Progestogen Profiling Over the Course of Diapause and Resumption of Embryo Development in the European Roe Deer

Progesterone (P4) plays a pivotal role in maintenance of pregnancy in many mammalian species. Species-specific P4 metabolites have been shown to function as primary acting progestogen and the receptor binding capacity varies between species. The European roe deer (Capreolus capreolus) displays a 4-5 month period of embryonic diapause, which decouples fertilization from implantation. The majority of roe deer have two corpora lutea that secrete P4. No changes in P4 concentrations have been observed during pre-implantation embryo development. As 5α-DHP is known to play a major role during pregnancy in elephants and horses, we hypothesized that 5α-DHP functions as additional progestogen facilitating embryo reactivation. The profile of 11 progestogens was quantified in roe deer plasma over the course of diapause and resumption of embryo development including P4, 3α- and 3β-DHP, 20α- and 20β-DHP, 5α- and 5β-DHP, 3α,5α- and 3α,5β-THP, as well as 3β,5α- and 3β,5β-THP. While P4 was most abundant during diapause and resumption of development, 20α-DHP was the most abundant P4 metabolite. This is different than in pregnant elephants, where 5α-DHP was most abundant, and the luteal phase in cattle, where 3α,5α-THP was most abundant. With the exception of a weak correlation of 3β,5α-THP, none of the progestogens significantly correlated with embryonic development in the roe deer. Thus, plasma 5α-DHP does not seem to play a role in embryo reactivation. We propose that progestogens might contribute to priming the endometrium for supporting embryo development and preparation for implantation

Do ovarian steroid hormones control the resumption of embryonic growth following the period of diapause in roe deer (Capreolus capreolus)?

Embryonic diapause in the European roe deer includes a period of five months from August to December in which embryonic development is extremely decelerated. Following exit from diapause, the embryo rapidly elongates and subsequently implants. In diapausing carnivores and marsupials, resumption of embryonic growth is regulated by ovarian steroid hormones. In the roe deer, the role of steroid hormones is not known to date. In the present study, progesterone (P4), estradiol-17β (E2) and total estrogens (Etot) were determined in blood plasma and endometrium of roe deer shot in the course of regular huntings between September and December. Steroid hormone concentrations were correlated to the corresponding size of the embryo derived from ex vivo uterine flushing and to the date of sampling. The mean plasma concentrations of P4 (5.4 ± 0.2 ng/ml, mean ± SE, N = 87), E2 (24.3 ± 2.6 pg/ml, N = 86) and Etot (21.7 ± 2.6 pg/ml, N = 78) remained constant over the sampling period and were not correlated to embryonic size. Likewise, endometrial concentrations of P4 (66.1 ± 6.5 ng/ml), E2 (284.0 ± 24.43 pg/ml) and, Etot (440.9 ± 24.43 pg/ml) showed no changes over time. Therefore, it was concluded that ovarian steroid hormones do not play a determining role in resumption of embryonic growth following the period of diapause in the roe deer

Uterine fluid proteome changes during diapause and resumption of embryo development in roe deer

The uterine microenvironment during pre-implantation presents a pro-survival milieu, and is essential for embryo elongation in ruminants. The European roe deer (Careolus capreolus) pre-implantation embryo development is characterised by a 4-month period of reduced development, embryonic diapause, after which the embryo rapidly elongates and implants. We investigated the uterine fluid proteome by label-free liquid chromatography tandem mass spectrometry at four defined stages covering the phase of reduced developmental pace (early diapause, mid diapause, and late diapause) and embryo elongation. We hypothesised that embryo development during diapause is halted by the lack of signals that support progression past the blastocyst stage. Three clusters of differentially abundant proteins were identified by a self-organizing tree algorithm: 1) gradual reduction over development; 2) stable abundance during diapause, followed by a sharp rise at elongation; and 3) gradual increase over development. Proteins in the different clusters were subjected to Gene Ontology analysis. ‘Cellular detoxification’ in cluster 1 was represented by alcohol dehydrogenase, glutathione S-transferase, and peroxiredoxin-2. ATP-citrate synthase, nucleolin, lamin A/C, and purine phosphorylase as cell proliferation regulators were found in cluster 2, and ‘cortical cytoskeleton’, ‘regulation of substrate adhesion-dependent cell spreading’, and ‘melanosome’ were present in cluster 3. Cell cycle promoters were higher abundant at elongation than during diapause, and polyamines presence indicates their role in diapause regulation. This study provides a comprehensive overview of proteins in the roe deer uterine fluid during diapause, and forms a basis for studies aiming at understanding the impact of the lack of cell cycle promoters during diapause

