1TITLE

2Specific locations and amounts of denatured collagen and collagen-specific3chaperone HSP47 in the oviducts and uteri of old cows as compared to those of4heifers

5

A

Raihana Nasrin Ferdousy, and ABHiroya Kadokawa

7

A

Faculty of Veterinary Medicine, Yamaguchi University, Yamaguchi-shi, Yamaguchi-ken

81677-1, Japan

9

Tel.: + 81 83 9335825; Fax: +81 83 9335938

6

10
11

B Corresponding author: E-mail address: hiroya@yamaguchi-u.ac.jp

12

Faculty of Veterinary Medicine, Yamaguchi University, Yamaguchi-shi, Yamaguchi-ken

13

1677-1, Japan

14

Tel.: + 81 83 9335825; Fax: +81 83 9335938

15

Running head: Denatured collagen in bovine oviducts and uteri

16

17

Abstract18

Collagen, the most abundant extra-cellular matrix in oviducts and uteri, performs critical

19

roles in pregnancies. We hypothesised that the locations and amounts of both denatured

20

collagen and the collagen-specific molecular chaperone, 47-kDa heat shock protein

21(HSP47), in the oviducts and uteri of old cows are different compared to those of young

22

heifers because of repeated pregnancies. Since detecting damaged collagen in tissues is23

challenging, in the first part of this study, we developed a new method that uses a24

denatured collagen detection reagent. Then, we compared damaged collagen in the25

oviducts and uteri between post-pubertal growing nulliparous heifers (22.1 ± 1.0 months

26old) and old multiparous cows (143.1 ± 15.6 months old). Further, we evaluated the27

relationship between denatured collagen and HSP47 by combining this method with28

fluorescence immunohistochemistry. Picro-sirius red staining showed collagen in almost

29

all parts of the oviducts and uteri. Expectedly, damaged collagen was increased in the30

oviducts and uteri of old cows. However, damaged collagen and HSP47 were not located31

in the same area in old cows. The number of fibroblasts increased, suggesting the presence

32

of fibrosis in the oviducts and uteri of old cows. These organs of old cows showed higher

33

HSP47 protein amounts than those of heifers. However, the uteri, but not oviducts, of old

34

cows showed lower HSP47 mRNA amounts than those of heifers. These findings revealed

35

the specific location and amounts of denatured collagen and HSP47 in the oviducts and36

uteri of old cows compared to those of heifers.

37

Additional keywords: aging, fibroblast, fibrosis, infertility, ruminants, serpin family H

38

member 1.

39

Introduction

40

Infertility increases after aging in various animals, including cows (Osoro and Wright411992). However, little is known about the exact pathophysiological mechanisms in42

oviducts and uteri. Collagen, one of the most abundant extra-cellular matrix (ECM)

43proteins, exerts a critical role in successful pregnancies, and abnormal collagen44

expression is associated with recurring miscarriages in women (Li et al. 2019, Shi et al.

452020). The bovine endometrium, similar to that of other mammalians, changes

46

morphologically throughout the oestrous cycle (Arai et al. 2013). Furthermore,

47

dysregulation of ECM remodelling in bovine endometrium may impair fertility (Scolari

48

et al. 2016).

49

Denaturation of collagen is increased due to various diseases, including cancer,

50

osteoporosis, and arthritis (Fields 2013, Ito and Nagata 2019). Denatured collagen was

51

studied by electron microscopy for corneal immune injury (Mohos and Wagner 1969). It

52

was technically challenging to detect damaged collagen in tissues until the recent

53development of a collagen hybridising peptide (Zitnay et al. 2017) and a denatured

54

collagen detection reagent (Takita et al. 2019). The latter is a biotin-labelled collagen-55

mimetic peptide that hybridises with the denatured portion of collagen. It enables the56

detection of denatured collagen via western blotting as well as through visualisation of57

heat damaged collagen fibrils in mouse fibroblasts (Takita et al. 2019).

58

Forty-seven kilodalton heat shock protein, HSP47, encoded by SERPINH1, is a sole

59

procollagen-specific molecular chaperone that is essential for correct folding of the60

unique, triple-helical structure of collagen (Ito and Nagata 2019). HSP47 also plays

61

important roles in the synthesis of collagen as well as in the prevention of procollagen62

aggregation (Duarte and Bonatto 2018). Therefore, HSP47 is central in detecting the

63

location of active collagen synthesis in the oviducts and uteri.

64

In this study, we hypothesised that the locations and amounts of both denatured

65

collagen and HSP47 in the oviducts and uteri of old cows are different compared to those66

of young heifers. In the first part of this study, we developed a new method using the67

denatured collagen detection reagent to compare young and old oviducts and uteri. Then,68

we evaluated the relationship between denatured collagen and HSP47 in heifers and old

69

cows utilising this method followed by fluorescence immunohistochemistry. Further, we

70

compared the amounts of the mRNA and protein of HSP47 in oviducts and uteri between

71

the old cows and heifers.

72
73

Materials and Methods

74

Sample collection

75

All experiments were performed according to the Guiding Principles for the Care76

and Use of Experimental Animals in the Field of Physiological Sciences (Physiological77

Society of Japan) and approved by the Committee on Animal Experiments of Yamaguchi

78

University (approval no. 301).

79

We obtained oviductal and uterine samples from cattle managed by contract

80

farmers in western Japan. All cattle born in Japan since 2003 are registered at birth with81

an individual identification number in a database of National Livestock Breeding Center82

of Japan. We utilised both individual identification numbers to search the database and83

information given by the contract farmers for the cattle in this study.

84

We used a previously reported method (Ferdousy et al. 2020) to obtain the ampulla,

85

isthmus, caruncular (CAR), and intercaruncular (ICAR) areas as well as the uterine

86

myometrium from healthy postpubertal, growing, young nulliparous heifers (22.1 ± 1.0

87months old; Young group), and old multiparous Japanese Black beef cows (143.1 ± 15.6

88

months old; 9 ± 1 parities; sacrificed at least 3 months after the last parturition; Old group)89

at a local abattoir. The heifers and cows were at days 2 to 3, 8 to 12, 15 to 17, or 19 to 21

90of the oestrous cycle (day 0 = day of oestrus), as determined via macroscopic examination

91of the ovaries and uterus (Miyamoto et al. 2000). The samples collected were from the92side ipsilateral to ovulation from days 2 to 3, 8 to 12, or 15 to 17 but were from the side93

ipsilateral to the dominant follicle from days 19 to 21. We collected at least five samples

94

per group per day. Half of the samples (whole tissue fragment; about 1 cm length of95

ampulla and isthmus; about 5 mm in width, 5 mm in length, and 2 mm in thickness of

96

CAR, ICAR, and myometrium) were frozen in liquid nitrogen and preserved at -80°C

97

until RNA or protein extraction was performed. The remaining half was embedded in a98

cryo-mould containing an optimum cutting temperature compound (Sakura Fintechnical99

Co. Ltd., Tokyo, Japan), and then the cryo-mould was wrapped with an aluminium foil.

100

The cryo-moulds were then frozen in liquid nitrogen and preserved at -80°C until in situ

101

detection of denatured collagen and immunohistochemistry studies were conducted.

102

Additionally, we measured the total collagen in tissue using picro-sirius red staining.

