scholarly journals 184 PRELIMINARY FINDINGS ON CARBOHYDRATE METABOLISM OF INTACT EQUINE CUMULUS-OOCYTE COMPLEXES DURING IN VITRO MATURATION

2016 ◽  
Vol 28 (2) ◽  
pp. 223 ◽  
Author(s):  
N. Lewis ◽  
K. Hinrichs ◽  
D. Brison ◽  
R. Sturmey ◽  
D. Grove-White ◽  
...  
Keyword(s):  
Carbohydrate Metabolism ◽  
Dna Content ◽  
In Vitro Maturation ◽  
Polar Body ◽  
Cumulus Cell ◽  
Glucose Consumption ◽  
Cumulus Cells ◽  
Significant Difference ◽  
Spent Media

Production of equine embryos in vitro is gaining popularity, and many differences exist in composition of in vitro maturation (IVM) media. Metabolism of the cumulus-oocyte complex (COC) is essentially unknown in the horse. Here, we describe preliminary data on carbohydrate metabolism of the equine COC during IVM. COC, collected by scraping of the granulosa layer of all visible follicles of abattoir-derived ovaries, were held overnight (12–18 h) at room temperature (~20°C) and then placed in Maturation Medium (M199 with Earle’s salts, 10% FBS, with 25 μg mL–1 gentamicin and 5 mU mL–1 FSH). They were incubated singly in 10-μL droplets under mineral oil for 30 h at 38.3°C in 5% CO2 in air. Control droplets without COC were incubated in the same dish. After incubation, COC were removed and spent media kept at –80°C for later analysis. Oocytes were denuded of cumulus cells by pipetting in the presence of hyaluronidase and evaluated by light microscopy at 500×. Those with a visible polar body were classified as metaphase II (MII); oocytes with an intact oolemma and no polar body were classified as immature intact (INT) and those with an irregular or unapparent oolemma, or shrunken cytoplasm, were classed as degenerating (DEG). To adjust for variation in cumulus cell number, the stripped cumulus cells were frozen at –20°C and later analysed for DNA content using Picogreene. The spent media was analysed for depletion of glucose and appearance of lactate on a BMG Fluostar spectrophotometer using an enzyme-linked ultrafluorometric method. Data were expressed as pmol/ng DNA/hr and analysed by t-test, x2 and logistic regression. Thirty COC were cultured and analysed; 14 were classified as MII, 2 INT and 14 DEG. Seven COC (23%) depleted all the available glucose, indicating that the rate of glucose consumption in those 7 complexes was ≥1866 pmol/COC per hour. DNA content was positively correlated with glucose depletion (P = 0.02). In the COC that did not deplete available glucose, the ratio of glucose consumption:lactate production was 1.82, indicating that the major fate of exogenous glucose was production of lactate by glycolysis. In the 7 oocytes that depleted all the glucose, the ratio of glucose consumption:lactate production was 1.22. One explanation for this may be that when glucose was no longer available, it was conserved for other pathways. It was noteworthy that these COC had more cumulus cells (P < 0.01) and the maturation rate was 4/7 (57%). In the group of COC that did not deplete all of the glucose, there was no significant difference in glucose consumption (13.17 v. 12.25 pmol/ng DNA per hour; P > 0.4) or lactate production (21.48 v. 20.28 pmol/ng DNA per hour; P > 0.4) between COC in which the oocyte reached MII (10/23; 43%), and those which contained a degenerated oocyte at the end of culture, respectively. To the best of our knowledge, this is the first report documenting the metabolism of equine COC. These data underline the importance of further studies to determine optimal conditions for in vitro maturation of equine COC, especially in terms of glucose availability.

scholarly journals The Number of LH Receptor could Predict the Success of Oocyte Maturity in the Process of In Vitro Maturation

2016 ◽  
Author(s):  
Adek Amansyah
Keyword(s):  
In Vitro Maturation ◽  
Clinical Laboratory ◽  
Polar Body ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Oocyte Maturity ◽  
Lh Receptor ◽  
First Polar Body ◽  
Significant Difference

