Ovarian parameters and fertility of dairy cows selected for one QTL located on BTA3

Recently, one Quantitative Trait Locus (QTL) of female fertility located on Bos Taurus chromosome 3 (BTA3), QTL-F-Fert-BTA3, has been identified in Holstein breed. It is implied in the success rate after the first AI (AI1) in cow. The failure of pregnancy can be due to several factors involved in the different steps of the reproductive process. The aim of our study was to finely phenotype heifers and primiparous cows selected for their haplotype at the QTL-F-Fert-BTA3. We specifically studied the ovarian follicular dynamic and several fertility parameters. Females carrying the favourable haplotype “fertil+” or unfavourable haplotype “fertil−” were monitored by transrectal ultrasonography during their cycle before the first AI (AI1). Follicular dynamic was similar between the two groups. However, the length of the estrus cycle was shorter in heifers than in primiparous cows and two-wave cycles were shorter than three-wave cycles, regardless of the age and the haplotype. The concentration of plasma anti-Müllerian hormone was correlated with the number of small antral follicles. It was higher in heifers than in primiparous cows, independently of their haplotype. The success rate at the AI1 was significantly higher in “fertil+” than in “fertil−” primiparous cows, 35 d after the AI1 (70% vs 39%). In both haplotypes, pregnancy failure occurred mainly before 21 d after AI1. The commencement of luteal activity after calving was significantly earlier in “fertil+” than in “fertil−” primiparous cows. Calving-AI1 and calving-calving intervals were similar between “fertil+” and “fertil−” primiparous cows. Taken together, “fertil+” and “fertil−” primiparous cows present a difference in the success rate after AI1 that is not explained by variations of ovarian dynamics.     

Functions of Fyn kinase in the completion of meiosis in mouse oocytes

Oocyte maturation invokes complex signaling pathways to achieve cytoplasmic and nuclear competencies for fertilization and development. The Src-family kinases FYN, YES and SRC are expressed in mammalian oocytes but their function during oocyte maturation remains an open question. Using chemical inhibitor, siRNA knockdown, and gene deletion strategies the function of Src-family kinases was evaluated in mouse oocytes during maturation under in vivo and in vitro conditions. Suppression of Src-family as a group with SKI606 greatly reduced meiotic cell cycle progression to metaphase-II. Knockdown of FYN kinase expression after injection of FYN siRNA resulted in an approximately 50% reduction in progression to metaphase-II similar to what was observed in oocytes isolated from FYN (−/−) mice matured in vitro. Meiotic cell cycle impairment due to a Fyn kinase deficiency was also evident during oocyte maturation in vivo since ovulated cumulus oocyte complexes collected from FYN (−/−) mice included immature metaphase-I oocytes (18%). Commonalities in meiotic spindle and chromosome alignment defects under these experimental conditions demonstrate a significant role for Fyn kinase activity in meiotic maturation.     

Improvement in in?vitro fertilization outcome following in?vivo synchronization of oocyte maturation in mice

Synchronization of oocyte maturation in vitro has been shown to produce higher in vitro fertilization (IVF) rates than those observed in oocytes matured in vitro without synchronization. However, the increased IVF rates never exceeded those observed in oocytes matured in vivo without synchronization. This study was therefore designed to define the effect of in vivo synchronization of oocyte maturation on IVF rates. Mice were superovulated and orally treated with 7.5 mg cilostazol (CLZ), a phosphodiesterase 3A (PDE3A) inhibitor, to induce ovulation of immature oocytes at different stages depending on frequency and time of administration of CLZ. Mice treated with CLZ ovulated germinal vesicle (GV) or metaphase I (MI) oocytes that underwent maturation in vitro or in vivo (i.e. in the oviduct) followed by IVF. Superovulated control mice ovulated mature oocytes that underwent IVF directly upon collection. Ovulated MI oocytes matured in vitro or in vivo had similar maturation rates but significantly higher IVF rates, 2–4 cell embryos, than those observed in control oocytes. Ovulated GV oocytes matured in vitro showed similar maturation rates but significantly higher IVF rates than those observed in control oocytes. However, ovulated GV oocytes matured in vivo had significantly lower IVF rates than those noted in control oocytes. It is concluded that CLZ is able to synchronize oocyte maturation and improve IVF rates in superovulated mice. CLZ may be capable of showing similar effects in humans, especially since temporal arrest of human oocyte maturation with other PDE3A inhibitors in vitro was found to improve oocyte competence level. The capability of a clinically approved PDE3A inhibitor to improve oocyte fertilization rates in mice at doses extrapolated from human therapeutic doses suggests the potential scenario of the inclusion of CLZ in superovulation programs. This may improve IVF outcomes in infertile patients.     

