Influence of prepubertal aflatoxicosis on pubertal reproductive activity in male rats

1. Prepubertal exposure to experimental induced aflatoxicosis affects the development of male genital organs in rats.
2. Prepubertal aflatoxicosis affects the reproductive efficiency in male rats.

Introduction

Materials and methods

Chemicals

Sigma Company, USA bought aflatoxin-B1 (from Aspergillus flavus 98% purity, Formula C17H12O6, Formula Weight 312.3 AMU). All the extra substances utilized in the study were highly analytical quality and obtained from neighboring commercial establishments.

Animal ethics and care

This research was conducted under to the guidelines provided by the National Research Council for the Care and Use of Laboratory Animals. The Ethical Council in the College of Veterinary Medicine, University of Al-Qadisiyah permitted the experimentation.

Experimental animals

Premature male rats (aged 35 days and weighted 75-88 g) were used in the present study. The animals were kept at 22 to 25 °C, 75-76% relative humidity, and 12:12 hours of dark and light cycle. The animals had free access to water and laboratory food at all times. After acclimatization, the animals were weighed before treatment (at day 35 of age) after each experiment period.

Experimental design

Sixty premature Wistar rats (35 days old) were allocated to control and treatment groups (30 males each). Control male rats were administered daily with distilled water per os. Treated male rats were administered daily with Aflatoxin-B1 per os (0.3mg/kg/day) (18,19). After 15, 25, and 35 days of treatment (prepubertal, pubertal, and postpubertal subgroups, respectively), ten males from each group were anesthetized, sacrificed, and blood samples were collected for assessment of the serum concentrations of GnRH, FSH, LH, and testosterone, by using ELISA kits, according to the instructions provided by the manufacturer (Cusabio, China). Tissue samples from the testis and epididymis were dissected and fixed in a formalin buffer solution, and microscopic slides were prepared at 5μM of thickness and stained with hematoxylin and eosin stains for histopathological examination (20). Additional tissue samples from the pituitary and testicles were also collected to analyze the gene expression levels of pituitary GnRHR, FSHβ and LHβ genes, and testicular FSHR, LHR, ANB, 3β-HSD, and 17β-HSD genes.

Molecular analysis of pituitary and testicular genes

According to the instructions from Bioneer company (Korea), the total RNA from pituitary and testicular tissues using an Accuzol® kit was extracted, and cDNA was synthesized. Quantitative real-time PCR (qRT-PCR) was used to quantitate the target specific genes, including pituitary GnRHR, FSHβ and LHβ genes, and testicular FSHR, LHR, ANB, 3β-HSD, and 17β-HSD genes, and normalize the results using the GAPDH gene as a housekeeping gene. The procedure followed the method previously described in a publication by Burow et al. (21). The qRT-PCR data were analyzed using the fold-change method for relative expression of the target genes and GAPDH, following the protocol outlined in a publication by Livak and Schmittgen (22).

Statistical analysis

The statistical analysis was conducted using GraphPad Prism version 5. The mean ± standard deviation (M±SD) was used to present the data. One-way ANOVA with Newman-Keuls posthoc analysis determined the significant differences between the periods within each group. The student’s t-test was also used to determine the significant differences between the groups at each period. The level of (p<0.05) was considered significant (23).

Results

Reproductive hormones

As shown in figure 1, male rats treated with AFB1 showed a significant decrease (P<0.05) in serum concentrations of GnRH, FSH, LH, and T in the three periods (50, 60, and 70 days) in comparison with control. When comparing the three periods for the control group, the concentration increased with age, whereas the concentrations of the treated group showed little increase at 70 days compared to 60 days old.

Figure 1: Serum concentration of GnRH, FSH, LH, and testosterone in AFB1 treated male rats. The values are depicted as Mean ± SD. The different uppercase letters indicate significant differences (p<0.05) between groups during each period. Lowercase letters indicate significant differences (p<0.05) between the three time periods for each group.

Pituitary GnRHR, FSHβ, and LHβ Genes

In all experimental periods, male rats treated with AFB1 showed a significant decrease (P<0.05) in the expression level (fold changes) of pituitary GnRHR, FSHβ, and LHβ genes compared with the corresponding periods of control male rats (Figure 2). Compared to the 50 days, the expression level of pituitary GnRHR, FSHβ, and LHβ genes of control males at 60 and 70 day periods showed a noticeable percentage of the increase, while in the treatment group, the increase was slight.

