D.M. Magalhães-Padilha

Quantification, morphology, and viability of equine preantral follicles obtained via the Biopsy Pick-Up method

A Biopsy Pick-Up (BPU) method was tested to determine the feasibility of retrieving preantral follicles from mare ovaries in vivo. A total of 33 ovarian biopsy procedures were performed on 18 mares during the breeding season. Mares were 5 to 21 years old and biopsies were performed during the estrous and/or diestrous phase, as confirmed by transrectal ultrasonography. Follicles were mechanically isolated using a tissue chopper, counted, and classified as normal or abnormal and primordial or primary. Viability of isolated follicles was determined by Trypan Blue dye. A total of 256 biopsy attempts were made resulting in 185 successful tissue sample collections (72% success rate). The mean weight of ovarian tissue collected per procedure was 25.0 ± 1.6 mg. Overall, 620 preantral follicles were collected and isolated (95% primordial and 5% primary). The mean (±SEM) number of follicles isolated per biopsy procedure was 18.8 ± 1.9. Primordial and primary follicles had an average diameter of 31.3 ± 6.2 and 41.1 ± 6.6 μm, respectively. Viability rate was higher (P < 0.001) for primordial follicles (91%) compared with primary follicles (50%). Primordial follicles tended (P < 0.06) to have a higher rate of morphological normality (96%) compared with primary follicles (80%). The total number of follicles isolated, amount of tissue harvested, and number of follicles per mg of tissue did not differ (P > 0.05) according to phase of the estrous cycle. Younger mares (5 to 7 years old) had more (P < 0.05) follicles isolated per procedure than older mares (14 to 21 years old). The length of the interovulatory interval was not affected (P > 0.05) by any biopsy procedure, and there were no adverse effects on cyclicity or general reproductive health. In conclusion, the BPU method provided large numbers of normal and viable preantral follicles for the study of early follicular development in mares. The BPU method might be used in the future to obtain preantral follicles for in vitro culture to enable the use of numerous oocytes present within the equine ovary. This could allow for the preservation of genetic material or large-scale embryo production.

Eight-Cell Parthenotes Originated From In Vitro Grown Sheep Preantral Follicles

We investigated the effect of the leukemia inhibitory factor (LIF) alone or in association with follicle-stimulating hormone (FSH) on the in vitro growth and antrum formation of sheep preantral follicles. To evaluate oocyte quality, parthenogenetic activation of the oocytes recovered from in vitro grown preantral follicles was performed. Preantral follicles >110 μm in diameter were isolated and cultured for 18 days in basic medium either alone (control) or supplemented with LIF (10 or 50 ng/mL) in the absence or presence of FSH. Every 6 days the follicular survival, growth, and antrum formation were evaluated. When compared to control (P < .05), antrum formation was increased in follicles cultured in the presence of LIF10 and FSH. At the end of the culture, the oocytes underwent in vitro maturation (IVM); their viability and chromatin configuration were assessed. Although IVM was not affect by the treatments (P > .05), the numerically highest maturation rates (29.63%) were obtained when follicles were cultured in 50 ng/mL LIF (LIF50). Therefore, their oocytes were submitted to parthenogenetic activation; from which 58.3% of the mature oocytes resulted in 8-cell stage parthenotes. In conclusion, although LIF10 + FSH increases antrum formation when compared to a nonsupplemented medium (minimum essential medium), oocytes from sheep preantral follicles are capable of growing and maturing in vitro independent of LIF addition to the medium, which resulted in the formation of 8-cell parthenotes.

In vitro culture of equine preantral follicles obtained via the Biopsy Pick-Up method

