scholarly journals In vitro maturation impacts cumulus–oocyte complex metabolism and stress in cattle

Reproduction ◽  
2017 ◽  
Vol 154 (6) ◽  
pp. 881-893 ◽  
Author(s):  
Maite del Collado ◽  
Juliano C da Silveira ◽  
Marcelo L F Oliveira ◽  
Bárbara M S M Alves ◽  
Rosineide C Simas ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Neutral Lipids ◽  
Cumulus Cells ◽  
Acid Synthesis ◽  
Cellular Stress Response ◽  
Mitochondrial Activity ◽  
Cumulus Oocyte Complex ◽  
Glycolysis Pathway

The influence of in vitro maturation (IVM) in oocytes is still not totally understood. The aim of this study was to determine the influence of IVM on the metabolism and homeostasis of bovine cumulus-oocyte complexes. In the present study, we demonstrated that IVM leads to accumulation of neutral lipids associated with differential levels of the mono-, di- and triacylglycerols in both cumulus cells and oocytes. We observed that in vitro-matured oocytes exhibited decreased glutathione and reactive oxygen species levels and a lower ATP/ADP ratio when compared to in vivo-matured oocytes, with no significant differences in metabolism and stress-related mRNA or miRNA levels. Moreover, in addition to an increase in lipids in in vitro-matured cumulus cells, fatty acid synthesis and accumulation as well as glycolysis pathway genes were upregulated, whereas those affiliated with the β-oxidation pathway were decreased. Our gene expression data in cumulus cells suggest the disruption of endoplasmic reticulum stress, apoptosis and cellular stress response pathways during IVM. Furthermore, a total of 19 miRNAs were significantly altered by the maturation process in cumulus cells. These results indicate some new negative influences of the in vitro system in cumulus-oocyte complexes, demonstrating the occurrence of functional disruption in lipid metabolism and stress pathways and showing evidences suggesting the occurrence of altered mitochondrial activity and energy metabolism during IVM, with a massive dysregulation of the corresponding transcripts in the surrounding cumulus cells.

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.

scholarly journals Mouse Oocytes Enable LH-Induced Maturation of the Cumulus-Oocyte Complex via Promoting EGF Receptor-Dependent Signaling

2010 ◽  
Vol 24 (6) ◽  
pp. 1230-1239 ◽  
Author(s):  
You-Qiang Su ◽  
Koji Sugiura ◽  
Qinglei Li ◽  
Karen Wigglesworth ◽  
Martin M. Matzuk ◽  
...  
Keyword(s):  
Growth Factors ◽  
Granulosa Cells ◽  
Egf Receptor ◽  
Cumulus Cells ◽  
Wild Type ◽  
Paracrine Factors ◽  
Cumulus Oocyte Complex ◽  
Egfr Expression

Abstract LH triggers the maturation of the cumulus-oocyte complex (COC), which is followed by ovulation. These ovarian follicular responses to LH are mediated by epidermal growth factor (EGF)-like growth factors produced by granulosa cells and require the participation of oocyte-derived paracrine factors. However, it is not clear how oocytes coordinate with the EGF receptor (EGFR) signaling to achieve COC maturation. The aim of the present study was to test the hypothesis that oocytes promote the expression of EGFR by cumulus cells, thus enabling them to respond to the LH-induced EGF-like peptides. Egfr mRNA and protein expression were dramatically reduced in cumulus cells of mutant mice deficient in the production of the oocyte-derived paracrine factors growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15). Moreover, microsurgical removal of oocytes from wild-type COCs dramatically reduced expression of Egfr mRNA and protein, and these levels were restored by either coculture with oocytes or treatment with recombinant GDF9 or GDF9 plus recombinant BMP15. Blocking Sma- and Mad-related protein (SMAD)2/3 phosphorylation in vitro inhibited Egfr expression in wild-type COCs and in GDF9-treated wild-type cumulus cells, and conditional deletion of Smad2 and Smad3 genes in granulosa cells in vivo resulted in the reduction of Egfr mRNA in cumulus cells. These results indicate that oocytes promote expression of Egfr in cumulus cells, and a SMAD2/3-dependent pathway is involved in this process. At least two oocyte-derived growth factors, GDF9 and BMP15, are required for EGFR expression by cumulus cells.

