ilable at ScienceDirect Theriogenology 86 (2016) 523–527 Contents lists ava Theriogenology journal homepage: www.ther io journal .com Expression of androgen-producing enzyme genes and testosterone concentration in Angus and Nellore heifers with high and low ovarian follicle count Bárbara Loureiro a, Ronaldo L. Ereno b, Mauricio G. Favoreto a, Ciro M. Barros b,* a Laboratory of Animal Reproductive Physiology, University of Vila Velha (UVV), Vila Velha, Espírito Santo, Brazil bDepartment of Pharmacology, Institute of Biosciences, São Paulo State University (UNESP), Botucatu, São Paulo, Brazil a r t i c l e i n f o Article history: Received 3 August 2015 Received in revised form 12 January 2016 Accepted 1 February 2016 Keywords: Ovary Follicle Testosterone Androgen enzymes * Corresponding author. Tel.: þ55 14 997765624; f E-mail address: barrosciro@gmail.com (C.M. Bar 0093-691X/$ – see front matter � 2016 Elsevier Inc http://dx.doi.org/10.1016/j.theriogenology.2016.02.0 a b s t r a c t Follicle population is important when animals are used in assisted reproductive programs. Bos indicus animals have more follicles per follicular wave than Bos taurus animals. On the other hand, B taurus animals present better fertility when compared with B indicus ani- mals. Androgens are positively related with the number of antral follicles; moreover, they increase growth factor expression in granulose cells and oocytes. Experimentation was designed to compare testosterone concentration in plasma, and follicular fluid and androgen enzymes mRNA expression (CYP11A1, CYP17A1, 3BHSD, and 17BHSD) in follicles from Angus and Nellore heifers. Heifers were assigned into two groups according to the number of follicles: low and high follicle count groups. Increased testosterone concen- tration was measured in both plasma and follicular fluid of Angus heifers. However, there was no difference within groups. Expression of CYP11A1 gene was higher in follicles from Angus heifers; however, there was no difference within groups. Expression of CYP17A1, 3BHSD, and 17BHSD genes was higher in follicles from Nellore heifers, and expression of CYP17A1 and 3BHSD genes was also higher in HFC groups from both breeds. It was found that Nellore heifers have more antral follicles than Angus heifers. Testosterone concen- tration was higher in Angus heifers; this increase could be associated with the increased mRNA expression of CYP11A1. Increased expression of androgen-producing enzyme genes (CYP17A1, 3BHSD, and 17BHSD) was detected in Nellore heifers. It can be suggested that testosterone is acting through different mechanisms to increase follicle development in Nellore and improve fertility in Angus heifers. � 2016 Elsevier Inc. All rights reserved. 1. Introduction It is well known that Bos indicus and Bos taurus animals behave differently when referring to reproduction [1]. Taurine animals reach sexual maturity earlier [2,3] and have smaller calving intervals than indicine females [4]. More- over, their estrus ismore evident and last formore hours [5]. ax: þ55 14 38116253. ros). . All rights reserved. 01 On the other hand, B indicus animals develop considerably more follicles per follicular wave than B taurus animals [6,7]. This feature is especially important for animals that are used in assisted reproductive technologies such as superovula- tion, ovum pick-up, and in vitro embryo production. The estrus cycle and follicular development are stimu- lated by hormones such as follicle-stimulating hormone (FSH), luteinizing hormones, and estrogen. Although in reduced concentrations, androgens are also important to promote follicular growth. They are synthesized by the thecal cells under the influence of luteinizing hormone mailto:barrosciro@gmail.com http://crossmark.crossref.org/dialog/?doi=10.1016/j.theriogenology.2016.02.001&domain=pdf www.sciencedirect.com/science/journal/0093691X http://www.theriojournal.com http://dx.doi.org/10.1016/j.theriogenology.2016.02.001 http://dx.doi.org/10.1016/j.theriogenology.2016.02.001 B. Loureiro et al. / Theriogenology 