In vitro maturation of blue fox oocytes and cAMP production in oocyte-cumulus cell complexes

This study aimed to examine the effects of treatment with glucuronic acid (GA) and N-acetyl-D-glucosamine (AG), which are components of hyaluronic acid (HA), during porcine oocyte in vitro maturation (IVM). We measured the diameter of the oocyte, the thickness of the perivitelline space (PVS), the reactive oxygen species (ROS) level, and the expression of cumulus cell expansion and ROS-related genes and examined the cortical granule (CG) reaction of oocytes. The addition of 0.05 mM GA and 0.05 mM AG during the first 22 h of oocyte IVM significantly increased oocyte diameter and PVS size compared with the control (non-treatment). The addition of GA and AG reduced the intra-oocyte ROS content and improved the CG of the oocyte. GA and AG treatment increased the expression of CD44 and CX43 in cumulus cells and PRDX1 and TXN2 in oocytes. In both the chemically defined and the complex medium (Medium-199 + porcine follicular fluid), oocytes derived from the GA and AG treatments presented significantly higher blastocyst rates than the control after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT). In conclusion, the addition of GA and AG during IVM in pig oocytes has beneficial effects on oocyte IVM and early embryonic development after PA and SCNT.

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Effects of BMP15 and GDF9 on Ovine Oocytes in an In Vitro Maturation System

10.26686/wgtn.17147732 ◽

2021 ◽

Author(s):

◽

Aanchal Singh

Keyword(s):

Cell Death ◽

Litter Size ◽

In Vitro Maturation ◽

Cumulus Cell ◽

Oocyte Quality ◽

Mature Oocyte ◽

High Ratio ◽

High Fecundity ◽

Species Specific

<p>Oocyte developmental competency is the intrinsic measure of oocyte quality and the capacity for a mature oocyte to support the early stages of embryo development and implantation. Oocyte-secreted factors (OSFs), such as growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), play a pivotal role in regulating the synchrony of various complex maturation events within the cumulus-oocyte complex (COC) through the induction of paracrine and endocrine signalling. These proteins act synergistically to influence the proliferation and differentiation of granulosa cells (GCs), cumulus cell (CC) expansion, promote survival, ovulation, the attainment of developmental competency and fertility. Species-specific ratios suggest that poly-ovulatory mammals have increased fecundity due to high ratios of GDF9:BMP15, which is directly reflected in their large litter size. Interestingly, it has also been found that higher ratios of GDF9:BMP15 also increased blastocyst rate in sheep implying that these embryos develop from oocytes that are more developmentally competent. In this study, I investigated the hypothesis that supplementing a commercial in vitro maturation (IVM) system with a high ratio of GDF9:BMP15 would increase the developmental competency sheep oocytes; a species with low-moderate litter size. To test this hypothesis, ovine oocytes were matured in a biphasic IVM system containing GDF9 and BMP15 at three divergent ratios (1:6, 1:1, 6:1). The results herein show that the 6:1 ratio resulted in higher levels of reagent transfer to the ovine oocyte through gap junctions (GJs) after 24 hours of incubation. Similarly, it was also observed that at the higher ratio, glutathione (GSH) levels were higher at 7.5 hours of incubation. The high GDF9:BMP15 ratio also facilitated the increased consumption of pyruvate by the COC consistently throughout the culture period. Importantly, the high GDF9:BMP15 ratio showed higher expression of the gene that encodes GJ (CX43) at 24 hours relative to the control. It was also demonstrated through decreased apoptotic factor (BAX:BCL2) ratios, that the addition of OSFs, regardless of ratio, protected against cell death. In summary, this study provides novel results that support the notion that a high GDF9:BMP15 ratio improves oocyte quality by delaying the timing of meiotic resumption. This subsequently improves the transport of key metabolites and antioxidants to protect against oxidative stress and cell death and aid in the completion of maturation, ultimately resulting in the increased developmental competency observed in high fecundity poly-ovulatory species.</p>

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α-Tocopherol modifies the expression of genes related to oxidative stress and apoptosis during in vitro maturation and enhances the developmental competence of rabbit oocytes

Reproduction Fertility and Development ◽

10.1071/rd17525 ◽

2018 ◽

Vol 30 (12) ◽

pp. 1728 ◽

Cited By ~ 3

Author(s):

M. Arias-Álvarez ◽

R. M. García-García ◽

J. López-Tello ◽

P. G. Rebollar ◽

A. Gutiérrez-Adán ◽

...

