Effects of TLR4 overexpression on sperm quality, seminal plasma biomarkers, sperm DNA methylation and pregnancy rate in sheep

Journal Pre-proof Effects of TLR4 overexpression on sperm quality, seminal plasma biomarkers, sperm DNA methylation and pregnancy rate in sheep Yi Fang, Wei Xia, Wentao Cai, Xiaosheng Zhang, Jinlong Zhang, Xiangwei Fu, Sa Li, Xiaohuan Fang, Shuchun Sun, Zhigang Wang, Xiaolei Zhang, Shien Zhu, Junjie Li PII:

S0093-691X(19)30456-X

DOI:

https://doi.org/10.1016/j.theriogenology.2019.10.009

Reference:

THE 15204

To appear in:

Theriogenology

Received Date: 14 May 2019 Revised Date:

4 October 2019

Accepted Date: 9 October 2019

Please cite this article as: Fang Y, Xia W, Cai W, Zhang X, Zhang J, Fu X, Li S, Fang X, Sun S, Wang Z, Zhang X, Zhu S, Li J, Effects of TLR4 overexpression on sperm quality, seminal plasma biomarkers, sperm DNA methylation and pregnancy rate in sheep, Theriogenology (2019), doi: https:// doi.org/10.1016/j.theriogenology.2019.10.009. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Inc.

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Effects of TLR4 Overexpression on Sperm Quality, Seminal Plasma Biomarkers,

2

Sperm DNA Methylation and Pregnancy rate in Sheep

3

Yi Fang1,2,5#, Wei Xia3#, Wentao Cai1, Xiaosheng Zhang4, Jinlong Zhang4, Xiangwei Fu2, Sa Li1,

4

Xiaohuan Fang1, Shuchun Sun1,6, Zhigang Wang1,6, Xiaolei Zhang3, Shien Zhu2*, Junjie Li1,6*

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1. College of Animal Science and Technology, Hebei Agricultural University, Baoding, China;

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2. College of Animal Science and Technology, China Agricultural University, Beijing 100193,

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China

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3. College of Life Science and Technology, Southwest Minzu University, Chengdu, China;

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4. Animal Husbandry and Veterinary Research Institute of Tianjin, Tianjin, China;

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5. Jilin Provincial Key Laboratory of Grassland Farming, Northeast Institute of Geography and

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Agoecology, Chinese Academy of Sciences, Changchun, Jilin 130062, China

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6.Research Center of Cattle and Sheep Embryo Engineering Technique of Hebei Province,

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Baoding, China

14

#

15

*

16

University, Baoding 071000, China

17

E-mail: [email protected]

18

*

19

University, Beijing 100193, China

20

E-mail: [email protected]

21 22 23 24 25 26 27

These authors contributed equally to this work.

Corresponding author at: College of Animal Science and Technology, Hebei Agricultural

Corresponding author at: College of Animal Science and Technology, China Agricultural

28

Abstract

29

Genetic modification provides a means to enhancing disease resistance in animals. In this

30

study, the first generation of genetically modified (GM) sheep overexpressing TLR4 was

31

produced by microinjection for better disease resistance. To compare semen characteristics

32

including sperm quality, seminal plasma biochemical index, sperm DNA methylation and

33

pregnancy rate of three-year old transgenic sheep with TLR4 overexpressed (toll like receptor

34

4, TLR4) and non-transgenic ram. Sixteen transgenic ram of F0 generation were produced by

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microinjection of the TLR4 plasmid into the pronucleus of fertilized ova. Seven transgenic

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sheep of F1 generation was produced by breeding F0 transgenic founders with non-transgenic

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sheep of the same breed. There were no significant differences between transgenic and control

38

rams for all semen quality parameters, including semen volume, sperm concentration, sperm

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viability, and percentages of sperm with an intact plasma membrane, acrosomal integrity, and

40

viable sperm with high mitochondrial membrane potential in both F0 and F1 generation.

