Occupational exposures to pesticides may increase parental risk of infertility and adverse pregnancy outcomes such as spontaneous abortion, preterm delivery, and congenital anomalies. Less is known about residential use of pesticides and the risks they pose to reproduction and development. In the present study we evaluate environmentally relevant, low-dose exposures to agrochemicals and lawn-care pesticides for their direct effects on mouse preimplantation embryo development, a period corresponding to the first 5-7 days after human conception. Agents tested were those commonly used in the upper midwestern United States, including six herbicides [atrazine, dicamba, metolachlor, 2,4-dichlorophenoxyacetic acid (2,4-D)], pendimethalin, and mecoprop), three insecticides (chlorpyrifos, terbufos, and permethrin), two fungicides (chlorothalonil and mancozeb), a desiccant (diquat), and a fertilizer (ammonium nitrate). Groups of 20-25 embryos were incubated 96 hr in vitro with either individual chemicals or mixtures of chemicals simulating exposures encountered by handling pesticides, inhaling drift, or ingesting contaminated groundwater. Incubating embryos with individual pesticides increased the percentage of apoptosis (cell death) for 11 of 13 chemicals (p less than or equal to 0.05) and reduced development to blastocyst and mean cell number per embryo for 3 of 13 agents (p less than or equal to 0.05). Mixtures simulating preemergent herbicides, postemergent herbicides, and fungicides increased the percentage of apoptosis in exposed embryos (p less than or equal to 0.05). Mixtures simulating groundwater contaminants, insecticide formulation, and lawn-care herbicides reduced development to blastocyst and mean cell number per embryo (p less than or equal to 0.05). Our data demonstrate that pesticide-induced injury can occur very early in development, with a variety of agents, and at concentrations assumed to be without adverse health consequences for humans. Key words: apoptosis, embryo, pesticide mixtures, pesticides, preimplantation development, reference dose.

Large numbers and large quantities of endocrine-disrupting chemicals have been released into the environment since World War II. Many of these chemicals can disturb development of the endocrine system and of the organs that respond to endocrine signals in organisms indirectly exposed during prenatal and/or early postnatal life; effects of exposure during development are permanent and irreversible. The risk to the developing organism can also stem from direct exposure of the offspring after birth or hatching. In addition, transgenerational exposure can result from the exposure of the mother to a chemical at any time throughout her life before producing offspring due to persistence of endocrine-disrupting chemicals in body fat, which is mobilized during egg laying or pregnancy and lactation. Mechanisms underlying the disruption of the development of vital systems, such as the endocrine, reproductive, and immune systems, are discussed with reference to wildlife, laboratory animals, and humans.

A number of studies have suggested that the active derivative of vitamin A, retinoic acid (RA), may be important for early development of mammalian embryos. Severe vitamin A deprivation in rodents results in maternal infertility, precluding a thorough investigation of the role of RA during embryogenesis. Here we show that production of RA by the retinaldehyde dehydrogenase-2 (Raldh2) enzyme is required for mouse embryo survival and early morphogenesis. Raldh2 is an NAD-dependent aldehyde dehydrogenase with high substrate specificity for retinaldehyde. Its pattern of expression during mouse development has suggested that it may be responsible for embryonic RA synthesis. We generated a targeted disruption of the mouse Raldh2 gene and found that Raldh2-/- embryos, which die at midgestation without undergoing axial rotation (body turning), exhibit shortening along the anterioposterior axis and do not form limb buds. Their heart consists of a single, medial, dilated cavity. Their frontonasal region is truncated and their otocysts are severely reduced. These defects result from a block in embryonic RA synthesis, as shown by the lack of activity of RA-responsive transgenes, the altered expression of an RA-target homeobox gene and the near full rescue of the mutant phenotype by maternal RA administration. Our data establish that RA synthesized by the post-implantation mammalian embryo is an essential developmental hormone whose lack leads to early embryo death.