Objective: To evaluate the relationship between the number of LH receptor and the success of oocyte maturity in the process of in vitro maturation (IVM). Method: This experimental study was conducted in the Permata Hati Infertility Clinical Laboratory, Dr. Sardjito General Hospital, Yogyakarta, with the samples of 300 oocytes obtained through collecting immature cow’s oocytes from the abattoir and grouped the oocytes into 3 (three) groups based on the pattern of oocyte cumulus cells on the vesicle germinal stage 2 - 8 mm with three layers of cumulus cell. The sample of the cumulus cells from these three groups were taken and the LH receptor examination was done with immunohistochemistry. After that, the IVM process was performed to the three groups and its development for 24 hours was evaluated. Its maturation quality was evaluated with the emergence of the first polar body (1PB) and compared to the other groups and related to the number of LH receptor in the three groups. Result: The result of this study indicated that the oocyte cumulus cells showed a difference of function during IVM process. The maturity rate in this study showed that the number of LH receptor was related to the morphological pattern of oocyte cumulus cells with oocyte maturity. The maturity of the cumulus cells which 100% covered the oocyte was higher than that of the cumulus cells which > 50% and < 30% covered the oocytes, namely, 74% compared to 60% and 12%. The result of this study also showed that the average number of LH receptors in the three groups (A, B, and C) was 183.4, 78.8, and 24.0 respectively. A significant difference was found in the three groups (p < 0.0001). When related to IVM maturity, this difference showed that the bigger number of oocyte cumulus cells influenced the oocyte maturity. Conclusion: The number of LH receptor can be used as a prediction to determine the success of oocyte maturation in the process of in vitro maturation. [Indones J Obstet Gynecol 2013; 1-4:183-7] Keywords: IVM, LH receptor, oocyte cumulus cell

326 EVALUATION OF CULTURE DURATION AND PARTIAL CUMULUS CELL REMOVAL DURING CANINE OOCYTE MATURATION

2006 ◽  
Vol 18 (2) ◽  
pp. 270
Author(s):  
C. Hanna ◽  
C. Long ◽  
M. Westhusin ◽  
D. Kraemer
Keyword(s):  
In Vitro Maturation ◽  
Calf Serum ◽  
Cumulus Cell ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Culture Conditions ◽  
Germinal Vesicle Breakdown ◽  
Significant Difference ◽  
Culture Duration

The objectives of this study were to determine whether the percentage of canine oocytes that resume meiosis during in vitro maturation could be increased by either increasing culture duration or by removing approximately one-half of the cumulus cells 24 h after oocytes were placed into culture. Canine female reproductive tracts were collected from a local clinic and ovaries were minced in warm TL-HEPES. Oocytes with a consistently dark ooplasm and at least two layers of cumulus cells were selected, cultured in a basic canine oocyte in vitro maturation medium consisting of TCM-199 with Earl's salts, 2.92 mM Ca-lactate, 20 mM pyruvic acid, 4.43 mM HEPES, 10% fetal calf serum, 1% Penicillin/Streptomycin (GibcoBRL, Grand Island, NY, USA), and 5 μg/mL porcine somatotropin, and incubated at 38.5°C in 5% CO2 in humidified air. Treatment groups were randomly assigned and oocytes were cultured for 60, 84, or 132 h (Basic). From each of these groups, one-half of the oocytes were pipetted through a fine bore pipette to partially remove the cumulus cells 24 h after the start of culture (Basic–1/2). At the end of culture, all oocytes were denuded and the nuclear status was observed with Hoechst 33342 under ultraviolet fluorescence. All data were analyzed by ANOVA with P < 0.05. Since the canine oocyte is ovulated at the germinal vesicle (GV) stage of meiosis and requires up to five days to mature in the oviduct, it was hypothesized that an increased culture time would allow for more oocytes to undergo nuclear maturation to metaphase II (MII). It was also hypothesized that partial removal of cumulus cells would decrease the cumulus cell component in the ooplasm that sustains meiotic arrest, allowing for more oocytes to resume meiosis (RM = germinal vesicle breakdown to MII). Results within each treatment group indicate that there is no significant difference between culture duration and the percent of oocytes that mature to MII. Additionally, there was no significance in the percent of oocytes that resumed meiosis after partial cumulus cell removal. Taken together, these data suggest that neither treatment is effective in canine in vitro maturation systems, given the current maturation culture conditions. Table 1. Nuclear status* of oocytes for three time periods with or without partial cumulus cell removal

scholarly journals In vitro maturation of domestic cat oocytes subjected to different incubation times

