Morphological changes during pubertal Leydig cell differentiation following maternal DES exposure

Brief maternal exposure of rats to the xenobiotics dibutyl phthalate or diethylstilbestrol alters adult-type Leydig cell development in male offspring

2013 Study Abstract

Maternal exposure to estrogenic xenobiotics or phthalates has been implicated in the distortion of early male reproductive development, referred to in humans as the testicular dysgenesis syndrome. It is not known, however, whether such early gestational and/or lactational exposure can influence the later adult-type Leydig cell phenotype.

In this study, Sprague-Dawley rats were exposed to dibutyl phthalate (DBP; from gestational day (GD) 14.5 to postnatal day (PND) 6) or diethylstilbestrol (DES; from GD14.5 to GD16.5) during a short gestational/lactational window, and male offspring subsequently analysed for various postnatal testicular parameters.

All offspring remained in good health throughout the study. Maternal xenobiotic treatment appeared to modify specific Leydig cell gene expression in male offspring, particularly during the dynamic phase of mid-puberty, with serum INSL3 concentrations showing that these compounds led to a faster attainment of peak values, and a modest acceleration of the pubertal trajectory. Part of this effect appeared to be due to a treatment-specific impact on Leydig cell proliferation during puberty for both xenobiotics.

Taken together, these results support the notion that maternal exposure to certain xenobiotics can also influence the development of the adult-type Leydig cell population, possibly through an effect on the Leydig stem cell population.

Discussion

In this pilot study, and as in earlier studies, a dosing regimen for both DES and DBP was chosen, which had as the primary objective sufficient xenobiotic exposure to induce minimal though statistically significant disruption of the classic end point of a reduced AGD in male pups, though without obvious effects on general health parameters. Thus, any change observed in specific parameters is not attributable to a general health deficit, but should be due to a specific disruption of those features. For this reason also, the few maternally DBP-treated offspring exhibiting gross testicular dysgenesis or atrophy were also excluded, so as not to invoke effects which might be the consequences of gross anatomical aberration. These criteria were met and strongly supported by the generally low within- and between-group variance in most parameters.

This study has focused on the long-term effects of maternal exposure to DBP and DES in male rats and in particular on the phenotype of adult-type Leydig cells in the intact testes. It does not aim therefore to reassess the known acute effects of maternal exposure leading to gross testicular dysgenesis (DBP), or to perinatal death (DES) that have been previously reported. The observations relating to this pathology are essentially in agreement with those described by other authors. As measures of Leydig cell phenotype, we have assessed specific Leydig cell numbers and gene expression shortly after cessation of treatment at PND10, at a single dynamic time point during their pubertal differentiation (PND24), and after the establishment of adult endocrine stability at PND90. Additionally, we have assessed the secretion into the circulation of the Leydig cell peptide hormone INSL3, which we have shown is expressed constitutively, and accurately reflects the numbers and/or differentiation status of Leydig cells in humans and other species. Total circulating testosterone was also measured, as was circulating LH. Both parameters were assessed only for single time points per day (from 9:00 a.m. to 11:00 a.m.), and therefore being pulsatile hormones, such measures are of statistical value only for the sample population, where the T/LH ratio should reflect the average capacity of Leydig cells for steroidogenesis. Although on PND24 at the beginning of puberty, there appeared to be a trend for both DES and DBP groups to have lower individual T/LH ratios compared to controls (data not shown), this did not reach statistical significance (P=0.2), and was definitely not maintained at the later time point (PND90).

Although it is normal practice to assess testicular growth during puberty, as for most other organs, as relative to whole body weight, for the testis this may be spurious. Postnatal growth of the testis is largely due to specific proliferation of Leydig and Sertoli cells, as well as of the germinal epithelium, as a consequence of the activation of the hormones of the HPG axis during puberty, and thus, is not necessarily governed by the same factors as for the general postnatal growth of other organs and tissues. Thus, the absolute testis weight for a cohort of identically aged rats appears to be a better assessment of the impact of maternal exposure to xenobiotics, compared to the relative testis weight. Importantly, these results indicate an effect on testis weight only of gestational DES treatment, and only at PND24, the most dynamic phase of testicular growth. This observation is also in full agreement with the estimate of total Leydig cell numbers per testis, as well as with the secretion profiles for the peptide hormone INSL3. Leydig cell numbers were close to those previously reported by Zirkin and Ewing and Hardy et al. Close inspection of the testicular histology (data not shown) indicated that the increase in Leydig cell numbers did not appear to be associated with any kind of focal hyperplasia as has been reported for the fetal testis under DBP treatment. Nor was there any evidence for focal atrophy in any of the testes examined, which, however, did exclude any of the grossly dysgenic testes resulting from maternal DBP treatment. Taken together, all of these data strongly suggest that particularly maternal estrogen exposure serves to accelerate postnatal Leydig cell differentiation and proliferation, resulting in an advance in the median value of INSL3 secretion from PND42 (control) to PND37 (DES treatment). The values for circulating LH at PND10 and PND24 would support this argument, suggesting an impact of gestational xenoestrogen exposure also on the development of the HPG axis. In contrast, maternal DBP treatment has little if any effect on either testis growth or Leydig cell numbers, although the circulating INSL3 profiles do suggest modest acceleration in Leydig cell differentiation, leading to earlier and higher circulating levels of INSL3, for example, at PND35, and which is reflected in the trends to increased Insl3 mRNA at PND24. The lack of xenobiotic impact on adult testosterone level is not surprising given its high within-animal fluctuation, and has been reported previously.

