Effects of hypergravity on mammary metabolic function: gravity acts as a continuum.

Mammary metabolic activity in pregnant rats is significantly increased in response to spaceflight. To determine whether changes in mammary metabolism are related to gravity load, we exposed pregnant rats to hypergravity and measured mammary metabolic activity. From days 11-20 of gestation (G), animals were centrifuged (20 rpm; 1.5, 1.75, or 2.0 x gravity) or were maintained at 1 G. On G20, five rats from each group were removed from the centrifuge and euthanized. The remaining dams (n = 5/treatment) were housed at 1 G until parturition. After 2 h of nursing by the pups, the postpartum dams were euthanized (G22). Glucose oxidation to CO2 and incorporation into lipids was measured. Mammary glands from dams euthanized on G20 revealed a strong negative correlation between metabolic rate and increased G load. Approximately 98% of the variation in glucose oxidation and 94% of the variation in glucose incorporation into lipids can be accounted for by differences in G load. Differences in metabolic activity disappeared in the postpartum dams. When we combined previous data from the microgravity with hypergravity environments and plotted the ratio of mammary metabolic rate vs. G load, there was a significant exponential relationship (r2 = 0.99). These data demonstrate a remarkable continuum of response across the microgravity and hypergravity environments and support the concept that gravitational load influences mammary tissue metabolism.

Maternal reproductive experience enhances early postnatal outcome following gestation and birth of rats in hypergravity.

A major goal of space life sciences research is to broaden scientific knowledge of the influence of gravity on living systems. Recent spaceflight and centrifugation studies demonstrate that reproduction and ontogenesis in mammals are amenable to study under gravitational conditions that deviate considerably from those typically experienced on Earth (1 x g). In the present study, we tested the hypothesis that maternal reproductive experience determines neonatal outcome following gestation and birth under increased (hyper) gravity. Primigravid and bigravid female rats and their offspring were exposed to 1.5 x g centrifugation from Gestational Day 11 either through birth or through the first postnatal week. On the day of birth, litter sizes were identical across gravity and parity conditions, although significantly fewer live neonates were observed among hypergravity-reared litters born to primigravid dams than among those born to bigravid dams (82% and 94%, respectively; 1.0 x g controls, 99%). Within the hypergravity groups, neonatal mortality was comparable across parity conditions from Postnatal Day 1 through Day 7, at which time litter sizes stabilized. Maternal reproductive experience ameliorated neonatal losses during the first 24 h after birth but not on subsequent days, and neonatal mortality was associated with changes in maternal care patterns. These results indicate that repeated maternal reproductive experience affords protection against neonatal losses during exposure to increased gravity. Differential mortality of neonates born to primigravid versus bigravid dams denotes gravitational load as one environmental mechanism enabling the expression of parity-related variations in birth outcome.

Effects of hypergravity exposure on the developing central nervous system: possible involvement of thyroid hormone.

The present study examined the effects of hypergravity exposure on the developing brain and specifically explored the possibility that these effects are mediated by altered thyroid status. Thirty-four timed-pregnant Sprague-Dawley rats were exposed to continuous centrifugation at 1.5 G (HG) from gestational Day 11 until one of three key developmental points: postnatal Day (P) 6, P15, or P21 (10 pups/dam: 5 males/5 females). During the 32-day centrifugation, stationary controls (SC, n = 25 dams) were housed in the same room as HG animals. Neonatal body, forebrain, and cerebellum mass and neonatal and maternal thyroid status were assessed at each time point. The body mass of centrifuged neonates was comparatively lower at each time point. The mass of the forebrain and the mass of the cerebellum were maximally reduced in hypergravity-exposed neonates at P6 by 15.9% and 25.6%, respectively. Analysis of neonatal plasma suggested a transient hypothyroid status, as indicated by increased thyroid stimulating hormone (TSH) level (38.6%) at P6, while maternal plasma TSH levels were maximally elevated at P15 (38.9%). Neither neonatal nor maternal plasma TH levels were altered, suggesting a moderate hypothyroid condition. Thus, continuous exposure of the developing rats to hypergravity during the embryonic and neonatal periods has a highly significant effect on the developing forebrain and cerebellum and neonatal thyroid status (P < 0.05, Bonferroni corrected). These data are consistent with the hypothesized role of the thyroid hormone in mediating the effect of hypergravity in the developing central nervous system and begin to define the role of TH in the overall response of the developing organism to altered gravity.

Altered gravity effects on mothers and offspring: the importance of maternal behavior.

In this paper, I review and discuss recent studies of pregnant, parturient and lactating rat mothers and neonates exposed to hypo- and hypergravity. These studies are revealing new insights into how deviations form Earth-normal gravity may affect fundamental reproductive and ontogenetic processes in mammals. By way of background, I will first briefly summarize the spaceflights that have carried mammalian mothers and their offspring into space.

