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Maternal Smoke Exposure Impairs the Long-Term Fertility of Female Offspring in a Murine Model1

ABSTRACT The theory of fetal origins of adult disease was first proposed in 1989, and in the decades since, a wide range of other diseases from obesity to asthma have been found to originate in early development. Because mammalian oocyte development begins in fetal life it has been suggested that environmental and lifestyle factors of the mother could directly impact the fertility of subsequent generations. Cigarette smoke is a known ovotoxicant in active smokers, yet disturbingly 13% of Australian and 12% of US women continue to smoke throughout pregnancy. The focus of our investigation was to characterize the adverse effects of smoking on ovary and oocyte quality in female offspring exposed in utero. Pregnant mice were nasally exposed to cigarette smoke for 12 wk throughout pregnancy/lactation, and ovary and oocyte quality of the F1 (maternal smoke exposed) generation was examined. Neonatal ovaries displayed abnormal somatic cell proliferation and increased apoptosis, leading to a reduction in follicle numbers. Further investigation found that altered somatic cell proliferation and reduced follicle number continued into adulthood; however, apoptosis did not. This reduction in follicles resulted in decreased oocyte numbers, with these oocytes found to have elevated levels of oxidative stress, altered metaphase II spindle, and reduced sperm-egg interaction. These ovarian and oocyte changes ultimately lead to subfertility, with maternal smoke-exposed animals having smaller litters and also taking longer to conceive. In conclusion, our results demonstrate that in utero and lactational exposure to cigarette smoke can have long-lasting effects on the fertility of the next generation of females.


INTRODUCTION
In utero exposure to cigarette smoke has been linked to adverse health effects in offspring, including an increased risk of premature birth, fetal growth retardation, and morbidity, and in later life obesity, asthma and congenital heart defects [1][2][3][4]. This is of particular concern, because despite increased public awareness of the adverse effects of smoking during pregnancy, 11%-14% of women at reproductive age (20-45 y) in Australia smoke daily [5]. Furthermore ;13% of Australian women and ;12% of women in the United States continue to smoke throughout their pregnancy [6,7]. Furthermore, a growing body of evidence suggests that gestational exposure to cigarette smoke negatively affects the fertility of female offspring, leading to early-onset menarche, reduced fecundability, and premature menopause [8][9][10][11][12][13]. A number of studies examining human ovaries have explored the potential mechanism underlying such altered fertility during early ovarian development (5-21 wk gestation); however, longer-term studies in humans have been limited [14][15][16].
Mammalian females are born with a finite number of germ cells that begin their development during fetal life [17]. At birth, these germ cells, known as ''oocytes,'' are meiotically arrested at prophase I within primordial follicles, with their number dictating the reproductive lifespan of an individual [18,19]. These quiescent primordial follicles consist of an immature oocyte surrounded by a single layer of granulosa cells. As folliculogenesis continues the number of granulosa cell layers increases, with these somatic cells providing important factors, such as inhibin, to the developing oocyte [20]. Once fully matured, a surge of luteinizing hormone causes ovulation and releases the oocyte from its prophase I arrest [21]. It is important to note, however, that most follicles are destined to die, with only a select few reaching ovulation, making them a precious resource.
In vivo and in vitro exposure of human and mouse fetal ovaries to cigarette smoke or its constituents nicotine, DMBA-DHD, DMBA, or BaP has been found to negatively affect oocyte and follicle development by altering proliferation and apoptosis of germ and granulosa cells [14,15,19,[22][23][24], and modifying hormone production of estrogen, progesterone, and inhibin [14,25]. In this study we used an animal model to examine the effects of in utero/lactational exposure to whole cigarette smoke on female fertility using our novel nose-only inhalation model [26][27][28][29][30][31]. Exposure of mice to cigarette smoke via this nose-only inhalation model for 8 wk leads to smokeinduced chronic obstructive pulmonary disease, which is evident in humans after years of cigarette smoking [26]. Our investigation focuses on both the short-term and long-term consequences of maternal cigarette exposure (MSE) on an offspring's ovarian and oocyte quality, and ultimately their fertility.

