BACKGROUND: Vitamin is a well-known co-factor for many metabolic processes and its roles in fertility and follicular growth have been studied. Vitamin supplementation is frequently achieved by daily ingestion in the form of a complex capsule. However, the role of single and complex vitamins in in vitro maturation of murine follicles is not fully elucidated. METHODS: In this study, we evaluated the effects of two forms of vitamins. Pure L-ascorbic acid, and multi-vitamin (vitamin C+vitamin B complex) was treated at two different concentrations (50 and 100 µg/ml), to pre-puberty murine follicles during in vitro maturation. To determine the specific stage of growth that is affected by treatment with vitamins, the vitamins were treated from day 0, 4, 9, and 13. Growth of each follicle was assessed by measuring diameters of whole expanded area and of the granulosa cells. Expression of follicular and oocyte growth-related genes and the effect of vitamin on the viability of follicles was assessed using senescence associated β-galactosidase staining. RESULTS: Treatment with vitamins promoted the in vitro growth of murine follicles and the upregulated the expression of granulosa cell- and oocyte-specific genes such as BMP15, Fsh receptor, and GDF9. The proliferation of the granulosa cells was enhanced by the treatment of vitamin. Fifty µg/ml concentration vitamin showed greater effects compared to higher concentration. The viability of in vitro grown follicles was also significantly improved in vitamin-treated follicles. The effects of single L-ascorbic acid and complex vitamin were not significantly different to those of day 4 and day 9 follicles. Vitamins promoted murine follicle development in vitro with different effects on specific growth stage. CONCLUSION: Supplementation of vitamins during in vitro maturation of murine follicles is an efficient strategy for in vitro expansion of follicular cells. These results could be customized to the sophisticated culture of follicles retrieved from aged or cancer-survived female that contain smaller number of follicles with reduced potential to develop into mature follicles.
Aging ; Ascorbic Acid ; Eating ; Female ; Fertility ; Granulosa Cells ; Humans ; In Vitro Techniques ; Metabolism ; Oocytes ; Ovarian Follicle ; Receptors, FSH ; Vitamins

BACKGROUNDFollicle stimulating hormone is necessary for normal reproduction in men. The biochemical actions of follicle stimulating hormone result from binding to the follicle stimulating hormone receptor in the plasma membrane of Sertoli cells. Here, we investigated the expression of the follicle stimulating hormone receptor in different testicular histological phenotypes of patients with idiopathic azoospermia.

METHODSFifty-seven cases of idiopathic azoospermia were classified into three groups according to the results of testicular biopsy: patients with hypospermatogenesis, patients with maturation arrest, and patients with Sertoli cell-only syndrome. Thirteen azoospermic patients identified by testicular biopsy as being capable of completing spermatogenesis acted as the control group. Immunohistochemistry and real-time quantitative reverse-transcriptase polymerase chain reaction were performed in each case, and the serum hormone level was also measured in all patients.

RESULTSThe serum follicle stimulating hormone level in patients with Sertoli cell-only syndrome was significantly higher than in patients with hypospermatogenesis, maturation arrest, and complete spermatogenesis (P < 0.01). The serum follicle stimulating hormone level in patients with maturation arrest was significantly higher than in patients with hypospermatogenesis and complete spermatogenesis (P < 0.05). There was no difference in serum follicle stimulating hormone levels in patients with hypospermatogenesis and complete spermatogenesis. The follicle stimulating hormone receptor expression level of testicular samples with Sertoli cell-only syndrome was significantly higher than in those with hypospermatogenesis, maturation arrest, and complete spermatogenesis (P < 0.05), but no significant difference was observed among hypospermatogenesis, maturation arrest, and complete spermatogenesis testicular samples.

CONCLUSIONSDifferent serum follicle stimulating hormone levels and follicle stimulating hormone receptor expression were found in the different testicular histology phenotypes in azoospermic patients. Differential follicle stimulating hormone receptor expression in testicular tissue of patients with idiopathic azoospermia may be associated with the degree of spermatogenesis.

