1 CONTACT Theresa Beachler beachler@iastate.edu © 2024 The Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Citation line: Clinical Theriogenology 2024, 16, 9400, http://dx.doi.org/10.58292/CT.v16.9400 Review Report Embryonic diapause: a unique stage of development Theresa Beachler Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA, USA Abstract Embryonic diapause is a unique stage of development that occurs in some mammalian species. Diapause serves as an evolutionary reproductive strategy to maximize the survivability of dam and offspring. Although individual mechanisms of control vary among species, the overarching physiology of diapause extends the length of pregnancy to delay parturition until either seasonal, environ- mental, nutritional, or internal physiologic constraints are optimal for parturition and rearing of young. This review discusses concepts and highlights control mechanisms unique to rodents, mink, and tammar wallabies. Keywords: Embryonic diapause, development, blastocyst, pregnancy Introduction Embryonic diapause, also known as delayed implantation, is a temporary state of arrested development that occurs in some mammals at the blastocyst stage. Embryonic diapause is an evolutionary strategy to delay parturition to ensure the optimal seasonal, nutritional, environmental, and physio- logic conditions occur for both survivability and the rearing of offspring. Diapause was first noted in roe deer (Capreolus capreolus) in the mid-19th century.1 Subsequently, it was observed in over 130 species across various orders ranging from rodents, marsupials, numerous carnivores, including minks, weasels, skunks, bears, seals, armadillos, bats, and some species of deer.2,3 The defining characteristic of dia- pause is the dramatic reduction or cessation of cellular activ- ity in the embryo at the G0/G1 stage.3 In some species (e.g. mice and marsupials) cellular activity stops in its entirety with cessation of all cell divisions, transcription, and transla- tion, whereas in others, such as the roe deer and some carni- vores (mink, weasels, and bears), growth and activity remains but at an incredibly slow rate.3,4 In roe deer, for example, ~ 10% of cells proliferate where the embryo expands from 300 to 20,000 cells over a period of 4 months, with the majority of growth occurring within the trophectoderm compared to the inner cell mass.4 Maintenance or length of delayed devel- opment may range from a brief halt lasting for a few days, as observed in mice and rats (4-10 days), to much longer extended period of ~ 10 months in the badger and up to 11 months in the tammar wallaby.2 Types of diapause Diapause is classified into 2 categories, either facultative or obligate diapause, with most species, excluding the tammar wallaby, experiencing 1 or the other.1-3,5 Facultative diapause occurs in response to a limiting internal physiological factor.2,3,5 Facultative diapause is also sometimes referred to as lactational diapause and is best understood in mice and some marsupials, where development is delayed in the presence of suckling young until weaning occurs.4 Obligate diapause or seasonal diapause has been best studied in carnivores, where delayed development occurs in every pregnancy under a seasonal pho- toperiod control.5 For example, in many carnivores residing in the Northern Hemisphere, such as the mink or skunk, and in bears, development is delayed until decreased melatonin is produced from the pineal gland in response to the onset of long days to support the birth and rearing of young during the most favorable environmental and nutritional conditions.2,5 Therefore, the hormone prolactin is a key player in both variet- ies of diapause with marked interspecies variability. Species-specific examples of lactational and seasonal control Mice Rodents such as rats and mice experience the first postpartum estrus within 24 hours of parturition.6,7 In mice mailto:beachler@iastate.edu http://dx.doi.org/10.58292/CT.v16.9400 2 Citation line: Clinical Theriogenology 2024, 16, 9400, http://dx.doi.org/10.58292/CT.v16.9400 (Mus muscularis) that were successfully mated, lactation in the presence of suckling young results in elevated concentra- tions of prolactin.6-8 High concentrations of prolactin pre- vent the estrogen surge that occurs at 3.5 days postovulation, initiating a facultative or lactational diapause for a period of 1 day to several weeks.5,8 Larger litter sizes additionally have been associated with longer periods of delayed develop- ment.7 Following weaning and the removal of the inhibitory effects of prolactin, embryos are reactivated by the estrogen surge and subsequent elevations in progesterone concentra- tions that prepare the endometrium for implantation.5 This has been supported experimentally by antagonist/dopamine agonist (bromocriptine) treatment, which terminated dia- pause and induced reactivation and implantation.9 Embryo transfer in ovariectomized female rats that were treated with progesterone prior to the natural estrogen surge, entered into an arrested state with reactivation occurring after estradiol treatment.10 Mink American mink (Neovison vison) is 1 of the best studied obli- gate or seasonal diapause family