Long-term effects of methamphetamine exposure in adolescent mice on the future ovarian reserve in adulthood

Toxicology Letters 242 (2016) 1–8

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Long-term effects of methamphetamine exposure in adolescent mice on the future ovarian reserve in adulthood Lan Wanga , Guoqiang Qub , Xiyuan Donga , Kai Huanga,c, Molly Kumard, Licheng Jia , Ya Wanga , Junning Yaoa , Shulin Yanga , Ruxing Wua , Hanwang Zhanga,* a

Reproductive Medicine Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030, China Criminal Science and Technology Institute of Public Security Bureau of Wuxi City, Wuxi, Jiangsu 214000, China Reproductive Medicine Center, The First Affiliated Hospital of Zheng Zhou University, Zhengzhou, Henan 450000, China d Laboratory of Reproductive Medicine, New York University Langone Medical Center, New York, NY 10014, USA b c

H I G H L I G H T S

 The long-term abuse of methamphetamine in the adolescent period causes mitochondrial damage in murine female ovarian tissue.  The long-term abuse of methamphetamine in the adolescent period activates the Bcl-2 and Bax apoptosis signaling pathways and leads to a decrease of anti-Mullerian hormone expression in the ovary.  Adolescent MA abuse results in a reduction of the ovarian follicle pool.

A R T I C L E I N F O

A B S T R A C T

Article history: Received 15 September 2015 Received in revised form 11 November 2015 Accepted 30 November 2015 Available online 2 December 2015

Currently, there is an increasing prevalence of adolescent exposure to methamphetamine (MA). However, there is a paucity of information concerning the long-term impact of early exposure to MA upon female fertility and ovarian reserve. The aim of this study was to investigate the effect of long-term MA exposure in adolescents on their ovarian reserve in adulthood. Adolescent mice received intraperitoneal injections of MA (5 mg/kg, three times per week) or saline from the 21st postnatal day for an 8 week period. Morphological, histological, biochemical, hormonal and ethological parameters were evaluated. An impaired ovarian reserve and vitality was found in the group treated with MA, manifesting in morphological-apparent mitochondrial damage, an activated apoptosis pathway in the ovarian tissue, a downward expression of ovarian anti-Mullerian hormone (AMH), a decreased number of primordial and growing follicles, an increased number of atretic follicles, and a depressed secretion of AMH, estradiol and progesterone from granulosa cells. However, no significant difference was noticed regarding the estrous cycle, the mating ability and the fertility outcome in the reproductive age of the mice after a period of non-medication. The present results confirmed that a long term exposure to methamphetamine in adolescent mice does have an adverse impact on their ovarian reserve, which indicates that such an early abuse of MA might influence the fertility lifespan of the female mouse. ã 2015 Elsevier Ireland Ltd. All rights reserved.

Keywords: Methamphetamine Adolescent Ovarian reserve Ovarian follicle counting Anti-Mullerian hormone

1. Introduction

Abbreviations: MA, methamphetamine; AMH, anti-Mullerian hormone; LH, luteinizing hormone; FSH, follicle stimulating hormone. * Corresponding author. E-mail address: [email protected] (H. Zhang). http://dx.doi.org/10.1016/j.toxlet.2015.11.029 0378-4274/ ã 2015 Elsevier Ireland Ltd. All rights reserved.

Methamphetamine (MA) is a central nervous system stimulant of the phenethylamine and amphetamine class, which is commonly used as a recreational drug (Scott et al., 2007). The rapid increase in the abuse of MA, particularly in adolescents, has become a public health problem (Herman-Stahl et al., 2006; Iritani et al., 2007; Marshall and Werb, 2010; Wu et al., 2006). MA abuse causes severe neurodegeneration (Brown et al., 2002; Callahan

