Introduction
The brown brocket deer (Mazama gouazoubira) is a species that adapts itself to a wide range of environments (Black-Décima et al., Reference Black-Décima, Rossi, Vogliotti, Cartes, Maffei, Duarte, Juliá, Duarte and González2010). According to the IUCN Red List of Threatened Species (IUCN, 2016), it is classified as ‘least concern’. However, in some regions of Brazil, it is considered as ‘endangered’ (SEMA, 1998). In situ and ex situ conservation programmes can benefit from reproductive biotechnologies, including artificial insemination and in vivo embryo production. Therefore, some recent studies have focused on its reproduction and physiology (Pereira et al., Reference Pereira, Polegato, Souza, Negrão and Duarte2006) as well as assisted reproductive techniques (ART; Zanetti and Duarte, Reference Zanetti and Duarte2011).
The determination of follicular fluid (FF) composition may be important to ascertain the microenvironment involved in follicle development and as an indication of the nutritional needs of the oocyte. In addition, selection of competent oocytes requires the identification of molecular markers present in the FF (Fahiminiya and Gérard, Reference Fahiminiya and Gérard2010), Several studies on the proteome of FF have been performed in bovine (Mortarino et al., Reference Mortarino, Vigo, Maffeo and Ronchi1999), canine (Fahiminiya et al., Reference Fahiminiya, Reynaud, Labas, Batard, Chastant-Maillard and Gérard2010), equine (Fahiminiya et al., Reference Fahiminiya, Labas, Roche, Dacheux and Gérard2011), swine (Sun et al., Reference Sun, Ping, Li, Sun, Yi, Chen, Li, Wang and Zhou2011), bubaline (Fu et al., Reference Fu, Huang, Wang, Chen, Huang, Lu, Liang and Zhang2016) and ovine (Wu et al., Reference Wu, Lin, Han, Wang, Chen, Liu and Huanget2018).
This work aimed to perform a preliminary study on the protein profile of FF of brown brocket deer and to verify possible differences between small/medium and large follicles.
Materials and methods
Experimental animals and FF collection
Five adult females received hormonal treatment by insertion of intravaginal devices impregnated with 330 mg progesterone (CIDR-G, Zoetis, Kalamazoo, MI, USA) for 8 days. On the day of CIDR insertion, females also received 0.25 mg of estradiol benzoate (Estrogin, Biofarm, Jaboticabal, Brazil) and on the fourth day of treatment 700 IU eCG (Folligon, Intervet, Boxmeer, The Netherlands). Females were fasted for 24 h, followed by anaesthesia with 5.0 mg/kg of ketamine hydrochloride (Vetbrans Saúde Animal, Jacareí, Brazil), 0.3 mg/kg of xylazine hydrochloride (Mallinckrodt Vet, Cotia, Brazil) and 0.5 mg/kg of medazolan (Cristália, Itapira, Brazil), by intravenous (i.v.) injection. Next, females were intubated and maintained on a superficial plane using isoflurane (Abbott, São Paulo, Brazil) according to Zanetti et al. (Reference Zanetti, Polegato and Duarte2010). Laparoscopy was performed using a 5-mm Hopkins laparoscope (Karl Storz, Tuttlingen, Germany). Follicles were visualized, punctured and fluid was collected using a 22-G needle connected to a vacuum pump (WTA, Cravinhos, Brazil) adjusted to 35 mmHg. For fluid collection, follicles were measured and classified as small/medium (≤ 3.5 mm) or large (>3.5 mm) (Figure 1). A skilled and experienced operator scrupulously avoided contamination with blood. Collection of FF without visible signs of blood (pink or red colour) was considered a success. Samples of FF were individualized (per animal and follicle size) and mixed with a protease inhibitor cocktail (10 μl/ml) (Sigma-Aldrich, St. Louis, MO, USA), according to Aquino-Cortez et al. (Reference Aquino-Cortez, Pinheiro, Lima, Silva, Mota-Filho, Martins, Rodriguez-Villamil, Moura and Silva2017). The samples was centrifuged at 3000 g at 4°C for 30 min and the supernatant was stored at −80°C until analysis.
Adult Mazama gouazoubira female (A), videolaparoscopy (B) and follicular puncture for fluid collection (C).