103

For this purpose, we obtained the ampulla, isthmus, CAR, and ICAR from four different

104

young nulliparous heifers (21.5 ± 0.6 months old), and four old multiparous Japanese

105Black beef cows (148.8 ± 12.0 months old; 9 ± 1 parities; sacrificed at least 3 months

106

after the last parturition) at the local abattoir. The samples were stored in 4%

107

paraformaldehyde (PFA) at 4°C for 16 h.

108
109
110
111

Development of the method for in situ detection of denatured collagenWe developed a new method for in situ detection of denatured collagen. As described
below, this method can be combined with fluorescent immunohistochemistry.

112

Unfixed tissue blocks were sectioned into 15-µm-thick sections using a cryostat

113(CM1950, Leica Microsystems, Wetzlar, Germany) and mounted on slides (MAS coat

114

Superfrost, Matsunami-Glass, Osaka, Japan). Next, the tissue was fixed with 4% PFA in

115

PBS for 15 min. Some sample sections from the young group were placed on slides

116

treated with 100 °C PBS for 2 min (pre-heated slides) or with 25 °C accutase cell117

detachment solution (Nacalai Tesque, Kyoto, Japan) for 30 min (digested slides) to118

denature collagen, whereas others were treated with ice-cold PBS (non-treated slides).

119

We used accutase because it is a proteolytic and collagenolytic enzyme, and it is not120

inhibited by the possible presence of calcium and magnesium in the tissues. We tried to121

use also collagenase, but the structures of the tissues were broken after staining, while122

accutase did not significantly damage the structure. Blocking was performed by123

incubating the tissue sections in PBS (0.5 mL) containing 10% normal goat serum (Wako

124

Pure Chemicals, Osaka, Japan) for 1 h at room temperature. Tissues were then treated125with an avidin/biotin blocking kit (Vector Laboratories, Burlingame, CA, USA) following

126

the manufacturer's protocol. After washing twice with PBS, the slide was loaded with 5127

μg/mL of denatured collagen detection reagent (Funakoshi, Tokyo, Japan) and incubated128

for 60 min at room temperature in a humid box. After washing twice with PBS, the tissues

129

were incubated with streptavidin, Alexa Fluor 546 conjugate (diluted to 1 μg/mL in PBS,

130

Thermo Fisher Scientific, Waltham, MA, USA), and 1 μg/mL of 4, 6-diamino-2-

131

phenylindole (DAPI; Wako Pure Chemicals) for 60 min at room temperature. After132

washing thrice, cover glasses were mounted using Vectashield HardSet Mounting

133

Medium (Vector Laboratories).

134

Sections were observed with a confocal microscope (LSM710; Carl Zeiss, Göttingen,

135

Germany) equipped with a 405 nm diode laser, 488 nm argon laser, 533 nm HeNe laser,136

and 633 nm HeNe laser. Images obtained by fluorescence microscopy were scanned with137

a 20× or 40× oil-immersion objective and recorded with a CCD camera system controlled

138

by ZEN2012 black edition software (Carl Zeiss). The exact same microscope settings

139

were used throughout immunofluorescence imaging of the ampulla, isthmus, CAR, ICAR,

140

or myometrium to compare between young and old cows. We distinguished between the141

layers or parts of oviducts and uteri according to Banu et al. (2005), Hayashi et al. (2017),

142

and Godoy-Guzman et al. (2018).

143
144

Picro-sirius red staining for total collagen145

The fixed tissues were dehydrated and embedded in paraffin using the Handed146

autokinette (model 1400P, Shiraimatsu Corp. Ltd., Osaka, Japan). Thin sections (10 µm

147

thick) were cut with a sliding microtome (Yamato Kohki, Saitama, Japan) attached onto

148

a slide glass. The paraffin-embedded sections were de-parafinised thrice in xylene, for 5

149

min each, followed by de-alcohol in 100%, 100%, 90%, and 70% ethanol and ultrapure

150

water for 5 min each. The slides were then covered in pico-sirius red staining solution

151(Picro-Sirius Red Stain Kit, ScyTek laboratories Inc., Logan, UT, USA) for 1 h. Following

152

staining, the slides were washed twice with 0.5% acetic acid solution. The sections were153

dehydrated through three changes of 100% ethanol and cleared in three changes of xylene.

154

After attaching the cover slip with Entellan new mounting medium (Sigma-Aldrich, St.

155

Lous, MO, USA), the stained sections were observed under a light microscope fitted with156

a digital camera (Eclipse Ci, Nikon, Tokyo, Japan). The staining and light microscopy

157

results showed collagen in red, muscle fibres and cytoplasm in yellow, and complex in

158

orange.

159
160

RT-PCR, sequencing of amplified products, and homology search in gene databases

161

Total RNA (at least four per tissue) was extracted using the RNAzol RT isolation

162

reagent (Molecular Research Centre Inc., Cincinnati, OH, USA) and treated with163

deoxyribonuclease. The concentration and purity of each RNA sample were evaluated by164

spectrophotometry (acceptable 260/280 nm ratio of absorbance, 1.8–2.1) and165

electrophoresis (28S:18S ratios were 2:1). Complementary DNA was synthesised using

166

the Verso cDNA Synthesis Kit (Thermo Fisher Scientific). No reverse transcription

167

controls (NRCs) were prepared for RT-PCR; they were generated by treating the168

extracted RNA with the same deoxyribonuclease but not with cDNA synthetase.

169

To determine the amount of HSP47 mRNA, a primer pair was designed by Primer

170

Express v3.0 (Thermo Fisher Scientific) based on the reference sequence of bovine

171

HSP47 [National Center for Biotechnology Information (NCBI) reference sequence of172

bovine HSP47 is NM_001046063]. Table 1 details the primers. PCR was performed using

173

20 ng of cDNA, 20 ng RNA as the NRC or water as the no template control (NTC), and174

polymerase (Tks Gflex DNA Polymerase, Takara Bio Inc., Shiga, Japan) under the175

following thermocycle conditions: 94 °C for 1 min for pre-denaturation followed by 35

176

cycles of 98°C for 10 s, 60°C for 15 s, and 68°C for 30 s. PCR products were separated

177

on 1.5% agarose gel by electrophoresis with a molecular marker (Nippon Gene, Tokyo,

178

Japan), stained with Gelstar (Lonza, Allendale, NJ, USA), and observed using a charge-

179

coupled device (CCD) imaging system (GelDoc; Bio-Rad, Hercules, CA, US). The PCR

180

products were purified with the NucleoSpin Extract II kit (Takara Bio Inc.) and then181

sequenced using one of the PCR primers and the Dye Terminator v3.1 Cycle Sequencing

182

Kit (Thermo Fisher Scientific). The sequences obtained were used as query terms with183

which to search the homology sequence using the basic nucleotide local alignment search

184

tool (BLAST) (available on the NCBI website).

185

186

Western blotting for HSP47 detection

187

G*Power 3 for windows (Faul et al. 2007) was used to estimate the required

188

number of samples with an alpha-error probability of 0.05 and a statistical power of 0.95.

