Molecular and cellular effects of insulin-like growth factor-1 and LongR3-IGF-1 on in vitro maturation of bovine oocytes: comparative study.

Addition effects of insulin-like growth factor-1 (IGF-1) and its synthetic analogue insulin-like growth factor-1 recombinant-3 (LongR3-IGF-1) after in vitro maturation (IVM) of cattle cumulus-oocyte complexes (COCs) were compared and evaluated on meiotic progression, apoptosis and profile genes of oocyte competence (GDF9, BMP15, BAX, BCL2, OOSP1, IGFBP2, IGBFP4 and IGFBP5), and their respective cumulus cells (AREG, EGFR, FSHR, COX2, BAX, BCL2, IGFBP2, IGFBP4 and IGFBP5). The 739 COCs (n = 10 pools) of bovine ovaries were collected, selected and matured with IGF-1 (100 ng/mL), LongR3-IGF-1 (100 ng/mL), and in two control groups with 0.1% polyvinyl alcohol (PVA) or 10% fetal bovine serum (FBS), for 22-24 h. The statistical analysis was performed by a linear mixed effects model, ANOVA and Tukey tests. There was no statistical difference between experimental groups taken into account the meiotic progression and apoptosis (P > 0.05). Nevertheless, there were statistical differences (P ≤ 0.05) among FBS, IGF-1 and LongR3-IGF-1 groups for IGFBP4 gene expression, and among PVA, IGF-1 and LongR3-IGF-1 for COX2 gene expression in cumulus cells. Moreover, statistical difference was found for BCL2 gene expression between IGF-1, FBS and PVA groups and for IGFBP4 gene expression between LongR3-IGF-1, PVA and FBS in oocytes. There was no statistical difference between experimental groups for other genes evaluated. These results showed a good performance of IVM of bovine oocytes in the presence of LongR3-IGF-1 and the possibility of replacement of IGF-1 and FBS.

Cryopreservation and characterization of canine preantral follicles.

The aim of this study was to define the population, morphological and ultrastructural characteristics of bitch preantral follicles (PAFs) and to compare the effects on the morphology of PAF of two cryopreservation techniques - slow freezing (SF) and vitrification (V) - of bitches' ovarian tissue. The average population (number per ovary) of PAFs was 48,541 ±â€¯18,366, where 94.25% were primordial (45,145 ±â€¯16,076). The average diameter of the primordial follicles was 27.5 ±â€¯4.2 µm. The overall percentage of morphologically normal PAFs was 93.66 ±â€¯6.81% for the control group, 86.16 ±â€¯11.05% after SF and 68.14 ±â€¯12.75% after V. The percentage of normal primordial follicles was 96.69 ±â€¯4.72% in control, 89.51 ±â€¯10.39% in SF and 75.32 ±â€¯9.23% in V. There was no significant difference in the overall percentage of normal PAFs among SF and the control. However, slow frozen follicles presented ultrastructural damage, while vitrified primordial and primary follicles were well preserved. In conclusion, although slow freezing seemed to be a good preserving method, vitrification was more effective than slow freezing in preserving the ultrastructure of primordial and primary follicles of bitches.

Further insights into the impact of mouse follicle stage on graft outcome in an artificial ovary environment.