2021 ◽  
Vol 10 (3) ◽  
pp. e15710313074
Author(s):  
Denilsa Pires Fernandes ◽  
Fernanda Araujo dos Santos ◽  
Luã Barbalho de Macêdo ◽  
Roberta Gonçalves Izzo ◽  
Brenna de Sousa Barbosa ◽  
...  
Keyword(s):  
Incubation Period ◽  
Cell Expansion ◽  
In Vitro Maturation ◽  
Polar Body ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Domestic Cat ◽  
First Polar Body ◽  
The Ideal

The aim of this study was to evaluate the effect of three different incubation times on in vitro maturation of domestic cat oocytes. Thus, ovaries (n = 42) were submitted to slicing procedure and the oocytes recovered were classified; only good quality oocytes (Grade I and II) underwent in vitro maturation for three different periods (24 vs. 30 vs. 36 h) in supplemented TCM-99 medium. After, oocytes were evaluated for cumulus cell expansion and presence of the first polar body. After six replicates (7 ± 1,7 ovaries per replicate), a total of 334 viable oocytes were recovered. Differences (p <0.05) were observed regarding the percentage of oocytes presenting expansion of the cumulus cells, where higher values were observed in the group of oocytes incubated for 36 h (84.3%), when compared to 30 (73.4%) and 24 h (71.0%). Moreover, differences were also observed regarding the presence of the first polar body (24 h: 29.7%; 30 h: 58.2%; 36 h: 69.8%). We conclude that the incubation period influenced the maturation rates, indicating 36 h as the ideal period for the in vitro maturation of domestic cat oocytes in supplemented TCM-199 medium.

165 In vitro maturation of ovine and caprine oocytes during breeding and nonbreeding seasons

2019 ◽  
Vol 31 (1) ◽  
pp. 207
Author(s):  
M. Markle ◽  
C. K. Mak ◽  
V. Medina ◽  
C. R. F. Pinto
Keyword(s):  
Breeding Season ◽  
In Vitro Maturation ◽  
Statistical Significance ◽  
Polar Body ◽  
Cumulus Cells ◽  
Nonbreeding Season ◽  
Nuclear Maturation ◽  
Significant Difference ◽  
Maturation Rate

The current study investigated the in vitro meiotic competence of ovine and caprine oocytes that underwent nuclear maturation during the breeding and nonbreeding seasons. We hypothesised that maturation rates of ovine and caprine oocyte would be significantly lower during the nonbreeding season. Ovine (Katahdin crossbred) and caprine (mainly Spanish crossbred) ovaries were collected from a local abattoir in the southern United States. Age of the animals was not determined. Cumulus-oocyte complexes (COC) were harvested by slicing the ovaries and searching using a stereomicroscope. Oocytes with more than 3 layers of unexpanded cumulus cells and with evenly granulated cytoplasm were selected for in vitro maturation (IVM). A commercial bovine IVM media (IVF Bioscience, Falmouth, United Kingdom) was used throughout the study. After 24h of IVM, ovine and caprine oocytes were denuded and oocytes with an extruded polar body (meiotic metaphase II oocytes) were considered to have reached nuclear maturation. The seasons in this study were defined as follows: breeding season=September to April and nonbreeding season=May to July. The presence of corpus hemorrhagicum or corpus luteum in at least 70% of the ovaries indicated the breeding season for the animals. Proportions of oocytes undergoing nuclear maturation were analysed using a two-tailed Chi-squared test. Statistical significance was set at P ≤ 0.05. The ovine maturation rate was 59% (65/111) and 49% (254/519) and the caprine maturation rate was 70% (39/56) and 40% (64/162) during the breeding and nonbreeding seasons, respectively. These results show a significant difference in nuclear maturation for caprine oocytes (P&lt;0.001) during the breeding and nonbreeding seasons; however, there was no significant difference in nuclear maturation for ovine oocytes (P=0.06) during the breeding and nonbreeding seasons. High environmental temperatures during the nonbreeding season may have had detrimental effects on oocyte nuclear maturation in caprine but not in ovine oocytes. Why oocytes from these 2 species differ on how they are adversely affected by season remains to be elucidated.

