Effects of pituitary-specific overexpression of FSHα/β on reproductive traits in transgenic boars

Background Follicle-stimulating hormone (FSH) is a gonadotropin synthesized and secreted by the pituitary gland. FSH stimulates follicle development and maturation in females. It also plays an important role in spermatogenesis in males, including humans and mice. However, the effects of FSH on male pigs are largely unknown. In this study, we generated transgenic pigs to investigate the effects of FSHα/β overexpression on reproductive traits in boars. Results After five transgenic F0 founders were crossed with wide-type pigs, 193 F1 animals were obtained. Of these, 96 were confirmed as transgenic. FSHα and FSHβ mRNAs were detected only in pituitary tissue. Transgenic boars exhibited significantly higher levels of FSHα and FSHβ mRNA, serum FSH, and serum testosterone, compared to full-sib non-transgenic boars. Significant increases in testis weight, vas deferens diameter, seminiferous tubule diameter, and the number of Leydig cells were observed, suggesting that the exogenous FSHα/β affects reproductive traits. Finally, transgenic and non-transgenic boars had similar growth performance and biochemical profiles. Conclusions Pituitary-specific overexpression of FSHα/β genes is likely to impact reproductive traits positively, as indicated by enhancements in serum testosterone level, testis weight, the development of vas deferens, seminiferous tubules, and Leydig cells in transgenic boars. A high level of serum FSH induces secretion of serum testosterone, possibly by boosting the number of Leydig cells, which presumably increases the libido and the frequency of sexual activity in transgenic boars. Our study provides a preliminary foundation for the genetic improvement of reproductive traits in male pigs.


Background
Follicle-stimulating hormone (FSH) is a gonadotropin and glycoprotein polypeptide hormone with a mass of 35.5 kDa [1]. As a member of the glycoprotein hormone superfamily, it consists of two subunits (α and β) that combine non-covalently to form an active heterodimer, as is also the case for luteinizing hormone (LH), thyroidstimulating hormone (TSH), and human chorionic gonadotropin (hCG) [1]. The synthesis and secretion of FSHα and FSHβ is regulated by gonadotropin-releasing hormone (GnRH). FSHβ is also regulated by inhibin, leptin, and activins derived from brain, pituitary, placenta, and other tissues [2][3][4]. In females, FSH plays a key role in antral follicle development and stimulates preovulatory follicular growth in cooperation with LH [5,6]. In males, FSH is required for the mitotic division of germ cells, and together with testosterone, is involved in spermatocyte maturation and spermatogenesis [7].
Transgenic mouse models incorporating human FSHα and FSHβ genes have been used to study the effect of FSH on reproductive function [8]. In transgenic mice carrying a 10 kb human FSHβ construct, the inserted gene is highly and specifically expressed in pituitary tissue and the mice exhibit normal fertility [9,10]. FSH-null (knockout) male mice are fertile and sire normal-sized litters, although they show reductions in epididymal sperm number, sperm motility, and testicle size, while female knockouts are infertile [5]. FSHβ has been verified to be an important gene controlling litter size in Chinese Erhualian pigs, one of the most prolific pig breeds in the world [11]. In transgenic mice exhibiting pituitaryspecific overexpression of the Chinese Erhualian FSH gene, ovulation rate and litter size increase markedly [12].
F 0 transgenic pigs, in which FSHα/β expression is pituitary-specific, were generated previously [13]. In this study, we obtained 193 F 1 transgenic animals derived from five F 0 founders crossed to wild-type Large White pigs. Integration of the exogenous FSHα/β genes and their expression were confirmed. Since genetic improvements are more efficiently transferred by males than by females in pig breeding, we focused on the effects of FSHα/β on reproductive traits in boars. As is typical in reproductive trait studies, multiple traits were assessed, including semen volume, sperm quality parameters, sperm per ejaculate, epididymis weight, reproductive tract weight, and seminiferous tubule diameters (Animal QTL database) [14]. Hormone assays and histological analyses were performed to investigate the effects of exogenous FSH expression on the reproductive traits of male offspring. In addition, the health status of transgenic pigs was evaluated based on growth and various biochemical criteria. The results are directly relevant to strategies for improving the fecundity of multiparous mammals.

