Toxoplasma gondii infection enhances testicular steroidogenesis in rats.This review covers pertinent literature regarding the synthesis of androgens by the testis of the rodent, the subhuman primate and man. By increasing the tren prop 100 of the cholesterol side-chain cleavage mitochondrial enzyme complex, LH is rate-limiting for the synthesis testicular steroidogenesis in rats hesticular androgens by regulating the conversion of cholesterol to pregnenolone. In the testicular steroidogenesis in rats, available information suggests that pregnenolone is converted to testosterone primarily through the 4-ene pathway. In subhuman primates, the 4-ene pathway also appears to be predominant for testosterone synthesis in more primitive species, such as the marmoset and the baboon. However, in higher primates, such as the orang-utan, both the 4-ene and the 5-ene pathways appear to be utilized for testosterone synthesis.
Toxoplasma gondii infection enhances testicular steroidogenesis in rats. - PubMed - NCBI
Howdeshell, Johnathan Furr, Robin M. The fungicide prochloraz PCZ induces malformations in androgen-dependent tissues in male rats when administered during sex differentiation. The sensitivity of fetal testicular steroidogenesis to PCZ was investigated to test the hypothesis that the reported morphological effects from maternal exposure were associated with reduced testosterone synthesis. Pregnant Sprague-Dawley rats were dosed by gavage with 0, 7.
On GD 18, the effects of PCZ on fetal steroidogenesis were assessed by measuring hormone production from ex vivo fetal testes after a 3-h incubation. These results suggest that PCZ inhibits the conversion of progesterone to testosterone through the inhibition of CYP These results demonstrate that PCZ lowers testicular testosterone synthesis by inhibiting CYP17 activity which likely contributes to the induced malformations in androgen-dependent tissues of male offspring. Development of the male reproductive system during gestation is dependent on androgen stimulation, and environmental chemicals that reduce androgen function have been shown to alter this development Gray et al.
Some phthalates such as di n -butyl phthalate and di ethylhexyl phthalate DEHP disrupt male rat development by reducing fetal testosterone and insl3 production in the fetal testis Mylchreest et al.
Although, it is unknown whether similar effects might be occurring in the human population, maternal levels of certain phthalates have been associated with reduced anogenital distance in male children, a marker of androgen function, which suggests these chemicals might affect human reproductive development Swan et al. The conazole pesticide prochloraz PCZ , which is an agricultural fungicide, also disrupts male rat differentiation.
Since PCZ displays two anti-androgenic mechanisms, it is unclear which of these modes of action contributes to the altered development. First, PCZ is reported to reduce fetal testosterone production in vivo and ex vivo Laier et al.
Studies examining the effects of PCZ on fetal testosterone production used a limited dose range, precluding assessment of the PCZ-testosterone production dose-response relationship Laier et al. Additionally, the relationship between reduced fetal testosterone production and morphological effects in adult males has not been investigated.
Furthermore, previous studies associating PCZ effects on male offspring measured fetal testosterone on gestational day GD 21, which would be after the testosterone surge within the fetal male Huhtaniemi, ; Laier et al. These uncertainties make it difficult to assess whether the changes in androgen-dependent tissues in adult males due to maternal PCZ exposure are the result of decreased testosterone or AR antagonism.
Therefore, this study first examined the sensitivity of fetal steroidogenesis during the testosterone surge to PCZ treatment by defining a five-point dose-response curve of ex vivo testosterone production. These data were compared to the reported dose-response of morphological effects in adult male rats Noriega et al.
Lastly, we measured amniotic fluid levels of PCZ to determine the concentration associated with reproductive malformations and to compare these values to the K i values for CYP17 inhibition and AR antagonism. Animals were provided Purina Rat Chow and watered ad libitum. Prior to dosing, animals were weight ranked and assigned to dose groups to minimize differences in means and variance among treatment groups.
Dams were dosed daily from GD 14 to 18 with 0, 7. Calculated dose was determined by daily weight measurements. On GD 18, dams were anesthetized with CO 2 , then decapitated between and h.
Trunk blood was collected for serum progesterone and estradiol measurement. Fetal testis testosterone production was evaluated using a method described previously Wilson et al. Briefly, the uterine tract was dissected and fetuses were quickly removed and placed in a tissue culture dish on ice. Each fetus was sexed under a dissecting microscope. The testes from the remaining males of each litter were removed for quantitative real-time polymerase chain reaction qRT-PCR.
Total RNA was extracted within 2—3 weeks using the method published by the manufacturer. RNA in the samples was quantified first using the Nanodrop spectrophotometer Nanodrop technologies Inc. The threshold cycle was chosen to ensure all reactions were within the range where the amplification remained exponential.
Copy number was determined from a standard curve generated by amplifying known cDNA quantities of each gene. PCZ inhibition of CYP17 hydroxylase activity was assessed using testis microsomes prepared from adult Sprague-Dawley rats, under the assumption that CYP17 activity is similar between adult and fetal Leydig cells.
The testes from each animal were removed and put into a cold mM potassium phosphate buffer solution pH 7. The final pellet was resuspended in mM potassium phosphate buffer pH 7. Enzymatic reactions with 0, 0. Reaction solutions consisted of mM potassium phosphate buffer pH 7. The multiple concentrations of progesterone substrate included 1. Reactions were stopped with 2 ml ethyl acetate at 10 min and placed in ice. Progesterone and its metabolites were extracted twice from the reaction mixture with 2 ml ethyl acetate, dried under nitrogen, and then spotted with a small volume of ethyl acetate onto a Whatman polyester-backed silica gel medium thin-layer chromatography TLC plate Fisher Scientific, Pittsburgh, PA.