297 EFFECT OF CUMULUS CELL REMOVAL DURING IN VITRO MATURATION OF PORCINE CUMULUS–OOCYTE COMPLEXES ON THE APOPTOTIC STATUS AND MEIOTIC PROGRESSION OF THE OOCYTES

2015 ◽  
Vol 27 (1) ◽  
pp. 237
Author(s):  
P. Ferré ◽  
H. Funahashi
Keyword(s):  
In Vitro Maturation ◽  
Cumulus Cell ◽  
Annexin V ◽  
Cumulus Cells ◽  
Dapi Staining ◽  
Meiotic Progression ◽  
Viability Assay ◽  
Significant Difference ◽  
Porcine Oocyte

This study was undertaken to examine the apoptotic status and meiotic progression of oocytes from small (SF) and medium follicles (MF) when the oocytes were denuded from cumulus cells (CC) before, during and after culture for in vitro maturation (IVM). Cumulus-oocyte complexes (COC) were aspirated from SF (0.5–2 mm in diameter) or MF (3–6 mm in diameter) of slaughtered prepubertal gilt ovaries. Only COC with a good morphology of the surrounding cumulus cells were cultured for IVM in modified porcine oocyte medium supplemented with 50 µM β-mercaptoethanol, 1 mM dibutyryl c-AMP, 10 IU mL–1 of eCG, and 10 IU mL–1 of hCG for 20 h at 39°C and 5% CO2 in air and then continued culture in the absence of dibutyryl c-AMP, eCG, and hCG in the same medium for another 24 h. Before and 20 h after the start of IVM culture, some of the oocytes were denuded of CC and the oocytes continued the IVM culture. After IVM culture, oocyte viability and meiotic progression were examined by the annexin V/PI viability assay and DAPI staining. Statistical analyses of 5 replicate data were performed with a 2-way ANOVA and a Tukey's multiple comparisons test. Before IVM culture, there was no significant difference between the viability of SF and MF oocytes, but the incidence of oocytes at the GV0 stage was higher in specimens from SF than MF (24.8 v. 3.3%), and that of oocytes at the GVI stage was the opposite (57.8 in MF v. 22.7% in SF). After IVM culture, apoptotic status of oocytes was only affected by the decumulation timing. The percentage of normal live oocytes was significantly higher when CC were removed after 20 and 44 h of IVM in both SF (39.7 and 39.3 v. 17.7%) and MF (45.4 and 37 v. 22.2%). The incidence of early and late apoptotic oocytes was significantly higher when the CC were removed before IVM culture in both SF (74.3 and 7.4%) and MF (69.4 and 6.7%). The incidence of mature live oocytes was significantly affected by both the origin of COC and the decumulation timing. Although the percentage of mature oocytes was higher in MF, maturation rates were significantly higher when oocytes were denuded at 20 h of IVM culture (SF 65.4%, MF 83.1%) as compared at 0 (SF 27.9%, MF 32.3%) and 44 h (SF 41%, MF 68.5%). However, the percentage of oocytes with normal spindle morphology was significantly higher when oocytes were denuded at 44 h of IVM culture (SF 70.6%, MF 91.5%) than 20 h (SF 66.8%, MF 73%). In summary, regardless of COC from SF and MF, removal of CC at 20 h of IVM culture seems to promote meiotic progression of the oocytes to the MII stage, but factor(s) from or communication with CC during the latter half of IVM culture may be needed to obtain a normal spindle morphology in mature oocytes.

229 EXPRESSION OF mRNA ENCODING GLYCOLYTIC ENZYMES IN BOVINE CUMULUS CELLS DURING IN VITRO MATURATION: EFFECTS OF TIME AND FSH

2010 ◽  
Vol 22 (1) ◽  
pp. 272
Author(s):  
E. S. Caixeta ◽  
P. Ripamonte ◽  
M. F. Machado ◽  
R. B. da Silva ◽  
C. Price ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Pattern ◽  
In Vitro Maturation ◽  
Housekeeping Gene ◽  
Cumulus Cells ◽  
Glycolytic Enzymes ◽  
Mrna Abundance ◽  
Relative Expression ◽  
Significant Difference