Figure 2: The expression levels of pituitary GnRHR, FSHβ, and LHβ genes in AFB1 treated male rats. The values are depicted as Mean ± SD. The different uppercase letters indicate significant differences (p<0.05) between groups during each period. Lowercase letters indicate significant differences (p<0.05) between the three time periods for each group.

Testicular FSHR, LHR, ANB, 3β-HSD and 17β-HSD

In all experimental periods, the expression levels (fold changes) of testicular FSHR, LHR, ANB, 3β-HSD, and 17β-HSD genes of treated male rats showed a significant decrease (P<0.05) compared with the corresponding periods of control male rats (Figure 3). When comparing to 50 days period, the expression level of testicular FSHR, LHR, ANB, 3β-HSD, and 17β-HSD genes of control males at 60 and 70 days periods showed a highly significant increase, while in the treatment group there is a slight increase.

Figure 3: The expression levels of testicular FSHR, LHR, ANB, 3β-HSD, and 17β-HSD genes in AFB1 treated male rats. The values are depicted as Mean ± SD. The different uppercase letters indicate significant differences (p<0.05) between groups during each period. Lowercase letters indicate significant differences (p<0.05) between the three time periods for each group.

Testes

The Histological findings of AF treated male rat’s testes of 50 days old (prepubertal period) showed atrophy of some seminiferous tubules, empty lumen, irregular shape of seminiferous tubules, a massive discharge of germ cell cytoplasm, and marked loss of cells, compared to the control group of the same period, that showed normalization of the seminiferous tubules, the presence of spermatogonia, spermatocytes, and Sertoli cells (Figure 4). At 60 days (pubertal period), the histological findings of the AF-treated male rat’s testes showed thickening of the wall of the seminiferous tubules, exfoliation of germ cells in the lumen of seminiferous tubules, irregular shape of seminiferous tubules, distributed vacuolation of germ cell, marked loss of cells compared with the control male rat’s testes, which showed the normal shape of seminiferous tubules, presence of spermatogonia, spermatocytes, and spermatids (Figure 5). At 70 days (postpubertal period), the histological findings of the AF-treated male rat’s testes showed thickening of the wall of the seminiferous tubules, atrophy of some seminiferous tubules, irregular shape of seminiferous tubules, huge vacuolation of germ cells, empty lumen, marked loss of cells compared with control male rat’s testes (Figure 6).

Epididymis

The Histological findings of the epididymis of AF treated male rats at 50 days old (prepubertal period) showed obliteration of the lumen, mild fibrosis, clumped spermatozoa, and necrosis of the epithelial layer compared with the control group (Figure 7). At 60 days (pubertal period), the histological findings of the AF-treated male rat’s epididymis showed a distorted lumen, mild fibrosis, hemorrhage in the lumen, and sloughed epithelial layer, compared with the control (Figure 8). At 70 days (postpubertal period), the histological findings of the AF-treated male rat’s epididymis showed a distorted lumen, mild fibrosis, and epithelial hypertrophy compared with the control group (Figure 9).

Figure 4: Histological section from AF-treated (A) and control (B) male rat testes at the prepubertal period (50 days period) showed atrophy of some seminiferous tubules (black arrow), empty lumen (red arrow), irregular shape of seminiferous tubules (blue arrow), vacuolation of germ cell cytoplasm (green arrows), marked loss of cells, whereas control male rat’s testes showed normal seminiferous tubules, presence of spermatogonia cells (SG), spermatocytes (SPC), spermatids (SPT), and Sertoli cells (SC). H&E, 100X and 400X.

Figure 5: Histological section from AF-treated (A) and control (B) male rat testes at the pubertal period (60 days period) showed thickening of the wall of the seminiferous tubules (black arrows), exfoliation of germ cells in the lumen of seminiferous tubules, irregular shape of seminiferous tubules, huge vacuolation of germ cell (red arrows), marked loss of cells compare with the group control (B) that normal showed shape seminiferous tubules, presence of spermatogonia cells (SG), spermatocytes (SPC), and spermatids (SPT). H&E, 100X and 400X.

