Nitrogen mustard exposure perturbs oocyte mitochondrial physiology and alters reproductive outcomes

Accepted Manuscript Title: Nitrogen mustard exposure perturbs oocyte mitochondrial physiology and alters reproductive outcomes Authors: Lynae M. Brayboy, Haley Clark, Laura O. Knapik, Ruby E. Schnirman, Gary M. Wessel PII: DOI: Reference:

S0890-6238(18)30194-1 https://doi.org/10.1016/j.reprotox.2018.10.002 RTX 7745

To appear in:

Reproductive Toxicology

Received date: Revised date: Accepted date:

15-5-2018 28-9-2018 4-10-2018

Please cite this article as: Brayboy LM, Clark H, Knapik LO, Schnirman RE, Wessel GM, Nitrogen mustard exposure perturbs oocyte mitochondrial physiology and alters reproductive outcomes, Reproductive Toxicology (2018), https://doi.org/10.1016/j.reprotox.2018.10.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Nitrogen mustard exposure perturbs oocyte mitochondrial physiology and alters reproductive outcomes

SC RI PT

Lynae M. Brayboy123*, Haley Clark3*, Laura O. Knapik1, Ruby E. Schnirman4, Gary M. Wessel3

1 Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology and

Infertility, Women &Infants Hospital of Rhode Island, Alpert Medical School of Brown University, 101 Dudley Street, Providence, RI 02905 USA

U

2 Alpert Medical School of Brown University, Providence, RI 02903

N

3 Department of Molecular Biology, Cell Biology and Biochemistry, Brown University,

A

Providence, RI 02912, USA

D

Article Type: Reproductive Toxicology

M

4 University of Chicago, 5801 South Ellis Avenue, Chicago, IL 60637

TE

Keywords: nitrogen mustard, ovary, oocyte, chemotherapy, oxidative stress,

EP

mitochondria, mitophagy.

Corresponding Authors: Lynae Brayboy [[email protected]] and Gary Wessel

CC

[[email protected]]

A

Corresponding Author's Institutions: Women & Infants Hospital of Rhode Island and Brown University *denotes shared first authorship

Highlights 

Molecular and longitudinal effects of NM exposure are transmitted to F1 generation, but not to the F2 generation.



Primary nitrogen mustard exposure increases reactive oxygen species, but does not change



SC RI PT

ATP abundance.

Maternal nitrogen mustard exposure prior to pregnancy resulted in lowered birth weights in PND3 pups

Abstract:

Nitrogen mustard (NM) is an alkylating chemical warfare agent, and its derivatives are used in

U

chemotherapy. Alkylating agents can cause mitochondrial damage, so exposed females may transmit

N

damaged genomes to their children, since mitochondria are maternally inherited and oocytes are not

A

thought to undergo mitophagy [1]. The objective of this study is to investigate NM’s effects on oocyte

M

mitochondria to understand risks facing female soldiers, cancer patients, and their children. Mice were injected intraperitoneally with NM, monitored for reproductive outcomes, and ovaries and oocytes were

D

isolated for analysis. Escalating doses of NM increased oxidative stress in parental and F1 generation

TE

oocytes, suggesting that mitochondrial damage by NM is enhanced by mitochondrial superoxide. NMtreated ovaries in vitro exhibited smaller mitochondrial volume, more electron-dense and multivesicular

EP

structures, and lower birth weight litters. These results demonstrate that females must be protected from

CC

alkylating agents for their health, and the health of their offspring.

A

1. Introduction:

1.1 What is nitrogen mustard? Soldiers actively deployed in military sites around the world face countless occupational hazards. Aside from combat fatality, casualties of war can include physical injury, trauma, disease contraction, and chemical exposure [2]. Active military personnel serving in the military today risk exposure specifically to the gonadotoxic chemical warfare agent, nitrogen mustard. Women make up 15.5% of the active

service and are typically in their prime reproductive years during their active duty [3]. Nitrogen mustard, a gaseous alkylating agent, was developed during World War I as a chemical weapon to be inhaled and absorbed by the skin, and remains in possession by radical militant groups today [4]. In addition to its use as a biologic warfare agent, nitrogen mustard and some of its chemical derivatives are used as

SC RI PT

chemotherapeutic drugs, most notably cyclophosphamide, because of their ability to disrupt DNA

structure and synthesis in fast replicating cells [5]. Nitrogen mustard, for example, is used clinically to treat Hodgkin’s Lymphoma, cancer of the lymphatic system, and other types of solid tumors with a standard therapeutic dose of 0.1mg/kg body weight [6].