Looking at specific testicular gene expression, it is also evident that maternal DBP treatment is having a similar positive impact on those gene transcripts involved in the first stages of the establishment of steroidogenesis, namely, Cyp11a1, Hsd17b3 and Star, though only at PND10. Interestingly, transcripts for the full-length LH receptor (Lhcgr) as well as for the side-chain cleavage enzyme (Cyp11a1), which are both relatively early-intermediate markers of Leydig cell differentiation, are both reduced for the DES-treated group at PND24. Note that Insl3 is a relatively late marker of Leydig cell differentiation. Taken together, these observations again support the notion that early estrogenic exposure is causing a temporal disruption of normal Leydig cell proliferation and differentiation, with early progenitor Leydig cells proliferating longer in an immature state (PND10), but then differentiating faster (PND24) to complete puberty somewhat earlier (median circulating INSL3 at PND37). The DBP effects are probably comparable to these, or intermediate between DES and control groups, though less intense, leading to few differences in the measured parameters compared to controls. These results are similar to those recently reported for maternal bisphenol A exposure, which appears to have comparable estrogenic effects on adult-type Leydig cell differentiation. There is also a good correlation between the findings reported here and our recent study on the influence of DBP and DES upon adult-type Leydig cell regeneration following ethane dimethane sulfonate ablation, where also the kinetics of Leydig cell differentiation was modulated by these xenobiotics.

This study has also highlighted the value of measuring circulating INSL3 during puberty, by emphasizing what has earlier been referred to as the ‘overshoot’ effect. As Leydig cells differentiate during puberty, concomitant with the large and initially not well regulated pulses of LH from the pituitary, they initially give rise to an overproduction of INSL3 as a measure of Leydig cell differentiation status. Unlike testosterone, whose enzymatic production is acutely regulated by feedback from the pituitary, INSL3 is constitutively expressed. Circulating testosterone, therefore, does not reveal such an overshoot effect, but once a maximum is attained, the testosterone level is held more or less constant by acute feedback regulation from the HPG axis. Following the overshoot at around day 40, circulating INSL3 production, and presumably Leydig cell activity, relaxes to yield stable and considerably lower adult levels by about days 60–90, when Leydig cell differentiation status has become regularized at a lower level as a consequence of chronic HPG control. This is reflected also in the reduced measure of INSL3 per individual Leydig cell. Interestingly, although this cannot be statistically supported, the results suggest that at PND90, both DBP and DES treatments lead to a modest reduction in LH and in total Leydig cell numbers per testis, which then appear to maintain their pubertal level of INSL3 production per individual cell.

The results of this study suggest that there may indeed be long-term though subtle consequences of maternal xenobiotic exposure for adult testis function, particularly during puberty and the establishment of the stable adult phenotype. Obviously, more detail is needed in order to explore precisely the mechanisms by which these long-term changes are achieved.

Sources and more information
  • Full text (free access) : Brief maternal exposure of rats to the xenobiotics dibutyl phthalate or diethylstilbestrol alters adult-type Leydig cell development in male offspring, Journal List, Asian J Androl PMC3739150, 2013 Jan 14.
  • Diagram to illustrate the treatment and sampling regimen applied (small dots above the bar indicate approximate blood sampling times). The morphological changes during pubertal Leydig cell differentiation (after,,) are illustrated at their corresponding times in the upper section of the figure. GD, gestational day; PND, postnatal day; DBP, dibutyl phthalate; DES, diethylstilbestrol featured image credit PMC3739150/figure/fig1.
DES DIETHYLSTILBESTROL RESOURCES

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