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et al., 2001; Ricaurte et al., 1980; Xie et al., 2000). In addition, MA abusers show risk increase of cerebral vascular accidents, even in young consumers, such as ischemic stroke and subarachnoid hemorrhage (Chaudhuri and Salahudeen, 1999; Jacobs, 1989). Besides its widely accepted impact on the nervous system, MA is also an important drug in the field of reproductive toxicology. It negatively affects male fertility by disrupting the function of the hypothalamic–pituitary–testicular axis, decreasing levels of luteinizing hormone (LH) and damaging sperm function as well as testicular structure (Fronczak et al., 2012; Yamamoto et al., 1999). One study showed that an acute injection of methamphetamine at different doses (5,10 or 15 mg/kg) induced apoptosis in seminiferous tubules in male mouse testis (Yamamoto et al., 2002). In addition to its adverse impact on male fertility and sperm quality, it is also reported that MA administration during pre-mating, gestational and lactational periods has a negative effect on maternal ethology and physiology (Slamberova et al., 2005). It decreases the blanket position of active nursing, as well as sniffing and rearing of rodents toward the pups (Slamberova et al., 2005). It also results in the disruption of the menstrual cycle and dysfunction of hypothalamic–pituitary–gonadal axis in women (Shen et al., 2014). Similar to other addictive drugs, several studies indicated that oxidative stress and mitochondria damage may play a crucial role in MA-induced genetic toxicity and cell apoptosis (Li et al., 2003; Potula et al., 2010). Such cytotoxicity and genetic toxicity could accelerate reproductive aging. However, the potential adverse effect of MA on fertility of young women has yet not been rigorously explored. It is widely accepted that ovarian follicle depletion is irreversible. So the evaluation of ovarian reserve and retardation of follicle depletion is of great importance to the lifespan of female fertility. Anti-Mullerian hormone (AMH), a member of the transforming growth factor-b (TGF-b) family, is produced by the granulosa cells of preantral and small antral follicles (Vigier et al., 1984). It has an inhibitory effect on primordial follicle recruitment as well as on the responsiveness of growing follicles to folliclestimulating hormone (FSH) (Visser et al., 2006). Therefore it is known as the ideal marker for ovarian reserve (Broekmans et al., 2006; Fanchin et al., 2003; van Rooij et al., 2002; Visser et al., 2006). Besides AMH there are also other parameters to evaluate ovarian reserve, such as ovarian follicle counting. To confirm the hypothesis, that adolescent MA abuse may influence ovarian reserve and reproductive ability later in

reproductive age, we established a mouse model to simulate the adolescent MA abuse and examined the related morphological, histological, ultra-structural, biochemical, hormonal and ethological parameters. 2. Materials and methods 2.1. Animals and MA administration The protocol of the animal experiments was approved by the ethic committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. Three weeks postnatal female ICR mice were purchased from the Center of Experimental Animals, Hubei Province, China. Mice were maintained at 21  2 C temperature, 55  5% humidity and a cycle of 12:12 h of light/dark. Methamphetamine at a medium-dose of 5 mg/kg or saline 0.9% was intraperitoneally administered (n = 24). The administration was performed once a day for three consecutive days per week from the 21st postnatal day for 8 consecutive weeks. Such an injection pattern emulated the human weekend associated consumption of MA (Barenys et al., 2009). The detailed protocol is described in Fig. 1. 2.2. Transmission electron microscope Ovaries were cut into thin slices and were fixed with a phosphate buffered (pH 7.3) 2.5% glutaraldehyde and 2% paraformaldehyde mixture solution for 2 h and then with 1% osmium tetraoxide for another 2 h. After washing, samples were embedded in Araldite 6005, and cut with a Leica EM FCS (Vien, Austria) ultramicrotome. Thin sections (60–70 nm) were stained with uranyl acetate and lead citrate, and examined and photographed using a LEO 906 E TEM (80kV; Oberkochen, Germany). 2.3. Western blot Ovarian tissue was milled in a grinder filled with 200 ml RIPA lysis buffer (Goodbio Technology Co., Wuhan, China) with freshly added protein inhibitors (Goodbio Technology Co.). The milling procedure was performed on ice. Then the lysed slurry was vibrated thoroughly by ultrasound. Solid tissue debris was removed by centrifugation at 1200  g for 10 min. Protein concentration was measured using BCA assay kit (Goodbio Technology Co.). Protein samples were subjected to 10% sodium

Fig. 1. Experimental structure and protocol schedule. Mice were injected with MA intraperitoneally at a medium dose of 5 mg/kg or the same amount of 0.9% saline for three consecutive days a week for 8 weeks starting from the 21st postnatal day. On completion of the 8 weeks animals from both groups were divided into three subgroups randomly. Ovaries from one third of the mice were used for ovarian morphological study and granulosa cell culture. Ovaries from another one third were used for follicle counting and to determine the expression of apoptosis and ovarian reserve relating genes. The remaining mice were used for mating and further testing of the offspring.