MATERIALS AND METHODS
All reagents were obtained from Sigma-Aldrich (St. Louis, MO) unless otherwise specified.

Animals and Ethics Approval
All experiments were performed with the approval of the University of Newcastle Animal Care and Ethics Committee. C57BL/6 mice were obtained from Australian BioResources and housed with ad libitum food and water under a 12L:12D lighting regimen.

Smoke Exposure
Six-week-old female mice were exposed to smoke as previously described [26,29,31]. Briefly animals were nasally exposed to mainstream cigarette smoke (twelve 3R4F reference cigarettes; University of Kentucky) for 75 min twice daily, 5 days a week for 12-13 wk, in individual mouse chambers, preventing passive smoking of breeding males and pups. On Week 5 females were housed with non-cigarette smoke-exposed males (two females per male) until visibly pregnant (abdominal bulge observable from approximately Gestational Day 14). Smoke exposure continued throughout pregnancy and lactation, ceasing once pups (F1 generation) were weaned on Postnatal Days 21-23 (PNDs 21-23) [28].

Fertility Trial
Four F1 females (age 6 wk) per treatment group were housed with noncigarette smoke-exposed males (age 8 wk) continuously. Date of birth and size of litters were recorded during a 3-mo period. Conception date was determined by subtracting 19 days from time of litter drop.

Follicle Counts and Staging
Three Bouin-fixed PND 6, 8-to 9-wk-old, and 9-mo-old ovaries per group were paraffin embedded and sectioned at 5 lm prior to hematoxylin and eosin staining. The PND 6 follicle counts were performed on every fifth section for half the ovary. The 8-to 9-wk-old and the 9-mo-old follicle counts were performed on every 15th section for the entire ovary. Only follicles with a visible oocyte nucleus were counted. The PND 6 follicles were staged through oocyte size. Oocyte diameters were measured using ImageJ software (freeware; National Institutes of Health); those with a diameter of ,15 lm were classed as primordial, 15-20 lm as activated follicles, and .20 lm as primary follicles. Morphological features were used to stage follicles in adult ovaries. Briefly, primordial follicles have a single layer of squamous pregranulosa cells surrounding the oocyte. Primary follicles are characterized by a single layer of cuboidal granulosa cells. By secondary stage, a zona pellucida (ZP) has formed around the growing oocyte, which is surrounded by multiple layers of granulosa cells with a theca externa. Finally, a follicle fluid-filled antral cavity forms in antral follicles [19].

TUNEL Analysis
Sections were treated with 20 lg/ml proteinase K (Promega) prior to TUNEL analysis using ApopTag Fluorescein In Situ Apoptosis Detection Kit (S7110; Merick Millipore) according to the manufacturer's protocol. DAPI was used to counterstain sections before mounting in citifluor (Citifluor) and imaging on an Axio Imager A1 epifluorescent microscope with an Olympus DP70 microscope camera. Total cells and TUNEL-positive cells were counted using ImageJ and used to calculate the proportion of TUNEL-positive cells.

Metaphase II Egg Collection and Fixation
Female mice were intraperitoneally injected with 7.5 IU of pregnant mares' gonadotropin (Intervet), followed by 5 IU of human chorionic gonadotropin (Intervet) 48 h later. Metaphase II arrested (MII) eggs were collected 12 h later from the ampulla into M2 media with BSA containing hyaluronidase (H4272) to remove cumulus cells. Oocytes were washed into M2 containing monastrol (200 lM; M8515) for 2 h to collapse the spindle for aneuploidy analysis or were fixed with 4% paraformaldehyde in PBS with 0.5% Triton X-100 for 30 min [33]. Following monastrol treatment, oocytes were fixed as described above.