U

1.2 Depletion of Ovarian Reserve

N

Mammalian females are born with a finite number of follicles that constitute her ovarian reserve

A

[7]. These follicles are comprised of an oocyte surrounded by granulosa cells and cumulus cells, which

M

perform cellular signaling and biochemical processing for the oocyte. From birth through menopause, these follicles are gradually lost through either ovulation of mature oocytes, through atresia, and/or

D

through programmed death of follicles [8]. Similar to humans, mice ovulate oocytes once an estrus cycle,

TE

incrementally decreasing their ovarian reserves of follicles until an insufficient number of follicles remain, resulting in menopause [9]. Gonadotoxic agents, such as chemotherapeutic drugs, prematurely

EP

deplete primordial follicles in mice and humans, causing amenorrhea and early onset of menopause [10]. This depletion of the ovarian reserve is thought to be one possible mechanism for the gondatoxicity of

CC

these chemotherapeutic agents [10]. Cyclophosphamide, a chemotherapeutic drug derived from nitrogen mustard, has been shown to more rapidly decrease the ovarian reserve of those exposed, and sometimes

A

results in ovarian failure in childhood survivors of cancer [10]. Specifically, the active metabolite of cyclophosphamide, phosphoramide mustard, depletes the ovarian reserve of primordial follicles in humans and rats [11]. Other alkylating agents also have been shown to be correlated with early-onset menopause in women exposed as children [5]. Toxicants in general, such as cigarette smoke, also have been implicated in decreased oocyte quality and subfertility that is transmitted to the F1 and F2

generations [12]. Thus, mixed gonadotoxic agents deplete ovarian reserves and worsen oocyte quality, both of which directly impair fertility of the exposed individual and their offspring as well as the children of these offspring.

SC RI PT

1.3 How does nitrogen mustard affect mitochondria?

Nitrogen mustard is an alkylating agent, promoting the formation of DNA adducts by binding directly to DNA strands. For example, mechlorethamide, a member of the nitrogen mustard family,

results in interstrand DNA cross-linking by preferentially cross-linking guanine residues between strands [13]. In ovaries exposed to alkylating mustard gas, this cross-linking can eventually induce single-strand

U

breaks in DNA, and results in the activation of ovarian DNA repair mechanisms in the granulosa cells

N

[14]. These DNA repair mechanisms deplete ovarian NAD+, an electron carrier that drives the electron

A

transport chain’s role in oxidative phosphorylation in the mitochondria, which is correlated to increases in

M

oxidative stress in mammalian tissues [15, 16]. Nitrogen mustard derivatives result in mitochondrial dysfunction and destruction associated with increased oxidative stress and decreased mitochondrial

D

protein content [17]. Damaged and dysfunctional mitochondria are also the primary sources of increased

TE

levels of reactive oxygen species (ROS) and free radicals in most cell types in the body, indicating that increased levels of oxidative stress most likely originates from mitochondrial dysfunction [18].

EP

Cyclophosphamide in particular, a derivative of nitrogen mustard, has been associated with increased ROS production and oxidative stress in mouse oocytes [19]. Additionally, cyclophosphamide has been

CC

observed to affect mouse ovaries 48 hours post administration [20]. Nitrogen mustard therefore has the

A

potential both to damage mitochondria and increase oxidative stress upon exposure.

1.4 Mitochondria in the Mammalian Oocyte Mitochondria are inherited maternally; all mitochondrial DNA (mtDNA) is derived solely from the mitochondria present in the oocyte upon fertilization [21]. Mature oocytes contain hundreds of thousands of mitochondria, more than any other cell type in the body. Mitochondria from the fertilizing

sperm do not contribute to the mtDNA of the offspring, and instead, these organelles are degraded by the oocyte upon fertilization [22]. Curiously, the mammalian oocyte does not appear capable of mitophagy, the process of degradation of dysfunctional or damaged mitochondria, resulting in the transmission of damaged mitochondria to the resulting embryo [1]. Mitophagy is most commonly regulated by proteins

SC RI PT

PINK1 and Parkin [23]. PINK1 is transported into the mitochondria in healthy organelles. However, it accumulates and stabilizes the membrane of damaged mitochondria if the proton gradient across the membrane is damaged. Accumulation of PINK1 then acts as a sensor for damaged mitochondria,

recruiting Parkin to promote the degradation of the damaged organelle [22]. Parkin is recruited to the

mitochondrial membrane in HEK293 (human embryonic kidney cells) within 12 hours after 10μM CCCP

U

mitochondrial uncoupling, a positive control for the presence of mitochondrial damage [24]. The specific

N

mechanism of mitochondrial degradation stimulated by PINK1 and Parkin is known as micromitophagy,

A

or type 3 mitophagy, in which mitochondria-derived vesicles are released from the organelles in response

M

to oxidative stress, conglomerate into multivesicular bodies, which then fuse with lysosomes to complete the degradation of the damaged mitochondria [25]. On the basis of the maternal inheritance pattern of

D

mitochondria, we sought to understand the transgenerational effects of nitrogen mustard exposure in the

A

CC

EP

TE

mouse.

