Equine Cumulus-oocyte Complexes Research Articles

Influence of secretome obtained from preovulatory follicular fluid on energy metabolism and meiotic competence of equine cumulus–oocyte complexes

Secretome is composed by proteins and microvesicles secreted by the granulosa cells that line the follicular wall. These bioactive compounds promote follicular growth and oocyte maturation, influencing oocyte metabolism. Hence, we hypothesized that addition of secretome from preovulatory follicular fluid (FF) to maturation medium may influence the meiotic competence and metabolism of cumulus-oocyte-complexes (COCs) obtained post mortem. FF was retrieved from preovulatory follicles (2 mares) after 32h of hCG administration. Three ml of FF from both mares were pooled, diluted 1:1 in PBS and centrifuged (4000g, 1h at 4°C) using a 10K Amicon® Ultra-15 Centrifugal Filter Unit and protein concentration was measured. A total of 186 COCs were retrieved from post mortem ovaries and were subjected to in vitro maturation (IVM) in TCM-199 medium, 10% of FBS and 5 mU/ml of FSH (Control; 59 oocytes) or with 20 µg/ml of secretome (Secretome20; 66 oocytes) or with 40 µg/ml of secretome (Secretome40; 61 oocytes) for 28 h in 5% CO2/95% air at 38.2°C. Oocytes were denuded, fixed in 4% formaldehyde and stained with Hoechst 33342 to assess chromatin conformation by fluorescence microscopy. Also, selected metabolites of the maturation medium prior to andafter IVM were assessed by magnetic proton nuclear resonance spectroscopy. After IVM, a trend towards glucose consumption and pyruvic acid production was observed compared to their controls prior IVM; a significant increase in lactic acid production in all treatments was observed (Table 1; p < 0.05). Our results show a tendency for quicker meiosis resumption in COCs incubated with Secretome40 (Table 2). In conclusion, secretome obtained from preovulatory follicular fluid added during IVM does not change COCs’ energy metabolism nor maturation rate of equine cumulus-oocyte complexes in our in vitro culture system. Funding: MCCIN/AEI (PID2020-112723RB-I00; RYC-2017-21545; RYC2020-028915-I), Junta de Extremadura-FEDER (IB20005).

Selection of frozen-thawed semen for standard in vitro fertilization

Since the report of a repeatable method for standard in vitro fertilization in the horse (IVF) using prolonged sperm pre-incubation (Felix et al., Biol. Reprod. 107:1551-1564, 2022), efforts have been directed toward applying the IVF procedure clinically. A major barrier to clinical use is the need for fresh semen. In this study, we compared three techniques – swim-up (SU), colloid centrifugation (CG), and microfluidics (MFL) – to select frozen-thawed sperm for standard IVF. Media were modified TALP (TALP-R and FT-PHE, Felix et al., 2022). Straws of frozen semen from one fertile stallion were used. For SU, 200 µL frozen-thawed semen was layered under 1 mL FT-PHE in each of two 5-mL tubes. The tubes were incubated for 30 min in 5% CO2 in air, then the uppermost 750 µL of medium was collected, combined, centrifuged and the pellet resuspended in TALP-R to ∼10 million/mL. The suspension was added to 45-µL droplets of FERT-TALP to a concentration of 1 million sperm/mL (in 50 µL) and the droplets were incubated in 5% CO2 in air. For CG, frozen-thawed semen (400µL) was layered on 3 mL of EquipureTM colloid and centrifuged for 20 min at 200g. The sperm pellet was washed once in FT-PHE, then resuspended in TALP-R and sperm added to droplets as above. For MFL, 400µL frozen-thawed semen was combined with 500µL of FT-PHE andthis suspension was placed into the loading chamber of a commercial microfluidic device. FT-PHE (700µL) was placed into the collection chamber. The device was incubated for 30 min in 5% CO2 in air, during which time sperm migrated through a porous membrane into the collection chamber. After incubation, 500µL fluid from the collection chamber was combined with 500µL FT-PHE then centrifuged for 5 minutes at 300g. The pellet was resuspended with TALP-R and sperm added to droplets as above. After droplets were incubated for 10h, in vitro-matured equine cumulus-oocyte complexes (COCs) were added. After 3h co-incubation, the COCs were transferred to embryo culture medium for 32h, then denuded and stained with DAPI. Cleaved embryos with two or more normal nuclei were considered to represent fertilization. Sperm selection by MFL resulted in a higher cleavage rate per co-cultured oocyte (23/32, 71.9%) than did the other two selection methods (8/35, 22.9% and 5/31, 16.1% for SU and CG, respectively). We conclude that standard equine IVF can be achieved with frozen-thawed semen and that sperm selection using a microfluidic device is associated with a higher cleavage rate than is selection using swim-up or colloid centrifugation.

