Impacts of Altered Gravity on Male
and Female Reproductive Health:
Joseph S. Tash, Ph.D.
Director, NIH Interdisciplinary Center for Male
Contraceptive Research & Drug Development
Department of Molecular & Integrative Physiology
Department of Urology
University of Kansas Medical Center
Kansas City, KS
1
Outline of Topics
• A hypothesis for a common mechanism underlying
space-flight induced declines in multiple physiological
systems
• Male reproductive physiology impacts
• Female reproductive physiology impacts
• Translational multidisciplinary approaches to discover
solutions
2
Thanks to 5 Shuttle Missions, Crews, and
Huge Ground Support Teams
STS-81
STS-84
STS-133
STS-131
STS-135
Key Acknowledgements
• Katherine F. Roby, PhD. , KUMC
• Current and former post-docs G. Bracho, V.
Gupta, L. Holets
• Numerous collaborators and ground and flight
support research facilities at NASA Ames, KSC,
DLR Numerous current and former lab staff*
• Multiple NASA ground and flight grants since
1996
• NASA Biospecimen Sharing Program
• NIH grants
*Detailed in slides attached at end of presentation
Space Flight as an Accelerated Model
for the Aging Process on Earth
• Parallels between space flight effects on physiology
and aging have been known since the 1960’s
• Systems that show similar changes include:
–
–
–
–
–
–
–
Cardiovascular (heart and circulation)
Immune System (infection and inflammatory response)
Vestibular (balance control, visual-motor coordination)
Bone (mass and strength)
Muscle (tone and strength)
Metabolism (nutrition and drug pharmacokinetics)
Wound healing (traumatic stress and injury)
5
Known Roles of Estrogen Receptor Signaling* in
Space-Flight Affected Physiological Systems (1)
• Bone*
– Bone repair and maintenance (receptors in
osteoblasts and osteoclasts)
– Osteoimmunology
• Muscle*
– Regulates glucose transport
– Insulin signaling
• Cardiovascular*
*In the male, testosterone is
converted to estradiol by the
enzyme aromatase in major
organs
– Vasodilation
– Glucose transport
6
Known Roles of Estrogen Receptor Signaling in
Space-Flight Affected Physiological Systems (2)
• Immune
– Ubiquitous receptor in immune system cells
– Pro-inflammatory response
– Auto-immune protection (immunosenescence)
• Wound healing
– Epithelial & endothelial repair
– Neuroprotection
– Capillary vessel formation
• Vestibular system (brain contains aromatase)
– Modulates synaptic transmission
7
Overall Hypothesis and Approach
The space-flight induced decline in multiple
physiological systems involve a common
mechanism in dysregulation of reproductive steroid
receptor-dependent signaling pathways.
If true, then space flight should produce declines in
gonad function in males and females.
8
Gonads Top the Table of
Radiation Tissue Sensitivity*
Single Dose (Gy)
Fractionated Dose (Gy)
Ovary
2–6
Testes
1–2
Bone marrow
2–10
Ovary
6–10
Testes
2–10
Eye (lens)
6–12
Eye (lens)
2–10
Kidney
20–30
Mucosa
5–20
Thyroid
20–40
Gastrointestinal
5–10
Lung
23–28
Lung
7–10
Skin
30–40
Colorectal
10–20
Liver
35–40
Kidney
10–20
Bone marrow
40–50
Vasculoconnective tissue
10–20
Heart
43–50
Liver
15–20
Gastrointestinal
50–55
Skin
15–20
Vasculoconnective tissue
50–60
Peripheral nerve
15–20
Spinal cord
50–60
Spinal cord
15–20
Brain
55–70
Brain
15–25
Peripheral nerve
65–77
Heart
18–20
Mucosa
65–77
Bone and cartilage
>30
Bone and cartilage
>70
Muscle
>70
Muscle
>70
*From Rubin P. Law and order of radiation sensitivity: absolute versus relative. In: Vaeth JM,
Meyer JL, eds. Frontiers of radiation therapy and oncology. Basel: Karger; 1989:7–40.
Male Reproductive Physiology
Impacts
10
Conclusions from Our STS-81 and
STS-84 Sperm Experiments*
• Sperm motility activation occurs more
quickly in microgravity and it is matched
with more rapid protein phosphorylation
• On the other hand, the sperm response to
egg chemotactic factors is significantly
delayed
• These data suggest that the fertilizing
capacity period of sperm may be
shortened in microgravity
*Already published: NASA, ESA, RSA collaborations
11
Long Term Hindlimb Unloading (HLS)
(Ground-based spaceflight model)
Inhibits Spermatogenesis in Adult
Male Rats
in the Absence Of Cryptorchidism
12
Testicular
WeightCOAfter
6 Week
HLS
SUS
O
S
Testicular Weight (gm + SEM)
2.0
1.8
b
a
a =P< 0.01
b =P< 0.001
1.6
1.4
a,b
1.2
1.0
0.8
0.6
0.4
0.2
0.0
Control
Tail
Animal Group
HLS
13
Is long term HLS-induced loss of
spermatogenesis reversible?