Mitochondrial distribution and meiotic progression in canine oocytes during in vivo and in vitro maturation

The objective was to evaluate mitochondrial distribution, and its relationship to meiotic development, in canine oocytes during in vitro maturation (IVM) at 48, 72, and 96 h, compared to those that were non-matured or in vivo matured (ovulated). The distribution of active mitochondria during canine oocyte maturation (both in vitro and in vivo) was assessed with fluorescence and confocal microscopy using MitoTracker Red (MT-Red), whereas chromatin configuration was concurrently evaluated with fluorescence microscopy and DAPI staining. During IVM, oocytes exhibited changes in mitochondrial organization, ranging from a fine uniform distribution (pattern A), to increasing clustering spread throughout the cytoplasm (pattern B), and to a more perinuclear and cortical distribution (pattern C). Pattern A was mainly observed in germinal vesicle (GV) oocytes (96.4%), primarily in the non-matured group (P < 0.05). Pattern B was seen in all ovulated oocytes which were fully in second metaphase (MII), whereas in IVM oocytes, ∼64% were pattern B, irrespective of duration of culture or stage of nuclear development (P > 0.05). Pattern C was detected in a minor percentage (P < 0.05) of oocytes (mainly those in first metaphase, MI) cultured for 72 or 96 h. In vitro matured oocytes had a minor percentage of pattern B (P < 0.05) and smaller mitochondrial clusters in IVM oocytes than ovulated oocytes, reaching only 4, 11, and 17% of MII at 48, 72, and 96 h, respectively. Thus, although IVM canine oocytes rearranged mitochondria, which could be related to nuclear maturation, they did not consistently proceed to MII, perhaps due to incomplete IVM, confirming that oocytes matured in vitro were less likely to be competent than those matured in vivo.     

Generation of mouse oocytes defective in cAMP synthesis and degradation: endogenous cyclic AMP is essential for meiotic arrest

Although it is established that cAMP accumulation plays a pivotal role in preventing meiotic resumption in mammalian oocytes, the mechanisms controlling cAMP levels in the female gamete have remained elusive. Both production of cAMP via GPCRs/Gs/adenylyl cyclases endogenous to the oocyte as well as diffusion from the somatic compartment through gap junctions have been implicated in maintaining cAMP at levels that preclude maturation. Here we have used a genetic approach to investigate the different biochemical pathways contributing to cAMP accumulation and maturation in mouse oocytes. Because cAMP hydrolysis is greatly decreased and cAMP accumulates above a threshold, oocytes deficient in PDE3A do not resume meiosis in vitro or in vivo, resulting in complete female infertility. In vitro, inactivation of Gs or downregulation of the GPCR GPR3 causes meiotic resumption in the Pde3a null oocytes. Crossing of Pde3a^−/− mice with Gpr3^−/− mice causes partial recovery of female fertility. Unlike the complete meiotic block of the Pde3a null mice, oocyte maturation is restored in the double knockout, although it occurs prematurely as described for the Gpr3^−/− mouse. The increase in cAMP that follows PDE3A ablation is not detected in double mutant oocytes, confirming that GPR3 functions upstream of PDE3A in the regulation of oocyte cAMP. Metabolic coupling between oocytes and granulosa cells was not affected in follicles from the single or double mutant mice, suggesting that diffusion of cAMP is not prevented. Finally, simultaneous ablation of GPR12, an additional receptor expressed in the oocyte, does not modify the Gpr3^−/− phenotype. Taken together, these findings demonstrate that Gpr3 is epistatic to Pde3a and that fertility as well as meiotic arrest in the PDE3A-deficient oocyte is dependent on the activity of GPR3. These findings also suggest that cAMP diffusion through gap junctions or the activity of additional receptors is not sufficient by itself to maintain the meiotic arrest in the mouse oocyte.     

