Premature ovarian failure (POF) is menopause before the age of 40 years. The frequency of POF is about 1% of all women. Recently inhibin alpha gene (INHalpha) has been indicated as candidate in POF pathogenesis. Inhibin, a glycoprotein, is a gonadal hormone, which can inhibit the synthesis and secretion of pituitary follicle-stimulating hormone (FSH), which has an important role in the recruitment and development of ovarian follicles during the folliculogenesis. G769A variation of INH alpha, alanine, is highly conserved across species, and has an important role of its receptor binding. We screened a G769A transition in the INHalpha from the total population of the patients of 84 women with POF and 100 normal fertile women. We found no variation between the normal subjects and the POF patients. G769A variation of INHalpha is rare in Korea women with POF.
Adult ; Female ; Follicle Stimulating Hormone/metabolism ; Human ; Infertility, Female/genetics ; Inhibins/*genetics/metabolism ; Korea ; Ovarian Failure, Premature/*genetics/metabolism ; *Polymorphism, Restriction Fragment Length ; Support, Non-U.S. Gov't

Inhibin, which is important for normal gonadal function, acts on the pituitary gonadotropins to suppress folliclestimulating hormone (FSH) secretion. The level and cellular localization of the inhibin isotypes, alpha, beta(A) and beta(B), in the testis of mice were examined during postnatal development in order to determine if inhibin expression is related to testicular maturation. Mouse testes were sampled on postnatal days (PNDs) 1, 3, 6, 18, 48 and 120, and analyzed by Western blotting and immunofluorescence. Western blot analysis showed very low levels of inhibin alpha, beta(A) and beta(B) expression in the testes at days 1 to 6 after birth. The levels then increased gradually from PND 18 to 48-120, and there were significant peaks at PND 48. Inhibin alpha, beta(A) and beta(B) were detected in testicular cells during postnatal development using immunohistochemistry. The immunoreactivity of inhibin alpha was rarely observed in testicular cells during PND 1 to 6, or in the cytoplasmic process of Sertoli cells surrounding the germ cells and interstitial cells during PND 18 to 120. Inhibin beta(A) and beta(B) immunoreactivity was rarely observed in the testis from PND 1 to 6. On the other hand, it was observed in some spermatogonial cells, as well as in the interstitial space between PND 48 and PND 120. We conclude that the expression of inhibin isotypes increases progressively in the testis of mice with increasing postnatal age, suggesting that inhibin is associated with a negative feedback signal for FSH in testicular maturation.
Aging/*physiology ; Animals ; Gene Expression Regulation/*physiology ; Inhibin-beta Subunits/genetics/*metabolism ; Inhibins/genetics/*metabolism ; Male ; Mice ; Mice, Inbred ICR ; Protein Isoforms/metabolism ; Protein Transport/*physiology ; Testis/*metabolism

OBJECTIVETo investigate the potential diagnostic value of A103 and inhibin-alpha in adrenocortical tumors and to evaluate the applicability of tissue microarray/tissue chip in pathological studies using immunohistochemistry.

METHODSA tissue microarray/tissue chip was constructed to contain 179 formalin-fixed, paraffin-embedded adrenal tissue samples which include 3 normal adrenal cortex, 2 fetal adrenal cortex, 2 nodular adrenocortical hyperplasia samples, 72 adrenocortical adenomas, 39 adrenocortical carcinomas, 3 adrenal medulla, 13 metastatic carcinomas, 4 metastatic malignant melanomas and 44 pheochromocytomas. Additional 20 cases of normal adult adrenal gland were used as controls. Immunohistochemical markers, including A103, inhibin-alpha, calretinin and Ki-67 were used on the tissue array sections by EnVision immunohistochemical staining methods.

RESULTSPositive staining of A103 was seen in all of the 23 (100%) adrenal cortex, 2 fetal adrenal cortex, 2 nodular adrenocortical hyperplasia samples, 60 of 66 (90.9%) adrenocortical adenomas samples, 35 of 37 (94.6%) adrenocortical carcinomas samples, 3 of 3 malignant melanomas, but in none of the adrenal medulla, pheochromocytomas or adrenal metastatic carcinoma samples. In all of the adrenal cortex, fetal adrenal cortex and nodular adrenocortical hyperplasia cases, inhibin-alpha immunoreactivity was limited to the zona reticularis and the innermost zona fasciculata. Fifty of the 66 (75.8%) adrenocortical adenomas, 28 of the 37 (75.7%) adrenocortical carcinomas were positive for inhibin-alpha. None of the adrenal medulla, pheochromocytoma, metastatic malignant melanoma or carcinoma samples showed a positive inhibin-alpha immunostain.

