Timing of transcriptomic and proteomic changes in the bovine placentome after parturition

Accepted Manuscript Timing of transcriptomic and proteomic changes in the bovine placentome after parturition Anthony K. McNeel, Jeff D. Ondrak, Olivia L. Amundson, Tara H. Fountain, Elane C. Wright, Katherine J. Whitman, Carol G. Chitko-McKown, Shuna A. Jones, Chadwick C. Chase, Jr., Robert A. Cushman PII:

S0093-691X(17)30259-5

DOI:

10.1016/j.theriogenology.2017.05.020

Reference:

THE 14125

To appear in:

Theriogenology

Received Date: 4 March 2016 Revised Date:

11 November 2016

Accepted Date: 24 May 2017

Please cite this article as: McNeel AK, Ondrak JD, Amundson OL, Fountain TH, Wright EC, Whitman KJ, Chitko-McKown CG, Jones SA, Chase Jr. CC, Cushman RA, Timing of transcriptomic and proteomic changes in the bovine placentome after parturition, Theriogenology (2017), doi: 10.1016/ j.theriogenology.2017.05.020. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.

ACCEPTED MANUSCRIPT RUNNING HEAD: Post-partum reproductive performance in beef cows

Timing of transcriptomic and proteomic changes in the bovine placentome after

2

parturition

3

Anthony K. McNeel*†‡, Jeff D. Ondrak‡, Olivia L. Amundson*, Tara H. Fountain*, Elane C.

4

Wright*, Katherine J. Whitman‡, Carol G. Chitko-McKown*, Shuna A. Jones*, Chadwick

5

C. Chase Jr.*, Robert A. Cushman2*

SC *

7

USDA3, ARS, U.S. Meat Animal Research Center, P.O. Box 166, Clay Center, NE 68933 †

8

Zoetis Genetics, Kalamazoo, MI 49007

‡

9

Great Plains Veterinary Educational Center, University of Nebraska-Lincoln, Clay Center, NE 68933

11 12

__________

13

1

Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of names by the USDA implies no approval of the product to the exclusion of others that may also be suitable.

2

17

Correspondence: P.O. Box 166, State Spur 18D (phone: 402-762-4186; fax 402-762-4382; e-

AC C

15

EP

14

TE D

10

18

M AN U

6

16

RI PT

1

mail: [email protected]

3

USDA is an equal opportunity provider and employer.

ACCEPTED MANUSCRIPT Post-partum reproductive performance in beef cows

2

ABSTRACT

20

Proper post-partum reproductive performance is important for reproductive efficiency in beef

21

cows, and dystocia decreases post-partum fertility. Crossbred beef cows (n = 1676) were

22

evaluated for lifetime performance based on degree of dystocia at presentation of the first calf.

23

Cows that experienced moderate or severe dystocia produced fewer calves during their

24

productive life (P < 0.01). The exact mechanism is unclear, but may be due to the contributions

25

of dystocia to abnormal placental separation. Proteolytic activity is hypothesized to contribute to

26

placental separation in ruminants; however, when ovine placentomes were collected following

27

caesarian section, no proteolytic activity was detected. We hypothesized that stage 2 of

28

parturition was necessary to stimulate proteolysis and initiate placental separation. Serial

29

placentome collections were performed on mature cows (n = 21 initiated; 7 with complete

30

sampling) at hourly intervals for the first 2 h after expulsion of the calf. An intact piece of each

31

placentome was fixed for histological evaluation, and a separate piece of caruncular and

32

cotyledonary tissue from each placentome was frozen for transcriptomic and proteolytic analysis.

33

A full set of placentomes was collected from only 7 of 21 cows at 0, 1, and 2 h, and all cows had

34

expelled fetal membranes by 6 h. Histological, transcriptomic and proteolytic analysis was

35

performed on placentomes from cows from which three placentomes were collected (n = 7). The

36

microscopic distance between maternal and fetal tissues increased at 1 h (P = 0.01). Relative

37

transcript abundance of matrix metalloprotease 14 (MMP14) tended to increase with time (P =

38

0.06). The relative transcript abundance of plasminogen activator urokinase-type (PLAU) was

39

greater in caruncles than cotyledons (P = 0.01), and tended (P = 0.10) to increase in the caruncle

40

between 0 and 2 h while remaining unchanged in the cotyledon over the same span of time.

41

Greater PLAU and plasminogen activator tissue-type (PLAT) proteolytic activity was detected

AC C

EP

TE D

M AN U

SC

RI PT

19

ACCEPTED MANUSCRIPT Post-partum reproductive performance in beef cows

3

by zymography in the caruncle than the cotyledon immediately post-partum (P < 0.01). From

43

these findings we conclude that 1) dystocia during the first parity decreases lifetime productivity

44

in beef cattle, 2) the PA system is present at both the transcript and protein level in the bovine

45

plactentome during parturition and 3) proteolytic activity is localized to the caruncular aspect of

46

the placentome.

47

Keywords: Cow, Parturition, Dystocia, Placental Separation, Proteolysis

SC

RI PT

42

AC C

EP

TE D

M AN U

48

ACCEPTED MANUSCRIPT Post-partum reproductive performance in beef cows 49 50

4

1. Introduction Repeat breeder beef cows were more likely to have experienced calving difficulty at some point in their life [1], and calving difficulty is a major contributor to retained fetal

52

membranes (RFM) in cows [2]. In beef cows, induction of parturition (e.g., with

53

dexamethasone) or caesarian section also increase the risk of RFM [2,3], and impair subsequent

54

reproductive performance [4]. Thus, a better understanding of the mechanisms controlling

55

placental separation could explain why cows with dystocia and without RFM suffer suppressed

56

reproductive performance which could lead to improved reproductive management of post-

57

partum beef cows.