The role of brain-derived neurotrophic factor in mouse oocyte maturation in vitro involves activation of protein kinase B

Brain-derived neurotrophic factor (BDNF) can promote developmental competence in mammalian oocytes during in vitro maturation, but the signal transduction pathways are not clear. In this study, we investigated (using western blots) the effects of BDNF on the phosphorylation of protein kinase B (PKB) and mitogen-activated protein kinase (MAPK) in mouse oocytes and cumulus cells cultured in vitro. Treatment with BDNF enhanced phosphorylation of PKB in oocytes at 2 h (P = 0.0006) and 3 h (P < 0.0001) of in vitro maturation, compared with control oocytes. However, the pan-specific tyrosine kinase (Trk) inhibitor K252a together with BDNF completely inhibited phosphorylation of PKB in the oocytes. Furthermore, BDNF increased phosphorylation of MAPK in oocytes at 16 h of in vitro maturation (P = 0.0041), but K252a together with BDNF did not reduce phosphorylation of MAPK in the oocytes. For cumulus cells, BDNF significantly prolonged the phosphorylation of PKB and MAPK and increased the total amounts of PKB and MAPK proteins after 16 h of in vitro maturation. However, BDNF did not affect apoptosis of the cumulus cells during oocyte maturation in vitro. In conclusion, the PKB pathway is likely to be one signaling cascade activated by BDNF in combination with the TrkB receptor, whereas the MAPK pathway is not involved. These findings may have relevance for BDNF-induced promotion of developmental capacity of in vitro-matured oocytes.     

Quercetin protects mouse oocytes against chromium-induced damage in vitro and in vivo

BackgroundChromium (Cr) is a naturally-occurring element that is used in various fields of industry. Humans may be exposed to hexavalent chromium [Cr(VI)], which is one of the stable valence states of the chromium through contaminated soil, air, and water. Exposure to Cr(VI) through contaminated drinking water, soil and air causes various cancers and also fertility problems in animals and humans. Quercetin (QCT), a common flavonoid compound, has numerous biological effects as an antioxidant and free radical scavenger, but its function and mechanisms in reproductive processes in various species remain unclear. This study aims to determine the chromium effects on mice oocyte quality and the ameliorative effect of QCT in both in vitro and in vivo experimental models.MethodsFor the in vitro experiment, oocytes were collected and divided into the control, sham, QCT-treated, Cr(VI) (potassium dichromate), and treatment [Cr(VI)+QCT] groups. Collected oocytes were cultured in maturation medium with or without 10 µM quercetin and 10 µM Cr(VI) for 14 h based on the defined experimental design. For the in vivo experiment, the mice were randomly divided into the control, sham, QCT-treated, Cr(VI), and Cr(VI) + QCT groups. Control and sham mice received regular drinking water and diet. Cr(VI) group received Cr(VI) (50 ppm in drinking water) and Cr(VI) + QCT group received 50 ppm Cr(VI) with QCT (20 mg/kg body wt, through i.p) for a period of 21 days and then oocytes were collected and cultured for 14 h for in vitro maturation. For both experiments, at the end of the culture period, we examined the ameliorative effect of QCT on oocyte maturation, spindle formation, ROS production, mitochondrial function, and apoptosis.ResultsOur in vitro and in vivo results showed that Cr(VI) disrupt the oocyte maturation and spindle formation (P < 0.001). Furthermore, we found that exposure to Cr(VI) significantly increased ROS levels and decreased mitochondrial membrane potential (P < 0.001). In addition, exposure to Cr(VI) induced early apoptosis and downregulated the Bcl-2 mRNA expression and upregulated the Caspase-3 and Bax mRNAs expression (P < 0.01). Finally, quercetin significantly restored the detrimental effects of Cr(VI).ConclusionThe results indicated that quercetin protects the oocytes against Cr(VI) toxicity through the suppression of oxidative stress and apoptosis. The conclusions drawn from our study's findings suggest that quercetin might be useful agent for oocyte maturation in case of possible exposure to toxic substances such as chromium.     

