Embryonic diapause, a condition of temporary suspension of development of the mammalian embryo, occurs due to suppression of cell proliferation at the blastocyst stage. It is an evolutionary strategy to ensure the survival of neonates. Obligate diapause occurs in every gestation of some species, while facultative diapause ensues in others, associated with metabolic stress, usually lactation. The onset, maintenance and escape from diapause are regulated by cascades of environmental, hypophyseal, ovarian and uterine mechanisms that vary among species and between the obligate and facultative condition. In the best-known models, the rodents, the uterine environment maintains the embryo in diapause, while estrogens, in combination with growth factors, reinitiate development. Mitotic arrest in the mammalian embryo occurs at the G0 or G1 phase of the cell cycle, and may be due to expression of a specific cell cycle inhibitor. Regulation of proliferation in non- mammalian models of diapause provide clues to orthologous genes whose expression may regulate the reprise of proliferation in the mammalian context.

Embryo implantation in the mink is preceded by a variable but obligate period of delay in development. Under the influence of progesterone and unknown luteal factors, the mink embryo implants 11–13 days following its exit from diapause. Recent work suggests that progranulin, a growth factor and secreted glycoprotein, is involved in trophoblast proliferation, placental development and endometrial differentiation in the mouse. Using the mink model of delayed implantation and endotheliochorial placentation, we examined the spatiotemporal distribution of progranulin in trophoblast and endometrium during pre- and early post-implantation gestation in vivo. A partial sequence of the mink progranulin gene was cloned and sequenced. Comparative sequence analysis revealed that exons 1 and 2 of mink progranulin share 86.6, 82.4, and 94.9% of nucleic acid sequence identity with the human, mouse, and dog sequences respectively, and indicated that the invariable residues of the cysteine-rich motifs of progranulin are well conserved in the mink sequence. By in situ hybridization, we show that mink progranulin transcript is present in the cytotrophoblast and in epithelial and stromal endometrial cells at the site of implantation and during early placental formation. Immunohistochemistry revealed the progranulin protein to be strongly expressed in endometrial luminal and glandular epithelium around the time of implantation. In the incipient labyrinth, progranulin expression is localized to cytotrophoblasts and fetal capillaries, as well as to the hypertrophied maternal endothelial cells. This study demonstrates that high levels of progranulin expression correspond to active cell proliferation, remodeling, and angiogenesis occurring during the establishment of the placenta in the mink.

Although putative horse embryonic stem (ES)-like cell lines have been obtained recently from in vivo-derived embryos, it is currently not known whether it is possible to obtain ES cell (ESC) lines from somatic cell nuclear transfer (SCNT) and parthenogenetic (PA) embryos. Our aim is to establish culture conditions for the derivation of autologous ESC lines for cell therapy studies in an equine model. Our results indicate that both the use of early-stage blastocysts with a clearly visible inner cell mass (ICM) and the use of pronase to dissect the ICM allow the derivation of a higher proportion of primary ICM outgrowths from PA and SCNT embryos. Primary ICM outgrowths express the molecular markers of pluripotency POU class 5 homeobox 1 (POU5F1) and (sex determining region-Y)-box2 (SOX2), and in some cases, NANOG. Cells obtained after the passages of PA primary ICM outgrowths display alkaline phosphatase (AP) activity and POU5F1, SOX2, caudal-related homeobox-2 (CDX2) and eomesodermin (EOMES) expression, but may lose NANOG. Cystic embryoid body-like structures expressing POU5F1, CDX2 and EOMES were produced from these cells. Immunohistochemical analysis of equine embryos reveals the presence of POU5F1 in trophectoderm, primitive endoderm and ICM. These results suggest that cells obtained after passages of primary ICM outgrowths are positive for trophoblast stem cell markers while expressing POU5F1 and displaying AP activity. Therefore, these cells most likely represent trophoblast cells rather than true ESCs. This study represents an important first step towards the production of autologous equine ESCs for pre-clinical cell therapy studies on large animal models.

Recent evidence points to a role for peroxisome proliferator-activated receptors (PPARs) δ and γ in embryo implantation and survival. In this study, we report the porcine PPARδ complete coding sequence and mRNA abundance of PPARδ, PPARγ1 and γ2, angiopoietin-like protein 4 (ANGPTL4) and adipocyte determination and differentiation-dependent factor 1 (ADD1) genes in the pregnant sow endometrium. Real-time PCR analysis was used to study the effect of parity (Yorkshire-Landrace multiparous (YL) and nulliparous (YLn)), site of endometrial tissue sampling (between and at embryo attachment sites) in crossbred Duroc×Yorkshire-Landrace (DYL) sows and stages of pregnancy (non-pregnant, day 15 and day 25 after mating) in Meishan-Landrace (ML) on mRNA levels. Parity effects were observed for PPARδ, ANGPTL4, and ADD1, with higher mRNA levels in YL than YLn sows. In DYL sows, lower mRNA levels were present at attachment sites compared to between attachment sites for PPARδ, PPARγ1, and ANGPTL4. Finally, day 15 pregnant ML sows had lower PPARδ mRNA levels compared to day 15 cycling ML sows. A significant increase of PPARγ1 mRNA levels was found on day 25 pregnant ML and DYL sows relative to day 15 ML or DYL pregnant sows. PPARδ and γ immunostaining was detected in endometrial tissue of day 15 cycling sows, day 15 and 25 pregnant sows and epithelial cells of day 25 embryos. Collectively, our results suggest a role for PPARδ, PPARγ1, and ANGPTL4, but not PPARγ2, during the peri-implantation period in pregnant sows.