41

Furthermore, no significant differences were found for seminal plasma concentrations of zinc,

42

neutral alpha-glucosidase, acid phosphatase or fructose, nor for levels of H19 and IGF2R

43

methylation in sperm DNA. In addition, pregnancy rate was also similar between these two

44

groups. In conclusion, there was no evidence that TLR4 overexpression altered the sperm

45

quality, seminal plasma or sperm DNA of transgenic sheep.

46

Key words: TLR4 overexpression; semen quality; DNA methylation; sheep

47

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1. Introduction

49

TLR4 (toll like receptor 4, TLR4) is an important Toll-like receptor in the innate immune

50

system which responds to common Gram-negative bacteria, e.g., Escherichia coli,

51

Proteusbacillus vulgaris, Shigella dysenteriae, and Brucella melitensis [1]. It is critical for the

52

recognition of lipopolysaccharide (LPS)/endotoxin from Gram-negative bacteria by different

53

host cells initiating cell activation and subsequent triggering of a proinflammatory response to

54

invading pathogens [2-5]. Overexpression of TLR4 in transgenic animals improved disease

55

resistance [6]. However, exogenous gene may insert at a suboptimal site, altering a balanced

56

genotype and producing unpredictable effects [7, 8].

57

Reproductive traits of transgenic male animals can affect creation of stable lines of

58

transgenic offspring. Interactions between the immune system and the reproductive system

59

have important consequences for fertility and reproductive health in general [9]. The TLR4

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gene is closely associated with ovulation, fertilization, pregnancy and delivery in animals and

61

can activate the innate immune system against reproductive diseases [10]. Although

62

reproductive disorders including decreased fertility, infertility and structural and functional

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defects of sperm have been reported in growth hormone transgenic mice and pigs [11-13],

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there were no effect in physiological and biochemical blood characteristic, oocyte methylation

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and reproductive performance in overexpressing Capra hircus TLR2 goats [14] and TLR4

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sheep [15]. Consequently, the reports on the biological safety of transgenic animals are

67

inconsistent.

68

The normal sperm morphology and motility are important for fertility potential of the male.

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Defected sperm has been associated with lowered fertility and following embryo development,

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and reduced capacity of binding to the ovum. Moreover, seminal plasma concentrations of

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zinc, fructose, acid phosphatase and neutral alpha-glucosidase were significantly correlated

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with sperm count, morphology, motility and semen volume [16, 17]. In addition, transgenic

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technology may change the stable equilibrium state of the genome and cause unpredictable

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effects [18], the interactions of endogenous and exogenous genes might cause epigenetic

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modifications in DNA [19], as frequently reported in studies of mice [20].

76

DNA methylation, a crucial impact in gene expression and chromosomal structure during

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early embryogenesis [21] or germ cell development [22] can regulate gene expression during

78

spermatogonial stem cell differentiation [23]. Various forms of assisted reproductive

79

treatments can alter DNA methylation and impact embryonic development [24] or random

80

insertion and expression of exogenous genes may alter DNA methylation [25, 26]. H19 and

81

Insulin-like growth factor 2 receptor (IGF2R) are frequently studied imprinted genes

82

regulating early fetal growth [27], and are also essential for normal spermatogenesis and

83

regulation [28-31]. Abnormal DNA methylation of differential methylated regions (DMR) in

84

imprinted genes may cause biallelic expression or silencing [32]. Therefore, analysis of H19

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and IGF2R methylation patterns of sperm can serve as important indicators of reproductive

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performance.

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In the present study, transgenic sheep with TLR4 overexpression were used as a model to

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assess reproductive safety by analyzing semen quality, seminal plasma biochemical markers

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and DNA methylation level of sperm.

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2. Materials and methods

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All experimental protocols and animal handling procedures were reviewed and approved

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by the Laboratory Animal Care and Use Committee of Hebei Province.

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2.1. Animals

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Transgenic males with the TLR4 gene overexpressed were produced as described in

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previous publications [14, 33, 34] and the production workflow of transgenic animals were

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summarized in Figure 1. For this study, thirty five rams of the F0 generation (transgenic rams:

97

n=16, non-transgenic rams: n=19) and eighteen rams of the F1 generation (transgenic rams:

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n=7, non-transgenic rams: n=11) were selected with the same breed and age. All males were

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raised in the same rearing environment and fed the same forage and commercial concentrate

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supplement year-round. All animal experiments in this study were approved by the

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Institutional Animal Care and Use Committee of China Agricultural University.

