Testicular development in male rats is sensitive to a soy-based diet in the neonatal period

Abstract

Approximately 30% of infants in the United States are exposed to high doses of isoflavones resulting from soy infant formula consumption. Soybeans contain the isoflavones genistin and daidzin, which are hydrolyzed in the gastrointestinal tract to their genistein and daidzein aglycones. Both aglycones possess hormonal activity and may interfere with male reproductive development. Testosterone, which supports male fertility, is mainly produced by testicular Leydig cells. Our previous studies indicated that perinatal exposure of male rats to isoflavones induced proliferative activity in Leydig cells and increased testosterone concentrations into adulthood. However, the relevance of the neonatal period as part of the perinatal window of isoflavone exposure remains to be established. The present study examined the effects of exposure to isoflavones on male offspring of dams maintained on a casein-based control or whole soybean diet in the neonatal period, that is, Days 2 to 21 postpartum. The results showed that the soybean diet stimulated proliferative activity in developing Leydig cells while suppressing their steroidogenic capacity in adulthood. In addition, isoflavone exposure decreased production of anti-Müllerian hormone by Sertoli cells. Similar to our previous in vitro studies of genistein action in Leydig cells, daidzein induced proliferation and interfered with signaling pathways to suppress steroidogenic activity. Overall, the data showed that the neonatal period is a sensitive window of exposure to isoflavones and support the view that both genistein and daidzein are responsible for biological effects associated with soy-based diets.

Keywords: Leydig cells; androgen; androgens/androgen receptor; daidzein; endocrine disruptors; genistein; phytoestrogen; sex steroids; testis; toxicology.

FIG. 1

Illustration of the experimental approach. I) Time-bred Long-Evans dams (10 or 11 animals per group) were fed control diet from Gestational Day 12 to PND 1 and were either continued on the control diet or were fed the SOY diet from PND 2 to PND 21, that is, during the neonatal period. Groups of male rats from control and SOY diet groups, selected randomly from each litter, were killed at 22, 35, and 96 days of age to assess testicular function, that is, Leydig cell proliferation, serum T and 17β-estradiol (E2) concentrations, and testicular and Leydig cell T secretion. II) In vitro experiments were performed with Leydig cells isolated from 21- and 35-day-old male rats not previously exposed to dietary soy for incubation in media containing genistein and/or daidzein and Sertoli cells isolated from 9-day-old rats.

FIG. 2

Leydig cells were isolated from male rats at 22 (26–28 animals per group) and 35 days of age (14–17 animals per group) after exposure to isoflavones in the neonatal period. Proliferative activity was determined by [³H] thymidine incorporation and measured by liquid scintillation counting (A, C). Expression of cell cycle cyclin D3 was analyzed in Western blots of Leydig cells (PND 22) not labeled with [³H] thymidine (B). Cyclin D3 = 35 kDA, ACTB = 42 kDa. Proteins were normalized to ACTB, and the results are representative of densitometric analysis of three different assays. PND, postnatal day; CTRL, control; SOY, soy diet. Bars represent means ± SEM. *P < 0.05 versus control; **P < 0.01 versus control.

FIG. 3

Testis samples and Leydig cells were obtained from male rats at 22 days of age after exposure to isoflavones in the neonatal period as in Figure 1. AMH protein expression (A) was analyzed in testis homogenates (n = 6), whereas AMHR2 protein (B) was analyzed in Western blots of pooled Leydig cells. AMH = 63 kDa, AMHR2 = 63 kDa, ACTB = 42 kDa. Proteins were normalized to ACTB and results are representative of densitometric analysis of three different assays. CTRL, control; SOY, soy diet. Bars represent means ± SEM. *P < 0.05 versus control; **P < 0.01 versus control.

FIG. 4

Serum was separated from blood after euthanasia of male rats at 22, 35, and 96 days of age after exposure to isoflavones in the neonatal period. Serum samples were assayed for 17β-estradiol (E2) (A–C) and testosterone (T) (D–F) concentrations by RIA. PND, postnatal day; CTRL, control; SOY, soy diet. Bars represent means ± SEM. * P < 0.05 versus control; **P < 0.01 versus control.

