Theriogenology 84 (2015) 1–10 Contents lists available at ScienceDirect Theriogenology journal homepage: www.theriojournal.com Steady-state level of messenger RNA and immunolocalization of aquaporins 3, 7, and 9 during in vitro growth of ovine preantral follicles A.D. Sales a, A.B.G. Duarte b, G.Q. Rodrigues a, L.F. Lima a, G.M. Silva a, A.A. Carvalho a, I.R. Brito a, R.M.S. da Maranguape c, C.H. Lobo d, J.A.S. Aragão c, A.A. Moura d, J.R. Figueiredo a, A.P.R. Rodrigues a,* a Laboratory of Manipulation of Oocytes and Preantral Follicles, Faculty of Veterinary Medicine, State University of CearádUECE, Fortaleza, Ceará, Brazil b Department of Morphology, Federal University of CearádUFC, Fortaleza, Ceará, Brazil c Center for Biotechnology Sobral, NUBIS, Laboratory of Molecular Biology, Acaraú Valley State UniversitydUVA, Sobral, Ceará, Brazil d Department of Animal Science, Group of Research in Biology of Reproduction, Federal University of CearádUFC, Fortaleza, Ceará, Brazil article info abstract Article history: Aquaporins (AQPs) are a well-conserved family of small (approximately 30 kDa) membrane Received 10 October 2014 channel proteins that facilitate rapid movement of fluids and have a unique tissue-specific Received in revised form 3 December 2014 pattern of expression. These proteins have been found in the female reproductive systems Accepted 6 January 2015 of humans, rats, and mice. However, the expression and cellular localization of AQPs have not extensively been studied in the female reproductive system of sheep. Therefore, this Keywords: study aimed to evaluate, by real-time polymerase chain reaction and immunohistochem- Aquaporin istry respectively, the levels of messenger RNA and the immunolocalization of AQP3, AQP7, Folliculogenesis Antrum formation and AQP9 in large isolated ovine secondary follicles over a period of IVC. Our analysis Ovine revealed that AQP3 and AQP9 were present predominately in follicles that exhibited antrum formation, suggesting a crucial role of these AQPs in the formation of the antrum. Inter- estingly, AQP7 was only expressed in follicles that had not formed an antrum by Day 12 of culture. In conclusion, the presence of protein channels (AQP3 and AQP9) seems to be essential for the formation of the antrum in isolated ovine secondary follicles cultured in vitro and thus plays an important role during folliculogenesis in this species. Ó 2015 Elsevier Inc. All rights reserved. 1. Introduction [3], mouse [4], and sheep granulosa cells from different follicular categories [5]. Furthermore, there is evidence that Aquaporins (AQPs) are a well-conserved family of small steroid hormones produced by follicles regulate the (approximately 30 kDa) membrane channel proteins that expression of ovarian AQPs [6]. There is increasing evidence facilitate rapid movement of fluids [1]. A number of studies that AQPs play an important role during folliculogenesis, in ovaries have confirmed the presence of AQP1, AQP2, contributing to follicular development by facilitating the AQP3, AQP4, AQP7, AQP8, and AQP9 in the human [2],rat transport of water into the interior of the follicle. It has been reported in rat granulosa cells that the influx of water during the formation of the antral cavity does not occur exclusively by intercellular transport, but instead, pre- * Corresponding author. Tel.: þ55 8531019852; fax: þ55 8531019840. dominantly by transcellular transport, aided by the action E-mail address: [email protected] (A.P.R. Rodrigues). of these AQPs [3]. 0093-691X/$ – see front matter Ó 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2015.01.005 2 A.D. Sales et al. / Theriogenology 84 (2015) 1–10 Subsequent studies in murine secondary follicles 200 mm in diameter were visualized under a stereomicro- cultured in vitro by using an alginate hydrogel showed a scope (SMZ645; Nikon, Tokyo, Japan) and manually correlation between the expression of AQP7, AQP8, and dissected from the strips of ovarian cortex using 26-ga AQP9 and steroid production in healthy follicles [4]. needles. After isolation, follicles were transferred to Expression of AQP3 has been reported in mature mouse 100-mL drops of fresh medium under mineral oil for further oocytes [7], and an increased expression of this protein evaluation of follicular quality. Follicles with a visible during in vitro follicular development promoted cyto- oocyte surrounded by granulosa cells, an intact basement plasmic maturation, making them competent [8]. membrane, and no antral cavity were selected for culture Various IVC systems have been developed to maintain (secondary follicles). viability and promote growth of preantral follicles (PFs), to After selection, the secondary follicles were individually obtain mature oocytes for in vitro embryo production [9].In cultured in 100-mL drops of culture medium in Petri dishes sheep, studies have revealed that isolated secondary folli- (60 Â 15 mm; Corning, USA) under mineral oil for 6 or cles develop