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2.2. Semen collection and processing

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Semen samples from each ram (transgenic and non-transgenic) were collected once a week

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using an artificial vagina (AV) containing water maintained at approximately 38～40°C. A

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graduated collecting tube attached to the disposable sleeve inside the AV was used to evaluate

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semen volume. Semen parameters analyzed included volume, concentration, viability,

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percentages of sperm with plasma membrane integrity, acrosomal integrity, and high

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mitochondrion membrane potential. Five ejaculates per male were analyzed.

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2.3. Sperm concentration

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After collection, semen was kept in an incubator at 41°C [35]. A portion of the semen was

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extended 1:99 with physiological saline and sperm concentration (109 sperm/mL ejaculate)

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was determined using a hemocytometer and evaluation with a microscope at 400×. Two

113

counts were made for each individual and averaged. If counts differed by 30% or more,

114

concentration was re-analyzed.

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2.4. Fluorescent staining to assess sperm parameters

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2.4.1. Apparatus

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Flow cytometer analyses were done with a FACSCalibur (Becton Dickinson, Mountain

118

View, USA) equipped with 15 mW air-cooled argon laser and a 488 nm excitation filter

119

(Supplementary figure 1). After acquisition, data were evaluated with CELLQuest (Becton

120

Dickinson) software.

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2.4.2. Sperm viability

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Sperm viability was analyzed, as described previously [36], using a LIVE/DEAD

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Spermatozoa Viability Kit (Invitrogen, L-7011). Briefly, SYBR-14 was diluted 1:50 with

124

dimethyl sulfoxide (DMSO: SYBR-14 working solution) and 5 µL of SYBR-14 working

125

solution was added to 1 mL of diluted semen to achieve a final concentration of 1×106

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cell/mL. Then, 5 µL of propidium iodide (PI) solution (Molecular Probes Inc.，Eugene, OR,

127

USA) was added. After 10 min of incubation at 37°C, sperm viability was evaluated by flow

128

cytometry. The SYBR-14 and PI, excited at 488 nm, were read with 530/30nm bandpass

129

emission filter (FL1) and 650/13 nm bandpass emission filter (FL3), respectively. On

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FL1/FL3 dot-plot, percentages of live sperm (SYBR-14+) and dead sperm (PI+) were

131

evaluated.

132

2.4.3. Acrosome integrity

133

Acrosomal status was assessed using FITC-PNA (lectin agglutinin of Arachis hypogaea,

134

L-7381, Sigma Chemical, St. Louis, MO, USA) and PI. The FITC-PNA was dissolved in

135

DMSO to achieve a storage solution at 1 mg/mL and stored at -20°C avoiding light. Then,

136

aliquots of 100 µL of each semen sample (1×106 sperm/mL) were incubated at room

137

temperature in the dark for 5 min with 1 µg/mL FITC-PNA (marker for acrosomal status) and

138

6 µM PI (marker for cell death), as described [37]. On a FL1/FL3 dot-plot, percentages of

139

acrosome-reacted live (ARL) and acrosome-reacted dead (ARD) sperm were evaluated as

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populations with high green fluorescence (FL1), without or with high red fluorescence (FL3),

141

respectively.

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2.4.4. Mitochondrial membrane potential

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The 5,5',6,6'-tetra-chloro-1,1',3,3'-tetraethylben-zimidazolyl-carbocyanine iodide (JC-1,

144

T3168, Molecular Probes, Eugene, OR, USA) and PI were used to assess mitochondrial

145

membrane potential status of sperm, as described [38], with some modifications. The JC-1

146

was dissolved in DMSO to achieve a stock solution (3 mM), and it was diluted 10-fold with

147

PBS to create a working solution. Semen was diluted to a concentration of 1×106 sperm/mL in

148

PBS and 100 µL of the suspension was incubated with 10 µL of JC-1 for 30 min at 37°C in a

149

water bath shielded from light. When mitochondrial membrane potential is high, JC-1

150

reversibly changes its fluorescence from green (monomeric status) to orange (multimeric

151

status) [39]. Emission filters of 585/42 nm bandpass were used to measure green (FL1) and

152

orange (FL2) fluorescence, indicating low and high mitochondrial membrane potential (lMMP

153

or hMMP, respectively), and percentage of cells with hMMP was assessed with CELL QUEST

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software on FL1/FL2 dot-plot.

