Adeline de Andrade Carvalho

Influence of vitrification techniques and solutions on the morphology and survival of preantral follicles after in vitro culture of caprine ovarian tissue

The objective was to compare the efficiency of various vitrification techniques and solutions for preserving morphology and viability of preantral caprine follicles enclosed in ovarian tissue. Fragments of ovarian cortex were cryopreserved by conventional vitrification (CV) in French straws, vitrification in macrotubes (MTV), or solid-surface vitrification (SSV). Six solutions containing 6 M ethylene glycol, with or without sucrose (SUC; 0.25 or 0.50 M) and/or 10% fetal calf serum (FCS) were tested (Experiment I). After 1 wk, samples were warmed and preantral follicles were examined histologically. To evaluate follicular viability (Experiment II), ovarian fragments were vitrified with the three techniques listed above, in a solution containing 0.25 M SUC and 10% FCS. After warming, follicles were assessed by the trypan blue dye exclusion test. In Experiment III, preantral follicles enclosed in ovarian tissue were vitrified using the protocol which yielded the highest percentage of viable preantral follicles (SSV with 0.25 M SUC and 10% SFB). After warming, the preantral follicles enclosed in ovarian tissue were cultured in vitro and then, were analyzed by histology and fluorescence microscopy (calcein-AM and ethidium homodimer-1). Every vitrification protocol significantly reduced the percentages of morphologically normal follicles relative to the control (88.0%); however, the addition of 0.25 M SUC and 10% FCS to the vitrification solution improved preservation of follicular morphology (67.4, 67.4, and 72.0% for CV, MTV, and SSV, respectively). Although follicular viability after SSV (80.7%) did not differ from that in fresh (non-vitrified) ovarian tissues (88.0%), after in vitro culture, percentages of viable follicles were significantly reduced (70.0%). Percentages of morphologically normal follicles after in vitro culture of vitrified ovarian tissue were similar (76.0%) to those in ovarian cortex fragments cultured without previous vitrification (83.2%). In conclusion, SSV using a solution containing 0.25 M SUC and 10% FCS, was the most efficient method for vitrifying caprine ovarian tissue.

Novel wide-capacity method for vitrification of caprine ovaries: Ovarian Tissue Cryosystem (OTC).

In this study we aimed testing the efficiency of a newly developed device for vitrification of ovaries without contact with liquid nitrogen, Ovarian Tissue Cryosystem (OTC). From each ovarian pair, fragments were recovered and immediately fixed for analysis (fresh control) or submitted to vitrification (fragments, hemi-ovary or whole ovary), either or not followed by in vitro culture for two days. Vitrification was performed using the OTC system. The OTC is a cylindrical structure made by stainless steel and composed by three pieces (basis, insert and cover), which can be hermetically closed avoiding contact of the tissue with liquid nitrogen during vitrification. Before and after culture, the ovarian tissue was histologically evaluated. Independently from the size of the ovarian tissue, it was observed a decrease (P<0.05) in the rates of normal preantral follicles when fragments (58.1%), hemi-ovary (54.4%) and whole ovary (54.3%) were vitrified, in comparison with fresh control (68.1%). These data were confirmed by ultrastructural analysis, which showed a great extension of degeneration in follicles vitrified in the whole ovary. Follicular survival after vitrification followed by culture was higher (P<0.05) when ovarian fragments were vitrified (36.1%) than in those enclosed in vitrified hemi-ovary (22.3%) or whole ovary (18.4%). In conclusion, the Ovarian Tissue Cryosystem (OTC) opens a new possibility for successful vitrification of caprine ovarian fragments.

Steady-state level of messenger RNA and immunolocalization of aquaporins 3, 7, and 9 during in vitro growth of ovine preantral follicles

Aquaporins (AQPs) are a well-conserved family of small (approximately 30 kDa) membrane channel proteins that facilitate rapid movement of fluids and have a unique tissue-specific pattern of expression. These proteins have been found in the female reproductive systems of humans, rats, and mice. However, the expression and cellular localization of AQPs have not extensively been studied in the female reproductive system of sheep. Therefore, this study aimed to evaluate, by real-time polymerase chain reaction and immunohistochemistry respectively, the levels of messenger RNA and the immunolocalization of AQP3, AQP7, and AQP9 in large isolated ovine secondary follicles over a period of IVC. Our analysis revealed that AQP3 and AQP9 were present predominately in follicles that exhibited antrum formation, suggesting a crucial role of these AQPs in the formation of the antrum. Interestingly, AQP7 was only expressed in follicles that had not formed an antrum by Day 12 of culture. In conclusion, the presence of protein channels (AQP3 and AQP9) seems to be essential for the formation of the antrum in isolated ovine secondary follicles cultured in vitro and thus plays an important role during folliculogenesis in this species.