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Fig. 2. The ultrastructure of ovaries after MA administration. Ultrastructure of ovarian granulosa cells of the control group (A, B) and the MA treated group (C, D). Black arrows refer to the swelling of mitochondria marked by the obvious enlargement of the interval of mitochondria cristae. A, C 1700; B, D 5000.

dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and were transferred on to nitrocellulose membranes using BioRad electroblot apparatus. Non-specific binding sites were blocked in 5% non-fat dry milk in 0.05% TBS-Tween. The following primary

antibodies were used: anti-rabbit AMH monoclonal antibody (1:300, 4  C overnight, Santa Cruz, CA, USA), anti-rabbit AMHR2 monoclonal antibody (1:200, 4  C overnight, Santa Cruz), antirabbit BAX antibody (1:400, 4  C overnight, Santa Cruz), anti-rabbit

Fig. 3. Western blot analysis of AMH, AMHR2, BAX and Bcl-2 protein level. Protein levels were calculated as a ratio of their densitometric value to that of the b-actin reference gene, and are represented in the right panel. The densitometric values for the b-actin were comparable for all samples. *P < 0.05; **P < 0.01; One-way ANOVA.

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2.5. Ovarian tissue dissociation and short-term granulosa cell culture Granulosa cells from the ovaries were dissected using a traditional mechanical method (Zhang et al., 2014). Cells (1 105 viable cells/well) were seeded in the six well culture plate and cells from different ovaries were seeded into different wells. Granulosa cell were cultured in McCoy’s 5A culure medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% FBS (Thermo Fisher Scientific), 1% antibiotics (Thermo Fisher Scientific) and rFSH (25 ng/ml, Sigma–Aldrich Co.) at 37  C in a humidified atmosphere of 5% CO2 for 2 days. After two days of short-term culture, the supernatant was centrifuged and collected for ELISA assay and the cells were trypsinized and counted by the automated Cell Counter (Alit International Trade Co., Ltd., Shanghai, China) using Trypan Blue (Goodbio Technology Co.) following the manufacture’s instruction. 2.6. Hormone assay of cell culture supernatant After two days of cell culture, the supernatant in each well was centrifuged and collected. Concentrations of AMH, estrogen and progesterone were measured by a commercially available enzymelinked immunosorbent assay kit (Cloud-Clone Corp., Houston, USA) and electrochemiluminescence immunoassay using the ADVIA Centaur XP immunoassay system (Siemens, Germany) according to the manufacturer’s instructions. 2.7. Determination of estrous cycle, mating ability, litter size and body weight of the offspring Fig. 4. Follicle counting. The percentage of primordial follicles, growth follicles (including primary follicles, secondary follicles and antral follicles) and atretic follicles in the ovarian sections of the control group were compared to that of the treatment group (A). The percentage of primordial and growth follicles are significantly decreased while the atretic follicles showed a significant increase in the MA group compared to the control group. *P < 0.05; **P < 0.01; One-way ANOVA; Primordial follicles are easy to find in the ovaries from the control group (B) as there are relatively fewer in the MA group (C) as the thin arrows show. Thick arrows display primary follicles (D). Also a typical secondary follicle is shown (E). Hematoxylin and eosin staining.

Such determinations were performed 2 weeks after the cessation of the MA administration. Females were checked by vaginal smear lavage to see whether or not MA altered the regularity and duration of the estrous cycle. Estrous cycles were considered irregular if they were shorter than 4 days or longer than 5 days. Animals were allowed to mate once every other day. Two females with one untreated male were housed in each cage. Successful mating was confirmed by the presence of a sperm plug observed in female mice the following morning. At birth the pups were weighed and the litter size was counted.

Bcl-2 antibody (1:400, 4  C overnight, Santa Cruz) and anti-rabbit b-actin monoclonal antibody (1:800, 4  C overnight, Goodbio Technology Co.). The secondary antibodies used were as follows: rabbit HRP-conjugated anti-goat IgG (1:2000, 37  C for 1 h; Goodbio Technology Co.). Protein bands were visualized by enhanced chemiluminescence (Pierce Biotechnology, Inc., Rockford, PO, USA).

