Reproductive status of male rat offspring following exposure to methamphetamine during intrauterine life: An experimental study

Abstract Background Methamphetamine abuse during pregnancy is associated with maternal and fetal adverse outcomes. Methamphetamine induces reproductive damage in adults; however, its effect has not been studied during pregnancy. Objective To investigate the effects of methamphetamine exposure during pregnancy on the reproductive system. Materials and Methods 15 pregnant Wistar rats were divided into 3 groups (n = 5/group), they received daily intraperitoneal injections of saline or methamphetamine (5, and 10 mg/kg) from day 10 until the end of pregnancy. One adult male offspring was selected from each dam. Subjects were euthanized, and their testis was removed. Sperm samples from cauda epididymis were analyzed for sperm concentration, morphology, and motility. Terminal deoxynucleotidyl transferase dUTP nick-end labeling assay was used to detect apoptotic cells. Levels of B-cell lymphoma 2 protein (Bcl-2) and Bcl-2 associated X-protein were measured using Western blot. Results Methamphetamine significantly decreased sperm concentration (5 mg vs. saline: p = 0.001, 10 mg vs. saline: p < 0.001), normal sperm morphology (saline vs. 10 mg: p = 0.001), and motility (p: saline vs. 5 mg = 0.004, 5 mg vs. 10 mg = 0.011, saline vs. 10 mg < 0.001) in a dose-dependent manner. There was a significantly higher number of terminal deoxynucleotidyl transferase dUTP nick-end labeling -positive cells and higher exposure. Moreover, Bcl-2 associated X-protein was increased, and Bcl-2 was decreased in these rats. Conclusion The present study shows that chronic methamphetamine exposure during intrauterine period can induce apoptosis of seminiferous tubules and decrease sperm quality in adult rats. Moreover, we showed that the intrinsic apoptotic pathway is involved in this process. Further studies are required to identify the complete molecular pathway of these results.


Introduction
Methamphetamine is a central nervous system stimulant and recreational drug that induces a sense of euphoria, increased energy, sexual desire, and risky sexual behavior in users (1)(2)(3). It is associated with neurological damage, cognitive dysfunction, and impaired judgment (1). Methamphetamine use during pregnancy has been associated with birth defects, low birth weight, small head circumference, and intrauterine growth restriction in humans (2) and exencephaly, premature death, and subarachnoid hemorrhage in mice (4,5). The adverse neurological outcomes of fetal methamphetamine exposure are well documented (2,6,7), but the research about other organ systems is limited.
Animal experiments have documented the adverse effects of methamphetamine on the adult male testis such as increased apoptosis, decreased cell proliferation, and a gap in the epithelium between the spermatogonia and other layers (8,9). These effects have been reported in doses as low as 4 mg/kg (10,11).
Methamphetamine can affect apoptosis by increasing apoptotic index, oxidative stress, and cleaved caspase-3 (12). It is shown to diffuse into mitochondria and decrease membrane potential, disrupting the electrochemical gradient, and causing the release of cytochrome c from mitochondria, followed by caspase activation (1).
Intrinsic apoptosis is mediated by proapoptotic proteins such as B-cell lymphoma 2 (BCL-2) associated X-protein (BAX), which increases the mitochondrial outer membrane permeability to allow the release of proteins such as cytochrome c and activating caspases (14,15). Membrane permeabilization is considered "the point of no return" in this process, after which the cell is committed to the fate of apoptosis (14). The action of BAX is antagonized by antiapoptotic proteins such as Bcl-2, which binds to BAX. Cell shift toward apoptosis is determined by the balance between proapoptotic and antiapoptotic Bcl-2 family proteins (14). Therefore, upregulation of BAX and downregulation of Bcl-2 are considered an initial step toward apoptosis (14). Changes in apoptosis regulator proteins can result from internal or external stimuli such as DNA damage (15).
While methamphetamine has been associated with damage to the male reproductive system in adults, its effects on the fetus's reproductive system are less known. Previous articles have focused solely on methamphetamine users themselves, and their offspring have not been studied. We aimed to investigate the potential effects of chronic methamphetamine exposure on the reproductive system of children born to methamphetamine-abusing mothers. To this end, the present study evaluates the reproductivity of a generation of adult rats chronically exposed to methamphetamine in the intrauterine period.

