Cyclin-dependent kinase 6 (CDK6) as a potent regulator of the ovarian primordial-to-primary follicle transition.

Introduction: Ovarian follicle development requires tight coordination between several factors to initiate folliculogenesis to generate a mature and fertile egg. Studies have shown that cell cycle factors might contribute to follicle development, hover specific knowledge on individual CDKs and follicle activation has not been investigated. Among cell cycle regulators, CDK6 is a key player through binding to cyclin D resulting DNA synthesis and genome duplication. Interestingly, the CDK6 gene is differentially expressed in oocytes and granulosa cells from human primordial and primary follicles, which suggest a potential role of CDK6 in the primordial-to-primary transition. In this study, we investigated the potential regulatory role of CDK6 in progression of primordial to primary follicle transition using BSJ-03-123 (BSJ), a CDK6-specific degrader. Methods: In mouse ovarian in vitro culture, BSJ reduced the activation of primordial follicles, and reduced follicle development. As a next step, we examined the egg maturation read-out and found that BSJ-treated follicles matured to competent MII eggs with resumption of first meiosis, comparable with the control group. Results: Noteworthy, it appears that inhibition of CDK6 did increase number of apotoptic cells, articular in the granulosa cells, but had no impact on ROS level of cultured ovaries compared to control group, indicating that the cells were not stressed. Oocyte quality thus appeared safe. Discussion: The results of this study indicate that CDK6 plays a role in the primordial-to-primary transition, suggesting that cell cycle regulation is an essential part of ovarian follicle development.

Lecithin nanoparticles enhance the cryosurvival of caprine sperm.

This study was designed to compare the effects on goat spermatozoa cryosurvival of nano-lecithin-based (NL), lecithin-based (L) and egg yolk-based (EY) extenders. Ejaculates were collected from four fertile goats using artificial vagina and diluted with nine extenders. NL and L were tested at concentrations 1%, 2%, 3% and 4% (w/v), versus 15% (v/v) egg yolk-based extender. Overall, sperm quality (higher motility, viability and HOST, and lower apoptosis) was higher for NL than for L treatments (P < 0.05 for most cases, except for 1%). NL at 1% and especially at 4% showed lower motility and viability than 2% or 3% NL. NL at 2% achieved a better performance (P < 0.05) than EY for VCL (131.5 ±â€¯1.3 vs. 120.3 ±â€¯1.9 µm/s), VSL (43.9 ±â€¯1.5 vs. 35.8 ±â€¯1.4 µm/s), LIN (35.7 ±â€¯0.6 vs. 29.3 ±â€¯0.8%), WOB (47.0 ±â€¯0.5 vs. 43.9 ±â€¯0.9%) and viability (66.4 ±â€¯1.7 vs. 52.7 ±â€¯1.9%). Late apoptotic spermatozoa were also lower in 2% NL compared to EY (16.0 ±â€¯0.5 vs. 26.3 ±â€¯1.1%, P < 0.001). EY and 2% NL were compared in an IVF trial, with no significant differences in cleavage (68.8 vs. 70.8%) or blastocyst ratios (21.3 vs. 20.8%). In conclusion, using 2% nanolecithin in semen dilution could improve sperm cryosurvival of goat.

Metabolic and molecular responses to calcium soap of fish oil fed to ewes during peripartal period.

It has been shown that n-3 long chain fatty acids (n-3 LCFA) are involved in energy/lipid metabolisms, reproductive parameters, and molecular regulations leading to maintained homeostasis. We hypothesized that supplementation of peripartal diets with fish oil (FO), as a source of n-3 LCFA, could improve energy balance and modulate metabolic pressure in a sheep model. Prepartum ewes (n = 24) were fed control (CON) or calcium soap of fish oil (FO) supplemented-diet from four weeks before until three weeks after parturation. Feed intake, body weight (BW) change, plasma metabolites, colostrums/milk composition, and fatty acids profile of milk along with the expression of core microRNAs in glucose and lipid metabolism were evaluated. Prepartal feed intake decreased in FO group (1674 ± 33.26 vs. 1812 ± 35.56) though post-partal intake was similar. Differences in BW were not also significant (55.47 ± 2.07 in CON vs. 53.69 ± 1.94 in FO). No differences were observed in plasma metabolites except for cholesterol that was lower in FO group (56.25 ± 0.71 vs. 53.09 ± 0.61). Milk fat percentage was reduced (8.82 ± 0.49 vs. 7.03 ± 0.45) while the percentage of milk total n-3 LCFA increased in FO group. In accordance, the relative transcript abundance of miR-101 (0.215 ± 0.08) and miR-103 (0.37 ± 0.15) decreased by FO supplementation. Results showed that FO supplementation during peripartal period decreased milk fat, feed intake, plasma cholesterol, milk n-6:n-3 ratio and the expression of miR-101. Although the trend indicated that FO could alter lipid metabolism during transition period, further studies are needed to fully address its effect on energy balance and homeorhetic processes.

