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RESEARCH |
Department of Animal Sciences, The Ohio State University, 2027 Coffey Road, Columbus, Ohio 43210, USA
Correspondence should be addressed to H Cárdenas; Email: cardenas.21{at}osu.edu
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Abstract |
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-dihydrotestosterone (DHT, 10 mg), flutamide (1.5 g, an androgen receptor inhibitor), DHT (10 mg) plus flutamide (1.5 g), testosterone (10 mg), and testosterone (10 mg) plus flutamide (1.5 g). Ovarian follicles 
5 mm in diameter were evaluated on day 17 or 19, 24 h after receiving the last treatment dose. Follicles were classified as healthy (H), moderately atretic (MA), or very atretic (VA). Treatment with DHT increased (P<0.05) the numbers of H follicles relative to control gilts on days 17 and 19. DHT administration from days 13 to 16 diminished (P<0.05) the amounts of LH receptor (LHR) mRNA in H follicles from day 17 (relative amounts: 1.45±0.33 and 2.72±0.33 for DHT- and vehicle-treated gilts respectively). The effects of DHT on numbers of H follicles and LHR mRNA were not observed in gilts receiving DHT plus flutamide. Androgens did not influence numbers of MA, VA, and total follicles, or follicular estradiol-17β concentrations and amounts of FSHR mRNA. Treating gilts with DHT during follicular recruitment and selection did not induce changes in the numbers of total follicles 5 mm, but rather increased the numbers of healthy follicles in this follicular population in association with decreased amounts of LHR mRNA. |
Introduction |
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Among the factors that influence follicular development and ovulation rate, follicle-stimulating hormone (FSH) is important because it stimulates granulosa cell differentiation, steroidogenic function, and survival (Richards 1994, Chun et al. 1996, Markström et al. 2002) and follicular recruitment (Driancourt 2001, Hunter et al. 2004). Luteinizing hormone (LH) also appears important during late follicular development in pigs. Evaluations of follicular growth, serum concentrations of gonadotropins, and amounts of gonadotropin receptors suggested that ovarian follicles of pigs were highly dependent on LH for developing from 
5 mm in diameter onward (Lucy et al. 2001). Like FSH and LH, any factor that influences follicular processes such as cell proliferation, oocyte development, atresia, and responses to ovulatory stimuli might be able to modulate ovulation rate. These factors are numerous and among them are the steroid hormones, progesterone, estrogens, and androgens.
Androgens have been demonstrated to influence early follicular development in nonhuman primates (Weil et al. 1999) and cattle (Yang & Fortune 2006). Female mice lacking a functional androgen receptor (AR) gene had fewer ovarian follicles and offspring during early reproductive life than the wild type, and became infertile later because of complete loss of follicles (Shiina et al. 2006). Previous research from our laboratory demonstrated that administration of testosterone (T) or 5-dihydrotestosterone (DHT) to gilts during the follicular phase of the estrous cycle increased ovulation rate (Cárdenas & Pope 1994, Cárdenas et al. 2002a). The effects of DHT suggested stimulation through the AR and possible exclusion of estrogenic actions. It is not known, however, what organ, cell type, or molecular pathway is actually influenced by androgenic treatment. Whatever the mechanism, it is conceivable that the effects of androgens must be related, directly or indirectly, to at least one of the physiological processes that define ovulation rate; follicular recruitment and selection. In the present experiments, androgens were administered to gilts during follicular recruitment and selection (follicular phase of the estrous cycle) to determine androgenic effects on follicular numbers, health, and expression of the FSH receptor (FSHR) and LH receptor (LHR) genes.
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5 mm were not altered by the treatments and day of the estrous cycle (Table 1). The numbers of H follicles were influenced by treatment and day (no interaction was observed). DHT increased (P<0.05) the mean number of H follicles on days 17 and 19. The effect of DHT on numbers of H follicles was blocked when administered to gilts simultaneously with flutamide (F). The numbers of H follicles in gilts receiving T, T+F, or F were not different from vehicle-treated gilts (Fig. 1 and Table 1). Treatments did not influence the numbers of VA and MA follicles or the proportions of H, MA, or VA follicles (Fig. 1 and Table 1). Regarding day effects, numbers and proportions of H follicles were less (P<0.05) on day 17 than on day 19 of the estrous cycle, possibly because only follicles 5 mm were considered. Differently from H follicles, greater (P<0.05) numbers and proportions of MA follicles were observed on day 17 than on day 19. However, the numbers of VA follicles did not differ between days (Table 1).