scholarly journals Quercetin influences in vitro maturation, apoptosis and metabolically active mitochondria of goat oocytes

Zygote ◽  
2018 ◽  
Vol 26 (6) ◽  
pp. 465-470 ◽  
Author(s):  
A.A.A. Silva ◽  
M.N.P. Silva ◽  
L.B.F. Figueiredo ◽  
J.D. Gonçalves ◽  
M.J.S. Silva ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Cumulus Cells ◽  
Lower Proportion ◽  
The Other ◽  
Mitochondrial Activity ◽  
Base Medium

SummaryThe present study aimed to investigate the effect of quercetin as an alternative antioxidant to cysteamine on in vitro maturation. Oocytes were collected from goat ovaries, destined for in vitro maturation and distributed into three groups: CIS group, oocytes were immersed in MIV base medium; in Groups Q4 and Q8, oocytes were immersed in the medium of the CIS group, adding 4 μM or 8 μM of quercetin, respectively, and cultured for 24 h at 38.5°C with 5% CO2. The CIS and Q4 groups presented the same percentage of expanded cumulus cells, but the per cent in the Q8 group was significantly lower than that of the other groups (P<0.05). The oocyte retraction rate in the Q8 group was higher (P<0.05) than in the CIS and Q4 groups. Treatment with 8 μM of quercetin presented a lower proportion of expanded oocytes than the CIS group and 4 μM of quercetin (P<0.05). The percentage of MII oocytes was higher in the Q4 group than in the CIS group (P<0.05), but the percentages in the CIS and Q8 groups were similar. The rate of apoptosis was higher in the CIS group than in the other groups (P<0.05). In addition, oocytes matured with 4 μM quercetin showed higher mitochondrial activity than matured oocytes in the CIS and Q8 groups (P<0.05). In conclusion, 4 μM of quercetin can be used as an alternative to cysteamine in the in vitro maturation of goat oocytes.

176 IN VITRO MATURATION OF DOG OOCYTES IN CANINE FOLLICULAR FLUID

2014 ◽  
Vol 26 (1) ◽  
pp. 202
Author(s):  
K. Reynaud ◽  
S. Canguilhem ◽  
S. Thoumire ◽  
S. Chastant-Maillard
Keyword(s):  
Follicular Fluid ◽  
In Vitro Maturation ◽  
Assisted Reproductive Technologies ◽  
Cumulus Cells ◽  
Reproductive Technologies ◽  
Control Group ◽  
Limiting Factor ◽  
Cytoplasmic Maturation