Relative transcript level of BMP15, BAX and CASP3 with qRT-PCR in vitrified equine immature cumulus-oocytes complexes

Research focused on female gamete vitrification has increased attention to develop a reliable cryopreservation method to preserve immature equine oocytes. Despite the intensive implementation of biotechnological procedures for horse breeding, vitrification of immature equine cumulus-oocyte complexes (COCs) remain to be clearly elucidated. We aimed to determine the relative transcript level of target genes Bone morphogenetic protein 15 (BMP15); Bcl-2-associated X protein (BAX); and Caspase 3 (CASP3) in equine COCs prior to and after vitrification. Ovarian follicles were aspirated from ovaries collected from an abattoir. A total of 240 COCs were collected and distributed into vitrified COCs (VIT, n=120) and non-vitrified (Non-VIT, n=120) groups. Then, COCs were preserved and relative transcript expressions of BMP15, BAX, CASP3 were measured and normalized against GAPDH performed by qRT-PCR. In addition, 38 COCs were evaluated to assess chromatin configuration of germinal vesicl e stage prior and after vitrification by exposure to 10 μg/ml of bisbenzimide. A difference was observed in the COCs’ mRNA level of abundance for the BAX gene between the VIT (2.05 ± 0.47) and (0.85 ± 0.08) Non-VIT groups. There was no difference in mRNA relative transcript level of CASP3 and BMP15 in Non-VIT (0.63 ± 0.20 and 1.55 ± 0.73, respectively) compared to VIT (0.64 ± 0.01 and 2.84 ± 2.20, respectively) equine COCs. All COCs where considered at immature stage of development even though COCs in Non-VIT group showed higher condensed chromatin configuration compared to VIT (100% vs 60.7%, respectively). We demonstrate that BMP15 and CASP3 are detected in VIT and Non-VIT immature COCs. In conclusion, BAX is expressed highly in vitrified immature equine COCs and indicates that activation of apoptosis signaling cascades in cells exposed to vitrification.

The Phosphodiesterase Inhibitor, Isobutyl-1-Methylxanthine Prevents the Sudden Drop in Cyclic Adenosine Monophosphate Concentration and Modulates Glucose Metabolism of Equine Cumulus–Oocyte Complexes Matured in Vitro

Spontaneous nuclear maturation of mammalian oocytes can occur when physically removed from the ovarian follicle during in vitro oocyte maturation (IVM), largely because of a decrease in cyclic adenosine monophosphate (cAMP) concentration. Modulation of oocyte cAMP during IVM by using phosphodiesterase inhibitors has been shown to maintain elevated oocyte cAMP concentrations and control meiotic resumption of bovine and ovine oocytes. This study determined the effect of inclusion of isobutyl-1-methylxanthine (IBMX) during collection and the first 12 hours of incubation of equine oocytes on cAMP concentration and glucose metabolism of cumulus–oocyte complexes (COCs). Abattoir-derived COCs were collected in aspiration medium with (Asp-IBMX) or without (Asp) IBMX. Cumulus–oocyte complexes were then incubated for 12 hours in IVM medium with (Mat-IBMX) or without (Mat) IBMX, followed by additional 24 hours in Mat medium. The cAMP concentration, glucose consumption, lactate production, and metaphase II rates of the COCs were assessed. Cumulus–oocyte complexes aspirated into Asp-IBMX (62.2 ± 2.6 fmol per COC) medium had higher cAMP concentration than Asp (31.8 ± 2.8 fmol per COC) control group (P < .05). Likewise, at 12 hours of IVM, Mat-IBMX group (33.2 ± 2.1 fmol per COC) had higher cAMP concentration than the Mat group (7.68 ± 0.5 fmol per COC; P < .05). Glucose consumption and lactate production were lower during the first 12 hours of incubation in COCs cultured in Mat-IBMX (P < .05). Isobutyl-1-methylxanthine prevented the rapid drop in cAMP concentration and altered metabolism of glucose by the COC. Preventing the sudden drop in cAMP prevents the premature nuclear maturation of in vitro–matured oocytes causing poor developmental competence.