2. Materials and Methods: 2.1 Animals: An animal protocol was obtained from the Institutional Animal Care and Use Committee (IACUC) Protocol numbers 1407000080 and 1710000312. Wild type FVBN female mice at 7 weeks of age were

SC RI PT

obtained from Taconic Biosciences, Hudson, NY. A treatment group of females was injected

intraperitoneally with 0.1mg/kg nitrogen mustard (Mechlorethamine hydrochloride, Sigma Aldrich 55-867), while a control group was injected with sterile saline. The mice were bred with untreated FVBN males at 7 weeks, also obtained from Taconic, to F2 generation. They were euthanized via successive carbon

dioxide inhalation and cervical dislocation and the ovaries were removed. Control mice were compared to

N

U

NM treated mice for superoxide detection, ATP amounts, morphology, and mitophagy.

A

2.2 Nitrogen mustard treatment for molecular investigation:

M

Female mice were injected intraperitoneally with sterile saline, the therapeutic dose of 0.1 mg/kg NM, or non-therapeutic doses of 0.5mg/kg or 1.0mg/kg NM. At 48 hours following injection, the mice were

D

sacrificed, and the ovaries were removed. Germinal vesicle (GV) oocytes from mechanical extraction of

TE

whole ovaries were isolated for analysis.

EP

2.3 Reactive Oxygen Species Detection: GV oocytes were isolated from control mouse ovaries, and ovaries from mice treated with 0.1mg/kg,

CC

0.5mg/kg, or 1.0mg/kg NM. Oocytes were incubated in MitoSOX™ Red Mitochondrial Superoxide Indicator for 10 minutes. 14-27 oocytes were then imaged with an EVOS® FL Auto Cell Imaging System

A

at 40X magnification. ImageJ was used to quantify intensity of red fluorescence, and correct total cell fluorescence was calculated using the equation CTCF = integrated density - (area of cell x mean background fluorescence) [26].

2.4 Transmission Electron Microscopy:

Oocytes were fixed in 2% paraformaldehyde and 2.5% glutaraldehyde with 0.15M sodium cacodylate buffer and 0.1M sucrose at 4˚C. Ovaries were fixed in Dietrich’s solution at room temperature for 24 hours, followed by 10% neutral buffered formalin (Fisher) at 4˚C. The samples were washed in 0.15M sodium cacodylate buffer with 2nM calcium chloride. They were incubated for 20 minutes with filtered

SC RI PT

thiocarbohydrazide (TCH) solution, followed by a 30-minute incubation with 2% OsO4 in ddH2O, all at room temperature. After washing in ddH2O, samples were placed in 1% uranyl acetate over night at 4˚C. The following day, Walton’s lead aspartate pH adjusted to 5.5 was heated to 60˚C. After washing samples in ddH2O, they were incubated with the lead aspartate at 60% for 30 minutes. Following another wash, samples were dehydrated by graded ethanol in 70%, 90%, and 95% respectively for 20 minutes each,

U

followed by 100% ethanol 3 times for 10 minutes each. Lastly, the samples were infiltrated with 1 part

N

ethanol and 1 part embedding medium on a rotator overnight, followed by 100% embedding medium

M

A

overnight. Samples were rotated with fresh embedding medium for at least 2 hours and then cured.

2.5 3D Electron Microscopy:

D

Blocks of embedded samples were imaged using an Apreo™ VS in Volumescope mode (ThermoFisher

TE

Scientific, Hillsboro, OR) at 3.0 KV accelerating voltage, with water vapor at 40 Pa to mitigate charging, and using the dedicated VS DBS detector with a 100pA probe current and 3μs dwell time. A large

EP

overview region was collected with a 400μm HFW and 130μm pixels to use the debris checker to prevent lost slices due to sectioning debris. Two or three ROIs were used for each sample, all were collected with

CC

10nm pixel size and 40μm HFW. 300 serial sections were cut for each block at 40nm each for a total volume of 40μm x 40μm x 12μm.

A

Amira™ 3D Image analysis was performed using greatest mitochondrial diameter to measure circumference each 200 slices of 40 nanometers each, one sample per condition. Mitochondrial volume was calculated using measured circumference. The number of mitochondria, as well as the unidentified, electron dense objects, were quantitated.

