Assessment of DNA damage in goat preantral follicles after vitrification of the ovarian cortex.

Effective methods for gamete preservation should have low impact on DNA integrity. The present study investigated the effects of vitrification of goat ovarian tissues on the occurrence of DNA fragmentation and DNA double-stand breaks using the terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling (TUNEL) assay and detection of phosphorylated histone H2AX (γH2AX), respectively. Goat ovaries were collected at a local abattoir and 12 tissue fragments were prepared from each ovarian pair. Tissue fragments were used as fresh control samples or were cultured in vitro, vitrified or vitrified and cultured. Vitrification was performed using the Ovarian Tissue Cryosystem. Fragments from all groups (control and treatments) were processed for histology, transmission electron microscopy, TUNEL assay and immunofluorescence. Compared with fresh control samples, a lower percentage of morphologically normal follicles was detected in the vitrification followed by culture treatment group (P<0.05). Normal follicular ultrastructure was observed in all groups. Immunofluorescence revealed the presence of γH2AX foci in few oocytes and ovarian stromal cells. TUNEL-positive follicles were found in samples without significant differences among groups (P>0.05). In conclusion, the vitrification protocol used in the present study did not increase DNA damage in preantral follicles enclosed in goat ovarian tissues.

Characterization of Oogonial Stem Cells in Adult Mouse Ovaries with Age and Comparison to In Silico Data on Human Ovarian Aging.

Many adult somatic stem cell lineages are comprised of subpopulations that differ in gene expression, mitotic activity, and differentiation status. In this study, we explored if cellular heterogeneity also exists within oogonial stem cells (OSCs), and how chronological aging impacts OSCs. In OSCs isolated from mouse ovaries by flow cytometry and established in culture, we identified subpopulations of OSCs that could be separated based on differential expression of stage-specific embryonic antigen 1 (SSEA1) and cluster of differentiation 61 (CD61). Levels of aldehyde dehydrogenase (ALDH) activity were inversely related to OSC differentiation, whereas commitment of OSCs to differentiation through transcriptional activation of stimulated by retinoic acid gene 8 was marked by a decline in ALDH activity and in SSEA1 expression. Analysis of OSCs freshly isolated from ovaries of mice between 3 and 20 months of age revealed that these subpopulations were present and persisted throughout adult life. However, expression of developmental pluripotency associated 3 (Dppa3), an epigenetic modifier that promotes OSC differentiation into oocytes, was lost as the mice transitioned from a time of reproductive compromise (10 months) to reproductive failure (15 months). Further analysis showed that OSCs from aged females could be established in culture, and that once established the cultured cells reactivated Dppa3 expression and the capacity for oogenesis. Analysis of single-nucleus RNA sequence data sets generated from ovaries of women in their 20s versus those in their late 40s to early 50s showed that the frequency of DPPA3-expressing cells decreased with advancing age, and this was paralleled by reduced expression of several key meiotic differentiation genes. These data support the existence of OSC subpopulations that differ in gene expression profiles and differentiation status. In addition, an age-related decrease in Dppa3/DPPA3 expression, which is conserved between mice and humans, may play a role in loss of the ability of OSCs to maintain oogenesis with age.

Catalase prevents lipid peroxidation and enhances survival of caprine preantral follicles cryopreserved in a 1,2-propanediol-freezing medium.

The objectives of this study were to determine: 1) the optimal concentration (1.0 or 1.5 M) and duration of exposure (5, 10, or 20 min) of ovarian tissue to 1,2-propanediol (PROH) on morphology and viability of caprine preantral follicles; and 2) the effect of supplementing cryopreservation medium supplementation with Trolox(®) (0.1, 0.5, or 1.0 mM) or catalase (5, 10, or 20 IU/mL) on follicular morphology, viability, and lipid peroxidation. Cryopreservation decreased (p<0.05) percentages of normal follicles relative to the control (84%). Although supplementation of the cryopreservation medium (1.0 M PROH) with catalase (10 or 20 IU/mL) or Trolox(®) (0.1 mM) resulted in follicular morphology and viability similar to that in the controls (P>0.05), lipid peroxidation was reduced only when 20 IU/mL catalase was added to the cryopreservation medium.

Quantification of dimethyl sulfoxide perfusion in sheep ovarian tissue: a predictive parameter for follicular survival to cryopreservation.

The aim of the present study was to determine the amount of dimethyl sulfoxide (DMSO) present in sheep ovarian tissue after exposure to cryoprotectant at different times (5, 10, 20, or 30 min) and at different concentrations (1.0, 1.5, or 2.0 M). To quantify the levels of DMSO in the ovarian tissue, the high-performance liquid chromatography (HPLC) method was applied. In addition, viability of preantral follicles after toxicity test and cryopreservation of ovarian tissue using the above mentioned concentrations of DMSO and exposure times was evaluated. We have observed that the presence of ∼0.6 mg of DMSO into the ovarian tissue may be deleterious to the sheep preantral follicles. In addition, the application of a short exposure time (5 min at 1.5 or 2.0 M DMSO) or low concentration (1.0 M for 10 min) of DMSO successfully preserves sheep preantral follicles following cryopreservation.