The European roe deer (Capreolus capreolus) was the first mammal in which embryonic diapause has been described. While diapause is characterized by a complete developmental arrest in some species, roe deer blastocysts show a very slow, yet continuous growth. To date, it is neither known whether this growth is accompanied by developmental progression nor whether it is uniform in both, the trophectoderm (TE) and the inner cell mass (ICM). We collected roe deer blastocysts during the regular hunting season from September 2018 to January 2019, and quantified the fraction of cells expressing the proliferation marker Ki67 by immunofluorescence and light-sheet microscopy. We found that the cell number increased from around 300 cells in September to over 20'000 cells per blastocyst in December before elongation occured. Concurrently, we observed considerable morphological changes, i.e. cavity formation and transition to a disk-like shape of the inner cell mass. During diapause, less than 10% of all cells displayed positive Ki67 staining. Strikingly, the relative increase in cell number was lower in the ICM compared to the TE, whereas the fraction of Ki67 positive cells appeared to be lower in the TE than in the ICM. Our findings thus confirm that roe deer blastocysts display developmental progression in the course of diapause. We hypothesize that while the overall duration of the cell cycle is longer in the ICM than in the TE, the fractional distribution of cell cycle phases differs, with TE cells having a longer G1 phase than cells of the ICM.

During mammalian diapause, the embryo is arrested at the blastocyst stage. In diapausing carnivores and marsupials, growth arrest of the blastocyst is coincident with a quiescent corpus luteum (CL). In the roe deer, progesterone is produced throughout diapause and the blastocyst exhibits very slow growth. To date, early roe deer embryos of known age have not been described. Therefore, it is not known at which stage and at what pace the roe deer embryo enters diapause. To collect multiple embryos of known gestational age, we performed cycle synchronization and superovulation in captive roe deer prior to artificial insemination (AI) and natural breeding (NB), respectively. Superovulatory responses were evaluated based on ultrasonographic detection of CL formation on the day of in vivo embryo flushing and on plasma progesterone (P4) analysis. In total, 33 superovulation treatments resulted in 6.6  2.2 (mean  SD) CL per doe on the day of embryo flushing. At days 6 and 7 after AI, 37 multicellular embryos up to the morula stage were retrieved from 8 does. At day 12 and 13 after natural breeding, 28 embryos, mainly at the blastocyst stage (n=20), were collected from 7 does. Multiple embryos could be collected from single animals, proving the feasibility of superovulation in this species. Compared to other diapausing and non-diapausing species where blastocysts have formed at days 6 -8, roe deer embryo development appeared to be already decelerated prior to the blastocyst stage. The roe deer might therefore be well suited as a model to investigate mechanisms regulating embryonic growth velocity with respect to cell cycle control.

No abstract

The first written reports on embryonic diapause in the European roe deer (Capreolus capreolus) date back to 1843 and 1854. While the rut takes place in July/August, rapid embryo elongation and implantation take place in December/January, after a five month period of embryonic diapause. The post-implantation development lasts another five months and an average of two fawns are born in May. As fertilization and subsequent implantation are decoupled, the roe deer offers the opportunity to study changes in hormones, embryonic and endometrial morphology and transcriptome dynamics, as well as the uterine microenvironment in a high time-dimension. So far, it has been shown that peripheral increases in prolactin, estradiol-17β and progesterone are a consequence, rather than the cause of reactivation of embryo development. Increased uterine secretions and the presence of embryonic cell organelles coincide with embryo elongation. This suggests that the "quiet embryo" hypothesis might hold for diapausing embryos. The factors controlling embryonic developmental pace in the roe deer are yet to be identified. We propose a multi-omics approach to determine the temporal molecular changes during diapause, prior to embryo elongation, which morphologically marks the reactivation of embryo development, and upon elongation. Thereby, we will obtain insights into the molecular mechanisms and environmental cues that inhibit and drive cell proliferation. By data integration, we aim at identifying the origin of the factors associated with reduced embryonic developmental pace. The knowledge of a species-independent mechanism driving embryo development past the blastocyst stage is of high interest to reproductive biologists and for species conservation programs.

The capacity of the mammalian embryo to arrest development during early gestation is a topic that has fascinated biologists for over 150 years. The first known observation of this phenomenon was in a ruminant, the roe deer (Capreolus capreolus) in 1854, later confirmed in a number of studies in the last century [1]. The phenomenon, now known as embryonic diapause, was then found to be present in a wide range of species and across multiple taxa. Since that time, its biological mystery has attracted studies by scientists from around the globe.