Generation of transgenic pigs
BAC DNA used for the production of transgenic animals in this study was described previously [12]. BAC clones for FSHα (BAC412H8) and FSHβ (BAC183O11) were isolated from a BAC library constructed using genomic DNA from a male Erhualian pig [15]. The LoxP-neo-LoxP cassette was introduced into two BAC constructs (FSHα and FSHβ) by homologous recombination (Fig. 1). BAC DNAs were linearized with NotI and co-transfected into fetal fibroblast cells. Positive cells were used as donors to produce transgenic founder pigs following standard procedures [16]. Transgenic F 0 pigs were mated with non-transgenic Large White pigs to produce F 1 pigs.

Phenotype measurements Body weight
Body weight of 20 F 1 pigs (10 Tg and 10 NTg half-sib individuals) was recorded at the ages of 1 d (birth weight), 10 d and 21 d (weaning weight), 60 d, 90 d, and 150 d.

Hormone assays
Serum from three pairs of randomly chosen Tg and NTg full-sib boars was collected 3 times within one week at 300 d of age. Levels of FSH, LH, testosterone, and estradiol (E2) were measured in triplicate using a standard radioimmunoassay. Assays were conducted at the Beijing North Institute of Biological Technology, China.

Assessment of sperm quality
Semen collection and quality assessments were performed as described [18]. Briefly, semen was collected from five pairs of Tg and NTg half-sib boars at an approximate age of 300 d. Three successive collections were performed at 7-day intervals. Semen volume was measured using graduated semen collection jars. Sperm concentration and motility were analyzed using the Sperm Quality Analyzer (Beijing, China). Total sperm number per ejaculate was calculated using the formula: sperm concentration × semen volume. The fraction of sperm exhibiting teratospermia, intact acrosomes, and normal mitochondrial function was assessed using methods described previously [19]. Seminal plasma quality was assessed by measuring levels of zinc, fructose, neutral α-glucosidase (NAG), and acid phosphatase (ACP), using a ChemWell BRED Analyzer (Guangdong, China) at the Beijing North Institute of Biological Technology.

Histological analysis
After slaughter, testes and epididymis were isolated and weighed. Testes tissue and vas deferens was fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. Tissue sections were stained with hematoxylin-eosin (H&E) and observed with a light microscope (Nikon, Japan). The diameters of vas deferens and seminiferous tubules were measured in~30 fields. Leydig cells were counted in~10 fields for each pig at 200× magnification and the average value was calculated.

Statistical analysis
Student's t-test was performed using SPSS Statistics (IBM Corporation, USA). All values are presented as mean ± standard error (SEM). P < 0.05 was the threshold for statistical significance.

Results
Transgenic pigs exhibiting pituitary-specific overexpression of the FSHα/β genes were generated using the BAC DNAs (FSHα and FSHβ) shown in Fig. 1. Five F 0 transgenic animals (two boars and three sows), in which both BACs were intact, were identified by PCR and Southern blot analysis, as described by Bi [13].

Integration and expression of exogenous FSH
Five founders were crossed with wild-type Large White pigs to obtain 193 F 1 progenies, of which nearly half (43 boars and 53 sows) were positive for the exogenous FSHα, FSHβ and Neo genes, as determined by PCR (Fig. 2a). The Neo gene was also detected by Southern blot in all 96 F 1 pigs (Fig. 2b). These data confirm that the integrated FSHα, FSHβ and Neo genes were transmitted to both male and female F 1 pigs with the expected Mendelian ratio. To determine whether the exogenous FSHα and FSHβ genes in the F 1 transgenic pigs were expressed in a tissue specific manner, FSH mRNA from pituitary gland and 13 other tissues was subjected to RT-PCR. FSHα and FSHβ expression was observed only in pituitary tissue (Fig. 2c). Because this experiment does not distinguish between contributions made by exogenous and endogenous FSH genes, FSHα and FSHβ expression in the pituitary glands of three pairs of full-sib transgenic and non-transgenic boars was compared by RT-PCR (Fig. 2d), and total FSH mRNA expression was quantified in the same samples using qPCR (Fig. 2e). As expected, mRNA levels of both FSHα and FSHβ were significantly higher in transgenic animals (P < 0.001).

Effect of FSH overexpression on reproductive traits
Several semen quality indicators and seminal plasma components were compared between transgenic and non-transgenic boars at~300 d of age. No significant differences were observed in any of the seven semen quality indicators (P > 0.05, Table 1). Transgenic and non-transgenic boars exhibited similar values for all four seminal plasma components (P > 0.05, Table 2).