Germinal Vesicle Oocyte Collection and In Vitro Maturation
Ovaries were dissected from 9-mo-old females, with one ovary per animal used for germinal vesicle (GV) oocyte collection. Briefly, preantral follicles were punctured with a 33.5-gauge needle, releasing cumulus-oocyte complexes into M2 with BSA and milrinone (M4659). Oocytes were denuded of cumulus cells, washed into MEM media (11900-024; Life Technologies), and allowed to undergo in vitro maturation in 5% CO 2 at 378C. After 16 h oocytes were scored for the presences of a polar body (MII oocytes), with MII oocytes treated with monastrol and fixed as described above.

Oocyte Immunocytochemistry
Immunocytochemistry was performed on oocytes from three to four animals per treatment using anti-a-tubulin (1:400; A11126; Life Technologies) or anti-CREST (1:400; 90C-CS1058; Bioclone Australia) overnight at 48C after blocking in 7% goat serum/PBS-0.1% Tween-20. Oocytes were washed through 1% BSA in PBS-0.1% Tween-20 prior to incubation with secondary antibodies conjugated with Alexa 555 or Alexa 633 (1:1000; Life Technologies). Oocytes were counterstained in Hoechst (20 lg/ml) and mounted in citifluor. Imaging was performed on an Olympus FV1000 using a 603/1.2 N.A. UPLSAPO oil immersion objective lens (Olympus). ImageJ was used to measure spindle size and count kinetochores using a macro designed by Dr. Simon I.R. Lane [34]. For ZP analyses, four measurements were taken from the transmission image of each egg to calculate mean thickness (Supplemental Fig. S1; Supplemental Data are available online at www.biolreprod.org), as previously described by Jennings et al. [35].

Sperm-Egg Binding Assays
Freshly collected MII eggs were examined for sperm-zona and spermoolemma binding as previously described by Sobinoff et al. [27] on three CAMLIN ET AL. animals per treatment. Briefly, MII oocytes were collected in M2 media with hyaluronidase to remove cumulus cells. For sperm-oolemma binding assays, the zona was removed from eggs using Acid Tyrode solution (T1788). Sperm was collected from the cauda epididymides of a mature untreated male mice and morphology analyzed. Morphologically normal samples were allowed to capacitate for 3 h at 378C. Eggs with or without their ZP were incubated with capacitated sperm at 2 3 10 5 sperm per milliliter in M2 medium for 15 min before being washed into sperm-free media. Phase microscopy was used to count sperm heads bound to the zona or oolemma.

Statistics
Statistical analysis was performed using GraphPad Prism 6.0 software (GraphPad Software Inc.). For categorical data, Fisher exact test was used. All other data were tested from normal distribution using D'Agnostino-Pearson omnibus normality test. For data found to follow a normal distribution, Student t-test or ANOVA with Tukey post hoc was used. For all other data, Mann-Whitney test or Kruskal-Wallis test with Dunn post hoc statistical test was performed. A P value ,0.05 was considered statistically significant. Further information on statistics tests used for each data set can be found within the figure legends.

Maternal Smoke Exposure Alters Neonatal Follicle Population
Initially we investigated the neonatal ovarian consequences for female mice whose ovaries had been exposed to the products of inhaled cigarette smoke in utero (maternally smoke exposed, herein referred to as ''MSE mice'').

MSE Neonatal Ovaries Display Abnormal Levels of Cellular Apoptosis and Proliferation
To elucidate the mechanisms behind the altered follicle population observed at PND 6, examination of neonatal ovaries for markers of apoptosis revealed that DNA fragmentation as examined by TUNEL assay was significantly elevated in MSE ovaries at PNDs 3 and 6 (positive cells, respectively: PND 3, 12.7% 6 8.6 vs. 1.0% 6 0.2%, P ¼ 0.0090; and PND 6, 7.4% 6 3.5% vs. 1.3% 6 1.7%, P ¼ 0.0013; Fig. 2A). To confirm this, a second marker of apoptosis, active caspase 3, was examined and was also found to be elevated in both the oocytes and granulosa cells of MSE females at PNDs 3 and 6 (;8-fold and ;52-fold, P ¼ 0.0088 and P ¼ 0.0007, respectively; Fig. 2B).
To determine whether proliferation of ovarian somatic cells, including granulosa cells, was affected by MSE, we analyzed PCNA expression, which has previously been correlated with the initiation of follicle growth [36]. Immunosignal, found primarily in ovarian somatic cells, was decreased in MSE ovaries at PND 3 (;0.5-fold, P ¼ 0.0102; Fig. 2C); however, by PND 6, the signal was significantly increased relative to controls (;1.5-fold, P ¼ 0.0194; Fig. 2C).