In the canine species, assisted reproductive technologies, especially in vitro maturation (IVM) and IVF, are still ineffective. The main limiting factor remains the immaturity of the oocytes collected from anestrus ovaries. The ability of an oocyte to reach the MII stage in vitro is linked to the diameter of its follicle and anestrus oocytes, collected from small (<1 mm) follicles, are profoundly immature (De Lesegno et al. 2008). The objective of this study was to improve cytoplasmic quality by mimicking in vivo conditions; that is, to test the effect of pure preovulatory follicular fluid (FF) on survival and IVM rates of anestrus dog oocytes, in order to improve the nuclear and cytoplasmic maturation of these immature oocytes. Follicular fluids samples were collected from 54 Beagle bitches at 2 stages: before the LH peak (n = 23 bitches) and after the LH peak (n = 31 bitches). Only follicular fluid samples from large (>4 mm) follicles were collected and pooled by stage. Control oocytes were matured in 20% FCS/M199 medium. Groups of 5 oocytes were in vitro matured in 30 μL of follicular fluid, in half-area 96-well plates (5% CO2, 38°C). After 72 h of IVM, oocytes were denuded, fixed, and stained for DNA and tubulin before observation by confocal microscopy, and nuclear stages were classified as GV-A to GV-E, MI, and MII (Reynaud et al. 2012). A total of 460 oocytes were collected from 13 anestrus bitches and allocated to either the control medium (n = 155), the Pre-LH FF (n = 145) or the Post-LH FF (n = 160) groups. After 72 h of IVM, the morphology of the cumulus–oocyte complexes (COC) in the post-LH group was different from that of the others: cumulus cells appeared more compact and darker. Analysis of the nuclear stages showed that the degeneration rate was significantly higher (P < 0.05) in the post-LH group (58.7%) than in the pre-LH (40.9%) or in the control group (34.4%). No significant differences (P > 0.05) were observed between the 3 groups in the rate of immature GVA-B oocytes (36.4, 28.5, and 25.3% in the control, Pre-LH, and Post-LH groups, respectively), in the rate of meiotic resumption (GV-C/D/E, MI, MII stages, 44.4, 51.9, and 38.7% in the control, Pre-LH, and Post-LH groups, respectively). Metaphase II rates were not significantly different (12.1, 8.6, and 4.8% in the control, Pre-LH, and Post-LH groups, respectively). In conclusion, canine COC may survive when exposed to IVM in pure follicular fluid, but the degeneration rate was higher in the post-LH group. The presence of follicular fluid did not inhibit meiosis resumption, but did not significantly improve IVM rates. To better mimic in vivo conditions, IVM in a sequence of media, such as IVM in follicular fluid followed by IVM in oviducal fluid remains to be tested.

Thymosins β-4 and β-10 are expressed in bovine ovarian follicles and upregulated in cumulus cells during meiotic maturation

2010 ◽  
Vol 22 (8) ◽  
pp. 1206 ◽  
Author(s):  
Mohamad Salhab ◽  
Pascal Papillier ◽  
Christine Perreau ◽  
Catherine Guyader-Joly ◽  
Joelle Dupont ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Expression Patterns ◽  
Cumulus Cells ◽  
Blastocyst Stage ◽  
Small Proteins ◽  
Progesterone Secretion ◽  
Inflammatory Processes ◽  
Polymerase Chain

β-Thymosins are small proteins that regulate the actin cytoskeleton and are involved in cell motility, differentiation, the induction of metalloproteinases, in anti-inflammatory processes and tumourigenesis. However, their roles in the ovary have not yet been elucidated. Using transcriptomics and real time reverse transcription–polymerase chain reaction validation, the present study demonstrates that thymosin β-4 (TMSB4) and thymosin β-10 (TMSB10) are upregulated in bovine cumulus cells (CCs) during in vitro maturation of cumulus–oocyte complexes (COCs) in parallel with an increase in mRNA expression of HAS2, COX2 and PGR genes. Using immunocytochemistry, both proteins were found to be localised mainly in granulosa cells, CCs and oocytes, in both the cytoplasm and nucleus, as well as being colocalised with F-actin stress fibres in CCs. Using different maturation mediums, we showed that the expression of TMSB10, but not TMSB4, was positively correlated with COC expansion and progesterone secretion and negatively correlated with apoptosis. Immunofluorescence, coupled with terminal deoxyribonucleotidyl transferase-mediated dUTP–digoxigenin nick end-labelling (TUNEL), demonstrated the absence of TMSB4 and/or TMSB10 in apoptotic cells. TMSB10 expression was higher in COCs matured in vivo than in vitro, and differences related to the age of the animal were observed. TMSB4 and/or TMSB10 expression was unchanged, whereas HAS2 overexpressed in CCs from oocytes that developed to the blastocyst stage in vitro compared with those that did not. Thus, TMSB4 and/or TMSB10 ovarian expression patterns suggest that these two thymosins may be involved in cumulus modifications during maturation.