CONCLUSIONSThe tissue microarray/tissue chip technique provides a reliable method to investigate marker expression by offering a rapid, economic and accurate screening of tissue specimens on a large scale. The combined use of A103 and inhibin-alpha is valuable in distinguishing adrenocortical tumor from pheochromocytoma and other metastatic neoplasms.

Adrenal Cortex ; metabolism ; Adrenal Cortex Neoplasms ; diagnosis ; metabolism ; secondary ; Adrenocortical Adenoma ; diagnosis ; metabolism ; Adult ; Antigens, Neoplasm ; biosynthesis ; genetics ; Female ; Humans ; Immunohistochemistry ; Inhibins ; biosynthesis ; genetics ; MART-1 Antigen ; Male ; Neoplasm Proteins ; biosynthesis ; genetics ; Oligonucleotide Array Sequence Analysis ; Pheochromocytoma ; diagnosis ; metabolism

A cDNA sequence coding for ovine inhibin N terminal 1-33 AA residue fragment (INH) was inserted between BamHI\SacI sites in plasmid pRSET-A to generate plasmid pR-INH. By utilizing a pair of isocaudamer BamHI and Bgl II sites and another downstream Hind III site, following simple double digestions and combination ligation of the resultant products, 2 to 6-repeat INH genes were constructed respectively. Each plamids containing 3 to 6 repeated INH fragment genes, pR-3INH, pR-4INH, pR-5INH and pR-6INH, directed expression of the target proteins in E. coli. BL21 (DE3) under induction of ITPG, which respectively accounted for 6%, 6%, 7% and 8% of the total bacterial protein. The expressed target proteins were all in the form of inclusion bodies. The above results implied that utilization of isocaudamer restriction disgetion sites in expression plasmid is capable of rapidly and correctly constructing repeat fragment polymer of short peptides, which may become a new method in construction of high immunogenic recombinant vaccines of short peptides.
Animals ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Inhibins ; biosynthesis ; genetics ; Peptide Fragments ; biosynthesis ; genetics ; Plasmids ; genetics ; Polymers ; chemistry ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification ; Repetitive Sequences, Nucleic Acid ; genetics ; Sheep

OBJECTIVETo explore effects of p, p'DDE on the expression of androgen binding protein (ABP), transferrin (Tf) and inhibin B (INH B) mRNA in testis Sertoli cells of Sprague Dawley rats.

METHODSA method has been set up to obtain a large number of viable Sertoli cells from SD rats of 18-20 days of age. With a series of concentration p,p'-DDE (10, 30, and 50 micromol/L) co-incubating the Sertoli cells in vitro, the expression of ABP, Tf and INH B mRNA were determined by RT-PCR.

RESULTSa) With increase of the incubated p, p'-DDE, the expression of ABP mRNA in Sertoli cells went up while that of Tf and INH B dropped in a dose-dependent manner (P < 0. 05). b) The correlation analysis among ABP, Tf and INH B showed that negative relationships were found between ABP and Tf or INH B, respectively (r = - 0. 391 3, P = 0. 032 5; r = - 0.235 2, P = 0.0158), and that positive correlation was indicated between Tf and INH B (r =0.4516, P =0.0047).

CONCLUSIONp,p'-DDE is a reproductive toxicant which disrupts the transcription of ABP, Tf and INH B in rat Sertoli cells so as to result in reproductive dysfunction.

Androgen-Binding Protein ; biosynthesis ; genetics ; Animals ; Dichlorodiphenyl Dichloroethylene ; toxicity ; Inhibins ; biosynthesis ; genetics ; Male ; RNA, Messenger ; genetics ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction ; Sertoli Cells ; drug effects ; metabolism ; Transferrin ; biosynthesis ; genetics

OBJECTIVEThe present study was performed to examine functional and structural impairment of rat sertoli cells following dibutyl phthalate (DBP) exposure.

METHODSThe 6-week-old healthy male Sprague Dawley rats were randomly divided into 4 groups with 16 animals in each group. DBP dissolved in peanut oil was administered by gavage at doses of 0, 250, 500 and 1 000 mg/kg. After 2-week DBP treatment, half of the rats were sacrificed. The rest were killed following 4-week DBP exposure. Follicle stimulating hormone (FSH) was analysed by radioimmunoassay. The relative expression levels of androgen binding protein (ABP) mRNA and inhibin (INH)alpha mRNA were determined by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). The sertoli cell ultrastructures were observed by using transmission electron microscope (TEM).