Mammalian oocytes require pyruvate as an energy source for growth and resumption of meiosis. Because oocytes are not competent to carry out glycolysis, cumulus cells (CC) are responsible for metabolizing glucose into pyruvate and providing it to the oocyte through gap junctions. The understanding of the energetic metabolism of CC in culture conditions might provide basis for the improvement of COC in vitro maturation. The aim of this study was to determine the temporal patterns of mRNA expression of glycolytic enzymes [phosphofructokinase (PFKP), aldolase (ALDOA), triosephosphate isomerase (TPI), enolase (ENO1), pyruvate kinase (PKM2), and lactate dehydrogenase (LDHA)] in bovine CC during COC in vitro maturation with or without FSH. Immature COC (grades 1 and 2) were obtained from 2- to 8-mm follicles from abattoir ovaries (predominantly Bos indicus). Cumulus cells were separated from COC and frozen before (immature group) or after COC culture for 4, 8, 12, 16, and 20 hours with (10 ng/mL) or without FSH. Total RNA was extracted using RNeasy® (Qiagen, Valencia, CA, USA), and 100 ng of RNA was reverse transcribed using oligo dT primers and Omniscript® (Qiagen). Relative expression of target genes was assessed by real-time PCR using bovine-specific primers and Power SYBR green master mix in an ABI Prism® 7300. To select the most stable housekeeping gene for expression normalization, cyclophilin-A (CYC-A), GAPDH, and histone H2AFZ amplification profiles were compared using the geNorm applet for Microsoft Excel (Vandesompele J et al. 2002 Genome Biol. 3, 1-11); the most stable housekeeping gene was CYC-A. Relative expression values were calculated using the AACt method with efficiency correction (Pfaffl MW 2001 Nucleic Acids Res. 29, 2002-2007). Effects of time in culture and of FSH treatment were tested by ANOVA, and groups were compared by Tukey-Kramer Honestly Significant Difference test. Nonparametric analysis was used when data were not normally distributed. Abundance of mRNA of all glycolytic enzymes decreased during in vitro maturation with or without FSH. Expression of PFKP, ALDOA, TPI1, ENO1, and LDHA genes was decreased to around half of the initial value (time 0) by 4 to 8 h of culture (P < 0.05) and did not increase thereafter. A similar expression pattern was observed for PKM2, although mRNA abundance was reduced later in comparison with other enzymes; levels were decreased by 16 (without FSH) to 20 h (with FSH) of culture. The presence of FSH did not alter the overall temporal pattern of gene expression but decreased mRNA abundance for PFKP, ALDOA, and TPI1 at 20, 16 and 16 h of culture, respectively. In conclusion, gene expression of glycolytic enzymes decreased with time during COC in vitro maturation in cattle, and FSH did not have a major influence on this expression pattern. This study was supported by CAPES and FAPESP.

205 CUMULUS CELL MORPHOLOGY BEFORE AND AFTER IN VITRO MATURATION AS AN INDICATOR OF PORCINE OOCYTE DEVELOPMENTAL COMPETENCE

2010 ◽  
Vol 22 (1) ◽  
pp. 260
Author(s):  
M. Bertoldo ◽  
P. K. Holyoake ◽  
G. Evans ◽  
C. G. Grupen
Keyword(s):  
Cell Morphology ◽  
In Vitro Maturation ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Oocyte Quality ◽  
Developmental Competence ◽  
Oocyte Developmental Competence ◽  
Before And After ◽  
Follicle Size

Effective in vitro maturation (IVM) is essential for successful in vitro embryo production. The morphology of the cumulus investment before and after IVM may be a useful noninvasive indicator of oocyte quality. In pigs, oocyte developmental competence is reduced during the summer months. The aim of this study was to determine whether the morphology of cumulus-oocyte complexes (COC) before and after IVM are associated with oocyte quality, using COC collected from small and large follicles in summer and winter as models of poor and good oocyte quality. Ovaries were collected from sows slaughtered 4 days after weaning. The COC recovered from small (3-4 mm) and large (5-8 mm) antral follicles were morphologically graded and parthenogenetically activated following IVM during winter (n = 1419; 10 replicates) and summer (n = 2803; 10 replicates). Grade 1 and 2 COC had >2 layers of compact cumulus cells and a homogenous cytoplasm. Grade 3 COC were either partially or fully denuded, had a heterogeneous cytoplasm, or were vacuolated or dark in color. Grade 4 COC had expanded cumulus cells. Cumulus expansion was also assessed subsequent to IVM. The COC recorded as having a cumulus expansion index (CEI) of 1 had the poorest expansion with no detectable response to IVM, whereas those with a CEI of 4 had the greatest amount of expansion, including that of the corona radiata. Data were analyzed using a generalized linear mixed model in GenStat® (release 10, VSN International, Hemel Hempstead, UK). There was an effect of follicle size for Grade 1 COC, with COC from large follicles in both seasons yielding better quality COC (P < 0.05). The proportion of COC in Grade 2 was higher in small follicles during winter compared with large follicles, but there were no differences between follicle sizes during summer (P < 0.05). The proportion of COC with CEI 1 was highest in COC from small follicles during summer (P < 0.05). The proportion of COC from large follicles with CEI 2 was higher during summer compared with winter (P < 0.05). There were no seasonal or follicle size effects on COC with CEI 3 or 4 (P > 0.05). The proportion of oocytes that developed to blastocysts was greater in winter than in summer (39.06% ± 5.67 v. 22.27% ± 4.01; P < 0.05). Oocytes derived from large follicles had a greater ability to form blastocysts compared with those from small follicles (37.13% ± 5.65 v. 23.32% ± 4.56; P < 0.06). Morphological assessment of cumulus cells before and after IVM may be a useful tool to evaluate the effects of follicle size on oocyte developmental competence. However, the results of the present study indicate that cumulus cell morphology is not a good indicator of the effect of season on oocyte developmental competence.

