Assessment of vascular endothelial growth factor expression and apoptosis in the ovarian graft: can exogenous gonadotropin promote angiogenesis after ovarian transplantation?

OBJECTIVE: To evaluate the effects of gonadotropin on angiogenesis by assessing vascular endothelial growth factor (VEGF) expression in rat ovaries transplanted after freezing and thawing. DESIGN: In vitro laboratory experiments. SETTING: Academic research institute. ANIMAL(S): Sixty immature female rats. INTERVENTION(S): Frozen-thawed ovaries were autotransplanted into the SC tissue of 60 rats (ages between 21 and 28 days). After transplantation, either pregnant mare's serum gonadotropin (PMSG) or saline was administered. The grafted ovaries were collected 2, 7, and 30 days after transplantation for evaluation. MAIN OUTCOME MEASURE(S): Assessment of the morphology and number of follicles, evaluation of apoptosis, and analysis of VEGF expression in the grafted ovaries. RESULT(S): Most follicles in the grafts were apoptotic on day 2 but recovered by day 7. The proportion of antral follicles and corpora lutea in the graft correlated with the duration after transplantation. A significant increase in the expression of VEGF188 mRNA was noticed in the grafted ovaries on day 2. Moreover, the mRNA expression in the PMSG group was higher than that in the control group. The increased VEGF protein production in the graft was confirmed by Western blot analysis. CONCLUSION(S): In ovariectomized animals, gonadotropin treatment may not provide any added benefits for folliculogenesis and angiogenesis. Nevertheless, a significant increase in the VEGF188 isoform in the gonadotropin-treated group may suggest the positive effect of exogenous gonadotropin therapy in the early stages of angiogenesis.

Assessment of the integrity of human oocytes retrieved from cryopreserved ovarian tissue after xenotransplantation.

BACKGROUND: Previous studies showed that immature oocytes stored in ovarian tissue could develop to the mature stage after transplantation. However, the quality and competency of the oocytes developed in xenografted ovarian tissue have never been investigated. As a pilot study to investigate this uncharted issue, we evaluated microtubule organization and chromatin configuration of human oocytes harvested from xenografted frozen-thawed ovarian tissue. METHODS: Frozen-thawed human ovarian tissue was transplanted into severe combined immunodeficient mice. All animals were stimulated with gonadotrophin from 20 weeks after transplantation. Grafts were recovered 36 h after hCG administration. The oocytes were retrieved from the antral follicles (>2 mm diameter), cultured in vitro, stained for microtubule and chromatin localization. RESULTS: Five oocytes from 21 female mice and seven oocytes from nine male mice were retrieved. Immunocytochemical examinations of these oocytes after in vitro maturation revealed only two developed to the metaphase II stage. Most oocytes were between prophase and metaphase with abnormal microtubule organization and chromatin configuration. CONCLUSIONS: Immature oocytes in stored human ovarian tissue can grow to maturity in host animals after xenotransplantation. Retrieval of oocytes from the xenograft can be carried out and is reproducible. However, many oocytes, grown in host animals and further matured in vitro, showed aberrant microtubule organization and chromatin patterns.

Quantitative assessment of ischemic tissue damage in ovarian cortical tissue with or without antioxidant (ascorbic acid) treatment.

OBJECTIVE: To estimate ischemic tissue damage in ovarian cortex and to evaluate the effectiveness of ascorbic acid, an antioxidant, to protect ovarian tissue from apoptosis caused by ischemia. DESIGN: In vitro laboratory experiments. SETTING: Academic research institute. INTERVENTION(S): Fresh and frozen/thawed cortical sections of bovine ovaries were incubated in MEM medium with or without ascorbic acid for a duration of 3, 24, and 48 hours at 37 degrees C. MAIN OUTCOME MEASURE(S): Oxygen consumption rates, lactate dehydrogenase concentrations, apoptosis rates determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining, and DNA fragmentation analysis. RESULT(S): The oxygen consumption rates were correlated inversely with the duration of incubation. When the rates of apoptosis in primordial follicles with or without ascorbic acid treatment were compared, there was no statistically significant difference between the two groups. However, the ascorbic acid treatment group showed significantly decreased apoptosis in ovarian cortex (stromal cells) with 24 hours of incubation. CONCLUSION(S): The correlation between ischemic tissue damage and the duration of ischemia was verified. Ovarian cortex could tolerate ischemia at least for 3 hours. Ascorbic acid treatment reduced apoptosis in ovarian cortex up to 24 hours of incubation in vitro. It appeared that stromal cells were more vulnerable to ischemia compared to primordial follicles.