Isadora Machado Teixeira Lima

In vitro production of a caprine embryo from a preantral follicle cultured in media supplemented with growth hormone

The objective was to evaluate the effects of growth hormone (GH) on the survival, growth, maturation, and fertilization of oocytes derived from caprine preantral ovarian follicles cultured in vitro. Preantral follicles were isolated from the cortex of caprine ovaries and individually cultured for 18 d in the absence (control) or presence of bovine GH at concentrations of 10 or 50 ng/mL (GH10 and GH50, respectively). Follicle development was evaluated on the basis of survival, antral cavity formation, diameter increase, and the presence of healthy cumulus-oocyte complexes and mature oocytes. After culture, oocytes were subjected to in vitro maturation (IVM) and in vitro fertilization (IVF). The rate of antrum formation after Day 6 of culture was higher in both GH10 and GH50 than in the control (81.0, 92.7, and 47.6%, respectively, P < 0.05). Percentages of grown oocytes that were acceptable for IVM were also higher (P < 0.05) in GH-treated groups than in the control (54.8, 48.8, and 11.9% for GH10, GH50, and Control). A higher percentage of oocytes in the GH50 treatment underwent meiotic resumption (50.0%), produced mature oocytes, and enabled production of an embryo after IVF than in the control group (0.0%; P < 0.05). In conclusion, GH promoted in vitro growth and maturation of goat preantral follicle oocytes and enabled production of an embryo. Furthermore, this study was apparently the first to produce a caprine embryo by in vitro fertilization of oocytes derived from preantral follicles grown in vitro.

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.

Dynamic medium containing kit ligand and follicle-stimulating hormone promotes follicular survival, activation, and growth during long-term in vitro culture of caprine preantral follicles.

The aim of this study was to evaluate the effects of a dynamic medium containing kit ligand (KL) and follicle-stimulating hormone (FSH) on the in vitro culture of caprine preantral follicles for 16 days. Ovarian fragments were cultured in α-MEM+ containing or not containing KL (50 ng/ml) and/or FSH (50 ng/ml) added during the first (days 0–8) and/or second half (days 8–16) of the culture period. Noncultured (control) and cultured fragments were processed for histological and ultrastructural evaluation. After 1 day of culture, only the treatments performed with KL or FSH maintained a percentage of normal follicles similar to that of the control. After 16 days, all treatments using KL until day 8 (KL/KL, KL/FSH, and KL/FSH+KL) and only FSH during the entire culture period (FSH/FSH) showed higher rates of follicular survival compared to α-MEM+ alone. After 1 and 8 days, the treatments initially cultured with KL increased the percentage of follicular activation in comparison to α-MEM+ alone and other treatments. The highest follicular diameter after 16 days was observed in follicles cultured with KL until day 8 followed by FSH (KL/FSH). Furthermore, this treatment promoted, as early as after 1 day of culture, an increase in oocyte growth compared to α-MEM+ alone. Ultrastructural analysis confirmed the integrity of follicles cultured in KL/FSH after 16 days. In conclusion, a dynamic medium containing KL and FSH maintained follicular integrity and promoted follicular activation and growth during the long-term in vitro culture of caprine preantral follicles.

Expression of keratinocyte growth factor in goat ovaries and its effects on preantral follicles within cultured ovarian cortex.

