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),thyroidstimulatinghormone(TSH),andhuman　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[2-4].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].F0transgenic pigs,in which FSHα/β expression is pituitary-specific,were generated previously[13].In this study,we obtained 193 F1transgenic animals derived from five F0founders 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.

Methods

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 F0pigs were mated with non-transgenic Large White pigs to produce F1pigs.

Identification of transgenic pigs and detection of gene expression

Transgenic pigs were identified by PCR and Southern blot using genomic DNA extracted from ear tissue.Three pairs of primers,FSHα-5-453-F/R(453 bp product),FSHβ-5-737-F/R(737 bp product)[13],and Neo-382-F/R(382 bp product),were used to amplify FSHα,FSHβ,and Neo,respectively.PCR products were digested with AvaII and PstI prior to gel electrophoresis.The primers Neo-382-F/R(forward 5′-GTTGTCACTGAAGCGGGAAG-3′and reverse 5′-CACAGTCGATGAATCCAGAAAA-3′)were used to generate a digoxigenin(DIG)-labeled probe for　the　Southern　blotassay　(Roche　Diagnostics,Mannheim,Germany).All primers were synthesized by the Sangon Company(Shanghai,China).

Three F1transgenic(Tg)boars and three non-transgenic(NTg)full-sib boars were slaughtered at approximately 300 d of age.Tissue samples from hypothalamus,pituitary,testis,epididymis,vas deferens,seminal vesicle,prostate,Cowper’s gland,heart,liver,spleen,lung,kidney,and pancreas were collected,rapidly frozen in liquid nitrogen,and stored at-80°C.Tissue-specific expression of the FSHα and FSHβ transgenes was determined by reverse transcription PCR(RT-PCR)and quantified by real-time PCR[17].Total RNA was extracted using an animal total RNA extraction kit according to the manufacturer’s instructions(Tiangen,Beijing,China).cDNA synthesis was performed with 1 μg total RNA following the protocol accompanying the FastQuant RT Kit(Tiangen,Beijing,China).GADPH expression was used for normalization.The specific primers used for quantifying expression were:FSH-α (forward:5′-GGGTGCCCCAATCTATCAGTG-3′,reverse:5′-GTGGCATTCGGTGTGGTTCTC-3′),FSH-β(forward:5′-CACCCCAAGATGAAGTCGCTG-3′,reverse:5′-GCCAGGTACTTTCACGGTCTCG-3′),and GADPH(forward:5′-GTTTGTGATGGGCGTGAAC-3′,reverse:5′-ATGGACCTGGGTCATGAGT-3′).

Phenotype measurements Body weight

螺钉断裂8例，7例发生于术后4 ～ 6个月，其中5例出现胸腰部疼痛，行开放手术翻修并增加伤椎固定螺钉，随访12个月骨折愈合良好；2例无明显不适采用胸腰背支具保护定期复查，术后12 个月骨折愈合良好拆除内固定。1例发生于术后8个月，行腰椎CT检查，明确骨折愈合行内固定取出。

Serum biochemistry

Serum was separated from blood samples obtained from F1pigs(5 Tg and 5 of NTg half-sib individuals)at 300 d,307 d,and 315 d.The following compounds were measured:glucose(GLU),urea(UREA),creatinine(CREA),blood urea nitrogen/creatinine(BUN/CREA),phosphorus(PHOS),calcium(CA),total protein(TP),albumin(ALB),globulin(GLB),alanine aminotransferase(ALT),alkaline phosphatase(ALKP),γ-glutamyl transpeptidase(GGT),cholesterol(CHOL),triglyceride(TRIG),amylase(AMYL),lipase(LIPA),and creatine kinase(CK).All assays were conducted at Beijing Tianzewanwu Veterinary Hospital,China.

Hormone assays

Fig.1　Schematic view of FSHα and FSHβ expression vectors.The vectors include the complete DNA sequences of the FSHα and FSHβ genes,along with the Neo gene and its promoter and terminator.Solid boxes represent exons.The red arrows represent LoxP,and the homologous arms are represented in blue.PCR primers(FSHα-5-453-F/R,FSHβ-5-737-F/R and Neo-382-F/R)are represented by black arrows.Southern blot probes are indicated by the label“probe”.NotI was the restriction enzyme cutting site

Growth and biochemical analysis

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 normalmitochondrialfunction　wasassessed　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.

