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Abstract
Background
We wanted to identify the presence of the estrogen receptor (ER) α in Sertoli cells and gain insight on the regulation of the ERα gene expression by testosterone in Sertoli cells. The transcriptional regulation of the ERα gene was investigated in primary Sertoli cell cultures by in situ hybridization and reverse transcription-polymerase chain reaction (RT-PCR).
Methods
Primary Sertoli cell culture was performed. The expression levels of ERα and ERβ mRNA in Sertoli cells were detected by Northern blot, RT-PCR, immunocytochemistry and in situ hybridization.
Results
The ovary, testis and epididymis showed a moderate to high expression of ERα while the prostate, ovary and LNCap cells showed the ERβ expression. ERα mRNA and protein were detected in the germ cells and Sertoli cells by in situ hybridization and immunocytochemistry. The level of ERα mRNA was gradually decreased in a time-dependent manner after testosterone treatment, and the changes of ERα mRNA were dependent on the concentration of testosterone. Androgen binding protein and testosterone-repressive prostate message-2 (TRPM-2) mRNA were reduced at 24 hour by estradiol, while the transferrin mRNA was not affected. ERα mRNA was strongly detectable in the testes of 7 days-old-rats, but it was gradually decreased from 14 to 21 days of age. The primary Sertoli cells also showed the same pattern. The ERα gene expression was also regulated by testosterone in the Sertoli cells prepared from the 14- and 21-day old rats.
Figures and Tables
Fig. 1
Morphology of primary Sertoli cell and peritubular cell. Sertoli cell and peritubular cell culture was performed as described in Materials and Methods. A, Sertoli cells; B, peritubular cells (×400)[18].
Fig. 2
Rat tissue and cell distribution of ERα and ERβ mRNA. The ERα and ERβ mRNA levels were measured by RT-PCR. RT-PCR products were separated on 1.2% agarose gel containing ethidium bromide. The other assays were performed as described in Materials and Methods.
Fig. 3
Identification of ERα mRNA in primary Sertoli-spermatogenic coculture and peritubular cells by in situ hybridization. The Sertoli, spermatogenic cells and peritubular cells were hybridized to ERα cDNA probes labeled with digoxigenin-labeled dUTP by random prime DNA labeling kit. A, Sertoli/spermatogenic cocultures hybridized with ERα cDNA; B, Sertoli1 spermatogenic cocultures hybridized with pBR322; C, MCF-7 cells hybridized with ERα cDNA; D, MCF-7 cells hybridized with pBR322.
Fig. 4
Immunocytochemistry of ER in the Sertoli/spermatogenic cells (A) and mouse embryo fibroblast 10T1/2 cells (B). Immunostaining was performed by avidin-biotin complex method using monoclonal antibody against human estrogen receptor (H222).
Fig. 5
Effect of the testosterone on the expression of ERα mRNA in the primary Sertoli cell culture, and effect of cycloheximide (CHX) on the testosterone-dependent repression of ERα mRNA levels. The Sertoli cell cultures were prepared from testes of 21-day-old rats. On the third day of cultures, the cells were rinsed and preincubated with fresh medium for 30 minutes. A, Testosterone (10-6 M) was added at time zero, and incubation continued up to 48 hrs. At times indicated, the cells were harvested and total RNA was prepared. B, Various concentration of testosterone (10-5~10-8 M) were added and the cells were incubated for 24 hours. C, The cells were treated for 12 hours with 10-6 M testosterone. Cycloheximide at a concentration of 5 µg/mL was added at 33 hours of incubation. The ERα mRNA level was measured by RT-PCR. Two separate experiments were performed repeatedly.
Fig. 6
Effect of estradiol-17β on the expression ABP, TRPM-2 and transferrin mRNAs in the primary Sertoli cell culture. On the third day of culture, Sertoli cells were rinsed and preincubated with fresh medium for 30 minutes. Estradiol-17β (10-6 M) was added at time zero and incubation continued up to 36 hours. At the indicated times cells were harvested and total RNA was extracted. ABP, TRPM-2 and transferrin mRNAs were measured by Northern blot hybridization.
Fig. 7
Changes of ERα mRNA level in testes (A) and testosterone-dependent repression of ERα mRNA levels in Sertoli cells (B) according to age. A, Total RNA was prepared from 7-, 14-, and 21-day-old rat testes. B, The primary Sertoli cell cultures were prepared from 14- and 21-day-old rat testes. Testosterone (10-6 M) was added on the third day after plating and total RNA was prepared. ERα mRNA levels were measured by RT-PCR.
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