189

Five samples per day in each tissue of each group (total of 20 samples in each tissue of

190

each group) were assessed and analysed statistically. The samples were ground in liquid

191

nitrogen and homogenised using tissue protein extraction reagent (T-PER; Thermo Fisher

192

Scientific) containing Halt protease inhibitor cocktail (Thermo Scientific). The total

193

protein content of each tissue homogenate was estimated using a bicinchoninic acid kit

194(Thermo Fisher Scientific). The extracted protein sample was boiled with Sample Buffer

195

Solution with Reducing Reagent (6x) for SDS-PAGE (09499-14; Nacalai Tesque) for 3

196

min at 100 °C. The protein samples (8,000 ng of total protein) were loaded onto a

197polyacrylamide gel (Any KD Criterion TGX gel, Bio-Rad) along with a molecular weight198

marker (Precision Plus Protein All Blue Standards; Bio-Rad). Then, the proteins were199

resolved using sodium dodecyl sulphate–polyacrylamide gel electrophoresis at 100 V for

200

90 min. The proteins were transferred onto polyvinylidene fluoride (PVDF) membranes

201(Trans-blot turbo PVDF, Bio-Rad) with electroblotting at 2.5 A and 25 V for 7 min using

202

a Trans-blot Turbo system (Bio-Rad).

203

We used a Can Get Signal Immunoreaction Enhancer kit (Toyobo Co. Ltd, Osaka,204

Japan) for membrane blocking (1 h at 25 °C), primary antibody reaction (1 h at 25 °C)

205with an anti-HSP47 rabbit polyclonal antibody (1:400,000 dilution with immunoreaction

206

enhancer solution; AP7366B; Abcepta Inc., San Diego, CA, USA), and secondary

207

antibody reaction (1 h at 25 °C) with goat anti-rabbit IgG horseradish peroxidase-

208

conjugated antibody (Bethyl Laboratories Inc., Montgomery, TX, USA; 1:400,000

209

dilution with immunoreaction enhancer solution). The anti-human HSP47 rabbit antibody

210

recognizes the mature C-terminal form of human HSP47 (corresponding to amino acids

211

390 to 418; FLVRDTQSGSLLFIGRLVRPKGDKMRDEL). This sequence had 100%212

homology to amino acids 390 to 418 of the mature C-terminal form of bovine HSP47 but

213

no homology to other bovine proteins, as determined using protein BLAST (NCBI

214

reference sequences of human and bovine HSP47 are NP_001193943.1 and215

NP_001039528.1, respectively).

216

The protein bands were visualised using an ECL-Prime chemiluminescence kit

217(GE Healthcare, Amersham, UK) and a CCD imaging system (LAS-3000 Mini; Fujifilm,

218

Tokyo, Japan). The images were exported using the Multigauge (version 3.0; Fujifilm)

219

software. To verify the specificity of the signals, we included several negative controls in

220

which the primary antibodies had been omitted or normal rabbit IgG (Wako Pure

221

Chemicals) antibodies were used instead of the primary antibodies. Signal specificity was222

also confirmed using negative controls in which the primary antibodies were pre-223

absorbed with 5 nM antigen peptide (Scrum Inc., Tokyo, Japan).

224

The antibodies were removed from the PVDF membrane with a stripping solution

225(Nacalai Tesque) prior to the blocking and subsequent immunoblotting with an anti-β-226

actin mouse monoclonal antibody (1:400,000 dilution; Sigma-Aldrich).

227

All relevant bands were cropped from the exported file using Adobe Photoshop

228

element ver. 2020 (Adobe, San Jose, CA, USA) and pasted onto a graph created using

229

DeltaGraph ver. 7.5.2J (Red rock software, Salt Lake, UT, USA). ImageQuant TL

230(version 8.2; Cytiva, Marlborough, MA, USA) software was used to measure the band

231

sizes and volumes (calculated using rolling ball background subtraction). The expression

232

protein amount of HSP47 was normalised against β-actin.

233
234

RT-qPCR analysis of HSP47

235

After preparation of high-quality total RNA and cDNA synthesis using the236

previously described protocol, HSP47 mRNA amount was compared among the young237

and old groups via the relative standard curve method of RT-qPCR and data analyses, as

238

described previously (Nahar and Kadokawa 2017, Ferdousy et al. 2020). G*Power 3 for239

windows (Faul et al. 2007) was used to estimate the required number of samples with an240

alpha-error probability of 0.05 and a statistical power of 0.95. Five cDNA samples per

241

day in each tissue of each group (total of 20 cDNA samples in each tissue of each group)242

were assessed and analysed statistically.

243

To prepare external standards for amplified fragments of cDNA products

244

containing target sequences for RT-qPCR of HSP47(800 bp) and two housekeeping genes,

245

C2orf29 (562 bp) and SUZ12 (1169 bp), PCRs were conducted as described previously

246(Nahar and Kadokawa 2017). The primers were designed by Primer Express Software247

v3.0 (Table 1). The PCR-amplified products were purified to prepare the standards, as

248

well as to verify the DNA sequence. Then, we prepared a 6-point relative standard by 10-249

fold diluting the PCR products for the relative standard method.

250

HSP47 mRNA levels were normalised to the geometric mean of the levels of two

251

house-keeping genes, C2orf29 and SUZ12, selected using Normfinder program

252(Vandesompele et al. 2002) and amplified using previously reported primers (Rekawiecki

253

et al. 2012; Nahar and Kadokawa 2017), since they are among the most stable and reliable

254

housekeeping genes in the bovine oviducts and uterus (Walker et al. 2009; Nahar and255

Kadokawa 2017).

256

The mRNA level was measured in duplicate by RT-qPCR analyses with 20 ng

257

cDNA, using CFX96 Real Time PCR System (Bio-Rad) and Power SYBR Green PCR

258

Master Mix (Thermo Fisher Scientific), together with the 6-point relative standards, NTC,

259

and NRC to generate the standard curve by plotting the log of the starting quantity of the260

dilution factor against the Cq value using appropriate software (CFXmanagerV3.1, Bio-

261

Rad). Temperature conditions for all genes were as follows: 95°C for 10 min for pre-262

denaturation; five cycles each of 95°C for 15s and 66°C for 30s; and 40 cycles each of

263

95°C for 15s and 60°C for 60 s. Melting curve analyses were performed at 95°C for each

264

amplicon and each annealing temperature to ensure the absence of smaller non-specific265

products, such as dimers. All the Cq values of the unknown samples (22.85 ± 0.15) were266

between the highest (8.00) and lowest (30.33) standards for HSP47 in RT-qPCR. Further,267

all the Cq values of the unknown samples were between the highest and lowest standards

268

for C2orf29 or SUZ12 in RT-qPCR. Reactions with a coefficient of determination (R2) >269

0.98 and efficiency between 95 and 105% were considered optimised. The coefficients

270of variation of RT-qPCRs were less than 6%. The concentration of PCR products was271

calculated by comparing Cq values of unknown samples with the standard curve using

272

CFXmanagerV3.1. Then, the HSP47 amount was divided by the geometric mean of273

C2orf29 and SUZ12 in each sample.

