Estrogen promotes germ cell and seminiferous tubule development in the baboon fetal testis

Abstract

The foundation for development of the male reproduction system occurs in utero, but relatively little is known about the regulation of primate fetal testis maturation. Our laboratories have shown that estrogen regulates key aspects of the physiology of pregnancy and fetal development. Therefore, in the present study, we characterized and quantified germ cells and Sertoli cells in the fetal baboon testis in late normal gestation (i.e., Day 165; term is 184 days) and in baboons administered the aromatase inhibitor letrozole throughout the second half of gestation to assess the impact of endogenous estrogen on fetal testis development. In untreated baboons, the seminiferous cords were comprised of undifferentiated (i.e., type A) spermatogonia classified by their morphology as dark (Ad) or pale (Ap), gonocytes (precursors of type A spermatogonia), unidentified cells (UI), and Sertoli cells. In letrozole-treated baboons, serum estradiol levels were decreased by 95%. The number per milligram of fetal testis (x10(4)) of Ad spermatogonia (0.42 +/- 0.11) was 45% lower (P = 0.03), and that of gonocytes (0.58 +/- 0.06) and UI (0.45 +/- 0.12) was twofold greater (P < 0.01 and P = 0.06, respectively), than in untreated baboons. Moreover, in the seminiferous cords of estrogen-deprived baboons, the basement membrane appeared fragmented, the germ cells and Sertoli cells appeared disorganized, and vacuoles were present. We conclude that endogenous estrogen promotes fetal testis development and that the changes in the germ cell population in the estrogen-deprived baboon fetus may impair spermatogenesis and fertility in adulthood.

FIG. 1.

Maternal peripheral serum estradiol concentrations in baboons left untreated (closed circles; each data point is the mean of n = 4–8 animals) or treated daily with letrozole (115 μg (kg body wt)⁻¹ day⁻¹, s.c.; open circles; n = 6) between Days 100 and 164 of gestation (term, 184 days).

FIG. 2.

Serum estradiol levels (mean ± SEM) in male fetuses (i.e., umbilical artery; A) and maternal uterine vein (B) and serum testosterone (T) in male fetuses (C) on Day 165 in baboons left untreated (n = 6) or treated with letrozole (n = 5) on Days 100–164. *P < 0.01 vs. untreated animals.

FIG. 3.

Fetal serum FSH (A) and LH (B) levels (mean ± SEM) in the baboons on which estradiol and testosterone levels are shown in Figure 2.

FIG. 4.

Histology of seminiferous cords within the fetal testis of untreated baboons on Day 165 showing type A dark spermatogonia (Ad), type A pale spermatogonia (Ap), unidentified cell (UI), Sertoli cell (SC), and gonocyte (G; inset). Bar = 10 μm.

FIG. 5.

Representative morphology of seminiferous cords of fetal testis on Day 165 in baboons left untreated (A) or treated on Days 100–164 with letrozole (B). BM, basement membrane; FBM, fragmented basement membrane. Bar = 10 μm.

FIG. 6.

Numbers of type A dark spermatogonia (Ad), gonocytes, and unidentified cells (UI) in the fetal testis on Day 165 of gestation in baboons untreated or treated with letrozole on Days 100–164. *P ≤ 0.03, **P < 0.01, and ⁺P = 0.06 vs. untreated baboons.

FIG. 7.

Caspase 3 immunocytochemistry in the fetal testis on Day 165 of gestation in baboons left untreated (A, C, and E) or treated on Days 100–164 with letrozole (B, D, and F). Replacement of caspase 3 primary antibody with species irrelevant immunoglobulin also is shown (G). Ad, type A dark spermatogonia; Ap, type A pale spermatogonia; UI, unidentified cell. Bar = 10 μm.

FIG. 8.

MKI67 immunocytochemistry in the fetal testis on Day 165 of gestation in baboons left untreated (A) or treated on Days 100–164 with letrozole (B) and in the testis of an untreated adult baboon. Bar = 25 μm.