Analysis of Chromosome Segregation, Histone Acetylation, and Spindle Morphology in Horse Oocytes

The field of assisted reproduction has been developed to treat infertility in women, companion animals, and endangered species. In the horse, assisted ...

Université de Liège

Oviduct epithelial cells (OEC) co-culture promotes in vitro fertilization (IVF) in human, bovine and porcine species, but no data are available from equine species. Yet, despite numerous attempts, equine IVF rates remain low. Our first aim was to verify a beneficial effect of the OEC on equine IVF. In mammals, oviductal proteins have been shown to interact with gametes and play a role in fertilization. Thus, our second aim was to identify the proteins involved in fertilization in the horse.

The secretions of oviduct epithelial cells increase the equine in vitro fertilization rate: are osteopontin, atrial natriuretic peptide A and oviductin involved?

Epigenetic modifications are established during gametogenesis and preimplantation embryonic development. Any disturbance of the normal natural environment during these critical phases could cause alterations of the epigenetic signature. Histone acetylation is an important epigenetic modification involved in the regulation of chromatin organization and gene expression. The present study was aimed to determine whether the proper establishment of post-translational histone H4 acetylation at lysine 8 (AcH4K8), 12 (AcH4K12) and 16 (AcH4K16) of equine oocytes is adversely affected during in vitro maturation (IVM) when compared with in vivo matured oocytes collected from naturally cycling mares not undergoing ovarian hyperstimulation. The acetylation patterns were investigated by means of indirect immunofluorescence staining with specific antibodies directed against the acetylated lysine residues. Our results indicate that the acetylation state of H4 is dependent on the chromatin configuration in immature germinal vesicle (GV) stage oocytes and it changes in a residue-specific manner along with the increase of chromatin condensation. In particular, the levels of AcH4K8 and AcH4K12 increased significantly, while AcH4K16 decreased significantly from the fibrillar to the condensed state of chromatin configuration within the GV. Moreover, during meiosis, K8 and K12 were substantially deacetylated without any differences between in vivo and in vitro conditions, while K16 displayed a strong acetylation in oocytes matured in vivo, and in contrast, it was markedly deacetylated following IVM. Although the functional meaning of residue-specific acetylation during oocyte differentiation and meiotic resumption needs further investigation, our results support the hypothesis that IVM conditions can adversely affect oocyte ability to regulate the epigenetic reprogramming, critical for successful meiosis and subsequent embryonic development.

Changes in histone H4 acetylation during in vivo versus in vitro maturation of equine oocytes.

Pulsed wave (PW) Doppler echocardiography has become a routine non invasive cardiac diagnostic tool in most species. However, evaluation of intracardiac blood flow requires reference values, which are poorly documented in goats. The aim of this study was to test the repeatability, the variability, and to establish the reference values of PW measurements in healthy adult Saanen goats. Using a standardised PW Doppler echocardiographic protocol, 10 healthy adult unsedated female Saanen goats were investigated three times at one day intervals by the same observer. Mitral, tricuspid, aortic and pulmonary flows were measured from a right parasternal view, and mitral and aortic flows were also measured from a left parasternal view. The difference between left and right side measurements and the intra-observer inter-day repeatability were tested and then the reference values of PW Doppler echocardiographic parameters in healthy adult female Saanen goats were established.

Repeatability, variability and reference values of pulsed wave Doppler echocardiographic measurements in healthy Saanen goats.

Myeloperoxidase (MPO) is a pro-oxidant enzyme contained in and released by neutrophils during degranulation or after lysis. Post-thaw semen contains MPO and its concentration is associated with decreased sperm motility. Recently, MPO concentration in post-thaw semen was shown to be associated with the presence of non-sperm cells (NSC). The objective of this study was to evaluate the effect of a single-layer colloidal centrifugation before cryopreservation on NSC and MPO concentrations in equine semen. The experimental design consisted of freezing semen with or without previous centrifugation through two concentrations of single-layer colloid media. Non-sperm cells and MPO concentrations were assessed in pellet and upper layer at each step of the procedure and MPO was detected in cells by immunocytochemistry. Single-layer colloid centrifugation decreased NSC and MPO concentrations in post-thaw semen. The MPO concentration was correlated with concentration of NSC in the upper layer of the supernatant. In post-thaw semen, with or without previous single-layer colloid centrifugation, MPO concentration was correlated with concentration of NSC. Overall, neutrophils were rarely observed and NSC were mainly epithelial cells or cellular debris, as demonstrated by MPO immunocytochemistry. At all steps of the semen processing and cryopreservation, MPO immunostaining was clearly identified only on NSC. In conclusion, our study shows that NSC present in fresh semen release MPO during freezing.