2.6 ATP Luminescence: MII oocytes from superovulation and GV oocytes from mechanical extraction from whole ovaries were isolated, as well as whole ovary, kidney, heart, and spleen. 50 Oocytes and ovaries were added directly into 100uL PBS in a white, 96-well luminescence assay plate. Whole kidney, heart, and spleen were

SC RI PT

homogenized in 200uL of PBS, and 10uL of the homogenized tissue solution was added to 90uL PBS in the plate. ATP standards were prepared in PBS, for final concentrations of 10μM, 1μM, 100nM, 10nM, and 1nM. 100uL of CellTiter-Glo® Reagent was added to each well, and the plate was then mixed for 2min on an orbital shaker and incubated at room temperature for 10min. Luminescence was recorded

U

BioTek Syngergy HT at 37˚C.

N

2.7 Nitrogen mustard treatment and transgenerational investigation:

A

Female mice were injected intraperitoneally with sterile saline (6 mice) or 0.1 mg/kg NM (6 mice). At 48

M

hours following injection, females were placed in mating pair cages with an untreated male mouse, along with enrichment huts and DietGel® Prenatal vitamins (ClearH2O Westbrook, ME). Females were

D

palpated for pregnancy after two weeks and monitored daily. Day of delivery and number of F1

TE

generation pups delivered were recorded. PND3 pups were weighed, and length was measured in cm. Litters were observed daily until day 10, when they were observed every other day until weaning age (25-

EP

28 days). Weaned pups were separated from their mother and then segregated by sex until sexual maturity (6 weeks). Then, they were either mated with non-siblings control group males and monitored for

CC

pregnancy and birth of F2 generation or sacrificed for molecular studies. The original sires were

A

reintroduced to F0 female cages upon weaning of litters in order to do successive matings.

2.8 Data Analysis: Differences between measurements were analyzed by two-sample unpaired t-tests, with significance cutoffs of P<0.05 or P<0.001.

3. Theory/Calculations This study aims to determine a phenotype of NM treated mouse oocytes, as well as to test for any transgenerational effects of exposure. We predict that ovarian and oocyte mitochondria will be compromised following exposure, and for these metabolic effects to be passed on to future generations

SC RI PT

via damaged oocyte mitochondria.

4. Results

4.1 Primary nitrogen mustard exposure increases ROS, but does not change ATP abundance

Mice were administered NM intraperitoneally and 48 hours after the injection, germinal vesicle (GV)

U

immature oocytes were mechanically separated from the ovary. The oocytes were then incubated with

N

MitoSox  and fluorescence was quantified. Nitrogen mustard treated oocytes had more total cell

A

fluorescence compared to controls, indicating the presence of more superoxide (Figure 1). However,

M

when we evaluated ATP amounts in those same oocytes, no difference was detectable between treated

D

oocytes and controls.

TE

4.2 Electron microscopy of nitrogen mustard treated oocytes show markers of mitophagy Ovaries of mice treated with saline and nitrogen mustard were fixed for electron microscopy. Nitrogen

EP

mustard-treated oocytes examined with both Transmission and 3D Electron microscopy showed

CC

mitochondria that were smaller and matted morphologically compared to controls (Figure 2a). The volume of the mitochondria was also lower in the nitrogen mustard treated oocytes (Figure 2b). We

A

observed that the treated oocytes had increased electron dense structures compared to untreated oocytes (Figure 2c). Some of these structures appear to be morphologically mitochondria (blue arrows). Others seem to be structurally similiar to multivesicular bodies (yellow asterisk) (Figure 2d), consistent with lysosomes charged with Type III mitochondrial autophagy (micromitophagy) known to occur in mammals [25].

4.3 Pups of nitrogen mustard treated female mice have lower birth weights Non-pregnant mice were injected with nitrogen mustard or saline and 48 hours after treatment were mated to untreated wild type males. The resulting F1 pups of mothers treated with nitrogen mustard before

SC RI PT

pregnancy had lower birth weights when compared to controls injected with saline (Figure 3a). The F2

grandmaternal exposure to nitrogen mustard pups did not display any differences compared to controls.

The non-pregnant females treated with saline and nitrogen mustard had similar numbers of pups in each successive litter (Figure 3b).

U

4.4 Maternal but not grandmaternal exposure to nitrogen mustard leads to increased ROS in oocytes

N

After breeding the parental generation of mice exposed to nitrogen mustard and saline the F1 females’

A

oocytes were observed to have increased superoxide, but not significantly in the F2 generation (Figure 4a

A

CC

EP

TE

D

M

and 4b).