Acetylation of H4K12 in porcine oocytes during in vitro aging: potential role of ooplasmic reactive oxygen species

Deterioration in the quality of mammalian mature oocytes during metaphase-II (M-II) arrest is called “oocyte aging”. Although histone acetylation may affect the progression of aging in murine oocytes, the mechanism is unknown. The objective was to determine the role of ooplasmic reactive oxygen species (ROS) in acetylation of histone H4 at lysine 12 (acH4K12) in porcine aged oocytes in vitro. Based on immunostaining with a specific antibody, acetylation of H4K12 in porcine oocytes increased during in vitro aging, which coincided with changing patterns of ooplasmic ROS content. Furthermore, both hydrogen peroxide (H₂O₂), and the mitochondrial membrane potential disrupter, carbonyl cyanide 3-chlorophenylhydrazone (CCCP), which can moderately elevate oocyte ROS content, significantly increased acetylation levels of H4K12 in porcine oocytes. It was noteworthy that acetylation in the CCCP group was decreased when ROS was counteracted by cysteine, a common antioxidant. In addition, the intracellular mRNA abundance of acetyltransferase gene HAT1 in aged and H₂O₂ treated oocytes was higher than in M-II phase oocytes, suggesting that HAT1 was involved in this reaction. After parthenogenetic activation, a lower proportion of oocytes developed to the blastocyst stage after CCCP or H₂O₂ treatment when compared with M-II phase oocytes (20 and 0% for CCCP and H₂O₂ groups, respectively, versus 42% for the M-II group, P < 0.05). In conclusion, elevated levels of H4K12 acetylation were attributed to increased ooplasmic ROS content during porcine oocyte aging in vitro.     

BIM EL-mediated apoptosis in cumulus cells contributes to degenerative changes in aged porcine oocytes via a paracrine action

Whether cumulus cells (CCs) contribute to oocyte aging remains controversial; in that regard, little is known about biochemical processes of gene expression in CCs surrounding aged oocytes. The objective was to elucidate contributions of CCs to porcine oocyte aging and degeneration, apoptosis and BIM expression in CCs during oocyte aging in vitro. When culture of cumulus oocyte complexes (COCs) was prolonged (68 h, which resulted in 24 h of aging), the rate of blastocyst formation following electro-activation was lower than that of oocytes aged without CCs (2.6 ± 0.1 vs 13.5 ± 1.3%, mean ± SEM; P < 0.05). In addition, the presence of CCs significantly accelerated spontaneous fragmentation of oocytes following prolonged (92 h) culture. Apoptotic CCs were present in COCs cultured for 68 h, and the abundance of Bim mRNA in CCs progressively increased after 56 h of culture (P < 0.05). Based on immunofluorescence, BIM protein expression was up-regulated in CCs surrounding aged oocytes; furthermore, quantification (Western blot) of BIM_EL protein progressively increased after 56 h of culture. Lastly, in a series of experiments to elucidate the signal pathway, blocking gap junctions (with 1-octanol) during aging did not eliminate the effect of CCs on accelerating oocyte aging, but prolonged co-culture of denuded oocytes with COCs after in vitro maturation reduced blastocyst rate relative to culture of denuded oocytes aged alone (4.15 ± 0.1 vs 11.0 ± 0.7%, P < 0.05). We concluded that apoptotic CCs, in which BIM_EL up-regulation was involved, accelerated oocyte aging and degeneration in vitro via a paracrine action.     

