Vitrified sheep isolated secondary follicles are able to grow and form antrum after a short period of in vitro culture.

The risk of reintroducing malignant cells after ovarian graft into patients following post-cancer treatment is an obstacle for clinical applications (autotransplantation). In this context, in vitro follicle culture would be an alternative to transplantation in order to minimize such risks. Therefore, the aim of this study was to compare the development of secondary follicles after vitrification in isolated form (without stroma) with vitrification in in situ form (within fragments of ovarian tissue). Follicles were first isolated from ovarian fragments from mixed-breed ewes and then vitrified; these comprised the Follicle-Vitrification group (Follicle-Vit), or fragments of ovarian tissue were first vitrified, followed by isolation of the follicles, resulting in the Tissue-Vitrification group (Tissue-Vit). Control and vitrified groups were submitted to in vitro culture (6 days) and follicular morphology, viability, antrum formation, follicle and oocyte diameter, growth rate, ultrastructural characteristics and cell proliferation were evaluated. The percentages of morphologically normal follicles and antrum formation were similar among groups. Follicular viability and oocyte diameter were similar between Follicle-Vit and Tissue-Vit. The follicular diameter and growth rate of Follicle-Vit were similar to the Control, while those of Tissue-Vit were significantly lower compared to the Control. Both vitrified groups had an augmented rate of granulosa cellular proliferation compared to Control. Secondary follicles can be successfully vitrified before or after isolation from the ovarian tissue without impairing their ability to survive and grow during in vitro culture.

Ewe Ovarian Tissue Vitrification: A Model for the Study of Fertility Preservation in Women.

Emergency in vitro fertilization followed by embryo vitrification is one feasible fertility preservation option for cancer patients. However, its clinical application has several limitations. Hormonal stimulation delays the initiation of oncotherapy and it is contraindicated in hormone-sensitive cancers or for use in pre-pubertal females. Vitrification of ovarian cortical tissue prior to the start of cancer treatment could be utilized for autotransplantation or for in vitro maturation of follicles enclosed in ovarian tissue. Nevertheless, the main concern associated with autotransplantation is the risk of malignant cell re-introduction to the patient, which is non-existent with the use of follicular in vitro culture. Since obtaining ovarian tissues from women for research is challenging and experimental studies are difficult to complete due to ethical issues, exploring the alternative usage of animal models for fertility preservation may provide beneficial insight into the prospects of follicular culture as an alternative for fertility restoration following ovarian tissue vitrification. Similarities between ewe and human ovary structures, as well as in ovarian follicular development dynamics, make the ewe a possible animal model for the study of female fertility preservation. As vitrification of ovarian tissue has the potential to cryopreserve preantral ovarian follicles, the present review will describe the progress of ovarian tissue vitrification studies completed in ewes.

Insulin-like growth factor II (IGF-II) and follicle stimulating hormone (FSH) combinations can improve the in vitro development of grown oocytes enclosed in caprine preantral follicles.

OBJECTIVE: Evaluate the possible role of IGF-II alone or in association with FSH on in vitro development of isolated caprine preantral follicles. METHODS: Preantral follicles (≥150 µm) were isolated from goat ovaries and cultured for 18 days in basic αMEM medium (control) or supplemented with IGF-II alone at 20 or 50 ng/ml, named IGF20 and IGF50, respectively, or in combination with recombinant FSH (FSH, IGF20F or IGF50F). During in vitro culture, the follicles were analyzed by using morphology criteria, antrum formation and growth rate as parameters. After 18 days of follicular culture, oocytes equal to or larger than 110 µm were used for in vitro maturation (IVM). Oocyte viability and meiosis resumption were assessed by fluorescence microscopy after labeling with calcein-AM, ethidium homodimer and Hoechst 33342. RESULTS: The IGF20 treatment was the only treatment capable of maintaining the percentage of morphologically normal follicles from D0 until D6 and from D12 to D18 (p>0.05), while in all other treatments the percentage of morphologically normal follicles decreased progressively during 18 days of in vitro culture (p<0.05). At D18, all treatments with IGF-II or FSH resulted in a significantly higher percentage of normal follicles when compared to αMEM alone. The IGF50F treatment provided a significantly higher early antrum formation rate when compared to αMEM and FSH alone. The addition of IGF-II alone (20 or 50 ng/ml) or in combination with FSH prevented oocyte degeneration after IVM. Moreover, the FSH treatment demonstrated a lower percentage of oocyte degeneration when compared to control (4.35% vs. 26.3%, respectively; p<0.05). Regarding meiosis resumption, the IGF20F treatment was the only treatment that significantly differed from αMEM alone. All treatments except the control (αMEM alone) presented oocytes at metaphase II. CONCLUSION: IGF-II associated with FSH stimulated in vitro follicular development, oocyte viability and meiotic resumption of caprine oocytes after IVM.