274
275

Immunofluorescence staining and confocal microscopy

276

For immunofluorescence staining, we randomly selected at least five tissue samples277

per day in each tissue of each group. To evaluate the association between HSP47-rich278

areas and denatured collage-rich areas, the tissues on slides were washed once after

279

incubation with streptavidin, Alexa Fluor 546 conjugate, and DAPI. Then, the sections

280

were treated with 0.3% Triton X-100 for 15 min, blocked with 10% normal goat serum

281

in PBS for 1 h at room temperature, and subjected to a primary antibody reaction with the282

anti-HSP47 antibody (1:1,000 dilution for overnight at 4°C). Subsequently, the samples

283

were subjected to a secondary antibody reaction with Alexa Fluor 647 goat anti-rabbit284

IgG (diluted to 1 µg/mL, Thermo Fisher Scientific) and 1 µg/mL DAPI for 2 h at room285

temperature. After washing the sections four times, the slides were mounted in

286

preparation for confocal microscopy. To verify the specificity of signals, we included

287

several negative controls in which the primary antiserum had been omitted or pre-288

absorbed with 5 nM antigen peptide, or in which normal rabbit IgG was used instead of

289

the primary antibody. We also included negative controls in which the secondary290

antibodies had been omitted or normal goat IgG (Wako Pure Chemicals) antibodies were291

used instead of the secondary antibodies. Signal specificity was also confirmed using292

negative controls in which the secondary antibodies were pre-absorbed with 5 nM normal

293

rabbit IgG.

294

Additionally,

we

performed

immunohistochemistry to

identify whether

295

cytokeratin-rich areas or vimentin-rich areas express HSP47. For this purpose, we

296

included anti-bovine pancytokeratin mouse monoclonal antibody or anti-bovine vimentin

297

mouse monoclonal antibody (both from Sigma-Aldrich, and diluted as 1:1,000) in the298

primary antibody reaction, and Alexa Fluor 488 goat anti-mouse IgG (diluted to 1 µg/mL,

299

Thermo Fisher Scientific) in the secondary antibody reaction.

300
301

Analysis of the 5′-flanking region of SERPINH1

302

The 5000-nucleotide sequence of the 5′-flanking region of the SERPINH1 gene303

(chromosome 15: 54,737,997-54,748,417) was obtained using the online Ensembl Search

304

Genome program (http://www.ensembl.org) against the bovine genome database (April.

305

2018, ARS_UCD_1.2/bosTau9). The sequence was analysed using Genetyx software v.13

306

(Genetyx, Tokyo, Japan) for the presence of consensus response element (RE) sequences307

for oestrogen—i.e., ERE (5′-GGTCANNNTGACC-3′), ERE-like sequence (5′-308

TGACCCCTGGGTCA-3′) (Gruber et al., 2004), and half ERE (GGTCA, TGACC, or

309

TGACT) (Liu et al., 1995), as well as for progesterone—i.e., PRE (5′-G/A G G/T AC

310

A/GTGGTGTTCT-3′) (Geserick et al., 2005) and half PRE (5′-TGTTCT-3′) (Tsai et al.,311

1988).

312

313

Statistical analysis

314

The statistical analyses were performed using StatView version 5.0 for Windows

315(SAS Institute, Inc., Cary, NC, USA). Grubb’s test verified the absence of outliers. The

316

Shapiro-Wilk’s test and Kolmogorov-Smirnov Lilliefors test verified the normality of

317

distribution of each variable. Two-factor analysis of variance (ANOVA) was used to318

evaluate the effect of age (young or old), stage (days 2 to 3, 8 to 12, 15 to 17, or 19 to 21),319

and interaction followed by post-hoc comparisons using Fisher’s protected least320

significant difference (PLSD) test for the data of RT-qPCR or western blotting for HSP47.

321

The statistical significance of differences among stages was assessed by one-factor

322

ANOVA followed by post-hoc comparisons using Fisher’s PLSD test using a model

323

consisting of variance from the effect of stage and the residual. The level of significance324

was set at P<0.05. Data are expressed as means ± standard errors of the mean (SEM).

325
326
327
328

Results
Picro-sirius red staining showed red or orange colour in almost all parts of theampullae, isthmuses, CAR, ICAR, and myometria (Fig. 1).

329

We successfully developed a new in situ assay for the detection of denatured collagen330(Fig. 2). The non-treated young tissues showed a very subtle signal indicating the331

presence of denatured collagen (red). By contrast, the old samples, similar to the heated332

and enzyme-treated young tissues, showed a strong signal indicating the presence of333

denatured collagen in the lamina propria and muscular layer of the ampullae and334

isthmuses. In addition, the heated young uteri showed strong signals in the luminal

335

epithelia and uterine stroma of CAR as well as in the glandular epithelia and uterine

336

stroma of ICAR, and various cells in the uterine myometria.

337

PCR products of a size corresponding to that of HSP47 (470 bp) were obtained from338

the ampullae, isthmuses, CAR, ICAR, and myometria, as revealed by agarose gel

339

electrophoresis (Fig. 3). Neither the NTC nor any of the NRCs yielded any PCR-amplified

340

products. A homology search against gene databases for the sequenced amplified products341

revealed bovine HSP47 (NM_001046063) as the best match, with a query coverage of

342

100%, an e-value of 0.0, and a maximum alignment identity of 99%. No other bovine

343

genes displayed homology with the PCR product, indicating that the amplified product344

was indeed HSP47.

345

We combined the in situ detection method for denatured collagen with346

immunofluorescence staining of HSP47 in the ampulla (Fig. 4A), isthmus (Fig. 4B), CAR

347(Fig. 5A), ICAR (Fig. 5B), and myometrium (Fig. 5C). Robust, high-intensity

348

fluorescence signals of denatured collagen were localised again in the above-mentioned349

areas of old samples but not in those of young samples. In addition, HSP47 signals were350

localised in the epithelial layer and superficial stroma very near to the epithelial layer of351

oviducts of old individuals but only weakly in those of young individuals (Fig.6). HSP47

352

signals were localised in the epithelia and stroma of the CAR and ICAR of old and young353

individuals. HSP47 signals were localised in the myometrium of old individuals but not354

in those of young individuals. More importantly, HSP47 rich-areas (green) and denatured

355

collagen-rich area (red) were different because there was little colocalisation (yellow) in356

the merged panels. There was no signal indicating that HSP47 had colocalised with357

denatured collagen in the lamina propria and muscular layers of ampullae and isthmuses.

358

Therefore, HSP47-rich and denatured collagen-rich areas were different.

359

Figure 7 shows the results of western blot and outcomes of two-factor ANOVA. The

360

western blot revealed HSP47 protein in all five specimens obtained from old cows, but361

only weak expression in those from young heifers (Fig. 4). Unexpectedly, an extra band362

at 25 kDa appeared only in old samples. The ANOVA revealed that the age effect was

363

significant for the 47 kDa or 25 kDa bands in the ampulla, isthmus, CAR, ICAR, and364

myometrium, and old samples contained a higher amount of HSP47 than the young

365

samples. The effect of stage was significant in almost all samples, except for the 47 kDa

366

band in ampulla and isthmus, and 25 kDa band in the myometrium. The interaction

367

between the effects of age and stage was significant only for 47 kDa and 25 kDa bands in368

the CAR.

369

Figure 8 shows the results of RT-qPCR, and outcomes of two-factor ANOVA. The age

370

effect was significant in the CAR, ICAR, and myometrium, but not in the ampulla and371

isthmus. The effect of stage was significant in the ampulla, isthmus, and ICAR, but not372

in CAR and myometrium. The interaction between the effects of age and stage was373

significant only in the isthmus.

374

The 5′-flanking region of the bovine SERPINH1 gene was analysed for EREs, PREs,

375

and similar sequences. Although there were no ERE, ERE-like, or PRE sequences, 19376

half ERE (7 GGTCA, 7 TGACC, and 5 TGACT) and 3 half PRE sequences were377

identified.