2.8. Statistical analysis

2.4. Histological study and follicle counting

Statistical analyses were performed using the SPSS 13.0 software package (SPSS Inc., Chicago, IL, USA). Data of protein expression, follicle counting, hormone levels, cycle days, litter size and the weight of pups was analyzed using one-way ANOVA and presented as means  S.E.M. Number of mice with irregular estrogen cycle was compared using Chi-square test. P-value <0.05 was considered statistically significant. 3. Results

Ovaries were embedded in paraffin and longitudinally cut into sections. The sections were further stained by hematoxylin and eosin (HE). Follicles at each developmental stage were counted and averaged in three serial sections from the largest cross-section through the center of the ovary as previously described (Jin et al., 2010; Nilsson et al., 2007; Winkler-Crepaz et al., 2014) under a light microscope (Leica, Germany). Sections were taken at a thickness of 5 mm, at intervals of 25 mm. Only follicles that contained an oocyte nucleus were counted to avoid double-counting. Follicles were classified according to Myers et al. (2004). The average number of different follicles in each section was calculated for each mouse. And the whole procedure was performed twice by two individual technicians.

No deaths were observed during the whole study. No significant variations of final body weight (38.0  1.1 g versus 37.4  1.2 g, P > 0.05) and body weight gain (406.4%  39.5% versus 395.4%  39.6%, P > 0.05) were observed between control and MA-treated group. Ovarian weight was not affected by the longterm MA administration either (23.9  1.9 mg versus 23.0  2.0 mg, P > 0.05). 3.1. Impact of MA abuse on the ultrastructure of ovarian granulosa cells The granulosa cells are rich in mitochondria. The ovaries treated with MA show marked swelling and degeneration of mitochondria

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Fig. 5. Hormone secretion profile. The secretion profile of AMH, estradiol and progesterone by granulosa cells were valued after a short term in vitro culture. The granulosa cells from the MA treated ovaries showed a significantly lower production of AMH, estradiol and progesterone. *P < 0.05, **P < 0.01. One-way ANOVA.

Fig. 6. The morphology of granulosa cells under light microscope after 2 days of in vitro culture. There are no visible morphological difference of the granulosa cells between the control group (A) and the treatment group (B). 200.

and an obvious enlargement of the interval of mitochondria cristae compared to the ovaries of the control group. No obvious difference was observed in other organelles in the cells, such as endoplasmic reticulum and cell nucleus, between the treatment group and the control group (Fig. 2). 3.2. Impact of MA abuse on the expression of apoptosis relating proteins and ovarian reserve relating proteins in mice ovaries In the adolescent MA treated mice BAX shows a significantly higher expression (P < 0.01) and Bcl-2 shows a significantly lower expression (P < 0.01). As a crucial signaling pathway in ovarian function and important markers of ovarian reserve and vitality AMH and its receptor (AMHR2) were also tested in the current study. The AMH expression of the ovary was significantly reduced after 8 weeks of MA treatment from the adolescent period (P < 0.05). No significant changes were found in the expression of AMHR2 between MA treated and the control groups (Fig. 3). 3.3. Adolescent MA abuse leads to a deletion of ovarian follicle pool There are no significant morphological changes between the MA treated group and the control group. However, the MA treated group showed a deficient pool of primordial follicles (P < 0.01) as well as a significantly lower percentage of growth follicles (P < 0.01) compared to the ovaries from the control group. The percentage of atretic follicles of the MA group was significantly higher than that of the control group (P < 0.05) (Fig. 4).

3.4. Long-term MA abuse adversely influences the ability of hormone secretion of granulosa cells Ovarian granulosa cells are capable of hormone secretion. They are the primary source of AMH, estradiol and progesterone production in the female. After short term in vitro cell culture the ovarian granulosa cells from the MA treated ovaries show a significantly lower secretion ability of AMH (P < 0.05) and estradiol (P < 0.01) as well as progesterone (P < 0.01) (Fig. 5). However, no obvious differences were found in the morphology of granulosa cells under optical microscope (Figs. 5 and 6). 3.5. Long-term MA abuse in the adolescent period has no obvious impact on the estrous cycle after sexual maturity, as well as the mating outcomes, litter sizes and the weight of the pups Estrous cycle and mating outcomes were observed after two weeks of rest from the MA administration. As is shown in Table 1, there was no significant difference between groups in the regularity of the estrous cycle (P > 0.05, Table 1). Similarly, no markedly gross differences in cumulative number of vaginal Table 1 Estrous cycles variation. Estrous cycle (n = 8)

MA Control

Regular/irregular number of females

Days in one cycle

5/3 7/1

5.00  0.33 4.37  0.26

Values are shown as mean  S.E.M. P > 0.05; Chi-square test for the estrous cycle and one-way ANOVA for the cycle days.