Glucose Metabolism Characterization During Mouse In Vitro Maturation Identifies Alterations In Cumulus Cells†

2021 ◽  
Author(s):  
Nazli Akin ◽  
Lucia von Mengden ◽  
Anamaria-Cristina Herta ◽  
Katy Billooye ◽  
Julia Leersum ◽  
...  
Keyword(s):  
Glucose Metabolism ◽  
In Vitro Maturation ◽  
Culture Media ◽  
Cumulus Cells ◽  
Routine Practice ◽  
Glycolytic Activity ◽  
Direct Measurements ◽  
Final Maturation

Abstract In vitro maturation (IVM) is an assisted reproduction technique with reduced hormone-related side effects. Several attempts to implement IVM in routine practice have failed, primarily due to its relatively low efficiency compared to conventional in vitro fertilization (IVF). Recently, capacitation (CAPA)-IVM, a novel two-step IVM method, has improved the embryology outcomes through synchronizing the oocyte nuclear and cytoplasmic maturation. However, the efficiency gap between CAPA-IVM and conventional IVF is still noticeable especially in the numerical production of good quality embryos. Considering the importance of glucose for oocyte competence, its metabolization is studied within both in vivo and CAPA-IVM matured mouse cumulus-oocyte-complexes (COCs) through direct measurements in both cellular compartments, from transcriptional and translational perspectives, to reveal metabolic shortcomings within the CAPA-IVM COCs. These results confirmed that within in vivo COC, cumulus cells are highly glycolytic, whereas oocytes, with low glycolytic activity, are deviating their glucose towards pentose phosphate pathway. No significant differences were observed in the CAPA-IVM oocytes compared to their in vivo counterparts. However, their cumulus cells exhibited a precocious increase of glycolytic activity during the pre-maturation culture step and activity was decreased during the IVM step. Here, specific alterations in mouse COC glucose metabolism due to CAPA-IVM culture were characterized using direct measurements for the first time. Present data show that, while CAPA-IVM cumulus cells are able to utilize glucose, their ability to support oocytes during final maturation is impaired. Future CAPA-IVM optimization strategies could focus on adjusting culture media energy substrate concentrations and/or implementing co-culture strategies.

scholarly journals Oocyte–cumulus cell interactions regulate free intracellular zinc in mouse oocytes

Reproduction ◽  
2013 ◽  
Vol 145 (4) ◽  
pp. 381-390 ◽  
Author(s):  
R S Lisle ◽  
K Anthony ◽  
M A Randall ◽  
F J Diaz
Keyword(s):  
Cumulus Cell ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Zinc Homeostasis ◽  
Relative Level ◽  
Intracellular Zinc ◽  
Fourfold Increase ◽  
Cumulus Oocyte Complex

Zinc increases in the oocyte during maturation and is required for progression and completion of meiosis. The objective of this study was to determine whether cumulus cells regulate the levels of free intracellular zinc in the oocyte during maturation. In the cumulus–oocyte complex (COC) the relative level of free intracellular zinc was almost fourfold higher in cumulus cells compared with the resident germinal vesicle-stage oocyte. Removal of cumulus cells caused a fourfold increase in intracellular zinc in the oocyte by 1 h after cumulus cell removal, but subsequent coculture of denuded oocytes with COC decreased free intracellular zinc in the oocyte by 65%. Thus, cumulus cells suppress free intracellular zinc in the oocyte. The mRNA transcripts for the zinc transporter proteins Slc39a6, Slc39a8, Slc39a9, Slc39a10, Slc39a12, Slc30a2, Slc30a4, Slc30a5 and Slc30a8 mRNAs were higher in oocytes, while Slc39a1, Slc39a7, Slc39a13, Slc39a14, Slc30a6, Slc30a7 and Slc30a9 mRNAs were higher in cumulus cells. Thus a complex zinc transport network is present in the COC. Pretreatment with epidermal growth factor for 4 h abolished the ability of COCs to restrict free intracellular zinc in denuded oocytes. Coculture of denuded metaphase II oocytes with COC lowers free intracellular zinc in mature oocytes. Oocytes matured in vivo or oocytes from older mice had lower levels of free intracellular zinc than oocytes matured in vitro or from younger mice. Thus, a precise mechanism for regulating oocyte zinc homeostasis has been uncovered in the COC that is disrupted with increasing age or by removal of cumulus cells.