378
379

Discussion

380

We developed a new method that uses a denatured collagen detection reagent to381

identify denatured collagen-rich areas. The picro-sirius red stain showed red or orange

382

colour in almost all parts of oviduct and uterus, including the epithelium. Boos (2000)

383

reported the presence of collagen types I, III, IV, and VI in various parts of the uterus and384

the presence of collagen types IV and VI in the epithelium. We did not identify the385

collagen type in this study, but collagen was present in almost all parts of the oviduct and386

uteri. The results indicated that the levels of denatured collagen in old oviducts and uteri

387

were higher than those in the oviducts and uteri of young ones. Thus, it is possible that388

repeated pregnancy and parturition increase the denatured collagen. Another possible389

reason for such an increase in the levels of denatured collagen may be infection and390

inflammation because lipopolysaccharides decrease collagen synthesis in myometrial

391

explants from women (Wendremaire et al. 2013).

392

The tunica mucosa of oviducts and epithelia of the endometrium are located adjacent393

to the lumen, and thus, the structure of these layers may be susceptible to damage or394

frequent changes. Ovarian steroid hormones drive ECM remodelling in the bovine

395

oviduct (Gonella-Diaza et al. 2018). Therefore, even young heifers may require collagen396

biosynthesis because these layers provide growth factors and nutrients for embryogenesis

397(Hugentobler et al. 2010; Besenfelder et al. 2012).

398

Little is known regarding HSP47 expression in the oviducts and uteri of all species.

399

Therefore, we were unable to compare the data obtained by the current study with those400

of previous studies. The high HSP47 protein amounts observed in old oviducts and uteri

401

compared with those in the young oviducts and uteri was somewhat unexpected because402

body weights of heifers were still in the phase of increasing (Inoue et al. 2020), and thus,403

we speculated that increased size of the uteri and oviducts may also play a role in HSP47

404

expression. Little is known regarding postnatal changes associated with the sizes of405

bovine oviducts and uteri, as growth continues at least until 15 months of age in beef406

heifers (Honaramooz et al. 2004). However, the minimum age at first calving among407

about 2600 Japanese Black heifers was 21.4 months old (Inoue et al. 2020). Therefore, it408

was possible that the growth of oviducts and uteri of the young group was completed409

before sampling in this study.

410

We must be careful in interpreting the data obtained using the anti-HSP47

411

antibody

fortworeasons.

First,

theantibody

isnotrecommended

for

412

immunohistochemistry in frozen sections by the supplier. However, we observed

413

increased denatured collagen in the paraffin section in our preliminary trial using the414

denatured collagen detection reagent because the steps of paraffin embedding and415

deparaffinisation increased the denatured collagen. Second, we observed an extra 25 kDa

416

band in the western blot. We could not find any paper reporting another size of HSP47.

417

Therefore, we must be careful as the HSP47 signal in the immunohistochemistry results418

may have been caused by another protein. However, we performed western blotting using419

another anti-HSP47 mouse monoclonal antibody, clone M16.10A1 (Enzo Life Sciences,

420

Inc., Farmingdale, New York. USA) and we observed both the 25 kDa and 47 kDa bands

421(Supplementary Figure 1). We tried to search similar proteins using the amino acid422

sequence of bovine HSP47 using protein BLAST. However, we could not find any protein423with high homology. Therefore, further studies are required to identify the protein that the424

25 kDa band corresponds to.

425

Importantly, HSP47 protein amount was increased in areas other than the426

denatured collagen-rich areas in old cows. These results suggested a possibility that427

collagen synthesis may not occur in denatured collagen-rich areas, indicating that a428

damaged structure may remain uncured. This may explain the increased infertility429

observed in older beef cows (Osoro and Wright 1992).

430

However, this brings into question the physiological significance of increased

431

HSP47 protein amount observed in other areas with less denatured collagen. Notably, an

432

abnormal increase in collagen synthesis by fibroblasts induced by transforming growth433

factor (TGF)-β1 and its isoforms may lead to adenomyosis and ectopic endometria in

434

women (Cheong et al. 2019). TGF-β1 may also exert important pathological effects on

435

fibroblasts during equine endometriosis (Szostek-Mioduchowska et al. 2019). Although

436

HSP47 is a key regulator of cell homeostasis, it also plays a role in fibrogenesis and437

fibrotic disorders in the liver, kidneys, and lungs. Excess HSP47 expression is an

438

important step in collagen-related diseases, including keloids and fibrosis (Ito and Nagata,4392019). In addition, this study showed an increase in the number of fibroblasts in various

440

parts of the oviducts and uteri, thereby suggesting fibrosis. Therefore, any increase in441

HSP47 protein amount may play an important role in inducing infertility in old cows.

442

The bovine endometrium is thicker on days 19‒21 or days 2‒3 than on other days

443

(Sugiura et al. 2018). The highest amount of HSP47 mRNA was observed in the ampulla,444

isthmus, CAR, and ICAR on either days 19‒21 or days 2‒3 in this study. Young oviducts

445

and uteri showed a higher amount of HSP47 mRNA than did old ones, whereas young446

oviducts and uteri showed a lower amount of HSP47 protein than did old ones. Based on

447

these results, high levels of HSP47 protein may be required only temporarily in oviducts

448

and uteri to avoid excess collagen synthesis, a possible cause for inflexibility. The HSP47

449

protein is bound to a ubiquitin-like protein, UBIN (Matsuda et al. 2001) and is degraded450

via the ubiquitin-proteasome system (Ito and Nagata 2016). Therefore, further studies are451

required to clarify the possibility that HSP47 protein is degraded via the ubiquitin-

452

proteasome system shortly after translation in young organs but is not degraded smoothly

453

in old organs, resulting in abnormal collagen synthesis.

454

HSP47 is induced by cellular stresses, but it is also constitutively expressed, and455

its expression is always up- or down-regulated concomitantly with changes in the456

expression of various types of collagens, as reviewed by Ito and Nagata (2017). Therefore,457

a possible reason for the lower amount of HSP47 mRNA observed in old organs is the458

increased collagen. We found 19 half ERE and 3 half PRE sequences in the 5'-flanking459

region of the bovine SERPINH1 gene. A caveat here is that half ERE and PRE-like460

sequences are only five or six nucleotides long; such short sequence can appear at random461

at every 1024 (= 45) or 2048 (= 46) nucleotides. Thus, some of the identified sequences462

may not be involved in the control of gene expression. Detailed studies are required to463

clarify the mechanisms regulating HSP47 expression and the roles of oestradiol and464

progesterone.

465

HSP47 signals were observed in the epithelial layer and superficial stroma very466

near to the epithelial layer of oviducts and endometria of old individuals. HSP47 is

467

expressed also in the surface epithelial cells of pneumonia (Kakugawa et al. 2005) and468

desmin-positive glomerular epithelium cells of the kidney (Razzaque and Taguchi 1999).

469

Moreover, HSP47 expression in human nasal mucosa and lacrimal sac (obtained

470

surgically) is associated with surgical outcome (Park et al. 2018). HSP47 induces471

mesenchymal phenotypes in mammary epithelial cells for cancer metastasis, and HSP47472

is a hub of the ECM transcription network (Xiong et al. 2020). Therefore, further studies473

are required to clarify the relationship between HSP47expression in the layers and fertility474

in cows.