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Fig. 7. Mating outcomes and further test of the pups. Animals were allowed to mate once every other day. Two females were mated with one single non-treated male. Successful mating was confirmed by the presence of sperm plug observed on the following morning at 08:00. At birth the pups were weighed and the litter size was also counted. Cumulative numbers of vaginal plugs from the first day of mating are similar in the two groups (A). The litter size and the weight of pups are also similar (B, C) with or without maternal MA abuse history in adolescent years. P > 0.05; One-way ANOVA.

plug-positive animals were observed (Fig. 7). There were also no significant differences in regard to the litter size (P > 0.05) and the weight of the pups (P > 0.05) (Fig. 7). 4. Discussion Several studies showed that MA injected during gestational or lactational periods may disrupt maternal behavior and the well being of the offspring (Inoue et al., 2004; Slamberova et al., 2005). However, as far as we know, there are few studies focusing on the potential toxicity of adolescent abuse of MA to female reproductive capability after sexual maturation. The current study focuses on the fertile toxicity of long-term early exposure of MA on female mice. We found, as far as we know, for the first time that long-term early exposure of MA could adversely impact the ovarian reserve in the female’s future life, although its influence on the female’s fertility is not obvious after a period of rest. Previous findings indicate that chronic MA administration can cause serious damage in mitochondrial function and lead to mitochondrial dependent apoptosis (Liou et al., 2014; Steinkellner et al., 2011). Mitochondrial dysfunction plays an important role in cellular apoptosis and the aging process (Parameyong et al., 2015). Our data show that long-term MA abuse in the adolescent period results in mitochondria damage of the ovarian granulosa cells in mice, manifesting in marked mitochondrial swelling and obvious enlargements of the interval of mitochondria cristae. Such damage is crucial to the growth and development of the female reproductive system as ovarian granulosa cells are the most important functional cells in the mammalian ovary. Bcl-2 proteins are functionally categorized into death-inhibiting or death-inducing members. Bcl-2, an inner mitochondrial membrane protein, inhibits apoptotic cell death by decreasing the net cellular generation of reactive oxygen species (Karch and Molkentin, 2015). BAX also belongs to the Bcl-2 family and is known to accelerate the cell apoptosis (Raineri et al., 2012). Studies by different investigations have provided unimpeachable evidence of a role for Bcl-2 and BAX related oxygen-based free radicals in MA-induced neurotoxicity (Galinato et al., 2015; Jayanthi et al., 2001). However, such a similar impact on the female reproductive system is still seldom studied. In our study, we found a similar trend in the mice ovarian tissue when treated with MA for 8 weeks starting in the adolescent days. After the 8 weeks of early MA exposure, the expression of Bcl-2 in the mice ovarian tissue is significantly decreased while the expression of BAX is significantly up-regulated. Such an enhanced cell apoptosis might adversely impact the follicle recruitment and follicle growth of the young female mice. The ovarian granulosa cell is the main constituent of the mammalian ovary and the only source of AMH production in

females (Falorni et al., 2012). AMH is absent in primordial follicles, it appears in granulosa cells of primary follicles and is highly expressed in growing preantral and small antral follicles until dominance (Falorni et al., 2012). A decreased AMH level is normally related to a depletion of the ovarian follicle pool and an impaired ovarian reserve. AMHR2 is the most important and confirmed receptor of the AMH signaling pathway and it is only expressed in ovarian granulosa cells. In the current study, the ovarian AMH expression of the MA treated group is significantly lower than the control group, although the AMHR2 expression of the two groups has no significant difference. Such a finding indicates that the MA abuse from the adolescent days will adversely affect the future ovarian reserve of the female mice. Besides AMH, another more intuitive factor in evaluating ovarian reserve is the primordial follicle counting. As current knowledge states, the adult mammalian ovary is devoid of definitive germline stem cells (Grive and Freiman, 2015). So the female reproductive senescence is mainly due to the gradual depletion of a finite ovarian primordial follicle pool that has already been produced in the embryonic period (Grive and Freiman, 2015). Several studies show that many medications or drugs can perturb primordial follicle assembly or lead to an accelerated depletion of primordial follicles, for example nicotine (Fowler et al., 2014; Grive and Freiman, 2015). In the current study, a significant decrease of primordial follicles was found in the ovaries of the MA treated group. A decline of growing follicle number was also noticed, while the number of atretic follicles was raised. Such a finding strongly indicates that long-term MA abuse during adolescent periods adversely affects the ovarian reserve in mice. The ability of the granulosa cells to secrete sexual hormones is essential to the normal growth of follicles and the function of ovary. In the current study, impaired hormone secretion is found in the granulosa cells from the ovaries treated by MA in vivo. After a short period of in vitro culture, cells from the MA group show a significantly lower secretion profile of AMH, estrogen and progesterone. Such a phenomenon might result from a lower cellular vitality or a lower cellular sensitivity to exogenous FSH after long-term treatment with MA in vivo. In contrast to other studies which demonstrated that MA administration impairs the estrous cycle both in human and in rodents (Shen et al., 2014; Slamberova et al., 2005), we found no significant difference in the estrous cycle of mice between the MA administration group and the control group. Such a result could be interpreted by the fact that we evaluated the estrous cycle after two weeks’ rest of non-medication and that the possible adverse effect are reversible, or the administration during the adolescent period simply has no obvious impact on the future ovarian cycle of female mice. Several studies showed that a prenatal MA exposure