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Discussion |
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5 mm in diameter, soon after the estimated completion of recruitment (day 17), provided no apparent indication that androgens induced incorporation of more follicles into the recruited pool.
Overall effects of androgens on follicular health were assessed by initially categorizing follicles according to health status and then comparing their numbers and proportions. DHT increased the numbers of H follicles by 5 on day 17 and 3 on day 19 (13% average increase across days). This effect was absent in gilts treated with DHT+F, which would be consistent with effects mediated by the AR. An increase in numbers of H follicles without a change in total numbers of follicles might be expected to occur concurrently with a decrease in numbers of atretic follicles. This decrease was observed only in terms of absolute values (no statistical significance) of numbers and proportions of MA and VA follicles in DHT- versus vehicle-treated gilts (Fig. 1 and Table 1). The numbers of H follicles in gilts treated with T were similar to those receiving DHT but compared with control gilts the difference only approached significance (P=0.09). Responses of different magnitude to DHT and T (also observed on LHR mRNA, Table 4) might be explained by the differences in biological activity between these two androgens (Grino et al. 1990) or possible interactions of androgenic and estrogenic effects elicited by T due to its conversion into estrogens.
Treatments did not induce changes in follicular size (diameter) within the categories of H, MA, and VA follicles. Therefore, any treatment effect including that of DHT on numbers of H follicles was not related to changes in follicular size. Associations between follicular size and ovulation rate have been described in pigs. For instance, smaller size of day 19 preovulatory follicles and greater ovulation rate were observed in Meishan sows when compared with Large-White hybrid sows (Miller et al. 1998). Smaller size of preovulatory follicles was interpreted as relatively fast follicular differentiation in preparation for ovulation (Hunter 2004). The difference in average diameter of H follicles between days 17 and 19 was 2 mm, which agrees with previous estimations of growth during late follicular development in the pig (1.1 mm/day, Dailey et al. 1976). Although the average diameter of MA follicles on day 19 was greater than on day 17, the difference between days (1.3 mm) was not as large as for H follicles. This might indicate that MA follicles on day 17 can continue to grow but at a slower rate than H follicles or that atresia started in a proportion of follicles after day 17. Unlike H and MA follicles, VA follicles from days 17 and 19 differed only slightly in average size.
In the present study, follicular fluid concentrations of E2 in follicles from the different categories of health were not influenced by treatments. Noteworthy is that the concentrations of E2 were measured 24 h after the last treatment and that E2 concentrations could have changed before 24 h and then returned to normal. Indeed, the previous research described a transient increase in serum concentrations of E2 after administration of 1 mg T on day 17 of the estrous cycle in gilts (Cárdenas & Pope 1994). Consequently, it might be assumed that enhancement of ovulation rate by androgens probably occurs independently of factors influencing the synthesis of E2 by ovarian follicles in a substantial and extended manner. Other researchers did not observe significant changes in plasma E2 concentrations in gilts having increased ovulation rate as a result of genetic selection (Mariscal et al. 1998).
Results on follicular numbers and health presented above suggest that androgen treatment increases the ovulation rate in gilts possibly by enhancing health among follicles of the recruited pool resulting in increased follicular selection. Results of a previous experiment in which DHT increased ovulation rate when administered during the post-recruitment period (day 17 to estrus) also suggested enhancement of follicular selection as a possible mechanism (Cárdenas et al. 2002a). Follicular health is regulated by interactions among multiple physiological processes that stimulate cell proliferation and survival or induce apoptotic cell death (Tsafriri & Braw 1984). Apoptosis-inducing factors that have been identified in pig granulosa cells include tumor necrosis factor (TNF)-, Fas ligand (TNF receptor superfamily member 6 or APO1), TNF related apoptosis-inducing ligand (also known as apoptosis-2, APO2), and APO3 (Manabe et al. 2004). Whether androgens influenced the actions of one of these factors, other components of the complex intracellular apoptotic pathways, or apoptosis inhibitory mechanisms (survival factors), is not known.
