The polyamines are ubiquitous polycationic compounds. Over the past 40 yr, investigation has shown that some of these, namely spermine, spermidine, and putrescine, are essential to male and female reproductive processes and to embryo/fetal development. Indeed, their absence is characterized by infertility and arrest in embryogenesis. Mammals synthesize polyamines de novo from amino acids or import these compounds from the diet. Information collected recently has shown that polyamines are essential regulators of cell growth and gene expression, and they have been implicated in both mitosis and meiosis. In male reproduction, polyamine expression correlates with stages of spermatogenesis, and polyamines appear to function in promoting sperm motility. There is evidence for polyamine involvement in ovarian follicle development and ovulation in female mammals, and polyamine synthesis is required for steroidogenesis in the ovary. Studies of the embryo indicate a polyamine requirement that can be met from maternal sources before implantation, whereas elimination of polyamine synthesis abrogates embryo development at gastrulation. Polyamines play roles in embryo implantation, in decidualization, and in placental formation and function, and polyamine privation during gestation results in intrauterine growth retardation. Emerging information implicates dietary arginine and dietary polyamines as nutritional regulators of fertility. The mechanisms by which polyamines regulate these multiple and diverse processes are not yet well explored; thus, there is fertile ground for further productive investigation.

Mammalian embryonic diapause is a phenomenon defined by the temporary arrest in blastocyst growth and metabolic activity within the uterus which synchronously becomes quiescent to blastocyst activation and implantation. This reproductive strategy temporally uncouples conception from parturition until environmental or maternal conditions are favourable for the survival of the mother and newborn. The underlying molecular mechanism by which the uterus and embryo temporarily achieve quiescence, maintain blastocyst survival and then resume blastocyst activation with subsequent implantation remains unknown. Here, we show that uterine expression of Msx1 or Msx2, members of an ancient, highly conserved homeobox gene family, persists in three unrelated mammalian species during diapause, followed by rapid downregulation with blastocyst activation and implantation. Mice with uterine inactivation of Msx1 and Msx2 fail to achieve diapause and reactivation. Remarkably, the North American mink and Australian tammar wallaby share similar expression patterns of MSX1 or MSX2 as in mice—it persists during diapause and is rapidly downregulated upon blastocyst activation and implantation. Evidence from mouse studies suggests that the effects of Msx genes in diapause are mediated through Wnt5a, a known transcriptional target of uterine Msx. These studies provide strong evidence that the Msx gene family constitutes a common conserved molecular mediator in the uterus during embryonic diapause to improve female reproductive fitness.

Embryonic diapause is a poorly understood phenomenon of reversible arrest of embryo development prior to implantation. In many carnivores, such as the mink (Neovison vison), obligate diapause characterizes each gestation. Embryo reactivation is controlled by the uterus by mechanisms that remain elusive. Because polyamines are essential regulators of cell proliferation and growth, it was hypothesized that they trigger embryo reactivation. To test this, mated mink females were treated with α-difluoromethylornithine, an inhibitor of ornithine decarboxylase 1, the rate-limiting enzyme in polyamine biosynthesis, or saline as a control during the first 5 d of reactivation. This treatment induced polyamine deprivation with the consequence of rearrest in embryo cell proliferation. A mink trophoblast cell line in vitro subjected to α-difluoromethylornithine treatment likewise displayed an arrest in cell proliferation, morphological changes, and intracellular translocation of ornithine decarboxylase 1 protein. The arrest in embryo development deferred implantation for a period consistent with the length of treatment. Successful implantation and parturition ensued. We conclude that polyamine deprivation brought about a reversible rearrest of embryo development, which returned the mink embryo to diapause and induced a second delay in embryo implantation. The results are the first demonstration of a factor essential to reactivation of embryos in obligate diapause.

Embryonic diapause is an evolutionary strategy to ensure that offspring are born when maternal and environmental conditions are optimal for survival. In many species of carnivores, obligate embryonic diapause occurs in every gestation. Reciprocal embryo transplant studies indicate that embryo arrest during diapause is conferred by uterine conditions and is due to a lack of specific factors necessary for continued development. In previous studies, global gene expression analysis revealed reduced uterine expression during diapause of a cluster of genes in the mink that regulate the abundance of polyamines, including ornithine decarboxylase 1 (ODC1). In addition, in vivo inhibition of the conversion of ornithine to the polyamine, putrescine, induced a reversible arrest in mink embryonic development and an arrest in trophoblast cell proliferation in vitro. Previous studies have implicated prolactin as the principal endocrine signal to terminate diapause. In this study, uterine expression of both the progesterone and estrogen receptors remained low at reactivation whilst the prolactin receptor was expressed at all times. Treatment of mink uterine epithelial cells with varying doses of prolactin indicated that this hormone induces ODC1 expression in the uterus via pSTAT1 and mTOR, thereby regulating uterine polyamine levels. In addition, we performed global gene expression analysis on mink embryos to further explore dynamic changes during diapause and found 94 genes upregulated at reactivation from diapause. Three polyamine-related genes, including ODC1, were also upregulated at reactivation from diapause. To establish whether polyamines mitigate escape from embryonic diapause, we collected mink embryos in diapause and incubated them in vitro with putrescine. Increase in embryo volume, the first indication of emergence from diapause, was observed within the first 5 days of culture in all viable embryos treated with putrescine, and the duration of embryo survival was increased threefold. Concomitant increases were also observed in both the total number of cells and the proportion of dividing cells in putrescine-treated embryos whilst control embryos remained in the diapause state. In further studies, inhibition of polyamine synthesis abrogated proliferation in cells derived from the inner cell mass of the mink embryo, while putrescine induced dose-dependent increases in cell division. We conclude that supplementation of embryos in diapause with putrescine results in their escape from developmental dormancy. These results provide strong evidence that obligate diapause in vivo is caused by the paucity of polyamines necessary for activation of the embryo after prolactin-induced termination of diapause.

Brominated flame retardants are incorporated into a wide variety of consumer products and are known to enter into the surrounding environment, leading to human exposure. There is accumulating evidence that these compounds have adverse effects on reproduction and development in humans and animal models. Animal studies have generally characterized the outcome of exposure to a single technical mixture or congener. Here, we determined the impact of exposure of rats prior to mating and during gestation to a mixture representative of congener levels found in North American household dust. Adult female Sprague-Dawley rats were fed a diet containing 0, 0.75, 250 or 750mg/kg of a mixture of flame retardants (polybrominated diphenyl ethers, hexabromocyclododecane) from two weeks prior to mating to gestation day 20. This formulation delivered nominal doses of 0, 0.06, 20 and 60mg/kg body weight/day. The lowest dose approximates high human exposures based on house dust levels and the dust ingestion rates of toddlers. Litter size and resorption sites were counted and fetal development evaluated. No effects on maternal health, litter size, fetal viability, weights, crown rump lengths or sex ratios were detected. The proportion of litters with fetuses with anomalies of the digits (soft tissue syndactyly or malposition of the distal phalanges) was increased significantly in the low (0.06mg/kg/day) dose group. Skeletal analysis revealed a decreased ossification of the sixth sternebra at all exposure levels. Thus, exposure to an environmentally relevant mixture of brominated flame retardants results in developmental abnormalities in the absence of apparent maternal toxicity. The relevance of these findings for predicting human risk is yet to be determined.