The objective was to conduct a preliminary evaluation of the efficacy of two media for in vitro culture of equine preantral follicles. Ovarian cortical strips were obtained from mares (N = 10) via the Biopsy Pick-Up method during the breeding season. Ovarian tissue was immediately submitted to histological analysis (noncultured control; D0) or cultured in situ for 1 day (D1) or 7 days (D7) in either α-MEM or TCM-199 and submitted to histological analysis, generating five treatment groups: noncultured control, α-MEM:D1, TCM-199:D1, α-MEM:D7, and TCM-199:D7. Preantral follicles were evaluated for follicle class (primordial, transitional, primary, and secondary) and morphology (normal vs. abnormal). A total of 142 preantral follicles were analyzed in five replicates. No follicles were observed in the TCM-199:D7 treatment group. The proportion of primordial follicles was higher (P < 0.03) in the control compared to the α-MEM:D7 treatment group. The proportion of primary follicles was higher (P < 0.04) in the α-MEM:D7 treatment group compared to the control. The proportion of developing follicles (transitional, primary, and secondary) was higher (P < 0.03) in the α-MEM:D7 treatment group compared to the control group. There was a greater (P < 0.004) percentage of morphologically normal developing follicles in the α-MEM:D1 treatment group compared to the TCM-199:D1 treatment group. Overall, the percentage of morphologically normal follicles was higher in the control group (72%; P < 0.02) and α-MEM:D1 group (84%; P < 0.0001) compared to the α-MEM:D7 (27%) treatment group. Mean follicle diameter was greater (P < 0.04) in the α-MEM:D7 treatment group (40.6 ± 1.1 μm) compared to the control group (37.3 ± 0.7 μm). Mean oocyte diameter was greater in the α-MEM:D1 (31.0 ± 0.7 μm; P < 0.006), TCM-199:D1 (30.7 ± 1.8 μm; P < 0.006), and α-MEM:D7 (33.2 ± 1.8 μm; P < 0.006) treatment groups compared to the control group (27.4 ± 0.9 μm). In conclusion, based on these preliminary data, in vitro culture of equine ovarian fragments obtained in vivo via the Biopsy Pick-Up method promoted preantral follicle development and follicle and oocyte growth in α-MEM for 7 days, with some follicles remaining morphologically normal throughout the culture period.

Equine preantral follicles obtained via the Biopsy Pick-Up method: histological evaluation and validation of a mechanical isolation technique.

The aims of this study in mares were to: (1) compare preantral follicle parameters between in vitro Biopsy Pick-Up (BPU) and scalpel blade collection methods and between histological and mechanical isolation processing (experiment 1); (2) histologically evaluate preantral follicles (experiment 2); and (3) compare histological analysis with a previously established mechanical isolation technique using a tissue chopper (experiment 3) for ovarian cortical fragments obtained in vivo using a BPU instrument. In experiment 1, preantral follicles were analyzed (N = 220; 90% primordial and 10% primary). Proportions of primordial and primary follicles did not differ (P > 0.05) between tissue collection (BPU vs. scalpel blade dissection) or processing (mechanical isolation vs. histology) methods. Follicle viability and morphology rates were similar (P > 0.05) between tissue collection methods, but mechanical isolation produced more (P < 0.05) morphologically normal follicles than histology. For experiment 2, preantral follicles (N = 332) were analyzed and primordial and transitional (combined) follicles and oocytes were 36.3 ± 0.3 and 26.1 ± 0.3 μm in diameter, respectively, and primary follicles and oocytes averaged 42.9 ± 1.8 and 31.8 ± 2.1 μm. For experiment 3 (188 preantral follicles), within the same animals, the proportion of primordial versus primary follicles was higher (P < 0.03) for histological analysis (98%) compared to tissue chopper analysis (94%), and number of follicles per mg of tissue was not affected (P > 0.05) by processing methods. In conclusion, most parameters evaluated for preantral follicles were similar between histological and tissue chopper processing techniques; hence, mechanical isolation efficiently dissociated equine preantral follicles from the ovarian cortex. Therefore, the tissue chopper could be used to isolate large numbers of morphologically normal equine preantral follicles for cryopreservation and/or in vitro culture.

Steady-state level of insulin-like growth factor-I (IGF-I) receptor mRNA and the effect of IGF-I on the in vitro culture of caprine preantral follicles