Ultrasonographic-guided retrieval of cumulus oocyte complexes after super-stimulation in dromedary camel (Camelus dromedarius)

In Experiment 1, studies were conducted to apply the transvaginal ultrasound guided ovum pick-up (OPU) technique in dromedary camels after their ovarian super-stimulation and in vivo oocyte maturation. In Experiment 2, the developmental potential of two commonly used oocyte types, i.e., in vivo matured oocytes collected by OPU and abattoir derived in vitro-matured oocytes was compared after their chemical activation. In Experiment 3, developmental competence of oocytes collected from super-stimulated camels by OPU, matured either in vivo or in vitro, was compared after their chemical activation. Mature female dromedary camels super-stimulated with a combination of eCG and pFSH were given an injection of 20 μg of the GnRH analogue, buserelin 24, 26, or 28 h before the scheduled OPU. For collection of cumulus oocyte complexes (COCs) the transducer was guided through the vulva into the cranial most portion of the vagina and 17-gauge, 55 cm single-lumen needle was placed in the needle guide of the ultrasound probe and advanced through the vaginal fornix and into the follicle. Follicular fluid was aspirated using a regulated vacuum pump into tubes containing embryo-flushing media. Aspirates were searched for COCs using a stereomicroscope, and they were then denuded of cumulus cells by hyaluronidase and repeated pipetting. The oocytes were classified as mature (with a visible polar body), immature (with no visible polar body), activated (with divided or fragmented ooplasm) and others (degenerated and abnormal).Overall an average of 12.12 ± 7.9 COCs were aspirated per animal with an oocyte recovery rate from the aspirated follicles of about 77%. The majority (> 90%) of the collected COCs by OPU were with loose and expanded cumulus cells. The proportion of matured oocytes obtained at 28-29 h (91.2 ± 4.1) and 26-27 h (82.1 ± 3.4) were higher (P < 0.005) when compared with those obtained at 24-25 h (40.4 ± 16.3) after GnRH administration. In Experiment 2, a higher proportion (P < 0.05) of in vivo matured oocytes cleaved (84.6 ± 2.1 vs. 60.9 ± 6.6) and developed to blastocyst stages (52.4 ± 4.1 vs. 30.5 ± 3.3) when compared with in vitro matured oocytes collected from slaughterhouse ovaries. In Experiment 3, no difference was observed between the developmental competences of oocytes, collected from super stimulated camels, matured in vitro with those collected after their in vivo maturation.In conclusion, about 80-90% mature oocytes can be collected by ultrasound guided transvaginal ovum pick-up from super-stimulated dromedary camels 26-28 h after GnRH administration. The developmental response, to chemical activation, of in vivo matured oocytes collected by ultrasound guided transvaginal OPU is better than in vitro matured oocytes obtained from slaughterhouse ovaries. However, no difference was observed in the developmental competence of oocytes collected by OPU whether they were matured in vivo or in vitro.     

Advancing maternal age predisposes to mitochondrial damage and loss during maturation of equine oocytes in vitro

In many mammalian species, reproductive success decreases with maternal age. One proposed contributor to this age-related decrease in fertility is a reduction in the quantity or functionality of mitochondria in oocytes. This study examined whether maternal age or (in vitro maturation). IVM affect the quantity of mitochondria in equine oocytes. Oocytes were collected from the ovaries of slaughtered mares categorized as young (<12 years) or aged (≥12 years) and either denuded and prepared for analysis immediately (not-IVM) or matured in vitro for 30 hours before preparation (IVM). The mean oocyte mitochondrial DNA copy number was estimated by quantitative polymerase chain reaction and found to be significantly lower in oocytes from aged mares and that had been subjected to IVM than in any other group. Transmission electron microscopy demonstrated that mitochondria in aged mare oocytes subjected to IVM experienced significantly more swelling and loss of cristae than in other groups. We conclude that maternal aging is associated with a heightened susceptibility to mitochondrial damage and loss in equine oocytes, which manifests during IVM. This predisposition to mitochondrial degeneration probably contributes to reduced fertility in aged mares.     