155

2.5. Seminal plasma markers assessment

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Seminal plasma was separated from sperm as described [40], with modifications. Each

157

semen sample was centrifuged at 3000 × g for 20 min at 4 °C within 1 h after collection. The

158

supernatant was decanted and stored at -80°C until used. Seminal plasma biochemical

159

markers assessed were zinc (Zn: Boruide Biotechnology Co., Ltd, China), neutral

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alpha-glucosidase (NAG: Boruide Biotechnology Co., Ltd, China), acid phosphatase (ACP:

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Boruide Biotechnology Co., Ltd, China) and fructose (Huakang Biomedical Engineering

162

Co Ltd, China). Concentrations of these markers were determined in clear 96-well plates

163

using a Multiskan MK3 (Thermo Fisher Scientific, USA).

164

2.6 Sperm motility

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For each sperm sample, a computer-aided sperm analysis system (CASA, Minitube,

166

Tiefenbach, Germany) was used to determine: total motility (TM, %), progressive

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motility (PM, %), curvilinear velocity (VCL, µm/s), progressive velocity (VSL, µm/s),

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and path velocity (VAP, µm/s). In brief, sperm concentration was calculated using a

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sperm density meter (Minitube) after dilution to 2.0 × 10 7 /mL in PBS in each group and

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incubation in a water bath at 37 °C for 5 min. Then, 5-µL of sample was placed on a

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preheated glass slide (37 °C). For each sample, five non-consecutive microscopic fields

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were randomly chosen on the slide and three slides per sample examined under 200 ×

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magnification using a phase contrast microscope (Axio Scope A1, Carl Zeiss,

174

Oberkochen, Germany).

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2.6. Sperm DNA methylation analysis

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2.6.1. Sperm genomic DNA extraction

177

Genomic DNA was extracted from sperm using a TIANamp Genomic DNA kit (Tiangen

178

Biotech, Beijing, China), according to the manufacturer’s instructions and as described [41].

179

Briefly, each 1.5 mL of semen sample was centrifuged at 1 000 g for 10 min. The sperm

180

pellet was re-suspended in 200 µL of buffer GA and mixed with 20 µL of proteinase K. The

181

solution was added with 200 µL of buffer GB and incubated at 70°C for 10 min before adding

182

200 µL of absolute ethanol. The mixture was transferred to a spin column CB3 and

183

centrifuged at 12 000 g for 30s. The supernatant was discarded, and 500 µL buffer GD was

184

added to the CB3 tube and it was centrifuged at 12 000 g for 30 s. Subsequently, 600 µL of

185

buffer PW was added to the tube and it was centrifuged at 12 000 g for 30 s. The entire

186

process was repeated. Afterward, the CB3 tube was placed in a new 2.0 mL collection tube

187

and 50 µL of buffer TE added to dissolve DNA, followed by centrifugation at 12 000 g for 2

188

min.

189

2.6.2. Bisulfite Conversion and DNA Recovery

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DNA samples were treated using the EpiTect Bisulfite Kit (Qiagen, Germany) for complete

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bisulfate conversion, as described [42], with some modifications. Briefly, 2 µg DNA in 20 µL

192

volume was used for each reaction and mixed with 85 µL bisulfate mix and 15 µL DNA

193

protect buffer. Bisulfite conversion was performed using the following thermal profile: 95°C

194

for 5 min, 60°C for 25 min, 95°C for 5 min, 60°C for 85 min, 95°C for 5 min, 60°C for 175

195

min and thereafter 20°C. Bisulfite-treated DNA was recovered with an EpiTect spin column

196

and sequenced to confirm the efficiency of bisulfite conversion. Bisulfite-converted DNA was

197

immediately used for PCR.