H2O2-induced mild stress in relation with in vitro ovine oocyte developmental competence: implications for blastocyst apoptosis and related genes expression.

In this study, in vitro maturation was performed in presence of various concentrations (0, 10, 100, or 1000 µM) of H2O2. The intracellular glutathione (GSH) level, fertilization, cleavage, and blastocyst rates, total cell number, and apoptotic cell number and expression of Bax, Bcl-2, and p53 genes in blastocyst-stage embryos were studied. At 10 µM H2O2 concentration, a higher GSH level was detected in comparison to the other groups while oocytes exposed to 1000 µM H2O2 had the lowest GSH level. Treatment of oocytes with 1000 µM H2O2 decreased the rate of two pronuclei formation as compared with other groups. A higher rate of blastocyst formation was seen in 100 µM H2O2 group as compared with the control group. However, exogenous H2O2 in maturation medium did not affect total cell numbers and apoptotic cell ratio at the blastocyst stage. Moreover, mRNA transcript abundance of Bax, Bcl-2, and p53 genes was similar between blastocysts derived from H2O2-induced oocytes and control blastocysts. Treatment of oocytes with H2O2 at mild level during in vitro maturation had a positive effect on GSH level and this, in turn, may lead to improvement in preimplantation embryonic development.

Comparative effects of zinc-nano complexes, zinc-sulphate and zinc-methionine on performance in broiler chickens.

Micronutrients, especially zinc, have an important role in normal metabolism and growth of broilers. Using novel technologies helps to synthesise novel zinc complexes to deliver this micronutrient more efficiently. In the present study, the effects of different zinc complexes and nano complexes on broiler performance were compared. Broilers in 6 groups were given basal diet (without zinc) and basal diet supplemented with zinc-sulphate, zinc-methionine, zinc-nano-sulphate, zinc-nano-methionine and zinc-nano-max (that was synthesised based on nanochelating technology) at a concentration of 80 mg/kg of diet. At 1-42 d of age, dietary zinc-nano-sulphate supplementation decreased weight gain and feed intake. However, feed conversion ratio was not influenced by treatments. Carcass yield (%) of birds in the zinc-nano-sulphate and control groups were dramatically reduced at 42 d of age and abdominal fat (%) increased in these groups. Relative to the control group, the antibody titre, spleen and bursa of Fabricius (%) were significantly higher in groups supplemented with zinc. Heterophil (%) was also significantly higher in the zinc-nano-methionine group in blood on d 42 compared to the control, zinc-sulphate and zinc-nano-sulphate. Compared to the controls, the mean malondialdehyde content in thigh tissue was significantly reduced in groups supplemented with zinc at the time 0, 50, 100 and 150 min after oxidation. Tibia zinc concentration in nanoparticle zinc samples was significantly higher relative to the control and zinc-sulphate groups. Taken together, our data indicate that delivery of zinc in the structure of zinc-nano-methionine and zinc-nano-max at concentrations of 80 mg/kg of diet improves growth performance. However, dietary zinc-nano-sulphate decreased growth performance in broilers.

Pluripotency properties of embryonic stem cells isolated from stage X blastoderm of Mazandaran native chicken.

The aim of this study was to isolate Embryonic Stem Cells (ESCs) from native chicken and to characterize their pluripotency properties through the cellular and molecular markers. Samples obtained from fertilized eggs from Mazandaran native hens. Cells were isolated from area of pellucida from stage X native hens' blastoderm. Then the cells were cultured on inactivated mouse SNL feeder cells in the presence of LIF, IGF-1, bFGF, CNTF, OSM, SCF, Il-6, and Il-11 growth factors. The native chickens' ESCs colonies were picked up and subsequently passaged. To characterize the cells, they were analyzed for their alkaline phosphatase activity, and also for the expression of SSEA-4, and TRA-1-60 as embryonic-specific markers at the protein level. Furthermore, the expression of pluripotency (cPouV, Sox2, and Nanog) and cell lineage specific (Cvh, Brachyury, and Gata6) gene markers was evaluated at the level of mRNA using quantitative RT-PCR. Isolated cells were passaged repeatedly and successfully up to ten passages. The stemness of embryonic cells has been approved by the activity of the alkaline phosphatase, presence of the SSEA-4, and TRA-1-60 protein, and expression of the molecular marker (cPouV, Nanog, and Sox-2) genes. The spontaneous differentiation of chicken ESCs confirmed the pluripotecy of the cells in differentiation into specialized cell lineages. Our observation showed that ESCs can be isolated successfully from stage X blastoderm of Mazandaran native chickens and these cells maintain their stemness properties during multi-passages in vitro.

Expression of microRNA and microRNA processing machinery genes during early quail (Coturnix japonica) embryo development.