Effects of prenatal spaceflight on vestibular responses in neonatal rats.

Ten pregnant Norway rats (Rattus norvegicus) were flown for 11 days on board the NASA space shuttle from gestational day 9 (launch) until gestational day 20 (landing) of the rats' 22-day pregnancy. After the birth of the pups, vestibular responses were analyzed from postnatal day (P) 0 until P5. In the first test, P0 neonates were supported on a platform in a side-lying position. Skyward head movements (i.e., movements performed against the gravity vector) were more frequent than head movements toward Earth in both flight and control neonates. In the second test, the contact-righting reflex, composed of stereotyped movements that rotate the body from supine to prone on a solid surface, was analyzed in P0 neonates. The frequency and latency of contact-righting responses did not differ in flight and control neonates. In the third test, vestibular head righting, with tactile and proprioceptive cues removed, was tested in neonates on P1, P3, and P5 by using a water-immersion test. Righting responses were observed less frequently in P1 and P3 flight neonates compared with controls. However, this deficit was transient, as evidenced by complete response recovery on P5. Collectively, these findings provide evidence for a selective disruption of vestibular-mediated responses after prenatal exposure to spaceflight.

Physiology of a microgravity environment selected contribution: effects of spaceflight during pregnancy on labor and birth at 1 G.

The events of parturition (labor, delivery, maternal care, placentophagia, and onset of nursing) were analyzed in female Norway rats (Rattus norvegicus) flown on either 11- or 9-day-long spaceflights beginning at the approximate midpoint of their pregnancies. Each space shuttle flight landed on the 20th day of the rats' pregnancies, just 48-72 h before parturition. After spaceflight, dams were continuously monitored and recorded by time-lapse videography throughout the completion of parturition and onset of nursing (days 22 and 23). Analyses of parturition revealed that, compared with ground controls, flight dams displayed twice the number of lordosis contractions, the predominant labor contraction type in rats. The number of vertical contractions (those that immediately precede expulsion of a pup from the womb), the duration of labor, fetal wastage, number of neonates born, neonatal birth weights, placentophagia, and maternal care during parturition, including the onset of nursing, were comparable in flight and ground control dams. Our findings indicate that, with the exception of labor contractions, mammalian pregnancy and parturition remain qualitatively and quantitatively intact after spaceflight during pregnancy.

Body mass, food and water intake, and activity of pregnant and lactating rat dams during 1.5-g centrifugation.

In the present study, we analyzed the relationship between body mass, food and water intake, and behavioral activity in pregnant and lactating rat dams exposed to continuous, 1.5-g centrifugation for 32 days. The period of centrifugation spanned from Gestational day (G) 11 of the rats' 22-day pregnancy until Postnatal day (P) 21, the time of weaning.

Effects of microgravity on vestibular development and function in rats: genetics and environment.

Our anatomical and behavioral studies of embryonic rats that developed in microgravity suggest that the vestibular sensory system, like the visual system, has genetically mediated processes of development that establish crude connections between the periphery and the brain. Environmental stimuli also regulate connection formation including terminal branch formation and fine-tuning of synaptic contacts. Axons of vestibular sensory neurons from gravistatic as well as linear acceleration receptors reach their targets in both microgravity and normal gravity, suggesting that this is a genetically regulated component of development. However, microgravity exposure delays the development of terminal branches and synapses in gravistatic but not linear acceleration-sensitive neurons and also produces behavioral changes. These latter changes reflect environmentally controlled processes of development.

Survival and growth of developing rats during centrifugation at 2G.

We studied the effects of 2G hypergravity on the survival, body mass and growth of postnatal rats (Rattus norvegicus). Nursing litters comprised of either neonatal (Postnatal day [P]7) or pre-weanling (P14) rats and their mothers were exposed to 16 days of continuous centrifugation. All of the offspring survived and gained body mass, indicating that mothers nursed their young. Following the onset of centrifugation, neonatal and pre-weanling rats showed a reduction in growth relative to age-matched environmental controls (EC). At the completion of testing, body mass of the hypergravity (HG) groups was significantly less than that of controls (p<0.05). Over the course of the test, the HG-exposed P7 group showed an overall 55% gain in body mass as compared to a 71% increase in controls, while the HG-exposed P14 group showed a 62% increase relative to 75% in controls. Neonatal offspring (P7) gained body mass during centrifugation, but at significantly slower rates as compared to EC controls (p<0.05). In contrast, growth rates of pre-weanling (P14) rats were not reduced relative to controls, possibly related to the initiation of weaning, around P18 in the rat. These findings raise key issues relevant to studies of nursing mammals reared in altered gravity.