Body weight of 20 F1pigs(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.

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.

新教特别是加尔文新教宣扬在世俗中履行来自上帝的召唤(Calling)才能获得拯救。这类新教的革命性观念，使人们人生的重心从脱离世俗的宗教禁欲生活转向世俗的日常工作，世俗生活和工作也具有了神圣的含义，以至于召唤(Calling)的另一个含义就是职业。

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 F0transgenic 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 F1progenies,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 F1pigs(Fig.2b).These data confirm that the integrated FSHα,FSHβ and Neo genes were transmitted to both male and female F1pigs with the expected Mendelian ratio.

Fig.2　Identification of exogenous FSHα/β insertion and expression analysis.(a)Identification of F1transgenic pigs by PCR using DNA obtained from ear tissue.P,a single F0transgenic pig as positive control;N,a single non-transgenic Large White pig as negative control;T416-27,28,29,31,41,T523-96,97,98,99,100,101,102,104,and T519-177,178 are identifiers for F1transgenic pigs.(b)Southern blot for transgenic pig identification.The Neo gene in transgenic pigs was detected using the probe shown in Fig.1.DNAs were digested with AvaII and PstI to generate a target fragment of 463 bp.(c)RT-PCR analysis of FSHα and FSHβ from pituitary and 13 other tissues.GADPH was used as a control.(d)RT-PCR analysis of FSHα and FSHβ expression using mRNA from the pituitaries of six transgenic pigs.(e).FSHα and FSHβ mRNA expression levels in the pituitaries of Tg and NTg pigs analyzed using qPCR.Relative expression was calculated relative to β-actin(reference gene).Values are expressed as means±SEM.***,P＜0.001

血痂儿结在嘴上 Upon lips, blood scab is ruthlessly growing

Serum concentrations of FSH,LH,testosterone,and E2

Fig.3　Hormone assays.(a)Serum FSH,(b)testosterone,(c)LH,and(d)E2 levels in F1pigs.All assays were conducted in triplicate.Bars represent means±SEM.*,P＜0.05

To examine the effects of FSHα/β overexpression on hormone levels,FSH,LH,testosterone and E2 levels were compared in full-sib transgenic and non-transgenic boars at an approximate age of 300 d(Fig.3).Serum levels of FSH were significantly higher in transgenic animals(2.25±0.18 mIU/mL vs.1.75±0.20 mIU/mL,P＜0.05,Fig.3a).Similarly,testosterone levels in transgenic boars were significantly higher than in nontransgenic boars(3.26±0.64 ng/mL vs.1.67±0.60 ng/mL,P＜0.05,Fig.3b).Although serum levels of both LH and E2 were higher in transgenic boars,the differences were not significant(LH:9.16±0.70 mIU/mL vs.8.19±0.67 mIU/mL,P＞0.05;E2:29.71±3.46 pg/mL vs.25.00±3.22 pg/mL,P＞0.05;Fig.3c-d).

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).

(4)一轴两极三片的空间格局基本成型。从中心度及结构洞分析来看，三峡地区旅游经过近20年发展，形成以解放碑(朝天门)、白帝城、小三峡、三峡大坝、神农溪、三峡人家、恩施大峡谷、武汉东湖等共12个景区为核心的旅游节点，其中以重庆解放碑、小三峡、三峡大坝、白帝城为最重要核心节点。据此，本文认为三峡旅游在空间形态上基本呈现一轴两极三片的空间格局，即长江轴线、成渝都市圈发展极、武汉都市圈发展极、奉节-巫山-宜昌发展片区、奉节—恩施—宜昌发展片区、万州-涪陵中线发展片区。

科学大洋钻探还显示，海洋沉积物中的细胞数量与海洋或土壤中的细胞数量大致相同。探险队不仅在8 000英尺深的沉积物中发现了生命，还在拥有8 600万年历史的沉积物和温度超过60摄氏度的沉积物中发现了生命。

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.