Effect of medium composition on the in vitro culture of bovine pre-antral follicles: morphology and viability do not guarantee functionality.

Summary This study investigated the effect of three different culture media (α minimum essential medium (α-MEM), McCoy or TCM199 during the in vitro culture (IVC) of bovine isolated pre-antral follicles. Pre-antral follicles greater than 150 µm in size were isolated and cultured for 0 (control), 8 or 16 days in one of the abovementioned culture media. Follicles were evaluated for survival, growth and antrum formation at days 8 and 16. The results showed that TCM199 was the most suitable medium to preserve follicular viability and ultrastructure, resulting in the highest rates of antrum formation. In conclusion, TCM199 promotes the in vitro development of isolated pre-antral follicles without hampering follicular functionality by sustaining in vitro growth and antrum formation.

The fibroblast growth factor family: involvement in the regulation of folliculogenesis.

Several growth factors have been identified as local regulators of follicle development and ovulation. Fibroblast growth factor (FGF) family members are potent mitogens and are involved in cell differentiation, cell migration and angiogenesis in many tissues and organs. In addition to FGF-2, which is the most-studied FGF, other important members are FGF-1, -5, -7, -8, -9 and -10. A number of studies have indicated that FGFs play important roles in regulating the initiation of primordial follicle growth, oocyte and follicle survival, granulosa and theca cell proliferation and differentiation, corpus luteum formation, steroidogenesis and angiogenesis. The purpose of this review is to highlight the importance of the FGFs on mammalian female reproduction, providing a better understanding of the roles of this family in ovarian physiology and female fertility.

Leukemia inhibitory factor stimulates the transition of primordial to primary follicle and supports the goat primordial follicle viability in vitro.

The aim of this study was to evaluate the effect of leukemia inhibitory factor (LIF) on the activation and survival of preantral follicles cultured in vitro enclosed in ovarian fragments (in situ). Goat ovarian cortex was divided into fragments to be used in this study. One fragment was immediately fixed (fresh control - FC) and the remaining fragments were cultured in supplemented minimum essential medium (MEM) without (cultured control - CC) or with different concentrations of LIF (1, 10, 50, 100 or 200 ng/ml) for 1 or 7 days, at 39°C in air with 5% CO2. Fresh control, CC and treated ovarian fragments were processed for histological and fluorescence analysis. The percentage of histological normal preantral follicles cultured for 7 days with 1 ng/ml (49.3%), 10 ng/ml (58.6%) and 50 ng/ml (58%) of LIF was higher than in the CC (32.6%; p < 0.05). After 7 days of culture, the percentage of primordial follicles in situ cultured with LIF decreased and primary follicles increased in all LIF concentrations compared with FC and CC (p < 0.05). In conclusion, LIF induced primordial follicle activation and supported preantral follicle viability of goat ovarian tissues cultured for 7 days.

Follicular interactions affect the in vitro development of isolated goat preantral follicles.