Energy metabolism of the equine cumulus oocyte complex during in vitro maturation

Horses are one of the few species, beside humans, in which assisted reproductive technology has important clinical applications. Furthermore, the horse can serve as a valuable model for the study of comparative reproductive biology. Here we present the first comprehensive characterisation of energy metabolism and mitochondrial efficiency in equine cumulus-oocyte complexes (COCs) during in vitro maturation (IVM), as determined using a combination of non-invasive consumption and release assays and mitochondrial function analysis. These data reveal notable species-specific differences in the rate and kinetics of glucose consumption and glycolysis throughout IVM. Approximately 95% of glucose consumed was accounted for by lactate production; however, high concurrent oxygen consumption indicated a comparatively increased role for non-glycolytic oxidative phosphorylation. Up to 38% of equine COC oxygen consumption could be attributed to non-mitochondrial activities and there was a significant loss of spare respiratory capacity over the course of IVM. Notably, our data also revealed that current IVM protocols may be failing to satisfy the metabolic demands of the equine COC. Our findings constitute the first report on mitochondrial efficiency in the equine COC and provide new insight into comparative gamete biology as well as metabolism of the COC during in vitro maturation.

Recovery and Maturation Rates, and Blastocyst Rates After ICSI, of Oocytes Aspirated From Follicles After Intrafollicular Oocyte Transfer

Previous studies have found low rates of blastocyst development (0–11%) after vitrification of germinal vesicle (GV)-stage equine oocytes. In this study, we systematically evaluated a short (non-equilibrating) system for GV-stage oocyte vitrification. In Exp. 1, we assessed oocyte volume in cumulus-oocyte complexes (COCs) exposed to components of a short protocol, using 2% each of ethylene glycol and propylene glycol in the first solution (VS1); 17.5% of each plus 0.3 M trehalose in the second solution (VS2); and fetal bovine serum as the base medium. Based on the time to oocyte minimum volume, we selected a 40-sec exposure to VS1. In Exp. 2, we evaluated exposure times to VS2 and, based on rates of subsequent maturation in vitro, we selected 65 s. In Exp. 3, we used the optimized vitrification system (40-VS1; 65-VS2) and evaluated three warming procedures. Blastocyst development after ICSI was equivalent (15%) for COCs warmed in either standard (trehalose stepwise dilution) or isotonic (base medium) solutions, but was reduced (0%) for COCs warmed in a highly hypertonic (1.5 M trehalose) solution. Exposure to the vitrification and warming solutions, without actual vitrification, was associated with reduced blastocyst development (0–5%; Exp. 4). We conclude that this optimized short protocol supports moderate blastocyst production after vitrification of GV-stage equine COCs. Oocytes can be warmed in isotonic medium, which simplifies the procedure. The systems used still showed a high level of toxicity and further work is needed on both vitrification and warming methods to increase the efficiency of this technique.

Effect of different shipping temperatures (∼22 °C vs. ∼7 °C) and holding media on blastocyst development after overnight holding of immature equine cumulus-oocyte complexes

Intracytoplasmic sperm injection (ICSI) is an important tool for equine embryo production in both clinical and research settings. In clinical ICSI programs, immature equine cumulus-oocyte complexes (COCs) are often collected at the mare's location and shipped to the ICSI laboratory. To simplify shipment and aid scheduling of subsequent procedures, COCs can be held overnight at room temperature (∼22 °C) before placement into maturation culture, with no detrimental effect on meiotic or developmental competence. A recent study indicated that it might be possible to hold COCs overnight at cold (∼4 °C) temperatures. If so, this might allow longer holding periods that would ease shipping requirements. In this study, we compared oocyte maturation rates, as well as cleavage and blastocyst rates after ICSI, for COCs held at either room or cold temperatures overnight before the onset of in vitro maturation. In Exp. 1, COCs were shipped overnight in a commercial embryo holding medium, ViGRO (Vg), in insulated containers designed to hold at either room temperature (RT, ∼22 °C) or cold temperatures (Cold, ∼7 °C). Subsequent rates of in vitro maturation, cleavage and blastocyst formation were significantly higher in the RT treatment (39%, 90% and 41%, respectively) than in the Cold treatment (23%, 60% and 17%, respectively, P < .05). In Exp. 2, we compared Vg medium with a second commercial embryo holding medium, SYNGRO (Sy). There was no significant difference between Vg and Sy groups in any evaluated parameter within either RT or Cold treatments. Within each medium group and for both media combined, the rates of in vitro maturation, cleavage and blastocyst formation were significantly higher in the RT treatment (42%, 81% and 42%, respectively for the combined media) than in the Cold treatment (29%, 54% and 10%, respectively for the combined media, P < .05). We conclude that shipment of immature equine COCs at cold temperatures (∼7 °C) is detrimental to subsequent in vitro maturation and embryo production.