5. Discussion 5.1 Nitrogen mustard affects mitochondrial physiology in mouse oocytes Nitrogen mustard caused changes in the oocytes of exposed animals. Likely the drug is affecting directly the oocytes, since its turnover is rapid (a reported half-life of 30-90 minutes) and is not present at the time

SC RI PT

of embryogenesis. Therefore, the effects that we observed likely occurred in the oocyte and associated granulosa cells. We witnessed that increasing doses of the chemical corresponds to increases in

mitochondrial superoxide, indicating that NM may be involved in mitochondrial damage or dysfunction. Mitochondrial superoxide ROS are produced as a byproduct of aerobic respiration when electron

transport chain function is dysregulated [27]. This observed increase in oxidative stress is therefore most

U

likely due to a disruption in aerobic respiration, a possible target of NM toxicity in the oocyte. However,

N

we did not detect any differences in steady state ATP levels upon NM exposure in various mouse tissues.

A

This result could reflect that the damaged mitochondria may have compromised bioenergetics of

M

oxidative phosphorylation and instead rely more on glycolysis to produce sufficient ATP. Increases in oxidative stress are indicative of damaged mitochondria that are transmittable to offspring of exposed

TE

D

mothers [21].

5.2 A mitophagy phenotype in NM-Exposed oocytes

EP

NM-exposed oocytes affected mitochondrial morphology in addition to levels of ROS production. Not only do exposed oocytes contain mitochondria that are smaller by volume when compared to

CC

controls, but they have more electron dense, unidentified structures. These structures are not consistent with the morphology of other oocyte organelles, and could potentially be damaged mitochondria, taking

A

up more of the electron dense stains used in this preparation for EM images [28]. NM-exposed oocytes also appeared to initiate mitophagy. The globular structures demonstrated in Figure 2d resemble multivesicular bodies and autolysosomes that have been shown to engage in damaged mitochondria degradation and destruction [29]. Identification of multivesicular bodies in oocytes is indicative of an override of this mechanism, indicating that NM exposure and the resulting mitochondrial damage may

cross a threshold required for the initiation of mitophagy in oocytes. Furthermore, decreased mitochondrial content as a result of successful mitophagy may effect fertility of exposed females, as critical oocyte mtDNA copy numbers are required [30]. Experiments are currently underway to detect the presence of other distinct markers of mitophagy such as PINK1 and Parkin, as well as co-localization

SC RI PT

with established mitochondrial proteins, in NM-exposed oocytes, to test further for the presence of mitophagy initiation.

5.3 Reproductive Outcomes

In breeding studies, injection of an F0 female mouse affected the birth weight of pups conceived post-

U

exposure. Mating pairs were assembled 48 hours post NM injection, to allow for the manifestation of the

N

metabolic and morphologic changes previously observed [20]. F1 pups of NM exposed mothers weighed

A

less than pups of control mothers. In humans, implications of low birth weight include increased incidents

M

of respiratory diseases, susceptibility to infections, and intraventricular hemorrhage [31]. NM therefore has the possibility of influencing the morbidity of children born to exposed mothers. NM did not exhibit

D

any other changes in reproductive outcomes in this study. All F1 pups were weaned at 28 days and

TE

successfully mated when placed in mating pairs at 6 weeks of age, indicating no difference in time to weaning age or sexual maturity between pups of control and NM-exposed mothers. Additionally, there

EP

was no difference between litter size of NM-exposed mother and control mothers after three successive matings. The given dose of NM did not deplete ovarian reserve significantly enough to affect the number

CC

of pups conceived in a third consecutive litter. Future successive mating to more than 3 consecutive litters, as well as follicle counting in isolated ovaries, will clarify if NM has any effect on mouse ovarian

A

reserve. The only affected reproductive outcome in NM-exposed F1 litters was a decreased birth weight, but was not replicated in the F2 generation. Pups of control and NM group F1 mice demonstrated no significant difference in reproductive outcomes, limiting the observed effects of 0.1mg/kg NM exposure to one generation in mice.

Increased levels of oxidative stress followed a similar trend to that of NM-associated reproductive outcomes. Oocytes of NM group F1 mice exhibited increased production of mitochondrial superoxide similar to that observed in F0 mice that were directly injected intraperitoneally. The continuation of this trend from F0 to F1 generation illustrated a transmission of mitochondrial defects from NM-exposed

SC RI PT

mothers to their offspring, consistent with the known maternal mode of mitochondrial inheritance.

Increased oxidative stress is not, however, observed in F2 mouse oocytes, limiting the observed molecular transmission to one generation. It is possible that mammalian DNA repair mechanisms, specifically base excision repair, could be responsible for repairing mtDNA damage that could result from increases in

ROS production [32]. Repair mechanisms could correct damaged mitochondria before transmission to F2

U

generation, resulting in the lack of any detectable phenotype. Alternatively, if mitophagy is occurring in

N

exposed oocytes, damaged mitochondria may be eliminated before a phenotype can by presented in the

A

F2 generation [25].