Adult ; Azoospermia ; blood ; metabolism ; Follicle Stimulating Hormone ; blood ; Humans ; Male ; Oligospermia ; blood ; metabolism ; Receptors, FSH ; genetics ; metabolism ; Spermatogenesis ; physiology ; Testis ; metabolism

OBJECTIVETo investigate the effect and mechanism of Zuoguiyin (ZGY, a Chinese medical remedy for regulating qi and nourishing yin) on expression of ovarian follicle-stimulating hormone receptor (FSHR) in rats during peri-menopausal period (PMP).

METHODSPMP rats were administered with low (13.78 g/kg), middle (20.67 g/kg) and high (31.00 g/kg) dose ZGY respectively by gastrogavage for eight weeks. FSHR mRNA and protein expressions were detected by RT-PCR and immunohistochemistry respectively. Besides, granulosa cells, collected from pregnant mare serum treated nonage rats, were incubated, and the level of FSHR mRNA expression in the cultured cells were detected after various disposals.

RESULTS(1) Ovarian levels of FSHR mRNA and protein expressions in PMP rats were significantly lower than those in young rats (P<0.01), but were up-regulated significantly by ZGY treatment (P<0.01). (2) As compared with the control, FSHR mRNA expression increased in cultured granulosa cells after treated by ZGY extract; the increasing effect of ZGY was enhanced by combined treatment with Forskolin and attenuated by Genistein (a tyrosine protein kinase inhibitor).

CONCLUSIONZGY could improve the ovarian functions during PMP by up-regulating the expression of FSHR and raise the ovarian responsibility to FSH, which may be possibly realized by activating cAMP-protein kinase and tyrosine protein kinase signal pathway.

Animals ; Cells, Cultured ; Drugs, Chinese Herbal ; pharmacology ; Female ; Ovary ; drug effects ; metabolism ; Perimenopause ; drug effects ; RNA, Messenger ; genetics ; Rats ; Rats, Wistar ; Receptors, FSH ; metabolism

OBJECTIVE: To determine the effect of soy isoflavones on cell proliferation and the transcription levels of follicle-stimulating hormone receptor (FSHR), inhibin α (INHα), INHβB, androgen binding protein (ABP), transferrin (Tf) and vimentin in testis sertoli cells in SD rats. METHODS: Sertoli cells were cultured in vitro, exposed to daidzein at 0.03, 0.3, 3, and 30 μmol/L and genistein at 0.05, 0.5, 5 and 50 μmol/L, respectively. MTT was used to detect the proliferation of sertoli cells. Real-time PCR was used to detect the relative mRNA expressions of FSHR, INHα, INHβB, ABP, Tf and vimentin. RESULTS: Compared with control groups, cell proliferation and the relative mRNA expression levels of INHβB and ABP in the treated cells showed no significant alternation. The INHα mRNA expression levels were increased in 0.3 and 3 μmol/L Dai and 0.05 μmol/L Gen, while the mRNA expression levels of FSHR were downregulated in 30 μmol/L Dai and Gen at all concentrations. Tf mRNA expression levels were downregulated in 30 μmol/L Dai and 5 μmol/L and 50 μmol/L Gen, and the mRNA expression levels of vimentin were downregulated in 3 and 30 μmol/L Dai and 50 μmol/L Gen. CONCLUSION Soy Isoflavones may have potential detrimental effect on the male reproductive system, as they may impact the function of sertoli cells by downregulating the transcription levels of some important proteins.
Androgen-Binding Protein ; metabolism ; Animals ; Inhibin-beta Subunits ; metabolism ; Inhibins ; metabolism ; Isoflavones ; adverse effects ; Male ; RNA, Messenger ; Rats ; Rats, Sprague-Dawley ; Receptors, FSH ; metabolism ; Sertoli Cells ; drug effects ; Soybeans ; chemistry ; Testis ; cytology ; drug effects ; Transferrin ; metabolism