MicroRNA (miRNA) are small regulatory RNA molecules that are implicated in regulating and controlling a wide range of physiological processes including cell division, differentiation, migration, apoptosis, morphogenesis, and organogenesis. The aim of this study was to determine the expression pattern of 32 miRNA and 18 miRNA processing machinery genes during somite formation in quail embryos. The embryos were collected at stages HH (Hamburger and Hamilton) 4, 6, and 9 of embryo development (19, 24, and 30 h of incubation, respectively). Total RNA including miRNA was isolated from 4 groups of embryos (each group consisting of 6 to 8 embryos) were collected at each of the 3 stages (19, 24, and 30 h). The expression pattern of candidate miRNA and miRNA processing machinery genes was performed using quantitative real-time PCR. The results demonstrated that 7 miRNA (let-7a, mir-122, mir-125b, mir-10b, P < 0.01; let-7b, mir-26a, and mir-126, P < 0.05) were differentially expressed during early quail embryo development. In addition, the expression profile of 18 miRNA processing machinery genes was not significantly increased at 30 h of incubation compared with both 19 and 24 h. Our results suggest that machinery genes for miRNA biogenetic pathways are functional, and hence, miRNA may be involved in the regulation of early quail development. These 7 differentially expressed miRNA are suggested to play critical roles in quail embryo somite formation.

G6PDH-activity in equine oocytes correlates with morphology, expression of candidate genes for viability, and preimplantative in vitro development.

Efficiencies for in vitro production of equine embryos are still low due to highly variable developmental competences of equine immature oocytes. In contrast to the equine, in vitro developmental competence of immature oocytes has been predicted successfully by the activity of glucose-6-phosphate dehydrogenase (G6PDH) indicated by brilliant cresyl blue (BCB) dye in a range of different species. Therefore, the aim of the present study was to test the association between G6PDH activity in equine oocytes with: (1) cumulus morphology and oocyte properties in terms of diameter and volume; (2) maturational competence; (3) gene expression of certain molecular markers; and (4) in vitro embryo development after intracytoplasmic sperm injection. Equine oocytes were exposed to BCB stain and were classified as BCB+ or BCB- according to their ability to convert the dye from blue to colorless. Additionally, BCB+ and BCB- oocytes were subclassified as having a compact (Cp) or expanded (Ex) cumulus complex. As a result, BCB+ oocytes had a greater proportion of expanded cumulus oocyte complexes compared with BCB- oocytes (71.2% vs. 49.5%). Moreover, we observed a significant difference in oocyte diameter and volume between BCB+ and BCB- oocytes irrespective of cumulus morphology. BCB+ oocytes reached a higher maturation rate compared with BCB- oocytes (59.0% vs. 28.7%). Regarding the analyzed candidate genes, relative transcript abundance was significantly different for nine genes. The expression of eight genes was significantly higher (P < 0.05) for BCB+ oocytes, including ATPV6E, IF-3, TFAM, DNMT1, STAT3, Aurora-A, ODC1, and CKS2 whereas BCB- oocytes showed higher in expression of COX1. These results are in line with the observed developmental competence. Cleavage rate (45.9% vs. 29.0%) and percentage of embryos that reached the blastocyst stage (9.2% vs. 1.4%) were significantly higher for embryos derived from BCB+ oocytes compared with BCB- oocytes. In conclusion, the present study provides evidence that G6PDH-activity in immature equine oocytes is a useful predictor for subsequent in vitro developmental competence.

Evaluation of bovine zona pellucida characteristics in polarized light as a prognostic marker for embryonic developmental potential.

It has previously been demonstrated that zona pellucida imaging of human oocytes using polarized light microscopy is a clinically applicable method for the noninvasive assessment of oocyte quality. This study was designed to investigate whether zona pellucida characteristics of bovine oocytes and zygotes in polarized light may similarly serve as a useful marker for developmental competence in bovine reproductive biotechnologies. Zona birefringence intensity parameters of 2862 oocytes/zygotes were objectively evaluated with an automatic analysis system and correlated with oocyte/zygote quality. In detail, immature oocytes of good quality assessed with brilliant cresyl blue staining showed significantly lower zona birefringence than poor-quality counterparts (P<0.001). After in vitro maturation and classification according to maturational status, the birefringence intensity parameters were significantly different in those oocytes that reached metaphase II compared with arrested stages (P<0.001). Following either parthenogenetic activation or IVF with subsequent in vitro culture in a well-of-the-well system until day 9, superior development as determined by cleavage, blastocyst formation, and hatching ability was associated with lower zona birefringence intensity parameters. When early zygote-stage embryos were selected and assorted in groups based on zona birefringence (high/medium/low), the group of embryos derived from high-birefringence zygotes displayed a significantly compromised developmental potential compared with low-birefringence zygotes. These results clearly show that developmentally competent bovine oocytes/zygotes exhibit lower zona birefringence intensity parameters. Therefore, birefringence imaging of zona pellucida is a suitable technique to predict bovine preimplantation embryo development.