146. MOLECULAR FILTRATION PROPERTIES OF THE EXPANDED CUMULUS MATRIX: CONTROLLED SUPPLY OF METABOLITES AND EXTRACELLULAR SIGNALS TO CUMULUS CELLS AND THE OOCYTE

2010 ◽  
Vol 22 (9) ◽  
pp. 64
Author(s):  
K. R. Dunning ◽  
L. N. Watson ◽  
J. G. Thompson ◽  
R. L. Robker ◽  
D. L. Russell
Keyword(s):  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Cumulus Cells ◽  
Oocyte Quality ◽  
Oocyte Competence ◽  
Signalling Molecules ◽  
The Matrix ◽  
Filtration Properties

Cumulus matrix genes are positively correlated with oocyte competence [1]. Formation of the expanded cumulus matrix during oocyte maturation is well described; however its function remains elusive. We investigated whether cumulus matrix acts as a molecular filter, based on recognised filtration properties of analogous matrices. We found that cumulus matrix controls metabolite supply to the oocyte and retains prostaglandin E2 (PGE2), which is critical in oocyte maturation. The uptake of fluorescently labelled hydrophilic and hydrophobic metabolites showed that cumulus matrix formation significantly impeded diffusion to the oocyte. Expanded in vivo matured cumulus oocyte complexes (COCs, eCG+hCG16h) resisted uptake of glucose and cholesterol compared to unexpanded (eCG44h, P < 0.05), as assessed by confocal microscopy and spatial quantitation of fluorescence (P < 0.05). In vitro maturation (IVM) results in pronounced compositional deficiency of cumulus matrix proteins [2] and poor oocyte quality. Glucose and cholesterol were transported more readily into cumulus cells and the oocyte of IVM COCs (matured in αMEM/5% FCS/50 mIU/mL FSH, 16 h) compared to in vivo matured COCs (P < 0.05 and P = 0.08, respectively). Taking the inverse approach we found that PGE2 synthesised by cumulus cells is retained within the matrix compartment of in vivo matured COCs but IVM COCs did not retain PGE2 and secreted 4.3-fold more into the media. The relationship of retained to secreted PGE2 was significantly higher after in vivo maturation vs IVM COCs (P < 0.0001). This property of the COC matrix reveals a potential mechanism whereby the prostaglandin signal intensifies through a physicochemical mechanism rather than gene regulation. This is the first demonstration that cumulus matrix regulates diffusion toward and secretion from the COC, thus excluding glucose, known to negatively affect oocyte quality, and trapping factors, including PGE2, with critical roles in oocyte maturation and fertilisation. Thus, IVM may reduce oocyte quality due to poor trafficking of metabolites and signalling molecules. (1) McKenzie LJ, et al. Human cumulus granulosa cell gene expression: a predictor of fertilization and embryo selection in women undergoing IVF. Hum Reprod 2004; 19: 2869–2874.(2) Dunning KR, et al. Altered composition of the cumulus-oocyte complex matrix during in vitro maturation of oocytes. Hum Reprod 2007; 22: 2842–2850.

scholarly journals Transcriptomic Pattern of Genes Regulating Protein Response and Status of Mitochondrial Activity Are Related to Oocyte Maturational Competence—A Transcriptomic Study

2019 ◽  
Vol 20 (9) ◽  
pp. 2238 ◽  
Author(s):  
Błażej Chermuła ◽  
Maciej Brązert ◽  
Michal Jeseta ◽  
Katarzyna Ożegowska ◽  
Ievgenia Kocherova ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
In Vitro Maturation ◽  
Transforming Growth Factor ◽  
Transmembrane Protein ◽  
Mitochondrial Activity ◽  
Maturation Process ◽  
Vitro Fertilization ◽  
Practical Applications