滑模负荷频率控制器设计包括切换面设计和控制器设计两个步骤,以保证系统在有限时间内达到切换面并稳定在滑模面。根据柴储混合电力系统负荷频率协调控制结构,将式(4)、式(6)和式(7)分别修改为

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 exhibitedhigher 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.

布鲁氏杆菌病阳性和阴性诊断血清由中牧股份成都药械厂提供，布鲁氏菌虎红平板凝集实验抗原由农业农村部动物检疫所国家外来动物疫病诊断中心提供，布鲁氏菌试管凝集实验抗原由中牧股份成都药械厂提供。

Table 1　Semen characteristics in transgenic and non-transgenic boars

Items　Tg　NTg　P-value Semen volume per ejaculate,mL　218.75±28.73　237.00±29.54　0.079 Sperm concentration,108/mL　3.58±0.09　3.46±0.08　0.619 Total sperm per ejaculate,108　794.74±28.35　832.78±25.36　0.314 Sperm mobility,%　77.11±2.63　73.57±2.36　0.411 Teratospermia,%　8.23±0.30　7.30±0.38　0.764 Acrosome intactness,%　81.68±0.25　81.34±0.23　0.890 Normal mitochondria function,%　80.18±1.52　82.22±1.36　0.930

Table 2　Biochemical indicators for seminal plasma in transgenic and non-transgenic boars

Items　Tg　NTg　P-value Seminal plasma zinc,μmol　0.76± 0.16　0.54± 0.15　0.248 Seminal plasma fructose,mIU　1.01±0.05　0.95±0.05　0.919 Neutral α-glucosidase,IU　0.95 ± 0.06　0.70 ± 0.05　0.324 Acid phosphatase,μmol　236.90± 14.17　217.89± 12.67　0.406

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 F1progenies,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].

Fig.4　Histological assessment of reproductive tissue from F1boars.(a)Comparison of testis and epididymis weight in Tg and NTg boars.(b)Vas deferens and seminiferous tubule diameters in Tg and NTg boars.(c-i)Histological sections of testis tissue.(c-d)Vas deferens at 50×magnification.Red arrows span the vas deferens diameter.(e-f)Seminiferous tubules at 200×magnification.Red arrows span tubule diameter.(g-h)Leydig cells at 200×magnification.Red arrows indicate Leydig cells between the seminiferous tubules.(i)The number of Leydig cells in Tg and NTg boars.Data are expressed as means±SEM.*,P＜0.05.**,P＜0.01

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.

Fig.5　Growth of F1Tg and NTg boars from birth to 150 d.Data is expressed as means±SEM

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.

2.2 教练员情况 教练员队伍的学历、职称的高低、对文化教育的态度、对运动员学习的关心程度与运动员的学习具有一定的相关作用。因为运动员平时与教练在一起的时间相对较长，教练员对运动员个体的关注程度相对更高，思想意识未成熟的运动员更易直接受到教练员言行与意识思想的影响。

Table 3　Blood biochemistry in transgenic and non-transgenic boars

Items　Tg　NTg　P-value Glucose,mmol/L　4.58±0.18　4.33±0.16　0.334 Urea,mmol/L　6.87±0.24　7.19±0.21　0.785 Creatinine,μmol/L　118.42± 3.17　122.67± 2.83　0.0629 Blood urea nitrogen/creatinine　16.50±0.52　15.80±0.47　0.254 Phosphorus,mmol/L　2.28±0.14　2.10±0.12　0.0516 Calcium,mmol/L　2.29±0.03　2.33±0.03　0.580 Total protein,g/L　70.75±0.36　70.27±0.32　0.491 Albumin,g/L　32.50±0.17　32.27±0.16　0.526 Globulin,g/L　38.25±0.19　38.00±0.17　0.831 Albumin/Globulin　0.84±0.02　0.87±0.02　0.545 Alanine aminotransferase,IU/L　59.17±0.62　60.00±0.56　0.809 Alkaline phosphatase,IU/L　76.75±4.61　82.93±4.12　0.366 γ-glutamyl transpeptidase,IU/L　32.92±1.25　34.60±1.22　0.802 Cholesterol,mmol/L　1.89±0.07　1.80±0.06　0.182 Triglyceride,mmol/L　0.59±0.06　0.50±0.06　0.406 Amylase,IU/L　434.03±30.35　474.80±27.14　0.617 Lipase,IU/L　23.50±1.78　21.08±1.63　0.496 Creatine kinase,IU/L　606.64±18.65　591.08±15.93　0.729

Conclusions

Abbreviations

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.

