R. Romaguera

Brilliant Cresyl Blue stain selects largest oocytes with highest mitochondrial activity, maturation-promoting factor activity and embryo developmental competence in prepubertal sheep

The aim of this study was to test the Brilliant Cresyl Blue (BCB) stain to select prepubertal sheep oocytes for in vitro blastocyst production. Oocyte diameter, mitochondrial activity, maturation-promoting factor (MPF) activity and mRNA relative expression (RE) of genes related to metabolism (ATPase Na(+)/K(+) transporting α 1 (ATP1A1) and cytochrome c oxidase subunit 1 (COX1)) and constitutive function of the cell (cytoplasmic polyadenylation-element-binding protein (CPEB) and S100A10) were assessed. Immature oocytes were exposed to different BCB concentrations (13, 26, 39 and 52  μM) and classified according to their cytoplasm colouration as grown BCB+ (blue cytoplasm) and growing BCB- (colourless cytoplasm). Staining oocytes with 13  μM BCB during 60  min allows selection of (BCB+) the largest (123.66  μm) and most competent oocytes to develop to the blastocyst stage (21%) with a higher number of cells (69.71 ± 6.19 s.e.m.) compared with non-stained BCB- oocytes (106.82  μm, 9% and 45.91 ± 3.35 s.e.m. respectively). Mitochondrial activity, assessed by MitoTracker Orange CMTMRos probe, was significantly higher in BCB+ than in BCB- oocytes after in vitro maturation (3369 and 1565  AU respectively). MPF activity was assessed by CDC2 kinase activity assay showing significantly higher activity at metaphase II stage in BCB+ than in BCB- oocytes (1.479 ± 0.09 and 1.184 ± 0.05 optical density respectively). The genes analysed in this work, ATP1A1, COX1, CPEB and S100A 10, did not show significant effect in mRNA RE between BCB selected oocytes. In conclusion, BCB stains larger and more competent oocytes to develop to the blastocyst stage with more active mitochondria and MPF activity and higher blastocyst cell number.

Oocyte secreted factors improve embryo developmental competence of COCs from small follicles in prepubertal goats

Oocytes secrete soluble paracrine factors called Oocyte Secreted Factors (OSFs) which regulate the cumulus cell phenotype. Follicle populations in ovaries from prepubertal females have smaller diameters than their adult counterparts. Oocytes from small follicles are less competent than those from large follicles. The aim of this study was to investigate, in prepubertal goats, the effect of OSFs secreted by denuded oocytes (DOs) from small (<3 mm) or large (>or=3 mm) follicles during IVM on embryo development and the blastocyst quality of cumulus-oocyte complexes (COCs) from small follicles and to determine if GDF9 participates in this process. Treatment groups were: (A) COCs non selected by their follicle size (control group); (B) cumulus oocytes complexes from small follicles (SFCOCs), (C) cumulus oocytes complexes from small follicles co-cultured with denuded oocytes from small follicles (SFCOCs + SFDOs), and (D) cumulus oocytes complexes from small follicles co-cultured with denuded oocytes from large follicles (SFCOCs + LFDOs). The effect of the addition of kinase inhibitor SB-431542, which antagonizes GDF9, was tested in A, C, and D treatment groups. Co-cultured SFCOCs with SFDOs or LFDOs significantly augmented the blastocyst rate in comparison to SFCOCs alone (15.77%, 17.39% vs. 10.31%, respectively). Blastocysts from SFCOCs + LFDOs group showed higher rates of tetraploid nuclei than blastocysts from SFCOCs and the control group (14.43% vs. 5.45% and 5.24%, respectively; P < 0.05). However, we did not observe differences in the hatching rate, mean cell number or embryo cryotolerance (P > 0.05) between the four treatment groups. The addition of SB-431542 during IVM did not have any effect on blastocyst rate (P > 0.05). In conclusion, in prepubertal goats, COCs with a low embryo developmental competence as a consequence of follicle size can be improved by coculturing them with denuded oocytes from both small and large follicles. GDF9 does not seem play a role in this improvement.

Effect of follicle diameter on oocyte apoptosis, embryo development and chromosomal ploidy in prepubertal goats

The aim of this study was to assess the following parameters in prepubertal goat oocytes of different follicle diameter (> or =3 mm, <3 mm, control): oocyte diameter, early (Annexin-V) and late (TUNEL) apoptosis, embryo development and chromosomal ploidy of these blastocysts using Fluorescence In Situ Hybridization (FISH). Before in vitro maturation, oocytes were measured and stained with Annexin-V or TUNEL. The rest of the oocytes were matured, fertilized, and cultured in vitro for 8 days. Oocytes from follicles of > or =3 mm showed greater mean oocyte diameter (128.27 +/- 7.20 microm vs. 125.35 +/- 7.59 microm), higher percentages of TUNEL positive (42.86 vs. 24.23%), higher cleavage (47.85 +/- 3.98 vs. 23.07 +/- 2.44 %) and blastocyst rates (19.77 +/- 3.04 vs. 4.11 +/- 1.10 %) than oocytes from follicles of <3 mm.. Blastocyst mean cell numbers did not show differences between follicular groups (123.83 +/- 49.62 vs. 104.29 +/- 36.09 for follicles of > or =3 mm and <3 mm, respectively). A total of 54 blastocysts with 7084 nuclei were hybridized with specific probes to chromosomes X and Y. Ninety-eight percent (98%) of the embryos presented at least one cell carrying an abnormal number of chromosomes, but 78% of them presented less than 25% of chromosomal abnormal cells. No differences in the percentage of blastocysts with abnormal ploidy were found in embryos produced from oocytes of different follicle diameter.