One-dimensional gel electrophoresis SDS-PAGE
The samples from one female were discarded due contamination with blood. Soluble protein content in FF samples was determined according to the Bradford method (Bradford, Reference Bradford1976). A volume of sample containing 15 μg of protein was mixed with sample buffer (0.125 M Tris–HCl, pH 6.8, 4% SDS, 20% (v/v) glycerol, 0.2 M DTT, 0.02% bromophenol blue), boiled for 90 s, and loaded into the wells of a stacking gel (4% acrylamide), laid on top of a 10% polyacrylamide resolving gel (GE Life Sciences, Piscataway, NJ, USA). In one well of the stacking gel, 10 μl of a 180–19 kDa molecular weight standard mix (BenchMark Prestained Protein Ladder, Invitrogen, CA, USA) was loaded to allow molecular weight estimation of the protein bands. An initial current at 150 V, 50 mA and 90 W was applied for approximately 1 h 45 min to enable proteins to migrate through the stacking gel. Gels were stained with colloidal Coomassie Blue G-250, rinsed with deionized water, and scanned using an Image Scanner II (GE Life Sciences). The gel images were analyzed using Quantity One 4.5 software (Bio-Rad, Hercules, CA, USA). After analysis of the digitalized gel images and identification of the bands of interest, bands were cut from the gels, sliced and subjected to in-gel digestion with trypsin (Promega, Madison, WI, USA). Briefly, bands were excised from the gels and transferred to clean tubes, and washed three times (15 min each wash) with 400 μl of a solution containing 50% acetonitrile and 50% ammonium bicarbonate (25 mM at pH 8.0). Then, the gel pieces were washed twice with 200 μl pure acetonitrile for 5 min and air dried at room temperature (20–25 ºC). Then, the gel pieces were incubated with trypsin (166 ng per band; Promega) for 20 h at 37 ºC. After incubation, peptides were extracted by three washes with 5% trifluoroacetic acid, and 50% acetonitrile in 50 mM ammonium bicarbonate. Supernatants containing the peptides were concentrated in the microtubes using a speed vacuum concentrator (Eppendorf, Hauppauge, NY, USA).
Mass spectrometry analysis and protein identification
Digested peptides were analyzed using an electrospray ionization-quadrupole-time of flight (ESI-Q-TOF) mass spectrometer (Thermo Scientific, Bremen, Germany). The peptides were injected the nano Acquity ultrapure liquid chromatography (UPLC) sample manager and chromatographic separation was performed using an EasySpray column (C18, 2 mm beads, 75 mm inner diameter) (Thermo Scientific) with a capillary of 25 cm bed length and flow rate of 0.3 ml/min. Mobile phases A and B consisted of 0.1% formic acid in water and 0.1% formic acid in acetonitrile, respectively. All solvents were mass spectrometry grade quality (Sigma-Aldrich). The flow rate was 0.3 ml/min, and the solvent gradient was 2% B to 30% B in 60 min, followed by 90% B for 20 min. Data were processed using a Protein Lynx Global Server (Waters Co., Milford, MA, USA) and were converted to peak list text files for database searching. Peptides were identified using the Mascot database (Matrix Science, Boston, MA, USA) by searching against the NCBI database. Searches were made according to the following criteria: maximum of one missed trypsin cleavage, mono-isotopic peptides with charge +1, +2 and +3, with variable modification of protein N-acetylation and oxidized methionine (M) residues and fixed variation of carbamidomethyl (C) residues using a tandem mass spectrometry (MS/MS) ion searching mode. Peptide mass tolerance and fragment mass tolerance were ±1.2 Da and ± 0.6 Da, respectively.
Statistical analysis
All data were compared by unpaired t-test. Data were expressed as mean ± standard deviation (SD) and a P-value < 0.05 was considered to be significant.
Results
From five females, 55 follicles were visualized and 44 (21 small/medium and 23 large) were punctured. The mean (± SD) number of small/medium and large follicles was 6.0 ± 4.4 and 5.0 ± 2.4, respectively (P > 0.05). Although care was taken to avoid blood contamination, for further analysis, samples from one female were discarded due to this condition.
On average, 71.5 µl of FF was obtained, distributed in small/medium (25.0 ± 15.0 µl) and large follicles (113.8 ± 98.1 µl). Protein concentration was similar (P > 0.05) in the two follicle categories: 49.2 ± 22.8 μg/μl (small/medium) and 56.7 ± 27.4 μg/μl (large).
Female #4, there did not have sufficient volume for small/medium follicles analysis. Therefore, for this female, visualization of the bands is not present in the gel (Figure 2). There were no significant differences in band intensities between the two follicular categories. The analysis of 1-D gels allowed the detection of 17 bands common to small/medium and large follicles. The best-defined bands (n = 13) were identified by MS/MS (Figure 2). Table S1 shows a detailed list of proteins with their accession numbers, scores, sequence coverage and molecular mass.