Vitrification of germinal-vesicle stage equine oocytes: Effect of cryoprotectant exposure time on in-vitro embryo production

Previous studies have found low rates of blastocyst development (0–11%) after vitrification of germinal vesicle (GV)-stage equine oocytes. In this study, we systematically evaluated a short (non-equilibrating) system for GV-stage oocyte vitrification. In Exp. 1, we assessed oocyte volume in cumulus-oocyte complexes (COCs) exposed to components of a short protocol, using 2% each of ethylene glycol and propylene glycol in the first solution (VS1); 17.5% of each plus 0.3 M trehalose in the second solution (VS2); and fetal bovine serum as the base medium. Based on the time to oocyte minimum volume, we selected a 40-sec exposure to VS1. In Exp. 2, we evaluated exposure times to VS2 and, based on rates of subsequent maturation in vitro, we selected 65 s. In Exp. 3, we used the optimized vitrification system (40-VS1; 65-VS2) and evaluated three warming procedures. Blastocyst development after ICSI was equivalent (15%) for COCs warmed in either standard (trehalose stepwise dilution) or isotonic (base medium) solutions, but was reduced (0%) for COCs warmed in a highly hypertonic (1.5 M trehalose) solution. Exposure to the vitrification and warming solutions, without actual vitrification, was associated with reduced blastocyst development (0–5%; Exp. 4). We conclude that this optimized short protocol supports moderate blastocyst production after vitrification of GV-stage equine COCs. Oocytes can be warmed in isotonic medium, which simplifies the procedure. The systems used still showed a high level of toxicity and further work is needed on both vitrification and warming methods to increase the efficiency of this technique.

Glucocorticoid metabolism in equine follicles and oocytes

The objective of this study was to determine whether (1) systemic and intrafollicular cortisol concentrations in horses are directly related and (2) supraphysiological levels of glucocorticoids affect in vitro maturation (IVM) rates of oocytes. Specifically, we studied the (1) changes in the intrafollicular cortisol and progesterone in context with granulosa cell gene expression during maturation of equine follicles (from 5–9 mm, 10–14 mm, 15–19 mm, 20–24 mm, and ≥25 mm in diameter) and (2) effects of cortisol supplementation on IVM rates and gene expression of equine cumulus–oocyte complexes (COCs). For these purposes, follicular fluid, granulosa cells, and COCs were collected from 12 mares (mean age 8.6 ± 0.5 yr) by transvaginal aspiration. Cortisol and progesterone concentrations in follicular fluid from follicles ≥25 mm were greater (P < 0.05) than in all other follicle classes and were positively correlated (r = 0.8; P < 0.001). Plasma concentrations of cortisol and progesterone did not differ before and after follicle aspiration (P > 0.05). In granulosa cells, gene expression of NR3C1, HSD11B1, HSD11B2, and CYP21A2 did not differ (P > 0.05) among different follicle classes. Maturation rates were similar (P > 0.05) among groups, regardless of the cortisol concentration in the IVM medium. In cumulus cells, messenger RNA expression of genes involved in glucocorticoid mechanism and apoptosis was either increased (NR3C1 and BCL2) or decreased (HSD11B2) by treatment (P < 0.01). In oocytes, gene expression of maturation markers (BMP15 and GDF9) was affected (P < 0.001) by cortisol treatment. This study demonstrates the involvement of glucocorticoids in follicle and oocyte maturation and cortisol modulation by HSD11B2 in equine COCs. Our data provide further information for understanding the normal ovarian endocrine physiology which might in turn also help improve equine assisted reproduction techniques.

Glucose uptake and lactate production of equine cumulus-oocyte complexes during in vitro maturation

Glucose uptake and lactate production of equine cumulus-oocyte complexes during in vitro maturation

Expression profiles of select genes in cumulus-oocyte complexes from young and aged mares.