Effect of the apoptosis rate observed in oocytes and cumulus cells on embryo development in prepubertal goats.

Oocyte quality is the main factor that determines blastocyst yield; any factor that could affect it, such as apoptosis, could impair subsequent embryonic development. Our aim was to investigate the incidence of apoptosis in prepubertal goat oocytes and cumulus cells, assessed by Annexin-V staining and TUNEL assay, and their effect on embryo development. Oocyte-cumulus complexes (COCs) from slaughtered females were collected and classified depending on COC morphology as: Healthy (H) and Early Atretic (EA). Each one of these groups was classified depending on oocyte diameter: A: 110-125microm, B: 125-135microm and C: >135microm. The COCs were IVM for 27h, IVF with fresh semen and IVC for 8 days after insemination. Apoptosis analyses were performed before and after maturation. Annexin-positive oocytes decreased with diameter in the EA class (immature oocytes: A: 42.6%; B: 30.3%; C: 21%; IVM-oocytes: A: 17.5%; B: 4.8%; C: 0%), while TUNEL assay showed a decrease of apoptosis in the largest oocytes before and after IVM only in Healthy oocytes (immature oocytes: A: 51.5%; B: 43.3%; C: 12.1%; IVM-oocytes: A: 31.7%; B: 12%; C: 0%). Blastocyst rate increased with increasing oocyte diameter, and it was higher in H than in EA oocytes (Healthy; A: 0%; B: 5.3%; C: 14.4%; Early atretic: A: 0.3%; B: 4.1%; C: 5.1%). Oocyte diameter and COC morphology had no effect on the percentage of apoptosis in blastocyst cells. In conclusion, oocyte developmental competence in prepubertal goats is influenced by oocyte diameter and COC morphology.

Vitrification of in vitro produced goat blastocysts: effects of oocyte donor age and development stage.

This study examines the effectiveness of the cryotop vitrification method for the cryopreservation of goat blastocysts. To determine the effects of embryo development stage and donor age on in vitro survival rates, good-quality blastocysts from adult and prepubertal goats were sorted into non-expanded, expanded, hatching and completely hatched. In vitro produced (IVP) blastocysts were derived from prepubertal goat oocytes by slicing of ovaries from slaughtered animals while adult goat oocytes were collected by the laparoscopic ovum pick up (LOPU) method. Blastocysts were vitrified/warmed using the cryotop technique. Survival rates were determined in terms of blastocoele re-expansion at 3 and 20 h post-warming. For prepubertal goats, survival rates at 3h post-warming were significantly higher when expanded blastocysts (78.3%) were vitrified/warmed compared to hatched blastocysts (57.4%), whereas non-expanded (62.5%) or hatching blastocysts (71.4%) showed similar rates. For adult goats, survival rates were significantly higher after warming in expanded (36.4%), hatching (75%) or hatched (50%) blastocysts when compared to non-expanded (0%) blastocysts. When survival rates were assessed at 20 h post-warming, no differences were observed when we compared non-expanded (45.8%), expanded (56.5%), hatching (64.3%) and hatched (50.5%) blastocysts from prepubertal goats; and for blastocysts from adult goats, survival rates were only significantly lower for the non-expanded stage (0%) compared to the other stages. For adult versus prepubertal blastocysts at the same developmental stage, our data indicate significantly higher survival rates at 3 h post-warming for non-expanded and expanded blastocysts from prepubertal goats over their counterparts from adult goats. At 20 h post warming, survival rates were only higher for non-expanded blastocysts from prepubertal goats versus adult goats. Collectively, our data reveal that blastocysts produced in vitro from prepubertal goats return similar survival rates regardless of their development stage, whereas blastocysts derived from adult goats are best for vitrification at the expanded, hatching or hatched stage.

Prepubertal goat oocytes from large follicles result in similar blastocyst production and embryo ploidy than those from adult goats