86 (2016) 523–527524 through the enzymatic conversion of androgen precursors [8]. In the follicular fluid, androgens can act by increasing FSH actions, or serve as a substrate to the production of estradiol by aromatase [9]. In bovines, androgen concentrations are positively related with the number of antral follicles in the ovary [10]. Furthermore, treatment with androgens increased the expression of growth factors in granulosa cells and oocytes of monkeys [11] and mice [8,12]. In vitro, treatment of pre- antral follicles with androgen increased follicle develop- ment and decreased apoptosis [13]. In addition, culture of granulosa cells with testosterone decrease mRNA and protein levels of anti-Mullerian hormone, a hormone that is known to hinder follicle development [14]. The present study aimed to evaluate expression of androgen-producing enzyme genes in antral follicles collected from Angus and Nellore heifers with low and high ovarian follicle count and measure plasma and intra- follicular testosterone concentration in these animals. 2. Materials and methods All reagents andmedia usedwere obtained from Sigma– Aldrich (St. Louis, MO, USA) unless stated otherwise. Animals were housed and cared for in accordance with the guidelines described by the ethics committee of the Universidade do Estado de São Paulo (Botucatu). 2.1. Animal selection To pre-select the animals an ultrasound (US) examina- tion (Mindray, 5–10 MHz, China) was performed in a random day of the estrus cycle in 100 Angus and 100 Nellore heifers. Only heifers that were cycling and did not have follicles greater than 5 mmwere selected. In this first evaluation, the total number of follicles in both ovaries was counted and the mean follicle number was determined for each breed. These heifers were synchronized with two doses of PGF2a 11 days apart. Four days after the second PGF2a (approximately 24 hours after follicle recruitment), a second US evaluation was performed to confirm the total number of follicles in each heifer. Considering the mean� standard deviation (SD) of the follicle population in each breed, the heifers were classified into two groups as follows: the low follicle count group (LFC; heifers that had the total number of follicles below the mean minus the SD) and high follicle count group (HFC; heifers that had the total number of follicles above the mean plus the SD). A total of 18 Nelore heifers were selected and allocated into two groups: eight with HFC (�40 follicles) and 10 with LHC (�20 follicles). For Angus heifers, 22 were selected and allocated into two groups: 13 with HFC (�20 follicles) and 09 with LFC (�13 follicles). Heifers were kept in Brachiaria brizantha grass ad libitum, were fed 2 kg of Cynodon spp hay and 4 kg of concentrate (16% crude protein and 70% total digestible nutrients)/per animal/day during 90 days. After this adap- tation period, all heifers were yet again synchronized using the same protocol (two doses of PGF2a 11 days apart). When heifers showed estrus, they were evaluated by US every 12 hours until ovulation. Twenty-four hours after ovulation, heifers were slaughtered in a local abattoir. After slaughter, ovaries were collected and placed in a saline solution at 4 �C to be transported to the laboratory. Three follicles from 2 to 4 mm in diameter were dissected from the ovary contralateral to the corpus luteum. Follicles were frozen individually in 1-mL Qiazol from the RNeasy Microarray Tissue Mini kit (Qiagen, Valencia, CA, USA) at �80 �C. 2.2. Real-time polymerase chain reaction This experiment was designed to evaluate the mRNA expression of androgen-producing enzymes. Twenty-two Angus heifers (LFC ¼ 09 and HFC ¼ 13) and 18 Nellore heifers (LFC ¼ 10 and HFC ¼ 08) were used in this experi- ment (these heifers were selected out of the 200 heifers US at the beginning). Total RNA was extracted individually from three follicles from each animal using the RNeasy Microarray Tissue Mini kit following the manufacturer’s instructions. Follicles were homogenized with an Ultra- Turrax (IKA, Campinas, SP, Brazil) for 1 minute in 1 mL of QIAzol. Total extracted RNAwas stored at �80 �C until real- time polymerase chain reaction analysis. RNA samples (1 mg) were incubated with DNAse I (1 U/mg; Invitrogen) and reverse transcribed with SuperScript III (Invitrogen) and oligo-dT primers. Quantitative real-time polymerase chain reaction anal- ysis of four androgen-producing enzyme genes (Cholesterol side chain cleavage enzyme [CYP11A1], 17-alpha-hydroxy- lase [CYP17A1], 3-beta-hydroxysteroid dehydrogenase [3BHSD], and 17-beta-hydroxysteroid dehydrogenase [17BHSD]) and a housekeeping gene (peptidylprolyl isom- erase A; PPIA) was performed on each follicle. Specific primers (Table 1) were designed using Integrated DNA Technologies software (http://idtdna.com). To select the most stable housekeeping gene for detailed analysis of each cell type, PPIA, glyceraldehyde-3-phosphate dehydrogenase, and histone H2AFZ (H2AFZ) amplification profiles were compared using the geNorm applet for Microsoft Excel (http://genorm.cmgg.be) [15]. On the basis of this compar- ison, the relative quantification was performed with PPIA. Power SybrGreen PCR Master Mix (Applied Biosystems) reaction chemistry and the ABI Prism 7500 Sequence Detection System (Applied Biosystems) were used to quantify mRNA concentrations, and the specificity of each polymerase chain reaction productwas determined through melting curve analysis. Negative controls (in which water replaced complementary DNA) were run in each plate. Duplicate reactions of each sample were analyzed. Target gene mRNA abundance is expressed relative to the level of PPIA mRNA. The relative expression of each gene was determined using the DDCt method. Results are expressed as fold change (2�DDCT). 2.3. Plasma and follicular fluid testosterone concentration Blood for the testosterone was drawn on the day of slaughter (24 hours after ovulation), centrifuged for 10 minutes at 900 � g, and plasma was frozen at �80 �C. The follicular fluid was collected from three 2 to 5 mm follicles http://idtdna.com http://genorm.cmgg.be Table 1 Primers sequences. Genes Primer sequence (50- 30) Amplicon size Annealing temperature CYP11A1 FdAGT CCA CAC CTC TTG CAC CTT TCT RdCGC CCA TCC CAT GAA GGC AAT AAA 140 pb 59 �C CYP17A1 FdGAA TGC CTT TGC CCT GTT CA RdCGC GTT TGA ACA CAA CCC TT 330 pb 62 �C 3BHSD FdGCC CAA CTC CTA CAG GGA GAT RdTTC AGA GCC CAC CCA TTA GCT 135 pb 59 �C 17BHSD FdAGT CCA CAC CTC TTG CAC CTT TCT RdCGC CCA TCC CAT GAA GGC AAT AAA 103 pb 58 �C PPIA FdGCC ATG GAG CGC TTT GG RdCCA CAG TCA GCA ATG GTG ATC T 65 pb 60 �C B. Loureiro et al. / Theriogenology 86 (2016) 523–527 525 from the corpus luteum contralateral ovary. It was diluted 1:100 in the dilution buffer from the kit. Testosterone concentration was measured by an enzyme-linked immu- nosorbent assay (Uscn Life Science Inc., Wuhan, China), following manufacture’s instruction, in 17 Angus heifers (LFC ¼ 08 and HFC ¼ 09) and 18 Nellore heifers (LFC ¼ 10 and HFC ¼ 08). Plate reading was done using Biotech Wave HTMicroplate Spectophotrometer (Bio Tek, Winooski, USA) with 450 nm. 2.4. Statistical analysis Gene expression was compared using the DDCt values with the Proc Mixed procedure of SAS (SAS for Windows, version 9.2, Cary, NC, USA). An initial analysis to test the effect of follicle diameter on gene expression was per- formed. Because follicle diameter did not influence gene expression between Nelore and Angus or between HFC and LFC groups, a second analysis was performed with the mathematical model including two main effects (breed and follicle count) and all interactions with the animal as the subject and follicle as the repeated measure. Differences in individual mean values were analyzed through pair-wise comparisons (probability of difference analysis; SAS). Data on the testosterone plasma and intrafollicular concentration were analyzed by least-squares analysis of variance using the general linear models procedure of SAS. Testosterone concentration was log transformed before analysis. All values are reported as the least-squares mean � standard error of the mean. Differences were considered significant when P < 0.05, and values of P � 0.05 and� 0.1 were taken to indicate a tendency. 