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first generation, designated fetal Leydig cells, differentiates height, 0.22 m) lined with wood chip bedding (Lab Products, Inc.), and water
from stromal cells of testis cords starting on Day 12 of was provided in glass bottles. Use of plastic cages and glass bottles was to
minimize background exposure of animals to estrogens, as occurs during use
gestation in the rat. Once formed, fetal Leydig cells are of cages made with resins [20]. Animals were maintained under constant
terminally differentiated, reaching their peak of steroidogenic conditions of light (12L:12D) and temperature (20–23.38C), with free access
capacity just before birth on Gestational Day (GD) 19 [15]. On to pelleted food. Diets containing casein (control) and whole soybeans as
the other hand, the progeny for the second generation, which sources of protein were used in the present study, and diets were formulated to
form adult Leydig cells in the postnatal period, first becomes be identical in terms of energy, micronutrients, cholesterol, calcium, and
apparent by Postnatal Day (PND) 11 [16]. These cells are phosphorus. The concentration of isoflavones in experimental diets was 0, 5,
50, or 1000 ppm based on the assayed content of genistin and daidzin and
designated progenitor Leydig cells and exhibit intense calculation of the equivalent aglycone as specified by the manufacturer
proliferative capacity. The transition to mature and steroido- (Harlan-Teklad). Pregnant dams were fed diets from GD 12 to PND 21, and
genically competent adult Leydig cells involves a dramatic all carried the pregnancy to term. Pregnancy outcome, including litter size,
decrease in proliferative capacity as progenitor cells enlarge to pup weight, and male:female ratio of pups, was assessed on PND 1. Although
form immature Leydig cells at approximately PND 35. it had been suggested that cross-fostering of pups achieves randomization of
Immature cells undergo a final round of cell division to form the genetic background or its interaction with maternal exposure [21], male
rats were reared with their natural littermates and not cross-fostered within
adult Leydig cells at approximately PND 56. Thus, the most groups in the present study. Pregnancy outcome (litter size, pup weight, and
rapid increases in Leydig cell numbers occur between PND 14 sex ratio) and of anogenital distance were measured and analyzed per litter up
and PND 56 in the rat [17]. The decrease in the rate of mitosis to PND 5. However, given the small population of Leydig cells in the
accompanying Leydig cell development is accompanied by a prepubertal rat testis, each isolation procedure required at least 25 rats from
gradual increase in the capacity for steroid hormone secretion. each group. Therefore, male rats were selected randomly from members of
Therefore, fully differentiated adult Leydig cells, which each litter per group for analysis of Leydig cell function and other parameters
from PND 21.
constitute the predominant population in the sexually mature At weaning (i.e., PND 21), animals were fed the soy-free, casein-based control
testis, lack mitotic activity but have great capacity for steroid diet until they were killed. The diets used in the present study were the same
hormone secretion. formulation as used in our earlier study [14]. Thus, serum levels of isoflavones
aliquots of 0.3–0.5 3 106 cells and lysed in microcentrifuge tubes containing Procedure for isolation of Leydig cells. Animals were killed by CO2
0.5 ml of hyamine hydroxide (catalog no. 802387, lot no. 8493J; MP asphyxiation. Isolation of Leydig cells involved collagenase digestion of testis
Biomedicals). Cellular [3H]thymidine uptake was quantified by liquid followed by Percoll density centrifugation according to a procedure described
scintillation counting. In addition, unlabeled control and genistein-treated previously [34] but excluding the elutriation step. After testis digestion but
Leydig cells were harvested and counted to determine cell numbers. To before Percoll density centrifugation, seminiferous tubules were removed by
determine whether proliferation was associated with cell-cycle progression, passage of testicular fractions through nylon mesh (pore size, 0.2 lm; Spectrum
Leydig cells were processed to obtain whole-cell lysates to measure levels of Laboratories, Inc.). Filtration of cell fractions was omitted if Leydig cells were
proliferating cell nuclear antigen (PCNA), cyclin D3, and pRBSer780. Activation isolated from adult rats (age, 60–90 days) when seminiferous tubules were
of RB is caused by cyclin-CDK complexes and results in the release of free E2F sedimented by gravity in 2 mg/ml of bovine serum albumin solution. Cell
transcription factors and activation of genes required for DNA synthesis, which fractions were loaded on to a Percoll gradient for 60 min to isolate bands of
are required for cell-cycle progression [29]. Measurement of cyclin D3 and Leydig cells. Leydig cell numbers were estimated with a hemocytometer,
pRBSer780 levels helped us to validate that increased Leydig cell numbers whereas purity was assessed by histochemical staining for HSD3B using 0.4
resulted primarily from cell-cycle progression and mitosis. mM etiocholan-3b-ol-17-one enzyme substrate (catalog no. E-5251, lot no.