The aims of this study were to evaluate the expression of keratinocyte growth factor (KGF) in goat ovaries and to study its effects on preantral follicle survival and development. The ovaries were used for immunohistochemistry or for in vitro culture for 1 or 7 days with KGF (0, 1, 10, 50, 100, 150, or 200 ng/mL). Noncultured (fresh control) and cultured ovarian slices were processed for histological analysis and transmission electron microscopy (TEM). The results showed that after 7 days of in vitro culture, all treatments had a significant reduction in the percentage of normal follicles compared with the fresh control. After 7 days of culture, the highest KGF concentrations (150 and 200 ng/mL) induced a significant reduction in the percentage of normal follicles compared with the tissues cultured in the absence (α-MEM+ alone) or presence of 1, 10, and 50 ng/mL KGF. Transmission electron microscopy confirmed follicular integrity after 7 days of culture in 1 ng/mL KGF. In addition, compared with the fresh control, the percentage of growing follicles was significantly increased in all treatments after 1 or 7 days of culture. Immunohistochemical analyses showed the expression of KGF in oocytes and granulosa cells in all follicle developmental stages as well as in thecal and stromal cells. In conclusion, this study demonstrated that, at the lowest concentration (1 ng/mL), KGF maintained the ultrastructure of goat preantral follicles cultured in vitro for up to 7 days. Furthermore, the KGF protein was widely distributed in goat ovaries, especially in ovarian follicles.

Presence of c-kit mRNA in goat ovaries and improvement of in vitro preantral follicle survival and development with kit ligand.

This study evaluated the levels of c-kit mRNA in goat follicles and the effects of kit ligand (KL) on the in vitro development of cultured preantral follicles. Preantral follicles isolated from goat ovarian cortex were cultured for 18 days in α-MEM(+) supplemented with KL (0, 50 or 100 ng/mL) in the absence or presence of follicle stimulating hormone (FSH). Real-time PCR showed that c-kit mRNA was higher in primordial and primary follicles than in secondary stage. Regarding the culture, KL addition in the absence of FSH improved the follicular survival, antrum formation, oocyte growth and meiotic resumption. KL-positive effects were not observed in the presence of FSH. In conclusion, c-kit mRNAs are detected in all follicular categories. KL promotes the survival and antral cavity formation of caprine preantral follicles after in vitro culture, as well as the growth and meiotic resumption of their oocytes in the absence of FSH.

Three-dimensional systems for in vitro follicular culture: Overview of alginate-based matrices

The in vitro culture of ovarian follicles has provided critical insight into the biology of the follicle and its enclosed oocyte and the physical interaction and communication between the theca and granulosa cells and the oocyte that is necessary to produce meiotically competent oocytes. Various two-dimensional (2D) and three-dimensional (3D) culture systems have been developed to evaluate the effect of growth factors, hormones, extracellular matrix components and culture conditions on follicle development and oocyte growth and maturation. Among these culture systems, 3D systems make it possible to maintain follicle structure and support communication between the various cell compartments within the follicle. In this review article, we will discuss the three main approaches to ovarian follicle culture: 2D attachment systems, 3D floating systems and 3D encapsulated systems. We will specifically emphasise the development of and advances in alginate-based encapsulated systems for in vitro follicle culture.

Ultrastructural features of agouti (Dasyprocta aguti) preantral follicles cryopreserved using dimethyl sulfoxide, ethylene glycol and propanediol

The objective was to develop an efficient protocol for cryopreservation of agouti (Dasyprocta aguti) ovarian tissue. Agouti ovarian fragments were placed, for 10 min, in a solution containing MEM and fetal bovine serum plus 1.5 M dimethyl sulfoxide (DMSO), ethylene glycol (EG) or propanediol (PROH); some of those fragments were subsequently cryopreserved in a programmable freezer. After exposure and/or thawing, all samples were fixed in Carnoy prior to histological analysis. To evaluate ultrastructure, follicles from the control and all cryopreserved treatments were fixed in Karnovsky and processed for transmission electron microscopy. After exposure and freezing, there was a significant decrease in the percentage of morphologically normal preantral follicles in all treatments when compared to the control (92.67 ± 2.79, mean ± SD). However, there were no significant difference when the exposure and freezing procedures were compared using the same cryoprotectant. Moreover, there was no significant difference among cryoprotectants at the time of exposure (DMSO: 64.7 ± 3.8; EG: 70.7 ± 11.2, PROH: 63.3 ± 8.5) or after freezing (DMSO: 60.6 ± 3.6, EG: 64.0 ± 11.9; PROH: 62.0 ± 6.9). However, only follicles frozen with PROH had normal ultrastructure. In conclusion, preantral follicles enclosed in agouti ovarian tissue were successfully cryopreserved using 1.5 M PROH, with satisfactory maintenance of follicle morphology and ultrastructure.