M AN U

SC

RI PT

51

Compared to placental development, surprisingly little is known about the molecular

59

mechanisms meditating placental separation during normal placental expulsion. Placental

60

development in ruminants is mediated by trophoblast adhesion mainly within the placentome.

61

Adhesion of trophoblasts to endometrial cells is mediated by the actions of integrins and their

62

interaction with extracellular matrix proteins, most notably osteopontin [5]. During parturition,

63

this integrin-mediated adhesion must be disrupted in order to facilitate proper separation and

64

expulsion of the fetal membranes, an important step for proper involution of the uterus following

65

pregnancy [6]. Studies that have investigated the molecular mechanism of placentome

66

separation have focused primarily on the collagenolytic family of proteases, and results of

67

several studies have produced conflicting results in terms of whether or not collagenolytic

68

activity is reduced in cases of RFM [7-9].

AC C

EP

TE D

58

69

Collagenases, also known as matrix-metalloproteases (MMPs), are a family of 26 Zn2+

70

dependent endoproteases that are synthesized and secreted as inactive zymogens. Four inhibitors

71

(tissue inhibitor of matrix-metalloproteases: TIMPs) of these proteases are known to play roles in

ACCEPTED MANUSCRIPT Post-partum reproductive performance in beef cows

5

modulating the activities of the MMP and their inhibition is not covalently mediated (i.e., not

73

SDS stable). Each MMP possesses a spectrum of specificities for different extracellular matrix

74

(ECM) proteins with some functional overlap, the most well studied being collagen [10].

75

Activation of MMP involves proteolytic removal of the N-terminus to expose the catalytic zinc

76

ion, referred to as the cysteine switch [11]. Once pro-MMPs are secreted, proteases within the

77

plasminogen activator (PA) family are responsible for MMP activation [12,13]. The PA family

78

consists of the enzymes plasmin/plasminogen (PLG), plasminogen activator tissue-type (PLAT),

79

plasminogen activator urokinase-type (PLAU) and its receptor (PLAUR). Inhibitors of the PA

80

family also exist including α2-antiplasmin, plasminogen activator inhibitor-1 (SERPINE1),

81

plasminogen activator inhibitor-2 (SERPINB2) and protein C inhibitor (SERPINA5).

82

Additionally, PA activation of MMP can result in the activation of pro-PA and other MMP,

83

further propagating the proteolytic cascade [12,14,15]. The known role of PA in activation of

84

MMP, combined with reports that placentomes from retained fetal membranes possess reduced

85

MMP activity, suggests PA activity may play a role in placental separation and the pathogenesis

86

of RFM of cattle.

SC

M AN U

TE D

When placentomes were collected from sheep at stage 2 of parturition following

EP

87

RI PT

72

delivery of the lambs by caesarian section, we were unable to detect PA activity, indicating that

89

there may be stimulatory signals that occur during parturition that are necessary to activate the

90

PA system [16]. This could explain why RFM occurs at a greater frequency after caesarian

91

sections are performed [3]. Surprisingly little is known, however, about placental morphology

92

during parturition and how MMP activation in vivo is mediated. To investigate this process we

93

refined a technique to perform the serial collection of placentomes in post-partum cows [17], and

94

in the present study, we compared PA transcript abundance and protease activity in placentomes

AC C

88

ACCEPTED MANUSCRIPT Post-partum reproductive performance in beef cows

6

collected at one h intervals after calf expulsion from spontaneously calving cows without any

96

difficulties. Given the lack of basic information regarding the physiologic mechanisms

97

responsible for normal placental separation, we chose to investigate the basic mechanisms

98

placental separation in lieu of contrasts affected and unaffected animals (eg dystocia, RFM). The

99

objective of this study was to a) quantify the effects of dystocia on lifetime productivity in beef

100

cattle and 2) determine if the PA system is present in the bovine placentome during parturition.

101

We hypothesized that: 1) experiencing calving difficulty during the first calving season would

102

decrease lifetime productivity in beef cows, and 2) in cases of normal parturition placental

103

morphology would change as a function of time after parturition, and would be associated with

104

increases in PA system transcript abundance and proteolytic activity after expulsion of the calf.

105

2. Materials and Methods

106

2.1. Influence of Calving Difficulty on Reproductive Longevity of Beef Cows

SC

M AN U

TE D

107

RI PT

95

All procedures were approved by the U.S. Meat Animal Research Center (USMARC) Animal Care and Use Committee and were in accordance with the FASS guidelines for the care

109

and use of agricultural animals in research. Standard management practices at the U.S. Meat

110

Animal Research Center include recording calf birth dates, calf sex, calving difficulty score

111

[18,19], calf birth weight, and calf weaning weights each year. In addition, each calf is provided

112

with an individual identification and all data are stored in the USMARC database. Data were

113

extracted for 2-yr-old crossbred cows (n = 1676) giving birth for the first time in the spring of

114

2000 – 2002. These years were chosen because they did not overlap with our previous published

115

reports of calving day and calving difficulty with heifers giving birth for the first time before

116

those years [18], and did not impinge on new experiments started in 2003 that examined the

AC C

EP

108

ACCEPTED MANUSCRIPT Post-partum reproductive performance in beef cows

7

117

influence of nutritional treatments on many of these endpoints in heifers giving birth for the first

118

time after those years [20-22].