members. In American mink residing in the Northern Hemisphere, embryo devel- opment is delayed until decreased melatonin production occurs from the pineal gland in response to the onset of long days or > 12 hours of daylight after the spring equi- nox.11-13 Mink breed during spring, with matings occurring between February and April. Subsequently, prolactin con- centrations remain low in response to high nocturnal mela- tonin production (< 12 hours of daylight). Regardless of the time of mating and ovulation, lengthening photoperiod and concurrent reduction in melatonin increases circulating prolactin concentrations, resulting in ovarian (corpora lutea) produced progesterone increase.11,12 On the contrary in rodents, experimental treatment with the dopamine ago- nist bromocriptine extended diapause, whereas treatment with the dopamine antagonist pimozide terminated dia- pause.14,15 Therefore, prolactin is directly luteotrophic, end- ing diapause and resuming embryonic development in mink. Wallabies Tammar wallabies (Macropus eugenii) are unique as they may undergo both a facultative and obligate diapause in response to elevated concentrations of prolactin for a total period last- ing up to 11 months.5 In the Southern Hemisphere, tammar wallabies typically give birth to a single joey in late January to early February and enter a postpartum estrus shortly after, with mating occurring as early as 1 hour after birth.16,17 In suc- cessfully bred females, high concentrations of prolactin from the suckling young contribute to the initiation of lactational diapause. If the joey is lost during the breeding season (January through May), embryonic development resumes in the absence of high prolactin concentrations.18 After the winter solstice (June), only the seasonal photoperiod is sufficient to cause reactivation, with no response if joey is lost after this period. After the subsequent summer solstice (December), prolactin concentrations decrease in response to decreased photoperiod and increase in melatonin, allowing for blasto- cyst reactivation.18,19 Tammar wallabies, therefore, are unique species as they may display either a single or a combination of a facultative or obligate diapause depending on the time of year and if a suckling joey is present during periods of pregnancy. Additional mechanisms for reactivation or cessation of diapause At the blastocyst stage, embryos of species that undergo dia- pause have not yet initiated implantation and remain within the uterine lumen either within (marsupials and carnivores) or hatched from the zona pellucida (rodents, roe deer, and arma- dillos). In addition to varied hormonal changes as discussed above under lactational or seasonal control, reactivation or the cessation of the period of diapause appears to also be mediated by a combination of cytokines, growth factors, and transcrip- tional factors produced from the uterine endometrium within uterine secretions and the embryo itself, all of which is not completely understood.2 At the arrested blastocyst stage, embryos have yet to initiate implantation and may not begin the process until multiple days after reactivation. Factors identi- fied with altered patterns of expression during reactivation are conserved across many species include epidermal growth factors (EGF and HB-EGF), leukemia inhibitory factor (LIF), insulin-like growth factor (IGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), platelet-activating fac- tor (PAF), transforming growth factor (TGF-β), interleukin 1β (IL1β), bone morphogenic protein-2 (BMP2), and prostaglan- din synthetase PTGS2 (COX2).1,5,20-22 In mice, leukemia inhibi- tory factor (LIF) has a critical molecule in reactivation and the onset of implantation as its expression is substantially upregu- lated within the endometrial glands of pregnant mice on days 4 and 5 of pregnancy.21,22 Female mice lacking functional LIF genes produced viable embryos that failed to implant at the blastocyst stage in vivo but underwent appropriate develop- ment when transferred to wild-type recipients.22 Similarly, epi- dermal growth factor (EGF) has been critical for implantation in mice and rats with knockout receptor dams failing all embry- onic development beyond the blastocyst stage with degrada- tion of the inner cell mass.23 Additionally, signaling molecules or transcription factors encoded by the wingless family (WNT) and muscle segment homeobox genes (MSX1 and MSX2) were involved in activation and implantation in mice, mink, and wallabies.1,2,24-27 WNT signaling may also contribute to the altered spacing of embryos which in some species occurs simul- taneously with embryo reactivation.24 Muscle segment homeo- box genes (MSX1 and MSX2) are further mediated by WNT5a signaling with marked downregulation of these genes and their products occurring at embryo reactivation and implantation in mice, mink, and wallabies.24,25 Conclusion Mammalian embryonic diapause is a phenomenon defined by a temporary arrest in blastocyst development as a reproduc- tive strategy to promote both the survival of the dam and neo- nate until either environmental or physiologic factors are favorable. 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