卡尔梅克草原位于亚欧草原的西端，是我们熟知的南俄草原的一部分。东面，伏尔加河由北向南静静地流过，南面是欧亚大陆的内海——里海，但卡尔梅克草原丝毫也享受不到来自里海的水汽，属于典型的大陆性气候，夏季酷热干旱，气温能达到+40多度。据说，五月是卡尔梅克最好的季节。

ACP:Acid phosphatase;ALB:Albumin;ALKP:Alkaline phosphatase;ALT:Alanine aminotransferase;AMYL:Amylase;BUN/CREA:Blood urea nitrogen/creatinine;CA:Calcium;CHOL:Cholesterol;CK:Creatine kinas;CREA:Creatinine;DIG:Digoxigenin;E2:Estradiol;ESR1:Estrogen receptor 1;FSH:Follicle-stimulating hormone;FSHα:Follicle-stimulating hormone subunit α;FSHβ:Follicle-stimulating hormone subunit β;GGT:γ-glutamyl transpeptidase;GLB:Globulin;GLU:Glucose;GnRH:Gonadotropin-releasing hormone;hCG:Human chorionic gonadotropin;LH:Luteinizing hormone;LIPA:Lipase;NAG:Neutral α-glucosidase;PHOS:Phosphorus;TP:Total protein;TRIG:Triglyceride;TSH:Thyroid-stimulating hormone

再次，在猪传染性胸膜肺炎发病的初期，可以适当实行群体给药。在该疫病的发病初期，生猪的病情尚且处于可控状态，因此应当严格坚持将专家所配置的药物混入饲料中，让整体猪群共同食用，以便增加猪群机体的抵抗力，避免病情加重甚至产生扩散的趋势，也有助于病猪的及早康复。

Acknowledgements

We thank for Mr.Qiao Xu,Mr.Guoying Hua,Mr.Chunzheng Fu,Mr.Yu Feng,and Mr.Linchao Wang for collecting the samples.

Funding

Funding for this study was provided by National Basic Research Program of China(973 Program,Grant 2014CB138501),the National Transgenic Animal Breeding Grand Project(2014ZX08006-005),and the Program for Changjiang Scholars and Innovation Research Teams in the University(IRT_15R62).The funding sources had no role in the study design.

We also compared testis and epididymis characteristics between transgenic and non-transgenic boars.As shown in Fig.4a,the testis weight in transgenic boars was significantly higher(501.6±35.6 g vs.355.2±32.8 g,P＜0.05).Transgenic boars exhibited higher epididymis weight but the levels were statistically indistinguishable(149.6±10.6 g vs.138.0±11.0 g,P＞0.05,Fig.4a).Vas deferens and seminiferous tubule diameters were also compared.Interestingly,both diameters were significantly　higher　in　transgenic　boars　(vas　deferens,2216.25　±　173.24　μm　vs.1894.72　±　270.86　μm,P ＜ 0.001;seminiferous tubules,117.30 ± 6.65 μm vs.107.79 ± 6.79 μm,P ＜ 0.001,Fig.4b-f).Enlargement of the vas deferens occurred mainly in the muscular layer of the wall.Finally,the number of Leydig cells in transgenic boars was significantly higher than in non-transgenic boars(337.6±14.3 vs.178.9±23.4,P＜0.01,Fig.4g-i).

To determine whether the exogenous FSHα and FSHβ genes in the F1transgenic 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 ofboth FSHα and FSHβ were significantly higher in transgenic animals(P＜0.001).

