In vitro Survival of Follicles Collected from Domestic Cats' Ovaries at Different Stages of Oestrous Cycle and Cultured with IGF-1
Author's address (for correspondence): Elisane Aracelle Alves, Departamento de Medicina Veterinária Preventiva e Reprodução Animal, Setor de Reprodução e Obstetrícia Veterinária, UNESP, Campus Jaboticabal, 14884-900 Jaboticabal-SP, Brazil. E-mail: email@example.com
Optimal conditions for in vitro culture of feline ovarian follicles have not yet been defined. Follicular development is regulated by intraovarian growth factors, as insulin-like growth factor (IGF-1), and during the different stages of the oestrous cycle, follicles are exposed to specific hormonal environments. The aim of this study was to investigate the effect of IGF-1 on in vitro growth and granulosa cell (GC) viability of preantral follicles collected from domestic cats at follicular and luteal phases of the oestrous cycle. Oestrus and ovulation were induced in 12 cats. A total of 39 and 32 follicles collected at the follicular and luteal phases, respectively, were individually cultured in vitro for 6 days in minimum essential medium media supplemented with or without IGF-1 (100 ng/ml). Follicles collected during the follicular phase and cultured without IGF-1 displayed a significant increase in size and higher GC viability (46.5 ± 22.1 μm, 66.7%, respectively) than that of follicles collected at the luteal phase and cultured without IGF-1 (26.7 ± 14.4 μm, 50%, respectively; p < 0.05). In contrast, when IGF-1 was added to the culture medium, no differences were observed in size or GC viability between follicles collected at the two phases of the cycle. Nonetheless, follicles collected at the luteal phase and cultured with IGF-1 had a significant increase in their diameter and GC viability (31.9 ± 15.9 μm, 63.6%, respectively) than that cultured without IGF-1 (26.7 ± 14.4 μm, 50%, respectively; p < 0.05). These data suggest that in vitro growth and GC survival of feline preantral follicles are affected by the oestrous cycle phase, and the IGF-1 exerts a positive effect on follicles collected at the luteal phase.
In vitro follicle culture is an important tool for supplying high numbers of competent oocytes that can be used for in vitro embryo production. However, in feline species, optimal culture systems that support follicular and oocyte development in vitro have not yet been defined.
Ovarian follicular development is regulated mainly by endocrine, autocrine and paracrine factors, and follicles are exposed to specific hormonal environments during different stages of the oestrous cycle. The intraovarian insulin-like growth factor-I (IGF-1) is involved in in vivo follicular development in mammals, but its effect on in vitro growth of preantral follicles has not yet been totally clarified.
It has been shown that IGF-1 stimulates the proliferation and differentiation of granulosa cells (GC) of preantral follicles, and both FSH and IGF-1 synergistically promote follicular survival (Giudice 1992). Nonetheless, the addition of IGF-1 and/or FSH to the culture media did not affect the in vitro development of caprine secondary follicles (Brito et al. 2012).
Although feline follicles have been previously cultured in vitro (Jewgenow and Pitra 1993; Jewgenow and Goritz 1995; Jewgenow 1996), there is no data available that analyse the response of feline follicles collected in different phases of the oestrous cycle to different culture conditions.
Thus, the aim of this study was to investigate the effect of IGF-1 on in vitro growth and viability of granulosa cells of preantral follicles collected from ovaries of domestic cats at follicular and luteal phases of the oestrous cycle.
Materials and Methods
All chemicals in this study were purchased from Sigma-Aldrich Chemical Company (St Louis, MO, USA) unless otherwise stated.
Oestrous induction and ovary retrieval
This study was conducted in accordance with federal legislation and was approved by the Animal Ethics Committee of Sao Paulo State University, protocol number 008064/12.
Twelve domestic cats aged between 1 and 5 years were housed with 12 h of light, and oestrus was induced by IM single injection of 100 UI eCG (Novormon®- Intervet do Brasil Veterinária Ltda, Cotia, São Paulo, Brazil), followed by single injection of 100 UI hCG (Vetecor®- Hertape Calier, Saude Animal S/A Juatuba, Minas Gerais, Brazil) at 82 h of the first injection (Villaverde et al. 2009).
For collecting ovaries at follicular phase, six domestic cats were spayed at 96 h after eCG, while for collecting ovaries at luteal phase, six domestic cats were spayed at 36 h after the hCG injection. Oestrous cycle phases were confirmed by vaginal cytology and macroscopic evaluation of ovarian structures after excision.
