Neonatal exposure to a glyphosate-based herbicide alters the uterine differentiation of prepubertal ewe lambs.

The exposure to endocrine-disrupting compounds (EDCs), such as glyphosate-based herbicides (GBHs), during early life might alter female fertility. The aim of the present study was to evaluate the effects of neonatal exposure to a GBH on sheep uterine development. To achieve this, Friesian ewe lambs were exposed to GBH (2 mg/kg of body weight/day; n = 12) or vehicle (controls; n = 10) through s.c. injections, from postnatal day (PND) 1 to PND14; on PND45, the uteri were obtained to evaluate histomorphological and molecular parameters. Morphological parameters were determined by picrosirius-hematoxylin staining. Protein expression of Ki67 (as a cell proliferation marker), p27, and molecules involved in uterine organogenetic differentiation was measured by immunohistochemistry. We also determined the mRNA expression of the IGF molecular pathway by RT-PCR. Although histomorphology was not modified, the uteri of GBH-exposed ewe lambs showed lower cell proliferation, together with higher p27 protein expression. In addition, the uteri of GBH-exposed ewe lambs showed increased gene expression of insulin-like growth factor binding protein 3 (IGFBP-3), decreased expression of ERα in the luminal (LE) and glandular (GE) epithelia and in the subepithelial stroma (SS), and lower PR expression in the LE but higher in the GE and SS. In addition, GBH treatment decreased the uterine expression of Wnt5a in the GE, of Wnt7a in the SS, of ß-catenin in the LE and GE, of Hoxa10 in the SS, and of Foxa2 in the GE as compared with controls. In conclusion, neonatal exposure to GBH decreased cell proliferation and altered the expression of molecules that control proliferation and development in the uterus. All these changes might have adverse consequences on uterine differentiation and functionality, affecting the female reproductive health of sheep. GBH may be responsible for uterine subfertility, acting as an EDC.

Neonatal exposure to a glyphosate-based herbicide alters the histofunctional differentiation of the ovaries and uterus in lambs.

The aim of the present study was to compare the effect of oral and subcutaneous exposure to a glyphosate-based herbicide (GBH) on the female reproductive system, specifically in the ovaries and uterus of prepubertal lambs. To this end, ewe lambs were exposed to a s.c. (n: 5) or an oral (n: 5) environmentally relevant dose of GBH (2 mg/kg/day) or to vehicle (controls, n: 12), from postnatal day (PND) 1 to PND14. Serum glyphosate and aminomethylphosphonic acid (AMPA) concentrations were measured on PND15 and PND45. The ovaries and uterus were obtained and weighed on PND45. Ovarian follicular dynamics and uterine morphological features were determined by picrosirius-hematoxylin staining. The proliferation marker Ki67 was evaluated by immunohistochemistry in ovarian and uterine samples. Glyphosate but not AMPA was detected in serum of exposed lambs on PND15, whereas neither glyphosate nor AMPA were detected on PND45. Controls were negative for glyphosate and AMPA on PND15 and PND45. GBH exposure did not affect ovarian or uterine weight. However, on PND45, the ovary of GBH-exposed lambs showed altered follicular dynamics, increased proliferation of granulosa and theca cells, and decreased mRNA expression of FSHR and GDF9, whereas their uterus showed decreased cell proliferation but no alterations in the histomorphology or gene expression. In conclusion, GBH exposure altered the ovarian follicular dynamics and gene expression, and the proliferative activity of the ovaries and uterus of lambs. It is noteworthy that all the adverse effects found in the ovaries and uterus of both GBH-exposed groups were similar, independently of the administration route.

Neonatal exposure to xenoestrogens impairs the ovarian response to gonadotropin treatment in lambs.

Bisphenol A (BPA) and diethylstilbestrol (DES) are xenoestrogens, which have been associated with altered effects on reproduction. We hypothesized that neonatal xenoestrogen exposure affects the ovarian functionality in lambs. Thus, we evaluated the ovarian response to exogenous ovine FSH (oFSH) administered from postnatal day 30 (PND30) to PND32 in female lambs previously exposed to low doses of DES or BPA (BPA50: 50 µg/kg per day, BPA0.5: 0.5 µg/kg per day) from PND1 to PND14. We determined: i) follicular growth, ii) circulating levels of 17ß-estradiol (E2), iii) steroid receptors (estrogen receptor alpha, estrogen receptor beta, and androgen receptor (AR)) and atresia, and iv) mRNA expression levels of the ovarian bone morphogenetic protein (BMPs) system (BMP6, BMP15, BMPR1B, and GDF9) and FSH receptor (FSHR). Lambs neonatally exposed to DES or BPA showed an impaired ovarian response to oFSH with a lower number of follicles ≥2 mm in diameter together with a lower number of atretic follicles and no increase in E2 serum levels in response to oFSH treatment. In addition, AR induction by oFSH was disrupted in granulosa and theca cells of lambs exposed to DES or BPA. An increase in GDF9 mRNA expression levels was observed in oFSH-primed lambs previously treated with DES or BPA50. In contrast, a decrease in BMPR1B was observed in BPA0.5-postnatally exposed lambs. The modifications in AR, GDF9, and BMPR1B may be associated with the altered ovarian function due to neonatal xenoestrogen exposure in response to an exogenous gonadotropin stimulus. These alterations may be the pathophysiological basis of subfertility syndrome in adulthood.

Neonatal exposure to bisphenol A or diethylstilbestrol alters the ovarian follicular dynamics in the lamb.

We hypothesized that neonatal xenoestrogen exposure affects the ovarian follicular dynamics in lambs. Female lambs were exposed from postnatal day (PND) 1-14 to low doses of diethylstilbestrol (DES) or bisphenol A (BPA). At PND 30, the follicular dynamics and ovarian biomarkers (ERα, ERß, AR, Ki67, p27) were evaluated. Lambs exposed to DES or BPA showed a decline in the stock of primordial follicles with stimulation of follicular development. BPA reduced ovarian weight and increased the number of multioocyte follicles. BPA promoted proliferation of granulosa/theca cells in antral follicles, and increased both the number of antral atretic follicles and p27 expression. Neonatal exposure to BPA or DES reduced the primordial follicle pool by stimulating their initial recruitment and subsequent follicle development until antral stage. In prepubertal lambs, the accelerated folliculogenesis resulted in increased incidence of atretic follicles. These alterations may affect the ovarian function in the adult.