The landscape of accessible chromatin in bovine oocytes and early embryos.

Chromatin reorganization governs the regulation of gene expression during preimplantation development. However, the landscape of chromatin dynamics in this period has not been explored in bovine. In this study, we constructed a genome-wide map of accessible chromatin in bovine oocytes and early embryos using an improved assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) which revealed unique features of the accessible chromatin during bovine early embryo development. We found that chromatin accessibility is low in oocytes and 2-/4-cell embryos, followed by a significant increase in embryos during major embryonic genome activation (EGA), and peaked in elongating day 14 embryos. Genome-wide characteristics of open chromatin showed that ATAC-seq signals in both transcription start sites (TSS) and transcription end sites (TES) were strong. Additionally, the distal ATAC-seq peaks were enriched in repeat elements in a type-specific and stage-specific manner. We further unveiled a series of transcription factor (TF) motifs with distinct variation of enrichment from distal ATAC-seq peaks. By integrated analysis of chromatin accessibility with transcriptomes and DNA methylomes in bovine early embryos, we showed that promoter accessibility was positively correlated with gene expression, especially during major EGA, and was strongly correlated to DNA methylation and CpG density. Finally, we identified the critical chromatin signatures and TFs that differ between in vivo and in vitro derived blastocysts, which provides insights to the potential mechanisms leading to low quality of embryos produced in vitro. Together, this comprehensive analysis revealed critical features of chromatin landscape and epigenetic reprogramming during bovine preimplantation embryo development.

Impact of the corpus luteum on survival of the developing embryo and early pregnancy in mares.

It has been more than a hundred years that studies aiming to elucidate the processes involved in cyclicity and pregnancy pointed out the requirement of ovaries and corpora lutea for embryo survival and pregnancy establishment. For horses, luteal progesterone is essential for pregnancy only during the first trimester. This progestational support is complex among domestic animals as ovarian luteal function is further enhanced by the LH-action role of equine chorionic gonadotropin (eCG) starting âˆ¼ on Day 35 of pregnancy. Increased eCG secretion leads to the formation of supplementary corpora lutea resulting from follicles that luteinize (accessory corpora lutea) or ovulate (secondary corpora lutea), thus increasing concentrations of blood progesterone. Physiological details of progesterone-driven embryo-maternal interactions continue to be elucidated. In recent years, researchers studying the transcriptomes and secretomes of uterine tubes, endometrium and early embryo provided insight into the composition of molecular and cellular events that enable embryo survival and remodeling of the endometrium before a functional placenta is formed. Aluteal pregnancy models have also shown that while fertilization and early embryo development until the early blastocyst stage can occur under a progesterone-deprived environment, dysregulation of important pregnancy-related genes occur; embryo development is compromised unless progestin supplementation is provided once the embryo arrives into the uterus. As the body of knowledge on embryo-maternal interactions in the horse continues to grow, a fact remains true: luteal support is essential for embryo survival mainly at the uterine stage, driving directly or indirectly gene expression that promotes adequate embryo-maternal physiological interactions until a full competent placenta is formed, resulting in optimal chances of delivering a live foal at term.

Effects of Combined Estradiol-Sulpiride Treatment and Follicle Ablation on Vernal Transition in Mares: Evaluation of Plasma and Follicular Fluid Hormones and Luteinizing Hormone Receptor Gene Expression.

This experiment assessed the hormonal production, secretory aspects, and changes in luteinizing hormone (LH) receptor gene expression of early induced ovulatory-sized follicles relative to the first ovulatory-sized follicles occurring naturally in the spring. Anovulatory mares were treated on January 21 with (1) 50 mg of estradiol cypionate (ECP, n = 8) alone or (2) with ECP followed by two 3-g sulpiride injections (n = 8), 5 and 12 days later. Half of each group also received complete follicle ablation via transvaginal aspiration before ECP treatment. Ovaries were scanned regularly until detection of a 32-35 mm follicle; follicular fluid was recovered via aspiration for analysis of hormonal concentrations. Blood was collected regularly to characterize plasma prolactin, LH, follicle stimulating hormone, progesterone, and estradiol concentrations. Mean date to first 35-mm follicle was earlier (P < .05) in sulpiride-treated mares: five of eight (63%) responded within 28 days of the first sulpiride injection. Ablation did not affect ovarian response. Plasma prolactin was stimulated (P < .0001) in ECP-sulpiride-treated mares for 16 days but did not dictate ovarian response. Estradiol stimulated plasma LH (P < .05), which was higher (P < .05) in treated mares that responded. There was no effect of treatment or ablation on follicular fluid concentrations of estradiol, progesterone, leptin, or insulin-like growth factor 1 or on LH receptor gene expression. These latter similarities indicate that ECP-sulpiride early induced follicles have apparently reached a degree of maturity equivalent to naturally occurring ovulatory-sized follicles later in the spring.

Bilateral occurrence of granulosa-theca cell tumors in an Arabian mare.

An Arabian mare was referred for right granulosa-theca cell tumor (GTCT) evaluation. The mare was presented 4.5 years later for a left GTCT, after successfully conceiving and delivering a normal foal in the interim. The concurrent or nonconcurrent occurrence of bilateral GTCT in mares appears to be rare.

Long-term efficacy following readministration of an adeno-associated virus vector in dogs with glycogen storage disease type Ia.

Glycogen storage disease type Ia (GSD-Ia) is the inherited deficiency of glucose-6-phosphatase (G6Pase), primarily found in liver and kidney, which causes life-threatening hypoglycemia. Dogs with GSD-Ia were treated with double-stranded adeno-associated virus (AAV) vectors encoding human G6Pase. Administration of an AAV9 pseudotyped (AAV2/9) vector to seven consecutive GSD-Ia neonates prevented hypoglycemia during fasting for up to 8 hr; however, efficacy eventually waned between 2 and 30 months of age, and readministration of a new pseudotype was eventually required to maintain control of hypoglycemia. Three of these dogs succumbed to acute hypoglycemia between 7 and 9 weeks of age; however, this demise could have been prevented by earlier readministration an AAV vector, as demonstrated by successful prevention of mortality of three dogs treated earlier in life. Over the course of this study, six out of nine dogs survived after readministration of an AAV vector. Of these, each dog required readministration on average every 9 months. However, two were not retreated until >34 months of age, while one with preexisting antibodies was re-treated three times in 10 months. Glycogen content was normalized in the liver following vector administration, and G6Pase activity was increased in the liver of vector-treated dogs in comparison with GSD-Ia dogs that received only with dietary treatment. G6Pase activity reached approximately 40% of normal in two female dogs following AAV2/9 vector administration. Elevated aspartate transaminase in absence of inflammation indicated that hepatocellular turnover in the liver might drive the loss of vector genomes. Survival was prolonged for up to 60 months in dogs treated by readministration, and all dogs treated by readministration continue to thrive despite the demonstrated risk for recurrent hypoglycemia and mortality from waning efficacy of the AAV2/9 vector. These preclinical data support the further translation of AAV vector-mediated gene therapy in GSD-Ia.