475

Takita et al. (2019) produced a standard curve based on an enzyme immunoassay

476(EIA) utilising the denatured collagen detection reagent and heated pure collagen (grade477

for culture dish coating). However, our attempts of immunoassays (HRP-conjugated478

streptavidin for EIA, fluorochrome-conjugated streptavidin for fluorescent immunoassay,

479

lanthanide-conjugated streptavidin for dissociation-enhanced lanthanide fluorescence

480

immunoassay) failed to obtain a good parallel between the standard curve and serially481

diluted bovine oviduct or uterine extracts that were extracted using various methods. This482

was due to the samples showing strong matrix effects unlike pure collagen. Therefore,483

our comparison could be performed only on photographic images.

484

In conclusion, these findings revealed the specific location and amounts of485

damaged collagen and HSP47 in the oviducts and uteri of old cows compared to those in486

heifers.

487
488

Acknowledgments

489

Raihana Nasrin Ferdousy was supported by a scholarship from MEXT (Ministry of490

Education, Culture, Sports, Science, and Technology). This research was partly supported

491

by a Grant-in Aid for Scientific Research (JSPS Kakenhi Grant Number 21H02345) from492

Japan Society for the Promotion of Science (Tokyo, Japan) to Hiroya Kadokawa. The

493

authors thank Prof. Ken Takeshi Kusakabe from the Laboratory of Veterinary Anatomy,

494

Yamaguhi University, Japan, for the advice regarding picro-sirius red staining.

495
496

Conflicts of Interest

497

The authors declare no conflicts of interest.

498

499

References

500

Arai, M., Yoshioka, S., Tasaki, Y., and Kiyoshi Okuda, K. (2013). Remodeling of bovine

501

endometrium throughout the estrous cycle. Anim. Reprod. Sci. 142, 1-9. doi:502

10.1016/j.anireprosci.2013.08.003

503

Banu, S.K., Arosh, J.A., Chapdelaine, P., and Fortier, M.A. (2005). Expression of504

prostaglandin transporter in the bovine uterus and fetal membranes during

505

pregnancy. Biol. Reprod. 73, 230-6. doi: 10.1095/biolreprod.105.039925

506

Besenfelder, U., Havlicek, V., and Brem, G. (2012). Role of the oviduct in early embryo

507

development. Reprod. Domest. Anim. 47, 156-63. doi:10. 1111/J.1439-

508

0531.2012.02070.X 50

509

Boos, A. (2000). Immunohistochemical assessment of collagen types I, III, IV and VI in510

biopsy samples of the bovine uterine wall collected during the oestrous cycle.

511

Cells Tissues Organs 167, 225-38. doi: 10.1159/000016799.

512

Cheong, M.L., Lai, T.H., and Wu, W.B. (2019). Connective tissue growth factor mediates

513

transforming growth factor β-induced collagen expression in human endometrial

514

stromal cells. PLoS One 14, e0210765. doi: 10.1371/journal.pone.0210765

515

Duarte, B.D.P., and Bonatto, D. (2018). The heat shock protein 47 as a potential

516

biomarker and a therapeutic agent in cancer research. J. Cancer. Res. Clin. Oncol.

517

144, 2319-28. doi: 10.1007/s00432-018-2739-9

518

Faul, F., Erdfelder, E., Lang, A.G., and Buchner, A. (2007). G*Power 3: a flexible

519

statistical power analysis program for the social, behavioral, and biomedical

520

sciences. Behav. Res. Methods 39, 175-91. doi: 10.3758/bf03193146

521

Ferdousy, R.N., Kereilwe, O., and Kadokawa, H. (2020). Anti-Müllerian hormone

522

receptor type 2 (AMHR2) expression in bovine oviducts and endometria:

523

comparison of AMHR2 mRNA and protein abundance between old Holstein and524

young and old Wagyu females. Reprod. Fertil. Dev. 32, 738-47. doi:

525

10.1071/RD19121

526
527

Fields, G.B. (2013). Interstitial collagen catabolism. J. Biol. Chem. 288, 8785-93. doi:
10.1074/jbc.R113.451211

528

Geserick, C., Meyer, H.A., and Haendler, B. (2005). The role of DNA response elements529

as allosteric modulators of steroid receptor function. Mol. Cell. Endocrinol. 236,530

1-7. doi: 10.1016/j.mce.2005.03.007

531

Godoy-Guzmán, C., Nuñez, C., Orihuela, P., Campos, A., and Carriel, V. (2018).

532

Distribution of extracellular matrix molecules in human uterine tubes during the533

menstrual cycle: a histological and immunohistochemical analysis. J. Anat. 233,

534

73-85. doi: 10.1111/joa.12814

535

Gonella-Diaza, A. M., Mesquita, F. S., Lopes, E., Silva, K. R. d., Cogliati, B., Strefezzi

536

R. D. F., and Binelli, M. (2018). Research Article Sex steroids drive the537

remodeling of oviductal extracellular matrix in cattle. Biol. Reprod. 99, 590-9.

538

doi:10.1093/biolre/ioy083

539

Gruber, C.J., Gruber, D.M., Gruber, I.M., Wieser, F., and Huber J.C. (2004). Anatomy of540

the estrogen response element. Trends Endocrinol. Metab. 15, 73-8. doi:

541

10.1016/j.tem.2004.01.008

542

Hayashi, K.G., Hosoe, M., Kizaki, K., Fujii, S., Kanahara, H., Takahashi, T., and543

Sakumoto, R. (2017). Differential gene expression profiling of endometrium

544

during the mid-luteal phase of the estrous cycle between a repeat breeder (RB)545

and non-RB cows. Reprod. Biol. Endocrinol. 15, 20. doi: 10.1186/s12958-017-

546

0237-6547

Honaramooz, A., Aravindakshan, J., Chandolia, R.K., Beard, A.P., Bartlewski, P.M.,

548

Pierson, R.A., and Rawlings, N.C. (2004) Ultrasonographic evaluation of the549

pre-pubertal development of the reproductive tract in beef heifers. Anim. Reprod.

550

Sci. 80, 15-29. doi: 10.1016/S0378-4320(03)00136-2

551

Hugentobler, S. A., Sreenan, J. M., Humpherson, P. G., Leese, H. J., Diskin, M. G., and552

Morris, D. G. (2010). Effects of changes in the concentration of systemic

553

progesterone on ions, amino acids and energy substrates in cattle oviduct and554

uterine fluid and blood. Reprod. Fertil. Dev. 22, 684-94. doi:10.1071/RD09129

555

Inoue, K., Hosono, M., Oyama, H., and Hirooka, H. (2020). Genetic associations between

556

reproductive traits for first calving and growth curve characteristics of Japanese

557

Black cattle. Anim. Sci. J. 91, e13467. doi: 10.1111/asj.13467

558

Ito, S., and Nagata, K. (2016). Mutants of collagen-specific molecular chaperone Hsp47

559

causing osteogenesis imperfecta are structurally unstable with weak binding

560

affinity to collagen. Biochem. Biophys. Res. Commun. 469, 437-42. doi:

561

10.1016/j.bbrc.2015.12.028

562

Ito, S., and Nagata, K. (2017). Biology of Hsp47 (Serpin H1), a collagen-specific563

molecularchaperone.

Semin.

564

10.1016/j.semcdb.2016.11.005

Cell.

Dev.

Biol.

62,

142-51.

doi:

565

Ito, S., and Nagata, K. (2019). Roles of the endoplasmic reticulum-resident, collagen-566

specific molecular chaperone Hsp47 in vertebrate cells and human disease. J.