There is compelling evidence that oocytes from mares >18 years of age have a high incidence of inherent defects that result in early embryonic loss. In women, an age-related decrease in oocyte quality is associated with an increased incidence of aneuploidy and it has recently been determined that the gene expression profile of human oocytes is altered with advancing age. We hypothesised that similar age-related aberrations in gene expression occur in equine oocytes. Therefore, the aim of the present study was to compare gene expression profiles of individual oocytes and cumulus cells from young and aged mares, specifically evaluating genes that have been identified as being differentially expressed with advancing maternal age and/or aneuploidy in human oocytes. Expression of 48 genes was compared between 14 cumulus-oocyte complexes (COCs) from mares aged 3-12 years and 10 COCs from mares ≥18 years of age. Three genes (mitochondrial translational initiation factor 3 (IF3), heat shock transcription factor 5 (HSF5) and Y box binding protein 2 (YBX2)) were differentially expressed in oocytes, with all being more abundant in oocytes from young mares. Three genes (ADP-ribosylation factor-like 6 interacting protein 6 (ARL6IP6), BCL2-associated X protein (BAX) and hypoxia upregulated 1 (HYOU1)) were differentially expressed in cumulus cells, with all being more abundant in aged mares. The results of the present study confirm there are age-related differences in gene expression in equine COCs, which may be associated with the lower quality and decreased developmental competence of oocytes from aged mares.

Lectin-binding sites in isolated equine cumulus-oocyte complexes: Differential expression of glycosidic residues in complexes recovered with compact or expanded cumulus

Equine cumulus-oocyte complexes (COCs) were analyzed by means of 13 lectins to evaluate their glycoconjugate patterns and to verify differences between COCs recovered with compact (Cp) and expanded (Exp) cumulus. Cumulus cells showed a similar staining pattern in both Cp and Exp COCs with all lectins used, except for a higher reactivity with SNA and GSA II in Cp COCs and SBA in Exp COCs. The zona pellucida (ZP) showed (1) uniform staining with MAL II, RCA 120, and SBA in both Cp and Exp COCs, (2) trilaminar binding pattern with WGA as well as higher Con A reactivity in the outer region of both types of COCs, (3) uniform staining with PNA only in Exp COCs, (4) uniform and trilaminar binding pattern with SNA in Cp and Exp COCs, respectively, and (5) major reactivity with GSA II in Exp COCs. Ooplasm showed similar staining intensity with Con A, HPA, GSA I-B 4, and WGA in both Cp and Exp COCs, with stronger reactivity to GSA II in Exp COCs, whereas SNA, UEA I, and LTA binding sites were present only in Cp COCs. Oocyte cortical granules of both Cp and Exp COCs reacted with Con A and WGA. These results suggest that, in the mare, viable (Cp) and atretic (Exp) COCs display different glycoconjugate staining pattern, which may account for the different maturation and developmental competence of COCs.

Expression and localization of the µ‐opioid receptor (MOR) in the equine cumulus–oocyte complex and its involvement in the seasonal regulation of oocyte meiotic competence

The micro-opioid receptor (MOR) was identified in equine oocytes, cumulus and granulosa cells. By RT-PCR, a 441bp fragment was observed. By immunoblotting, a 65 kDa band was detected in samples of winter anestrous whereas in cells recovered in breeding season, two bands, 65 and 50 kDa, were found. The 65 kDa band was significantly more intense in winter anestrous specimens. In samples recovered in the breeding season, this band significantly decreased with the raise of follicle size and was heavier in compact oocytes and cumulus cells. The protein was localized on the oolemma and within the cytoplasm of oocytes and cumulus cells. In vitro oocyte maturation rate (MR), analyzed by confocal microscopy for nuclear chromatin, microfilaments and microtubules, was reduced after the addition of 3 x 10(-8) M beta-endorphin in medium without additional hormones. Inhibitory effects of 10(-3) M Naloxone in oocytes collected in anestrous and spring transition were observed, both in presence and absence of hormones added to culture medium. Increased MRs were observed in oocytes collected in anestrous and cultured in presence of 10(-8) M Naloxone. The exposure to 10(-3) M Naloxone induced significant intracellular calcium increases in cumulus cells recovered all over the year. beta-Endorphin 3 x 10(-8) M induced significant calcium increases only in cumulus cells recovered in fall transition and anestrous. Naloxone 10(-8) M did not induce intracellular calcium modifications. We conclude that the MOR is differentially expressed in equine cumulus-oocyte complexes in the different seasons of the year and plays a role in the seasonal regulation of meiotic competence of equine oocytes.

