Abstracts

 

Roe Deer Research Group

 

A pregnancy-associated glycoprotein (PAG) unique to the roe deer (Capreolus capreolus) and its role in the termination of embryonic diapause and maternal recognition of pregnancy

Roger T.  Lambert

School of Biological Sciences, University of Aberdeen, Aberdeen, AB24 2TZ, UK

 

During reactivation of the roe deer blastocyst from embryonic diapause, the conceptus secretes a unique protein. The conceptuses were cultured individually in vitro with a radiolabel to establish de novo protein synthesis. Endometrial tissue from the same does was also cultured in vitro to establish radiolabel incorporation into endometrial secretions during blastocyst reactivation.  Incorporation into secretory proteins from the blastocysts showed a significant increase during reactivation (P <0.05) whereas incorporation into endometrial secretory proteins remained constant. The secretory proteins from the conceptus were identified using electrophoresis and fluorography as having the same molecular weight and pI as pregnancy associated glycoproteins (PAGs). Fluorography clearly identified a single PAG with a molecular weight of 66 kDa and an isoelectric point (pI) of 6.2.  Previous research shows these blastocyst secretions target endometrial receptors. The current study demonstrates this is followed by a dramatic increase in concentrations of maternal estradiol in serum, from 1.07 0.4 pg ml-1 during diapause to 49.17 0.37 pg ml-1 at trophoblast elongation. Estradiol is the hormone responsible for triggering the endocrine events that lead to implantation. PAGs are released from trophoblast cells and bind to specific cell surface receptors on maternal target cells. The role for PAGs in trophoblast cells had not previously been defined. Data from this study demonstrate that embryonic programming initiates the transcription of specific genes in the inner cell mass of the blastocyst, messenger RNA directs synthesis of PAG in the trophoblast, and the roe deer-specific PAG triggers the maternal response by targeting endometrial receptors.

Israel Journal of Zoology  2005,  Vol:51, No.1 pp 1-11

2005 LPPLtd.   

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6th European Roe Deer Meeting

Biology Department, University of Minho, Portugal 23rd April – 26th April 2003.

 

A pregnancy associated glycoprotein (PAG) unique to the roe deer (Capreolus capreolus) and its role in the termination of embryonic diapause and maternal recognition of pregnancy

 

Roger T. Lambert1, Paul A. Racey1and Cheryl J. Ashworth2

1Department of Zoology, University of Aberdeen, Aberdeen AB24 2TZ, UK

2Department of Applied Physiology, Scottish Agricultural College, Bucksburn Aberdeen AB21 9YA, UK

 

The study found during reactivation of the roe deer blastocyst from embryonic diapause that the conceptus secretes a unique protein. The conceptuses were cultured individually in vitro with a radiolabel to establish de novo protein synthesis. Endometrial tissue from the same does was also cultured in vitro to establish radiolabel incorporation into endometrial secretions during blastocyst reactivation. The blastocysts showed a significant increase in secretory proteins during blastocyst reactivation (P <0.05) whereas incorporation into endometrial secretions remained constant. The secretory proteins from the conceptus were identified using electrophoresis and fluorography as having the same molecular weight and pI as pregnancy associated glycoproteins (PAGs). The fluorograph cleary indentified a single PAG with a molecular weight of 66 000 Da and a pI of 6.2. The study also showed the presence of the PAG in roe deer serum after implantation. Once blastocyst secretions have targeted endometrial receptors there was a dramatic increase in concentrations of maternal oestradiol in serum, from 1.07 0.4 pg ml-1 during diapause to 49.17 0.37 pg ml-1 at trophoblast elongation. Oestradiol is the hormone responsible for triggering the endocrine events that lead to implantation. Previous studies have shown the properties of PAGs as belonging to the aspartic proteinase gene family, are highly polymorphous, are released from trophoblast cells and bind to specific cell surface receptors on maternal target cells. The role for PAGs in trophoblast cells had not previously been defined. Data from this study demonstrate that embryonic programming initiates the transcription of specific genes in the inner cell mass of the blastocyst, messenger RNA directs synthesis of PAG in the trophoblast and the roe deer specific PAG triggers the maternal response by targeting endometrial receptors.