Figure 6: Histological section from AF-treated (A) and control (B) male rat testes at the postpubertal period (70 days period) showed thickening of the wall of the seminiferous tubules (black arrows), atrophy of some seminiferous tubules, irregular shape of seminiferous tubules (red arrows), huge vacuolation of germ cell (red arrows), empty lumen (EL), marked loss of cells compare with the group control (B) that normal showed shape seminiferous tubules, presence of spermatogonia cells, spermatocytes, and spermatids. H&E, 100X and 400X.

Figure 7: Histological section from AF-treated (A) and control (B) male rat epididymis at the prepubertal period (50 days period) showed obliteration of the lumen (black arrows), mild fibrosis (red arrows), clumped spermatozoa (blue arrow), and necrosis of the epithelial layer (green arrow) compare with the control group (B) that showed normal shape tubules, normal muscular layer (red arrow), basement membrane (black arrow), columnar epithelial cells (green arrow), and thin connective tissue (pink arrow). H&E, 40X, 100X and 400X.

Figure 8: Histological section from AF-treated (A) and control (B) male rat epididymis at the pubertal period (60 days period) showed distorted lumen (red arrow), mild fibrosis (blue arrows), hemorrhage in the lumen (black arrow), and sloughed epithelial layer (green arrow) compare with the control group (B) that showed normal epididymis structures. H&E, 100X and 400X.

Figure 9: Histological section from AF-treated (A) and control (B) male rat epididymis at the postpubertal period (70 days period) showed distorted lumen (black arrow), and mild fibrosis (red arrow), and hypertrophy of the epithelial compared with the control group (B) that showed normal epididymis structures. H&E, 100X and 400X.

Discussion

In the current study, exposure to AFB1 at the prepubertal stage reduced the serum concentrations of GnRH, FSH, LH, and testosterone in both the prepubertal and postpubertal stages. This result indicates that exposure to aflatoxin in the prepubertal stage leads to structural and functional changes reflected in male’s reproductive efficiency. AFB1 was found to harm the reproductive activity, as the hypothalamic-pituitary-gonadal (HPG) axis is essential for gametogenesis, maintenance of gonadal hormone synthesis, and reproductive organ functions (24). Previous research has also suggested that AFB1 can be toxic to Leydig cells and affects testicular steroidogenesis (25). The study conducted by Owumi et al. (17) found that FSH and testosterone concentrations in rats treated with AFB1 were significantly reduced, which may affect the semen quality, as the composition of seminal fluid was significantly affected in A. flavus exposed male rats (26). These results are consistent with the findings of our study.

Moreover, the current decline in pituitary GnRHR, FSHβ, and LHβ gene expression corroborates the previous findings and indicates that the effect may be central via the hypothalamus and/or pituitary gland. This result was consistent with the decrease in serum FSH and LH concentrations, as their decrease came from a decline in the influence of GnRH. After administering AFB1 to premature male rats, the LH concentrations decreased significantly accompanied by decreased testosterone concentrations, indicating disruption of the HPGA axis. Previous studies have suggested that AFB1 can suppress LH in adult rats (15,17,27,28).

On the other hand, the decrease in the expression concentrations of testicular FSHR, LHR, ANB, 3β-HSD, and 17β-HSD genes may confirm that the effect of AFB1 is also on the concentration of testes, whether in the puberty stage, as indicated by Supriya et al. (29) and Mohammed et al. (11), or in the prepubertal stage, as indicated by the results of the current study. Moreover, the current pathological changes observed in the testicular and epididymal tissues of both immature and mature male rats as a result of AFB1 treatment are additional evidence that aflatoxicosis is effective in the prepubertal stage and continues its effect until after puberty. These findings were in agreement with those reported by Mukumu and Macharia (30), Owumi et al. (17), and Mohammed et al. (11), who found similar pathological changes in the adult testicular tissues.

Conclusion

The study suggests that prepubertal exposure to Aflatoxin B1 is exceptionally toxic to the male reproductive system of rats, as it can adversely affect the rat’s male reproductive organs resulting in structural and functional alteration of the HPG axis.

Acknowledgment

The authors would like to acknowledge the Department of Physiology, Pharmacology, and Biochemistry, College of Veterinary Medicine, University of Al-Qadisiyah for their provision to perform the current work.

Conflict of Interest

The authors declare that there are no conflicts of interest regarding the publication and or funding of this manuscript