198

2.6.3. Primers and PCR assay

199

Specific primers for amplification and sequencing of DMRs of H19 (Accession: AJ566210,

200

position: 2926-3070) and IGF2R (Accession: AY182033, position: 141-290) were designed

201

using the PyroMark Assay Design2.0 (Table 1). The PCR was continued for 40 cycles after

202

an initial denaturation at 95°C for 3 min. Each cycle of PCR consisted of 30 s at 94°C, 30 s at

203

annealing temperature (56°C), and 60 s at 72°C, and a final extension for 7 min at 72°C. The

204

H19 was analyzed by pyrosequencing using a pair of primers covering a total of 11 CpGs

205

(corresponds to the EMBL/Gen Bank accession number AJ566210 at the 6th CTCF-binding

206

site). However, Igf2r was designed three pair of primers covering a total of 15 CpGs

207

(corresponds to the EMBL/Gen Bank accession number AY182033 at the 2nd DMR) due to

208

the limitation to analyzing amplicons >80 bp by pyrosequencing.

209

2.6.4. Pyrosequencing

210

Pryosequencing was done as described [43], with some modifications. For this, 40µL of

211

PCR product was incubated for 10 min at room temperature with shaking in the presence of 38

212

µL of binding buffer (10 mM Tris, 2 M NaCl, 1 M EDTA, 0.1% Tween 20; pH 7.6; adjusted

213

with 1 M HCl) and 2 µL of streptavidin-coated Sepharose beads (GE Healthcare) after

214

verification by standard gel electrophoresis on 1.5% agarose gel. The binding mix was purified

215

and rendered single stranded using the Pyrosequencing Vacuum Prep Workstation

216

(Pyrosequencing AB), according to the manufacturer’s instructions. Beads were released into

217

40 µL annealing buffer (20 mM Tris, 2 mM magnesium acetate tetrahydrate; pH 7.6; adjusted

218

with 4 M acetic acid) containing 1.5 µL of the respective sequencing primer. The primers were

219

annealed to the target by incubation at 85°C for 2 min. Pyrosequencing was performed on a

220

PyroMark Q96ID Pyrosequence System (QIAGEN) and data analyzed with Pyro Q-CpG

221

Software (QIAGEN). Methylation analyses were done in duplicate.

222

2.7. Statistical analysis

223

Each experiment was repeated at least three times. All computations were performed using

224

SPSS (Version 20.0 for Windows; SPSS). Data for two groups was compared by one-way

225

ANOVA. Data are presented as the mean ± SD. In all cases, P<0.05 was considered

226

significant.

227

3. Results

228

3.1. Seminal parameters from transgenic and wild type sheep

229

There were no significant differences between transgenic and non-transgenic sheep for

230

ejaculate volume, sperm concentration, or for percentages of live sperm, viable sperm with an

231

intact plasma membrane, viable sperm with an intact acrosome and viable sperm with high

232

mitochondrial membrane potential in both F0 and F1 generation (Table 2).

233

No significant differences were found between transgenic and non-transgenic sheep

234

groups for mean concentrations of zinc, Neutral α-glucosidase, acid phosphatase, or fructose

235

(Table 3).

236

3.2 CASA Motility parameters from transgenic and wild type sheep

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The results of the motility parameters of the semen with TRL4 overexpression and wild

238

type, which were analyzed with CASA, are displayed in Figure 2. There was no significant

239

difference (P < 0.05) in all the motility parameters at transgenic sheep to any wild type

240

control in both F0 and F1 generation.

241

3.3. H19 and Igf2r methylation levels of spermatozoa DNA from transgenic and wild

242

type sheep

243

To determine whether insertion and expression of exogenous genes influenced DNA

244

methylation levels of imprinted genes in sperm, regions including 11 CpGs located in the H19

245

DMR, 15 CpGs located in the IGF2R DMR2 were chosen for analysis using pyrosequencing.