119

At parturition, cows were assigned a calving difficulty score based on a standard that has been previously reported for USMARC [18,19]. Briefly, calving was subjectively evaluated on

121

the following 6-point scale: 1 = no assistance; 2 = little difficulty, assistance given by hand; 3 =

122

little difficulty, mechanical pull; 4 = slight difficulty, mechanical pull; 5 = moderate mechanical

123

pull; 6 = hard mechanical pull. Cows with a calving score of 1 received no post calving care

124

after delivery. If a cow required some assistance (score of 2, 3, 4) they were treated with

125

oxytocin (5 ml, i.v.), and penicillin G benzathine (3.5 ml/100 lbs, s.q). Cows with more difficult

126

deliveries (score of 5, 6) were treated with oxytocin (5 ml, i.v.), penicillin G aqueous (3.5 ml/100

127

lbs, i.m) for three days, and given two oxytetracycline intrauterine boluses. Any cows with

128

additional medical needs were treated following USMARC protocols.

129

2.2. Placentomectomies

TE D

M AN U

SC

RI PT

120

Placentomes were collected from 3-yr-old MARCII (25% Angus, 25% Hereford, 25%

131

Simmental and 25% Gelbvieh) beef cows (n = 21), using the transvaginal collection technique

132

that we described previously [17]. The experiment was designed to collect placentomes at 0, 1,

133

and 2 h after expulsion of the calf without any difficulties to determine if normal progression

134

through stage 2 of parturition stimulated proteolytic activity or altered transcript abundance

135

within the bovine placentome. A single intact representative placentome was excised at each of

136

the three times, excess fetal membranes were trimmed and the placentomes separated into

137

cotyledonary and caruncular components. Samples for gene expression and proteolytic analysis

138

were placed into 2.0 ml cryovials and snap frozen in liquid nitrogen within 5 min of collection.

AC C

EP

130

ACCEPTED MANUSCRIPT Post-partum reproductive performance in beef cows

8

Samples were transported back to the laboratory in liquid nitrogen and stored at -80°C until total

140

cellular RNA and protein were extracted. Only seven cows maintained intact placentomes

141

through the 2 h collection and all cows completely expelled the fetal membranes within 6 h of

142

parturition. Therefore, further analysis was only performed using tissues from the seven cows

143

that provided intact placentomes at 0, 1, and 2 h after parturition.

144

2.3.2 Histology

SC

RI PT

139

Additional pieces of intact placentomes were fixed in 10% Neutral Buffered Formalin,

146

dehydrated in ethanol using a step-wise series of fluid changes (25% v:v to 100% v:v), cleared

147

using 4 changes of xylenes and embedded in paraffin and sectioned at 8 µm [23]. Slides were

148

stained using hemotoxalin and eosin and imaged at 40x, 100x, and 200x magnification on a Zeiss

149

Axioplan2 microscope. For each cow at each time point, a random 40x image was captured and

150

the average distance between maternal and fetal tissues was measured at three points on each

151

image using BioQuant Imaging software.

152

2.4. RNA Extraction and Quantification

TE D

EP

153

M AN U

145

Cotyledonary and caruncular tissues were homogenized and total cellular RNA was extracted using TRIzol (Invitrogen, Carlsbad, CA) according to manufacturer’s instructions.

155

Purified RNA was suspended in autoclaved distilled water and quantified on a Nanodrop 1000

156

(Thermo Scientific, Willmington, DE) and only samples with a 280/260 absorbance greater than

157

1.7 were used for reverse transcription.

158

2.5. Reverse Transcription

AC C

154

ACCEPTED MANUSCRIPT Post-partum reproductive performance in beef cows 159

9

One microgram of the total cellular RNA was reverse transcribed using oglio-dT as part of the Roche First Strand cDNA kit according to manufacturer’s instructions (Roche Diagnostics

161

Corporation, Indianapolis, IN). Briefly, one microgram of RNA was incubated with 1 µl of

162

oglio-dT and ultrapure water to 13 µl, 4 µl of 5x reaction buffer, 0.5 µl of RNase inhibitor, 2 µl

163

of dNTPs and 0.5 µl of reverse transcriptase at 55°C for 30 min. Reverse transcriptase was

164

denatured by incubating the reaction at 85°C for 5 min followed by immediate cooling to 4°C.

165

Resultant cDNA was diluted to 100 µl (10 ng of cDNA equivalents per µl) with sterile distilled

166

water and stored at -20°C for subsequent gene expression assays.

167

2.6. Real-Time Polymerase Chain Reactions for Relative Quantification of Transcript

168

Abundance

SC

M AN U

169

RI PT

160

Relative quantification of transcript abundance of genes of interest was performed using the 2-∆∆CT method [24] with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) serving as the

171

endogenous reference gene. Glyceraldehyde-3-phosphate dehydrogenase was not different

172

across collection time points or tissues (P > 0.92), providing validation that GAPDH was an

173

acceptable reference gene for these analyses. Primers spanning an exon-exon junction were

174

designed (Table 1) using Primer-BLAST [25,26] for each gene of interest. Two microliters of

175

diluted cDNA, assumed to be 20 ng of cDNA, were incubated in qPCR reactions performed in

176

duplicate using the Roche LightCycler 480 SYBR Green I Master mix according to

177

manufacturer’s instructions with the appropriate primers on a Roche LightCycler 480 (Roche

178

Diagnostics Corporation, Indianapolis, IN). A water reaction served as a negative control for

179

each gene of interest included in each run. Briefly, hot-start DNA polymerase was heat activated

180

by incubating the reactions at 95°C for 5 min followed by 45 cycles of the following program:

AC C

EP

TE D

170

ACCEPTED MANUSCRIPT Post-partum reproductive performance in beef cows

10

denature at 95°C for 10 s, anneal at 58°C for 10 s, extend at 72° for 10 s, and measure

182

florescence. A high resolution melt curve was performed to detect the presence of any non-

183

specific priming and visualization of the amplicons in a 2% agarose gel stained with ethidium

184

bromide revealed a single band of the predicted size for each gene of interest (data not shown).