332 GRANULOCYTE-MACROPHAGE COLONY STIMULATING FACTOR (GM-CSF) ENHANCES CUMULUS CELLS EXPANSION OF IN VITRO-MATURED BOVINE CUMULUS - OOCYTE COMPLEXES IN A CHEMICALLY DEFINED MEDIUM

2010 ◽  
Vol 22 (1) ◽  
pp. 322
Author(s):  
D. D. Bücher ◽  
M. A. Castro ◽  
M. E. Silva ◽  
M. A. Berland ◽  
I. I. Concha ◽  
...  
Keyword(s):  
Cell Viability ◽  
In Vitro Maturation ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Cell Number ◽  
Control Group ◽  
Cumulus Expansion ◽  
Gm Csf ◽  
Nuclear Stage

Granulocyte-macrophage colony stimulating factor (GM-CSF) is a pleiotropic cytokine that stimulates proliferation, differentiation and function in different cells types. We have previously demonstrated (Bücher DD et al. 2008 Reprod. Dom. Anim. 43 (Suppl. 3), 146 abst.) that both subunits of GM-CSF receptor are expressed in granulosa cells from antral follicles in bovine ovaries. Also, we determined that the cytokine enhances glucose uptake through facilitative hexose transporters in granulosa cells in primary culture. The goals of the present study were to characterize the expression of GM-CSF receptor in cumulus cells and oocytes from bovine antral follicles and to determine its effects on in vitro-matured bovine COCs in a chemically defined medium. To determine the presence of a and |5 subunits of GM-CSF receptor, COCs were aspirated from follicles <8 mm in diameter, fixed, and submitted to immunocytochemistry. To study the effect of GM-CSF on in vitro maturation of oocytes, COCs (n =481) were cultured using serum-free medium (SOF) containing 0, 1, 10, and 100 ng mL-1 of human recombinant GM-CSF (R&D Systems, Inc., Minneapolis, MN, USA) for 22 h at 39°C, 5% CO2 in humidified air. Nuclear stage, cumulus expansion, cumulus cell number, and viability were analyzed after in vitro maturation. Cumulus expansion was assessed using the cumulus expansion index (CEI) (Fagbohun C and Down S 1990 Biol. Reprod. 42, 413-423). Nuclear stage was evaluated using aceto-orcein stain. To determine cumulus cell viability and number, COCs (n = 10-12 per group) were transferred into an Eppendorf tube and cumulus cells were removed by vortexing for 3 min, stained with trypan blue and counted with a hemocytometer. The study was conducted in 6 replicates. Data from cumulus expansion and cell number were analyzed by Kruskal-Wallis analysis. Data for nuclear stage and cell viability were analyzed by chi-square analysis and one way ANOVA, respectively. Both receptor subunits were present in cumulus cells and oocytes from COCs. COCs cultured in 10 and 100 ng mL-1 GM-CSF had CEI scores (0.8 and 1.22, respectively) greater (P < 0.01) than controls (0.2), but the proportion of COCs displaying second metaphase did not differ (P = 0.5) among treatment groups. GM-CSF at a concentration of 100 ng mL-1 increased (P < 0.01) cumulus cell viability by more than 20% compared to the control group. Similarly, GM-CSF at concentrations of 10 and 100 ng mL-1 increased (P < 0.05) cumulus cell number by more than 20% and 45%, respectively, from the control group. The use of a specific inhibitor of PI3 kinase (Ly294002; 10 and 100 μM) blocked the stimulatory effect of GM-CSF on cumulus expansion, cell viability, and cell number. In conclusion, the results of the study suggest a plausible modulator role of GM-CSF in the metabolism and function of cumulus cells and oocytes during in vitro maturation. Funding from Faculty of Veterinary Sciences, Universidad Austral de Chile, MECESUP AUS-0005, AUS-0601, and DID D-2006-24 and from Universidad Católica de Temuco, research grant 2007 DGI-CDA-04.