Animal treatment and drug administration
This was an experimental study conducted at Islamic Azad University Tehran Medical Sciences Branch, Tehran, Iran. 30 adult Wistar rats (15 male and 15 female) were procured from Pasteur Institute (Tehran, Iran). All rats were in good health and were never exposed to methamphetamine or any other intervention before. After estrus detection, female and male rats were caged together for one night, after which they were separated and the females were smeared vaginally.
The detection of vaginal plug was designated as day 1 of gestation. Female rats were randomly assigned to 3 groups (n = 5/each): the saline group received an injection of 0.9% NaCl, 1 ml/kg. On day 20 of gestation, each pregnant rat was housed in a separate cage. All rats gave birth at days 21-22 of gestation. All offspring lived in the cage with their mother until weaning at 28 days of age, after which the male and female offspring were segregated. Each group of male offspring were housed in a separate cage. All groups were kept at the same conditions (food and water ad libitum, 12-hr light/dark cycle, and 20°C) and did not receive methamphetamine, saline, or any other intervention. One male offspring from each mother was randomly allocated to a group using computer-generated random numbers.

Sample collection
Subjects were euthanized at 12 wk of age. The literature suggests that Wistar rats are sexually mature by the end of 10 wk (16,17). Euthanasia was carried out by decapitation using guillotines after sedation. Rats were dissected using a midline incision, and the testes were removed. The epididymis was removed from the testis and used for sperm analysis. The testes were fixed using a 10% formalin solution and sent to a lab for analysis.
Both left and right testes (or epididymis) were used for each analysis, and their average was used for each variable.

Sperm analysis
The cauda epididymis was cut open and placed in 2 ml of phosphate-buffered saline (PBS) for a few minutes to allow its contents to disperse. A solution sample was collected and viewed under a bright-field microscope within 1 hr of obtaining the sample. The sperm concentration, morphology, and motility were examined under the microscope at 10 different random fields of view, and the average was used.
Normal sperm morphology was defined using the classification by Krzanowska (18). Sperms with progressive motility and non-progressive motility were counted as normal motile sperm. The researcher carrying out the sperm analysis was blinded to the group allocations.

Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay
The testes were sectioned, fixed using paraffin, and mounted on microscopic slides. TUNEL assay

Statistical analysis
The collected data was numerical which was described using mean and standard deviation. The normality of data was assessed using Kolmogorov-Smirnov test. The statistical differences between groups were tested by Student's t test using SPSS software (Version 25.0. Armonk, NY: IBM Corp). A p-value < 0.05 was considered statistically significant.

Results
There were 5 adult male rats in each group.

Discussion
The present study shows that intrauterine methamphetamine exposure in male rats can lower sperm quality and damage testis, the effects of which continue into adulthood. Amphetamines act by inducing the release and blocking the reuptake of catecholamines, mainly in the central nervous system (2,19).
Amphetamines are thought to have a chemical Therefore, they act as an agonist for monoamine transporters and block the reuptake of extracellular monoamine neurotransmitters (1,19,20). This, in turn, leads to an increase in these neurotransmitters in the central nervous system.
Other amphetamine-based drugs have also demonstrated effects similar to those of methamphetamine.
For example, significant DNA damage, decrease in sperm count, and motility was reported in the testis of rats exposed to intrauterine MDMA (21). Similar results were observed in adult male mice exposed to Ritalin such as a significant decrease in body weight, Leydig cells, and testosterone levels (22).
Substances used by drug-abusing pregnant mothers, such as Methamphetamine can cross the placenta and enter fetal circulation (2,23). This transfer's most common mechanism is passive diffusion resulting from concentration gradient between maternal and fetal circulation (23).
Because of the weak detoxification mechanisms in the fetus, these drugs tend to concentrate in fetal organs and gradually increase over time to the point that fetal drug levels are higher than maternal levels (24). Large concentrations of drugs have been found in the placenta and other organs such as the fetus's lung, kidney, intestines, liver, brain, and heart (25). Amphetamines and their metabolites are readily detected in the umbilical cord, placenta, and amniotic fluid of exposed fetuses (23). They can induce teratogenic and fetotoxic effects on the fetus (2,4,5).
Amphetamines in fetal circulation are thought to have effects similar to those observed in adults, namely blocking the reuptake of monoamine neurotransmitters (26). There was also no control group without injection in this study. However, this is one of the first studies to discuss the long-term effects of intrauterine methamphetamine exposure.
Most of the research about the offspring of methamphetamine-abusing mothers revolve around defects presenting at birth and/or neurological symptoms. This study sought to address this issue by assessing the reproductive system of adult mice whose mothers were chronically exposed to methamphetamine in a dosage consistent with the existing literature.
However, these results should be treated with caution when generalizing to other species, including humans.

Conclusion
In conclusion, we showed that chronic exposure to methamphetamine in the intrauterine period can induce reproductive damage in adult male rats. We also showed that the intrinsic mitochondrial pathway is one of the mechanisms responsible for this by causing apoptosis.