185

Average inter-assay CV for each primer pair set was < 20%.

186

2.7. Protein Extraction and Quantification

SC

187

RI PT

181

Protein was extracted from cotyledonary and caruncular tissues as described previously [16,27]. Briefly, 100 mg of a cotyledon or caruncle was weighed and homogenized in the

189

appropriate amount of extraction buffer (1 ml/500 mg) with a tissue homogenizer and

190

centrifuged at 9000 x g for 30 min at 4°C. The supernatant was removed and stored at -20°C

191

until later assay. Protein samples were subjected to a maximum of three freeze thaw cycles in

192

order to reduce variation in enzyme activity. Protein concentrations were measured using Pierce

193

BCA kit (Thermo Scientific) in a microplate and read at 550 nm on an Elx808IU ultra microplate

194

reader (Biotek Instruments, Inc, Winooski, VT).

195

2.8. Casein-Plasminogen Zymography

TE D

EP

Casein-plasminogen zymography was performed as described previously [16,27].

AC C

196

M AN U

188

197

Briefly, 80 µg of protein from caruncle or cotyledon and a standard curve of high molecular

198

weight human PLAU (American Diagnostica, Stamford, CT) were loaded onto castellated 4%

199

polyacrylamide gels (0.375 M Tris, 0.1 % SDS, pH 8.6) cast on top of a 10% polyacrylamide

200

gels (0.375 M Tris, 0.1 % SDS, pH 8.6) containing 0.025% Hammerstein-casein (Sigma-Aldrich,

201

St. Louis, MO) and 25 mU/ml bovine plasminogen (Sigma-Aldrich). Samples were

ACCEPTED MANUSCRIPT Post-partum reproductive performance in beef cows

11

electrophoresed for 1 h at 140 V in a Mini-Protean Tetra-cell (Bio-Rad, Hercules, CA). Gels

203

were removed from the apparatus, the stacking gel was removed, and the running gel was

204

washed in fresh 2.5% Triton X-100 for 45 min to remove SDS. Gels were then washed once in

205

incubation buffer (50 mM Tris, 100 mM NaCl, pH 7.6), placed in fresh incubation buffer, and

206

incubated for 17 h at 37°C. Gels were then washed once in distilled H2O, placed in staining

207

solution (40% methanol, 40% distilled H2O, 10% acetic acid, 0.025% Coomasie Brilliant Blue)

208

for 45 min, and de-stained in staining solution less Coomassie Brilliant Blue. Matrix-

209

metalloprotease mediated caseinolysis is not possible with this assay as both the electrophoresis

210

and incubation buffers lack the zinc ions necessary for matrix-metalloprotease activity [28]. This

211

has been demonstrated previously when we incubated samples that were negative for proteolytic

212

activity using the incubation buffer above subsequently revealed MMP activity when the same

213

samples were assayed for gelatinolytic activity in the presence of zinc ions [16].

214

2.9. Statistical Analyses

SC

M AN U

TE D

215

RI PT

202

For production traits, continuous measurements of reproductive performance were analyzed using the MIXED procedure of SAS with calving difficulty score as a fixed effect.

217

Calf weights and gains were analyzed using the MIXED procedure of SAS with sex of the calf

218

and calving difficulty score as fixed effects. Percent of calves weaned was analyzed using the

219

GLIMMIX procedure of SAS with a binomial distribution and a logit link. Calving difficulty

220

score was the fixed effect.

AC C

221

EP

216

Relative abundance of transcripts for proteolytic genes was analyzed using the MIXED

222

procedure of SAS with time (0, 1, or 2 h), tissue source (caruncle or cotyledon), and the

223

interaction as fixed effects. The distance between fetal and maternal tissues in histological

ACCEPTED MANUSCRIPT Post-partum reproductive performance in beef cows

12

samples was analyzed using the MIXED procedure of SAS with time as a fixed effect.

225

Proportion of caruncular and cotyledonary tissue demonstrating PLAU or PLAT activity was

226

analyzed by Chi-square analysis.

227

3. Results

228

3.1. Performance Data

There was a significant decrease in the number of calves that a cow produced after a

SC

229

RI PT

224

moderate or hard pull was required to extract the calf (P < 0.01; Table 2). As would be expected,

231

there was a positive association between calf birth weight and calving difficulty (P < 0.01), such

232

that calf birth weights were greater when a moderate or hard pull was required. This resulted in

233

a decrease in calf survival to weaning (P < 0.01). The pre-weaning average daily gains of the

234

calves exposed to a moderate or hard pull tended to be decreased (P = 0.10) and these same

235

calves had intermediate weaning weights, even though they had the greatest birth weights.

236

3.2. Gross Observations and Histology

TE D

As the final stages of parturition progressed, tissue sections from these time points

EP

237

M AN U

230

displayed morphological changes to the microanatomy that were isolated to the fetal aspect of

239

the placentome (Fig. 1A, 1B, and 1C). The changes between the 0 h collection and the 2 h

240

collection are best described as near complete loss of tissue integrity that corresponded with a

241

loss of cotyledonary adhesion in vivo. Additionally, there was an influence of progression of

242

stage 2 of labor on the average distance between the maternal and fetal tissues (P = 0.01; Fig. 2),

243

such that average distance between the tissues increased between 0 and 1 h but was not different

244

between 1 and 2 h of stage 2 of labor.