188 IMPROVEMENT OF IN VITRO CANINE OOCYTE MATURATION BY OVIDUCTAL SECRETOME

2017 ◽  
Vol 29 (1) ◽  
pp. 202 ◽  
Author(s):  
A. Lange-Consiglio ◽  
C. Perrini ◽  
P. Esposti ◽  
F. Cremonesi
Keyword(s):  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Cumulus Cell ◽  
Follicular Development ◽  
Cumulus Cells ◽  
Chi Square ◽  
Cell Layers ◽  
Hoechst Staining

The in vitro maturation of canine oocyte is problematic because it is difficult to reproduce the oviducal microenvironment where the in vivo maturation occurs. Because cells are able to communicate with each other by paracrine action, oviducal cells could be in vitro cultivated to obtain the conditioned medium (CM) consisting of soluble factors and microvesicles (MV), which represent a carrier for nonsoluble molecules including microRNA. The aim of the present work was to investigate the effect of the addition of CM or MV, secreted by oviducal cells, to the canine in vitro maturation medium. To generate CM, cells from oviducts of 3 animals in late oestrus were cultured for 5 days at 38.5°C in a humidified atmosphere of 5% CO2. Supernatants were collected, pooled, centrifuged at 2500 × g, and stored at −80°C. Microvesicles were obtained by ultracentrifugation of CM at 100,000 × g for 1 h at 4°C and measured for concentration and size by a Nanosight instrument. Ovaries were obtained from 50 healthy domestic bitches (1–4 years old) of different breeds that underwent ovariectomy regardless of the oestrous cycle. Cumulus-oocyte complexes were released by slicing the ovarian cortex with a scalpel blade, and only Grade 1 cumulus-oocyte complexes (darkly granulated cytoplasm and surrounded by 3 or more compact cumulus cell layers) 110 to 120 µm in diameter were selected for culture. Maturation was performed at 38.5°C in a humidified atmosphere of 5% CO2 and 5% of O2 in bi-phasic systems: 24 h in SOF with 5.0 μg mL−1 of LH followed by 48 h in SOF supplemented with 10% of oestrous bitch serum and 10% CM or 50, 75, 100, or 150 × 106 MV mL−1 labelled with PKH-26. Control was the same medium without CM or MV. Oocytes were observed under a fluorescent microscope to detect metaphase II (MII), by Hoechst staining, and the incorporation of MV. Statistical analysis was performed by chi-square test. Results show that canine oviducal cells secreted MV of 234 ± 23 nm in size, underling that these MV fall within the shedding vesicles category. The incorporation of labelled MV occurred at first in cumulus cells, at 48 h of maturation, and then, at 72 h, in oocyte cytoplasm. These MV had a positive effect on maturation rate (MII) at the concentration of 75 and 100 × 106 MV mL−1 compared with CM and control (20.34 and 21.82 v. 9.09 and 3.95%, respectively). The concentration of 150 × 106 MV mL−1 provided only 9.26% of MII. To understand the role of MV, we assessed the expression of 3 microRNA (miRNA-30b, miR-375, and miR-503) that are involved in some key pathways (WNT, MAPK, ERbB, and TGFβ) regulating follicular development and meiotic resumption. The lower rate of MII with the higher concentration of MV is possibly due to the high level of miR-375, which recent literature shows to suppress the TGFβ pathway, leading to impaired oocyte maturation. In conclusion, the oviducal MV, or specific microRNA, are involved in cellular trafficking during oocyte maturation, and their possible use in vitro could facilitate the exploitation of canine reproductive biotechnologies.

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