3. Results According to US analysis, Nellore heifers had more (P < 0.05) follicles (32 � 3.1) than Angus heifers (20 � 2.6). LFC Nellore heifers had 16 � 1.54 follicles, whereas HFC Nellore heifers had 52 � 1.70 follicles. LFC Angus heifers had 10 � 1.41, whereas HFC Angus heifers had 27 � 1.24. 3.1. Androgen-producing enzyme mRNA expression The results of the androgen-producing enzyme mRNA expression in follicles from Angus and Nellore heifers with LFC and HFC are expressed in the graphs as fold change normalized to one (Fig. 1). There was no effect of follicle size on mRNA expression in any of the genes studied. The mRNA expression of CYP11A1, the side chain cleav- age enzyme, was higher in follicles from Angus heifers when compared with follicles from Nellore heifers; how- ever, no difference was found within groups or an inter- action (Fig. 1A). The mRNA expression of the enzymes CYP17A1, 3BHSD, and 17BHSD was higher in follicles from Nellore heifers when compared with follicle dissected from Angus heifers (Fig. 1B–D). In addition, mRNA expression of CYP17A1 and 3BHSD was higher in follicles from the HFC groups in both breeds (Fig. 1B and C). There was no interaction between breeds and groups. 3.2. Plasma and intrafollicular testosterone concentration As listed in Table 2, plasma testosterone concentration was higher (P < 0.001) in Angus heifers when compared with Nellore heifers. There was no difference between follicle count groups within breeds. Intrafollicular testos- terone concentration was also higher in Angus animals (P< 0.02) when compared with Nellore animals. There was no difference between follicle count groups within breeds. 4. Discussion Androgens are important to promote follicle develop- ment. They are involved in the increased number of pre- antral and small antral follicles [16]. Furthermore, they affect the quality of the developing follicle and oocyte through the increase in the gene expression of important growth factors [8,11,12]. In this study, we found that Nellore heifers have more antral follicles than Angus heifers. This trait is important when using biotechnologies such as superovulation and follicle aspiration for in vitro embryo production. The suc- cess of these technologies is dependent on the number and quality of oocytes collected. Testosterone concentrationwas higher in Angus heifers, in both plasma and intrafollicular fluid. Expression of CYP11A1 gene was also higher in follicles from Angus heifers. This enzyme catalyzes the first step in the biosyn- thesis of all steroid hormones through oxidation of cholesterol [17]. However, there was no difference within low or high follicle heifers. Fig. 1. The mRNA expression (mRNA fold change, normalized to one) of androgen-producing enzymes CYP11A1, CYP17A1, 3BHSD, 17BHSD in follicles from Angus and Nellore heifers with low and high follicle count. B. Loureiro et al. / Theriogenology 86 (2016) 523–527526 The mRNA expression of enzyme CYP17A1 (responsible for conversion of pregnenolone into dehydroepiandroster- one and androstenedione) and 3BHSD (involved in the conversion of 3b-hydroxysteroids, pregnenolone, 17a- hydroxypregnenolone, and dehidroepiandrosterone into 3-ketosteroids, progesterone, 17a-hydroxyprogesterone, and androstenedione, respectively) was higher in follicles dissected fromNellore heiferswhen comparedwith follicles fromAngus heifers. In addition, in both breeds, follicles from HFC groups had higher mRNA expression of these enzymes whencomparedwith follicles fromtheLFCgroup. ThemRNA expressionof17BHSD, anenzymethat catalyzes thefinal step in the biosynthesis of testosterone, was also higher in folli- cles from Nellore heifers but not different within groups. Evenwith a higher mRNA expression of three androgen- converting enzymes (CYP17A1, 3BHSD, and 17BHSD), Nellore heifers had lower concentrations of plasma and intrafollicular