AC C

238

ACCEPTED MANUSCRIPT Post-partum reproductive performance in beef cows

245

13

3.3. Semi-Quantitative Real-Time Reverse Transcription Polymerase Chain Reaction There were no differences in the transcript abundance of MMP2, MMP9, TIMP2, TIMP3,

247

SERPINE1 or SERPINB2 in response to time, tissue type, or the interaction (P > 0.10). Relative

248

transcript abundance of MMP14 tended to increase with time post-partum (P = 0.06). The

249

relative transcript abundance of PLAU was greater in caruncles than cotyledons (P = 0.01), and

250

there was a tendency for an interaction between tissue type and time on relative transcript

251

abundance of PLAU (P = 0.10), such that PLAU increased in the caruncle between 0 and 2 h

252

(0.12 ± 0.30 vs. 1.09 ± 0.30 relative units) while remaining unchanged in the cotyledon over the

253

same span of time (0.12 ± 0.30 vs. 0.01 ± 0.30 relative units).

254

3.4. Zymography

M AN U

SC

RI PT

246

Based on molecular weight and band intensity, the primary plasminogen activator activity

256

detected by zymography was PLAU. Plasminogen activator tissue-type activity was also present

257

at lower intensity (Fig. 3). Plasminogen activator urokinase-type and PLAT activity were only

258

identified in the caruncle, this represented a significant difference between the maternal and fetal

259

component of the placentome in PLAU and PLAT activity (Chi-square, P < 0.01).

260

4. Discussion

EP

AC C

261

TE D

255

Cows that experienced calving difficulty in their first calving season produced fewer

262

calves as calving difficulty increased, and yet, among the cows that suffered the greatest calving

263

difficulty, some were able to produce three or more calves. This indicates that there are

264

differences in response to dystocia among cows, and that understanding the biological

ACCEPTED MANUSCRIPT Post-partum reproductive performance in beef cows

14

differences in placental separation may be the way to improve interventions and lifetime

266

productivity in these cows. This is important because research indicates that post-partum uterine

267

function may play a greater role in post-partum reproductive performance than the initiation of

268

reproductive cycles [1,29-32].

269

RI PT

265

In repeat breeder dairy cows the data are mixed. Pothmann et al. [33] reported no

decrease in the percent of repeat breeder cows that initiated reproductive cycles and minimal

271

impact of sub-clinical endometritis or uterine infection on fertility. In contrast, Salasel et al. [34]

272

reported that sub-clinical endometritis was a major contributor to repeat breeder syndrome in

273

dairy cows. Furthermore, inflammation in the uterus decreased pregnancy rates by reducing the

274

ability of oocytes to fertilize and develop to the morula stage and impairing elongation of the

275

conceptus, independent of cyclic status [35]. While dairy cows are managed differently, these

276

data indicate that further investigation of the factors contributing to placental separation and how

277

these factors contribute to post-partum uterine function in beef cows is warranted, given our data

278

that demonstrates minimal impact of cyclic status on post-partum reproductive performance [29].

TE D

M AN U

SC

270

The findings of the present study demonstrated that during the final stages of normal

280

bovine parturition, there are rapid appreciable changes in both the gross morphology and the

281

histology of the placentome. These changes are associated with increases in PLAU transcript

282

abundance and increases in PLAU proteolytic activity, and are in stark contrast to our previous

283

study where we induced parturition with dexamethasone and the placentomes remained firmly

284

attached for the first 3 h following completion of stage 2 of labor [17]. While it is probably

285

impossible to completely prevent cross-contamination of the caruncular and cotyledonary tissues

286

when separating them, the dramatic differences in both PLAU transcript abundance and PLAU

287

proteolytic activity between tissue types indicate that cross-contamination was minimal. Again,

AC C

EP

279

ACCEPTED MANUSCRIPT Post-partum reproductive performance in beef cows

15

288

compared to our previous study where very few of the placentomes separated easily even at 3 h

289

when dexamethasone was used, the current data support the concept that during normal

290

separation the process is very rapid. In contrast to our previous study, we observed increases in PLAU activity in the earliest

292

caruncular samples that we collected after parturition. This is different from ovine samples that

293

we collected at caesarian section because PLAU activity was undetectable [16]. Taken together,

294

these findings indicate that the stimulus to initiate PLAU activity may indeed be expulsion of the

295

calf and that the change in function happens rapidly in the caruncle, at or just before expulsion of

296

the calf. These changes appear to be happening too quickly to require traditional transcriptional

297

and translational machinery and would indicate that PLAU enzymatic activity is increased

298

immediately as a result of stage 2 of parturition. The increase in PLAU transcript abundance that

299

we observed in the caruncle with time may actually be part of the process that is responsible for

300

uterine involution.