567

Biol. Chem. 294, 2133-41. doi: 10.1074/jbc.TM118.002812

568

Kakugawa, T., Mukae, H., Hayashi, T., Ishii, H., Nakayama, S., Sakamoto, N., Yoshioka,

569

S., Sugiyama, K., Mine, M., Mizuta, Y., and Kohno, S. (2005). Expression of

570

HSP47 in usual interstitial pneumonia and nonspecific interstitial pneumonia.

571

Respir. Res. 6, 57. doi: 10.1186/1465-9921-6-57

572

Li, M., Yao, L., Xin M., and Gao, M. (2019). Dysregulation of collagen expression in573

peri-implantation endometrium of women with high ovarian response. J. Obstet.

574

Gynaecol. Res. 45, 1035-44. doi: 10.1111/jog.13936

575

Liu, D., Xiong, F., and Hew, C.L. (1995). Functional analysis of estrogen-responsive

576

elements in chinook salmon (Oncorhynchus tschawytscha) gonadotropin II beta

577

subunit gene. Endocrinology 136, 3486-93. Doi: 10.1210/endo.136.8.7628385

578

Matsuda, M., Koide, T., Yorihuzi, T., Hosokawa, N., and Nagata K. (2001). Molecular579

cloning of a novel ubiquitin-like protein, UBIN, that binds to ER targeting signal

580

sequence.

581

10.1006/bbrc.2000.4149

Biochem.

Biophys.

Res.

Commun.

280,

535-40.

doi:

582

Miyamoto, Y., Skarzynski, D. J., and Okuda, K. (2000). Is tumor necrosis factor alpha a583

trigger for the initiation of endometrial prostaglandin F(2alpha) release at584

luteolysis in cattle? Biol. Reprod. 62, 1109-15. doi: 10.1095/biolreprod62.5

585

Mohos, S.C., and Wagner, B.M. (1969). Damage to collagen in corneal immune injury.

586

Observation of connective tissue structure. Arch. Pathol. 88, 3-20. PMID:

587

4893570

588

Nahar, A., and Kadokawa, H. (2017). Expression of macrophage migration inhibitory589

factor (MIF) in bovine oviducts is higher in the postovulatory phase than during590

the oestrus and luteal phase. Reprod. Fertil. Dev. 29, 1521-9. doi:591

10.1071/RD15546

592

Osoro, K., and Wright, I. A. (1992). The effect of body condition, live weight, breed, age,593

calf performance, and calving date on reproductive performance of spring-594

calving beef cows. J. Anim. Sci. 70, 1661-6. doi: 10.2527/1992.7061661x

595

Park, J., Lee, J., and Baek, S. (2018). Pathologic features and expression of heat shock596

protein 47 in the nasal mucosa and lacrimal sac: does it influence the surgical597

outcome of endoscopic endonasal dacryocystorhinostomy? Eye (Lond). 32,

598

1432-9. doi: 10.1038/s41433-018-0115-2

599

Ray, I.L., Dumas, M., Sagot, P., and Bardou, M., (2013). Effects of leptin on

600

lipopolysaccharide-induced remodeling in an in vitro model of human601

myometrial

602

10.1095/biolreprod.112.104844

inflammation.

Biol.

Reprod.

88,

45.

doi:

603

Razzaque, M.S., and Taguchi, T. (1999). Role of glomerular epithelial cell-derived heat604

shock protein 47 in experimental lipid nephropathy. Kidney Int. Suppl. 71, S256-

605

9. doi: 10.1046/j.1523-1755.1999.07169.x

606

Rekawiecki, R., Rutkowska, J., and Kotwica, J. (2012). Identification of optimal

607

housekeeping genes for examination of gene expression in bovine corpus luteum.

608

Reprod. Biol. 12, 362-7. doi: 10.1016/j.repbio.2012.10.010

609

Scolari, S.C., Pugliesi, G., Strefezzi, R.F., Andrade, S.C., Coutinho, L.L., and Binelli, M.

610(2016). Dynamic remodeling of endometrial extracellular matrix regulates

611

embryo receptivity in cattle. Reproduction REP-16-0237. doi: 10.1530/REP-16-

612

0237

613

Shi, J.W., Lai Z.Z., Yang, H.L., Yang, S.L., Wang, C.J., Ao, D., Ruan, L.Y., Shen, H.H.,

614

Zhou, W.J., Mei, J., Fu, Q., and Li, M.Q. (2020). Collagen at the maternal-fetal

615interface in human pregnancy. Int. J. Biol. Sci. 16, 2220-34. doi:

616

10.7150/ijbs.45586

617

Sugiura, T., Akiyoshi, S., Inoue, F., Yanagawa, Y., Moriyoshi, M., Tajima, M., and618

Katagiri S. (2018). Relationship between bovine endometrial thickness and619

plasma progesterone and estradiol concentrations in natural and induced estrus.

620

J. Reprod. Dev. 64, 135-43. doi: 10.1262/jrd.2017-139.

621

Szóstek-Mioduchowska, A., Słowińska, M., Pacewicz, J., Skarzynski., D.J., and Okuda,

622

K. (2020). Matrix metallopeptidase expression and modulation by transforming

623

growth factor-β1 in equine endometrosis. Sci. Rep. 10, 1119. doi:624

10.1038/s41598-020-58109-0

625

Takita, K.K., Fujii, K.K., Ishii, K., and Koide, T. (2019). Structural optimization of cyclic

626

peptides that efficiently detect denatured collagen. Org. Biomol. Chem. 17, 7380-

627

7. doi: 10.1039/c9ob01042d

628

Tsai, S.Y., Carlstedt-Duke, J., Weigel, N.L., Dahlman, K., Gustafsson, J.A., Tsai, M.J.,

629

and O'Malley, B.W. (1988). Molecular interactions of steroid hormone receptor

630

with its enhancer element: evidence for receptor dimer formation. Cell 55, 361-

631

9. doi: 10.1016/0092-8674(88)90059-1

632

Vandesompele, J., De Preter, K., Pattyn, F., Poppe, B., Van Roy, N., De Paepe, A., and633

Speleman, F. (2002). Accurate normalisation of real-time quantitative RT-PCR

634

data by geometric averaging of multiple internal control genes. Genome Biol. 3,635

research0034.1–research0034.11. doi:10.1186/gb-2002-3-7-research0034

636

Walker, C.G., Meier, S., Mitchell, M.D., Roche, J.R., and Littlejohn, M. (2009).

637

Evaluation of real-time PCR endogenous control genes for analysis of gene638

expression in bovine endometrium. BMC Mol. Biol. 10, 100. doi: 10.1186/1471-

639

2199-10-100

640

Wendremaire, M., Mourtialon, P., Goirand, F., Lirussi, F., Barrichon, M., Hadi, T., Garrido,

641

C., Ray, I.L., Dumas, M., Sagot, P., and Bardou, M. (2013). Effects of leptin on

642

lipopolysaccharide-induced remodeling in an in vitro model of human643

myometrial

644

10.1095/biolreprod.112.104844

inflammation.

Biol.

Reprod.