Maternal obesity enhances oocyte chromosome abnormalities associated with aging

Obesity is increasing globally, and maternal obesity has adverse effects on pregnancy outcomes and the long-term health of offspring. Maternal obesity has been associated with pregnancy failure through impaired oogenesis and embryogenesis. However, whether maternal obesity causes chromosome abnormalities in oocytes has remained unclear. Here we show that chromosome abnormalities are increased in the oocytes of obese mice fed a high-fat diet and identify weakened sister-chromatid cohesion as the likely cause. Numbers of full-grown follicles retrieved from obese mice were the same as controls and the efficiency of in vitro oocyte maturation remained high. However, chromosome abnormalities presenting in both metaphase-I and metaphase-II were elevated, most prominently the premature separation of sister chromatids. Weakened sister-chromatid cohesion in oocytes from obese mice was manifested both as the terminalization of chiasmata in metaphase-I and as increased separation of sister centromeres in metaphase II. Obesity-associated abnormalities were elevated in older mice implying that maternal obesity exacerbates the deterioration of cohesion seen with advancing age.

     

Effect of blood plasma collected after adrenocorticotropic hormone administration during the preovulatory period in the sow on oocyte in vitro maturation

Reproduction may be affected by stressful events changing the female endocrine or metabolic profile. An altered environment during oocyte development could influence the delicate process of oocyte maturation. Here, the effect of simulated stress by media supplementation with blood plasma from sows after adrenocorticotropic hormone (ACTH) administration during the preovulatory period was assessed. Oocytes were matured for 46 hours in the presence of plasma from ACTH-treated sows, or plasma from NaCl-treated control sows, or medium without plasma (BSA group). The plasma used had been collected at 36 and 12 hours (±2 hours) before ovulation (for the first 24 hours + last 22 hours of maturation, respectively). Subsequent fertilization and embryo development were evaluated. Actin cytoskeleton and mitochondrial patterns were studied by confocal microscopy both in the oocytes and the resulting blastocysts. Nuclear maturation did not differ between treatments. Subtle differences were observed in the actin microfilaments in oocytes; however, mitochondrial patterns were associated with the treatment (P < 0.001). These differences in mitochondrial patterns were not reflected by in vitro outcomes, which were similar in all groups. In conclusion, an altered hormonal environment provided by a brief exposure to plasma from ACTH-treated sows during in vitro oocyte maturation could induce alterations in actin cytoskeleton and mitochondrial patterns in oocytes. However, these changes might not hamper the subsequent in vitro embryo development.     

Molecular differentiation of the rabbit ovum. I. During oocyte maturation in vivo and in vitro

The normality of nuclear and cytoplasmic maturation of rabbit oocytes, matured in vivo and in vitro, has been assessed by cytogenetic and electrophoretic criteria. The findings indicate not only that nuclear maturation in vivo and in vitro are directly comparable, but also, as observed by high-resolution, two-dimensional polyacrylamide gel electrophoresis, (1) that both qualitative and quantitative changes in the pattern of polypeptide synthesis occur during maturation, (2) that these patterns are directly comparable in oocytes that had been matured either in vivo or in vitro, and (3) that each stage of maturation is associated with the appearance of specific polypeptides in the autoradiographic patterns. The major differences observed between oocytes matured under these two conditions are (1) that several polypeptides fail to appear in in vitro matured oocytes at the time they are detected in vivo and (2) that the synthesis of some polypeptides is prolonged in vitro compared to in vivo matured oocytes.