Catalase addition to vitrification solutions maintains goat ovarian preantral follicles stability

The aim of this study was to verify whether the addition of catalase (20 IU/mL) at different steps of goat ovarian tissue vitrification affects ROS levels, follicular morphology and viability, stromal cell density, apoptosis and the expression of proteins related to DNA-damage signaling (γH2AX) and repair (53BP1). Goat ovarian tissues were analyzed fresh (control) or after vitrification: without catalase (VS-/WS-), with catalase in vitrification solutions (VS+/WS-), with catalase in washing solutions (VS-/WS+) or with catalase in both solutions (VS+/WS+). The vitrification without catalase had higher ROS levels than the control. The catalase, regardless the step of addition, maintained ROS levels similar to the control. There were no difference between treatments regarding follicular viability, stromal cell density and detection of γH2AX and 53BP1. There was no difference in follicular morphology and DNA fragmentation between groups vitrified. In conclusion, catalase addition to vitrification solutions prevents ROS formation in cryopreserved goat ovarian tissues.

Immunolocalization of the Anti‐Müllerian Hormone (AMH) in Caprine Follicles and the Effects of AMH on In Vitro Culture of Caprine Pre‐antral Follicles Enclosed in Ovarian Tissue

The aims of this study were to evaluate the localization, by immunohistochemistry, of the anti-Müllerian hormone (AMH) in goat ovaries and to investigate its effects on the in vitro survival and development of caprine pre-antral follicles enclosed in fragments of ovarian tissue. Pre-antral follicles were cultured in vitro for 1 or 7 days in α-MEM(+) in the absence or presence of kit ligand (KL; 50 ng/ml, positive control) or AMH (50 or 150 ng/ml). The results showed that AMH was localized in oocytes and granulosa cells from the primordial follicle to antral follicle stages. Addition of AMH maintained the percentage of developing follicles, similar to that in the uncultured control; however, the percentage of developing follicles was significantly lower than that in the cultured control and KL. Nonetheless, addition of AMH to the culture medium did not affect survival rates and follicular growth. In conclusion, this study demonstrated that the expression of AMH varies according to the compartment and stage of follicular development. Furthermore, AMH inhibits the activation of caprine primordial follicles.

Adding Ascorbic Acid to Vitrification and IVC Medium Influences Preantral Follicle Morphology, but Not Viability

In this study, we analysed the effect on morphology and viability of ovine primordial follicles, when ascorbic acid (AA) was added to vitrification and in vitro culture (IVC) media. For morphological analysis, ovarian tissue was vitrified using DMSO or ethylene glycol (EG), to which AA was added or omitted. After warming, the tissue was fixed for histology or 1-day cultured in the presence or absence of AA. Isolated primordial follicles from ovine ovarian tissue vitrified with DMSO or EG, both supplemented with AA were stained with trypan blue for viability analysis, or 5-day cultured with or without AA followed by a viability analysis. In this study, we report on the successful vitrification protocol developed for ovine ovarian tissue using EG. Vitrification using DMSO reduced the percentage of morphological normal primordial follicles, whereas addition of AA to the vitrification and culture media did enhance these results (p < 0.05). However, vitrification in a DMSO + AA medium followed by 5-day IVC resulted in a significant decrease in the follicular viability, independently of the presence of AA in the IVC medium.

Two Methods of Vitrification Followed by In Vitro Culture of the Ovine Ovary: Evaluation of the Follicular Development and Ovarian Extracellular Matrix

The aim of this study was to evaluate the influence of two vitrification techniques on the extra cellular matrix (ECM) and ovarian follicular development. The ovarian cortex was fragmented (9 mm(3)) and divided into six groups, viz. fresh control, cultured control, vitrified by the Ovarian Tissue Cryosystem (OTC) method, conventional solid surface vitrification (SSV) method, OTC/cultured and SSV/cultured. Follicles from all the fragments were analysed for morphology, development and viability. The ECM was evaluated based on the condition of collagen and reticular fibres and the immunolocalization of type I collagen and fibronectin. After 7 days of culture, the tissue vitrified by OTC revealed a higher percentage (p < 0.05) of morphologically normal (30.66%) and viable (60.00%) follicles when compared with those vitrified using the SSV technique (21.33% and 23.00%). In all the fragments cultured, regardless of the vitrification method, a significantly higher percentage of developing follicles was observed when compared with the non-cultured tissue. Analysis of the type I collagen showed increased immunostaining after the in vitro culture in the vitrified fragments. In conclusion, the OTC is better for preserving the follicular viability and morphology and maintaining the integrity of the extracellular matrix components of the ovine ovary.

