Cumulus Cell Transcripts Transit to the Bovine Oocyte in Preparation for Maturation.

So far, the characteristics of a good quality egg have been elusive, similar to the nature of the physiological, cellular, and molecular cues leading to its production both in vivo and in vitro. Current understanding highlights a strong and complex interdependence between the follicular cells and the gamete. Secreted factors induce cellular responses in the follicular cells, and direct exchange of small molecules from the cumulus cells to the oocyte through gap junctions controls meiotic arrest. Studying the interconnection between the cumulus cells and the oocyte, we previously demonstrated that the somatic cells also contribute transcripts to the gamete. Here, we show that these transcripts can be visualized moving down the transzonal projections (TZPs) to the oocyte, and that a time course analysis revealed progressive RNA accumulation in the TZPs, indicating that RNA transfer occurs before the initiation of meiosis resumption under a timetable fitting with the acquisition of developmental competence. A comparison of the identity of the nascent transcripts trafficking in the TZPs, with those in the oocyte increasing in abundance during maturation, and that are present on the oocyte's polyribosomes, revealed transcripts common to all three fractions, suggesting the use of transferred transcripts for translation. Furthermore, the removal of potential RNA trafficking by stripping the cumulus cells caused a significant reduction in maturation rates, indicating the need for the cumulus cell RNA transfer to the oocyte. These results offer a new perspective to the determinants of oocyte quality and female fertility, as well as provide insight that may eventually be used to improve in vitro maturation conditions.

Thyroid hormones alter the transcriptome of in vitro-produced bovine blastocysts.

Thyroid hormones (THs) have been shown to improve in vitro embryo production in cattle by increasing blastocyst formation rate, and the average cell number of blastocysts and by significantly decreasing apoptosis rate. To better understand those genetic aspects that may underlie enhanced early embryo development in the presence of THs, we characterized the bovine embryonic transcriptome at the blastocyst stage, and examined differential gene expression profiles using a bovine-specific microarray. We found that 1212 genes were differentially expressed in TH-treated embryos when compared with non-treated controls (>1.5-fold at P < 0.05). In addition 23 and eight genes were expressed uniquely in control and treated embryos, respectively. The expression of genes specifically associated with metabolism, mitochondrial function, cell differentiation and development were elevated. However, TH-related genes, including those encoding TH receptors and deiodinases, were not differentially expressed in treated embryos. Furthermore, the over-expression of 52 X-chromosome linked genes in treated embryos suggested a delay or escape from X-inactivation. This study highlights the significant impact of THs on differential gene expression in the early embryo; the identification of TH-responsive genes provides an insight into those regulatory pathways activated during development.

Thyroid hormone concentrations in systemic circulation and ovarian follicular fluid of cows.

The aim of this study was to determine and compare the concentrations of total (T) and free (F) fractions of thyroid hormones (T(3)-triiodithyronine and T(4)-thyroxin) in peripheral circulation and follicular fluid of cows in relation to ovarian follicular status in vivo (Experiment 1), and in the follicles from the slaughterhouse ovaries (Experiment 2). In Experiment 1, estrus was synchronized in 15 cows using two Estrumate (cloprostenol sodium) injections (250 mg cloprostenol intramuscular), the time of ovulation (Day 0) was confirmed by ultrasonography, and ovarian antral follicles were ablated on Day 5. The ensuing superovulatory treatment consisted of eight Folltropin-V injections (50 mg intramuscular) administered twice daily from Day 6 to Day 9, followed by two injections of Estrumate (Day 10 am and pm) and a single dose of Lutropin Alfa (Day 11; 750 IU intramuscular). On Day 5, both TT(3) and FT(3) concentrations were greater (P < 0.05) in serum than follicular fluid from dominant (DFs) or subordinate antral follicles (SFs), and TT(4) concentrations were greater (P < 0.05) in DFs compared with SFs. Serum concentrations of FT(4) were greater (P < 0.05) on Day 12 than on Day 5, and TT(4) concentrations in follicular fluid collected on Day 12 were higher than those in DFs and SFs on Day 5. In Experiment 2, there were no differences (P > 0.05) in thyroid hormone concentrations between the largest and all remaining antral follicles visible on the surface of the ovary (n = 20 ovaries). We concluded that: (i) physiological status of bovine antral follicles (i.e. dominant versus subordinate) may impinge on the accumulation of TT(4) in follicular fluid; and (ii) hormonal ovarian superstimulation increases circulating levels of FT(4) and follicular fluid content of TT(4).

Thyroid hormone supplementation improves bovine embryo development in vitro.

BACKGROUND: Early embryo development (EED) forms the basis of assisted reproductive technologies (ARTs), which are used to treat human infertility and to propagate other mammalian species. Thyroid hormones (THs) play an important role in the post-implantation development of the embryo in mammals; however, the effects of THs on pre-attachment embryos are not known. Currently utilized in-vitro embryo production media are devoid of THs and hence our main objective was to examine whether THs affected EED in a bovine model. METHODS: To determine if THs are present at the site of fertilization and EED in cattle, we evaluated the presence of the hormones in oviductal and uterine horn tissues. To assess the outcome of free TH supplementation (50 ng/ml of each hormone: triiodothyronine-T3 and thyroxin-T4), embryos were followed through standard and TH-supplemented in-vitro procedures, and evaluated for the cleavage rates, blastocyst formation rate and hatching rates. Embryo quality was assessed using TUNEL assay and post-cryopreservation survival was also evaluated. RESULTS: Although TH levels in in-vitro culture media were found to be approximately 60% of the administered doses, the TH-treated embryos exhibited significant increases in blastocyst formation and hatching rates (P < 0.05). Embryo quality was significantly improved in the treated groups as demonstrated by greater total cell counts and reduced proportions of apoptotic cells (P < 0.05). Finally, TH supplementation was associated with improved post-cryopreservation viability, defined by blastocyst re-expansion and hatching rates after frozen embryos had been thawed and cultured (P < 0.05). CONCLUSIONS: These findings not only provide a way of optimizing ART efficiency, but also further our understanding of how THs influence embryonic development in mammals.