246

As shown in Figure 3, there were no significant differences between TLR4 transgenic sheep

247

and control group for mean H19 (Fig.2A) or IGF2R (Fig. 2B) DMR methylation.

248

3.4 Pregnancy rates after artificial insemination with sperm from both the transgenic

249

and wild type sheep

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The pregnancy rates after artificial insemination with sperm from both the transgenic and

251

wild type control are displayed in Figure 4. There is no significant difference between the

252

TRL4 overexpression group and control.

253 254

4. Discussion

255

For successful applications of genetic engineering in animal production systems, animal

256

health and welfare should also be considered. The main aim of this study was to compare

257

semen characteristics, including sperm quality, seminal plasma biochemical index, and

258

methylation of sperm DNA of transgenic sheep with TLR4 and non-transgenic counterparts.

259

All values for the semen traits of control and transgenic sheep were within normal ranges [44],

260

with no significant differences between transgenic and wildtype sheep for any parameter

261

measured. Similar findings have been reported for transgenic cattle and rabbits [45, 46].

262

Antimicrobial protection of male reproductive organs is an essential aspect of

263

reproductive physiology [47]. Because of the role of the epididymis in sperm maturation and

264

storage, it is also critical that the epithelium of the male reproductive tract be protected from a

265

variety of pathogens that can invade the tract, including pathogens that cause sexually

266

transmitted diseases [48]. A number of viruses also infect the male reproductive tract [48].

267

Toll-like receptors (TLRs) are a large family of highly conserved proteins that are essential

268

pathogen-specific recognition sensors of the innate immune system, which are also involved

269

in induction of adaptive immune responses [5, 49]. Although little is known about the

270

significance of TLRs in the male reproductive tract, the abundant expression of a majority of

271

TLR family members together with expression of TLR adaptors and activation targets

272

provides strong evidence that TLRs play important roles in innate immunity of the male

273

reproductive tract [50] .

274

In addition, a number of studies have reported the presence of TLR family members in

275

the female reproductive tract of mice [51] and humans [52-54]. In humans, Tlr2 and Tlr4

276

appear to show differential expression patterns in the fallopian tube, endometrium, cervix, and

277

ectocervix [52] . As reviewed by Wira et al. [55], it is becoming clear that TLRs are important

278

for innate immunity of the female reproductive tract; yet, by comparison. TLR4 contributes to

279

seminal fluid modulation of the periconception immune environment. Activation of TLR4

280

signaling is thus implicated as a key element of the female tract response to seminal fluid at

281

the outset of pregnancy [56] and may thus plausibly contribute to the establishment of

282

maternal immune tolerance induced by seminal fluid [57].

283

Semen quantity (volume, concentration and total number of sperm per ejaculate) and

284

quality (percentage of motile sperm, sperm progressive motility and percentage of abnormal

285

sperm) of sheep are influenced by many factors, including breed [58], age [59], environment

286

[60-62] and nutrition [63]. Sperm viability, acrosome integrity, plasma membrane status and

287

high mitochondrial membrane potential have been correlated with fertilization capacity [64].

288

Similar results have been reported by Yao et al (2017) on the fact that the presence and

289

expression of the TLR4 transgene in the genome does not interfere with normal semen

290

production[65].

291

Seminal plasma biochemical markers are key factors that affect sperm life-span [66]. For

292

example, seminal plasma zinc, secreted by the prostate, is closely related to spermatogenesis

293

[67], sperm count and percentage of sperm with normal morphology [17]. Seminal plasma

294

acid phosphatase, also secreted by the prostate, is correlated to male fertility and often used as

295

a specific marker for determining the activity of the prostate gland [68]. Concentrations of

296

α-glucosidase in seminal plasma are positively correlated to sperm count [69] and percentage

297

of motile sperm [70], and they reflect the functional state of the epididymis. In addition,

298

α-glucosidase concentrations in seminal plasma may be useful for differential diagnosis of

299

certain cases with azoospermia [71]. Seminal plasma fructose, an energy source for sperm, is

300

derived from the seminal vesicles and is therefore a suitable marker for the secretory function

301

of this accessory sex gland [72]. Fructose is significantly correlated with semen volume,

302

spermatozoa count, motility and morphology [16]. In the present study, seminal plasma

303

concentrations of zinc, acid phosphatase, α-glucosidase and fructose were not significantly

304

altered between groups. Therefore, the TLR4 transgene in the genome of these rams did not

305

interfere with normal seminal plasma markers, or the secretion function of reproductive

306

organs, including prostate, epididymis and seminal vesicles.