Effect of sequential medium on in vitro culture of goat ovarian cortical tissue

A sequential medium was evaluated on the survival, activation and growth rates of caprine preantral follicles submitted to a long-term culture period, aiming to establish an ideal in vitro culture system. Ovarian fragments were cultured for 16 days in α-MEM(+) alone or supplemented with hormones (GH and/or FSH) added sequentially on different days of culture. Ovarian fragments were cultured in the first (days 0-8) and second (days 8-16) halves of the culture period, generating 10 treatments: α-MEM(+)/α-MEM(+), FSH/FSH, FSH/GH, FSH/FSH+GH, GH/GH, GH/FSH, GH/FSH+GH, FSH+GH/FSH+GH, FSH+GH/FSH and FSH+GH/GH. Follicle morphology, viability and ultrastructure were analyzed. After day 1 of culture, FSH treatments maintained the percentage of normal follicles similar to the fresh control. At day 16 of culture, the treatment FSH/GH showed the highest (P<0.05) percentage of normal follicles. The ultrastructure of follicles was preserved in the fresh control and FSH/GH treatment. Follicles cultured with FSH/GH had a higher (P<0.05) viability than α-MEM(+); however the viability was lower (P<0.05) when compared to the fresh control. The FSH/GH treatment showed the highest (P<0.05) percentage of follicular activation and secondary follicle formation and produced the largest (P<0.05) mean follicular diameter after 16 days of culture. In conclusion, a sequential medium supplemented with FSH followed by GH during a long-term culture maintains the survival, viability and ultrastructure of goat preantral follicles, and promotes activation and secondary follicles.

The effect of IGF-1 and FSH on the in vitro development of caprine secondary follicles and on the IGF-1, IGFR-I and FSHR mRNA levels

This study verified the in vitro effects of IGF-1, FSH or both on caprine preantral follicle development and mRNA levels encoding IGF-1, IGFR-1 and FSHR. Secondary follicles were cultured for six days with FSH, IGF-1 or IGF-1+FSH. The results showed that IGF-1 and/or FSH addition did not influence follicular development for six days. The interaction between IGF-1 and FSH increased the mRNA levels of IGF-1 and FSHR, and FSH increased the expression of the IGFR-1 mRNA. Thus, IGF-1 and/or FSH increased IGF-1, IGFR-1 and FSHR mRNA levels in in vitro cultured caprine secondary follicles, but they did not influence their development after six days of in vitro culture.

Transforming growth factor-β (TGF-β) maintains follicular ultrastructure and stimulates preantral follicle growth in caprine ovarian tissue cultured in vitro.

The objectives of this study were to investigate whether TGF-β affect the survival, activation and further growth of goat primordial follicles enclosed in ovarian cortex after in vitro culture. Goat ovaries were collected from an abattoir and pieces of ovarian tissues were cultured for one or seven days in a supplemented alpha Minimum Essential Medium, alone or containing TGF-β (1, 5, 10 or 50ng/mL). Ovarian tissues from the fresh control as well as those cultured were processed for histological and ultrastructural studies. The results showed that when compared with fresh control, there was decrease in the percentages of histologically normal follicles in all treatments only after seven days culture. TGF-β did not affect the activation of preantral follicles regardless of its concentration, however, larger follicles diameter (P<0.05) was observed using 10ng/mL TGF-β than in the fresh control and other treatments. Moreover, this concentration maintained the normal ultrastructure after seven days of culture. In conclusion, TGF-β showed additional effect on the follicle growth and the maintenance of ultrastructural integrity of goat preantral follicles enclosed in ovarian tissue when used at 10ng/mL during seven days of culture.