SC

M AN U

TE D

301

RI PT

291

During the course of this study, we collected placentomes from 21 cows and were able to collect the full complement of samples (0, 1, and 2 h) from only 7 of the cows. While losing

303

some cows to expedited placental expulsion was anticipated, the variation in the population was

304

revealing and raised some questions regarding the definition of retained fetal membranes. In this

305

study, all 21 cows expelled the fetal membranes by 6 h after expulsion of the calf, and there were

306

several instances from the herd of 400+ cows where this was accomplished before the cow could

307

be restrained for placentome collection (< 15 min). Based on the rapid morphological changes

308

that we observed and the elevation in PLAU activity, it may be time to revisit the definition of

309

retained fetal membranes. From our observations, it would seem that no more than 6 h are

310

required to indicate that the normal separation mechanisms have failed in beef cows. This is

AC C

EP

302

ACCEPTED MANUSCRIPT Post-partum reproductive performance in beef cows

16

currently the lower limit of retained placenta’s definition. While 12 and 24 hours appear to be

312

the most widespread definitions, the symmetry in the observation schedule would suggest that

313

these definitions were not empirically derived. Defining what is to be considered an abnormal

314

placental separation is a difficult proposition given the extensive nature of beef production.

315

More intensive production systems, such as dairy cattle, would provide the observational

316

opportunity to apply a statistically driven approach. Using technological advancements in

317

calving monitors and then deriving the mean time to placental expulsion following expulsion of

318

the calf in a large population of multiple parities and seasons would be informative. Such a

319

model would provide sufficient data to estimate what would be biologically “normal”.

SC

M AN U

320

RI PT

311

In conclusion, the current study indicates that during normal parturition PLAU is activated in the caruncle around the time of expulsion of the calf. We believe that this may

322

contribute to activation of MMPs and the rapid degradation observed in the fetal membranes in

323

the hour following parturition. Following calving difficulty, failures in this system may

324

contribute to the increased incidence of retain fetal membranes that are observed, resulting in

325

increased incidence of reproductive loss in post-partum cows. Further research will be necessary

326

to determine if and how the pathways are altered following calving difficulty.

327

Acknowledgements

EP

AC C

328

TE D

321

The authors gratefully acknowledge the assistance of Chad Engle, Arnie Svoboda, Doug

329

Barritt, and USMARC Cattle Operations for expert care and handling of the cows; Lillian Larsen

330

and Darrell Light for managing the database and assistance with data mining for the historical

331

data; and Donna Griess for assistance with manuscript preparation. This research was funded by

ACCEPTED MANUSCRIPT Post-partum reproductive performance in beef cows

17

332

ARS Project Plan number 3040-31000-093-00D entitled “Strategies to Improve Heifer Selection

333

and Heifer Development” (RAC).

AC C

EP

TE D

M AN U

SC

RI PT

334

ACCEPTED MANUSCRIPT Post-partum reproductive performance in beef cows

18

References

336

[1] Maurer RR, Echternkamp SE. Repeat-breeder females in beef cattle: Influences and causes. J

337

Anim Sci 1985;61:624 - 636.

338

[2] Lyons NA, Karvountzis S, Knight-Jones TJ. Aspects of bovine caesarean section associated

339

with calf mortality, dam survival and subsequent fertility. Vet J 2013;197:342-50.

340

[3] Beagley JC, Whitman KJ, Baptiste KE, Scherzer J. Physiology and treatment of retained

341

fetal membranes in cattle. J Vet Intern Med 2010;24:261-8.

342

[4] Patterson DJ, Bellows RA, Burfening PJ. Effects of caesarean section, retained placenta and

343

vaginal or uterine prolapse on subsequent fertility in beef cattle. J Anim Sci 1981;53:916-21.

344

[5] Johnson GA, Burghardt RC, Bazer FW, Spencer TE. Osteopontin: roles in implantation and

345

placentation. Biol Reprod 2003;69:1458-71.

346

[6] Kiracofe GH. Uterine involution: its role in regulating postpartum intervals. J Anim Sci

347

1980;51 Suppl 2:16-28.

348

[7] Maj JG, Kankofer M. Activity of 72-kDa and 92-kDa matrix metalloproteinases in placental

349

tissues of cows with and without retained fetal membranes. Placenta 1997;18:683-7.

350

[8] Walter I, Boos A. Matrix metalloproteinases (MMP-2 and MMP-9) and tissue inhibitor-2 of

351

matrix metalloproteinases (TIMP-2) in the placenta and interplacental uterine wall in normal

352

cows and in cattle with retention of fetal membranes. Placenta 2001;22:473-83.

353

[9] Dilly M, Hambruch N, Shenavai S, Schuler G, Froehlich R, Haeger JD, Ozalp GR, Pfarrer

354

C. Expression of matrix metalloproteinase (MMP)-2, MMP-14 and tissue inhibitor of matrix

355

metalloproteinase (TIMP)-2 during bovine placentation and at term with or without placental

356

retention. Theriogenology 2011;75:1104-14.

AC C

EP

TE D

M AN U

SC

RI PT

335

ACCEPTED MANUSCRIPT Post-partum reproductive performance in beef cows

19

[10] Overall CM. Molecular determinants of metalloproteinase substrate specificity: matrix

358

metalloproteinase substrate binding domains, modules, and exosites. Mol Biotechnol

359

2002;22:51-86.

360

[11] Chakraborti S, Mandal M, Das S, Mandal A, Chakraborti T. Regulation of matrix

361

metalloproteinases: an overview. Mol Cell Biochem 2003;253:269-85.

362

[12] Ny T, Wahlberg P, Brandstrom IJ. Matrix remodeling in the ovary: regulation and

363

functional role of the plasminogen activator and matrix metalloproteinase systems. Mol Cell

364

Endocrinol 2002;187:29-38.

365

[13] Curry TE, Jr., Osteen KG. The matrix metalloproteinase system: changes, regulation, and

366

impact throughout the ovarian and uterine reproductive cycle. Endocr Rev 2003;24:428-65.