Follicle-stimulating hormone (FSH) is a glycoprotein which regulates the development, growth, pubertal maturation and reproductive processes of the body. Exogenous FSH has been used to promote ovarian follicular growth and maturation in female and spermatogenesis in male. The relative short elimination half life and rapid metabolic clearance of current versions of FSH require a daily or twice-daily scheduled subcutaneous injection to maintain stable FSH level being not below the threshold during ovarian stimulation. The development of recombinant long-acting FSH with enhanced biological activities may be helpful for less injection therefore to improve patient compliance, while reducing patient stress and error rates. A number of technological strategies have been explored to develop recombinant longer-acting FSH. For examples, attachment of the C-terminal peptide (CTP) of the human chorionic gonadotropin beta subunit or a sequence containing potential glycosylation sites to either subunit of FSH, creation of a single chain containing the alpha and beta subunits of FSH combined with CTP or N-linked glycosylation signal sequence as a linker, or fusion of the Fc domain of IgGi to FSH. Based on the modifiable molecular structure and pharmacokinetic and pharmacodynamic properties of recombinant FSH, it is hopeful that more FSH drugs with prolonged half-life and increased bioactivity will be developed to meet the modern clinical demands.
Animals ; Follicle Stimulating Hormone, Human ; chemistry ; genetics ; metabolism ; pharmacology ; Glycosylation ; Half-Life ; Humans ; Immunoglobulin Fc Fragments ; chemistry ; metabolism ; Ovulation Induction ; methods ; Receptors, FSH ; chemistry ; metabolism ; Recombinant Fusion Proteins ; chemistry ; genetics ; metabolism ; pharmacology ; Reproduction ; drug effects

OBJECTIVETo simplify the method for separation and cultivation of rat testicular Sertoli cells with high viability, quantity and expression efficiency.

METHODSTesticular Sertoli cells from 2 to 3-week-old male Wistar rats were prepared by digestion with collagenase, trypsin and DNase and cultured together with active lymphocytes to observe their killing effect against lymphocytes. After cell culture for 72 h, the Sertoli cells were morphologically observed by different means and identified with transmission electron microscope. Fas ligand and follicle-stimulating hormone receptor (FSHR) were examined immunohistochemically to identify testicular Sertoli cells. SABC method was used for labeling the Fas ligand on the testicular Sertoli cells.

RESULTSThe viability of the isolated and cultured Sertoli cells was more than 90%, and in in vitro culture, Sertoli cells, which expressed the Fas ligand, could kill the active lymphocytes.

CONCLUSIONThis method improves the efficiency in acquisition of rat testicular Sertoli cells expressing Fas ligand, which are believed to be a potential donor for co-transplantation with parathyroid cells to offer immune privilege.

Animals ; Cell Communication ; immunology ; Cell Separation ; methods ; Cell Survival ; immunology ; Cells, Cultured ; Fas Ligand Protein ; metabolism ; Immunohistochemistry ; Lymphocytes ; cytology ; immunology ; Male ; Microscopy, Electron, Transmission ; Rats ; Rats, Wistar ; Receptors, FSH ; metabolism ; Sertoli Cells ; cytology ; metabolism ; ultrastructure ; Testis ; cytology