Keyword(s):

Oxidative Stress ◽

Cell Cycle ◽

In Vitro Maturation ◽

Cumulus Cell ◽

Rabbit Model ◽

Developmental Competence ◽

Antioxidant Agents ◽

Junction Protein

The developmental competence of in vitro maturation (IVM) oocytes can be enhanced by antioxidant agents. The present study investigated, for the first time in the rabbit model, the effect of adding α-tocopherol (0, 100, 200 and 400 µM) during IVM on putative transcripts involved in antioxidant defence (superoxide dismutase 2, mitochondrial (SOD2), glutathione peroxidase 1 (GPX1), catalase (CAT)), cell cycle regulation and apoptosis cascade (apoptosis tumour protein 53 (TP53), caspase 3, apoptosis-related cysteine protease (CASP3)), cell cycle progression (cellular cycle V-Akt murine thymoma viral oncogene homologue 1 (AKT1)), cumulus expansion (gap junction protein, alpha 1, 43 kDa (GJA1) and prostaglandin-endoperoxide synthase 2 (prostaglandin G/H synthase and cyclo-oxygenase) (PTGS2)) and metabolism (glucose-6-phosphate dehydrogenase (G6PD)). Meiotic progression, mitochondrial reallocation, cumulus cell apoptosis and the developmental competence of oocytes after IVF were also assessed. Expression of SOD2, CAT, TP53, CASP3 and GJA1 was downregulated in cumulus–oocyte complexes (COCs) after IVM with 100 μM α-tocopherol compared with the group without the antioxidant. The apoptotic rate and the percentage of a non-migrated mitochondrial pattern were lower in COCs cultured with 100 μM α-tocopherol, consistent with better-quality oocytes. In fact, early embryo development was improved when 100 μM α-tocopherol was included in the IVM medium, but remained low compared with in vivo-matured oocytes. In conclusion, the addition of 100 μM α-tocopherol to the maturation medium is a suitable approach to manage oxidative stress and apoptosis, as well as for increasing the in vitro developmental competence of rabbit oocytes.

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327 OOCYTE-SECRETED FACTORS DIRECTLY AFFECT OOCYTE DEVELOPMENTAL COMPETENCE DURING IN VITRO MATURATION OF THE BOVINE CUMULUS - OOCYTE COMPLEX

Reproduction Fertility and Development ◽

10.1071/rdv18n2ab327 ◽

2006 ◽

Vol 18 (2) ◽

pp. 271 ◽

Cited By ~ 1

Author(s):

T. S. Hussein ◽

R. B. Gilchrist ◽

J. G. Thompson

Keyword(s):

In Vitro Maturation ◽

Cumulus Cell ◽

Blastocyst Stage ◽

Developmental Competence ◽

Bovine Oocyte ◽

Paracrine Factors ◽

Cell Functions ◽

Oocyte Developmental Competence ◽

Secreted Factors

Paracrine factors secreted by the oocyte (oocyte-secreted factors, OSFs) regulate a broad range of cumulus cell functions including proliferation, differentiation, and apoptosis. The capacity of oocytes to regulate their own microenvironment by OSFs may in turn contribute to oocyte developmental competence. The aim of this study was to determine if OSFs have a direct influence on bovine oocyte developmental competence during in vitro maturation (IVM). Cumulus-oocyte complexes (COCs) were obtained by aspiration of >3-mm follicles from abattoir-derived ovaries. IVM was conducted in Bovine VitroMat (Cook Australia, Eight Mile Plains, Brisbane, Australia) supplemented with 0.1 IU/mL rhFSH for 24 h under 6% CO2 in air at 38.5�C. In the first experiment, COCs were co-cultured with denuded oocytes (DOs, 5/COC in 10 �L) beginning at either 0 or 9-h of IVM. To generate the 9-h DO group, COCs were first cultured intact for 9-h and then denuded. In the second experiment, specific OSFs, recombinant bone morphogenetic protein-15 (BMP-15) and growth differentiation factor 9 (GDF-9), were prepared as partially purified supernatants of transfected 293H cells, and used as 10% v/v supplements in Bovine VitroMat. Treatments were: (1) control (no supplement), (2) BMP-15, (3) GDF-9, (4) BMP-15 and GDF-9, and (5) untransfected 293H control. Following maturation, in vitro production of embryos was performed using the Bovine Vitro system (Cook Australia) and blastocysts were examined on Day 8 for development. Developmental data were arcsine-transformed and analyzed by ANOVA, followed by Tukey's test. Cell numbers were analyzed by ANOVA. Co-culturing intact COCs with DOs from 0 or 9 h did not affect cleavage rate, but increased (P < 0.001) the proportion of cleaved embryos that reached the blastocyst stage post-insemination (50.6 � 1.9 and 61.3 � 1.9%, respectively), compared to COCs cultured alone (40.7 � 1.4%). Therefore, paracrine factors secreted by DOs increased the developmental competence of oocytes matured as COCs. OSFs also improved embryo quality, as co-culture of COCs with DOs (0 or 9 h) significantly increased total cell (156.1 � 1.3 and 159.1 � 1.3, respectively) and trophectoderm (105.7 � 1.3 and 109.8 � 0.4, respectively) numbers, compared to control COCs (total = 148 � 1.2, trophectoderm = 98.2 � 0.8, P < 0.001). BMP-15 alone or with GDF-9 also significantly (P < 0.001) increased the proportion of oocytes that reached the blastocyst stage post insemination (57.5 � 2.4% and 55.1 � 4.5%, respectively), compared to control (41.0 � 0.9%) and 293H-treated (27.1 � 3.1%) COCs. GDF-9 also increased blastocyst yield (49.5 � 3.9%) but not significantly. These results are the first to demonstrate that OSFs, and particularly BMP-15 and GDF-9, directly affect bovine oocyte developmental competence. These results have far-reaching implications for improving the efficiency of IVM in domestic species and human infertility treatment, and support the role of OSF production by oocytes as a diagnostic marker for developmental competence.