Effect of non-sperm cells removal with single-layer colloidal centrifugation on myeloperoxidase concentration in post-thaw equine semen.

Intraluminal fluid and excessive abnormal hyperedema are regularly used for the diagnosis of endometritis in the mare, which is routinely confirmed by the presence of neutrophils on endometrial smears. Studies show a relation between neutrophils and myeloperoxidase (MPO), an enzyme contained in and released by neutrophils during degranulation or after cell lysis. This enzyme has been found in many fluids and tissues, and associated with different inflammatory pathologies in the horse. The aims of this study were to assess the presence and concentration of MPO in the equine uterus, and to investigate its relation with neutrophils, and other clinical signs of endometritis. Mares (n = 51) were evaluated for the presence of intraluminal fluid and excessive endometrial edema before breeding, and a small volume lavage and cytology samples were obtained. From 69 cycles, supernatant of the uterine flushes was analyzed with a specific equine MPO ELISA assay to measure MPO concentration. Cytology samples were used for the diagnosis of endometritis. Myeloperoxidase was present in the uterus of all estrus mares in highly variable concentrations. Myeloperoxidase concentrations were significantly (P < 0.05) higher in samples with positive cytologies and in the presence of intraluminal fluid. Occasionally, some samples with negative cytologies showed high MPO concentration, but the opposite was never observed. Cycles presenting hyperedema weren't associated with high concentration of MPO, intraluminal fluid, or positive cytology, making it a poor diagnostic tool of endometritis.

High concentrations of myeloperoxidase in the equine uterus as an indicator of endometritis.

Glycoprotein oligosaccharides play major roles during reproduction, yet their function in gamete interactions is not fully elucidated. Identification and comparison of the glycan pattern in cumulus-oocyte complexes (COCs) from species with different efficiencies of in vitro spermatozoa penetration through the zona pellucida (ZP) could help clarify how oligosaccharides affect gamete interactions. We compared the expression and localization of 12 glycosidic residues in equine and porcine in vitro-matured (IVM) and preovulatory COCs by means of lectin histochemistry. The COCs glycan pattern differed between animals and COC source (IVM versus preovulatory). Among the 12 carbohydrate residues investigated, the IVM COCs from these two species shared: (a) sialo- and βN-acetylgalactosamine (GalNAc)-terminating glycans in the ZP; (b) sialylated and fucosylated glycans in cumulus cells; and (c) GalNAc and N-acetylglucosamine (GlcNAc) glycans in the ooplasm. Differences in the preovulatory COCs of the two species included: (a) sialoglycans and GlcNAc terminating glycans in the equine ZP versus terminal GalNAc and internal GlcNAc in the porcine ZP; (b) terminal galactosides in equine cumulus cells versus terminal GlcNAc and fucose in porcine cohorts; and (c) fucose in the mare ooplasm versus lactosamine and internal GlcNAc in porcine oocyte cytoplasm. Furthermore, equine and porcine cumulus cells and oocytes contributed differently to the synthesis of ZP glycoproteins. These results could be attributed to the different in vitro fertilization efficiencies between these two divergent, large-animal models.

Differential expression and localization of glycosidic residues in in vitro- and in vivo-matured cumulus-oocyte complexes in equine and porcine species.

Implantation failure and genetic developmental disabilities in mammals are caused by errors in chromosome segregation originating mainly in the oocyte during meiosis I. Some conditions, like maternal ageing or in vitro maturation (IVM), increase the incidence of oocyte aneuploidy. Here oocytes from adult mares were used to investigate oocyte maturation in a monovulatory species. Experiments were conducted to compare: (1) the incidence of aneuploidy, (2) the morphology of the spindle, (3) the acetylation of lysine 16 on histone H4 (H4K16) and (4) the relative amount of histone acetyltransferase 1 (HAT1), K(lysine) acetyltransferase 8 (KAT8, also known as MYST1), histone deacetylase 1 (HDAC1) and NAD-dependent protein deacetylase sirtuin 1 (SIRT1) mRNA in metaphase II stage oocytes that were in vitro matured or collected from peri-ovulatory follicles. The frequency of aneuploidy and anomalies in spindle morphology was increased following IVM, along with a decrease in H4K16 acetylation that was in agreement with our previous observations. However, differences in the amount of the transcripts investigated were not detected. These results suggest that the degradation of transcripts encoding for histone deacetylases and acetyltransferases is not involved in the changes of H4K16 acetylation observed following IVM, while translational or post-translational mechanisms might have a role. Our study also suggests that epigenetic instabilities introduced by IVM may affect the oocyte and embryo genetic stability.

