• Reproductive and Developmental Biology
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• v.34 no.1
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• pp.33-40
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• 2010

Equine chorionic gonadotropin (eCG) is a heavily glycosylated glycoprotein composed of non-covalently linked $\alpha$- and $\beta$-subunits. To study the function and signal transduction of tethered recombinant-eCG (rec-eCG), a single chain eCG molecule was constructed, and the rec-eCG protein was prepared. In this study, we constructed 5 mutants (${\Delta}1$, ${\Delta}2$, ${\Delta}3$, ${\Delta}4$, and ${\Delta}5$) of rec-eCG using data about known glycoprotein hormones to analyze the role of specific follicle stimulating homone (FSH)-like activity. Three amino acids of certain specific sites were replaced with alanine. The expression vectors were transfected into CHO cells and subjected to G418 selection for 2~3 weeks. The media were collected and the quantity of secreted tethered rec-eCGs was quantified by ELISA. The LH- and FSH-like activities were assayed in terms of cAMP production by rat LH/CG and rat FSH receptors. Then, the metabolic clearance rate analyzed by the injection of rec-eCG (5 IU) into the tail vein was analyzed. The mutant eCGs (${\Delta}l$, ${\Delta}4$, and ${\Delta}5$) were transcripted, but not translated into proteins. Rec-eCG A2 was secreted in much lower amounts than the wild type. Only the rec-eCG ${\Delta}3$ ($\beta$-subunit: $Gln^{94}-Ile^{95}-Lys^{96}{\rightarrow}Ala^{94}-Ala^{95}-Ala^{96}$) was efficiently secreted. Although activity is low, its LH-like activity was similar to that of tethered $eCG{\beta\alpha}$. However, the FSH-like activity of rec-$eCG{\beta\alpha\Delta}3$ was completely flat. The result of the analysis of the metabolic clearance rate shoed the persistence of the mutant in the blood until 4 hours after the injection. After then, it almost disappeared at 8 hours. Taken together, these data suggest that 94~96 amino acid sequences in eCG $\beta$-subunit appear to be of utmost importance for signal transduction of the FSH receptor.

• Clinical and Experimental Reproductive Medicine
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• v.35 no.3
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• pp.181-191
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• 2008

Objective: Environmental chemicals alter reproduction, growth, and survival by changing the normal function of the endocrine system. Bisphenol A (BPA), one of the endocrine disruptors, is known to be an estrogen receptor agonist. Therefore, we hypothesized that BPA may affect male reproduction including spermatogenesis in the mouse testis. Methods: We used 7-week-old ICR mice. The first experiment group received BPA in sesame oil (vehicle, 1 mg/kg, 10 mg/kg, and 100 mg/kg) by i.p. injection and mice were sacrificed 24 hr later. The second experiment group received BPA (vehicle, 10 ${\mu}g/kg$, 1 mg/kg, and 100 mg/kg) daily for 14 days by subcutaneous injection. Expression of cell type-specific marker genes in the testis was evaluated by RT-PCR. Histological analysis, immunofluorescence staining, and TUNEL staining were also performed. Results: RT-PCR analyses showed that expression of luteinizing hormone receptor (LHR), a marker gene for the Leydig cell, was notably decreased in the testes of high dose-exposed mice. No obvious difference in the histology of testes was noted among treatment groups. Immunostaining of LHR in the first experiment group did not show noticeable difference in LHR protein expression in Leydig cells. Immunohistochemistry also revealed heightened expression of the immunoreactive Bax in the treatment group, and this was accompanied by positive TUNEL staining in the interstitial area within testis where Leydig cells reside. Conclusions: Our result suggests that BPA affects Leydig cell functions by altering gene expression and by increasing apoptosis in the mouse testis.