MSE Adult Ovaries Display Abnormal Levels of Cellular Proliferation but No Apoptosis
To determine if changes in ovarian somatic cell proliferation continued into adulthood, 8-to 9-wk-old and 9-mo-old ovaries were examined for PCNA expression that localized primarily to granulosa cells in secondary and antral follicles. At 8-9 wk, Downloaded from www.biolreprod.org.
MSE ovaries again had greatly decreased PCNA levels, presumably due to the lower numbers of proliferating follicles present, because PCNA at this age is most often associated with replicating granulosa cells of large preantral and antral follicles (;10-fold, P , 0.0001; Fig. 4A). Interestingly, however, by 9 mo MSE displayed an increase in PCNA fluorescence compared with controls (;2-fold, P ¼ 0.0011; Fig. 4A). To investigate the possible cause of this elevated PCNA proliferation at 9 mo, ovaries were examined for AMH because it has been shown to reduce follicle proliferation [37]. Immunosignal of AMH that localized to granulosa cells in secondary and early antral follicles was significantly reduced in MSE ovaries (;2-fold, P ¼ 0.0013; Fig. 4B). Further examination of ovaries for markers of apoptosis found no change in TUNEL-positive cells at 8-9 wk or 9 mo (Fig. 4C).

Reduced Oocyte Number and Quality in MSE Animals
Having established a reduction in preantral follicle number, as well as altered somatic cell proliferation and survival, in the ovaries of MSE females, we next assessed the number and quality of mature eggs isolated from these mice after hormonal stimulation. As expected, MSE females ovulated fewer MII eggs compared with controls at 8 wk (21 6 2.8 vs. 28 6 2.4, P ¼ 0.0001; Fig. 5A). Furthermore, at 9 mo a reduced number of mature GV oocytes were collected from MSE ovaries (8.0 6 0.8 vs. 13.2 6 2.6, P ¼ 0.0088; Fig. 5B). To determine the oxidative stress status of 8-wk-old eggs, we examined lipid peroxidation and mitochondrial superoxide leakage using the cell-permeable probes BODIPY and MitoSOX red, respectively. Superoxide leakage was increased approximately ;2-fold in MSE eggs (P ¼ 0.0113; Fig. 5C), as was lipid peroxidation, which was 1.5-fold higher (P , 0.0001; Fig. 5D). Oxidative stress has been hypothesized to lead to poor oocyte quality, including increased incidence of aneuploidy [38]. Interestingly, however, there was no change in the aneuploidy rate between MSE and control animals at 4 wk or 9 mo of age (Supplemental Fig. S2), indicating normal chromosome segregation was likely to have taken place during meiosis I in these eggs.
Aneuploidy can also arise during completion of meiosis II, so we next examined the shape and size of the MII spindle, which can provide an indication of whether this second meiotic division is likely to occur normally. Both spindle pole-to-pole length (13.9 6 1.2 vs. 15.1 6 1.3 lm, P , 0.0001; Fig. 6) and spindle width (8.9 6 1.0 vs. 10.7 6 1.1 lm, P , 0.0001; Fig.  6) were smaller in MSE oocytes compared with controls, which suggests that completion of meiosis II after fertilization could be compromised.