STUDY QUESTION: Are mouse preantral follicles differently affected by isolation, encapsulation and/or grafting procedures according to stage? SUMMARY ANSWER: Isolated secondary follicles showed superior ability to survive and grow after transplantation, which was not related to a particular effect of the isolation and/or grafting procedure, but rather to their own ability to induce neoangiogenesis. WHAT IS KNOWN ALREADY: Isolated and encapsulated mouse preantral follicles can survive (6-27%) and grow (80-100%) in a fibrin matrix with a low concentration of fibrinogen and thrombin (F12.5/T1) after short-term transplantation. STUDY DESIGN, SIZE, DURATION: An in vivo experimental model using 20 donor Naval Medical Research Institute (NMRI) mice (6-25 weeks of age) and 14 recipient severe combined immunodeficient (SCID) mice (11-39 weeks of age) was applied. Each NMRI mouse underwent mechanical disruption of both ovaries and isolation of primordial-primary and secondary follicles with ovarian stromal cells, in order to encapsulate them in an F12.5/T1 matrix. Twelve out of 40 fibrin clots were immediately fixed as controls (D0) (10 for histology and 2 for scanning electron microscopy [SEM]) and the others (n = 28) were grafted to the inner part of the peritoneum for 2 (16 fibrin clots) or 7 (12 fibrin clots) days (D2 and D7). PARTICIPANTS/MATERIALS, SETTING, METHODS: This study involved the participation of the Gynecology Research Unit (Universitè Catholique de Louvain) and the Physiological Sciences Department (University of Brasília). Specific techniques were used to analyze the follicle recovery rate (hematoxylin-eosin staining), vascularization (CD34) and follicle ultrastructure (transmission electron microscopy [TEM] and SEM). MAIN RESULTS AND THE ROLE OF CHANCE: After follicle isolation and encapsulation, a statistically higher percentage of normal follicles was observed in the secondary group (62%) than in the primordial-primary group (47%). Follicle recovery rates were 34% and 62% for primordial-primary and secondary follicles on D2, respectively, and 12% and 42% on D7, confirming that secondary follicles survive better than primordial-primary follicles after grafting. Concerning vascularization, both follicle stages exhibited similar vascularization to that seen in control mouse ovary on D7, but a significantly higher number of vessels and greater vessel surface area were detected in the secondary follicle group. Despite structural differences in fiber density between fibrin clots and ovarian tissue observed by SEM and TEM, preantral follicles appeared to be well encapsulated in the matrix, also showing a normal ultrastructure after grafting. LARGE SCALE DATA: Not applicable. LIMITATIONS, REASONS FOR CAUTION: As demonstrated by our results during the isolation procedure, we encapsulated a significantly higher number of round structures in the primordial-primary group than in the secondary group, which could partially explain the lower recovery rate of early-stage follicles in our previous study. However, it is not excluded that the physical and mechanical properties of the fibrin matrix may also play a role in follicle survival and growth, so further investigations are needed. WIDER IMPLICATIONS OF THE FINDINGS: This research represents one more key step in the creation of the artificial ovary. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by grants from the Fonds National de la Recherche Scientifique de Belgique (FNRS) to C.A. Amorim as a research associate at FRS-FNRS and (grant 5/4/150/5 awarded to M.M. Dolmans), Fonds Spéciaux de Recherche, Fondation St Luc, Foundation Against Cancer, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES-Brazil) (grant #013/14 CAPES/WBI awarded to C.M. Lucci, with F. Paulini receiving a post-doctoral fellowship), and Wallonie-Bruxelles International, and donations from the Ferrero family. None of the authors have any competing interests to declare in relation to the topic.

Influence of follicle stage on artificial ovary outcome using fibrin as a matrix.