The effect of high concentrations of testosterone on ovarian follicle development was investigated. Primary follicles and granulosa cells were cultured in vitro in media supplemented with a testosterone concentration gradient. The combined effects of testosterone and follicle-stimulating hormone (FSH) on follicular growth and granulosa cell gonadotropin receptor mRNA expression were also investigated. Follicle growth in the presence of high testosterone concentrations was promoted at early stages (days 1-7), but inhibited at later stage (days 7-14) of in vitro culture. Interestingly, testosterone-induced follicle development arrest was rescued by treatment with high concentrations of FSH (400 mIU/mL). In addition, in cultured granulosa cells, high testosterone concentrations induced cell proliferation, and increased the mRNA expression level of FSH receptor (FSHR), and luteinized hormone/choriogonadotropin receptor. It was concluded that high concentrations of testosterone inhibited follicle development, most likely through regulation of the FSH signaling pathway, although independently from FSHR downregulation. These findings are an important step in further understanding the pathogenesis of polycystic ovary syndrome.
Androgens ; pharmacology ; Animals ; Cell Proliferation ; drug effects ; Female ; Follicle Stimulating Hormone ; genetics ; metabolism ; pharmacology ; Gene Expression Regulation, Developmental ; Granulosa Cells ; cytology ; drug effects ; metabolism ; Mice ; Ovarian Follicle ; cytology ; drug effects ; growth & development ; metabolism ; Primary Cell Culture ; RNA, Messenger ; genetics ; metabolism ; Receptors, FSH ; genetics ; metabolism ; Receptors, Gonadotropin ; genetics ; metabolism ; Receptors, LH ; genetics ; metabolism ; Signal Transduction ; drug effects ; genetics ; Testosterone ; antagonists & inhibitors ; pharmacology

The homologous regulation of pituitary Gonadotropin Releasing Hormone Receptor (GnRH-R) mRNA expression by GnRH has been well demonstrated. However, the regulation of the ovarian GnRH-R is poorly understood. The present study was performed to demonstrate the presence of GnRH transcripts in addition to GnRH-R mRNA and the regulation of GnRH-R mRNA expression in the granulosa cells isolated from small antral follicles. The GnRH and GnRH-R mRNA levels were determined by a competitive reverse transcription-polymerase chain reaction (RT-PCR). The granulosa cells were obtained from immature rats implanted with diethylstilbestrol for 3 days. When GnRH transcript expression was examined in isolated granulosa cells by RT-PCR, the PCR products showed two bands. The larger band contained intronic sequences and the smaller band was a fully processed GnRH gene transcript identical to hypothalamic GnRH. This suggests that authentic GnRH gene transcripts are expressed in ovarian granulosa cells and may act on the granulosa cells in a paracrine or autocrine manner. Since GnRH action in the granulosa cells is mediated by specific GnRH-R, it is of interest to examine whether GnRH-R is synthesized in the granulosa cells. When the granulosa cells were cultured in media only, GnRH-R mRNA levels increased abruptly within 3 h and gradually decreased thereafter during the 24 h culture period. However, GnRH itself did not alter the GnRH-R mRNA expression levels in cultured granulosa cells. Interestingly, treatment with FSH decreased the GnRH-R mRNA levels in a dose-dependent manner. A time-course analysis revealed that the GnRH-R mRNA levels were significantly lower up to 9 h after FSH treatment, and returned to the basal level between 12 h-24 h. Activation of adenylate cyclase with forskolin also decreased the GnRH-R mRNA levels. It is therefore concluded that in the granulosa cells of the small antral follicles GnRH-R mRNA expression was not homologously regulated by GnRH, while FSH may negatively regulate GnRH-R mRNA expression in the granulosa cells possibly through a cAMP-protein kinase A pathway.
Animal ; Cells, Cultured ; FSH/pharmacology ; Female ; Gene Expression Regulation* ; Gonadorelin/pharmacology ; Granulosa Cells/metabolism* ; Granulosa Cells/drug effects ; RNA, Messenger/metabolism* ; Rats ; Rats, Sprague-Dawley ; Receptors, LHRH/genetics* ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo observe the effects of Yangjing Zhongyu Decoction (YJZYD) on the serum estradiol (E2), testosterone (T), 17-hydroxyprogesterone (17-OHP), ovarian follicle stimulating hormone receptor (FSHR), insulin-like growth factor I (IGF-1), steroid hormone acute regulator protein (StAR) mRNA expressions in female rats.