Effect of cumulus morphology and maturation stage on the cryopreservability of equine oocytes

Oocyte cryopreservation is a potentially valuable way of preserving the female germ line. However, the developmental competence of cryopreserved oocytes is presently poor. This study investigated whether the morphology of the cumulus complex surrounding an immature equine oocyte and/or the oocyte's stage of maturation affect its cryopreservability. Compact (Cp) and expanded (Ex) cumulus oocyte complexes (COCs) were vitrified either shortly after recovery (germinal vesicle stage, GV) or after maturation in vitro (IVM); cryoprotectant-treated and -untreated non-frozen oocytes served as controls. In Experiment I, oocytes matured in vitro and then vitrified, or vice versa, were examined for maturation stage and meiotic spindle quality. Cp and Ex COCs vitrified at the GV stage matured at similar rates during subsequent IVM (41 vs 46% MII), but meiotic spindle quality was better for Cp than Ex (63 vs 33% normal spindles). Vitrifying oocytes after IVM resulted in disappointing post-warming spindle quality (32 vs 28% normal for Cp vs Ex). In Experiment II, oocytes from Cp and Ex COCs vitrified at the GV or MII stages were fertilized by intracytoplasmic sperm injection (ICSI) and monitored for cleavage and blastocyst formation. Oocytes vitrified prior to IVM yielded higher cleavage rates (34 and 27% for Cp and Ex COCs) than those vitrified after IVM (16 and 4%). However, only one blastocyst was produced from a sperm-injected vitrified-warmed oocyte (0.4 vs 9.3% and 13% blastocysts for cryoprotectant-exposed and -untreated controls). It is concluded that, when vitrification is the chosen method of cryopreservation, Cp equine COCs at the GV stage offer the best chance of an MII oocyte with a normal spindle and the potential for fertilization; however, developmental competence is still reduced dramatically.

Glutathione content and glutathione peroxidase expression in in vivo and in vitro matured equine oocytes

The in vitro developmental competence of equine oocytes is still low in comparison with other domestic mammals. A major factor affecting the viability of cells during in vitro culture is the increased oxidative stress. Oxidative modifications could be responsible for oocyte incompetence for in vitro maturation (IVM). Cysteamine, a glutathione (GSH) synthesis enhancer, has been shown to increase intracellular GSH content and to improve embryo development when added during IVM of bovine, porcine, and ovine oocytes. The aim of the present study was (1) to determine whether equine cumulus-oocyte complexes (COCs) benefit from the addition of cysteamine during IVM, (2) to compare the GSH content of oocytes after in vivo maturation and IVM, (3) to assess whether cysteamine administration during IVM of equine oocyte enhances early embryonic developmental capability following ICSI, (4) to study the glutathione peroxidase (GPX) mRNA level in COCs. In vivo matured COCs were collected by aspiration from preovulatory follicles, and analyzed at collection. Immature COCs were collected in vivo or from slaughterhouse ovaries and matured in culture media supplemented or not with 100 microM cysteamine. After nuclear stage assessment, oocytes were analyzed for GSH concentration and both oocytes and cumulus cells were analyzed for GPX and GAPDH mRNA. Our data showed that the maturation capability was similar in both in vivo aspirated oocytes and in those isolated from slaughterhouse ovaries. Moreover, the addition of cysteamine during IVM affected neither GSH content nor maturation rate. At the time of collection, intra-oocyte GSH content was not influenced by the chromatin status. GSH concentration was similar in in vivo and in vitro matured metaphase II (MII) stage oocytes, and was significantly higher in MII than immature germinal vesicle stage oocytes. Moreover, the presence of serum inhibited whereas its absence stimulated the accumulation of GSH within oocytes during IVM. After ICSI, a similar proportion of zygotes in each group developed beyond the two-cell stage after 72 hr of culture. Cumulus cells expressed GPX mRNA, while GPX transcript was absent in both immature and mature oocytes. Cumulus expression of GPX mRNA was significantly higher when analyzed at collection than after IVM. Taken together, our results demonstrate that in equine oocytes, GSH increases during IVM but the relative intra-oocyte content of this thiol does not affect maturation and early development efficiency after fertilization. We hypothesize that factor(s) other than GSH/GPX are responsible for the limited in vitro early developmental capability of equine oocytes.