307

Imprinted genes methylation, especially methylation of H19 (related to the paternal allele),

308

is important for spermatogenesis [73]. Methylation of H19 first appeared in a subset of

309

spermatogonia and then was maintained in spermatocytes, spermatids and mature sperm [28].

310

Many previous studies indicated that abnormal semen parameters (e.g. sperm motility and

311

concentration) are associated with methylation of H19 in infertile patients [29, 74-76]. In the

312

present study, methylation levels of H19 DMR CpG1-11 sites were similar between

313

transgenic and non-transgenic sheep (Figure 3A), suggesting that TLR4 gene insertion did not

314

impact the DNA methylation level at H19. This was consistent with our findings of no effects

315

on semen quality.

316

Insulin-like growth factor-2 receptor (IGF2R), another extensively studied imprinted gene,

317

is generally imprinted on the paternally inherited allele and expressed from maternal allele

318

dependent to the imprinting control region (ICR) differentially methylated [77, 78].

319

Methylation of IGF2R DMRs may be an indicator to assess sperm reprogramming status.

320

There were overall low DNA methylation levels in the 25th and 26th CpG sites of the IGF2R

321

gene in Bos taurus sperm [79]. Results from sperm sex-sorted with flow cytometry suggested

322

that the overall DNA methylation level of the IGF2R gene was not affected by sex-sorting

323

[31]. Given the importance of epigenetic modifications in sperm, we investigated methylation

324

levels of IGF2R. Somatic cell nuclear transfer procedures in sheep can lead to abnormal DNA

325

methylation at IGF2R [80]. However, in the present study, the methylation level of IGF2R

326

DMR CpG1-15 sites in transgenic was not significantly different from the non-transgenic

327

group (Figure 3B). Therefore, we inferred that there was no change in IGF2R methylation

328

level due to insertion of an exogenous TLR4 gene.

329

To the best of our knowledge, this was the first report of DNA methylation of imprinted

330

genes in sperm of transgenic sheep. We confirmed that DNA methylation of H19 and IGF2R

331

imprinted genes in the transgenic sheep was similar with that of our control group. Based on

332

the biological functions of H19 and IGF2R [29, 31], we inferred that normal methylation

333

levels of H19 and IGF2R were suggestive that reproduction was not altered in TLR4

334

transgenic sheep. However, only two regions of the genome were assessed, and it is possible

335

that other methylation errors were not detected. In addition, compared with genome-wide

336

methylation

337

comprehensive although DMRs are regarded as possible functional regions involved in gene

338

transcriptional regulation[81]. Genome-wide methylation analysis is required for this kind of

339

study with the development of DNA sequencing technology.

340

5. Conclusions

analysis,

differentially

methylated

regions (DMRs)

analysis

are

not

341

Based on the results of semen quality, seminal plasma biochemical markers, imprinted

342

genes methylation and pregnancy rate between treated and control group, we concluded that

343

overexpression of TLR4 had no effect on reproductive potential of transgenic sheep.

344 345

Acknowledgements

346

This work was supported by Hebei Province Science and Technology Support Program

347

(17226613D), Natural Science Foundation of Hebei Province of China (C2019204260),

348

Tianjin Science and Technology Project (17ZXZYNC00040), Key Special Projects of

349

Breeding New Varieties of Genetically Engineered Organisms in China (2011ZX08011-004),

350

Young Scientists Fund of the National Natural Science Foundation of China (31900586).

351

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571 572

2011;39:e58.