In vitro maturation affects chromosome segregation, spindle morphology and acetylation of lysine 16 on histone H4 in horse oocytes.

Most wild and domestic donkey breeds are currently endangered or threatened. Their preservation includes the creation of a Genome Resource Bank. Embryos cryopreservation allows the preservation of genetics from both male and female and is the fastest method to restore a breed. Because embryo production in vivo is limited in equids, our objective was to establish conditions for in vitro production of embryos in donkey using ovum pick up (OPU), IVM, IVF, and in vitro culture of zygotes. Donkey cumulus-oocyte complexes (COCs) were collected by transvaginal ultrasound-guided aspirations OPU in adult cyclic jennies and in vitro matured in tissue culture medium 199 supplemented with fetal calf serum and epidermal growth factor for 24, 30, 34, or 38 hours. They were preincubated with oviductal fluid for 30 minutes, coincubated with frozen-thawed donkey semen treated with procaine for 18 hours, and cultured for 30 hours in Dulbecco's Modified Eagle Medium-F12 supplemented with NaHCO3, fetal calf serum, and gentamycin. From the five OPU sessions, we collected 92 COCs in 193 follicles (48%) with an average of 4.2 COCs per jenny. All COCs were expanded after more than 24-hour IVM. At collection, jennies oocytes contained a germinal vesicle. Metaphase 1 oocytes were observed after 30-hour IVM and 44% were in metaphase 2 after 34-hour IVM. In our conditions, IVM of donkey oocytes was slower than IVM of equine oocytes and optimal duration for donkey oocytes IVM may be 34 hours. Only 15% of jennies oocytes contained two pronuclei after coincubation with donkey spermatozoa and none of them developed further after 48 hours post-IVF. Moreover, some parthenogenetic activation occurred. Thus, the treatment of donkey sperm with procaine may not be efficient for IVF. In conclusion, we established for the first time conditions for OPU in jennies with high recovery rates. We reported that IVM of jennies oocytes can produce 44% of metaphase 2 oocytes after 34 hours in culture and we described for the first time the chronology of IVM of donkey oocytes. Further studies are in progress to establish efficient conditions for IVF and development of donkey zygotes.

Establishment of conditions for ovum pick up and IVM of jennies oocytes toward the setting up of efficient IVF and in vitro embryos culture procedures in donkey (Equus asinus).

Transvaginal ultrasound-guided follicular punctures are widely used in the mare for diagnosis, research, and commercial applications. The objective of our study was to determine their influence on pain, stress, and well-being in the mare, by evaluating heart rate, breath rate, facial expression changes, and salivary cortisol before, during, and after puncture. For this experiment, 21 pony mares were used. Transvaginal ultrasound-guided aspirations were performed on 11 mares. After injections for sedation, analgesia, and antispasmodia, the follicles from both ovaries were aspirated with a needle introduced through the vagina wall into the ovary. In the control group, 10 mares underwent similar treatments and injections, but no follicular aspiration. Along the session, heart rate and breath rate were evaluated by a trained veterinarian, ears position, eyelid closure, and contraction of facial muscles were evaluated, and salivary samples were taken for evaluation of cortisol concentration. A significant relaxation was observed after sedative injection in the punctured and control mares, according to ear position, eyelid closure, and contraction of facial muscles, but no difference between punctured and control animals was recorded. No significant modification of salivary cortisol concentration during puncture and no difference between punctured and control mares at any time were observed. No significant modification of the breath rate was observed along the procedure for the punctured and the control mares. Heart rate increased significantly but transiently when the needle was introduced in the ovary and was significantly higher at that time for the punctured mares than that for control mares. None of the other investigated parameters were affected at that time, suggesting discomfort is minimal and transient. Improving analgesia, e.g., through a multimodal approach, during that possibly more sensitive step could be recommended. The evaluation of facial expression changes and heart rate is easy-to-use and accurate tools to evaluate pain and well-being of the mare.

Influence of transvaginal ultrasound-guided follicular punctures in the mare on heart rate, respiratory rate, facial expression changes, and salivary cortisol as pain scoring.

Stefan Deleuze

Clinique des Animaux de Compagnie et des Équidés

Université de Liège

Analysis of Chromosome Segregation, Histone Acetylation, and Spindle Morphology in Horse Oocytes

Stefan Deleuze

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Clinique des Animaux de Compagnie et des Équidés

Université de Liège

Analysis of Chromosome Segregation, Histone Acetylation, and Spindle Morphology in Horse Oocytes

Clinique des Animaux de Compagnie et des Équidés