Growth and biochemical analysis
Body weight at six growth stages (from birth to 150 d) was compared between transgenic and non-transgenic boars. There were no significant differences, although transgenic boar body weight was slightly higher from birth to 90 d, while non-transgenic boars exhibited higher body weight at 150 d (Fig. 5). In addition, no significant differences in blood chemistry were observed (Table 3). We conclude that the transgenic boars in this study exhibited no detectable health defects relative to wild-type controls.

Discussion
Pig fecundity is one of the most economically important traits in pig production. Because pig reproductive traits have low heritability [20], only a few candidate genes affecting pig reproduction have been identified, such as estrogen receptor 1 (ESR1) and FSHβ [11,21]. Transgenic mice in which porcine FSH is overexpressed exhibit significantly increased female fertility [12]. In this study, we investigated the effects of porcine FSH on reproductive traits in male transgenic pigs.
In 193 F 1 progenies, 96 transgenic pigs were identified. The transmission rate was 49.74%, consistent with ordinary Mendelian inheritance. FSH expression occurred in a pituitary-specific pattern (Fig. 2c), similar to results reported for FSHβ-overexpressing mice [12]. Because the exogenous and endogenous porcine FSHα/β are nearly identical in sequence, we could not distinguish between them using molecular methods. However, when total FSHα/β mRNA and serum FSH were compared in transgenic and non-transgenic pigs, transgenic animals exhibited significantly higher levels. These results suggest that pituitary-specific overexpression of FSH was successfully established in our transgenic pig model. While FSHβ mRNA increased approximately 10-fold in the transgenic animals, and FSHα mRNA increased about 3-fold, we observed only a modest increase in serum FSH levels (Figs. 2e and 3a). This is expected because serum FSH is a heterodimer, consisting of two subunits of FSHα and FSHβ, and FSH levels are probably limited by the lower level of FSHα mRNA expression (Fig. 2d-e) [1].
Male fertility is important in reproductive performance [22], and growing evidence suggests that FSH may be an important factor. In our study, the diameter of vas deferens and seminiferous tubules (Fig. 4b-f ) increased with the increasing levels of serum FSH in transgenic boars (Fig. 3a). The enlargement of vas deferens mainly occurred in the muscular layer of the wall. In humans, the vas deferens wall is thinner after vasectomy [23].  We suggest that the thickened muscular layer of the vas deferens might affect sperm transportation and the ejaculation process, but the hypothesis has not yet been tested. Seminiferous tubule diameter correlates positively with semen quality parameters (sperm concentration, sperm motility, and total sperm per ejaculate) in rabbits [24]. In contrast, no improvement in semen quality was identified in transgenic boars in this study. In addition, semen quality in pigs does not change after treatment with FSH, although serum testosterone level increases [25]. Testosterone levels are enhanced in male mice that overexpress FSH [10]. In contrast, FSH and FSH receptor knockout mice have smaller testes and exhibit reduced numbers of germ and Leydig cells [5,26]. In this study, we also observed that the serum testosterone level (Fig. 3b), testis weight (Fig. 4a) and the number of Leydig cells (Fig. 4g-i) increased in transgenic boars. The main function of Leydig cells is testosterone synthesis and secretion [27], and serum testosterone concentration is strongly related to libido in humans [28], rams [29], rats [30], and mice [31]. Testosterone also enhances libido, frequency of sexual acts, and sleep-related erections in humans [32]. If the underlying biology is similar in pigs, the increased number of Leydig cells in transgenic boars would be expected to increase testosterone levels and thereby enhance libido, increase the frequency of sexual activity, and increase the frequency of semen collection. Because our results indicate that overexpression of FSH increases serum testosterone levels in boars, the effect is likely to be an improvement in the downstream reproductive traits. Finally, we evaluated whether the exogenous FSHβ gene exerts deleterious effects on the transgenic pigs. Body weight, levels of various biochemical components in blood plasma and semen plasma, and semen quality, were similar in transgenic and non-transgenic animals.
This suggests that FSH overexpression has no detectable adverse impact on pig health.

Conclusions
In summary, we successfully produced transgenic pigs in which exogenous FSHα/β genes were integrated and expressed at high levels in a pituitary-specific manner. The high level of serum FSH increases the level of serum testosterone, possibly by increasing the number Leydig cells. Higher levels of testosterone would be expected to enhance the libido and the frequency of sexual activity in transgenic boars. Nevertheless, augmented FSH levels did not improve semen quality, even though testis weight and seminiferous tubules diameter increased. Finally, the expression of exogenous FSHα/β genes resulted in no detectable adverse effects on growth or the overall health of transgenic boars.