Immediately after surgery, ovaries were transported to the laboratory at room temperature in minimum essential medium (MEM) supplemented with 100 mg/ml penicillin and 100 mg/ml streptomycin.
Follicle isolation, culture and evaluation
Ovarian cortical slices (1-mm thick) were cut from the ovarian surface using a surgical blade under sterile conditions and placed in MEM. Follicles were visualized under a stereomicroscope (SMZ 645 Nikon, Tokyo, Japan) and dissected mechanically using a 26-gauge needle.
Preantral follicles surrounded by complete basal membrane and containing more than one layer of granulosa cells (GC) were selected as previously described (Jewgenow and Pitra 1993; Lima et al. 2003; Reynaud et al. 2009), and the diameter was measured under an inverted phase-contrast microscope with an ocular micrometre (100× magnification).
Follicles were individually cultured in 100 μl of MEM supplemented with 1000 ng/ml FSH, 1.25 mg/ml bovine serum albumin (BSA), ITS (insulin 6.25 ng/ml, transferrin 6.25 ng/ml, selenium 6.25 ng/ml), 2 mm glutamine, 2 mm hypoxanthine, 50 μg/ml of ascorbic acid and with or without IGF-1 (100 ng/ml) under mineral oil at 38.5°C and 5% CO2 in air for 6 days. Fifteen microlitre of MEM was exchanged for fresh medium every other day.
At the end of culture (Day6), preantral follicles characterized by the integrity of basal membrane and of GC layers were considered as intact, whereas degenerated follicles showing breakdown or rupture of the basement membrane, opacity of GC or extrusion of the oocyte from within the follicle were recorded and discarded. The diameter of the intact follicles was measured, and the GC viability was assessed by fluorescein diacetate (FDA; Sigma, Sigma-Aldrich Co., St. Louis, MO, USA) staining. Follicles containing ≥75% of live GC were recorded.
Data from follicular diameters were submitted to Kolmogorov–Smirnov test to confirm normal distribution. Mean values of the increase in follicular size (difference between mean diameter at Day 6 and Day 0 of culture) were analysed by Student–Newman–Keuls test, whereas GC viability and follicular degeneration rates were analysed by chi-square test (p < 0.05).
A total of 71 preantral follicles collected from cats in follicular phase (n = 39) and in luteal phase (n = 32) were in vitro cultured. Data for follicle diameters before (D0) and after 6 days of culture (D6), GC viability and degeneration rates at D6 are summarized in Table 1.
Table 1. Mean size and increase (μm) ± SD, granulosa cell viability and follicular degeneration rates (%) of feline preantral follicles collected at follicular and luteal phases of the oestrous cycle and cultured for 6 days with or without IGF-1
|Follicle diameter (μm)|
|D0||202.6 ± 19.2||201.2 ± 19||204.4 ± 20||212.9 ± 30.3||208.6 ± 30.5|| 218 ± 30.2|
|D6||240.4 ± 22.3||247.7 ± 18.5||231.1 ± 23.7||248.4 ± 34.4|| 247 ± 35.3||249.9 ± 34.4|
|Increase|| 37.8 ± 21.3A|| 46.5 ± 22.1B|| 26.7 ± 14.4C|| 35.5 ± 21.7A|| 38.5 ± 25.7D|| 31.9 ± 15.9D|
|Follicle with ≥75% of live GC at Day 6 of culture, n (%)||9/15 (60.0)A||6/9 (66.7)A||3/6 (50.0)B||15/22 (68.2)A||8/11 (72.7)A||7/11 (63.6)A|
|Follicle degeneration at Day 6 of culture, n (%)||19/34 (55.9)A||10/19 (52.6)C||9/15 (60)A||15/37 (40.5)B||9/20 (45)B||6/17 (35.3)B|
Follicles collected during the follicular phase and cultured in MEM without IGF-1 compared with those collected in the luteal phase displayed a significant increase in size (46.5 ± 22.1 μm vs 26.7 ± 14.4 μm; p < 0.05), a higher GC viability rate (66.7% vs 50%; p = 0.0007) and a lower degeneration rate (52.6% vs 60%; p = 0.02). When IGF-1 was added to the culture medium, no differences were observed in the increase in size, GC viability and degeneration rates between follicles collected at the two phases of the cycle. A positive effect on increase in diameter and GC viability was observed when follicles collected at the luteal phase were cultured in IGF-1 (31.9 ± 15.9 μm, 63.6%, respectively), in comparison with that of non-cultured with IGF-1 (26.7 ± 14.4 μm, 50.0%, respectively; p < 0.05). In contrast, it was not observed the same positive effect when follicles were collected at the follicular phase and cultured with IGF-1. Nonetheless, the addition of IGF-1 decreased the degeneration rates, regardless of whether follicles were collected at luteal or follicular phase (35.3%, 45.0%, respectively), in contrast to that cultured without IGF-1 (60.0%, 52.6%, respectively; p < 0.05).