METHODSFifty PCOS rats were equally divided into 5 groups, i. e., the control group (C, normal PNA rats), the model group (M), the low dose YJZYD group (Y1), the medium dose YJZYD group (Y2), and the high dose YJZYD group (Y3), 10 in each. The levels of serum hormones were detected using radioimmunoassay. The morphological changes of the ovary were observed using HE method. The expressions of FSHR, IGF-1, and StAR mRNA were detected using RT PCR.

RESULTSCompared with Group C, serum T and 17-OHP significantly increased (P < 0.01), E2 significantly decreased (P < 0.01), the expressions of FSHR, IGF-1, and StAR mRNA significantly decreased in Group M (P < 0.01). Compared with Group M, the serum T level significantly decreased (P < 0.01), 17-OHP decreased (P < 0.05), and E2 significantly increased in Group Y3 (P < 0.01). The expressions of FSHR, IGF-1, and StAR mRNA increased in Group Y1, Y2, and Y3. The increase was most obvious in Group Y3 (P < 0.01).

CONCLUSIONSYJZYD could lower the hyperandrogenemia of PCOS rats. It also could increase the ovarian expressions of FSHR, IGF-1, and StAR mRNA, improve the ovarian functions, and promote the follicular development.

17-alpha-Hydroxyprogesterone ; blood ; Androgens ; blood ; Animals ; Drugs, Chinese Herbal ; pharmacology ; Estradiol ; blood ; Female ; Insulin-Like Growth Factor I ; metabolism ; Ovary ; drug effects ; metabolism ; Polycystic Ovary Syndrome ; blood ; Rats ; Rats, Wistar ; Receptors, FSH ; blood ; Testosterone ; blood

At present, there is no reliable in vitro assembled prepubertal testis-like biomimetic organ culture system designed to assess the functional effects of human gonadotropins on Sertoli and Leydig cells. Spermatogenesis is regulated by endocrine, paracrine, and juxtacrine factors (testicular cross-talk), mainly orchestrated by gonadotropins such as luteinizing hormone (LH) and follicle-stimulating hormone (FSH) that play a pivotal role by stimulating Leydig and Sertoli cells, respectively. The aim of our study was to set up an in vitro prepubertal porcine bioengineered construct as a new model for experimental studies on reassembled Sertoli and Leydig cells. We have evaluated Sertoli and Leydig cells obtained from 15- to 20-day-old neonatal pig testes in terms of purity and function. Subsequently, purified Sertoli and enriched Leydig cells were subjected to coincubation to obtain an in vitro prepubertal porcine testis-like culture system. We performed enzyme-linked immunosorbent assay (ELISA) for anti-Müllerian hormone (AMH), inhibin B, and testosterone secretion in the medium, and Real-Time PCR analysis of AMH, inhibin B, FSH-r, aromatase, LHr, and 3β-HSD mRNA expression levels. This in vitro testis-like system was highly responsive to the effects of human gonadotropins and testosterone. AMH mRNA expression and secretion declined, and inhibin-B increased, while FSH-receptor expression was downregulated upon FSH/LH exposure/treatment. Finally, the production of testosterone was increased selectively upon LH treatment. In summary, our proposed model could help to better determine the action of human gonadotropins on Sertoli and Leydig cells. The potential usefulness of the system for shedding light into male infertility-related issues is evident.
3-Hydroxysteroid Dehydrogenases/metabolism* ; Animals ; Animals, Newborn ; Anti-Mullerian Hormone/metabolism* ; Aromatase/metabolism* ; Cell Culture Techniques ; Enzyme-Linked Immunosorbent Assay ; Follicle Stimulating Hormone/pharmacology* ; Hormones/pharmacology* ; In Vitro Techniques ; Inhibins/metabolism* ; Leydig Cells/metabolism* ; Luteinizing Hormone/pharmacology* ; Male ; Models, Biological ; Real-Time Polymerase Chain Reaction ; Receptors, FSH/metabolism* ; Receptors, LH/metabolism* ; Sertoli Cells/metabolism* ; Swine ; Testis/metabolism* ; Testosterone/metabolism*