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could impair both the maternal behavior and the fetal growth, and even cause the histological, cellular and chromosomal defects in fetal mice (Inoue et al., 2004; Mirjalili et al., 2013; Slamberova et al., 2005; Smith et al., 2003; Won et al., 2002). In the present study, we found that early exposure of MA during adolescent days has no significant effect on the females’ mating behavior, their litter size and the weights of the pups. So the early exposure of MA in adolescent days followed by a period of rest before pregnancy might have no effect on the reproductive ability of the females in their sexual maturation period. It is emphasized more and more that an unhealthy lifestyle delivers an adverse impact on female fertility treatment and fertility lifespan (Burgo et al., 2015; Domar et al., 2015; Gormack et al., 2015; Nicolau et al., 2014). A recent clinical study shows that alcohol intake induces diminished ovarian reserve in women, manifesting in altered hormone levels, decreased ovarian volume and ovarian antral follicle number, as well as increased menstrual abnormalities (Li et al., 2013). Our study, though focused on murine sample, might raise some inspiration of the potential relationship between adolescent MA abuse and female fertility lifespan in human beings. The innovation point of the current research is that we studied the potential fertility toxicity of MA using the adolescent female mouse as a model. A recent clinical study shows that long-term MA abuse disrupts the menstrual cycles and hypothalamic– pituitary–ovarian axis (Shen et al., 2014). However, further studies on the ovarian toxicity of MA seem not as abundant as those focusing on the male fertility toxicity (Alavi et al., 2008; Lin et al., 2014; Nudmamud-Thanoi and Thanoi, 2011; Yamamoto et al., 1999). One possible reason for that is the spermatogenetic procedure is much shorter and less complicated than oogenesis, so the acute toxic effect on sperm is much easier to observe. Another possible explanation is the currently believed sexual difference of MA toxicity. It is reported that estrogen can act as a protector to the MA’s toxicological effects on nervous system (Dluzen and McDermott, 2000; Gajjar et al., 2003; Mickley and Dluzen, 2004; Myers et al., 2003). So estrogen might also be a protective factor on MA’s fertility toxicity. Therefore, we established a long-term exposure mouse model using the adolescent mice whose gonad axis is not yet fully developed. In this way, we aimed to explore the potential effect of MA on their future fertility lifespan. There are, however, still several limitations of our study. The long-term impact of methamphetamine on ovarian reserve of adolescents was only shown in mice with a relatively small sample size. A perspective RCT on humans is difficult because of ethical restrictions. Additionally, we only focused on the impact of MA on ovarian reserve and reproductive ability, but not on its impact directly on the development of the germ cells. Furthermore, it’s of great importance to support the preliminary finding by doing a dose related research in the future. It is also of great significance to make a follow-up study of the treated mice until they reach their perimenopause period in order to see whether the adolescent MA abuser will experience a shorter reproductive lifespan or an abnormal reproductive ability in their elder life. Further studies on these issues would be also interesting. In summary, long-term exposure of MA in the adolescent period does have an adverse impact on the murine ovarian reserve, while the estrous cycle, the mating ability and the fertility outcome in the reproductive age of the mice after a period of non-medication seem to have not been influenced by the adolescent MA abuse. Indicating that such an early abuse of MA may only influence the ovarian reserve and the related lifespan of reproductivity of the female mice.

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