Transcriptional downregulation of ABC transporters is related to follicular degeneration after vitrification and in vitro culture of ovine ovarian tissue

International audienceATP-binding cassette (ABC) transporters perform multiple functions in reproductive tissues. During ovarian tissue vitrification, the plasma membrane has important functions in the influx or efflux of water, and substances such as cryoprotectants and channel proteins that are required in this process. Thus, the present study aimed to verify the relative abundance of mRNA transcript of ABC transporters ABCB1, ABCG2, and MRP2 after vitrification and in vitro culture (IVC) of ovine ovarian tissue. For this study, the ovarian cortex fragments were proportioned into four groups as fresh control, vitrified control, fresh culture, and vitrified culture groups. After vitrification and in vitro culture, the ovarian tissue was evaluated using morphological procedures. Further, relative abundance of ABCB1, ABCG2, and MRP2 transporter mRNA transcripts in the ovarian cortex subjected to aforementioned treatment conditions were evaluated using qPCR. Our results showed a negative association between degenerated follicles and mRNA transcript abundances of ABCB1 and ABCG2. In addition, the percentage of growing follicles in the ovine ovarian cortex after vitrification was similar to that of the fresh control tissue without in vitro culture. The in vitro culture of fresh and vitrified tissue however, showed a significant decrease in the percentage of growing follicles. To the best of our knowledge, we believe that our data for the first time has studied the relative abundances of ABCB1 and ABCG2 mRNA transcripts in the ovine ovarian cortex. In addition, alterations of these protein channels may be indicative of a deleterious effect of osmotic stress on follicular survival during vitrification. Furthermore, these effects were detectable only after the IVC of the ovarian tissues. Nonetheless, further studies are required to investigate the functions of ABC trans-porters in ovine folliculogenesis, especially after in vitro culture of ovarian tissue

Expression of growth and differentiation factor 9 (GDF-9) and its effect on the in vitro culture of caprine preantral ovarian follicles

AbstractThis study examined the expression of growth and differentiation factor 9 (GDF-9) in caprine ovarian follicles, and the effect of GDF-9 with or without FSH on the in vitro culture of preantral follicles. To evaluate the expression of GDF-9 in Experiment 1, follicles were recovered from 32 goat ovaries and the total RNA isolated and transcribed for real-time polymerase chain reaction (PCR). Experiments 2 and 3 each used a further 32 goat ovaries to provide preantral follicles of ≥150μm. These follicles were isolated and cultured individually in 100μL drops. In each experiment at least 45 follicles were used per treatment. Every 6 days, follicles were evaluated for viability, antrum formation and growth rate. At the end of the culture period, oocytes were submitted to in vitro maturation (IVM), viability tests and chromatin evaluation. In Experiment 2, follicles were cultured in a basal medium (control) or this medium supplemented with GDF-9 at a concentration of 100ng/mL (GDF-9 100) or 200ng/mL (GDF-9 200). The same media were used in Experiment 3, supplemented with recombinant FSH at a level of 100ng/mL from day 0, 500ng/mL from day 6 to 12 and 1000ng/mL from day 12 to 18 of culture to form the three treatments: control FSH, GDF-9 (100) plus FSH and GDF-9 (200) plus FSH. Relative GDF-9 expression (Experiment 1) was greater in the secondary (18units) than the primordial (1unit) and the primary (1unit) preantral follicles (P<0.05). In the antral follicles, GDF-9 expression was significantly higher in the cumulus–oocyte complexes COC's<3mm (1.6units) than those of >3mm diameter (1unit; P<0.05), and in COC's<3mm and >3mm (319.2 and 200.1units, respectively), compared to their respective granulosa and theca cells (1unit for each category, P<0.05). In Experiment 2, GDF-9 supplementation significantly improved the survival of the follicles (60.8%, 66.0% and 77.4% for the control, GDF-9 100 and GDF-9 200, respectively; P<0.05), follicular growth rate and antrum formation following 18 days of culture. Oocyte survival was approximately 100% in all treatments. More oocytes were submitted to IVM from GDF-9 100 (78.0%; P<0.05), compared to GDF-9 200 (48.1%), but no suitable oocytes could be retrieved from the control (58.8%). The proportion of oocytes showing a resumption of meiosis, was not significantly different between treatments (41.4%, 35.9% and 36.0% for the control, GDF-9 100 and GDF-9 200, respectively). The addition of GDF-9 to the media supplemented with FSH (Experiment 3) did not significantly affect any of the variables studied. The proportion of oocytes submitted to IVM in Experiment 3 was 53.3%, 56.5% and 63.8% for the control FSH, GDF-9 100 plus FSH and GDF-9 200 plus FSH, respectively (no statistical differences). The resumption of meiosis was 75.0%, 60.9% and 60.7% for the control FSH, GDF-9 100 plus FSH and GDF-9 200 plus FSH, respectively (NS). The occurrence of metaphase II was very low in both experiments. It was concluded that the supplementation of a basal medium with GDF-9 had a positive effect on the survival and development of caprine preantral follicles, but had no real effect in the presence of FSH

Modulation of aquaporins 3 and 9 after exposure of ovine ovarian tissue to cryoprotectants followed by in vitro culture

International audienceOur aim has been to evaluate the effect of cryoprotective agents (CPAs) on the exposure, vitrification (VIT), and in vitro culture (IVC) of ovarian tissue with regard to the expression and immunolocalization of aquaporins (AQPs) 3 and 9 in ovine preantral follicles. Tissues were treated as follows: Experiment I: (1) control (without exposure to CPAs), (2) e-EG (exposure to ethylene glycol), (3) er-EG (exposure to and removal of EG), (4) e-DMSO (exposure to dimethyl sulfoxide), (5) er-DMSO (exposure to and removal of DMSO), (6) e-EG+DMSO (exposure to EG+DMSO), (7) er-EG+DMSO (exposure to and removal of EG+DMSO); Experiment II: (1) control, (2) VIT, (3) IVC, (4) VIT-IVC. In Experiment I, following er-EG or er-DMSO, tissue showed the down-regulation (P  0.05) to that of the control group. Nevertheless, VIT-IVC treatment led to the down-regulation of mRNA of AQP3 and AQP9. Thus, AQP3 and AQP9 act in a mutually dependent way, maintaining the cell homeostasis that is essential for the ovary cryopreservation process. Furthermore, the changes in the expression profiles of mRNA and protein after culture are a strong indicator that in vitro conditions have to be strictly controlled to ensure follicle viability and functionality

Analysis of aquaporins in ovine ovarian tissue after exposure to cryoprotectant agents, followed by vitrification and in vitro culture

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