Availability of data and materials

中国印刷及设备器材工业协会制定了“以源头治理和过程控制为重点，兼顾末端治理”的方针，引导使用环保型油墨，再统筹考虑过程控制和末端治理，尽量避免舍本逐末。为了更加直观准确地引导企业，协会计划在明年4月中国（广东）国际印刷技术展览会上系统展示以水醇体系为重点的治理方案，逐渐厘清盲目投、良莠不齐的局面，使软包装企业在VOCs治理上逐渐走上正确轨道。

Data sharing not applicable to this article.

Authors’contributions

KW conceived and designed the experiments.WL,YQ,MZ,MZ,YC and QL performed the experiments and collected the samples.WL and KW analyzed the data,wrote the main manuscript,and prepared the figures.All authors reviewed and approved the final manuscript.

Ethics approval

All experiments involving transgenic pig production and sample collection strictly followed protocols approved by the Animal Welfare Committee of China Agricultural University(Approval Number:XK257).

Consent for publication

Not applicable.

Competing interests

2010年7月19日00时,850 hPa上江淮地区东部有一中心位势高度为1 430 hPa的低涡存在,并伴随着西南暖湿气流向东北方向移动,移动过程中强度略有增大。

The authors declare that they have no competing interests.

生态清洁小流域建设是统筹水利、市政、环保、林业、交通、国土等多部门的综合性公益事业，是跨水利工程、环境科学、农学、林学、生态学等多学科的艰巨任务。强化部门联动工作机制、推动宏观与微观领域科学研究，是落实和发展生态清洁小流域建设的必备条件，也将不断推动北运河流域水土资源的可持续利用、经济社会的可持续发展和生态环境的可持续维护。

Author details

1College of Animal Science and Technology,China Agricultural University,Beijing 100193,China.2College of Animal Science and Veterinary Medicine,Henan Agricultural University,Zhengzhou 450002,China.3Institute of Zoology,Chinese Academy of Sciences,Beijing 100101,China.4The Department of Animal Husbandry,Rongchang Campus,Southwest University,Rongchang,Chongqing 402460,China.5State Key Laboratory for Agrobiotechnology,China Agricultural University,Beijing 100193,China.

References

1.　Pierce JG,Parsons TF.Glycoprotein hormones:structure and function.Annu Rev Biochem.1981;50:465-95.

2.　Matzuk MM,Kumar TR,Shou W,Coerver KA,Lau AL,Behringer RR,et al.Transgenic models to study the roles of inhibins and activins in reproduction,oncogenesis,and development.In:conn PM(ed.)Recent Prog Horm Res,vol.51;1996:123-157.

3.　Gharib SD,Wierman ME,Shupnik MA,Chin WW.Molecular-biology of the pituitary gonadotropins.Endocr Rev.1990;11:177-99.

4.　Kato Y,Imai K,Sakai T,Inoue K.Simultaneous effect of gonadotropin-releasing hormone(GnRH)on the expression of 2 gonadotropin-beta genes by passiveimmunization to GnRH.Mol Cell Endocrinol.1989;62:135-9.

5.　Kumar TR,Wang Y,Lu N,Matzuk MM.Follicle stimulating hormone is required for ovarian follicle maturation but not male fertility.Nat Genet.1997;15:201-4.

6.　McGee EA,Hsueh AJ.Initial and cyclic recruitment of ovarian follicles.Endocr Rev.2000;21:200-14.

7.　Ruwanpura SM,McLachlan RI,Meachem SJ.Hormonal regulation of male germ cell development.J Endocrinol.2010;205:117-31.

8.　Kumar TR.Mouse models for gonadotropins:a 15-year saga.Mol Cell Endocrinol.2007;260-262:249-54.

9.　Kumar TR,Fairchild-Huntress V,Low MJ.Gonadotrope-specific expression of the human follicle-stimulating hormone beta-subunit gene in pituitaries of transgenic mice.Mol Endocrinol.1992;6:81-90.

10.Kumar TR,Low MJ.Gonadal steroid hormone regulation of human and mouse follicle stimulating hormone beta-subunit gene expression in vivo.Mol Endocrinol.1993;7:898-906.

11.Zhao YF,Li N,Xiao L,Cao GS,Chen YZ,Zhang S,et al.FSH beta subunit gene is associated with major gene controlling litter size in commercial pig breeds.Science in China Series C-Life Sciences.1998;41:664-8.