This paper aims to identify and describe new genetic markers involved in the processes of protein expression and modification reflected in the change of mitochondrial activity before and after in vitro maturation of the oocyte. Porcine oocytes collected from the ovaries of slaughtered landrace gilts were subjected to the process of in vitro maturation. Transcriptomic changes in the expression profile of oocyte genes involved in response to hypoxia, the transmembrane protein receptor serine threonine kinase signaling pathway, the “transforming growth factor β receptor signaling pathway”, “response to protein stimulus”, and “response to organic substance” were investigated using microarrays. The expression values of these genes in oocytes was analyzed before (immature) and after (mature) in vitro maturation, with significant differences found. All the significantly altered genes showed downregulation after the maturation process. The most changed genes from these gene ontologies, FOS, ID2, VEGFA, BTG2, CYR61, ESR1, AR, TACR3, CCND2, CHRDL1, were chosen to be further validated, described and related to the literature. Additionally, the mitochondrial activity of the analyzed oocytes was measured using specific dyes. We found that the mitochondrial activity was higher before the maturation process. The analysis of these results and the available literature provides a novel insight on the processes that occur during in vitro oocyte maturation. While this knowledge may prove to be useful in further research of the procedures commonly associated with in vitro fertilization procedures, it serves mostly as a basic reference for further proteomic, in vivo, and clinical studies that are necessary to translate it into practical applications.

scholarly journals Localisation of the hyaluronan receptor CD44 in porcine cumulus cells during in vivo and in vitro maturation

Zygote ◽  
2002 ◽  
Vol 10 (4) ◽  
pp. 317-326 ◽  
Author(s):  
Masaki Yokoo ◽  
Paisan Tienthai ◽  
Naoko Kimura ◽  
Koji Niwa ◽  
Eimei Sato ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Cumulus Cells ◽  
Culture Conditions ◽  
Binding Ability ◽  
Migration Ability ◽  
And Migration ◽  
Boar Spermatozoa

Polyspermy is fairly common during porcine in vitro fertilisation (IVF), perhaps due to incomplete in vitro oocyte maturation (IVM). Porcine cumulus cells (CCs) layered around the oocyte produce large amounts of extracellular hyaluronan (HA) when forming an expanding cell cloud during the last phase of oocyte maturation. The specific actions of HA are mediated via HA-binding proteins (HABPs), such as CD44, which act as receptors. In this study using immunocytochemistry and western blotting we investigated the localisation of CD44 in CCs obtained from in vivo-matured pig cumulus-oocyte complexes (COCs) and compared it with that in CCs from immature COCs and of COCs subjected to IVM and IVF procedures. Immunolabelling of CD44 was absent or very weak in CCs from immature COCs but strongly present on the surface of the CCs obtained from in vivo, displaying a similar localisation in the in vitro-matured COCs. In the latter, the labelling decreased but did not disappear in CCs 4 h after sperm co-incubation during IVF. Immunoblotting detected bands of between 73 and 88 kDa, corresponding to CD44, in the protein extract from in vivo CCs collected immediately prior to, or following spontaneous ovulation. The in vitro-matured CCs, however, presented bands ranging from 81 kDa to 88 kDa. Also, the bands found in the in vivo-matured CCs showed a larger variation of intensity and migration among animals than did the batches of in vitro-matured CCs. No CD44 band was detected on aliquots of the frozen-thawed boar spermatozoa used for IVF. The results clearly demonstrate that the specific HA receptor CD44 is present in expanding CCs of in vivo-matured pig COCs, in relation to increasing amounts of inter-CC HA. The subtle differences in molecular weight and migration ability observed between in vivo and in vitro samples may relate to differences in glycosylation and thus explain differences in HA-binding ability, of consequence for optimising in vitro culture conditions.

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