Despite extensive adverse publicity, tobacco use continues in approximately 25% of all pregnancies in the United States, overshadowing illicit drugs of abuse, including cocaine. The societal cost of maternal smoking is seen most readily in underweight newborns, in high rates of perinatal morbidity, mortality and Sudden Infant Death Syndrome and in persistent deficits in learning and behavior. We have designed animal models of nicotine exposure to prove that nicotine itself is a neuroteratogen, thus providing a causative link between tobacco exposure and adverse perinatal outcomes. In particular, nicotine infusion paradigms that, like the transdermal patch used in man, produce drug exposure without the confounds of other components of tobacco or of episodic hypoxic-ischemic insult, have enabled a mechanistic dissection of the role played by nicotine in fetal brain damage. Nicotine targets specific neurotransmitter receptors in the fetal brain, eliciting abnormalities of cell proliferation and differentiation, leading to shortfalls in the number of cells and eventually to altered synaptic activity. Because of the close regulatory association of cholinergic and catecholaminergic systems, adverse effects of nicotine involve multiple transmitter pathways and influence not only the immediate developmental events in fetal brain, but also the eventual programming of synaptic competence. Accordingly, defects may appear after a prolonged period of apparent normality, leading to cognitive and learning defects that appear in childhood or adolescence. Comparable alterations occur in peripheral autonomic pathways, leading to increased susceptibility to hypoxia-induced brain damage, perinatal mortality and Sudden Infant Death. Identifying the receptor-driven mechanisms that underlie the neurobehavioral damage caused by fetal nicotine exposure provides a rational basis for decisions about nicotine substitution therapy for smoking cessation in pregnancy. In contrast to the effects of nicotine, animal models of crack cocaine use in pregnancy indicate a more restricted spectrum of effects, a reflection of differences both in pharmacokinetics and pharmacodynamics of the two drugs. Notably, although cocaine, like nicotine, also targets cell replication, its effects are short-lived, permitting recovery to occur in between doses, so that the eventual consequences are much less severe. To some extent, the effects of cocaine on brain development resemble those of nicotine because the two share cardiovascular actions (vasoconstriction) that, under some circumstances, elicit fetal hypoxia-ischemia. In light of the fact that nearly all crack cocaine users smoke cigarettes, the identification of specific developmental effects of cocaine may prove difficult to detect. Although scientists and the public continue to pay far more attention to fetal cocaine effects than to those of nicotine or tobacco use, a change of focus to concentrate on tobacco could have a disproportionately larger impact on human health.

Bone morphogenetic proteins (BMPs), which are members of the transforming growth factor beta (TGF-beta) superfamily, have been implicated in bone formation and the regulation of early development. To better understand the roles of BMPs in Xenopus laevis embryogenesis, we have cloned a cDNA coding for a serine/threonine kinase receptor that binds BMP-2 and BMP-4. To analyze its function, we attempted to block the BMP signaling pathway in Xenopus embryos by using a dominant-negative mutant of the BMP receptor. When the mutant receptor lacking the putative serine/threonine kinase domain was expressed in ventral blastomeres of Xenopus embryos, these blastomeres were respecified to dorsal mesoderm, eventually resulting in the formation of a secondary body axis. These findings suggest that endogenous BMP-2 and BMP-4 are involved in the dorsal-ventral specification in the embryo and that ventral fate requires induction rather than resulting from an absence of dorsal specification.

We have demonstrated previously that a combination of signals from the neural tube and the floor plate/notochord complex synergistically induce the expression of myogenic bHLH genes and myogenic differentiation markers in unspecified somites. In this study we demonstrate that Sonic hedgehog (Shh), which is expressed in the floor plate/notochord, and a subset of Wnt family members (Wnt-1, Wnt-3, and Wnt-4), which are expressed in dorsal regions of the neural tube, mimic the muscle inducing activity of these tissues. In combination, Shh and either Wnt-1 or Wnt-3 are sufficient to induce myogenesis in somitic tissue in vitro. Therefore, we propose that myotome formation in vivo may be directed by the combinatorial activity of Shh secreted by ventral midline tissues (floor plate and notochord) and Wnt ligands secreted by the dorsal neural tube.