The objectives were to quantify insulin-like growth factor receptor-1 (IGFR-1) mRNA in preantral follicles on Days 0 and 18 of in vitro culture in the presence or absence of FSH, and to evaluate the effects of IGF-I on the rate of normal follicles, antral cavity formation, and in vitro growth and maturation of caprine oocytes on Days 0, 6, 12, and 18 of culture. The expression of IGFR-1 was analyzed using real-time RT-PCR before and after follicle culture. Preantral follicles were isolated from the cortex of caprine ovaries and individually cultured for 18 d in the presence or absence of bovine IGF-I (50 or 100 ng/mL). At the end of the culture period, the oocytes were submitted to IVM. The expression of IGFR-1 mRNA in preantral follicles cultured in vitro only approached being significantly higher in follicles supplemented with FSH when compared to follicles immediately after recovery (P<0.06) and cultured without FSH (P<0.1). There was a higher (P<0.05) percentage of normal follicles on Days 6, 12, and 18 of culture in IGF-I 50 (97, 92, 67%, respectively) and IGF-I 100 (100, 90, 80%) groups versus the control (90, 64, 36%). In addition, the rate of antrum formation at 6 and 12 d of culture was higher (P<0.05) in IGF-I groups (IGF-I 50: 72 and 90% and IGF-I 100: 69 and 85%) than the control group (41 and 59%). After 18 d of culture, the percentages of grown oocytes acceptable for IVM were higher (P<0.05) in follicles cultured in the presence of IGF-I (82 vs 49%). Furthermore, follicles cultured in the presence of IGF-I 50 and IGF-I 100 had higher (P<0.05) meiotic resumption rates (63 and 66%, respectively) than the control group (11%). In conclusion, treatment with FSH tended to increase IGFR-1 mRNA expression during the in vitro culture of preantral follicles and the addition of IGF-I to the culture medium clearly improved the in vitro development of caprine preantral follicles.

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.

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.

Gene Expression During Early Folliculogenesis in Goats Using Microarray Analysis

ABSTRACT Understanding of gene expression and metabolic, biological, and physiological pathways in ovarian follicular development can have a significant impact on our understanding of the dynamics of follicular atresia or survival. In fact, some oocyte loss occurs during the transition from secondary to early tertiary follicles. This study aimed to understand, by microarray analysis, the temporal changes in transcriptional profiles of secondary and early antral (tertiary) follicles in caprine ovaries. Ovarian follicles were microdissected and pooled to extract total RNA. The RNA was cross-hybridized with the bovine array. Among 23 987 bovine genes, a total of 14 323 genes were hybridized with goat mRNAs while 9664 genes were not. Of all the hybridized genes, 2466 were stage-specific up- and down-regulated in the transition from secondary to early tertiary follicles. Gene expression profiles showed that three major metabolic pathways (lipid metabolism, cell death, and hematological system) were significantly differentiated between the two follicle stages. In conclusion, this study has identified important genes and pathways that may potentially be involved in the transition from secondary to early tertiary follicles in goats.

Effects of growth differentiation factor-9 and FSH on in vitro development, viability and mRNA expression in bovine preantral follicles.

The present study investigated the role of growth differentiation factor (GDF)-9 and FSH, alone or in combination, on the growth, viability and mRNA expression of FSH receptor, proliferating cell nuclear antigen (PCNA) and proteoglycan-related factors (i.e., hyaluronan synthase (HAS) 1, HAS2, versican, perlecan) in bovine secondary follicles before and after in vitro culture. After 12 days culture, sequential FSH (100 ng mL⁻¹) from Days 0 to 6 and 500 ng mL⁻¹ from Days 7 to 12) increased follicular diameter and resulted in increased antrum formation (P<0.05). Alone, 200 ng mL⁻¹ GDF-9 significantly reduced HAS1 mRNA levels, but increased versican and perlecan mRNA levels in whole follicles, which included the oocyte, theca and granulosa cells. Together, FSH and GDF-9 increased HAS2 and versican (VCAN) mRNA levels, but decreased PCNA mRNA expression, compared with levels in follicles cultured in α-minimum essential medium supplemented with 3.0 mg mL⁻¹ bovine serum albumin, 10 µg mL⁻¹ insulin, 5.5 µg mL⁻¹ transferrin, 5 ng mL⁻¹ selenium, 2 mM glutamine, 2mM hypoxanthine and 50 μg mL⁻¹ ascorbic acid (α-MEM⁺). Comparisons of uncultured (0.2 mm) and α-MEM⁺ cultured follicles revealed that HAS1 mRNA expression was higher, whereas VCAN expression was lower, in cultured follicles (P<0.05). Expression of HAS1, VCAN and perlecan (HSPG2) was higher in cultured than in vivo-grown (0.3 mm) follicles. In conclusion, FSH and/or GDF-9 promote follicular growth and antrum formation. Moreover, GDF-9 stimulates expression of versican and perlecan and interacts positively with FSH to increase HAS2 expression.

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.