Transient Hypothyroidism: Dual Effect on Adult-Type Leydig Cell and Sertoli Cell Development

Abstract

Transient neonatal 6-propyl-2-thiouracil (PTU) induced hypothyroidism affects Leydig and Sertoli cell numbers in the developing testis, resulting in increased adult testis size. The hypothyroid condition was thought to be responsible, an assumption questioned by studies showing that uninterrupted fetal/postnatal hypothyroidism did not affect adult testis size. Here, we investigated effects of transient hypothyroidism on Leydig and Sertoli cell development, employing a perinatal iodide-deficient diet in combination with sodium perchlorate. This hypothyroidism inducing diet was continued until days 1, 7, 14, or 28 postpartum (pp) respectively, when the rats were switched to a euthyroid diet and followed up to adulthood. Continuous euthyroid and hypothyroid, and neonatal PTU-treated rats switched to the euthyroid diet at 28 days pp, were included for comparison. No effects on formation of the adult-type Leydig cell population or on Sertoli cell proliferation and differentiation were observed when the diet switched at/or before day 14 pp. However, when the diet was discontinued at day 28 pp, Leydig cell development was delayed similarly to what was observed in chronic hypothyroid rats. Surprisingly, Sertoli cell proliferation was 6- to 8-fold increased 2 days after the diet switch and remained elevated the next days. In adulthood, Sertoli cell number per seminiferous tubule cross-section and consequently testis weight was increased in this group. These observations implicate that increased adult testis size in transiently hypothyroid rats is not caused by the hypothyroid condition per se, but originates from augmented Sertoli cell proliferation as a consequence of rapid normalization of thyroid hormone concentrations.

Keywords: Leydig cell; Sertoli cell; macroorchidism; proliferation; testis; testosterone; thyroid hormone.

Figure 1

Schematic overview of the experimental design. Dams were treated with an iodide poor diet supplemented with perchlorate for 2 weeks prior to mating to induce hypothyroidism. The diet switched to a control diet (euthyroidism is restored) on the day of birth (T1) or 7 (T7), 14 (T14), or 28 (T28) days postpartum. The offspring was sacrificed with regular intervals. A propylthiouracil (PTU; finely dotted line) treated (treatment from day of birth to day 28 postpartum), continuously hypothyroid (iodide poor diet and perchlorate induced; tickly dotted line) and euthyroid group served as controls. | represents a day of sacrifice.

Figure 2

Body (A) and testis (B) weights of euthyroid control, continuously hypothyroid and transiently (T28/PTU-treated) hypothyroid rats. The body weights of the euthyroid control rats are significantly higher for the entire experimental period (p < 0.05). Testis weights of T28 and PTU rats were significantly lower compared to the euthyroid controls up to day 49 pp, caught up by day 63 pp, to become significantly higher 2 weeks later (p < 0.05). Testis weights of the continuously hypothyroid animals are identical to the euthyroid controls at day 100 pp. Values represent means + SEM. # indicates a significant different from the euthyroid control group to all other groups (p < 0.05). Groups that do not share the same letter differ significantly on day 100 pp, n = 5–12.

Figure 3

Plasma thyroid stimulating hormone (TSH) (A), total thyroxine (T₄) (B), and total tri-iodothyronine (T₃) (C) concentrations of euthyroid control, continuously hypothyroid and transiently (T28/PTU-treated) hypothyroid rats. T₃ and T₄ levels are decreased in hypothyroid animals. After the switch to a euthyroid control diet, TSH values recover rapidly, whereas T₃ and T₄ levels follow soon after. Bars represent means + SEM. ^*Indicates a significant difference from the control group (p < 0.05), n = 5–8.

Figure 4

Seminiferous tubule diameter (in mm) (A), percentage of BrdU labeled Sertoli cells (B), and percentage of BrdU labeled Leydig cells (C) in euthyroid control, continuously hypothyroid and transiently (T28/PTU-treated) hypothyroid rat testes. Testes were double stained using BrdU and SOX9 (Sertoli cells) or HSD3B (Leydig cells) as markers. The Sertoli cells in the euthyroid control group already stopped proliferation well-before day 29 pp. Bars represent means + SEM. ^*Indicates a significant difference from the control group (p < 0.05), n = 5–8.

Figure 5

Representative histological testis sections of 30-day-old rat testes. (A–C) BrdU (brown nuclei)/hematoxylin staining of a euthyroid control (A), chronic hypothyroid (B), and transient hypothyroid (T28) rat testis. (D–E) Fluorescent staining for SOX9 (red nuclei) and BrdU (green nuclei) of a euthyroid control (D), chronic hypothyroid (E), and transient hypothyroid (T28) rat testis (F). In the transient (T28) testis several SOX9/BrdU positive Sertoli cells were observed (arrows). Bar (A–C) represents 30 μm, bar (D–E) represents 40 μm.

Figure 6

Plasma luteinizing hormone (LH) (A), follicle stimulating hormone (FSH) (B), and testosterone (C) concentrations in euthyroid control, continuously hypothyroid and transiently (T28/PTU-treated) hypothyroid rats. FSH concentrations have not been determined in PTU treated rats. Bars represent means + SEM. ^*Indicates a significant difference from the control group (p < 0.05), n = 5–8.