The aim of this study was to evaluate the influence of the number of follicles per drop (one or three) and antral follicles on in vitro development of isolated goat preantral follicles. Preantral follicles were isolated through microdissection and distributed individually (control) or in groups of three follicles (treatment) in microdroplets of α-MEM with or without 1000 ng/ml follicle stimulating hormone (FSH) for Experiments 1 and 2, respectively. Experiment 3 was divided into four treatments according to the presence of one or three preantral follicles, associated or not with antral follicles. After culture, oocytes were retrieved from morphologically normal follicles and submitted to in vitro maturation (IVM) and live/dead fluorescent labelling. Results of Experiment 1 (basic medium without FSH) showed that culture of preantral follicles in groups enhances viability, growth and antrum formation after 12 days. However, in the presence of FSH (Experiment 2), only the recovery rate of fully grown oocytes for IVM was significantly affected by grouping of follicles. In Experiment 3, in general, co-culture of preantral follicles with an early antral follicle had a detrimental effect on viability, antrum formation and production of oocytes for IVM. In conclusion, the performance of in vitro culture of goat preantral follicles is affected by the number of follicles per drop, the presence of an antral follicle and FSH.

Canine preantral follicles cultured with various concentrations of follicle-stimulating hormone (FSH).

The objective was to evaluate the effects of various concentrations of exogenous FSH during in vitro culture of isolated canine preantral follicles. Preantral secondary follicles (>200 microm) were isolated by microdissection and cultured for 18 d in supplemented alpha-Minimum Essential Medium (alpha-MEM). There were three treatment groups: 1) absence of FSH (control medium); 2) FSH100 (fixed concentration of 100 ng/mL throughout the entire culture period); and 3) sequential FSH (FSHSeq - 100, 500, and 1,000 ng/mL were added sequentially). Following culture, all follicles from all treatments were still viable (marked green by calcein-AM). The initial (D0) average follicle diameter for the control, FSH100, and FSHSeq was (mean +/- SEM) 298.96 +/- 7.02, 286.00 +/- 5.87, and 275.39 +/- 174 6.55 microm, respectively (P > 0.05). Mean diameter of follicles treated with FSHSeq on Day 18 (D18-439.80 +/- 14.08 microm) was greater than those of the other treatments (P < 0.05). Daily follicular growth rate (microm/d) of follicles in the FSHSeq treatment (6.47 +/- 0.55) was significantly faster than for both the control (3.67 +/- 0.32) and FSH100 (4.47 +/- 0.38) treatments. Furthermore, FSH100 and FSHSeq treatments had a significantly higher rate of antrum formation than the control group on D12 of culture, whereas after D12, FSH100 had a significantly higher rate of extrusion compared to the control (P < 0.05). In conclusion, the sequential addition of FSH to the culture medium maintained the survival of isolated canine preantral follicles and promoted an increased rate of follicular growth and antrum formation.

Short-term preservation of canine preantral follicles: Effects of temperature, medium and time.

The use of the large pool of preantral follicles is a promising alternative to provide high numbers of fertilizable oocytes to reproductive biotechnology. This issue is particularly important to canids, since current rates of success of in vitro techniques using oocytes are very limited, and many species within this family are threatened by extinction. The aim of this study was to evaluate effects of temperature, medium and time on morphology and viability of canine preantral follicles during short-term preservation. Canine ovaries were cut into fragments which were incubated in 0.9% NaCl solution or in minimum essential medium (MEM) at 4, 20 or 38 degrees C for 2, 6, 12 or 24 h. Afterwards, preantral follicles were analyzed by histology, transmission electron microscopy and viability testing using trypan blue, calcein-AM and ethidium homodimer-1. Percentages of morphological normal and viable follicles were maintained similar to control (time 0 h) after incubation in 0.9% NaCl at 4 or 20 degrees C for up to 6h and at 38 degrees C for 2 h. Using MEM, such preservation was possible for 12h at 4 or 20 degrees C, and for 6h at 38 degrees C. These results indicate that preservation of canine preantral follicles might be better accomplished through hypothermic (4 or 20 degrees C) storage in MEM, which ensures maintenance of morphology and viability for up to 12h.