 

The pre-oestrus signal, variations in pregnancy length and photoperiod manipulation of pregnant roe deer does (Capreolus capreolus)

 

Roger T. Lambert1, Paul A. Racey1and Cheryl J. Ashworth2

1Department of Zoology, University of Aberdeen, Aberdeen AB24 2TZ, UK

2Department of Applied Physiology, Scottish Agricultural College, Bucksburn Aberdeen AB21 9YA, UK

 

In the European roe deer the rut takes place during late July or early August.  Ten roe does ranging in age between 1 and 3 years at commencement of the study were used to observe mating behaviour and gestation length (July to following June) over a period of 4 years. Five pregnant does were exposed to long day photoperiod manipulation (16L: 8D between 17 October and 10 April) to assess the effect on the duration of embryonic diapause, compared to 5 control does. Two distinct phases of mating behaviour (courtship, chasing, attempting to mount and mounting) occurring a week apart were observed. Previously published studies show that the first phase is associated with the start of cyclicity and reflecting in general the maturation process of the hypothalamus-pituitary-ovary axis and the second phase by actual oestrus. The mean length of gestation was 292.7 7.2 days. There was considerable variation in gestation length with yearling does (n = 3) having a shorter gestation of ~280 days and older does a gestation length of up to 312 days. Examination of conceptuses in culled animals with known conception dates demonstrated that this variation in pregnancy length was attributed to a variation in the interval between blastocyst reactivation and implantation. These variations in gestation length were inversely related to the body condition of the doe (body condition score of 1.5 = 312 days gestation; condition score of 4 = 280 days gestation). Does experimentally exposed to long days gave birth three weeks earlier than the control group (gestation length: control group; 292.33 2.44; experimental group; 293.44 5.14) and showed a three-week advance in the timing of last oestrus behaviour. As there is no significant difference between gestation length in the control animals and experimental animals this demonstrates that the length of embryonic diapause was unaffected. Does subjected to long day photoperiod manipulation started moulting their winter coats in January (control animals started moulting in April). This study demonstrates that the period of diapause is genetically fixed and the variations in pregnancy length show that the doe, after blastocyst reactivation from diapause and prior to implantation, is controlling conceptus development in direct relationship to her body condition.

 

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Temporal changes in reproductive hormones and conceptus-endometrial interactions during embryonic diapause and reactivation of the blastocyst in European roe deer  (Capreolus capreolus)

R.T. Lambert 1,2*, C.J. Ashworth 2*, L. Beattie 2, F.E. Gebbie 3,
J.S.M. Hutchinson 4, D.J. Kyle 2 and P.A. Racey 1
1Department of Zoology, University of Aberdeen, Aberdeen, AB24 2TZ, UK
2 Rowett Research Institute, Bucksburn, Aberdeen, AB21 9SB, UK
3 Department of Applied Physiology, Scottish Agricultural College, Craibstone Estate, Aberdeen, AB21 9YA, UK
4 Department of Agriculture, University of Aberdeen, Aberdeen, AB24 5UA, UK
* Present address: Department of Applied Physiology, Scottish Agricultural College, Craibstone Estate,  Aberdeen

Summary
The roe deer blastocyst is in diapause between August and December, after which it expands and elongates rapidly before implantation. Blood samples were taken from 30 animals to define temporal changes in reproductively important hormones to investigate the physiological cues present at embryo reactivation. In 15 of these animals, changes in uterine and conceptus protein synthesis and secretion, and luteal progesterone release during diapause and reactivation, were assessed after culture of these tissues in vitro. Oestradiol concentrations remained low during diapause (1.07 sem 0.4 pg ml-1) and expansion (1.2 sem 0.4 pg ml-1) increasing by 30 times at trophoblast elongation (49.17 sem 0.37 pg ml-1). Prolactin remained at basal concentrations (4.69 sem 0.86 ng ml-1) and increased after implantation (12.34 sem 2.71 ng ml-1). Peripheral progesterone concentrations and luteal progesterone release remained constant throughout diapause, reactivation and implantation (peripheral progesterone: 3.82 sem 1.97 ng ml-1; luteal progesterone: 6.72 sem 0.81 ng mg-1 protein).  Incorporation of a radiolabel into conceptus secretory proteins increased by four times at expansion compared with diapause, whereas incorporation into endometrial secretions remained constant. At elongation, incorporation into endometrial secretions increased two times and conceptus secretions 32 times. Two-dimensional electrophoresis and fluorography showed that the profile of endometrial secretory proteins was constant until implantation when qualitative changes were evident. Although a role for an endocrine maternal trigger of reactivation from diapause cannot be dismissed, these data provide no supporting evidence and indicate that the conceptus itself may drive reactivation

Reproduction (2001)  121:6 pp 863-871.  You can read the full paper as a pdf file Click to go to download

(c) 2001 Journals of Reproduction and Fertility, Cambridge



5th European Roe Deer Meeting
Instituto Nazionale per la Fauna Selvatica, Tredozio, Italy, 24 – 27 April 2001

Embryonic Diapause in Roe Deer: The physiology and endocrinology of the pregnant roe deer between the period of late embryonic diapause and early fetal growth.
R.T. Lambert 1,2*, C.J. Ashworth 2*, L. Beattie 2, F.E. Gebbie 3,  J.S.M. Hutchinson 4, D.J. Kyle 2 and P.A. Racey
1Department of Zoology, University of Aberdeen, 2 Rowett Research Institute, Aberdeen
3 Department of Applied Physiology, Scottish Agricultural College, 4Department of Agriculture, University of Aberdeen,
*Present address: Department of Applied Physiology, Scottish Agricultural College, Aberdeen, AB21 9YA, United Kingdom