367

[14] Orgel D, Schroder W, Hecker-Kia A, Weithmann KU, Kolkenbrock H, Ulbrich N. The

368

cleavage of pro-urokinase type plasminogen activator by stromelysin-1. Clin Chem Lab Med

369

1998;36:697-702.

370

[15] Xu D, McKee CM, Cao Y, Ding Y, Kessler BM, Muschel RJ. Matrix metalloproteinase-9

371

regulates tumor cell invasion through cleavage of protease nexin-1. Cancer Res 2010;70:6988-

372

98.

373

[16] McNeel AK, Cushman RA, Vallet JL. The plasminogen activator system in the ovine

374

placentome during late gestation and stage-two of parturition. Mol Reprod Dev 2013;80:466-73.

375

[17] McNeel AK, Ondrak JD, Cushman RA. Technical report: serial collections of placentomes

376

during parturition in cattle and subsequent reproductive performance. J Anim Sci 2013;91:2119-

377

21.

378

[18] Cushman RA, Kill LK, Funston RN, Mousel EM, Perry GA. Heifer calving date positively

379

influences calf weaning weights through six parturitions. J Anim Sci 2013;91:4486-4491.

AC C

EP

TE D

M AN U

SC

RI PT

357

ACCEPTED MANUSCRIPT Post-partum reproductive performance in beef cows

20

[19] Bennett GL, Thallman RM, Snelling WM, Kuehn LA. Experimental selection for calving

381

ease and postnatal growth in seven cattle populations. II. Phenotypic differences. J Anim Sci

382

2008;86:2103-14.

383

[20] Cushman RA, McNeel AK, Freetly HC. The impact of cow nutrient status during the

384

second and third trimester on age at puberty, antral follicle count, and fertility of daughters.

385

Livestock Sci 2014;162:252-258.

386

[21] Eborn DR, Cushman RA, Echternkamp SE. Effect of postweaning diet on ovarian

387

development and fertility in replacement beef heifers. J Anim Sci 2013;91:4168-79.

388

[22] Freetly HC, Vonnahme KA, McNeel AK, Camacho LE, Amundson OL, Forbes ED, Lents

389

CA, Cushman RA. The consequence of level of nutrition on heifer ovarian and mammary

390

development. J Anim Sci 2014;92:5437-43.

391

[23] Cushman RA, DeSouza JC, Hedgpeth VS, Britt JH. Alteration of activation, growth and

392

atresia in bovine preantral follicles by long-term treatment of cows with estradiol and

393

recombinant bovine somatotropin. Biol Reprod 2001;65:581-586.

394

[24] Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time

395

quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001;25:402-8.

396

[25] Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool.

397

J Mol Biol 1990;215:403-10.

398

[26] Ye J, Coulouris G, Zaretskaya I, Cutcutache I, Rozen S, Madden TL. Primer-BLAST: a

399

tool to design target-specific primers for polymerase chain reaction. BMC Bioinformatics

400

2012;13:134.

AC C

EP

TE D

M AN U

SC

RI PT

380

ACCEPTED MANUSCRIPT Post-partum reproductive performance in beef cows

21

[27] Dow MP, Bakke LJ, Cassar CA, Peters MW, Pursley JR, Smith GW. Gonadotrophin surge-

402

induced upregulation of mRNA for plasminogen activator inhibitors 1 and 2 within bovine

403

periovulatory follicular and luteal tissue. Reproduction 2002;123:711-9.

404

[28] Hidalgo M, Eckhardt SG. Development of matrix metalloproteinase inhibitors in cancer

405

therapy. J Natl Cancer Inst 2001;93:178-93.

406

[29] Cushman RA, Allan MF, Thallman RM, Cundiff LV. Characterization of biological types

407

of cattle (Cycle VII): Influence of postpartum interval and estrous cycle length on fertility. J

408

Anim Sci 2007;85:2156-62.

409

[30] Echternkamp SE, Cushman RA, Allan MF. Size of ovulatory follicles in cattle expressing

410

multiple ovulations naturally and its influence on corpus luteum development and fertility. J

411

Anim Sci 2009;87:3556-68.

412

[31] Warnick AC, Hansen PJ. Comparison of ovulation, fertilization and embryonic survival in

413

low-fertility beef cows compared to fertile females. Theriogenology 2010;73:1306-10.

414

[32] Echternkamp SE, Thallman RM, Cushman RA, Allan MF, Gregory KE. Increased calf

415

production in cattle selected for twin ovulations. J Anim Sci 2007;85:3239-48.

416

[33] Pothmann H, Prunner I, Wagener K, Jaureguiberry M, de la Sota RL, Erber R, Aurich C,

417

Ehling-Schulz M, Drillich M. The prevalence of subclinical endometritis and intrauterine

418

infections in repeat breeder cows. Theriogenology 2015;83:1249-53.

419

[34] Salasel B, Mokhtari A, Taktaz T. Prevalence, risk factors for and impact of subclinical

420

endometritis in repeat breeder dairy cows. Theriogenology 2010;74:1271-8.

421

[35] Ribeiro ES, Gomes G, Greco LF, Cerri RL, Vieira-Neto A, Monteiro PL, Jr., Lima FS,

422

Bisinotto RS, Thatcher WW, Santos JE. Carryover effect of postpartum inflammatory diseases

423

on developmental biology and fertility in lactating dairy cows. J Dairy Sci 2016;99:2201-20.

