Mammalian Reproduction and Development on the International Space Station (ISS): Proceedings of the Rodent Mark III Habitat Workshop

Animal Enclosure Modules (AEMs) onboard the shuttle adequately supported late-pregnant rats. Pregnant rats that experienced spaceflight and were subsequently returned to Earth within 48-72 hrs of normal birth underwent delivery at the expected time. The duration of the birth process was similar in spaceflight-exposed and ground control rats although spaceflight dams exhibited two times more labor contractions.

Pups suckled on anesthetized dams during 25 seconds of weightlessness during parabolic flight on NASA’s KC-135 airplane and stayed on the nipple during parabolic maneuvers, obtaining milk. Dams were injected with oxytocin that successfully caused milk letdowns during the parabolic flight. Pups showed milk-letdown reflexes, stretching and extending their hindlimbs, but they remained attached – with their bodies “out in space” around the mother’s ventrum during periods of microgravity and hypergravity that occur in the parabolic flight. Suckling also was demonstrated during the Neurolab mission, STS-90, in 1998.

STS-72 (NIH.R3) 5-day-olds (housed in the Nursing Facility AEM-NF) and STS-90 (Neurolab) 8-day-olds held in the Research Animal Holding Facility (RAHF) within the Shuttle/Spacelab had high mortality rates and low body weights. Habitat design played a crucial role in neonatal survival in microgravity, particularly in the youngest neonates (Maese and Ostrach, 2002; Ronca, 2003).

Males mated within 5 days of landing with new females produced offspring, but pups were developmentally delayed: (evidence of epigenetic influence on males?). Males mated 2 months post-flight produced normal pregnancies and offspring (no developmental anomalies support epigenetic effect on sperm). No data are available from follow-on assessments of females. There was no video monitoring during this flight. Therefore, it was not possible to distinguish failed fertilization from failure to mate. Ground controls may have also failed to become pregnant (Keefe, 1985). There was no assessment of hardware design to ascertain whether mating could be accomplished in the habitat/caging provided.

The three experiments used female C57BL/6 or BalbC mice. Microgravity negatively impacted ovarian histology in mice as evidenced by lower numbers of corpora lutea, and unhealthy oocytes. Estrogen receptor levels were lower in flight mice, while gene expression of the HSPH-1 stress marker was down regulated. Oocyte maturation and production were blocked or terminated after 12-15 days (3 estrous cycles) of spaceflight (post-flight recovery has not been ascertained). These findings raise the possibility that COSMOS 1129 rats had no eggs to fertilize and/or refused to mate in absence of estrogen-dependent mating behavior, though the sensitivity of female mouse and rat reproductive factors to spaceflight is unknown.

Since its inception at the NASA Ames Research Center in the mid-1970s, many laboratories around the world have used the rat hindlimb unloading (HLU) model to mimic the effects of weightlessness and to study various aspects of musculoskeletal loading (Morey-Holton et al., 2005). In this model, the hindlimbs of rodents are elevated to produce a 30 degrees head-down tilt resulting in a cephalad fluid shift and to avoid weight-bearing by the hindquarters. Long-term HLU inhibits spermatogenesis in adult male rats in the absence of cryptorchidism, changes in testicular function due to hyperthermic testes (Tash et al., 2002). All HLU animals were sterile in 2 mating attempts whereas controls were 100% fertile. Chronic testicular hyperthermia was observed with temperature elevated above normal by 2.2°C, P<.00001). Other findings included: (1) Invasion of inflammatory cells (≥3 weeks), (2) Catastrophic apoptosis in the testes, (3) These factors cause aspermatogenic dysfunction.

Less information is known for mice – much more is known for rats.

Rats live in colonies with multiple males and females. They are characterized by a promiscuous mating system – males mate with multiple females; females mate with multiple males. Mice live in demes with a single territorial male maintaining a range that encompasses homes of multiple females with which it mates. Females may mate with multiple, territorial males. Both species rely on maternal behavior only for rearing young (compared to a few rodents, such as gerbils and some voles that exhibit biparental care). Mice are known for both maternal aggression, with which lactating dams vigorously defend nest and young against males, and for their willingness to engage in “communal rearing” of young, in which they brood and nurse each others’ offspring along with their own.

Colonial life of rats is associated with high levels of tolerance in close proximity (a “contact species”), seen in both females and males, although males are larger and tend to be more aggressive. Social organization of mice is associated with lower levels of tolerance among unrelated, unfamiliar adult males. Amicable contact behaviors are frequently seen among female mice but are not well understood.

Females exhibit an estrous cycle, 4–5 days continuously. Some seasonality is suspected but cycling occurs throughout the year in the lab. Females are sexually “receptive” during the estrus phase of the cycle, but they are more accurately defined as proceptive—because the estrus female actively solicits the male’s sexual attention. Female proceptive behavior includes: Moving into vicinity of male, making available their arousing, and estrus odors, which attract male approach behavior and sniffing. The female then “darts” away and stops, the male approaches again, the female darts and “ear wiggles.” The “dance” continues and escalates until copulation occurs.

During copulation: The male mounts and the female exhibits lordosis, making the pudenda more accessible and intromission more likely. Males may intromit and thrust, and multiple intromissions and thrusts culminate in ejaculation. Number and timing of intromissions contributes to fertile matings and reproductive success. Females control timing by retreating and regulating access and modulating their bouts of solicitation. Females show a host of mechanisms and suspected adaptations related to copulation and reproductive success including: (1) Odor-induced pregnancy blockades, (2) Odor-induced resorption, (3) Estrus synchrony (questionable by recent analyses), and (4) Delayed implantation.