SDS-PAGE analysis of follicular fluid of Mazama gouazoubira females (♯1–4). S/M (small/medium) and L (large) follicles. Numbers in italic shows the different bands identified on the gel. MM: molecular mass (180–186 kDa).
Discussion
The study of FF components may contribute to an understanding of reproductive physiology of deer and for further use in ART. To our knowledge, there has been no previous information on the protein profile of FF in any species of deer. Therefore, this is the first study using SDS-PAGE combined with LC-MS/MS.
Follicular puncture by laparoscopy was efficient for FF collection. According to Ginther et al. (Reference Ginther, Kot, Kulick and Wiltbank1997), an unsuccessful aspiration was the result of blood contamination of the FF. However, visual inspection provided a fast and relatively reliable method for determination of blood-contaminated FF (Levay et al., Reference Levay, Huyser, Fourie and Rossouw1997).
Proteins derived from blood serum (albumin, serotransferrin, inter-alpha-trypsin, haptoglobin, apolipoprotein and ceruloplasmin) were found in M. gouazoubira and had previously been described in FF of other species, such as bubaline (Fu et al., Reference Fu, Huang, Wang, Chen, Huang, Lu, Liang and Zhang2016), bovine (Ferrazza et al., Reference Ferrazza, Garcia, Schmidt, Mihm Carmichael, Souza, Burchmore, Sartori, Eckersall and Ferreira2017), caprine (Paula Junior et al., Reference Paula Junior, Van Tilburg, Lobo, Monteiro-Moreira, Moreira, Melo, Souza-Fabjan, Araújo, Melo, Teixeira, Moura and Freitas2018) and ovine (Wu et al., Reference Wu, Lin, Han, Wang, Chen, Liu and Huanget2018). During the final stage of folliculogenesis, the composition of FF is influenced by the blood–follicle barrier, which allows passage of low-molecular-weight proteins (Fahiminiya and Gérard, Reference Fahiminiya and Gérard2010).
Other detected proteins were plasma proteins from the acute phase of inflammation, such as complement factor (complement C3). In goats, this protein significantly increased as follicles developed from medium to large size (Paula Junior et al., Reference Paula Junior, Van Tilburg, Lobo, Monteiro-Moreira, Moreira, Melo, Souza-Fabjan, Araújo, Melo, Teixeira, Moura and Freitas2018), explaining the inflammation process promoting follicle wall breakdown during ovulation (Fu et al., Reference Fu, Huang, Wang, Chen, Huang, Lu, Liang and Zhang2016).
Alpha-2-macroglobulin and haptoglobin, which are expressed during the physiological response to inflammation (Handrieder et al., Reference Handrieder, Nyakas, Naessén and Bergquist2008) were also observed in the FF of bubaline (Fu et al., Reference Fu, Huang, Wang, Chen, Huang, Lu, Liang and Zhang2016) and bovine (Ferrazza et al., Reference Ferrazza, Garcia, Schmidt, Mihm Carmichael, Souza, Burchmore, Sartori, Eckersall and Ferreira2017). Proteins with enzyme inhibitory function (serpin A3 and inter-alpha trypsin) were detected in this study as observed in buffalo females (Fu et al., Reference Fu, Huang, Wang, Chen, Huang, Lu, Liang and Zhang2016).
Interestingly, the FF of M. gouazoubira contained thyroxine-binding globulin and lactotransferrin, respectively, the principal carrier of thyroid hormone in serum (Murata et al., Reference Murata, Magner and Refetoff1986) and a multifunctional protein occurring in many biological secretions (Sánchez et al., Reference Sánchez, Calvo and Brock1992). However, these two proteins have not been described in the FF of farm animals.
This study was not able to detect any significant difference in protein content in the FF as follicles developed from small/medium to large size. We hypothesized that this result may be due to: (i) joining of small and medium follicles into one group; or (ii) if such differences occurred, it would probably be at a time very close to ovulation.
Finally, the current study may contribute to a further understanding of the physiological mechanisms underlying folliculogenesis. It would be interesting to use quantitative techniques such as shotgun proteomics to obtain more detailed information on the FF of brown brocket deer.
Supplementary material
To view supplementary material for this article, please visit https://doi.org/10.1017/S0967199419000741.
Financial support
This work was financially supported by a CAPES scholarship CNPq (grant number 461330/2014- 8) and FUNCAP) (grant number CI3-0093-0005580100/14).
Conflicts of interest
The authors hereby declare that there are no conflicting interests.
Ethics statement
This study was approved by the Ethical Committee for the Animal Use of the State University of Ceará (2437412/2016) and the Biodiversity Information and Authorization System (45727-1).
