely defined system.*All chemicals were purchased from Sigma, St. Louis, MO, USA, unless otherwise stated. Table 1 Maturation of equine oocytes in different media

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The presence of acylated ghrelin duringin vitromaturation of bovine oocytes induces cumulus cell DNA damage and apoptosis, and impairs early embryo development

Zygote ◽

10.1017/s0967199417000478 ◽

2017 ◽

Vol 25 (5) ◽

pp. 601-611 ◽

Cited By ~ 2

Author(s):

Matias A. Sirini ◽

Juan Mateo Anchordoquy ◽

Juan Patricio Anchordoquy ◽

Ana M. Pascua ◽

Noelia Nikoloff ◽

...

Keyword(s):

Dna Damage ◽

Embryo Quality ◽

Cumulus Cell ◽

Early Embryo ◽

Acylated Ghrelin ◽

Cumulus Expansion ◽

Nuclear Maturation ◽

Developmental Capacity ◽

Bovine Oocytes

SummaryThe aim of this study was to investigate the effects of acylated ghrelin supplementation duringin vitromaturation (IVM) of bovine oocytes. IVM medium was supplemented with 20, 40 or 60 pM acylated ghrelin concentrations. Cumulus expansion area and oocyte nuclear maturation were studied as maturation parameters. Cumulus–oocyte complexes (COC) were assessed with the comet, apoptosis and viability assays. Thein vitroeffects of acylated ghrelin on embryo developmental capacity and embryo quality were also evaluated. Results demonstrated that acylated ghrelin did not affect oocyte nuclear maturation and cumulus expansion area. However, it induced cumulus cell (CC) death, apoptosis and DNA damage. The damage increased as a function of the concentration employed. Additionally, the percentages of blastocyst yield, hatching and embryo quality decreased with all acylated ghrelin concentrations tested. Our study highlights the importance of acylated ghrelin in bovine reproduction, suggesting that this metabolic hormone could function as a signal that prevents the progress to reproductive processes.

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172 DETERMINING THE REQUIREMENTS FOR LH AND FSH DURING SHEEP IN VITRO OOCYTE MATURATION

Reproduction Fertility and Development ◽

10.1071/rdv26n1ab172 ◽

2014 ◽

Vol 26 (1) ◽

pp. 200 ◽

Cited By ~ 5

Author(s):

C. de Frutos ◽

R. Vicente-Perez ◽

P. J. Ross

Keyword(s):

Oocyte Maturation ◽

In Vitro Maturation ◽

Mixed Logit ◽

Cumulus Cells ◽

Pituitary Hormones ◽

Developmental Competence ◽

Cumulus Expansion ◽

Nuclear Maturation ◽

Grade 3

In vitro maturation (IVM) of oocytes in domestic animals is a widespread practice of research and commercial relevance. Gonadotropic hormones are typically supplemented to the IVM medium to stimulate resumption of meiosis, progression to metaphase II (MII), and oocyte developmental competence. The common use of pituitary-derived products presents 2 problems: contamination from other pituitary hormones and inconsistences from batch-to-batch variation. Recombinant hormones can help circumvent these issues and identify specific gonadotropin requirements for in vitro maturation. The aim of the present study was to determine the effect of supplementing recombinant bovine LH and/or FSH (AspenBio) to the maturation of ovine oocytes in terms of cumulus expansion and progression to the MII stage. Abattoir-derived sheep cumulus–oocyte complexes (COC) were obtained from 1- to 5-mm-diameter antral follicles by ovary slicing. Oocytes with a homogeneous cytoplasm surrounded by at least 3 layers of cumulus cells were selected and cultured in serum-free IVM medium (Cotterill et al. 2012 Reproduction 144, 195–207) at 38.5°C and 5% CO2. The COC obtained from 8 replicates were allocated into 4 experimental groups: (1) no hormones; (2) 1.5 μg mL–1 recombinant bovine LH (rbLH); (3) 1.5 μg mL–1 recombinant bovine FSH (rbFSH); and (4) rbLH and rbFSH. The expansion of cumulus cells was recorded in each group after 24 h of IVM and COC classified as (1) very poor or no cumulus expansion (grade 1); (2) limited cumulus expansion (grade 2); and (3) full cumulus expansion (grade 3). Nuclear maturation in the 4 treatments was evaluated by assessing progression to the MII stage via DNA staining with Hoechst 33342 and fluorescence imaging. The effect of treatment on the observed proportion of MII oocytes was evaluated using a mixed logit model including treatment and replicate as fixed and random effects, respectively. Culture in IVM medium in the absence of gonadotropins or in the presence of rbLH resulted in poor cumulus expansion (grade 1). The supplementation of IVM medium with rbFSH (with or without rbLH) yielded a high degree of cumulus expansion (grades 2–3). Likewise, addition of rbFSH enhanced progression of oocytes to the MII stage, whereas use of rbLH, although it had an effect on progression to MII, did not augment the effect of rbFSH (Table 1). These results indicate that rbFSH is necessary and sufficient to induce sheep oocyte maturation in a high proportion of oocytes. Table 1.Cumulus expansion and oocyte nuclear stage after IVM

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Porcine follicular fluid derived from > 8 mm sized follicles improves oocyte maturation and embryo development during in vitro maturation of pigs

Zygote ◽

10.1017/s0967199420000398 ◽

2020 ◽

pp. 1-6

Author(s):

Ji-Eun Park ◽

Sang-Hee Lee ◽

Yong Hwangbo ◽

Choon-Keun Park

Keyword(s):