424

AC C

EP

TE D

M AN U

SC

RI PT

401

ACCEPTED MANUSCRIPT Post-partum reproductive performance in beef cows

22

Figure Legends

426

Fig. 1. Representative photomicrographs of the fetal maternal interface within the placentome

427

collected at 0 (A), 1 (B) and 2 (C) h post expulsion of the calf. Histological changes in the

428

organization of the fetal aspect of the placentome are readily apparent as time progresses,

429

whereas the maternal aspect remained unaffected.

SC

430

RI PT

425

Fig. 2. Changes in the average microscopic distance between fetal and maternal over time

432

during stage 2 of labor. The average distance between fetal and maternal tissues increased

433

between 0 and 1 h, but did not differ between 1 and 2 h (P = 0.01). abBars with different

434

superscripts are significantly different.

435

Fig. 3. Representative zymograph of caruncular extracts (in duplicate) at zero (0), one (1), and

436

two (2) h after expulsion of the calf demonstrating plasmionogen activator (lytic zones).

437

Plasminogen activator urokinase-type activity is the prominent band below the numbers while

438

faint PLAT activity can be seen above.

TE D

EP AC C

439

M AN U

431

ACCEPTED MANUSCRIPT Post-partum reproductive performance in beef cows 440

Table 1

441

Primer sequences for semi-quantitative real-time RT-PCR. Gene Symbol

Direction

23

TM (°C)a

Sequence

Reference

GAPDH

Forward

5’-ACAGTCAAGGCAGAGAACGG-3’

Reverse

5’-CCAGCATCACCCCACTTG-3’

Forward

5’-ATCGTCTTCGACGGCATCTC-3’

Reverse

5’-GTGGGTCTTCGTACACAGCA-3’

Forward

5’-AGGGTAAGGTGCTGCTGTTC-3’

Reverse

5’-AGGAGGTCGAAGGTCACGTA-3’

Forward

5’-ACAGTCGCGGACCATGTCTC-3’

Reverse

5’-CTGAGAGGGACTGGGGTGAG-3’

Forward

5’-TGAGCGACTCTGATGGCAGC-3’

Reverse

5’-TTGGGCAACTGCATCGCTGA-3’

Forward Reverse

5’-TACCAGGACCAGGCGCGTCA-3’

Forward

5’-GGGCGCCCGGGGAAATACTG-3’

Reverse

5’-GGACGCGTTGATGGCGTTGC-3’

NM_174390.2

58

NM_174147.2

5’-GGTCCGCGGTTTCATGCCCA-3’

58

NM_174137.2

TE D

58

NM_001192079.1

58

NM_174472.4

58

NM_174473.4

442 443

a

M AN U

EP

Forward

5’-GCAACAGGGGTTTTGCAATG-3’

Reverse

5’-GATGTCGTTGCCAGAGTCCA-3’

Forward

5’-GGATTCACCAAGATGCCCCA-3’

Reverse

5’-GAGCTGGTCCCACCTCTGTA-3’

AC C

TIMP3

Melting temperature.

NM_174745.2

58

PLAU

TIMP2

58

NM_174744.2

MMP14

SERPINB2

NM_001034034.2

58

MMP9

SERPINE1

58

SC

MMP2

RI PT

Sequence (GI)

ACCEPTED MANUSCRIPT Post-partum reproductive performance in beef cows

24

444

Table 2

445

Influence of calving difficulty on lifetime productivity and first calf performance. Difficulty Scorea 3

4

1306

24

125

167

Years Bred

5.5±0.1a

4.9±0.4ab

5.4±0.2ab

5.1±0.2b

Years Calved

4.3±0.1a

4.0±0.4ab

4.2±0.2

3.9±0.2b

Julian Calving Day

75.3±0.4

79.7±3.2

73.2±1.4

75.1±1.2

Birth Weight, kg

34.5±0.1a

37.3±0.9b

36.5±0.4b

39.3±0.3b

Percent Weaned

93.5±1.0a

67.2±9.6c

88.0±2.9b

Weaning Weight, kg

189.8±0.8a

189.6±6.9a

0.9±0.01

0.9±0.03

Heifers

Calf ADG, kg/d

5

6

RI PT

2

P

41

13

-

4.2±0.3c

3.6±0.3c

< 0.01

3.0±0.3c

2.6±0.6c

< 0.01

78.0±2.4

81.1±4.4

0.22

40.9±0.7c

44.8±1.2c

< 0.01

88.7±2.4b

73.4±6.9c

44.6±13.9d

< 0.01

196.3±2.7b

SC

1

198.1±2.3b

188.5±5.1ab

178.9±11.4ab

< 0.01

0.9±0.01

0.9±0.01

0.8±0.02

0.8±0.05

0.10

M AN U

Traits

446

a

447

difficulty, mechanical pull; 5 = moderate mechanical pull; 6 = hard mechanical pull.

Difficulty Score: 1 = no assistance; 2 = little difficulty, assistance given by hand; 3 = little difficulty, mechanical pull; 4 = slight

AC C

EP

TE D

448

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

Fig 1A

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

Fig. 1B

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

Fig. 1C

ACCEPTED MANUSCRIPT

Fig 2.

12

b

8 6

a

2 0 1

2

M AN U

0

SC

4

EP

TE D

Time postpartum, h

AC C

Distance, um

RI PT

b

10

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

Fig 3

ACCEPTED MANUSCRIPT

Highlights

AC C

EP

TE D

M AN U

SC

RI PT

1) Calving difficulty was as a 2-year-old was associated with decreased reproductive longevity 2) Protease activity increases at or around the time of calf expulsion in the absence of calving difficulty 3) Protease activity and transcription are greater in the caruncle than the cotelydon