12.Bi M,Tong J,Chang F,Wang J,Wei H,Dai Y,et al.Pituitary-specific overexpression of porcine follicle-stimulating hormone leads to improvement of female fecundity in BAC transgenic mice.PLoS One.2012;7:e42335.

13.Bi M.Construction of BAC transgenic mice and pigs harboring porcine follicle-stimulating hormone gene.Beijing:China Agricultural University;2013.

14.Hu Z,Park CA,Wu X,Reecy JM.Animal QTLdb:an improved database tool for livestock animal QTL/association data dissemination in the post-genome era.Nucleic Acids Res.2013;41:D871-9.

15.Liu W,Zhang Y,Liu Z,Guo L,Wang X,Fei J,et al.A five-fold pig bacterial artificial chromosome library:a resource for positional cloning and physical mapping.Prog Nat Sci.2006;16:889-92.

16.Gong G,Dai Y,Fan B,Zhu H,Wang H,Wang L,et al.Production of transgenic blastocyst by nuclear transfer from different types of somatic cells in cattle.Sci China C Life Sci.2004;47:183-9.

17.Bai Y,Zhang JB,Xue Y,Peng YL,Chen G,Fang MY.Differential expression of CYB5A in Chinese and European pig breeds due to genetic variations in the promoter region.Anim Genet.2015;46:16-22.

18.Hu JH.Study on cryoperservation of boar semen.Yangling:Northwest A&F university;2006.

19.Cai W,Su W,Fang Y,Chang Q,Sun S,Zhao Y,et al.Analysis of semen quality,routine blood,biochemical indexes of serum and seminal plasma in transgenic goats.Chinese.J Anim Sci.2014;50:28-32.

20.Zhang Y.Animal breeding.Beijing:China Agricultural University Press;2001.

21.Rothschild M,Jacobson C,Vaske D,Tuggle C,Wang L,Short T,et al.The estrogen receptor locus is associated with a major gene influencing litter size in pigs.Proc Natl Acad Sci U S A.1996;93:201-5.

22.Berger T.Male Effects on Reproductive Performance.J.Anim.Sci.1998;(Suppl 3):47-51.

23.Schmidt SS,Brueschke EE.Anatomical sizes of the human vas deferens after vasectomy.Fertil Steril.1976;27:271-4.

24.Princewill Ogbuewu I,Charles Okoli I,Uwaezuoke Iloeje M.Semen quality characteristics,reaction time,testis weight and seminiferous tubule diameter of buck rabbits fed neem(Azadirachta Indica a.Juss)leaf meal based diets.International journal of reproductive.Biomedicine.2009;7:23-8.

25.Wagner A,Claus R.The effects of postnatal FSH substitution on Sertoli cell number and the sperm production capacity of the adult boar.Anim Reprod Sci.2009;110:269-82.

26.O'Shaughnessy PJ,Monteiro A,Abel M.Testicular development in mice lacking receptors for follicle stimulating hormone and androgen.PLoS One.2012;7:e35136.

27.Hodgson Y,Hudson B.Leydig cell function.In:The pituitary and testis:springer;1983.p.107-32.

28.Travison TG,Morley JE,Araujo AB,Donnell AB O,JB MK.The relationship between libido and testosterone levels in aging men.J Clin Endocrinol Metab.2006;91:2509-13.

29.Aguirre V,Orihuela A,Vázquez R.Effect of semen collection frequency on seasonal variation in sexual behaviour,testosterone,testicular size and semen characteristics of tropical hair rams(Ovis Aries).Trop Anim Health Prod.2007;39:271.

30.Damassa DA,Smith ER,Tennent B,Davidson JM.The relationship between circulating testosterone levels and male sexual behavior in rats.Horm Behav.1977;8:275-86.

31.Luttge WG,Hall NR.Differential effectiveness of testosterone and its metabolites in the induction of male sexual behavior in two strains of albino mice.Horm Behav.1973;4:31-43.

32.Shabsigh R.The effects of testosterone on the cavernous tissue and erectile function.World J Urol.1997;15:21-6.