Decreased Sperm-Egg Interaction with MSE
The interaction of eggs from MSE ovaries with wild-type sperm was also investigated to determine the likelihood of normal conception. The MSE animals had a reduction in both sperm-zona binding (14 6 3.5 vs. 23 6 5.2 bound sperm per egg, P ¼ 0.0007; Fig. 7A) and sperm-oolemma binding (17 6 4.6 vs. 24 6 3.5 bound sperm per egg, P , 0.0001; Fig. 7B) compared with controls. We also observed that ZP thickness was reduced in MSE animals at 4 wk (5.7 6 0.6 vs. 7.0 6 0.8 lm, P , 0.0001; Fig. 7C). By 9 mo there was no difference between control and MSE ZP thickness (7.8 6 0.6 vs. 7.9 6 0.6 lm); however, ZPs of both groups were significantly thicker compared with 4-wk animals (P , 0.0001; Fig. 7C). Together these results suggest that maternal smoke exposure leads to reduced quality of the egg plasma membrane and glycoprotein extracellular matrix, which may impact the success of fertilization.

MSE Decreases F1 Female Fertility
Finally, we sought to establish the overall reproductive consequences for MSE females. Control and MSE female adult mice (age 6 wk) were mated with non-smoke-exposed males during a period of 3 mo, and the days to conception, litter sizes, and pup weights were recorded. Time to conception was significantly increased in MSE females (8.2 6 7.7 vs. 3.3 6 2.5 days, P ¼ 0.0206; Fig. 8). Additionally, a small but significant reduction in litter size was also found in MSE mice compared with controls (6 6 1.6 vs. 7 6 1.8 pups per litter, P ¼ 0.0190; Fig. 8) with no significant change in pup weights, suggesting phenotypically normal litters (Supplemental Fig.  S3).

