Stem cell-conditioned medium improves methylation patterns and quality of caprine preantral follicles.

This study investigated the methylation patterns of H3K4me3 and H3K9me3, as well as the mRNA expression of genes encoding the epigenetic regulators KDM1AX1, KDM1AX2, and KDM3A in goat preantral follicles developed in vivo (Uncultured control) or after in vitro culture for 7 days in either the absence (α-MEM+) or presence of conditioned medium (α-MEM+ + CM) from Wharton's jelly mesenchymal stem cells (WJ-MSCs). In the in vivo setting, all follicular categories exhibited similar H3K4me3 and H3K9me3 patterns, and transcripts of KDM1AX1, KDM1AX2, and KDM3A were detected in all samples. During in vitro culture, α-MEM+ + CM enhanced several important aspects. It increased the percentage of normal growing follicles, oocyte diameters across all categories, stromal cell density, and the H3K4me3 methylation pattern in preantral follicles. Simultaneously, it decreased the levels of reduced thiols and reactive oxygen species in the spent media, diminished the presence of lipofuscin aggresomes, lowered granulosa cell apoptotic rates, and reduced the H3K9me3 methylation pattern in preantral follicles. In conclusion, the findings from this study provide compelling evidence that supplementing the in vitro culture medium (α-MEM+) with CM from WJ-MSCs has a protective effect on goat preantral follicles. Notably, CM supplementation preserved follicular survival, as evidenced by enhanced follicular and oocyte growth and increased stromal cell density when compared to the standard culture conditions in the α-MEM+ medium. Furthermore, CM reduced oxidative stress and apoptosis and promoted alterations in H3K4me3 and H3K9me3 patterns.

Equine ovarian tissue xenografting: impacts of cooling, vitrification, and VEGF.

Ovarian tissue transplantation methods using cooled and cryopreserved samples have been attractive options for fertility preservation in animal models and humans. The aim of this study was to evaluate the impact of previous exposure to cooling, cryopreservation, and VEGF on the overall efficiency of equine ovarian tissue after heterotopic xenotransplantation in mice. The end points evaluated were follicular morphology and development, follicular and stromal cell densities, angiogenesis (i.e. the density of new and mature blood vessels), collagen types I and III fiber densities, and total fibrosis. Ovaries of adult mares were harvested after ovariectomy, and ovarian fragments were xenografted in the i.p. wall of BALB nude mice. Ten types of treatments involving different combinations of cooling, cryopreservation, xenografting procedures, and VEGF exposure were compared. The novel aspect of this study was the use of equine ovarian tissue xenotransplantation in mice, challenging the fragments with different combinations of treatments. The main findings were (i) cooling but not cryopreservation was effective in preserving the follicular morphology, (ii) a greater percentage of developing follicles but lower follicular and stromal cell densities were observed after ovarian tissue engraftment, (iii) exposure to VEGF increased new and mature vessels in cryopreserved-transplanted tissue, and (iv) an appropriate balance in the collagen types I and III fiber ratio in cooling-transplanted tissue was observed after exposure to VEGF. This study contributes to advancing knowledge in the preservation of ovarian tissue after cooling-cryopreservation and transplantation aiming to be applied to genetically superior/valuable horses, livestock, endangered animals, and, possibly, humans. LAY SUMMARY: Due to ethical limitations involving humans, the female horse (mare) has recently emerged as an alternative model for reproductive comparisons with women to optimize fertility restoration using ovarian tissue transplantation techniques. This study determined if ovarian tissue from donor mares (n = 3), exposed or not to vascular endothelial growth factor (VEGF) before transplantation, better survives for 7 days after transplantation into mouse hosts (n = 12). Tissues submitted to different combinations of cooling, freezing, and transplanting treatments, along with control groups, were evaluated using the parameters morphology, development, the density of immature eggs (follicles), the density of supportive (stromal) cells, collagen protein proportions, and density of blood vessels. Frozen-thawed treatments had lower percentages of normal follicles. Exposure to VEGF increased blood vessel densities in frozen tissue and favored adequate collagen levels in cooled-transplanted treatments. In conclusion, VEGF exposure seems to be beneficial for mare ovarian tissue transplantation and warrants further investigation.

Oocyte maturation with royal jelly increases embryo development and reduces apoptosis in goats.

Royal jelly (RJ) was supplemented to goat oocyte in vitro maturation (IVM) medium at three different concentrations (2.5, 5, and 10 mg/ml). Maturation rate, embryo cleavage, and blastocyst rate were recorded. Gene expression of apoptosis-related transcripts was investigated in matured oocytes. Percentage of oocytes that reached MII-stage was increased in RJ-treated groups compared to the control group. Glutathione (GSH) content of mature oocytes was enhanced when RJ was added to IVM medium at any supplementation compared with control. Percentage of cleaved embryos and blastocysts was higher in the RJ-treated groups at a concentration of 5 than in the 2.5 mg/ml and control group. Total number of cells per blastocyst was not different in the control and RJ-treated group at 5 mg/ml. However, number of apoptotic cells per blastocyst was higher in the control group than in the RJ-treated group at 5 mg/ml. Expression profile of Bax, and p53 was down-regulated while Bcl-2 was up-regulated in oocytes treated with RJ at 5 and 10 mg/ml compared with the control group. Addition of RJ at concentrations of 5 mg/ml improved embryo production through increasing maturation rate. RJ seems to improve the IVM microenvironment by reducing expression of genes inducing apoptosis, enhancing GSH content, and reducing incidence of apoptosis in blastocysts.

Organogenesis of the Musculoskeletal System in Horse Embryos and Early Fetuses.

Musculoskeletal system development involves heterotypical inductive interactions between tendons, muscles, and cartilage and knowledge on organogenesis is required for clarification of its function. The aim of this study was to describe the organogenesis of horse musculoskeletal system between 21 and 105 days of gestation, using detailed macroscopic and histological analyses focusing on essential developmental steps. At day 21 of gestation the skin was translucid, but epithelial condensation and fibrocartilaginous tissues were observed on day 25 of pregnancy. Smooth muscle was seen in lymphatic and blood vessel walls and the beginning of cartilaginous chondrocranium was detected at day 30 of gestation. At day 45, typical chondroblasts and chondrocytes were observed and at day 55, mandibular processes expanded toward the ventral midline of the pharynx. At day 75, muscles became thicker and muscle fibers were seen developing in carpal and metacarpal joints with the beginning of the ossification process. At day 105, major muscle groups, similar to those seen in an adult equine, were observed. The caudal area of the nasal capsule and trabecular cartilages increased in size and became ossified, developing into the ethmoid bone. The presence of nasal, frontal, parietal, and occipital bones was observed. In conclusion, novel features of equine musculoskeletal system development have been described here and each process was linked with an early musculoskeletal event. Data presented herein will facilitate a better understanding of the equine muscular system organogenesis and aid in the detection of congenital deformities. Anat Rec, 299:722-729, 2016. © 2016 Wiley Periodicals, Inc.