Introduction
Maturation of oocytes and the subsequent outcome of the embryo in vitro production are affected by the composition of the maturation medium (Rose & Bavister, Reference Rose and Bavister1992; Ali & Sirard, Reference Ali and Sirard2002a). The use of commercially available serum as a macromolecular component to improve oocyte maturation is a common practice in the in vitro production of bovine embryos. Although serum contains beneficial substances for embryo development, such as growth factors, chelators of heavy metals and components that support cumulus cell expansion (Abe & Hoshi, Reference Abe and Hoshi2003), its presence in in vitro culture medium has been associated with decreased granulosa cells steroidogenesis (Gutierrez et al., Reference Gutierrez, Campbell and Webb1997). As inhibition of endogenous steroid production drastically decreased the percentage of metaphase-II oocytes and completely abolishes cumulus expansion in bovine cumulus–oocyte complexes (COCs) (Wang et al., Reference Wang, Isobe, Kumamoto, Yamashiro, Yamashita and Terada2006), the maintenance of an in vivo-like steroidal secretion might be beneficial for the full in vitro development of oocytes. As it is not entirely clear whether COCs aromatase activity is compromised by the presence of the serum in in vitro maturation (IVM) medium or by the absence of an adequate substrate milieu, we evaluated here if an androgen-supplemented medium supports bovine COCs steroidogenesis after 24 h of culture in the presence of serum.
Materials and methods
All the chemicals used in this study were purchased from Sigma Chemical (St Louis, MO, USA), unless otherwise indicated.
Ovaries
Bovine ovaries from random stages of the estrous cycle were collected from a local abattoir immediately after slaughter and transported to the laboratory within 2 h. During transit, they were maintained at 25–30 °C in 0.9% normal saline supplemented with penicillin G (100 IU/ml) and streptomycin (100 μg/ml). Upon arrival at the laboratory, ovaries were freed from ligaments and rinsed several times in pre-warmed phosphate-buffered saline (PBS) supplemented with penicillin G (100 IU/ml) and streptomycin (100 μg/ml).
Selection of cumulus–oocyte complexes (COCs)
COCs collection and selection was based on a previously published protocol (Viana et al., Reference Viana, Camargo, Ferreira, Sa, Fernandes and Junior2004). Briefly, under sterile conditions, COCs were aspirated with a syringe and needle from 2–8-mm follicles, washed in HEPES-TALP medium (Gibco/BRL, Grand Island, NY, USA) and morphologically classified according to oocyte cytoplasm aspect and number and morphology of cumulus cell layers, as follows: degree I – compact COCs, more than three layers of cumulus cells and oocyte with homogeneous cytoplasm; degree II – compact COCs with three or less layers of cumulus cells and oocyte with slightly heterogeneous cytoplasm.
In vitro maturation (IVM)
The basic maturation medium was HEPES-buffered (5 mM) tissue culture medium-199 (TCM-199, Gibco/BRL, Grand Island, NY, USA) supplemented with 10% fetal calf serum (FCS; Invitrogen Gibco/BRL), 0.2 M sodium pyruvate, 100 IU/ml penicillin G, 100 μg/ml streptomycin, 0.5 μg/ml follicle-stimulating hormone (FSH; Follitropin-Bioniche, Canada) and 5 μg/ml luteinizing hormone (LH; Lutropin-Bioniche, Canada) as previously published (Vireque et al., Reference Vireque, Camargol, Serapião, Rosa e Silva, Watanabe, Ferreira, Navarro, Martins and Ferriani2009). Degrees I and II selected COCs were randomly divided into two experimental groups: (a) non-androgen group, COCs cultured in the absence of any androgen substrate; and (b) androgen group, COCs cultured in the presence of 1 μM androstenedione. The concentration of androstenedione used herein was chosen based on the dose known to induce in vitro estradiol production by bovine granulosa cells (Hamel et al., Reference Hamel, Vanselow, Nicola and Price2005) similar to the concentration found in the follicular fluid of bovine antral follicles (Ireland & Roche, Reference Ireland and Roche1983). For culture, four-well plastic tissue culture dish (Nunclon®, Nunc, AS Roskilde, Denmark) containing 400 μl of maturation medium and 40 COCs per well were used. The COCs (40 COCs in each treatment/replicate) were cultured for 24 h in a humid atmosphere containing 5% CO2 in air at 38.5 °C. At the end of the culture period medium from 11 replicates were collected and stored at –20 °C for steroid measurement.
Measurement of steroids
Estradiol (E2) and progesterone (P4) levels that accumulated in the maturation culture medium during 24 h of culture were determined using commercially available chemiluminescence kits (DPC; Immulite System, Los Angeles, CA, USA). Assays for steroids were carried out in accordance with the manufacturer's protocol using duplicate samples. All samples for each steroid hormone were determined in a single assay in order to avoid inter-assay variability. The intra-assays coefficients of variation were less than 15% for estradiol and 10% for progesterone. The analytical sensitivities of the estradiol and progesterone kits were 15 pg/ml and 0.1 ng/ml, respectively.