DISCUSSION
In utero exposure to cigarette smoke in humans has been linked to adverse health effects in offspring, including increased risk of obesity, asthma, and congenital heart defects, and reduced female reproductive capacity [1][2][3][8][9][10][11][12][13]. Through the use of our novel animal model we have been able to avoid external confounding factors present in human epidemiological studies of smoking and have begun to unravel the effects of maternal smoke exposure directly on the fertility of the subsequent generation of females. In the current study, we have identified ovarian and oocyte changes associated with pregnancy and lactational smoke exposure in the mouse that may underlie the reduced fecundability and early-onset menopause observed following human maternal exposure. Interestingly, changes in oocyte and ovarian quality observed in MSE mice are similar to alterations detected in their mothers [27].
Smoke exposure during the gestational/weaning period resulted in the loss of nearly half the ovarian follicle population at PND 6. In the mouse, the entire cohort of primordial follicles, from which all mature oocytes are ultimately derived, is established during early postnatal life, with some follicles beginning to enter the growing pool. Therefore, the time after birth is crucial in determining the reproductive capacity of the adult female. At PNDs 3-6 we observed increased levels of TUNEL, which suggests that loss of primordial and/or activated follicles is occurring at this time through an apoptosis-mediated mechanism. Such a mechanism of germ cell depletion has been observed in in vitro studies where neonatal ovaries were exposed to individual chemical constituents of cigarette smoke. For example, culture of PND 3-4 ovaries in the presence of benzo[a]pyrene, 3-methylcholanthrene, or 7,12-dimethylbenz[a] anthracene resulted in increased follicle apoptosis with an elevation in activated caspase 2, caspase 3, and DNA strand breaks (TUNEL) [39][40][41]. It is possible that germ cell apoptosis in MSE females may be initiated prior to birth, in the developing fetal ovary when early meiotic events are taking place. This could contribute to the reduction in follicle numbers observed in neonatal MSE ovaries. In support of this concept, the culture of mouse fetal ovaries with polycyclic aromatic hydrocarbons or in utero exposure of human fetuses to cigarette smoke has been shown to significantly elevate proapoptotic markers Bax and HRK [14,24]. In addition, data from our male MSE model indicate Downloaded from www.biolreprod.org.
that meiotic spermatocytes are particularly susceptible to apoptosis following smoke exposure [28]. Future investigations examining the female MSE model at embryonic stages may assist in our understanding of how smoke exposure impacts early female meiosis. Together with increased apoptosis in the neonatal ovary, we noted reduced proliferation of ovarian somatic cells at PND 3. Elevations in granulosa PCNA have previously been correlated with the initiation of follicle growth [36]. This reduced PCNA expression at PND 3, in conjunction with the increased proportion of primordial follicles at PND 6, indicated that in MSE ovaries, primordial follicles are failing to activate and transition to the growing pool in a timely manner. Interestingly, PCNA expression in both stromal and granulosa cells was elevated in MSE ovaries by PND 6. In addition to being a marker of S-phase, PCNA is involved in DNA repair [42], and so the elevation observed in MSE neonatal ovaries may reflect increased DNA repair activity. Our previous work in MSE males also revealed an increase in PCNA and DNA repair protein DCM1 in testis, indicating that gestational/neonatal exposure to cigarette smoke can lead to significant DNA damage and subsequent activation of repair and/or apoptotic pathways [28]. PCNA signal in control 8-to 9-wk ovaries was detected as expected, with numerous immunopositive granulosa cells present in preantral and antral follicles. We predict that the severe reduction in PCNA levels in adult MSE ovaries reflects abnormal granulosa cell proliferation, and therefore abnormal follicle growth. Despite this decreased PCNA expression, only a slight, nonsignificant reduction in the follicle population was observed at 8-9 wk. Because the ovarian follicle pool is established in neonatal life, it is therefore possible that this normalization in the follicle pool is a result of delayed follicle activation. During follicle activation and maturation the vast majority of follicles undergo atresia [43,44]. The delay in follicle activation observed in PND 6 ovaries could result in the apparent follicular population catchup in MSE ovaries at 8-9 wk; however, by late adult life (9 mo) follicle numbers were significantly reduced. Folliclestimulating hormone (FSH) has been shown to increase PCNA expression in granulosa cells, and it is therefore possible that despite the apparently normal histological follicle population, the MSE ovaries are less sensitive to hormonal cues, resulting in the reduction of ovulated eggs observed [45,46]. By 9 mo of age MSE ovaries contained significantly fewer follicles, resulting in a reduction of mature GV oocytes. Interestingly, however, PCNA expression was increased in MSE ovaries. We hypothesize that the elevation in follicular proliferation observed was the result of altered hormone dynamics within the ovary. One hormone in particular, AMH, which reduces follicular sensitivity to FSH and subsequently prevents follicle growth, is known to decrease in women near the end of their reproductive life [37,47,48]. In contrast to this, FSH levels are found to increase in these women [48]. Our investigation of AMH levels in reproductively aged females (9 mo) revealed decreased AMH levels in MSE ovaries. It is therefore possible that MSE animals are displaying a more severe aging phenotype than controls. Reduced AMH levels may be leading to elevated granulosa proliferation as a result of increased sensitivity to raised FSH levels.
It is important to note, however, that a previous study indicated that smoke-exposed F0 females are significantly smaller than controls [26]. Several human studies have found altered serum cholesterol levels in women with premature ovarian failure [49,50]. Cholesterol is an essential precursor for estrogen and is therefore important for normal fetal ovarian development [51]. As such, it is possible that the reduction in the ovarian reserve of MSE animals could be the result of exposure to ovotoxic cigarette smoke constituents in combination with reduced maternal body mass.
In addition to the loss of both immature and mature oocyte numbers, gestational/neonatal exposure to cigarette smoke also reduced mature oocyte quality, with respect to oxidative stress status. The key source of oxidative stress in MSE oocytes may well be endogenous from damaged mitochondria, which FIG. 6. Maternal cigarette smoke exposure decreases the metaphase II spindle size in ovulated oocytes. Representative confocal images of control and MSE oocytes immunostained for a-tubulin (green) at 600x magnification. Chromosomes are counterstained with Hoechst (blue). Graphical representations of median MII spindle length/width (centerline); box outline 25th-75th percentiles and whiskers, 10th-90th percentiles. n, number of oocytes examined from three control/MSE animals. *P , 0.0001, Student t-test, Bar ¼ 5lm. MATERNAL SMOKE EXPOSURE AND FEMALE FERTILITY displayed increased rates of superoxide leakage. Oxidative stress has the potential to impact numerous aspects of oocyte maturation and development, such as meiotic spindle formation. Previous work has shown that exposure of oocytes to the pro-oxidant tertiary butyl hydroperoxide, or H 2 O 2 , significantly reduces spindle size in a manner similar to that observed in MSE mice [38,52]. Correct spindle formation is critical for chromosome separation, and changes to normal MII spindle morphology in MSE oocytes could potentially lead to chromosome segregation errors during MII completion, causing aneuploidy, and ultimately embryo loss.
Furthermore, the increased lipid peroxidation in MSE oocytes, predicted to be another outcome of increased oxidative stress, may be the causative factor behind the decreased sperm-egg interactions we observed. We hypothesize that increased lipid peroxidation may have led to decreased fluidity of the oolemmal membrane, potentially altering its protein composition and impacting proteins involved in spermegg interaction, such as CD9 or GM1 [53,54]. Such scenarios have been previously reported in other cell systems, including in macrophages, whereby exposure to H 2 O 2 decreased plasma membrane fluidity through increasing the frequency of lipid rafts [55]. Representative confocal images of MII oocytes highlighting the varying thickness of ZP (red arrows) at 3600 magnification. Graphical representation shows median zona thickness between 4-wk and 9-mo oocytes. n, number of oocytes examined from four to six control/MSE animals, P , 0.0001, ANOVA with Tukey post hoc. Box plot shows mean (centerline) with box outline 25th-75th percentiles and whiskers 10th-90th percentiles. Bar ¼ 20 lm.