AKT and genistein action. Evidence indicates that AKT acts as a cell 11K4058; Sigma) [35].
survival factor and increases the cell population in several tissues [30]. Immunohistochemistry and stereology. Testes were obtained after whole-
Therefore, experiments were performed to determine whether genistein-induced body perfusion of rats with 4% paraformaldehyde (catalog no. 19200, lot no.
Leydig cell proliferation involves AKT. After exposure to isoflavones in vivo 090820; Electron Microscopy Sciences) and stored until embedded in paraffin.
or genistein treatment in vitro (0.1 lM, 3 h), Leydig cells were processed to Testes from four animals per group were analyzed in this procedure. To
obtain whole-cell lysates, which were analyzed for AKT activation (i.e., enumerate Leydig cell numbers, sampling of testicular tissue was done
phosphorylation on serine residue 473 [pAKTSer473]). In separate experiments, according to the fractionator technique [36]. In every fractionation step, the
aliquots of Leydig cells were incubated for 1 h in culture medium containing a selection of a set of blocks or 6-lm-thick sections was performed with
specific AKT kinase inhibitor (1L6-hydroxymethyl-chiro-inositol-2-(R)-2-O- systematic random sampling. Identification of Leydig cells was facilitated by
methyl-3-O-octadecyl-sn-glycerocarbonate; catalog no. 124005, lot no.
processing tissue for immunocytochemistry and staining with a polyclonal
D00031576, 0.1 lM; Calbiochem) and then in fresh medium containing
antibody specific for the Cyp17a1 enzyme (sc-46081; Santa Cruz Biotechnol-
genistein (0.1 lM, 3 h). Leydig cells were then processed to measure cellular
ogy). Sections were examined using a Nikon Eclipse E600 microscope (Nikon
[3H]thymidine uptake. The concentration of kinase inhibitor was determined in
Instruments) equipped with epifluoresence, bright-field, and differential
pilot experiments not to affect Leydig cell proliferation when acting alone. In
interference optics. Images were made with a Spot RT Slider digital camera
Parameter 0 5 50 1000
a
Litter size (number of pups) 13.2 6 0.8 12.2 6 0.6 12.6 6 0.5 13.1 6 0.2
Pup sex ratio (male:female)a 1.5 6 0.3 1.1 6 0.2 1.8 6 0.4 1.2 6 0.2
Body weights (g, PND 1)a 6 6 0.1 6.2 6 0.1 6.3 6 0.1 6 6 0.1
Anogenital distance (mm, PND 5)a 7.29 6 0.25c 7.95 6 0.3c 8.74 6 0.3d 7.46 6 0.33c
Body weights (g, PND 21)b 64.2 6 0.8 62.1 6 1.0 64.3 6 1.3 61.9 6 0.9
Paired testis weights (g, PND 21)b 0.29 6 0.01 0.31 6 0.01 0.31 6 0.01 0.29 6 0.01
Body weights (g, PND 90)b 510 6 11 538 6 17 529 6 10 522 6 15
Paired testis weights (g, PND 90)b 3.5 6 0.1 3.7 6 0.1 3.6 6 0.1 3.8 6 0.1
Paired epididymal weight (g, PND 90)b 1.12 6 0.1 1.23 6 0.03 1.16 6 0.04 1.19 6 0.07
Dorsolateral prostate (g, PND 90)b 0.63 6 0.06 0.55 6 0.01 0.59 6 0.04 0.61 6 0.04
Ventral prostate (g, PND 90)b 0.71 6 0.04 0.69 6 0.04 0.74 6 0.06 0.68 6 0.03
Seminal vesicles (g, PND 90)b 0.81 6 0.06 0.77 6 0.06 0.86 6 0.03 0.85 6 0.04
a
Values were based on the litter as a unit of measurement with n ¼ 14.
b
Values were based on measurements from randomly chosen male rats from each group with n ¼ 10–12.
c,d
Anogenital values with different superscripts are significantly different (P , 0.05).