In a recent experiment, DHT and T administered to gilts during the follicular phase of the estrous cycle increased serum concentrations of FSH (Jiménez et al. 2006). Considering the effects of FSH on follicular development (see Introduction), an increase in its concentrations could mediate, at least in part, an increase in ovulation rate that is supported by enhancement of follicular health. However, other regulators of follicular development, whether or not related to FSH, could also be affected by DHT. Two important receptors were examined in Experiment 2, FSHR and LHR. No differences were observed among treatments in relative amounts of FSHR mRNA in H follicles collected on day 17 or 19. These results were somewhat unexpected because of a previous report of increased amounts of FSHR mRNA in preovulatory follicles from DHT-treated gilts (Cárdenas et al. 2002a). In this previous experiment, gilts were treated with DHT longer (day 13 to first day of estrus) and follicles were more developed at the time of evaluation (first day of estrus) than in the present experiment. Duration of treatment and advancement of follicular development might be important for the effects of DHT on FSHR mRNA because FSHR is actively regulated and its mRNA amounts remarkably decrease during the final days of follicular development in pigs (Lucy et al. 2001, Cárdenas et al. 2002a, present experiment). Although the influence of DHT on FSHR appears variable in our pig model, other investigators have observed positive effects of androgens on FSHR mRNA in monkey granulosa cells in vivo (Weil et al. 1999) and bovine granulosa cells in vitro (Luo & Wiltbank 2006).
Contrary to FSHR, which was unaltered by the treatments, amounts of LHR mRNA were 50% less in H follicles collected on day 17 from DHT-treated gilts compared with those receiving vehicle. Consistent with androgenic actions mediated by the AR, the inhibitory effect on LHR mRNA did not occur when gilts received DHT+F. This is a novel observation on regulation of LHR mRNA in ovarian follicles by administration of androgens. It is not known whether the effect of DHT was directly exerted on transcription of the LHR gene or indirectly by influencing other regulators. It is likely that DHT might have exerted its effects indirectly because no androgen response element has been reported in the LHR promoter. Some factors influencing LHR in ovarian follicles have been identified. LHR mRNA was enhanced synergistically by FSH and E2 in granulosa cells of hypophysectomized rats (Segaloff et al. 1990). Likewise, FSH increased LHR mRNA in pig cumulus cells (Okazaki et al. 2003). In addition, the LHR gene can be regulated by repression. For example, orphan receptors and histone deacetylase (Zhang & Dufau 2000, 2003) and an upstream initiator-like element (Youn et al. 2005) have been determined to inhibit expression of the LHR gene.
The temporary nature of the effect of DHT on LHR mRNA (the effect was observed on day 17 but not on day 19) indicates that other factor(s) might have influenced LHR mRNA as follicles approached final maturation, overcoming the effects observed on day 17. The factor(s) is as yet undetermined, but based on the results of Segaloff et al. (1990, see previous paragraph), FSH (increased after DHT treatment, Jiménez et al. 2006), and E2 (increased twofold from day 17 to 19 in follicular fluid, Table 3), might be candidate modulators of the LHR mRNA pattern observed in DHT-treated gilts.
Treatment of gilts with F alone also decreased the amounts of LHR mRNA. In rats, F treatment produced a significant decrease in testicular LH receptors (Marchetti & Labrie 1988) but had no effects on ovarian LH receptors (Luthy et al. 1987). It is expected that inhibition of AR by F would block, at least partially, the actions of endogenous androgens. If this was the case, then androgens might be necessary to maintain normal amounts of LHR mRNA in pig ovarian follicles. The reasons by which both inhibition of endogenous androgen signaling (F treatment) and increased androgen stimulation (DHT treatment) produced inhibitory effects on LHR mRNA are not clear. A similar change in the type of action of androgens on follicular development, from stimulatory to inhibitory, and apparently in direct association with the magnitude of AR stimulation has been observed in Rhesus monkeys (Zeleznik et al. 2004).
Lesser amounts of LHR mRNA might presumably result in decreased LHR protein synthesis and subsequent inhibition of LH actions in ovarian follicles. An important function of LH is the stimulation of androgen synthesis (pigs, Evans et al. 1981). Decreased amounts of androgens might result in decreased synthesis of E2. In Experiment 1, concentrations of E2 in follicular fluid were not affected by DHT treatment. In a previous experiment, plasma concentrations of E2, T, and androstenedione in gilts were not altered by DHT treatment (authors' observations, unpublished). These results suggest that androgen synthesis might not have been affected by a possible decrease in LH stimulation.