Progesterone and metabolites were resolved using a 3: Hydroxylase activity was calculated as the sum of the produced progesterone metabolites resolved on the TLC plates: The sum was used under the assumption that all progesterone metabolites were derived from CYP17 hydroxylase activity e.
The values for K m , V max , and K i were the average of the two experimental runs. Maternal serum and amniotic fluid pooled by litter were collected from half of the control and treated dams 0, Serum and amniotic fluid samples were weighed in the 2-ml centrifuge tubes in which they were stored, and then transferred with acetonitrile rinse to ml polypropylene centrifuge tubes and diluted to 5 ml with acetonitrile.
The empty 2-ml tubes were reweighed to determine sample weights. Samples were vortexed, sonicated for 30 min, vortexed again, and then placed in a refrigerator overnight. Each concentrate was diluted to 1. Samples were transferred to 0. The filtrate was adjusted to 1. PCZ concentrations were determined using the response at wavelength nm or nm and an external standard method of quantitation. An Agilent MS was used to confirm the presence of PCZ in the samples using an atmospheric pressure electrospray interface and selective ion monitoring acquisition of the molecular ion positive polarity.
Hormone data from the right and left fetal testis of each male were averaged and then a litter mean was generated for analysis. Since testes were pooled by litter for RNA extraction, the individual data points were litter means. Heterogeneous data having SDs that increased with the means e. PCZ treatment had no statistically significant effect on dam body weight at necropsy, but the pesticide did significantly decrease maternal weight gain Table 2 , which suggests some minimal maternal toxicity.
Maternal serum progesterone levels were unaffected by PCZ. PCZ also significantly affected ex vivo fetal hormone production. PCZ decreased testosterone and androstenedione production but only at doses of Reduced hormone production in the fetus could result from one of several mechanisms including inhibition of steroidogenic gene expression or from inhibition of enzyme activity.
As suspected, PCZ did not alter testis gene expression. While PCZ did not alter the expression levels of these genes, it did inhibit in vitro CYP17 hydroxylase activity in a dose-dependent manner Fig. The K m and V max values determined from the Lineweaver-Burke plot were 1. Plotting the data in Hanes or Eadie-Hofstee plots did not aid in explaining the increased V max app. Lower or higher substrate concentrations were excluded from analysis to determine if they contributed to the increased V max app but removing them did not affect the result.
The intersection of the lines before the y-axis Fig. Lineweaver-Burke double reciprocal plot of the same data B. The average recovery from six PCZ-spiked samples was The PCZ concentration within the amniotic fluid was generally about half that in the maternal serum, except at the highest dose implying that placenta might have been a barrier to PCZ distribution or there was metabolism by the fetus Fig. PCZ was not detected in any of the control serum or amniotic fluid samples.
The variability of PCZ concentration within treatment groups did not correlate with the order of necropsy, indicating that the variability was not due to time between dosing and necropsy. The results of the current study support the hypothesis that PCZ suppression of testosterone production contributes to the developmental effects of PCZ in the male fetus.
Herein, this is the first study to show that PCZ inhibits testosterone production during the testosterone surge at the same dose levels as those that demasculinize the male rat in utero Noriega et al. The reported permanent morphological effects from gestational exposure to PCZ occurred at doses of A low incidence of nipple retention in adult males was reported in the There was no evidence that the elevated progestin levels contributed toward any fetal pathology, but this needs to be examined more thoroughly.
Summary of PCZ effects on fetal testosterone production from this study and reported incidence of altered development in adult males after maternal exposure. Effects in testis histology These results are consistent with those of a previous PCZ study which found that these genes, among others, were not affected at GD 21 or PND 16 after maternal exposure Laier et al.
These findings differ from those obtained with maternal di n -butyl phthalate exposure or DEHP exposure, compounds which produce anti-androgenic effects in male rats by inhibiting steroidogenic gene expression rather than directly inhibiting the enzymes Barlow et al. The herbicide linuron is similar to PCZ in that it reduces testosterone production ex vivo Hotchkiss et al.
The reason for the differences in adverse effects within male offspring, although both chemicals appear to work through similar mechanisms, is unknown. The enzymatic activity of CYP17 was significantly affected in the present study. How much of the PCZ inhibition of either the hydroxylase or lyase activity contributes to the reduced androgen production is not clear.
The amniotic fluid concentrations of PCZ at an effective dose for producing reproductive tract malformations were similar to the K i for CYP17 hydroxylase inhibition. However, one would need to also know what the testosterone and dihydrotestosterone levels were in the developing tissues to be more certain about the role of AR antagonism versus inhibition of testosterone synthesis inhibition by PCZ.
It is possible that the reproductive effects in the male rat result are due to a cumulative effect between the two anti-androgen mechanisms. However, more work is needed to model these effects and sort out the potential roles of these two mechanisms that disrupt the androgen-signaling pathway.
Maternal PCZ treatment causes a significant delay in parturition and at high-dose levels results in whole litter loss during delivery and some maternal death Laier et al. One of the maternal hormones critical in triggering parturition is estradiol.
Here we show that maternal serum estradiol levels are reduced at GD 18, which may be indicative of PCZ affecting the estradiol increase leading up to parturition Fang et al. Although it has been reported previously that PCZ inhibits aromatase in vitro Andersen et al. In conclusion, the results of this study indicate that PCZ inhibition of testosterone production contributes to the reproductive malformations in the male rat after maternal exposure.
The mechanism of reduced testosterone production does not involve down regulation of genes involved in steroidogenesis, but instead the direct inhibition of CYP17 enzyme activity. We would also like to thank Dr William Kelce for his comments on the manuscript. Oxford University Press is a department of the University of Oxford.