M

NM has been established to have cellular-levels effects on oocytes of exposed female mice that are able to be transmitted to offspring. While no other reproductive outcomes were altered by the short

D

NM exposure in this study, the transmission of oxidative stress from parental to F1 generation has

TE

implications for human female veterans and children they may have following chemical exposure. NMinduced mitochondrial damage and oxidative stress have the potential to influence metabolic and

EP

mitochondria-associated cellular pathways, and ultimately lead to mitophagy and programmed cell death of various tissue types, in veterans and their children [33]. Additionally, non-pregnant women in the

CC

military who are actively deployed in areas of the world where NM is used may unknowingly be endangering the health of their future children. Effects of NM were observed in exposed mice 48 hours

A

following exposure, a period of time well exceeding the half-life of the drug. The offspring of these mice also exhibited oxidative stress, indicating that the effects of NM exposure last well past the time of drug clearance and can still be transmitted to offspring. Exposure to NM in warfare then has the potential to affect future children of pre-pregnant female soldiers.

Further experiments must be performed to clarify that mitochondrial damage specifically plays a role in the toxicity of NM, and that the phenotype observed in treated mice is not a result of other mechanistic pathways. Distinct markers of mitochondrial stress have been observed in response to NM exposure, so now we seek to further investigate NM-induced metabolic compromise or status of

SC RI PT

mitochondria in exposed individuals and their offspring, in order to determine their long-term effects.

Acknowledgments:

We want to thank Nathalie Oulhen for manuscript feedback. We also appreciate Sokunvichet Long’s

technical support. Thank you to Stephen Helfand and Jason Wood for use of their luminometer and plates.

U

The authors would like to acknowledge Paula Weston for performing transmission electron microscopy,

N

Geoff Williams for performing 3D electron microscopy on ovary samples and Melinda Golde for

A

embedding fixed tissue samples. The authors would also like to thank Rebecca Hamelin and Danielle

M

Papa for their veterinary technician support. This work was funded by NIH 4K12HD00084929 awarded to the Reproductive Scientist Development Program by the Eunice Kennedy Shriver National Institute of

D

Child Health & Human Development and by the American Board of Obstetrics & Gynecology and

TE

National Institute of General Medical Sciences P20 GM121298-01 (LMB) and NIH- 2RO1HD028152

A

CC

EP

(GMW).

Figure 1. Brightfield

MitoSOX Red

B)

SC RI PT

0.1mg/kg NM

Control

A)

Control

*

K

id n

ve r

ea rt

le en

H

Sp

U

G

V

O

O

va ry

0

Li

500

ey

NM

1000

oc yt es

1.6mg/kg NM

C)

ATP Concentration (nM)

0.4mg/kg NM

ATP Amount 1500

N

Cell/Tissue Type

* P<0.05, ** P<0.001

A

Figure 1: Nitrogen mustard increases oxidative stress in oocytes, but does not alter ATP

M

production

A. Images of oocytes isolated from mice 48 hours post injection. Scale bar: 100 m B. Quantification of

D

fluorescence of oocytes treated with MitoSOX™. Oocytes from mice (n=25) of each increasing nitrogen

TE

mustard dose group exhibited significantly increased oxidative stress when compared to controls, as well as when compared to lower doses. C. Concentration of ATP produced in mice oocytes (n=20) and various

EP

tissue types of mice injected with sterile saline (control group) or 0.1mg/kg nitrogen mustard. Nitrogen

CC

mustard exposure does not exhibit a consistent trend amongst tissue and cell types. Exposure significantly

A

decreases ATP production in spleen, but does not significantly affect production of ATP in oocytes.

Figure 2. A)

Mitochondrial Volume

B)

**

2.0×106 1.5×106 1.0×106 5.0×105

C

on

N

M

tr ol

0.0

SC RI PT

Volume (nm3)

2.5×106

Treatment Group

50

Number per area

C)

Oocyte Mitochondria and Unidentified Objects Number

40 30 20 10

Mitochondria

Unidentified Objects

0. 1

m

C

g/

on

kg N M

tr ol

0

*

* P<0.05, ** P<0.001

A

N

U

Treatment Group

M

Figure 2: 3D Electron Microscopy reconstruction of control and NM-treated ovaries, and quantification of mitochondrial volume, count, and number of unidentified objects.

D

A. Mitochondria in control ovaries appear larger and darker than those in NM-treated tissue. B. and C.

TE

NM treated ovaries have a significantly smaller volume than mitochondria of control ovaries, and contain more unidentified, fairly electron dense objects (UFO) than controls. Volume of ovaries: Control image:

EP

30 x 20 x 12 um NM image: 40 x 40 x 12 um.