573 574

Table 1: Designed Primer Sequence Genes

IGF2R -1

Primer

Sequence (5’-3’)

Forward

GGGGTAGGAGTTAGAGTAGAAATTTT

Reverse

ACCTTCTCAACACCTTACTCA

Sequencing

TTTTTTTGGTGAAGGAA

Amplicon

CACGCGAAACAGTACACGTCGAAAGACACG

Number

Amplicon

of CpGs

length (bp)

5

41

5

32

8

60

11

71

ATACAACAGAA

IGF2R -2

Forward

GTAGGGGGTTTTTTTTTTGGTGAAGGAATA

Reverse

ACCTTCTCAACACCTTACTCA

Sequencing

GAAAGATAAGATATAATAGA

Amplicon

ACGGGGCGTGTTCCGCGAGGGGGCGGCCTG GC

IGF2R -3

Forward

TGGAGTGTTTATAGAAAGAGGAGTAG

Reverse

ACCTTCTCAACACCTTACTCA

Sequencing

ACACCTTACTCAAAACCTA

Amplicon

GTGTTCCGCGAGGGGGCGGCCTGGCCG GAGCACGTCGGAGAGGGCTAGCGGCCC GGCTGG

H19

Forward

GGTTGTGGGTGTGGAGATA

Reverse

AACTCTCAAATCTAAATCCACCTCAAT

Sequencing

GGTGTGGAGATAGATG

Amplicon

CGGCCGCGAGGCGGCAGTGCGGGCGCGA GCATCGCCGCCTGCGGCCGCTGTGCCTGA AGTCTGATTATGGC

575 576 577 578 579

580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604

Figure 1 the flowchart of transgenic animal production

605

Table 2: The parameters of sperm quality from transgenic (TG) and wild type (WT) sheep in both

606

F0 and F1 generation F0

F1

Control a (n=19)

Transgenica (n=16)

Control a (n=11)

Transgenic a (n=7)

1.25±0.20

1.02±0.17

1.19±0.19

1.06±0.21

Semen concentration (10 /mL)

2.28±0.27

2.41±0.09

2.18±0.23

2.11±0.15

Percentage of live spermatozoa (%)

77.28±1.28

78.73±5.02

75.12±1.07

72.65±2.13

66.51±1.73

69.84±5.08

63.43±1.96

62.37±3.37

76.72±1.40

76.87±3.74

74.12±1.25

73.54±2.52

51.46±2.70

52.55±3.69

50.39±1.99

49.78±2.66

Parameters

Ejaculate volume (mL) 9

Percentage of viable spermatozoa with an intact plasma membrane（%） Percentage of viable spermatozoa with intact acrosome（%） Percentage of viable spermatozoa with high mitochondrial membrane potential（%） 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622

Note: a No significant differences were detected between control and transgenic animals (P>0.05)

623

Table 3 Seminal plasma biochemical parameters in transgenic and wild type sheep Zinc

Groups

Neutral α-glucosidase

Acid phosphatase

（mM）

（mM） （U/L）

Control a (n=19) a

Transgenic (n=16) 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639

Fructose

（U/mL）

0.37±0.01

0.72±0.03

4.07±1.33

28.68±2.31

0.33±0.06

0.72±0.17

4.49±1.12

20.77±5.29

Note: a No significant differences were detected between control and transgenic animals (P>0.05)

640 641

Figure 2 CASA Motility parameters from transgenic (TG) and wild type (WT) sheep in both F0 and F1

642

generation

643 644 645 646 647 648 649 650 651 652 653 654

655

656 657

Figure 3 Statistical methylation analysis of H19 and IGF2R DMR in sperm achieved via pyrosequencing

658

for TLR4 transgenic sheep and non-transgenic sheep. A) Mean percentage methylation levels of H19

659

DMR (CpG1-11). B) Mean percentage methylation levels of IGF2R DMR (CpG1-15). Mean±SD values

660

are plotted.

661 662 663

664 665

Figure 4 Pregnancy rate of transgenic (TG) and wild type (WT) sheep in both F0 and F1 generations

No significant difference semen quality parameters for transgenic sheep No significant difference of CASA motility parameters for transgenic sheep No significant difference of H19 and IGF2R methylation levels in sperm DNA. Pregnancy rate were also similar between transgenic and control sheep.