testosterone. It is possible that the increased Table 2 Number of animals in each breed and follicle count group (mean � SD), plasma analyzed by ELISA. Variables Angus Total LFC HF Animals (n) 17 08 09 Mean follicle 20 � 2.6a 10 � 1.41 Intrafollicular testo (ng/mL) 46.08 � 4.6c 42.95 � 7.4 49 Plasma testo (ng/mL) 6.80 � 0.84e 7.49 � 1.34 6 Different letters in the same line indicate significant differences. Abbreviations: HFC, high follicle count; LFC, low follicle count; LS, least squares; mRNA expression of CYP11A1, the first enzyme in the ste- roids pathway and the only one that was increased in Angus heifers in this study, is responsible for the increased plasma and intrafollicular concentration in testosterone seen in Angus heifers. Androgens are important for follicle and oocyte quality. Female monkeys treated with androgen showed increased mRNA expression of FSH and IGF1 receptors and IGF1 in the granulose cells, and increased mRNA expression of IGF1 and IGF1 receptor in theoocyte [11].Mice treatedwithandrogens also showed increased mRNA expression of IGF1 receptor in oocytes of primordial follicles [12]. In addition, mRNA expression of GDF9 and TGFB was induced by androgens in granulose cells and oocytes [8]. These growth factors can improve quality and fertility capacity of the oocytes [18]. The higher testosterone concentration seen in this study might be responsible for the greater reproductive qualities reported for B taurus animals. B taurus and crossbred and intrafollicular testosterone (testo) concentratrion (LS mean � SEM) Nellore C Total LFC HFC 18 10 08 27 � 1.24 32 � 3.1b 16 � 1.54 52 � 1.70 .20 � 5.7 29.25 � 4.8d 22.77 � 6.4 35.73 � 7.4 .10 � 1.02 1.50 � 0.92f 1.78 � 1.12 1.22 � 1.45 SD, standard deviation; SEM, standard error of the mean. B. Loureiro et al. / Theriogenology 86 (2016) 523–527 527 taurus � indicus animals usually present younger age at puberty, longer estrus duration, shorter calving interval, and better pregnancy rates when compared with pure B indicus animals [1,4]. On the other hand, Nellore heifers presented greater folliclenumbers. Androgenshavebeenpreviouslyassociated with pre-antral and antral follicle growth [10]. Treatment of female monkeys with androgens increased the number of pre-antral follicles and small antral follicles through androgens receptors [16]. Mossa et al. [10] also showed that mRNA expressions of CYP17A1 were higher in HFC animals. However, in their study, androstenedione concentration in the follicular fluid and testosterone concentration in the plasma were higher in HFC cows, although we found no difference between groups within breeds. It is worth mentioning that study by Mossa et al. used only crossbred Hereford � Angus � Charolais animals. Furthermore, it has been shown in women that testosterone concentration is directly related to the follicle sensitivity to FSH [19]. We concluded that Nellore heifers have more antral follicles than Angus heifers, and that in both breeds heifers can be classified in LFC or HFC groups. Moreover, Nellore heifers have increased mRNA expression of androgen- producing enzymes (CYP17A1, 3BHSD, and 17BHSD), with CYP17A1 and 3BHSD also higher in HFC heifers. On the other hand, testosterone concentration was higher in Angus heifers; this increase can be associated with the increased expression of CYP11A1. It could be suggested that testos- terone is acting through different mechanisms to increase follicle development in Nellore and improve fertility in Angus heifers. Acknowledgments This workwas supported by FAPESP grant number 2011/ 50964-0. B.L., R.L.E., and M.G.F. were granted a FAPESP scholarship. 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Introduction 2. Materials and methods 2.1. Animal selection 2.2. Real-time polymerase chain reaction 2.3. Plasma and follicular fluid testosterone concentration 2.4. Statistical analysis 3. Results 3.1. Androgen-producing enzyme mRNA expression 3.2. Plasma and intrafollicular testosterone concentration 4. Discussion Acknowledgments Competing Interests References