88,

45.

doi:

645

Xiong, G., Chen, J., Zhang, G., Wang, S., Kawasaki, K., Zhu, J., Zhang, Y., Nagata, K.,

646

Li, Z., Zhou, B.P., and Xu, R. (2020). Hsp47 promotes cancer metastasis by647

enhancing collagen-dependent cancer cell-platelet interaction. Proc. Natl. Acad.

648

Sci. U. S. A. 117, 3748-58. doi: 10.1073/pnas.1911951117

649

Zitnay, J.L., Li, Y., Qin, Z., San, B.H., Depalle, B., Reese, S.P., Buehler, M.J., Yu, S.M.,

650

and Weiss, J.A. (2017). Molecular level detection and localization of mechanical651

damage in collagen enabled by collagen hybridizing peptides. Nat. Commun. 8,65214913. doi: 10.1038/ncomms14913

653

654

Table 1. Details of the primers used for PCRs.
Gene

HSP47

C2orf29

SUZ12

HSP47

HSP47

C2orf29

SUZ12

655

PrimerSequence 5ʹ-3ʹ

Size

Position(bp)

Nucleotide

Exon

ForwardGACAACCGAGGCTTCATGGT

770

4

ReverseAGCTCCTCACGCCCGTAGAT

1239

6

Forward

AAGTTTTTTCTTTCCCAGCTCATG

666–688

2

ReverseCAGGAAGTTTGGCTGGAGTGA

1207–1227

5

ForwardGGAAGAGACTGCCTCCATTTGA

1019–1040

10

Reverse

CCCTGAGACACCATCTGTTTCC

2166–2187

16

ForwardTGTCGGGCAAGAAGGACCTA

1131

5

Reverse

AAAATGGGGAGGAAAGTGGG

1930

6

ForwardACAAGATGCGAGACGAGTTGT

1347

5

Reverse

CCCTGTTTTCCCACCCATGT

1439

5&6

ForwardTCAGTGGACCAAAGCCACCTA

928–948

3

ReverseCTCCACACCGGTGCTGTTCT

1077–1097

4

ForwardCATCCAAAAGGTGCTAGGATAGATG

1441–1465

13

ReverseTTGGCCTGCACACAAGAATG

1581–1600

14

470

562

1169

800

93

170

160

656
657
658
659
660
661

Fig. 1. Picro-sirius red staining for total collagen in ampulla, isthmus, and caruncular(CAR) and intercaruncular (ICAR) areas of the endometrium and myometrium in healthy
postpubertal, growing and young nulliparous heifers (Young), and old multiparous
Japanese Black beef cows (Old). The Picro-sirius red staining shows collagen in red,muscle fibres and cytoplasm in yellow, and complex in orange.

662

667

Fig. 2. In situ detection of denatured collagen. The no-heated, non-digested young tissues
show very subtle signals of denatured collagen (red). In contrast, the old samples, in amanner similar to the heated or enzyme-digested young tissues, show strong signals
corresponding to denatured collagen in the lamina propria and muscular layers of
ampullae and isthmuses; the stroma of CAR and ICAR; the glandular epithelium of

668

ICAR; and various myometrial cells.

663
664
665
666

669
670
671
672
673
674
675
676

Fig. 3. Expression of heat shock protein (HSP47) detected via RT-PCR analysis. The
electropherogram shows the size (470 bp) expected from PCR products of bovine HSP47
in the ampulla, isthmus, CAR, ICAR, and myometrium (Myo) in old cows. Bands were
not present in the no template control (NTC) and no reverse transcription control (NRCs)
lanes.

677
678
679
680
681
682
683
684
685

Fig. 4. Comparison of denatured collagen-rich areas (red) and HSP47-rich areas (green)in the ampullae or isthmuses (collected on day 3 of the oestrous cycle) of Young and Oldgroups. Nuclei are counterstained with DAPI (dark blue). Unlike the Young, the Old
specimens showed strong signal corresponding to denatured collagen in the lamina
propria and muscular layers. The Old specimens showed strong signal corresponding to
HSP47 in the epithelia of tunica mucosa and superficial stroma. Not many signals
corresponding to colocalisation (yellow) were present in the Merge panels; scale bars are
50 µm.

686
687

Fig. 5. Comparison of denatured collagen-rich areas (red) and HSP47-rich areas (green)

688
689
690
691
692
693
694
695
696

in the CAR, ICAR, or Myo (collected on day 13 of the oestrous cycle) of Young and Oldgroups. Nuclei are counterstained with DAPI (dark blue). Both the Young and Old
specimens showed signals corresponding to both denatured collagen and HSP47 inluminal epithelia, glandular epithelia, and stroma. Unlike the Young, the Old specimens
also showed strong signals corresponding to both denatured collagen and HSP47 in thestroma and myometria. Particularly, the number of fibroblasts (green dots) increased.
However, not many signals corresponding to colocalisation (yellow) were present in the
Merge panels; scale bars are 50 µm. The dark patches in old myometria are broken parts.

697
698

701

Fig. 6. Comparison of HSP47-rich areas (green) and cytokeratin- or vimentin-rich areas(red) in the ampullae or isthmuses (collected on day 3 of the oestrous cycle), or the CAR,
ICAR, or Myo (collected on day 13 of the oestrous cycle) of Old groups. Nuclei are702

counterstained with DAPI (dark blue); scale bars are 50 µm.

699
700

703

704
705
706
707

Fig. 7. Representative photos of western blotting analysis for HSP47 or anti-actinmouse antibodies in extracts from ampulla, isthmus, CAR, ICAR, and myometrium(Myo) in the Young and Old groups (A). Relative HSP47 protein levels normalised to

708
709
710
711
712
713
714
715
716
717

those of β-actin in the ampulla (B), isthmus (C), CAR (D), ICAR (E), or myometrium (F)in the Young and Old groups. The header in the upper corner of each graph represents theresults of two-factor ANOVA followed by Fisher’s PLSD test, including the effect of age(Young or Old) and effect of stage and interaction. Greek letters (,, or ) above thegrey left-side bars indicate significant between-stage differences in expression in Youngsamples; letters (a, b, or c above the white right-side bars indicate significant betweenstage differences in expression in Old samples (one-factor ANOVA followed by Fisher’s
PLSD test).
N.S., non-significant.

718
719

724

Fig. 8. Relative HSP47 mRNA levels (mean ± SEM) in the ampulla (A), isthmus (B),CAR (C), ICAR (D), or Myo (E) in healthy, post-pubertal growing nulliparous heifers(Young group) and old multiparous cows (Old group), as determined via RT-qPCR. Data
were normalised to the geometric means of C2orf29 and SUZ12 levels. The header in theupper corner of each graph represents the results of two-factor ANOVA followed by725

Fisher’s PLSD test, including the effect of age (Young or Old) and effect of stage and726interaction. Greek letters (,, or ) above the grey left-side bars indicate significant
between-stage differences in expression in Young samples; letters (a, b or c above thewhite right-side bars indicate significant between-stage differences in expression in Old
samples (one-factor ANOVA followed by Fisher’s PLSD test).
N.S. is abbreviation of non-significant.

720
721
722
723

727
728
729
730
731

732

733
734
735
736
737
738

Supplementary Fig. 1. Representative photos of western blotting using another antiHSP47 mouse monoclonal antibody, clone M16.10A1 (Enzo Life Sciences, Inc.,
Farmingdale, New York. USA) or anti-betaactin mouse antibodies in extracts from
ampulla, isthmus, CAR, ICAR, and myometrium (Myo) in the Young and Old groups.