Effects of gravity on development: the importance of behavior.

Organisms live in four dimensions. With the dimension of time, we recognize that each organism is a lifecycle--an expression of its development. The cycles of differentiation expressed in the timeframe of biotic evolution yields the diversity of life on Earth. Thus, phylogenesis, can be understood as alterations in developmental processes. Species of organisms are thus groupings of individuals that share developmental processes. Natural selection acts throughout the lifecycle to select for developmental features that are adoptively appropriate for the organism's immediate environment. The process of ontogeny occupies an appropriately central position in gravitational biology. The evolution of all life on Earth, and thus the evolution development took place in the presence of gravitational forces. It is thus natural and necessary for biologists to wonder and to hypothesize about the role of gravity in development. We shall discuss some of the ideas and concepts that we believe are fundamental to appreciating the place of developmental analyses in gravitational physiology. We focus on problems relevant to the effects of gravity on vestibular development, and then emphasize issues pertinent to mammalian development.

Perinatal stimulation facilitates suckling onset in newborn rats.

The fetus' experience of birth derives from a sequence of stimulation provided by the mother's labor contractions, her licking and handling, and the contrasting environmental conditions of the uterus and outside world. In the present investigation, Day 21 fetal rats were externalized from the dam's body; subjects in one uterine horn were compressed by simulated uterine contractions while control subjects in the opposite horn were not compressed. All pups were Cesarean-delivered, stroked, and exposed to a thermal environment simulating either room (21 degrees C), nest (33 degrees C), or intrauterine (36 degrees C) temperature. After 1-hr exposure to the experimental temperature, all pups were maintained at 33 degrees C and tested for their suckling response to an anesthetized dam. When newborns were tested at 120 min postpartum, simulated contractions increased the probability of nipple attachment in pups exposed to 21 degrees C relative to noncompressed littermates maintained at the same temperature. Atypically warm postpartum conditions (nestlike or intrauterine) obviated the effects of compression by increasing suckling above the levels seen in noncompressed newborns exposed to the cool condition. Thus, compressions facilitate the achievement of suckling under thermal conditions resembling those typically encountered by the newborn rat.

Rat pregnancy and parturition survive spaceflight challenge: new considerations of developmental consequences.

NASA: Results of the NASA-NIH.R1 and NASA-NIH.R2 pregnant rat studies are reported and compared with results of Cosmos-1514 study. Similarities and differences between the Cosmos and STS flights are reviewed. STS rats were videotaped so that in-flight and post-flight behavior could be observed. Rats were observed during readaptation to 1-g and labor and delivery. Results indicate that pregnancy can proceed after exposure to microgravity and that vaginal delivery can occur despite readaptation to 1-g. Analysis of videotape revealed that flight dams experienced almost twice as many labor contractions as controls.^ieng

Altered vestibular function in fetal and newborn rats gestated in space.

NASA: Researchers evaluated vestibular development and function in rat pups flown during gestation on the NASA-NIH R1 and R2 missions. Fetal and postnatal vestibular function were examined. Altered vestibular-mediated responses in the experimental fetal pups are attributed to either direct effect of gravity on the vestibular system or indirect effects of microgravity transduced through the mother. The postnatal tests confirmed the hypothesis that the vestibular system continually adapts and responds to tonic stimulation.^ieng

Sampling of prenatal and postnatal offspring from individual rat dams enhances animal use without compromising development.

To assess prenatal and postnatal developmental status in the offspring of a group of animals, it is typical to examine fetuses from some of the dams as well as infants born to the remaining dams. Statistical limitations often arise, particularly when the animals are rare or especially precious, because all offspring of the dam represent only a single statistical observation; littermates are not independent observations (biologically or statistically). We describe a study in which pregnant laboratory rats were laparotomized on day 7 of gestation (GD7) to ascertain the number and distribution of uterine implantation sites and were subjected to a simulated experience on a 10-day space shuttle flight. After the simulated landing on GD18, rats were unilaterally hysterectomized, thus providing a sample of fetuses from 10 independent uteruses, followed by successful vaginal delivery on GD22, yielding postnatal samples from 10 uteruses. A broad profile of maternal and offspring morphologic and physiologic measures indicated that these novel sampling procedures did not compromise maternal well-being and maintained normal offspring development and function. Measures included maternal organ weights and hormone concentrations, offspring body size, growth, organ weights, sexual differentiation, and catecholamine concentrations.

Perinatal stimulation and adaptation of the neonate.