antral cavities from Day 6 of culture, regard- 12 days, at 39 C and 5% CO2 in air. The basic culture me- less of the composition of the culture medium [10]. Thus, dium (control) consisted of a-MEM (pH 7.2–7.4) supple- we believe that not only hormones such as FSH and growth mented with 3 mg/mL BSA, ITS (insulin, 10 mg/mL; factors such as leukemia inhibitory factor (LIF) and kit transferrin, 5.5 mg/mL; and selenium, 5 ng/mL), 2-mM ligand (KL) but also AQPs may be directly involved in the glutamine, 2-mM hypoxanthine, 50 mg/mL of ascorbic development of the antrum during folliculogenesis in acid, 50 ng/mL of LIF, and 50 ng/mL of KL, as described sheep. Recently, we have observed the presence of AQP3 previously [11]. Fresh medium was prepared immediately messenger RNA (mRNA) in antral follicles and a progressive before use and was incubated at 39 C for at least 2 hours expression of this protein in sheep ovarian follicles at before use. Every 6 days, 60 mL of each drop was replaced different stages of development (primordial, primary, sec- with the fresh medium [10]. The culture was replicated six ondary, and antral), and therefore, this provides a good times, and a mean of 50 follicles were used for each starting point to find the correlation between protein follicular category established after the period of IVC (6 and channels and antrum formation in this species [5]. 12 days), i.e., no antrum, antral, and extrused follicles. To contribute to a more comprehensive understanding Analysis of the RNA and protein expression of AQP3, AQP7, of the role of AQPs during follicular development, the and AQP9 was carried out using live follicles from these present study was conducted to evaluate the expression of three categories, after 6 and 12 days of culture. genes encoding AQP3, AQP7, and AQP9, and the immuno- localization of their respective proteins in isolated ovine 2.4. Morphologic evaluation of follicle development secondary follicles cultured in vitro for 6 and 12 days. Follicles were classified according to their morphology, 2. Materials and methods and only those showing an absence of morphologic signs of degeneration (such as darkness of the oocytes and sur- 2.1. Chemicals rounding cumulus cells, or misshapen oocytes) were clas- sified as surviving follicles. Every 6 days (Days 0, 6, and 12), Unless mentioned otherwise, culture media and other in addition to those mentioned above, the following char- chemicals used in the present study were purchased from acteristics were analyzed only in surviving follicles: (1) Sigma Chemical Co. (St. Louis, USA). antral cavity formation, defined as a visible translucent cavity within the granulosa cell layers and (2) the follicular 2.2. Source of ovaries diameter, measured as the mean of two perpendicular measures of each follicle using an ocular micrometer Ovaries (n ¼ 60) were collected at a local slaughter- (magnification: Â 100) inserted into a stereomicroscope house from 30 adult crossbreed ewes, making a total of six (SMZ645; Nikon). experimental repeats (five ewes/repeat). Immediately postmortem, the ovaries were washed in 70% ethanol fol- 2.5. Assessment of follicle viability by fluorescence lowed by two rinses with minimum essential medium microscopy (MEM) containing HEPES and antibiotics (100 mg/mL penicillin and 100 mg/mL streptomycin). The ovaries were Follicular viability was determined using live/dead transported to the laboratory within 1 hour in MEM at 4 C. fluorescence labeling. Those follicles cultured for 12 days (n ¼ 15) were incubated in 100-mL drops containing 2-mM 2.3. Isolation of secondary follicles and IVC conditions ethidium homodimer-1 and 4-mM calcein AM (Molecular Probes, Invitrogen, Karlsruhe, Germany) at 37C for 15 mi- In the laboratory, the surrounding fat tissue and liga- nutes. Then, the follicles were washed in MEM HEPES and ments were stripped from the ovaries. Ovarian cortical analyzed using an inverted fluorescence microscope slices (1–2 mm in diameter) were cut from the ovarian (Eclipse 80i; Nikon). The fluorescence signals emitted by surface using a surgical blade under sterile conditions. calcein AM and ethidium homodimer-1 were monitored at Then, the ovarian cortex was placed in a fragmentation 488 and 568 nm, respectively. The oocytes and granulosa medium, consisting of MEM supplemented with HEPES and cells were considered viable when the cytoplasm was antibiotics (100 mg/mL penicillin and 100 mg/mL strepto- positively marked by calcein AM (green), and chromatin mycin). Preantral follicles that were approximately at least was not marked by ethidium homodimer-1 (red). A.D. Sales et al. / Theriogenology 84 (2015) 1–10 3 2.6. Real-time PCR 60 C, 1 minute at 72 C) and a final extension step for 10 minutes at 72 C. The specificity of each primer set was 2.6.1. Total RNA isolation determined using melt curve analysis, carried out between For RNA isolation, three pools of 10 follicles (no antrum, 60 C and 95 C for each primer pair.