The total gestation period in roe deer is approximately 290 days (10 months). When the blastocyst reaches the 20 –30 cell stage it enters a 5-month period of diapause. During the last six weeks of diapause the blastocyst undergoes a degree of mitosis and reactivates in late December or early January at the 100-cell stage. The conceptus (embryo and associated membranes) undergoes a period of rapid growth, which last between 10 and 25 days, before placental attachment to the endometrium followed by normal fetal growth. We studied the period between late diapause and early fetal development to monitor changes in the reproductively important hormones, progesterone, oestradiol, pregnancy associated glycoprotein (PAG) and prolactin. Secretion of important signalling proteins by the conceptus and uterus during this period was also studied. We also investigated whether a regimen of long day photoperiod manipulation would effect the length of the period of diapause. Both peripheral concentrations and luteal release of progesterone remained consistent throughout the period studied. Oestradiol showed a dramatic rise at the time of rapid elongation of the trophoblast and both prolactin and PAGs were consistently low until after implantation.  The secretion of newly synthesised protein by the endometrium and the conceptus was consistent during the latter period of diapause, but at reactivation conceptus secretion increased by a factor of 4, whereas endometrial secretion was constant until trophoblast elongation, when it doubled. At trophoblast elongation conceptus secretions had increased by a factor of 32. Endometrial protein secretion remained consistent until implantation. Thereafter they decreased linearly as fetal development progressed. The dominant conceptus secretory protein appeared to be a roe deer specific PAG.  Long day photoperiod manipulation advanced the timing of the rut by three to four weeks. Subsequent parturition was advanced by the same period. The total gestation length for the animals studied was 291 sem 2.5 days. Our data suggest that there is no role for a maternal endocrine trigger for reactivation of the blastocyst and that the conceptus drives reactivation from diapause.



Society for the Study of Fertility and
Deutsche Gesellschaft Zum Studien Der Fertilitat Und Sterilitat
Joint Winter Meeting 1998
17th-19th December 1998 at Aachen, Germany

Temporal changes in reproductive hormones during embryonic diapause and
reactivation of the blastocyst in the European roe deer (Capreolus capreolus)
RT Lambert1,2, CJ Ashworth2, L Beattie2, FE Gebbie3, JSM Hutchinson4, DJ Kyle2
and PA Racey1.
1Department of Zoology, University of Aberdeen; 2Rowett Research Institute; 3Scottish Agricultural College;
4Department of Agriculture, University of Aberdeen, Aberdeen, UK.

Roe deer blastocysts exhibit obligate embryonic diapause between early August and late December. The blastocyst then expands and elongates rapidly before implantation. The objective of this study was to establish the cues for reactivation of the diapausing blastocyst. Previous results from our group have shown that both endometrial and conceptus tissues exhibit a dramatic increase in de novo protein synthesis and secretion between expansion and implantation. The increase in conceptus protein synthesis precedes that of the endometrium (1) suggesting that the conceptus may provide the cue. Peripheral progesterone (P4) and oestradiol-17b concentrations were determined in 22 blood samples from pregnant does between 11 Nov and 29 Jan. Luteal P4 release was determined following in vitro incubation of 18 minced corpora lutea obtained from 9 of these does. Oestradiol-17b was consistently low throughout diapause and early expansion (1.226 pg ml-1 SD 0.326 pg ml-1; n=18) but increased at the elongated trophoblast stage and implantation (37.04 pg ml-1 SD 7.89 ng ml-1; n=4). P4 concentrations declined from diapause (2.9 ng ml-1 SD 0.802 ng ml-1 n=15) to 1.305 ng ml-1 SD 0.657 ng ml-1 (n=4) during expansion and elongation and did not rise until implantation (3.92 ng ml-1 SD 3.56 ng ml-1 n=3). Luteal P4 decreased from 7.03 ng ml-1 SD 0.81 ng mg-1 (n=6) during diapause to 3.73-1 ng ml SD 0.69 ng mg-1 (n=4) at expansion, increased to 7.16 ng ml-1 SD 0.285 ng mg-1 (n=2) at elongated trophoblast stage and to 10.27 ng ml-1 SD 0.616 ng mg-1 (n=6) at implantation. The consistently low luteal P4 release and peripheral P4 and oestradiol-17b concentrations at early expansion, coupled with our earlier data showing that endometrial protein synthesis and secretion did not change during late diapause suggest that conceptus reactivation is not in response to a maternal trigger. One possibility is that the embryo is genetically programmed to commence development at a given time. Alternative hypotheses include a role for photoperiod or intra-uterine prolactin.

1. Lambert et al, (1998) Proceedings of the 4th International Biology of Deer Congress,
Advances in Deer Biology. (ed) Z. Zomborszky.

Abstract published 1998, Journal of Reproduction and Fertility 22: 33.
ISSN:0954-0725

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