The present report describes psychobiological studies of behavior around the time of birth. An adaptive, ecological perspective is presented in which stimulation of the fetus and newborn is purported to instigate adaptive postpartum behavior. Studies describing the perinatal sensory environment are reviewed, with a consideration of emergent sensory function of the fetus. It is asserted that afferent input associated with parturition perturbs the fetus and neonate, producing a general arousal state that facilitates breathing, suckling, and early learning. The view developed herein is that perinatal sensory input induces and canalizes the newborn's behavior, thereby regulating adaptive postpartum function. Deviations in afferent input may alter ontogenetic trajectories and compromise developmental outcome by reducing availability of conditions necessary for adequate postpartum adaptation.

Simulated uterine contractions facilitate fetal and newborn respiratory behavior in rats.

We tested the hypothesis that sensory and nonsensory factors associated with birth stimulate respiratory behavior in the fetal and newborn rat. Late gestation (Day 21) rat fetuses were externalized from the uterus with intact umbilical connections to the dam and exposed to stimuli normally associated with labor and delivery. Onset and maintenance of respiratory movements were monitored. In the first experiment, rat fetuses were exposed to either: (i) simulated uterine contractions; (ii) cooling (26 degrees C); (iii) umbilical cord occlusion; or (iv) air heated to intrauterine temperature (37.5 degrees C). Subjects were videotaped for 1 h, and respiratory movements counted during tape review. Fetuses in each group showed some respiratory behavior although compression significantly elevated respiratory rate compared to other experimental conditions. All subjects in each group were respiring after 1 h, except for pups that received umbilical cord occlusion. The 100% attrition rate of the cord occlusion-alone group was reversed by combining cord occlusion with compression, or with compression and cooling, but not by combining cord occlusion with cooling. Simulated birth pups (i.e., those exposed to compression, cooling and umbilical cord occlusion) and normal, vaginally delivered pups breathed at identical rates and showed a similar pattern of postpartum breathing. These results suggest that mechanical stimulation of the fetus associated with uterine contractions plays a critical role in the maintenance of continuous respiration at birth. Possible mechanisms for the facilitative effects of compression on perinatal breathing are discussed.

Proximal control of fetal rat behavior.

We examined the influence of the amniotic sac on spontaneous movement in late gestation fetal rats. Using techniques for in vivo observation of fetal behavior, Day 21 rat fetuses were exteriorized from the uterus, with umbilical connections to the dam intact, and videotaped for 15 min either: (a) through the intact amniotic membranes, or (b) following removal of the membranes. Analysis of fetal behavior categories replicated the findings of previous investigators: Movements of the head, forelimbs, and rearlimbs were significantly increased by sac removal, as was the total frequency of behavior categories and the simultaneous occurrence of different behavior categories. Frame-by-frame analysis of videotaped behavior revealed that amniotic sac removal increased the frequency of movement bouts without altering the overall amount of time that fetuses spent moving. Movement bout durations ranged from 50 msec to 70 s. The average duration of movement bouts was significantly reduced for fetuses lacking the amniotic sac as compared to fetuses within the sac, as was the overall distribution of movement bout durations. Frequency distributions of movement bout durations and interbout interval (IBIs) revealed that sac removal significantly increased the occurrence of short (1-2 s) movement bouts and reduced the frequency of protracted movement bouts and interbout intervals (> 10-s duration). Taken together, these findings indicate that the quantitative dimensions of fetal rat movements are influenced by proximal features of the uterine environment. During prenatal life, the amniotic sac appears to sustain movement, possibly by providing proprioceptive feedback or physical support to the fetus, or by regulating the chemical milieu.

Fetal experience revealed by rats: psychobiological insights.

Psychobiological studies of fetal ontogenesis in non-human species, particularly in laboratory rat, now have much to offer students of human development. Psychobiological approaches are 'ecologically' oriented which, for fetal research, implies consideration of the adaptive significance of behavior within the uterine habitat. Consistent with this orientation is a concern for the sensory capabilities of the fetus, especially in relation to the specific forms and levels of stimulation that occur within the uterine milieu. We describe recent advances in these areas, and the impressive range of fetal responses to naturally-occurring (often mother-induced) forms of sensory stimulation. To the psychobiologist, sensory-evoked responses of all kinds constitute the fetus' experience and much has been learned about the roles of such experience in regulating the rate, direction, and form of development. Indeed, experience can be considered a significant, if not neglected, mechanism of development. Finally, we briefly survey some contemporary analyses of the interrelations between developmental and evolutionary processes. We use onset of sensory function (which occurs in a stereotyped sequence in every vertebrate species for which there are data) as a developmental phenotype. Phylogenetic considerations of sensory development are discussed. By identifying and clarifying some of the intellectual questions that guide perinatal research with non-human species, we hope to improve the appreciation of novel insights and interpretive strategies that derive from different approaches. This is bound to enhance the study of human development and to improve the design and use of animal models.