Embryonic Development ◽

Follicular Fluid ◽

In Vitro Maturation ◽

Developmental Competence ◽

Control Group ◽

Cumulus Expansion ◽

Treatment Groups ◽

Nuclear Maturation ◽

Cytoplasmic Maturation

Summary The aim of the present study was to investigate the effects of porcine follicular fluid (pFF) from large-sized (LFF; >8 mm in diameter) and medium-sized (MFF; 3–6 mm in diameter) follicles on the maturation and developmental competence of porcine oocytes. Cumulus–oocyte complexes (COCs) were collected from follicles 3–6 mm in diameter. The collected COCs were incubated for 22 h with LFF or MFF (in vitro maturation (IVM)-I stage) and were incubated subsequently for 22 h with LFF or MFF (IVM-II stage). Cumulus expansion was confirmed after the IVM-I stage and nuclear maturation was evaluated after the IVM-II stage. Intracellular glutathione (GSH) and reactive oxygen species (ROS) levels were measured and embryonic development was evaluated. Relative cumulus expansion and GSH levels were higher in the LFF group compared with in the MFF group after the IVM-I stage (P < 0.05). After the IVM-II stage, the numbers of oocytes in metaphase-II were increased in the LFF group and GSH content was higher in all of the LFF treatment groups compared with in the MFF treatment groups during both IVM stages (P < 0.05). ROS levels were reduced by LFF treatment regardless of IVM stage (P < 0.05). Blastocyst formation and the total numbers of cells in blastocysts were increased in all LFF treatment groups compared with the control group (P < 0.05). These results suggested that pFF from large follicles at the IVM stage could improve nucleic and cytoplasmic maturation status and further embryonic development through reducing ROS levels and enhancing responsiveness to gonadotropins.

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Metabolic markers of developmental competence for in vitro-matured mouse oocytes

Reproduction ◽

10.1530/rep.1.00831 ◽

2005 ◽

Vol 130 (4) ◽

pp. 475-483 ◽

Cited By ~ 31

Author(s):

Kimberly A Preis ◽

George Seidel ◽

David K Gardner

Keyword(s):

Lactate Production ◽

Glucose Consumption ◽

Cumulus Cells ◽

Defined Medium ◽

Developmental Competence ◽

Metabolic Markers ◽

Oocyte Competence ◽

Carbohydrate Consumption ◽

Mouse Oocytes

In vitro maturation of oocytes has enormous potential in assisted reproductive technology, but its use has been limited due to insufficient knowledge of oocyte physiology during this dynamic period and lack of an adequate maturation system. The aim of this study was to characterize the metabolic profiles of three groups of oocytes throughout maturation: cumulus–oocyte complexes (COCs), denuded oocytes, and denuded oocytes co-cultured with cumulus cells. Mouse oocytes were collected from 28-day-old unstimulated females and matured in a defined medium. Oocytes were matured individually and transferred into fresh 0.5 μl drops of medium at 4 h intervals until 16 h. Ultramicrofluorimetry was used to quantitate carbohydrate consumption from and metabolite release into the medium. Glucose consumption and lactate production of COCs increased (P < 0.001) over the maturation interval (0–16 h). Glucose consumption by COCs that subsequently fertilized was higher between 8–12 h of maturation than by COCs that did not fertilize (38 versus 29 pmol/COC per h, respectively; P < 0.01). Lactate production by COCs that subsequently fertilized was higher between 8–16 h of maturation, than by oocytes that did not fertilize (8–12 h, 66 versus 46 pmol/COC per h, P < 0.01; 12–16 h, 56 versus 40 pmol/COC per h, respectively; P < 0.05). These data indicate that the final hours of maturation may hold a unique marker of oocyte competence, as during this time fertilizable COCs take up more glucose and produce more lactate than those not subsequently fertilized.

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RELATIVE POTENCY OF ANTI-DIABETIC COSTUS SPECIOSUSPLANT EXTRACT IN MOUSE FIBROBLAST CELL LINE

International Journal of Research -GRANTHAALAYAH ◽

10.29121/granthaalayah.v4.i3.2016.2790 ◽

2016 ◽

Vol 4 (3) ◽

pp. 99-107

Author(s):

Bindu ◽

Rama Bhat ◽

Girish ◽

Krishna Prasad

Keyword(s):

Glucose Uptake ◽

Biological Activities ◽

Glycogen Synthesis ◽

In Vitro Study ◽

Significant Inhibition ◽

Mouse Fibroblast ◽

Relative Potency ◽

Major Pathway ◽

Chemical Structures

Plant-derived compounds have been used clinically to treat type 2 diabetes for many years as they also exert additional beneficial effects on various other disorders. PI3K pathway is the major pathway activated by insulin receptor (IR). It induces glucose uptake, glycogen synthesis, protein synthesis, cell growth and differentiation. Hence metabolic assay was employed to assess glucose uptake based on the property of 3T3-L 1 cells to differentiate into adipocytes which can take up the glucose in medium due to the effect of insulin or insulin like molecules. The results of the current study showed that plants extract probably exerts its anti-diabetic properties by stimulating glucose uptake in adipocytes with significant inhibition of adipogenesis demonstrating reliable relative potency in comparision to the commercial insulin. The ability of existing therapies to target various aspects of the insulin resistance syndrome induces other metabolic abnormalities, chiefly those involved in lipid metabolism. In this preliminary in-vitro study Costusspeciosus plant extract demonstrated to have significant relative potency in comparison to commercial Insulin which can be exploited to treat diabetes using natural herbal extracts Current study leads researchers to elucidate the chemical structures, isolate active ingrediatents in the crude extract for such biological activities in reference to commercial and recombinant insulins.

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