Regardless of the stage of the oestrous cycle, the overall results showed that an increase in diameter and GC viability was not different between follicles cultured with or without IGF-1, but degeneration rates were lower when IGF-1 was added to the culture medium.
Ovarian follicles develop in vivo under the control of several factors, and follicular culture systems should mimic, as much as possible, the physiological conditions for their growth up to the antral stage. IGF-I plays a crucial role as potent stimulator of cellular proliferation, differentiation and development and regulates GC steroidogenesis and apoptosis during follicular development (Mani et al. 2010).
Follicles in vivo are exposed to dramatic hormonal environment changes during the different stages of the oestrous cycle, and their retrieval during follicular or luteal phase might affect their in vitro developmental competence.
This study evaluated for the first time the in vitro growth of feline follicles collected from cat donors at different phases of the oestrous cycle. In this study, we demonstrated that feline preantral follicles collected during follicular phase of the cycle have a higher growing potential and GC viability compared with those retrieved in the luteal phase. During the in vivo follicular phase, up-regulation of FSH receptors on GC occurs, and interaction of these receptors with FSH at a high concentration induces follicular growth, through GC proliferation/differentiation and antrum formation (Richards et al. 2002).
Previous reports had indicated that the presence of FSH in the culture medium induces a significant increase in the diameter of feline secondary follicles recovered at random stages of the oestrous cycle (Jewgenow and Pitra 1993).
Therefore, it is possible that the higher growing potential and GC viability of follicles collected at follicular stage of the cycle might be due to exposure to gonadotropin priming in the ovarian environment before collection, which benefit from the presence of FSH into the culture medium, than those retrieved at the luteal phase.
It has also been shown that IGF-1 can act synergistically with FSH to enhance follicular development, although the mechanisms underlying this interaction remain uncertain (Richards et al. 2002). Because of this synergistic effect, improved results in growth and GC viability of feline preantral follicles were expected by adding IGF-1 to the culture medium.
However, these results showed that IGF-1 addition to the culture medium does not exert a beneficial effect on in vitro development and GC survival of follicles derived from follicular phase. In contrast, follicles collected at the luteal phase achieve similar GC viability rates and increase in diameter to those collected in the follicular phase and cultured with IGF-1.
Overall results indicated that similar increase in diameter and GC viability is obtained after 6 days of in vitro culture, regardless of the stage of the oestrous cycle, but a lower degeneration rate was observed in follicles cultured in presence of IGF-1 compared with those cultured in its absence.
The addition of IGF-I to the culture medium for in vitro culture of follicles of different mammalian species is still controversial. Some authors had reported an increased follicular growth and preservation of GC viability, while others have reported no beneficial effects of IGF-1 and/or FSH supplementation (Brito et al. 2012).
Supplementation of culture medium with growth factors for in vitro culture of preantral follicles collected from domestic cats at random stages of the oestrous cycle showed that a combination of IGF-1 and bFGF (basic fibroblast growth factor) increased GC proliferation (Jewgenow 1996). The authors suggest that the positive effect was mainly because of the presence of bFGF, while IGF-1 was involved in cellular activation of oocytes, which is modulated by EGF.
Complex interactions among growth factors are also involved in in vivo and in vitro follicular development, and the effect of these growth factors on ovarian follicles may vary between species, oestrous stage and culture conditions.
It should be also pointed out that in vivo follicular development in non-rodent species is a long-lasting process (Jewgenow and Paris 2006). Therefore, additional studies that evaluate the viability and functional integrity of the enclosed oocyte are needed for a better understanding of the requirements of feline follicles to growth in a long-term culture, and the role of IGF-1 and other growth factors in inducing the follicular transition from secondary to tertiary stage.
In conclusion, the present data suggest that in vitro growth and GC survival of feline preantral follicles are affected by the oestrous stage of the donor, and IGF-1 exerts a positive effect on follicles retrieved in the luteal phase of the oestrous cycle. Thus, hormonal environment of the follicles within the ovary might impact their potential development when isolated and cultured.
Further investigations on culture requirements for in vitro growth of follicles as a source of competent oocytes that can be used for in vitro embryo production are required.
The authors thank CNPq (Brazil) for financial support.
Conflicts of interest
None of the authors have any conflicts of interest to declare.