STUDY QUESTION: Do primordial-primary versus secondary follicles embedded inside a fibrin matrix have different capabilities to survive and grow after isolation and transplantation? SUMMARY ANSWER: Mouse primordial-primary follicles showed a lower recovery rate than secondary follicles, but both were able to grow. WHAT IS KNOWN ALREADY: Fresh isolated mouse follicles and ovarian stromal cells embedded in a fibrin matrix are capable of surviving and developing after short-term autografting. STUDY DESIGN, SIZE, DURATION: In vivo experimental model using 11 donor Naval Medical Research Institute (NMRI) mice and 11 recipient severe combined immunodeficiency (SCID) mice. Both ovaries from all NMRI mice were mechanically disrupted and primordial-primary and secondary follicles were isolated with ovarian stromal cells. They were then encapsulated in a fibrin matrix composed of 12.5 mg/ml of fibrinogen (F12.5) and 1 IU/ml of thrombin (T1) (F12.5/T1), and grafted to the inner part of the peritoneum of SCID mice for 2 and 7 days. PARTICIPANTS/MATERIALS, SETTING, METHODS: This study was conducted at the Gynecology Research Unit, Université Catholique de Louvain. All materials were used to conduct histological (H-E staining) and immunohistochemical (Ki67, TUNEL) analyses. MAIN RESULTS AND THE ROLE OF CHANCE: Although all grafted fibrin clots were recovered, the follicle recovery rate on day 2 was 16 and 40% for primordial-primary and secondary follicles respectively, while on day 7, it was 6 and 28%. The secondary group showed a significantly higher recovery rate than the primordial-primary group (23%, P-value <0.001). Follicles found in both groups were viable, as demonstrated by live/dead assays, and no difference was observed in the apoptosis rate between groups, as evidenced by TUNEL. Their growth to further stages was confirmed by Ki67 immunostaining. LIMITATIONS, REASONS FOR CAUTION: As demonstrated by our results, secondary follicles appear to be more likely to survive and develop than primordial-primary follicles in a fibrin matrix after both periods of grafting. These findings may also be attributed to the specific features of the fibrin matrix, which could benefit larger follicles, but not smaller follicles. WIDER IMPLICATIONS OF THE FINDINGS: This study is essential to understanding possible impairment caused by factors such as the isolation procedure or fibrin matrix composition to the survival and development of different follicle stages. It therefore provides the basis for further investigations with longer periods of grafting. STUDY FUNDING/COMPETING INTERESTS: This study was supported by grants from the Fonds National de la Recherche Scientifique de Belgique (grant Télévie No. 7.4578.14 and 7.4627.13, grant 5/4/150/5 awarded to Marie-Madeleine Dolmans), Fonds Spéciaux de Recherche, Fondation St Luc, the Foundation Against Cancer, and the Region Wallone (Convention N°6519-OVART) and donations from Mr Pietro Ferrero, Baron Frère and Viscount Philippe de Spoelberch. None of the authors have any competing interests to declare.

Post-hatching development of in vitro bovine embryos from day 7 to 14 in vivo versus in vitro.

This study evaluates the post-hatching development of in vitro-produced (IVP) embryos until Day 14. On Day 7, IVP embryos were either transferred to recipient uteruses or placed in a post-hatching development (PHD) system. As a control group, in vivo-produced (IVV), Day-7 embryos were also transferred to recipient uteruses. All groups were collected on Day 14 and were morphologically evaluated. Day-7 and Day-14 IVV and IVP embryos were used for quantification of eight genes (PLAC8, CD9, SLC2A1, SLC2A3, KRT8, SOD2, HSP1A1, and IFNT2) by reverse transcriptase qPCR. Day-14 embryos from the PHD system were smaller (2.92 ± 0.45 mm) and had a lower embryonic disk diameter (0.14 ± 0.00 mm) than those produced by IVV (24.18 ± 3.71; 0.29 ± 0.03 mm, respectively) or IVP (19.06 ± 2.43; 0.28 ± 0.01 mm) culture and transferred to the uterus (P > 0.05). Day-7 IVP embryos had a higher expression of the HSP1A1, SCL2A1, and SCL2A3 genes than IVV embryos. When these embryos were cultured in the uterus, no differences in gene expression were observed on Day 14. Conversely, Day-14 IVP embryos cultured in the PHD system showed a higher expression of PLAC8, SOD2, and SLC2A3 genes. It is concluded that Day-7 IVP embryos are different from IVV embryos in regards to gene expression, although exposure to the uterine environment during the elongation period allowed the IVP embryos to overcome this difference. In contrast, IVP embryos cultured in the PHD system were morphologically and molecularly different, being of poorer quality than those cultured in the uterus.