Testis Weight 6 Months
After 6 Week HLS
*
*All HLS animals were
sterile, but showed
normal testosterone
levels , and normal
mating behavior
15
Severe Testicular and Epididymal Degeneration
in Male Rats 6 Months After Release
From 6 Week HLS Treatment
TO Control1
1Inguinal
HLS1,2
canals were partially ligated to prevent abdominal descending testes.
2All HLS animals were sterile in 2 mating attempts, controls were 100% fertile.
16
MRI of Partial Inguinal Ligated HLS Confirms
Testes Remain Scrotal During Unloading
Sagittal
view
Axial
view
Standing
HLS
Mean Scrotal Surface Temperature is
Significantly Elevated in Hindlimb
Unloaded vs Normal Standing Rats
Treatment
n
Free roaming
control (FRC)
4
Mean
∆T vs
temp.
FR Control
(°C ± SD)
28.5 ± 0.3
--
Tail only (TO)
elevated control
8
29.2 ± 0.4
0.7
0.007 vs FRC
Hindlimb
10 30.7 ± 0.6
unloaded (HLS)
2.2
0.00001 vs TO
0.00002 vs FRC
Probability
(t-Test)
Tunel
H&E
Evidence of Massive Testicular Apoptosis
6 Months After 6 Week HLS Treatment
Invasion of ROS-Producing Inflammatory
Cells* Precedes Loss of
Spermatogenesis and Apoptosis in HLS
*Myeloperoxidase immunohistochemistry
Epididymal Weight and Plasma
Hormone Levels 6 Months After 6 Week
HLS Treatment
Parameter
FR Controls
TO Controls
HLS
0.52 ± 0.03
0.62 ± 0.06
0.56 ± 0.03
146 ± 12
124 ± 27
140 ± 30
FSH (ng/ml)
6.57 ± 0.46
8.59 ± 0.52
17.9 ± 1.64ab
LH (ng/ml)
1.60 ± 0.52
1.71 ± 0.29
5.60 ± 1.70
524 ± 79
537 ± 123
648 ± 104
Cauda epididymal
weight (gm)
Testosterone (ng/ml)
Corticosterone (ng/ml)
a= P<0.001 vs. FR, b= P< 0.001 vs. TO
Mechanisms Driving Testicular
Degeneration During the 6 Week HLS
Period
• Chronic testicular hyperthermia (elevated
temperature: increased by 2.2ºC, P<.00001)
• Invasion of inflammatory cells (≥3 weeks)
• Catastrophic apoptosis
• These cause aspermatogenic dysfunction
22
Similarities and Consequences:
HLS and µG in the Male Mammal
(Rodent predicts human?) (1)
• In 1G, the gravitational vector combined with
the cremasteric muscle tonus determines
testicular position relative to body to regulate
testis temperature (identical in humans and
rats)
• In µG and HLS, the gravity vector is reduced,
thus no counteracting force against the
cremaster tonus to lower the testis: causing
chronic hyperthermia.
23
Similarities and Consequences:
HLS and µG in the Male Mammal
(Rodent predicts human?) (2)
• These processes are the same in humans
and rodents.
• When combined with known increased
radiation risk, this could significantly elevate
the risk for testicular cancer (at least 4-10 fold
based on the cryptorchid cancer model).
24
Artificial Gravity (2G for 6 wks)¥
Maintains or Slightly Increases Male
Fertility Outcomes
Group
SC
AG
RC
VIV
Pups/
Litter
Pup Wt Pups lost
(5-day) (1d:5d/No.
litters)
10.9 ±1.1*
7.56
7 / 7 (1.0)
12.3 ± 1.0* 7.53
6 / 7 (0.86)
10.5 ± 1.4
7.16
1 / 2 (0.5)
11.6 ± 2.0
7.59 5 / 7 (0.71)
*P = 0.02
¥ARC
24ft Centrifuge
Facility
% ♂ Pups
0.38 ± 0.13
0.52 ± 0.15
0.34 ± 0.16
0.39 ± 0.19
Male Reproductive Health
Conclusions
• Sperm function is sensitive across the gravity
spectrum (± depends on parameter)*
• Testicular function is negatively impacted by
the hindlimb unloading model of µG, and
unaltered or positively affected in artificial
gravity (2G)*
• Multiple factors are involved in male sterility in
the HLS model including chronic hyperthermia
leading to catastrophic inflammatory invasion,
reactive oxygen species (ROS), and apoptosis.