Effect of homologous follicular fluid from medium-sized and large follicles on in vitro maturation of equine cumulus - oocyte complexes

The in vitro maturation (IVM) of equine oocytes is typically performed using various synthetic media; however, an optimal IVM system for equine oocytes has not been developed. The aim of the present study was to evaluate the effects of two types of follicular fluid (FF) obtained from cyclic mares and two incubation intervals for the IVM of equine cumulus-oocyte complexes (COCs). Follicular fluid was collected from medium-sized (20-29 mm diameter) and large (e30 mm; post-human chorionic gonadotrophin administration) follicles using transvaginal ultrasound-guided follicle aspiration. Compact (n = 232) and non-compact (n = 183) COCs obtained from a slaughterhouse were incubated separately in the following groups: (1) FF from medium follicles for 24 h; (2) FF from large follicles for 24 h; (3) control (synthetic) medium for 24 h; (4) FF from medium follicles for 24 h then FF from large follicles for an additional 24 h; (5) FF from large follicles for 48 h; and (6) control medium for 48 h. For compact COCs, there was a tendency (P = 0.06) for more COCs incubated in FF from large follicles for 24 h to reach metaphase II compared with those incubated in control medium for 24 h (58% v. 35%, respectively). More (P < 0.05) compact COCs had degenerated after incubation in control medium for 48 h compared with all other groups (51% v. 14-24%, respectively). For non-compact COCs, incubation in FF from medium follicles for 24 h resulted in more (P = 0.05) COCs at metaphase II compared with control medium for 48 h (58% v. 29%, respectively). These results indicate that homologous FF from cyclic mares is a suitable alternative for the IVM of equine COCs and that it may be superior to conventional media for longer (i.e. >24 h) incubation intervals.

Cumulus expansion, nuclear maturation and connexin 43, cyclooxygenase-2 and FSH receptor mRNA expression in equine cumulus-oocyte complexes cultured in vitro in the presence of FSH and precursors for hyaluronic acid synthesis

The aim of this study was to investigate cumulus expansion, nuclear maturation and expression of connexin 43, cyclooxygenase-2 and FSH receptor transcripts in equine cumuli oophori during in vivo and in vitro maturation in the presence of equine FSH (eFSH) and precursors for hyaluronic acid synthesis. Equine cumulus-oocyte complexes (COC) were cultured in a control defined medium supplemented with eFSH (0 to 5 micrograms/ml), Fetal Calf Serum (FCS), precursors for hyaluronic acid synthesis or glutamine according to the experiments. After in vitro maturation, the cumulus expansion rate was increased with 1 microgram/ml eFSH, and was the highest with 20% FCS. It was not influenced by precursors for hyaluronic acid synthesis or glutamine. The expression of transcripts related to cumulus expansion was analyzed in equine cumulus cells before maturation, and after in vivo and in vitro maturation, by using reverse transcription-polymerase chain reaction (RT-PCR) with specific primers. Connexin 43, cyclooxygenase-2 (COX-2) and FSH receptor (FSHr) mRNA were detected in equine cumulus cells before and after maturation. Their level did not vary during in vivo or in vitro maturation and was influenced neither by FSH nor by precursors for hyaluronic acid synthesis. Results indicate that previously reported regulation of connexin 43 and COX-2 proteins during equine COC maturation may involve post-transcriptional mechanisms.

Expression of interleukin-1 (IL-1) system genes in equine cumulus-oocyte complexes and influence of IL-1beta during in vitro maturation.

A growing body of evidence suggests that the ovary is a site of inflammatory reactions, and thus, ovarian cells could represent sources and targets of the interleukin-1 (IL-1) system. The aim of the present work was to investigate the expression of IL-1alpha, IL-1beta, IL-1ra, IL-1R1, and IL-1R2 genes in equine cumulus cells and oocytes. Moreover, the influence of IL-1beta on in vitro maturation of cumulus-oocytes complexes (COCs) was examined. COCs were collected using ultrasound-guided follicular puncture in vivo. Oocytes and cumulus cells were isolated from preovulatory and subordinate follicles and were analyzed either at collection or after in vitro culture. An ultrasensitive reverse transcription-polymerase chain reaction was used to study the expression of IL-1 system members. In order to study the in vitro effect of IL-1beta on oocyte nuclear maturation and cumulus expansion, immature COCs collected from subordinate follicles were cultured for 30 h in media containing or not containing IL-1beta alone, or in combination with equine luteinizing hormone. Our results indicated that equine oocytes expressed IL-1beta and IL-1R2 genes, and that cumulus cells expressed all IL-1 system members except IL-1alpha. In oocytes, IL-1beta expression significantly decreased during in vitro culture compared with immature and mature oocytes analyzed at collection. Similarly, in cumulus cells, in vitro culture decreased IL-1ra and IL-1R1 expression. IL-1beta was shown to significantly decrease the gonadotropin-induced nuclear maturation of oocytes. In conclusion, we demonstrated for the first time, in mares, the presence of IL-1beta and IL-1R2 transcripts in oocytes and of IL-1beta, IL-1ra, IL-1R1, and IL-1R2 in cumulus cells. The regulatory effect of IL-1beta on COCs during maturation suggests that the IL-1 system could be of crucial importance for ovarian physiology in the mare.