FIG. 5

Testicular explants and Leydig cells were obtained from male rats at 22, 35, and 96 days of age after exposure to isoflavones in the neonatal period. Testicular explants (A–C) and Leydig cells (D–F) were incubated in triplicate in culture medium containing ovine LH (100 ng/ml) and assayed for testosterone (T) secretion by RIA. PND, postnatal day; CTRL, control; SOY, soy diet. Bars represent means ± SEM. *P < 0.05 versus control; **P < 0.01 versus control.

FIG. 6

Leydig cells, obtained at euthanasia from adult male rats (8–10 animals/group) exposed to isoflavones in the neonatal period were analyzed for expression of the StAR protein and steroidogenic enzymes by Western blot analysis using anti-StAR (A), anti-CYP11A1 (B), anti-HSD3B (C), anti-CYP17A1 (D), and anti-HSD17B3 (E) antibodies and appropriate secondary antibodies. Protein concentrations were normalized to ACTB, and the data for each graph represent the results from at least three Western blot assays. CTRL, control; SOY, soy diet. Bars represent means ± SEM **P < 0.01 versus control.

FIG. 7

Leydig cells were isolated and pooled from isoflavone-free male rats at 21 days of age (n = 35) and incubated in culture medium containing ovine LH (10 ng/ml) and genistein (GEN) and/or daidzein (DAID) (100 nM, 18 h). After treatment, Leydig cells were analyzed for thymidine incorporation by scintillation counting (A). In separate experiments using Leydig cells pooled from 35-day-old male rats, Leydig cells were treated with GEN and/or DAID (18 h) and analyzed for T production 3h posttreatment by RIA (B). In addition, protein expression of AMH and transferrin were analyzed in primary cultures of Sertoli cells after incubation in culture medium containing GEN and/or DAID (100 nM, 18 h) (C, D). Protein levels were measured by Western blot analysis using anti-AMH and anti-transferrin antibodies and appropriate secondary antibodies. The data represent the results of three separate and independent experiments and at least three Western blot procedures for each parameter per experiment. Protein levels were normalized to ACTB. AMH = 63 kDa, transferrin = 79 kDa, ACTB = 42 kDa. Bars represent means ± SEM. *P < 0.05 versus control; **P < 0.01 versus control.

FIG. 8

Leydig cells were isolated and pooled from isoflavone-free male rats at 21 days of age (n = 35). Proliferative activity was assessed by [³H] thymidine incorporation after incubation of Leydig cells in culture medium containing daidzein (0, 100, or 200 nM, 18 h) and ovine LH (10 ng/ml) (A). Expression of proliferating cell nuclear antigen (PCNA) (B) and protein kinase activation of protein kinase B (Akt) and extracellular regulated kinase (MAPK3/1) were analyzed in Western blots (C, D). In separate experiments, Leydig cells isolated from isoflavone-free 35-day-old male rats were incubated in culture medium containing daidzein (18 h) and ovine LH (100 ng/ml) followed by measurement of T secretion in aliquots of spent media by RIA (E). Leydig cells were processed for Western blot analysis of steroidogenic acute regulatory protein (StAR) levels (F). PCNA and StAR protein were analyzed in Western blots of Leydig cells probed with anti-PCNA, anti-Akt, anti-p-Akt^Ser473, anti-MAPK3/1, anti-p-MAPK3/1, and anti-StAR primary antibodies and the appropriate secondary antibodies. The data for each parameter represent the results from three separate and independent experiments and at least three Western blot procedures. PCNA and StAR protein were normalized to ACTB, whereas the levels of p-MAPK3/1 and p-Akt^Ser473 were normalized to total MAPK3/1 and total Akt levels, respectively. PCNA = 35 kDa, AKT/p-AKT^Ser473 = 56/60 kDa, (p-) MAPK3/1 = 42/44 kDa, StAR = 30 kDa, ACTB = 42 kDa. Bars represent means ± SEM. *P < 0.05 versus control; **P < 0.01 versus control.