*Collaborations with ESA, ARC, DLR
26
Near Term Solutions
• Cryopreservation of semen before long term
space flight
– Offers peace-of-mind in case impacts are
realized with longer term habitation in space
– Significantly reduces the risk for insemination
with damaged (including mutated) sperm later on
• Dietary anti-oxidants and ROS scavengers?
27
Future Discovery of Mechanisms and
Solutions Will Require Long Duration Flight
Experiments
• The complete spermatogenic cycle takes ~5060 day from spermatogonium to sperm.
• This processes take much longer than the
duration of space shuttle and previous
satellite experiments to complete,
• Thus ISS and free-flyer satellite (i.e. BION
M1) options are required to determine
mechanisms and solutions
Female Reproductive Health
Impacts
29
Background Related to Female
Reproductive Health and Microgravity (1)
• Does space flight alter ovarian function and how
does this relate to estrogen receptor signaling?
– Paucity of flight data for normal adult estrous
cycling female rodents has been published
• Previous female rodent flight data exists for pregnant,
nursing, or pre-pubertal animals
• One RSA satellite experiment showed no pregnancies
produced in adult female rats exposed to adult male
rats in flight.
30
Background Related to Female
Reproductive Health and Microgravity (2)
– No detailed human data has been published
on effects of space flight on normal ovarian
function.
• Female astronauts are recommended to take
contraceptives during space flight to prevent
cycling and pregnancy
• There is published data on female astronauts
experiencing post-flight difficulties getting
pregnant (age+drug effects makes interpretation
difficult).
31
Main Hypothesis & Objective
• Main Hypothesis:
– Microgravity has a negative impact on female
reproductive health via effects on the ovary
• Objective:
– To determine the effect of space flight on function
and endocrine hormone receptor signaling in
ovaries and uterine horn of “normal” mice flown
12-15 days* in microgravity in LOE, compared to
ground controls
*Estrous cycle in mice is 4-5
days vs 28 days in humans.
32
Flight Ovaries Were Significantly* Smaller
and Had Atretic (Dying) Follicles
Ground
Flight
Ovary
Uterine
Horn
Avg ovary wt
5.5 ± 0.8 mg
4.0 ± 1.5 mg *P < 0.001
Normal
oocytes
Normal
follicle
Atretic
follicles
Abnormal
oocytes
33
Flight Mice Had Significantly Lower
Number of Corpora Lutea*
*
P=0.02
Ground = 3.8 ± 0.6 (n=8)
Flight = 1.6 ± 0.6 (n=7)
*Lower number of corpora lutea reveal compromised egg
production
34
Space Flight Induced Declines in
Ovarian Structure/Function Repeatable
in 3 Space Flights*
Parameter
Control
STS-131**
(n=8)
STS-133**
(n=2)
STS-135**
(n=8)
% change
Ovarian
weight
5.5 ± 0.8
4.0 ± 1.5
(P<0.001)
n too low
1.9 ± 0.6
(P<0.001)
-27.2%
n too low
-29.6%
-58%
-100%
tbd
2.7 ± 0.6
Corpora
lutea
3.8 ± 0.6
4.3 ± 1.5
1.6 ± 0.6
(P<0.02)
0.0 ± 0.0
(P<0.02)
tbd
Follicle
atresia
No
Yes
Yes
tbd
**Data are color coded
to each flight.
35
Estrogen Receptors, ERa and ERb,
Significantly Down-regulated in Flight Uteri
Relative mRNA abundance ERa/GAPDH
Ovary
Uterus
ERa
0.9
9
0.8
8
0.7
7
0.6
6
0.5
5
0.4
4
0.3
3
0.2
2
0.1
1
P<0.05
0
0
GROUND
GROUND
FLIGHT
FLIGHT
ERb
1.2
2
1.8
1
1.6
1.4
0.8
P<0.05
1.2
0.6
1
0.8
0.4
0.6
0.4
0.2
0.2
0
0
GROUND
FLIGHT
GROUND
FLIGHT
36
Estrogen Signaling Marker, Lactoferrin,
Down-regulated in Uterus by Space
Flight
Relative mRNA abundance
LF/GAPDH
Ovary
Uterus
25
1.4
1.2
20
1
15
0.8
c
0.6
10
0.4
P<0.05
5
0.2
0
0
GROUND
FLIGHT
GROUND
FLIGHT
Female Rodent Study Conclusions (1)
• Microgravity negatively impacts ovarian histology in
mice: lower numbers of corpora lutea, mainly atretic
follicles, and unhealthy oocytes
• Estrogen receptor levels were lower in flight mice,
while stress markers were not increased
• The data suggest that oocyte maturation and
production were blocked or terminated after 12-15
days (3 estrous cycles) of space flight (post-flight
recovery has not been ascertained)
38
Female Rodent Study Conclusions (2)
• Since ovaries have a limited population of maturation
competent follicles, and atretic follicles do not recover,
space flight may shorten reproductive lifespan.