RESULTSFSH levels were increased after 4-week DBP exposure with significance at doses of 250 and 1 000 mg/kg. Sperm head count and daily sperm product were decreased significantly in 500 and 1 000 mg/kg groups. The expression levels of ABP mRNA were 0.89 +/- 0.15, 0.85 +/- 0.23, 0.54 +/- 0.17, 0.52 +/- 0.16 and 0.88 +/- 0.16, 0.61 +/- 0.12, 0.48 +/- 0.15, 0.47 +/- 0.11 for 0, 250, 500 and 1 000 mg/kg after 2- and 4-week DBP treatments respectively with significance at doses of 500 and 1 000 mg/kg (P < 0.01), while the levels of INHalpha mRNA were 0.88 +/- 0.16, 0.61 +/- 0.12, 0.48 +/- 0.15, 0.47 +/- 0.11 and 0.75 +/- 0.19, 0.56 +/- 0.16, 0.53 +/- 0.08, 0.45 +/- 0.10 with significance at all exposure groups (P < 0.01 or P < 0.05). In sertoli cells of rats exposed to 1 000 mg/kg DBP, TEM photos showed more lysosomes in cytoplasm, proliferated and expanded endoplasmic reticulum and nuclei malformation.

CONCLUSIONSSertoli cell should be one of the major toxic targets. Impairment of spermatogenesis caused by DBP should be partly due to the suppression of ABP and INHalpha biosynthesis.

Androgen-Binding Protein ; genetics ; Animals ; Dibutyl Phthalate ; administration & dosage ; toxicity ; Dose-Response Relationship, Drug ; Follicle Stimulating Hormone ; metabolism ; Inhibins ; genetics ; Male ; RNA, Messenger ; genetics ; metabolism ; Radioimmunoassay ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction ; Sertoli Cells ; drug effects ; metabolism ; pathology ; Testis ; drug effects ; metabolism ; pathology

OBJECTIVETo evaluate the effects of the Chinese herbal formula Wuzi Yanzong Pill (, WYP) on the spermatogenesis and specific secretory functions of Sertoli cells in rat model and to investigate the underlying mechanism.

METHODSFive groups of male Sprague-Dawley rats including the control group, the model group, the low-dose WYP group, the medium-dose WYP group and the high-dose WYP group (5 in each group) were treated daily with vehicle, multiglycosides of Tripterygium wilfordii Hook f (GTW) either alone (20 mg/kg) or followed by WYP (0.5, 1.0, or 2.0 g/kg daily), respectively for 30 days. Serum levels of follicle-stimulating hormone (FSH), inhibin B (INHB) and testosterone (T) were evaluated using enzyme-linked immunosorbent assay. Androgen-binding protein (ABP) gene expression and transferrin (TF) protein expression in testis tissue specimens of all rats were determined using real-time reverse transcriptase polymerase chain reaction and Western blotting analysis, respectively. Histopathological alterations in the testis were determined using Johnsen's score.

RESULTSThe toxicity of GTW towards Sertoli cell secretory functions and spermatogenesis was accompanied by increased serum FSH concentrations and decreased INHB and T concentrations. Upregulated ABP mRNA levels, and decreased TF protein expression and Johnsen's scores were detected in the model group compared with the control group P<0.05 or P<0.01). Oral high-dose WYP administrations to GTW-treated rats effectively alleviated all of the GTW-induced changes in specific secretory functions of Sertoli cells (ABP, INHB and TF). Furthermore, serum T level and Johnsen's score of the testis increased greatly compared with the model group (P<0.01).

CONCLUSIONWYP has the ability to improve the spermatogenesis, possibly through modulating the secretory proteins expression of Sertoli cells.

Androgen-Binding Protein ; genetics ; metabolism ; Animals ; Blotting, Western ; Drugs, Chinese Herbal ; pharmacology ; Follicle Stimulating Hormone ; blood ; Gene Expression Regulation ; drug effects ; Inhibins ; blood ; Male ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Sertoli Cells ; drug effects ; secretion ; Spermatogenesis ; drug effects ; Tablets ; Testis ; cytology ; metabolism ; Testosterone ; blood ; Transferrin ; metabolism