Within the Hoxb homeobox gene complex, Hoxb-1 is the earliest member expressed in the mesoderm and neuroectoderm of primitive streak and presomite embryos, preceding rhombomere-restricted expression in the hindbrain. Ectopic exposure of embryos to retinoic acid alters spatial aspects of Hox gene expression patterns. However, the role of retinoids in regulating these genes during normal development is unclear. We have now identified two enhancers, 3' of the mouse Hoxb-1 gene, which together reconstruct the early endogenous expression pattern and mediate the early ectopic response to retinoic acid. Furthermore, these regions are functionally conserved in both chicken and pufferfish (Fugu rubripes) Hoxb-1 genes. The enhancer that controls the retinoic acid response, and regulates expression predominantly in neuroectoderm, contains a retinoic acid response element (RARE). Point mutations in the RARE abolish expression in neuroectoderm. Therefore, this RARE is not only involved in the ectopic response to retinoic acid, but is also essential for establishing aspects of the early Hoxb-1 expression pattern.

Since morpholino oligos were first introduced as a means to inhibit gene function in embryos, in the Spring of 2000, they have been tested in a range of model organisms, including sea urchin, ascidian, zebrafish, frog, chick, and mouse. This review surveys the results of these studies and examines the successes and limitations of the approach for targeting maternal and zygotic gene function. The evidence so far suggests that, with careful controls, morpholinos provide a relatively simple and rapid method to study gene function.

In recent years, the Cre integrase from bacteriophage P1 has become an essential tool for conditional gene activation and/or inactivation in mouse. In an earlier report, we described a fusion protein between Cre and a mutated form of the ligand binding domain of the estrogen receptor (Cre-ER) that renders Cre activity tamoxifen (TM) inducible, allowing for conditional modification of gene activity in the mammalian neural tube in utero. In the current work, we have generated a transgenic mouse line in which Cre-ER is ubiquitously expressed to permit temporally regulated Cre-mediated recombination in diverse tissues of the mouse at embryonic and adult stages. We demonstrate that a single, intraperitoneal injection of TM into a pregnant mouse at 8.5 days postcoitum leads to detectable recombination in the developing embryo within 6 h of injection and efficient recombination of a reporter gene in derivatives of all three germ layers within 24 h of injection. In addition, by varying the dose of TM injected, the percentage of cells undergoing a recombination event in the embryo can be controlled. Dose-dependent excision induced by TM was also possible in diverse tissues in the adult mouse, including the central nervous system, and in cultured cells derived from the transgenic mouse line. This inducible Cre system will be a broadly useful tool to modulate gene activity in mouse embryos, adults, and culture systems where temporal control is an important consideration.

In humans and rodents, exposure to hormonally active chemicals during sex differentiation can produce a wide range of abnormal sexual phenotypes including masculinized and defeminized females and feminized and demasculinized males. Although numerous "environmental estrogens," including pesticides, toxic substances (PCBs), and plant and fungal estrogens, have been shown to alter mammalian sex differentiation, similar information on environmental androgens is lacking. Recently, the fungicide vinclozolin (V) was found to inhibit sexual differentiation in male rats in an antiandrogenic manner. In the present study, V was administered to pregnant rats (p.o.) at 0, 100, or 200 mg/kg/day in corn oil during the period of sex differentiation (Gestational Day 14 to Postnatal Day 3) to examine the demasculinizing effect of this fungicide more closely. In both groups of V-treated male offspring, anogenital distance was female like at birth, and nipple development was prominent at 2 weeks of age. After puberty, most of the V-treated male offspring were unable to attain intromission even though they all mounted sexually receptive females. The V-treated male offspring that appeared to achieve intromission, failed to ejaculate normally, as no sperm were found in the uterus after overnight matings. A factor in the abnormal ejaculation was that all V-treated male offspring had cleft phallus with hypospadias. In addition, a number of unusual reproductive malformations were noted when the males were necropsied at 1 year. Many V-treated male offspring had suprainguinal ectopic scrota/testes, a vaginal pouch, epididymal granulomas, and small to absent sex accessory glands. During the study, about 25% of the V-treated males died as a result of bladder stones, hydroureter, or hydronephrosis, while other males displayed these lesions at necropsy. While some of the above malformations in male offspring can also be produced by perinatal administration of a potent estrogen, like DES, V-treated female offspring did not display any estrogen-like alterations of reproductive development or fecundity. The only change seen in the female offspring was a reduced anogenital distance during neonatal life. Our observation of perinatal-induced agenesis of the prostate and blocked testicular descent, a pattern of malformations nearly identical to that reported for the antiandrogen flutamide, is consistent with other recent evidence that this fungicide is an androgen-receptor antagonist.