• In contrast to male, female mechanisms and solutions
could be defined in shorter duration microgravity
flights <12-15 days duration
39
Near Term Solutions
• Oocyte vitrification or cryopreservation
– Offers peace-of-mind in case impacts are
realized with longer term habitation in space
– Significantly reduces the risk for pregnancy
with damaged (including mutated) oocyte later
on
40
Translational Research to Address
Reproductive Health Risks for Long Duration
Space Exploration (1)
• Until recently, most male and female astronauts on
ISS have been beyond family planning years
• Younger astronauts are now beginning to have
longer missions in µG – human reproductive health
risks are more likely to be realized
• What level of post-flight astronaut cancer and/or
infertility risk is acceptable?
• Are the rodent data suggestive of any existing data
available from LONG TERM human exposures to
microgravity?
41
Translational Research to Address
Reproductive Health Risks for Long Duration
Space Exploration (2)
• Do the changes in the reproductive systems and
their endocrine hormone signaling mechanisms
underlie the broader declines in physiological
systems during space flight?
• Is there corroborating related human data?
• Explore mechanisms to partner/collaborate with to
discover mechanisms and develop effective
countermeasures?
42
Translating Space Flight
Discoveries to Earth Benefit (1)
• Identify common mechanism of space flight
impacts on physiological systems that similarly
change during aging
• Since “space flight aging” occurs much more
rapidly, underlying mechanisms could be
identified more rapidly in microgravity than on
earth
43
Translating Space Flight
Discoveries to Earth Benefit (2)
• Elucidating mechanisms reveals targets for
drug discovery and development, as well as
novel palliative care protocols
• Resulting drugs will improve aging populations
on earth and provide countermeasures for
space flight
44
END
STS-135 and BION M1 BSP Team at
SLSL KSC*
*Largest team of biological scientists (~45) ever assembled for a Space
Shuttle experiment, including Bioserve, BSP, Amgen
46
Additional Acknowledgements
(KUMC)
• Tash lab: GE Bracho, PhD, J Brann, R Beard, E Cambron, K
Denklefsen, JJ Fritch, S Kim, ME Landis, S Macheras, S
McDonald, L Tash, B Timmerberg, MA, S Wolfe, M Wulser,
• GC Enders, T Gleason, DC Johnson, JD Pierce, RN, S Platt,
MA, P Terranova, PhD
• M Bilgen, PhD, Hoglund Brain Imaging Center
• F Sun, DVM, D Pinson, DVM, N Culley, DVM
• NIH Center for Reproductive Sciences (U54 and NIH
Specialized Cooperative Centers Program in Reproduction
Research, U54 Interdisciplinary Center for Male
Contraceptive Research & Drug Development)
Acknowledgments
(STS-81 & STS-84 Flight)
• Mission Specialists: John Grunsfeld, PhD (STS-81), and
Ed Lu, PhD (STS-84)
• ESA Biorack Ground Unit: JM Merle, U Kuebler, N Hiesgen,
D Narbonne
• J Fishman , R Schaefer, PhD - Lockheed/Martin
• T Savage - NASA AMES, V Schneider, MD - NASA HQ
• E Brinckmann, PhD C Brioullet, PhD, P Genzel - ESTEC
• CNES / COMAT (Flight Hardware)
• J Hatton and D Schmitt, MD - Toulouse
• M Lewis, PhD - UA-Huntsville
• NASA and Biorack Ground Support Team
48
STS-131, STS-133 & STS-135
Acknowledgements
• Dr. Viju Gupta, Dr. Lesya Holets, Dr. Ajay Nangia,
Brian Kern, Anne Hoffman, Stanton Fernald,
Barbara Shull, Shari Standiferd, Jennifer Wallace,
KUMC
• NASA Grant NNX09AP04G to JST
• Richard Boyle, Paula Dumars, Vera Vizir, GwoShing, Kenny Vassigh: Ames Research Center,
NASA
• Stacy Engel & Ashleigh Ruggles: Kennedy Space
Center, Florida
DLR Acknowledgements
(NASA and Related)
• NASA grants to JST: NAG-2-1016 and
NAG-2-1491, NNA04CC54A
• M Schuber, MD, D Seibt: MUSC at DLR
• SM Claasen - Bonn University
ARC 24ft Centrifuge
Acknowledgements
• Dr. April Ronca, Dr. Charles Wade
• Danielle Galindo, Tianna Shaw, Stephen
Voels (NASA Ames)
• 24 ft Centrifuge Facility at NASA Ames
Gravitational Biology Research Branch
• Center for Reproductive Sciences
• Grants from NASA and NIH