Low Doses of Glyphosate/Roundup Alter Blood-Testis Barrier Integrity in Juvenile Rats

Abstract

It has been postulated that glyphosate (G) or its commercial formulation Roundup (R) might lead to male fertility impairment. In this study, we investigated the possible effects of G or R treatment of juvenile male rats on blood-testis barrier function and on adult male sperm production. Pups were randomly assigned to the following groups: control group (C), receiving water; G2 and G50 groups, receiving 2 and 50 mg/kg/day G respectively; and R2 and R50 groups receiving 2 and 50 mg/kg/day R respectively. Treatments were performed orally from postnatal day (PND) 14 to 30, period of life that is essential to complete a functional blood-testis barrier. Evaluation was done on PND 31. No differences in body and testis weight were observed between groups. Testis histological analysis showed disorganized seminiferous epithelium, with apparent low cellular adhesion in treated animals. Blood-testis barrier permeability to a biotin tracer was examined. A significant increase in permeable tubules was observed in treated groups. To evaluate possible mechanisms that could explain the effects on blood-testis barrier permeability, intratesticular testosterone levels, androgen receptor expression, thiobarbituric acid reactive substances (TBARS) and the expression of intercellular junction proteins (claudin11, occludin, ZO-1, connexin43, 46, and 50 which are components of the blood-testis barrier) were examined. No modifications in the above-mentioned parameters were detected. To evaluate whether juvenile exposure to G and R could have consequences during adulthood, a set of animals of the R50 group was allowed to grow up until PND 90. Histological analysis showed that control and R50 groups had normal cellular associations and complete spermatogenesis. Also, blood-testis barrier function was recovered and testicular weight, daily sperm production, and epididymal sperm motility and morphology did not seem to be modified by juvenile treatment. In conclusion, the results presented herein show that continuous exposure to low doses of G or R alters blood-testis barrier permeability in juvenile rats. However, considering that adult animals treated during the juvenile stage showed no differences in daily sperm production compared with control animals, it is feasible to think that blood-testis barrier impairment is a reversible phenomenon. More studies are needed to determine possible damage in the reproductive function of human juvenile populations exposed to low doses of G or R.

Keywords: blood–testis barrier; glyphosate; male reproduction; roundup; testis.

Figure 1

Experimental protocol used to evaluate the effect of different doses of G and R treatment on rats at PND 31 and PND 90.

Figure 2

Effect of G and R treatment on testicular histology. Animals (n = 5/group) were treated with 2 and 50 mg/kg/day of G or R from PND 14 to 30. At PND 31, animals were euthanized, and testes were removed and fixed in Bouin solution. Sections (3–5 μm) obtained from the poles and equatorial areas were stained with hematoxylin/eosin and examined by light microscopy. Representative pictures are shown. Upper panels: scale bar, 50 µm. Higher magnifications images are shown in lower panels: scale bar, 10 µm.

Figure 3

Effect of G and R treatment on BTB permeability. Animals (n=7/group) were treated with 2 and 50 mg/kg/day of G or R from PND 14 to 30. At PND 31, animals were euthanized, and testis were dissected and used for BTB integrity assay. Testes were injected with a biotin tracer (red) and cell nuclei were dyed with 4′-6-diamidino-2-phenylindole (DAPI, blue). (A) Representative pictures are shown in upper panels. Scale bar, 50µm. Higher magnifications images are shown in lower panels. Scale bar, 10 µm. (B) Quantification of permeable tubules in each experimental group. At least 50 seminiferous tubules from three nonconsecutive testis sections from each rat were examined. Results are presented as mean ± SD, different letters indicate statistically significant differences P<0.05.

Figure 4

Analysis of possible mechanisms involved in G and R effects on BTB integrity. Animals (n=5/group) were treated with 2 or 50 mg/kg/day of G or R from PND 14 to 30. At PND 31, animals were euthanized, and testes were homogenized to evaluate (A) intratesticular testosterone and (B) Thiobarbituric Acid Reactive Substances (TBARS). Additionally, testes were used to evaluate mRNA levels of genes encoding for (A) Androgen Receptor (AR) and (C) BTB proteins (claudin11, occludin, ZO-1, connexin43, 46, and 50). HPRT1 was used as internal control. Results are presented as mean ± SD, different letters indicate statistically significant differences P<0.05.

Figure 5

Effect of juvenile R treatment on adult testicular histology and BTB integrity. Animals (n = 7/group) were treated with 50 mg/kg/day of R from PND 14 to 30. At PND 90, animals were euthanized. (A) Testes were removed and fixed in Bouin solution. Sections (3–5 μm) obtained from the poles and equatorial areas were stained with hematoxylin/eosin and examined by light microscope. Representative pictures are shown. Higher magnification images are shown in lower panels. Scale bar, 50 µm. (B) Quantitative analysis of the frequency of the stages of the cycle of the seminiferous tubule epithelium in each experimental group. The stages are indicated by roman numerals I–XIV and expressed as percentage. Results are presented as mean ± SD. No statistically significant differences were found between Control and R 50 mg/kg/day group. (C) Testes were injected with a biotin tracer (red) and cell nuclei were dyed with 4′-6-diamidino-2-phenylindole (DAPI, blue). Representative pictures are shown in upper panels. Scale bar, 50 µm. Higher magnification images are shown in lower panels. Scale bar, 10 µm.(D) Quantification of permeable tubules in each experimental group. At least 50 seminiferous tubules from three nonconsecutive testis sections from each rat were examined. Results are presented as mean ± SD, different letters indicate statistically significant differences P < 0.05.

Figure 6

Effect of juvenile R treatment on daily sperm production (DSP) and sperm parameters. Animals (n = 7/group) were treated with 50 mg/kg/day of R from PND 14 to 30. At PND 90, (A) testes were collected to determine DSP; (B, C) epididymal sperm were used to analyze sperm motility and morphology. (A, B) Results are presented as mean ± SD, different letters indicate statistically significant differences P < 0.05. (C) Representative microphotographs are shown.