Statistical analysis
Data are presented as mean ± standard error of the mean (SEM). Differences in E2 and P4 levels between groups were analyzed by Student's t-test. A P-value < 0.05 was considered to be statistically significant.
Results
Steroidal profile of bovine COCs cultured in the presence of aromatizable androgen. Figure 1 shows E2 (A) and P4 (B) concentrations in the IVM medium of bovine COCs cultured in the presence or absence of 1 μM androstenedione. Medium E2 content significantly increased (P < 0.0001) after incubation of COCs with androstenedione (17.52 ± 1.86 ng/ml to androgen group; 0.32 ± 0.05 ng/ml to non-androgen group; Fig. 1A). No significant difference in the P4 content (P = 0.4088) was detected despite the presence of androstenedione (21.83 ± 1.61 ng/ml to androgen group; 21.73 ± 1.67 ng/ml to non-androgen group, Fig. 1B).
Figure 1 Steroidal profile of bovine cumulus–oocyte complexes (COCs) cultured in the presence of aromatizable androgen. Concentrations of estradiol (A) and progesterone (B) after 24 h of bovine COCs cultured in serum-supplemented IVM medium in the presence (androgen group) or absence (non-androgen group) of 1 μM androstenedione. Data are shown as mean ± standard error of the mean (SEM); ***P < 0.0001.
Discussion
In this short report we showed that the addition of androstenedione to the bovine COCs IVM medium provides an aromatizable androgen source to estrogen production. These data may suggest that serum-decreased COC steroidogenesis in vitro may be caused by deficiency of androgen substrate rather than by disturbed steroidal enzyme activity. As serum decreases FSH receptor mRNA in the bovine COCs during IVM (Calder et al., Reference Calder, Caveney, Sirard and Watson2005), it has been proposed that serum-induced low COC steroid secretion may occur through inhibition of FSH action, as cytochrome P450 aromatase (P450arom) in the granulosa cells converts androgens to 17β-estradiol under the regulation of FSH (Xu et al., Reference Xu, Garverick, Smith, Smith, Hamilton and Youngquist1995). The fact that bovine COCs cultured in androgen-supplemented medium with 0.5 μg/ml FSH produce considerable amounts of E2, as shown presently, suggests that the FSH-dependent aromatase activity remains functional after 24 h of culture despite the presence of serum. We can also suggest that androstenedione addition into the IVM medium may restore serum-decreased COCs steroidogenesis acting as an aromatizable androgen source for estradiol synthesis or inducing augments in the P450arom activity. In fact, androstenedione has been shown to increase estradiol secretion by bovine non-luteinizing granulosa cells through induction of increased P450arom expression (Hamel et al., Reference Hamel, Vanselow, Nicola and Price2005). According to these authors, this action is specifically directed towards P450arom, as androstenedione had no effect on P450 side-chain cleavage or 3β-hydroxysteroid dehydrogenase enzyme mRNA levels as well as on progesterone secretion. Our findings of unchanged progesterone levels in the bovine COCs IVM medium also confirms that the route of steroidogenesis process within supplementation with aromatizable androgen is directed to estradiol production. Further studies are needed to demonstrate a role for androstenedione in the activity of P450arom during culture of bovine COCs in serum-supplemented medium. As the inclusion of exogenous estradiol to the bovine IVM medium has been positively associated with improvement of oocyte competence and embryonic development (Beker-van Woudenberg et al., Reference Beker-van Woudenberg, Zeinstra, Roelen, Colenbrander and Bevers2006; Ali & Sirard, Reference Ali and Sirard2002b), we suggest that the maintenance of an active COCs steroidal function during IVM might be beneficial for the in vitro bovine oocyte maturation. In addition, it is unclear whether a low or high capacity of COCs aromatization during IVM can distinctly affect the acquisition of oocyte competence or embryonic development. These assessments are being studied and are underway in our laboratory. In conclusion, we have provided evidence that the aromatase activity of bovine COCs retains high estrogenic capacity after 24 h of IVM in the presence of serum and that decreased estradiol production by COCs is mainly caused by deficiency of an aromatizable androgen source. These findings are of importance and are relevant to the application of in vitro oocyte maturation methods in all species, including human, as serum is a common additive of oocyte maturation medium.
Financial support
This study was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo–FAPESP.
Acknowledgements
We are grateful for the technical support of the ART core and the Endocrine Services at the Department of Obstetrics and Gynecology, Faculty of Medicine of Ribeirão Preto, Brazil.