Developmental progression continues during embryonic diapause in the roe deer

Abstract Embryonic diapause in mammals is a temporary developmental delay occurring at the blastocyst stage. In contrast to other diapausing species displaying a full arrest, the blastocyst of the European roe deer (Capreolus capreolus) proliferates continuously and displays considerable morphological changes in the inner cell mass. We hypothesised that developmental progression also continues during this period. Here we evaluate the mRNA abundance of developmental marker genes in embryos during diapause and elongation. Our results show that morphological rearrangements of the epiblast during diapause correlate with gene expression patterns and changes in cell polarity. Immunohistochemical staining further supports these findings. Primitive endoderm formation occurs during diapause in embryos composed of around 3,000 cells. Gastrulation coincides with elongation and thus takes place after embryo reactivation. The slow developmental progression makes the roe deer an interesting model for unravelling the link between proliferation and differentiation and requirements for embryo survival

Developmental progression continues during embryonic diapause in the roe deer

Embryonic diapause in mammals is a temporary developmental delay occurring at the blastocyst stage. In contrast to other diapausing species displaying a full arrest, the blastocyst of the European roe deer (Capreolus capreolus) proliferates continuously and displays considerable morphological changes in the inner cell mass. We hypothesised that developmental progression also continues during this period. Here we evaluate the mRNA abundance of developmental marker genes in embryos during diapause and elongation. Our results show that morphological rearrangements of the epiblast during diapause correlate with gene expression patterns and changes in cell polarity. Immunohistochemical staining further supports these findings. Primitive endoderm formation occurs during diapause in embryos composed of around 3,000 cells. Gastrulation coincides with elongation and thus takes place after embryo reactivation. The slow developmental progression makes the roe deer an interesting model for unravelling the link between proliferation and differentiation and requirements for embryo survival.ISSN:2399-364

Embryonic diapause modulates ovarian follicular dynamics and oocyte quality in the European roe deer (Capreolus capreolus)

The phenomenon of embryonic diapause has been reported to possibly affect ovarian follicular development. The aim of the current study was to identify the impact of the embryonic diapause period on follicle number and oocyte quality in the European roe deer (Capreolus capreolus). In this species, the embryo undergoes five months of developmental delay. We have determined the antral follicle count (AFC), oocyte morphological quality, oocyte meiotic resumption capacity and the transcriptome profile in immature (GV) and in vitro matured (MII) oocytes obtained during embryonic diapause and after embryo reactivation. A drop in AFC, number of oocytes suitable for in vitro maturation and oocytes reaching the MII stage was observed after embryonic reactivation. A large impact of the diapause period was evident when MII oocytes were compared against GV oocytes using an RNA-seq approach. A total of 1028 genes significantly differed between MII and GV oocytes during diapause and 1795 genes after reactivation. These genes included PDE3A and SOD2, known for being involved in oocyte meiotic arrest and oxidative stress. In addition, tumour-related genes included NDRG3, NCSTN and UGDH, which have been associated with cell proliferation, migration and metabolism, were found. Comparing diapause versus reactivation, differential transcript abundance in both GV and MII oocytes was observed (44 and 32 differentially expressed genes, respectively). Our results suggest that the embryonic diapause and reactivation periods modulate follicular dynamics, influencing the oocyte towards lower quality after reactivation. This is may be related to increased apoptosis, abnormal mitochondria function/structure and metabolic dysfunction in the maturing oocyte. © 2020 Third International Symposium on Embryonic Diapause

Amino acids activate mTORC1 to release roe deer embryos from decelerated proliferation during diapause