CC

D. Electron Microscopy of Control and Treated Oocyte Mitochondria, and Possible Evidence of Mitophagy. Columns A. and B. depict oocyte mitochondria in isolated ovaries of mice injected with

A

sterile saline (Figure 2D Column A) or 0.1mg/kg nitrogen mustard (Figure 2D Column B). Mitochondria observed in control oocytes exhibit typical, light structures with visible cristae (red arrows). Treated oocyte mitochondria are visibly more sparse, dark in color, oblong in shape, and lacking in cristae (blue arrows). They are also surrounded by more cellular debris when compared to controls. Columns C. and D. show globular structures exhibited only in the Nitrogen mustard treated group. The unidentified structures

potentially resemble multivesicular bodies (yellow asterisk), structures that form in the process of PINK1 and Parkin mediated mitophagy. Scale bar: 200 nm-600nm as listed Magnifications ( Direct

SC RI PT

Magnification D1: 21000x, D2: 38000x, D3: 21000x, D4: 21000x, D5: 38000x, D6: 16900x)

Figure 3.

* P<0.05

TE

D

M

A

N

U

A)

Figure 3: A. Reproductive outcomes of the F1 generation.

EP

n=6 litters for both control and NM groups. Pups of nitrogen mustard treated mice weigh significantly

CC

less than pups of control mice. No other reproductive outcomes measured were affected significantly. The p-values were calculated by unpaired t-test. Additionally, all F1 pups that were later placed in mating

A

pairs with untreated males successfully mated and produced litters, indicating that pups of both control and NM treated mothers reached sexual maturity at the expected 6 weeks. Reproductive outcomes of successive matings to F1 generation. n=3 litters for both control and NM groups. There is no difference in the average number of pups per litter after 3 successive matings in control and NM treated mice.

Reproductive outcomes of the F2 generation. n=5 control litters, and n=6 NM litters. No significant differences were seen in reproductive outcomes between control and NM-exposed lineages by the F2 generation.

of pups produced with each litter.

Figure 4. MitoSOX Red

Brightfield F1 Control

B)

** P<0.001

TE

D

F2 NM

M

A

F2 Control

N

U

F1 NM

A)

SC RI PT

B. After three successive matings of the parental F0 generation no differences were found in the number

EP

Figure 4: MitoSOX™ Fluorescence in Oocytes of F1 and F2 Generation Pups. A. Images of germinal

CC

vesicle (GV) oocytes isolated from mice 48 hours post injection treated with MitoSOX™ post B. Quantification of fluorescence of oocytes treated with MitoSOX™. F1 oocytes of pups from NM

A

treated mothers exhibited a significant increase in oxidative stress when compared to F1 controls. F2 pup oocytes did not differ between control and NM groups. The NM-associated increase in oxidative stress observed in F0 female mice seems to be transmitted to immediate F1 offspring, but the effect is no longer seen by F2 generation.

References:

8. 9.

10. 11.

SC RI PT

U

CC

12.

N

7.

A

5. 6.

M

4.

D

3.

TE

2.

Boudoures, A.L., et al., Obesity-exposed oocytes accumulate and transmit damaged mitochondria due to an inability to activate mitophagy. Dev Biol, 2017. 426(1): p. 126138. Gawande, A., Casualties of war--military care for the wounded from Iraq and Afghanistan. N Engl J Med, 2004. 351(24): p. 2471-5. Moore, B.L., Introduction to Armed Forces & Society: Special Issue on Women in the Military. Armed Forces & Society, 2017. 43(2): p. 191-201. Hoffman, Y., Who is afraid of ISIS? ISIS anxiety and its correlates. Stress Health, 2018. 34(1): p. 84-92. Rebar, R.W., Premature ovarian failure. Obstet Gynecol, 2009. 113(6): p. 1355-63. Goodman, L.S., M.M. Wintrobe, and et al., Nitrogen mustard therapy; use of methyl-bis (beta-chloroethyl) amine hydrochloride and tris (beta-chloroethyl) amine hydrochloride for Hodgkin's disease, lymphosarcoma, leukemia and certain allied and miscellaneous disorders. J Am Med Assoc, 1946. 132: p. 126-32. Markstrom, E., et al., Survival factors regulating ovarian apoptosis -- dependence on follicle differentiation. Reproduction, 2002. 123(1): p. 23-30. te Velde, E.R. and P.L. Pearson, The variability of female reproductive ageing. Hum Reprod Update, 2002. 8(2): p. 141-54. Smith, E.R. and X.X. Xu, Ovarian ageing, follicle depletion, and cancer: a hypothesis for the aetiology of epithelial ovarian cancer involving follicle depletion. Lancet Oncol, 2008. 9(11): p. 1108-11. Larsen, E.C., et al., Diminished ovarian reserve in female childhood cancer survivors with regular menstrual cycles and basal FSH <10 IU/l. Hum Reprod, 2003. 18(2): p. 417-22. Madden, J.A., P.Q. Thomas, and A.F. Keating, Phosphoramide mustard induces autophagy markers and mTOR inhibition prevents follicle loss due to phosphoramide mustard exposure. Reprod Toxicol, 2017. 67: p. 65-78. Camlin, N.J., et al., Grandmaternal smoke exposure reduces female fertility in a murine model, with great-grandmaternal smoke exposure unlikely to have an effect. Hum Reprod, 2017. 32(6): p. 1270-1281. Hopkins, P.B., et al., Sequence Preferences of DNA Interstrand Cross-Linking Agents Importance of Minimal DNA Structural Reorganization in the Cross-Linking Reactions of Mechlorethamine, Cisplatin, and Mitomycin-C. Tetrahedron, 1991. 47(14-15): p. 24752489. Ganesan, S. and A.F. Keating, Phosphoramide mustard exposure induces DNA adduct formation and the DNA damage repair response in rat ovarian granulosa cells. Toxicol Appl Pharmacol, 2015. 282(3): p. 252-8. Pearson, G.S., Veterans at Risk: The Health Effects of Mustard Gas and Lewisite, edited by Constance M. Pechura and David P. Rall. Nature, 1993. 365(6443): p. 218. Chandra, J., A. Samali, and S. Orrenius, Triggering and modulation of apoptosis by oxidative stress. Free Radic Biol Med, 2000. 29(3-4): p. 323-33.