In conclusion, androgenic stimulation through the AR during the follicular phase of the estrous cycle enhanced the numbers of healthy follicles among a numerically unaltered pool of total recruited follicles. How a simultaneous decrease in amounts of LHR mRNA might be related to this increase in healthy follicles is interesting but at present obscure.
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Materials and Methods |
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0800 and 1700 h). To reduce possible influences of the variability in estrous cycle duration, only gilts that exhibited two consecutive estrous cycles of 19.5–21.0 days (day 0=first day of estrus) were included in the experiments. The mean (±S.D.) estrous cycle duration for these gilts was 20.2±0.5 days. At the time, treatments were given gilts ranged from 120 to 130 kg. Gilts (n=5 per treatment) received daily (at 0800 h) injections (i.m.) of the corresponding treatment on days 13 to 16 (estimated period of follicular recruitment) or days 13 to 18 (follicular recruitment and selection). Treatments were as follows: corn oil (vehicle), DHT (10 mg), flutamide (F, 1.5 g, nonsteroidal AR inhibitor), DHT (10 mg) plus F (1.5 g), T (10 mg), and T (10 mg) plus F (1.5 g). Hormones and F were obtained from Sigma. Gilts were ovariectomized by mid-ventral laparotomy on day 17 or 19, 24 h after receiving their last treatment. Two experimental protocols having the same treatments and days of ovarian evaluations were conducted. All the experimental procedures involving animals were approved by the University Animal Care and Use Committee.
Experiment 1
The objectives were to determine the effects of androgens on numbers of follicles, follicular health, and concentrations of estradiol-17β (E2) in follicular fluid on days 17 and 19 of the estrous cycle. All evaluations were performed on follicles 5 mm in diameter. It was determined that among follicles 2 mm on day 16 (end of recruitment) most were 4 mm or larger (Grant et al. 1989). We considered that follicles 4 mm represented the majority of the recruited pool and that they would be 5 mm by day 17. The group of follicles 5 mm evaluated on day 19 would include those selected for ovulation and most that were initially recruited but had become atretic.
Immediately after ovariectomy, follicles were dissected individually or in groups of two to six if clustered together. Diagrams were drawn for groups of follicles to ensure follicle identification. A total of 1469 follicles were collected. Follicular fluid (10–50 µl depending on the size of the follicle) was obtained from each follicle using a tuberculin syringe. Follicular fluid was transferred to a 1.5 ml tube and stored at –20 °C until E2 concentrations were determined. Follicles were then fixed in 4% paraformaldehyde for 20 h at 4 °C and embedded in paraffin (Paraplast Plus; Fisher Scientific, Florence, KY, USA). Tissue sections (7 µm) were cut and attached to positively charged slides (Superfrost Plus, Fisher Scientific) for subsequent staining and morphological assessment of follicular health.
Follicular health
Tissue sections (two to three per follicle) were stained using the Mason Trichrome procedure, which included sequential staining with hematoxylin, acid fuchsin, ponceau de xylidine, and fast green (Humason 1972). Sections were examined using a light microscope (Nikon Labophot) at magnifications up to 400x. Follicular health was determined based on morphological integrity of the membrana granulosa, presence of granulosa cells with pyknotic nuclei, and observation of cells and/or apoptotic bodies in the follicular antrum (Maxson et al. 1985, Cárdenas & Pope 1994, Clark et al. 2004). Pyknotic nuclei appeared intensely stained, frequently compacted, and sometimes crescent in shape. Apoptotic bodies resulting from cell disintegration following apoptotic death were round or irregularly shaped structures, intensely stained, and smaller than healthy nuclei. Healthy granulosa and theca cells had light-blue or red-blue nuclei with darker dots in the nucleoplasm and were abundant in the membranes having good morphological integrity.
