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Pubertal sexual development and endpoints for disrupted spermatogenesis in the model Xenopus tropicalis.
Svanholm S
,
Roza M
,
Marini D
,
Brouard V
,
Karlsson O
,
Berg C
.
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Peripubertal models to determine effects of anti-androgenic endocrine disrupting chemicals are needed. Using the toxicological model species Xenopus tropicalis, the aims of the study were to 1) provide data on sexual maturation and 2) characterise effects of short-term exposure to an anti-androgenic model substance. Juvenile (2.5 weeks post metamorphosis old) X. tropicalis were exposed to 0, 250, 500 or 1000 µg flutamide/L (nominal) for 2.5 weeks. Upon exposure termination, histology of gonads and Müllerian ducts was characterised in detail. New sperm stages were identified: pale and dark spermatogonial stem cells (SSCs). The testes of control males contained spermatozoa, indicating pubertal onset. The ovaries were immature, and composed of non-follicular and pre-vitellogenic follicular oocytes. The Müllerian ducts were more mature in females than males indicating development/regression in the females and males, respectively. In the 500 µg/L group, the number of dark SSCs per testis area was decreased and the number of secondary spermatogonia was increased. No treatment effects on ovaries or Müllerian ducts were detected. To conclude, our present data provide new knowledge on spermatogenesis, and pubertal onset in X. tropicalis. New endpoints for evaluating spermatogenesis are suggested to be added to existing assays used in endocrine and reproductive toxicology.
Fig. 1. Histomicrographs of testes from juvenile Xenopus tropicalis, five weeks post metamorphosis, showing A) seminiferous chords (dotted line) and seminiferous tubuli containing a lumen (solid line), B) spermatogonial stem cells (SSCs); pale SSCs (P) and dark SSCs (D), C) nests with proliferating secondary spermatogonia, D) nest with primary spermatocytes, E) secondary spermatocytes starting to differentiate into spermatids (arrow), F) spermatids (arrow). G) Fully mature spermatozoa (arrow). Sections are stained with haematoxylin-eosin. Dashed lines encircle germ cell nests.
Fig. 2. Testis maturity as determined by the most mature germ cell stage observed per male Xenopus tropicalis after short-term peripubertal exposure to 0, (Control), 250 (Low), 500 (Mid) or 1000 (High) µg flutamide/L. SSCs: spermatogonial stem cells, spg: spermatogonia, spc: spermatocytes. Number of individuals (n) is shown within the parenthesis.
Supplementary figure S1. Ovary from juvenile
female Xenopus tropicalis containing both non-follicular
and follicular pre-vitellogenic oocytes.
Supplementary figure S2. Müllerian duct (MD) stages from juvenile Xenopus tropicalis based on the scoring by Jansson et al. (2016). A) Stage 1: “Small rounded bulge of irregularly packed mesenchymal cells protruding from the kidney”. B) Stage 2: “Small bud protruding from the kidney” C) Stage 3: “Distinct structure attached to the lateral side of the kidney” D) Stage 4: “Distinct tubular structure without a cavity” E) Stage 5: “Distinct tubular structure with a cavity lined by elongated epithelial cells”.
Supplementary figure S3. Relationship between testis cross-section area and A) pale spermatogonial stem cells (SSCs, r2 = 0.02296), B) dark SSCs (r2 = 0.2760), C) total number of SSCs (r2 = 0.1311), D) secondary spermatogonia (spg, r2 = 0.05437) and E) primary spermatocytes (spc, r2 = 0.06203) in control males. *Statistical significant correlation (p<0.05), linear regression.
Supplementary figure S4. Relationship between ovary cross-section area and A) number of non-follicular oocytes (r2 = 0.01304), B) number of follicular oocytes (r2 = 0.8788) and C) total number of oocytes (r2 = 0.9258) in control females. ***Statistical significant correlation (p<0.0001), linear regression.