EP

1.

A

13.

14.

15. 16.

25. 26. 27. 28. 29. 30. 31.

CC

32.

SC RI PT

24.

U

23.

N

22.

A

21.

M

20.

D

19.

TE

18.

Sarkadi, B., et al., Human multidrug resistance ABCB and ABCG transporters: participation in a chemoimmunity defense system. Physiol Rev, 2006. 86(4): p. 1179-236. Brookes, P.S., et al., Calcium, ATP, and ROS: a mitochondrial love-hate triangle. Am J Physiol Cell Physiol, 2004. 287(4): p. C817-33. Jeelani, R., et al., Cyclophosphamide and acrolein induced oxidative stress leading to deterioration of metaphase II mouse oocyte quality. Free Radic Biol Med, 2017. 110: p. 11-18. Brayboy, L.M., et al., Multidrug resistance transporter-1 and breast cancer resistance protein protect against ovarian toxicity, and are essential in ovarian physiology. Reprod Toxicol, 2017. 69: p. 121-131. Cummins, J.M., The role of maternal mitochondria during oogenesis, fertilization and embryogenesis. Reprod Biomed Online, 2002. 4(2): p. 176-82. Ashrafi, G. and T.L. Schwarz, The pathways of mitophagy for quality control and clearance of mitochondria. Cell Death Differ, 2013. 20(1): p. 31-42. Vives-Bauza, C., et al., PINK1-dependent recruitment of Parkin to mitochondria in mitophagy. Proc Natl Acad Sci U S A, 2010. 107(1): p. 378-83. Narendra, D., et al., Parkin is recruited selectively to impaired mitochondria and promotes their autophagy. J Cell Biol, 2008. 183(5): p. 795-803. Lemasters, J.J., Variants of mitochondrial autophagy: Types 1 and 2 mitophagy and micromitophagy (Type 3). Redox Biol, 2014. 2: p. 749-54. McCloy, R.A., et al., Partial inhibition of Cdk1 in G 2 phase overrides the SAC and decouples mitotic events. Cell Cycle, 2014. 13(9): p. 1400-12. Brand, M.D., et al., Mitochondrial superoxide: production, biological effects, and activation of uncoupling proteins. Free Radic Biol Med, 2004. 37(6): p. 755-67. Li, N., et al., Ultrafine particulate pollutants induce oxidative stress and mitochondrial damage. Environ Health Perspect, 2003. 111(4): p. 455-60. Coticchio, G., et al., Ultrastructure of human oocytes after in vitro maturation. Mol Hum Reprod, 2016. 22(2): p. 110-8. Wai, T., et al., The role of mitochondrial DNA copy number in mammalian fertility. Biol Reprod, 2010. 83(1): p. 52-62. McCormick, M.C., The contribution of low birth weight to infant mortality and childhood morbidity. N Engl J Med, 1985. 312(2): p. 82-90. Bohr, V.A., Repair of oxidative DNA damage in nuclear and mitochondrial DNA, and some changes with aging in mammalian cells. Free Radic Biol Med, 2002. 32(9): p. 80412. Kowaltowski, A.J. and A.E. Vercesi, Mitochondrial damage induced by conditions of oxidative stress. Free Radic Biol Med, 1999. 26(3-4): p. 463-71.

EP

17.

A

33.