Postnatal testis development, Sertoli cell proliferation and number of different spermatogonial types in C57BL/6J mice made transiently hypo- and hyperthyroidic during the neonatal period

Abstract

The role of thyroid hormones in testis structure and function has been fairly well studied in laboratory rodents. However, there are no comprehensive data in the literature for mice regarding the effects of transiently induced neonatal hypo- and hyperthyroidism on testis and spermatogonial cell development from birth to adulthood. Our goals were to evaluate the effects of propylthiouracil (PTU) and triidothyronine (T3) on Sertoli cell proliferation/differentiation and to correlate these events with the evolution of the spermatogenic process, tubular lumen formation, blood vessel volume density, and size and number of different spermatogonial types. Although Sertoli cell maturation was accelerated or delayed, respectively, in T3- and PTU-treated mice, the pace of the germ cell maturation was only slightly altered before puberty and the period of Sertoli cell proliferation was apparently not affected by the treatments. However, compared with controls, the total number of Sertoli cells per testis from 10 days of age to adulthood was significantly increased and decreased in PTU- and T3-treated mice, respectively. In comparison to all other spermatogonia, type A(2) was the largest cell in all ages and groups investigated. The PTU-treated mice had a significantly increased total number of undifferentiated spermatogonia as well as volume and percentage of vessels/capillaries, probably due to the higher number of Sertoli cells, particularly at 10 days of age. Taken together, our results suggest that neonatal hypothyroidism may be a valuable tool for studying spermatogonial biology as well as a means for providing more spermatogonial stem cells that could potentially be used for spermatogonial transplantation, thereby optimizing the efficiency of this technique when young mice are used as donors.

Fig. 1

Body weight (A), testis weight (B) and gonadosomatic index (C) in controls, propylthiouracil PTU- and T3-treated animals killed at different ages after birth (mean + SEM). Different letters for the same age denote significant differences (P < 0.05).

Fig. 3

Seminiferous cord/tubule sections of controls (A, B, E, H, K, N and Q), propylthiouracil PTU-(C, F, I, L, O and R) and T3-treated mice (D, G, J, M, P and S) killed between 1 and 100 days of age. Bar = 20 μm (A–J) and 40 μm (K–S). sc, seminiferous cords; st, seminiferous tubules; asterisk, lumen; arrows, Sertoli cells. The insert indicates the most advanced cell type: G, gonocyte; A₃, type A₃ spermatogonia; Pl, pre-leptotene spermatocyte; P, pachytene spermatocyte; Ar 1, step 1 round spermatid; Al 7/8, step 7/8 elongated spermatid; Al 13, step 13 elongated spermatid.

Fig. 2

The gonocyte and spermatogonia morphology in young animals followed criteria similar to those described by Vergouwen et al. (1991) and Chiarini-Garcia & Russell (2001). Briefly, gonocytes have the largest volume with one to three nucleoli. The undifferentiated spermatogonial population shows a nucleus with mottled chromatin and nuclear vacuoles are often present in the plane of the section (arrows). Differentiated type A₁ spermatogonia have a nucleus clearly demarcated from the surrounding nucleoplasm; differentiated type A₂ spermatogonia have small flakes (< 10%) of heterochromatin rimming the nuclear envelope; the differentiated type A₃ spermatogonia nucleus is slightly increased compared with A₂ and the nucleoli are generally reticulated and not well defined but extend deeply into the nucleoplasm; and differentiated type A₄ spermatogonia have 30–70% of the nuclear envelope encrusted with heterochromatin. Only small differences are noted when these cells are compared with A₃. Intermediate spermatogonia present shallow heterochromatin lining 70–100% of the nucleus, making the nuclear/cytoplasmic boundary well delineated. Type B spermatogonia (B) have small, densely stained spots of heterochromatin attached to the nuclear envelope. Bar = 7 μm.

Fig. 4

Diameter of seminiferous cords/tubules in controls, propylthiouracil PTU- and T3-treated animals killed at different ages after birth (mean ± SEM). Different letters for the same age denote significant differences (P < 0.05).

Development of lumen in the seminiferous cords/tubules in controls, propylthiouracil PTU- and T3-treated animals killed at different ages after birth (mean + SEM). Different letters for the same age denote significant differences (P < 0.05).

Fig. 6

Individual germ cell volume in control, propylthiouracil PTU- and T3-treated mice killed between 5 and 100 days of age. Different letters for the same age denote significant differences (P < 0.05) between groups. Undifferentiated type A (A_und), differentiated type A (A_1–4), intermediate (In) and type B (B) spermatogonia (mean ± SEM). Different letters for the same age germ cell type indicate significant differences (P < 0.05).

Fig. 7

Total number of germ cells per testis in control, propylthiouracil PTU- and T3-treated mice killed between 5 and 100 days of age. Different letters for the same age denote significant differences (P < 0.05) between groups. Undifferentiated type A (A_und), differentiated type A (A_1–4), intermediate (In) and type B (B) spermatogonia (mean + SEM). Different letters for the same age germ cell type indicate significant differences (P < 0.05).

Fig. 8

Total number of undifferentiated (A) and differentiated (B) spermatogonia, and total number of combined undifferentiated and differentiated spermatogonia (C) per testis in control, propylthiouracil PTU- and T3-treated mice killed between 5 and 100 days of age. Different letters for the same age indicate significant differences (P < 0.05) between groups (mean + SEM).

Fig. 9

Sertoli cell nuclear volume (A) and total number of Sertoli cells per testis (B) in controls, propylthiouracil PTU- and T3-treated mice killed at different ages. Different lowercase letters for the same age indicate significant differences (P < 0.05) between treatments and different uppercase letters indicate significant differences between the ages investigated (P < 0.05) (mean + SEM).