Analysis of MUC-1 and integrin subunit expression in equine uterine epithelium and trophectoderm

Intracytoplasmic sperm injection (ICSI) is an important tool for equine embryo production in both clinical and research settings. In clinical ICSI programs, immature equine cumulus-oocyte complexes (COCs) are often collected at the mare's location and shipped to the ICSI laboratory. To simplify shipment and aid scheduling of subsequent procedures, COCs can be held overnight at room temperature (∼22 °C) before placement into maturation culture, with no detrimental effect on meiotic or developmental competence. A recent study indicated that it might be possible to hold COCs overnight at cold (∼4 °C) temperatures. If so, this might allow longer holding periods that would ease shipping requirements. In this study, we compared oocyte maturation rates, as well as cleavage and blastocyst rates after ICSI, for COCs held at either room or cold temperatures overnight before the onset of in vitro maturation. In Exp. 1, COCs were shipped overnight in a commercial embryo holding medium, ViGRO (Vg), in insulated containers designed to hold at either room temperature (RT, ∼22 °C) or cold temperatures (Cold, ∼7 °C). Subsequent rates of in vitro maturation, cleavage and blastocyst formation were significantly higher in the RT treatment (39%, 90% and 41%, respectively) than in the Cold treatment (23%, 60% and 17%, respectively, P < .05). In Exp. 2, we compared Vg medium with a second commercial embryo holding medium, SYNGRO (Sy). There was no significant difference between Vg and Sy groups in any evaluated parameter within either RT or Cold treatments. Within each medium group and for both media combined, the rates of in vitro maturation, cleavage and blastocyst formation were significantly higher in the RT treatment (42%, 81% and 42%, respectively for the combined media) than in the Cold treatment (29%, 54% and 10%, respectively for the combined media, P < .05). We conclude that shipment of immature equine COCs at cold temperatures (∼7 °C) is detrimental to subsequent in vitro maturation and embryo production.

Influence of insulin-like growth factor-I and its interaction with gonadotropins, estradiol, and fetal calf serum on in vitro maturation and parthenogenic development in equine oocytes.

The effects of insulin-like growth factor-I (IGF-I) and its interaction with gonadotropins, estradiol, and fetal calf serum (FCS) on in vitro maturation (IVM) of equine oocytes were investigated in this study. We also examined the role of IGF-I in the presence or absence of gonadotropins, estradiol, and FCS in parthenogenic cleavage after oocyte activation with calcium ionophore combined with 6-dimethylaminopurine (6-DMAP), using cleavage rate as a measure of cytoplasmic maturation. Only equine cumulus-oocyte complexes with compact cumulus and homogenous ooplasm (n = 817) were used. In experiment 1, oocytes were cultured in TCM-199 supplemented with BSA, antibiotics, and IGF-I at 0 (control), 50, 100, 200 ng/ml, at 39 degrees C in air with 5% CO(2), 95% humidity for 36 or 48 h. In experiment 2, oocytes were cultured with FSH, LH, estradiol, and FCS with IGF-I at the concentration that promoted the highest nuclear maturation rate in experiment 1. In experiment 3, oocytes from the three experimental groups (IGF-I; hormones; and IGF-I + hormones) were chemically activated by exposure to calcium ionophore followed by culture in 6-DMAP. In experiment 1, IGF-I stimulated equine oocyte maturation in a dose-dependent manner with the highest nuclear maturation rate at a concentration of 200 ng/ml. No significant effect of IGF-I on nuclear maturation was observed in experiment 2. In experiment 3, a significant difference in cleavage rate was observed between the hormone + IGF-I group (15 of 33; 45.4%) compared with IGF-I (10 of 36; 27.8%) and hormone (4 of 31; 12.9%) alone (P < 0.05). These results demonstrated that IGF-I has a positive effect on nuclear maturation rate of equine oocytes in vitro. The addition of IGF-I to an IVM medium containing hormones and FCS did not increase nuclear maturation, but resulted in a positive effect on cytoplasmic maturation of equine oocytes measured by parthenogenic cleavage.