Follicles were classified as H, MA, or VA. Examples of follicles within these classifications are depicted in Fig. 2. H follicles had sound membrana granulosa with none or only few isolated cells with pyknotic nuclei. MA follicles had numerous granulosa cells with pyknotic nuclei detached from the membrana granulosa. Additionally, apoptotic bodies were observed within the clusters of detached cells. Granulosas of MA follicles had partial integrity (at least three layers) but their antral border appeared rough due to cell detachment. VA follicles had nearly all their granulosa cells with pyknotic nuclei, and granulosas were completely or mostly (
2 cell layers remaining) disintegrated. Consequently, in VA follicles most granulosa cells and apoptotic bodies were observed dispersed, individually or in small clusters, within the follicular antrum. Evaluations of follicular health were performed using a person unaware of treatment allocation of gilts.
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Experiment 2
The objective of this experiment was to evaluate the influence of androgens on amounts of the FSHR and LHR mRNAs in the tissues of H follicles. After ovariectomy, six to eight healthy follicles (pink walls and abundant capillary bed) measuring 5–6 mm on day 17, or 7–8 mm on day 19, were quickly dissected. Follicles were punctured with a needle, gently squeezed to eliminate follicular fluid, pooled, and then frozen in liquid nitrogen. Samples were frozen no later than 15 min after ovariectomy. Follicular tissues were stored at –80 °C until isolation of total RNA.
Measurement of mRNAs
Total cellular RNA was isolated using TRI reagent as described by the manufacturer (Molecular Research Center, Cincinnati, OH, USA). RNA integrity was verified by estimating the 28s to 18s rRNA ratio following agarose gel electrophoresis. RNA purity and concentration were evaluated by u.v. spectroscopy.
Relative amounts of FSHR and LHR mRNAs were determined using SYBR Green-based real-time reverse transcription PCR. RT (2 µg total RNA per 100 µl reaction) was performed using a kit and random hexamers as primers (TaqMan RT Reagents; Applied Biosystems, Foster City, CA, USA). Aliquots of the RT reactions, SYBR Green PCR master mix (Applied Biosystems), and specific primers (300 nM) were used for PCR amplification of the first-strand cDNAs for LHR, FSHR, and ribosomal protein L19 (endogenous control used as normalizer). Volume of PCRs was 25 µl and reactions were run in triplicate for each gene. The forward and reverse primers (5'–3' sequences) and size (bps) of products for FSHR, LHR, and L19 were: TCGAGGCAAATGTGTTCTCC, AAGGTTCTGGAAGGCATCAG, 101; TCTCCCTATCAAAGTAATCC, GTTCTGGATCAGTATTTCAG, 147; TACTGCCAATGCTCGAATGC, ACATGTGGCGGTCAATCTTC, 110 respectively.
PCR mixtures were incubated for 10 min at 95 °C and then 40 cycles at 95 °C for 15 s (DNA denaturing) and 60 °C for 1 min (annealing/extension). Default incubation parameters for generation of dissociation curves were included at the end of each run. Reactions in which reverse transcriptase was replaced by water were used as negative controls. All assays were performed in 96-well plates using an ABI Prism 7700 SDS real-time PCR machine. Amounts of RNA for the target and normalizer genes were estimated by the relative standard curve method using the ABI 7500 System SDS software. cDNA for standards (1 to 1x10–4 relative concentrations) was synthesized using a pool of total RNA from six experimental samples. Authenticity of the PCR products was determined by sequencing. No significant differences in relative amounts of the normalizer (L19) among treatments were detected. Single products were present in all the reactions as indicated by the dissociation curves, which was verified in some samples by agarose gel electrophoresis. Results of the amounts of FSHR and LHR mRNAs are presented as ratios to the amounts of the normalizer (L19) in the same sample.
Statistical analysis
Data were analyzed by ANOVA using general linear models of the SAS (SAS Institute, Cary, NC, USA) or SPSS (SPSS Inc., Chicago, IL, USA) software. Analyses for completely randomized two-way factorial designs were applied to follicular numbers, proportions, diameters, and also to the amounts of mRNA. Analysis of LHR mRNA was performed independently for each day because heterogeneity of variance could not be eliminated. Amounts of LHR mRNA on day 17 were analyzed by ANOVA as described above, but data for day 19 were analyzed using a nonparametric test (Kruskal–Wallis). In this case, means and standard errors of original data are presented. Repeated measurements analysis was used for the concentrations of E2. Means were compared using least significant difference tests. Statistical effects and differences among means were considered significant when P<0.05.
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Received 21 September 2007
First decision 19 October 2007
Accepted 4 December 2007
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References |
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