International audienceEmbryonic diapause in mammals leads to a reversible developmental arrest. While completely halted in many species, European roe deer ( Capreolus capreolus ) embryos display a continuous deceleration of proliferation. During a 4-mo period, the cell doubling time is 2 to 3 wk. During this period, the preimplantation blastocyst reaches a diameter of 4 mm, after which it resumes a fast developmental pace to subsequently implant. The mechanisms regulating this notable deceleration and reacceleration upon developmental resumption are unclear. We propose that amino acids of maternal origin drive the embryonic developmental pace. A pronounced change in the abundance of uterine fluid mTORC1-activating amino acids coincided with an increase in embryonic mTORC1 activity prior to the resumption of development. Concurrently, genes related to the glycolytic and phosphate pentose pathway, the TCA cycle, and one carbon metabolism were up-regulated. Furthermore, the uterine luminal epithelial transcriptome indicated increased estradiol-17β signaling, which likely regulates the endometrial secretions adapting to the embryonic needs. While mTORC1 was predicted to be inactive during diapause, the residual embryonic mTORC2 activity may indicate its involvement in maintaining the low yet continuous proliferation rate during diapause. Collectively, we emphasize the role of nutrient signaling in preimplantation embryo development. We propose selective mTORC1 inhibition via uterine catecholestrogens and let-7 as a mechanism regulating slow stem cell cycle progression

Amino acids activate mTORC1 to release roe deer embryos from decelerated proliferation during diapause

Embryonic diapause in mammals leads to a reversible developmental arrest. While completely halted in many species, European roe deer (Capreolus capreolus) embryos display a continuous deceleration of proliferation. During a 4-mo period, the cell doubling time is 2 to 3 wk. During this period, the preimplantation blastocyst reaches a diameter of 4 mm, after which it resumes a fast developmental pace to subsequently implant. The mechanisms regulating this notable deceleration and reacceleration upon developmental resumption are unclear. We propose that amino acids of maternal origin drive the embryonic developmental pace. A pronounced change in the abundance of uterine fluid mTORC1-activating amino acids coincided with an increase in embryonic mTORC1 activity prior to the resumption of development. Concurrently, genes related to the glycolytic and phosphate pentose pathway, the TCA cycle, and one carbon metabolism were up-regulated. Furthermore, the uterine luminal epithelial transcriptome indicated increased estradiol-17β signaling, which likely regulates the endometrial secretions adapting to the embryonic needs. While mTORC1 was predicted to be inactive during diapause, the residual embryonic mTORC2 activity may indicate its involvement in maintaining the low yet continuous proliferation rate during diapause. Collectively, we emphasize the role of nutrient signaling in preimplantation embryo development. We propose selective mTORC1 inhibition via uterine catecholestrogens and let-7 as a mechanism regulating slow stem cell cycle progression

An international outbreak of Salmonella enterica serotype Enteritidis linked to eggs from Poland: a microbiological and epidemiological study

Background: Salmonella spp are a major cause of food-borne outbreaks in Europe. We investigated a large multi-country outbreak of Salmonella enterica serotype Enteritidis in the EU and European Economic Area (EEA). Methods: A confirmed case was defined as a laboratory-confirmed infection with the outbreak strains of S Enteritidis based on whole-genome sequencing (WGS), occurring between May 1, 2015, and Oct 31, 2018. A probable case was defined as laboratory-confirmed infection with S Enteritidis with the multiple-locus variable-number tandem repeat analysis outbreak profile. Multi-country epidemiological, trace-back, trace-forward, and environmental investigations were done. We did a case-control study including confirmed and probable cases and controls randomly sampled from the population registry (frequency matched by age, sex, and postal code). Odds ratios (ORs) for exposure rates between cases and controls were calculated with unmatched univariable and multivariable logistic regression. Findings: 18 EU and EEA countries reported 838 confirmed and 371 probable cases. 509 (42%) cases were reported in 2016, after which the number of cases steadily increased. The case-control study results showed that cases more often ate in food establishments than did controls (OR 3·4 [95% CI 1·6–7·3]), but no specific food item was identified. Recipe-based food trace-back investigations among cases who ate in food establishments identified eggs from Poland as the vehicle of infection in October, 2016. Phylogenetic analysis identified two strains of S Enteritidis in human cases that were subsequently identified in salmonella-positive eggs and primary production premises in Poland, confirming the source of the outbreak. After control measures were implemented, the number of cases decreased, but increased again in March, 2017, and the increase continued into 2018. Interpretation: This outbreak highlights the public health value of multi-country sharing of epidemiological, trace-back, and microbiological data. The re-emergence of cases suggests that outbreak strains have continued to enter the food chain, although changes in strain population dynamics and fewer cases indicate that control measures had some effect. Routine use of WGS in salmonella surveillance and outbreak response promises to identify and stop outbreaks in the future. Funding: European Centre for Disease Prevention and Control; Directorate General for Health and Food Safety, European Commission; and National Public Health and Food Safety Institutes of the authors' countries (see Acknowledgments for full list)