Reproduction and Lactation
|The in vitro embryo production (IVEP) is an Assisted Reproduction Technology aimed to increase the reproductive potential of selected females by using the pool of immature oocytes found in the ovary. Our studies are focussed in the study of oocyte competence, testing different in vitro culture media, assessing molecular cytoplasmic parameters and searching for biomarkers of oocyte quality. The last goal is to obtain good quality embryos produced in the laboratory and suitable to be transferred to receptors females.
María Teresa Paramino Nieto
Maria Dolors Izquierdo Tugas
Effect of the caprine oocyte competences on in vitro embryo production in biphasic maturation system with OSF (Oocytes Secreted Factors) (Reference: PID2020-113266RB-I00)
Goats, especially dairy breeds, could be an interesting animal for food production in difficult and dry environmental conditions.
In vitro embryo production (PIVE) is a tool of great interest for animal genetic improvement programs. In previous studies, we have observed a direct and positive relationship between oocyte diameter, follicle diameter and its development to blastocyst. In our previous project (AGL2017-85837-R) we have studied a biphasic maturation system based on 2 steps: 1) pre-in vitro maturation (IVM) medium with 200 nM CNP (C-type Natriuretic Peptides) plus Estradiol in order to stop meiosis and 2) conventional IVM medium. With this biphasic system, we have significantly increased blastocyst development of oocytes from juvenile goats (1 to 2 months old) (from 17% to 30% blastocysts). In this project we are going to study the response of oocytes of different qualities (oocytes from adult and from prepubertal females recovered from follicles larger and smaller than 3 mm) to biphasic IVM treatments enriched with 2 Oocyte Secreted Factors (OSF), such as bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9). These OSFs act improving meiotic arrest and communication between cumulus cells and oocyte.
According to the hypothesis of Richani and Gilchrist (2018), oocytes increase their developmental competence during their and this is observed because they become responsive to EGF (Epidermal Growth Factor) peptide through cAMP modulators (such as CNP), together with GDF9 and/or BMP15. Consequently, the aim of the present project is to study the effect of the biphasic IVM system, together with the addition of BMP15 and GDF9 at concentrations of 50 and 100 ng/mL in the pre-IVM , on the ability to produce embryos from the 3 qualities of goat oocytes. In these studies we will analyze the following parameters: nuclear stage with Hoescht; growth of oocytes by staining with Brilliant Cresyl Blue (BCB); concentration of cAMP in COCs and cumulus cells by RIA; oocyte-cumulus cells junctions (GAPJ) by microinjection of Fluorescent Lucifer Yellow; concentrations of ROS and GSH (glutathione) in the oocytes; total RNA, mitochondrial DNA by qPCR, and apoptosis of the cumulus cells and oocytes by Annexin. Finally, the competence of the oocytes will be studied, after in vitro fertilization (IVF) / Parthenogenic Activation (AP) and embryo culture, by the percentage of blastocysts at 8 days post-infection / post-activation. The last objective will be to know if different oocytes need different protocols of in vitro maturation media.
Biphasic maturation systems to improve embryo competence of goat oocytes (AGL2017-85837-R)
In spite of the improvements in IVEP in farm animals, the results of blastocyst production are still low and inconsistensts between laboratories and experiments. It is widely accepted that the low efficiencies of in vitro maturation (IVM) is in part due to precocious oocyte meiotic resumption following artificial removal of cumulus-oocyte complexes (COCs) from antral follicles and subsequent culture. Moreover, oocytes from small- and mid-sized antral follicles (such as in prepubertal females) have inherently low developmental competence. These incompetent oocytes exhibit an impaired cytoplasmic maturation. Both factors: unknown nuclear stage plus an immature cytoplasm give a pool of oocytes with low embryo development. The present project aims to prepare a protocol of biphasic IVM: Step 1) prematuration medium with CNP (Natriuretic Ppeptides type C) to improve oocyte developmental competence by inhibiting spontaneous oocyte meiotic maturation in vitro and by adding Forskolin and IBMX, compounds which aim is to increase the cAMP in COCs and to preserve cumulus cells–oocyte GAP junctions (GAPJ). After this prematuration the oocytes will be placed in a conventional IVM. We test these prematuration systems after, IVF, ICSI and PA (Parthenogenic Activation) on blastocyst development. Moreover, and because the longer process of these 2 steps IVM systems, we will study the effect of melatonin added at the IVF and IVC of embryos to avoid the large amount of ROS in these media.
The central objective is to improve oocyte developmental competence by inhibiting spontaneous oocyte meiotic maturation, increasing cAMP and preserve CC–oocyte communication in vitro (pre-maturation), allowing the oocyte to culminate the whole growth process before being fertilized, as maturation is critical to generating a healthy mature oocyte capable of sustaining embryo development.
Contact person: María Teresa Paramio and Dolors Izquierdo (Teresa.Paramio@uab.cat , Dolors.Izquierdo@uab.cat)