Restoring fertility after ovarian tissue cryopreservation: a half century of research

Tissue transplantation and in vitro ovarian follicle culture have been investigated as alternative techniques to restore fertility in young women who are facing fertility-threatening diseases or treatments following ovarian tissue cryopreservation. Although transplants of fresh or frozen ovarian tissue have successfully yielded healthy live births in different species including humans, the risks of reintroducing cancer cells back into the patient, post treatment, have limited its clinical purpose. The in vitro ovarian follicle culture minimizes these risks and provides a way to harvest more mature oocytes, however its clinical translation has yet to be determined. Not only is it possible for tissue cryopreservation to safeguard fertility in cancer patients, this technique also allows the maintenance of germplasm banks for animals of high commercial value or for those animals that are at risk of extinction. Given the importance of managing female genetic material, this paper reviews the progress of the methods used to preserve and restore female fertility in different species to demonstrate the results obtained in the past 50 years of research, the current achievements and the future directions on this field.

In situ cultured preantral follicles is a useful model to evaluate the effect of anticancer drugs on caprine folliculogenesis.

Despite the increase in the incidence of cancer, the number of women who survive cancer treatment is growing. However, one of the principal results of chemotherapy is premature ovarian failure (POF). The aim of this study was to use the in situ culture preantral follicles as an in vitro model to evaluate the toxicity of two anticancer drugs, doxorubicin (DXR) and paclitaxel (PTX), on the integrity and development of ovarian follicles. Fragments of the ovarian cortex of goats were cultured in vitro for 1 or 7 days in α‐MEM+ supplemented with different concentrations of DXR (0.003, 0.03, or 0.3 µg/mL) and PTX (0.001, 0.01, or 0.1 µg/mL). Analyses were performed before and after culture to evaluate tissue integrity by classical histology, apoptosis by TUNEL assay, DNA laddering kit and the detection of activated caspase 3, and DNA damage by the immune detection of phosphorylated histone H2A.x (H2AXph139). Both DXR and PTX reduced the number of morphologically normal primordial and developing follicles. Positive staining for TUNEL and active caspase 3 was detected in all the samples (P < 0.05). Therefore, we propose the in situ culture of caprine preantral follicles as a useful experimental model for assessing the toxic effects of the chemotherapeutic agents on ovarian folliculogenesis. Microsc. Res. Tech. 79:773–781, 2016.

Comparison between the additive effects of diluted (rFSH) and diluted/dynamized (FSH 6 cH) recombinant follicle-stimulating hormone on the in vitro culture of ovine preantral follicles enclosed in ovarian tissue

OBJECTIVE This study compared 2 types of recombinant follicle stimulating hormone (rFSH): diluted and diluted/dynamized, on in vitro development of ovine follicles. METHODS In experiment 1, ovarian fragments were cultured for 1 or 7 days in α-MEM(+) in the absence or presence of different concentrations of diluted rFSH to determine the best concentration. In experiment 2, the effect of diluted and diluted/dynamized rFSH (rFSH 6 cH--ultradiluted and succussioned), alone or in combination, was studied. RESULTS In experiment 1, compared to control, 50ng/mL of diluted rFSH induced higher rates of follicular survival after 7 days of culture and higher percentages of growing follicles at day 1 of culture (P<0.05). In experiment 2, compared to control, diluted/dynamized rFSH induced higher follicular diameter and survival rate after 7 days and early follicle activation at day 1 of culture (P<0.05). Compared to diluted rFSH, diluted/dynamized rFSH induced higher rates of follicle activation at day 1 of culture (P<0.05). CONCLUSION In conclusion, compared to the control medium, diluted/dynamized rFSH promoted survival and early activation of follicles, while diluted rFSH promoted higher activation later in the culture. Thus, diluted/dynamized rFSH may be used as an alternative to diluted rFSH for the in vitro culture of ovine preantral follicles.