The effects of spaceflight on development ranged from unimpaired to permanently altered. Interpretation of these results as to whether gravity is required for normal development must be tempered by the facts that all of the pregnant flight animals experienced at least one week of gravity just prior to launch, and the flight duration of 16-day gravity deprivation was likely too brief to have a major impact on some systems.

Effects of spaceflight on development and growth—the space-flown rodents were pre-exposed to gravity, and the exposures to the spaceflight environment were too short to complete development, growth, maturation, and aging such that deficiencies would manifest. Some findings indicate existence of critical periods requiring gravity environment stimulation for normal (1g) development and growth of systems. Studies of rodents generated in space and never exposed to one-gravity are necessary to elucidate the full impact of the spaceflight environment on development, maturation, and aging (i.e., studies across the lifespan and multiple generations in space). Return of live space-reared organisms is necessary for assessment of exposure to 1g stress to ascertain system weaknesses and repair capacities and the existence of irrevocable structure and function. An on-orbit centrifuge is needed to determine the level of gravity exposure (0-1g) sufficient for 1g comparable development. Science return is enhanced by forming integrated-discipline teams and by judicious sharing of litters. On-orbit animal microsurgery, complex tissue processing, and stable storage of specimens is feasible. Sample return is required for full analysis requiring techniques unavailable in space. Rodent habitat redesign is essential to maintain the neonate and dam interactions that facilitate adequate nutrition, sleep, body temperature control, and other factors of viability provided in a nest environment on Earth (Ronca, 2003).

Life on Earth evolved with a single environmental constant, gravity (1g). Chronic acceleration or hypergravity is the resultant of 1g Earth gravity combined with centrifugal force. The UC Davis Chronic Acceleration Research Unit (CARU) provides an experimental facility for exposing animals to short or long duration hypergravity.

Hypergravity has been used to establish the “Principle of Continuity” (Wade, 2005), the idea that gravitational fields are continuous above and below the gravitational field on Earth, and that biological responses to changes across the spectrum of gravity exhibit a similar continuity. On Earth (1g), fractional increments in g-load exceeding 1g can be continuously applied for extended periods, and dose-response relationship established. While the principle of continuity has not been rigorously tested and validated across the gravity continuum, there is a sizeable corpus of data suggesting that the principle is valid across multiple systems. Reproduction and development, size/growth, energy and metabolism, musculoskeletal, cardiovascular, immune, and CNS sensory/vestibular responses are among those that have been studied in space.

The lack of definitive data on exposure to centrifugation in space for rats and mice makes the extrapolation of the continuum to levels below Earth-gravity problematic. However, in systems where responses are detected for both spaceflight and acceleration by centrifugation, a gravitational continuum is present, supporting the Principle of Continuity or a systematic dose-response relationship between gravity load and the magnitude of physiological response. Accordingly, the use of hypergravity holds significant potential for predicting responses to spaceflight.

This model simulates certain features of microgravity exposure by elevating the hindquarters 30 to produce head-down tilt. Support structures are unloaded (bone, muscle), and cephalic fluid shifts are induced. Thus HLU mimics spaceflight effects for certain key systems. Limitations of the technique include immobility of the animals, no limitations or alterations of CNS sensory input such as that which would typically occur in spaceflight.

Will females hormonally cycle in the novel space environment? If females cycle, will they solicit and will males respond? In microgravity, can pairs manage mechanics of repeated intromissions needed for ejaculation?

Use proven breeders. Cage design should incorporate the biomechanics of mating. Test specific strains of each species in a flight-like habitat. Include some relevant environmental stressors (noise, thermal spikes, lighting) and evaluate breeding success.

Detrimental stress effects on the gestating offspring seem likely for both rats and mice. Disrupted onset and initiation of maternal behavior patterns is a major risk (unaddressed by Neurolab experiences), although maternal behavior was intact in late-pregnant dams exposed to spaceflight, then returned to Earth just prior to birth. If there is an absence of nesting material, then we must understand implications of depriving dam of nest building experience as part of maternal behavior and, importantly, the thermal consequences of no nest insulation!

It is vital to collect data on thermal requirements of both rats and mice in absence of nesting material. Collect quantitative and qualitative data on mouse parturition in selected strains.

For the dam—the energetic demands of lactation are considerable, so there must be an adequate balance of energy intake and output (including that for milk production). Adequate diet (enriched in most labs) is essential, as well as feeders capable of delivering more food than is reasonable to expect. For the first 12-14 days postpartum, the dam approaches a unified group of pups. This is important because all nipples acquire milk at once per letdown and thus all pups should be attached to get their fair share. Neurolab revealed the difficulties of maintaining litter integrity. For pups—ready access to dam from Day 14 to 28, as well as appropriate presentation of food for weaning.

Collect data on energetics of lactation cycle and pup growth under flight-like thermal conditions. Anticipate food presentation and quantities appropriate for dams and for weanlings.

Two additional presentations were made. Mike Hines presented the current rodent habitat designs for transport to the ISS on the Space X Dragon, the Animal Enclosure Module–Transfer (AEM-T) and housing on the ISS (AEM-X). Cecelia Wigley presented ISS Mission Characteristics including launch, concept of operations, and Recovery. These presentations can be viewed at URL (http://spacebiosciences.arc.nasa.gov/media/Mark3_v1.14_signed_rotated.pdf)