Developmental competence of oocytes from prepubertal females is lower than those from adult females. Oocyte development competence is positively related to follicular diameter. Most of the follicles of prepubertal goat ovaries are smaller than 3 mm. The aim of this study was to compare oocytes of two follicle sizes (< 3 mm and ≥ 3 mm) from prepubertal goats with oocytes from adult goats in relation to their in vitro production and quality of blastocysts. Oocytes from prepubertal goats were obtained from slaughterhouse ovaries and selected according to the follicle diameter whereas oocytes from adult goats were recovered in vivo by LOPU technique without prior selection of follicle size. COCs were IVM for 27 h, IVF at the conventional conditions with fresh semen and presumptive zygotes were cultured in SOF medium for 8 days. Blastocysts obtained were vitrified and after warming their blastocoele re-expansion and the ploidy by FISH technique were assessed. We found significant differences between blastocysts yield of oocytes recovered from follicles smaller than 3 mm of prepubertal goats compared to those from adult goats (5.45% vs 20. 83%, respectively) however, these differences disappear if oocytes were recovered form large follicles (18.07%). A total of 28 blastocysts were analysed and 96.43% showed mixoploidy. Age did not affect the number of embryos with abnormal ploidy or blastocyst re-expansion after warming. Furthermore, the percentage of diploid blastomeres per embryo was similar in the 3 groups studied, adult, prepubertal from follicles ≥ 3 mm and < 3 mm (68.6%, 80.8% and 73.6%, respectively). In conclusion, IVP of blastocysts coming from follicles larger than 3 mm of goats 45 days old were not different to the blastocysts produced from adult goats, both in terms of quantity and quality.

Developmental competence and embryo quality of small oocytes from pre-pubertal goats cultured in IVM medium supplemented with low level of hormones, insulin-transferrin-selenium and ascorbic acid.

The aim of this study was to test the effect of insulin-transferrin-selenium (ITS) and L-ascorbic acid (AA) supplementation and the hormonal level during in vitro maturation (IVM) of small oocytes from pre-pubertal goat on the blastocyst yield and quality. Concretely, we used four maturation media: conventional IVM medium (CM), growth medium (GM: CM+ITS+AA and low level of hormones), modified CM (mCM: CM with low level of hormones) and modified GM (mGM: CM+ITS+AA and normal level of hormones). Cumulus-oocyte complexes (COCs) were classified into two categories according to oocyte diameter: <125 μm and ≥ 125 μm. Large oocytes were matured 24 h in CM (Treatment A). Small oocytes were matured randomly in six experimental groups: Treatment B: 24 h in CM; Treatment C: 12 h in GM and 12 h in CM; Treatment D: 24 h in mGM; Treatment E: 12 h in mGM and 12 h in CM; Treatment F: 12 h in mCM and 12 h in CM; and Treatment G: 12 h in GM and 12 h in mGM. After IVM, oocytes were fertilized and cultured for 8 days. The blastocyst quality was assessed by the survival following vitrification/warming and the mean cell number. When different maturation media were combined, the blastocyst rate did not improve. The large oocytes produced the highest blastocysts yield. However, the culture of small oocytes in GM (53.3%) enhanced the post-warming survival of blastocysts compared to large oocytes matured in CM (35.7%). In conclusion, IVM of pre-pubertal goat small oocytes in GM would be useful to improve the quality of in vitro-produced blastocysts.

354 EFFECT OF OOCYTE-SECRETED FACTORS (OSF) ON EMBRYO DEVELOPMENT OF OOCYTES FROM SMALL FOLLICLES OF PREPUBERTAL GOATS

Prepubertal goat ovaries contain a great number of small follicles, between 2.5 and 3 mm in diameter (Martino A et al. 1994 Theriogenology 41, 969-980). Oocyte-secreted factors (OSFs) act on granulosa cells to perform multiple functions required for an appropriate development of the oocyte. In bovines, co-culture of COCs with denuded oocytes (DOs) during IVM improved COCs embryo development dueto OSFs secreted byDOs (Hussein T et al. 2006 Dev Biol. 296, 514-521). In each step of follicle growth, the OSFs secreted are different (Eppig JJ et al. 2001 Reproduction. 122, 829-838). Our hypothesis is that oocytes from small follicles could improve their embryo development by co-culturing with DOs which secrete additional OSFs. Oocytes from prepubertal goats were classified according to their follicular origin: large follicles (>3 mm), small follicles (<3 mm), and control (oocytes recovered by slicing technique and selected by their morphology). The COCs from small follicles were divided in 3 groups and each one transferred into 150-μL droplets of IVM (TCM-199 with serum, hormones and cysteamine): 1) 30 COCs alone (small group); 2) 30 COCs co-cultured with 75 denuded oocytes from small follicles (SDO group); 3) 30 COCs co-cultured with 75 denuded oocytes from large follicles (LDO group). After 27 h of IVM, COCs were fertilized in vitro and the presumptive zygotes were cultured for 8 days in SOF with 10% FCS. Two samples of oocytes were used as control groups: oocytes fertilized (IVF group) and oocytes activated (activated group).At 48 h and 8 days post-insemination (pi), respectively, cleavage and blastocyst rates were recorded. Results are shown in Table 1. Differences between treatment groups were assessed using Fisher’s exact test (Graph-Pad software, San Diego, CA, USA). Values with P < 0.05 were considered statistically significant. Oocytes from small follicle had a significantly lower cleavage and blastocyst rates than IVF and activation oocyte groups. The co-cultured of COCs with SDOs and LDOs increased significantly the blastocyst rate compared to COCs from small follicles. In conclusion, additional OSFs secreted by DOs, improved blastocyst yield of oocytes coming from small follicles. However, not differences were found between OSFs secreted by small or large follicles of prepubertal goat oocytes. Table 1.