DISCUSSION
The present results demonstrate that soy isoflavones
stimulate proliferative activity in developing Leydig cells at
concentrations approximating those achieved after consump-
tion of soy-based diets. This finding has implication for the use
of soy-based diets by pregnant mothers and in infant formulas.
For example, genistein and daidzein were detected, measuring
in the nanomolar range, in amniotic fluid obtained from
pregnant mothers [38, 39]. Also, approximately 750 000 infants
fed soy-based cereals in the United States each year are
exposed to genistein and daidzein, which attain blood
concentrations in the range between 300 and 600 nM [40,
41]. These blood isoflavone levels can be achieved by
consumption of diets containing isoflavones at concentrations
between 30 and 1000 mg/kg diet [42]. Induction of
proliferative activity was linked to increased expression of
FIG. 6. Serum testosterone concentrations were measured as a marker cell-cycle proteins, in agreement with reports indicating that
for Leydig cell repopulation of testis following administration of the mitotically active Leydig cells express high levels of proteins
cytotoxin ethane dimethylsulfonate (EDS) to 60-day-old rats exposed to that support cell division [43, 44]. For example, PCNA is
dietary isoflavones in the perinatal period. Serum testosterone levels known to function as a cofactor of DNA polymerase during
were measured 7 days (A) and 28 days (B) after EDS administration. DNA synthesis in the nucleus [45], and phosphorylation of RB
The numbers of Leydig cells recovered from testis of male rats exposed
to dietary isoflavones compared to control animals was determined in on serine residue 780 activates genes required for cell-cycle
sexually mature rats at 90 days (n ¼ 9–10; C). *P , 0.05 versus progression [46]. Our findings agree with suggestions that low
control. concentrations of genistein (10 lM) tend to induce cell
PHYTOESTROGENS REGULATE LEYDIG CELL DEVELOPMENT 495
abrogated AKT phosphorylation and the antiapoptotic effect of target for mitogenic factors regulating Leydig cells during the
insulin-like growth factor 1 in immature Leydig cells [75]. In peripubertal period of reproductive tract development.
the present study, we obtained two pieces of evidence implying The finding that the action of genistein in Leydig cells was
a role for AKT in the regulation of Leydig cell division. First, abrogated equally by the AKT kinase inhibitor and the
the levels of pAKTSer473 were greater in proliferating Leydig antiestrogen ICI 182,780 seems to imply that molecules in
cells after in vivo and in vitro exposure to isoflavones. Second, the signaling pathways mediated by the ESR and AKT are
age-dependent decreases occurred in pAKTSer473 levels during involved in genistein regulation of Leydig cell division, and it
the transition from progenitor to adult Leydig cells. These suggests that MAPK possibly facilitates cross-talk between
findings tend to support the general view that the signaling ESR- and AKT-mediated pathways. Cross-talk between
pathway mediated by phosphatidylinositol 3-kinase and AKT signaling pathways in estrogen-sensitive tissues is thought to
regulates survival signals and enhances cellular resistance to synergize and enhance plasticity of estrogen action [79]. In this
programmed cell death [30, 76–78]. Therefore, AKT is likely a regard, we obtained data indicating that MAPK3/1 are involved
498 SHERRILL ET AL.
in the mitogenic action of genistein, thereby providing a Alternatively, genistein may act independently, albeit simulta-
putative mechanism for cross-talk between AKT- and ESR- neously, through ESR- and AKT-mediated pathways, which
mediated signaling. This interpretation is in line with then converge on MAPK3/1 as downstream effector molecules
suggestions that genistein binds to ESRs and causes down- [33, 80]. Indeed, it has been proposed that the biphasic effect of
stream activation of AKT, which in turn activates MAPK3/1. genistein on cell division results from differential MAPK
PHYTOESTROGENS REGULATE LEYDIG CELL DEVELOPMENT 499
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