CAMLIN ET AL.
Sperm-zona interaction also appeared to be reduced in MSE oocytes, and interestingly these oocytes possessed a thinner ZP matrix at 4 wk. In the mouse, the ZP consists of three proteins: ZP1, ZP2, and ZP3 [56]. The importance of this structure is indicated by the fact that deletion of the ZP2 or ZP3 genes in mice causes infertility due to abnormal follicle and oocyte development [57,58]. Although ZP1-null females display relatively normal oocyte development, similar to our observations these oocytes had a significantly thinner ZP and females had smaller litters due to embryonic loss [59]. We predict that maternal smoke exposure might influence either 1) the production of ZP components through transcriptional or translational changes, or 2) ZP glycoprotein stability or turnover. It would be of particular interest to determine whether MSE influences the expression of the ZP or other key genes involved in oocyte development, which may impact the final quality of the mature oocyte. Interestingly, however, by 9 mo zona thickness was increased in both groups. In humans a correlation between zona thickening and increasing age has been found [60]. It is therefore possible that our aging MSE mice are showing a more severe aging phenotype with respect to zona thickness such that there is negligible difference from controls by 9 mo. To support this idea, we find that MSE oocytes have a 36% increase in zona thickness with age compared with the 12% increase observed in controls.
In summary, by using a model that avoids the external confounders present in human epidemiological studies, such as lifestyle and environmental factors, we have been able to provide strong evidence that maternal smoke exposure adversely affects the reproductive health of female offspring. Our results indicate that such smoke exposure results in reduced female fertility in adult life, which can be attributed to both a reduction in the number and quality of ovulated oocytes. These findings provide an important basis for understanding the decreased fecundability and early-onset menopause observed in humans following maternal smoking. Further studies using our MSE model could be instrumental in pinpointing the effects that smoking cessation prior to conception or at different embryonic stages of pregnancy has on the fertility of the subsequent generation, and our future work will focus on the potential of transgenerational effects of MSE. Such knowledge will hopefully facilitate smoking cessation cam-paigns targeting young women, particularly those who are pregnant and/or breast-feeding.