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

Reproduction Fertility and Development ◽

10.1071/rdv18n2ab326 ◽

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

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254 9-cis RETINOIC ACID INHIBITS CUMULUS CELL APOPTOSIS DURING IN VITRO MATURATION OF BOVINE OOCYTES THROUGH INHIBITION OF AP-1 PATHWAY

Reproduction Fertility and Development ◽

10.1071/rdv23n1ab254 ◽

2011 ◽

Vol 23 (1) ◽

pp. 225

Author(s):

G. K. Deb ◽

S. R. Dey ◽

J. I. Bang ◽

S. J. Cho ◽

T. H. Kwon ◽

...

Keyword(s):

Retinoic Acid ◽

Tumor Necrosis Factor ◽

Oocyte Maturation ◽

Tumor Necrosis ◽

In Vitro Maturation ◽

Tumor Necrosis Factor Α ◽

Cumulus Cell ◽

Factor Α ◽

Necrosis Factor

Cumulus cells (CC) play a critical role in oocyte maturation and fertilization via gap junctions. The oocyte itself maintains CC health to favour oocyte maturation via the secretion of paracrine growth factors. However, the antiapoptotic effects of oocyte-secreted factors follow a gradient from the site of the oocytes. Moreover, degrees of CC apoptosis are inversely related to the in vitro embryo development. Therefore, inhibition of CC apoptosis is important for efficient in vitro embryo development. The beneficial effects of retinoic acid (RA) during in vitro embryo production are well known in different species. However, the effect of RA on CC apoptosis is yet to be elucidated. All-trans RA and 9-cis RA are the natural components of retinoids, and all-trans RA are metabolized to 9-cis RA for physiological function. Therefore, the objective of the present study was to evaluate the effect of 9-cis RA on the mechanism for inhibition of apoptosis in CC. Slaughterhouse cumulus–oocyte complexes (COC) were matured in vitro in TCM-199-based in vitro maturation medium containing 0 or 5 mM 9-cis RA for 23 to 24 h (15 COC/100 μL droplet) at 38.5°C and 5% CO2 in air with maximum humidity. Following in vitro maturation, COC of a droplet were fixed in 4% paraformaldehyde for TUNEL staining using In Situ Cell Death Detection Kit (Roche, Budapest, Hungary). The proportion of apoptotic cells was estimated using Olympus Soft Imaging Solutions GmBH (Olympus, Münster, Germany). The COC of the remaining droplet were denuded. The CC were frozen and stored at –80°C. The CC of 3 different cultures were pooled, and total RNA was extracted using RNeasy Mini Kit (Qiagen, Valencia, CA, USA). Total RNA was reverse transcribed into cDNA using Omniscript Reverse Transcription kit (Qiagen). Relative expression of candidate genes was quantified using SYBER green real-time PCR with ΔΔ CT method. The expression was normalized against β-actin, glyceraldehyde 3-phosphate dehydrogenase, and 18s rRNA genes expression. The PCR efficiencies were calculated using relative calibration curves following 10-fold dilution series at 5 measuring points. Data were analysed for one-way ANOVA. The proportion of apoptotic cells was low in the 9-cis RA group (1.3 v. 3.3% of total CC; P < 0.05). Expression of tumor necrosis factor-α (11.1 v. 1.0; P < 0.001), caspase9 (2.0 v. 1.0; P < 0.01), and caspase3 (2.1 v. 1.0; P < 0.001) genes was down-regulated in the 9-cis RA group, whereas expression of Bcl2 gene was increased (1.0 v. 2.6 fold; P < 0.05). Moreover, the expression of c-fos gene of AP-1 pathway was down-regulated (1.9 v. 1 fold; P < 0.05) in the 9-cis RA group. Retinoic acid suppressed the expression of NF-kB, which in turn inhibits tumor necrosis factor-α-mediated caspase activity. However, the expression of NF-kB in CC was not affected by 9-cis RA (1.1 v. 1.0; P > 0.05). In conclusion, the present study indicated that 9-cis RA may inhibit cumulus cell apoptosis through suppression of AP-1 pathway. This work was partly supported by a scholarship from the BK21 program, the KRF (KRF-2008-211-F00011), the IPET (108068-03-1-SB010), and the KOSEF (10525010001-05N2501-00110).

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

Reproduction Fertility and Development ◽

10.1071/rdv22n1ab205 ◽

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.

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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

Reproduction Fertility and Development ◽

10.1071/rdv22n1ab332 ◽

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.

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188 IMPROVEMENT OF IN VITRO CANINE OOCYTE MATURATION BY OVIDUCTAL SECRETOME

Reproduction Fertility and Development ◽

10.1071/rdv29n1ab188 ◽

2017 ◽

Vol 29 (1) ◽

pp. 202 ◽

Cited By ~ 1

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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