This study aims to examine the effect of superfine powder and aqueous extract of Polygonati Rhizomaon on natural perimenopausal syndrome in rats and explore the underlying mechanism. To be specific, a total of 60 female SD rats(14-15 months old) with estrous cycle disorder were screened by the vaginal smear and randomized into model control group, β-estradiol 3-benzoate group(0.1 mg·kg~(-1)), superfine powder of Polygonati Rhizoma group(0.25, 0.5 g·kg~(-1)) and aqueous extract of Polygonati Rhizoma group(0.25, 0.5 g·kg~(-1)), and another 10 female SD rats(14-15 months old) were selected as the youth control group. The administration lasted 6 weeks. Then the perimenopausal syndrome-related indexes such as body temperature, microcirculatory blood flow of face and ear, vertigo period, salivary secretion, grip force, and bone strength were determined and open field test was conducted. The immune system-related indexes such as the wet weight and index of thymus and spleen, percentage of T lymphocytes and subgroups in peripheral blood, and hematological indexes were measured. In addition, the ovary-related indexes such as estrous cycle, the wet weight and index of uterus and ovary, ovarian tissue morphology, and cell apoptosis were determined. Moreover, hypothalamus-pituitary-ovary axis(HPO)-related indexes such as serum sex hormone levels, cytochrome P450 family 11 subfamily A member 1(CYP11A1), cytochrome P450 family 19 subfamily A member 1(CYP19A1), and cytochrome P450 family 17 subfamily A member 1(P450 17A1) in ovarian tissue were measured. The results showed that the superfine powder and aqueous extract of Polygonati Rhizoma significantly decreased body temperature(anal, facial and dorsal temperature), microcirculatory blood flow in the ear, and vertigo period, increased salivary secretion, grip force, bone strength, total distance and total speed in the open field test, wet weight and index of thymus and spleen, lymphocyte ratio, CD3~+ level, and CD4~+/CD8~+ ratio, reduced neutrophil number and ratio, estrous cycle disorder ratio, and number of ovarian apoptotic cells, raised wet weight and index of uterus, wet weight of ovary, levels of inhibin B(INHB), estradiol(E_2), anti-müllerian hormone(AMH), and ovarian CYP11A1 and CYP19A1, decreased follicle-stimulating hormone(FSH) and luteinizing hormone(LH) content, and improved ovarian tissue morphology. It is suggested that the superfine powder and aqueous extract of Polygonati Rhizoma can improve the symptoms associated with natural perimenopausal syndrome in rats and enhance ovarian function and immune function. The mechanism is that they regulate HPO axis function by increasing estrogen synthesis.
Female ; Animals ; Rats ; Rats, Sprague-Dawley ; Microcirculation ; Cholesterol Side-Chain Cleavage Enzyme ; Perimenopause ; Powders ; Cytochrome P-450 CYP1A1

ObjectiveTo explore the feasibility of inducing human umbilical cord mesenchymal stem cells (HUMSCs) to differentiate into Leydig cells in the interstitial tissue of the rat testis.

METHODSHUMSCs were obtained by tissue blocks culture attachment and their purity and multi-lineage differentiation ability were verified by flow cytometry and chondrogenic/adipogenic/osteogenic differentiation. Then the HUMSCs were marked by CM-Dil and transplanted into the interstitial tissue of the rat testis. At 4 and 8 weeks after transplantation, the survival and differentiation status of the HUMSCs were observed by immunofluorescence staining and flow cytometry. The suspension of the rat Leydig cells was obtained at 8 weeks for determining the expression of the Leydig cell marker 3β-HSD in the HUMSCs, the cells labeled with CM-Dil were sorted and cultured, and the medium collected after 3 days of culture for measurement of the testosterone level.

RESULTSThe expression of the Leydig cell marker CYPllal was not observed in the HUMSCs at 4 weeks but found at 8 weeks after transplantation and the differentiation rate of 3β-HSD was about 14.5% at 8 weeks. CM-Dil labeled cells survived after sorting and testosterone was detected in the medium.

CONCLUSIONSHUMSCs are likely to differentiate into Leydig cells in the interstitium of the rat testis.

Animals ; Biomarkers ; metabolism ; Carbocyanines ; Cell Differentiation ; Cholesterol Side-Chain Cleavage Enzyme ; metabolism ; Feasibility Studies ; Humans ; Leydig Cells ; cytology ; metabolism ; Male ; Mesenchymal Stromal Cells ; cytology ; Rats ; Testis ; cytology ; Time Factors ; Umbilical Cord ; cytology

OBJECTIVETo establish an in vitro model of cultured mouse testis using rotary aerobic culture.

METHODSRotary aerobic incubation with optimized culture conditions was used for in vitro culture of mouse testis, and the morphology of the cultured testicular tissues was compared with that cultured in Transwell chambers. The changes in the testicular tissue structure were examined using HE staining, and the cell proliferation was assessed with BrdU staining. Testosterone concentrations in the culture medium were tested with radioimmunoassay and the expression of the functionally related proteins in the testis was detected using immunohistochemistry.

RESULTSThe testicular tissue cultured by optimized rotary aerobic culture presented with more intact histological structure with the size of the testis ranged from 0.3 to 0.8 mm(3). In the two culture systems, the prolifeation index of the spermatogonia increased and that of Sertoli cells decreased with time, and such changes in spermatogonia and Sertoli cell proliferation indices became statistically significant at 3 days (P<0.05) and 5 days (P<0.05) of culture, respectively, as compared with those at 1 day. The concentration of testoerone in the culture media decreased significantly with incubation time (P<0.05). At 3 days of culture, the protein expression of 3β-hydroxysteroid dehydrogenase, cytochrome P450 17α-hydroxylase and cholesterol side-chain cleavage enzyme was detected in Leydig cell cytoplasm and vimentin expression in Sertoli cell cytoplasm.

CONCLUSIONAn in vitro model of cultured mouse testis has been successfully established using rotary aerobic incubation.

17-Hydroxysteroid Dehydrogenases ; metabolism ; Animals ; Cholesterol Side-Chain Cleavage Enzyme ; metabolism ; Culture Media ; chemistry ; Leydig Cells ; cytology ; Male ; Mice ; Organ Culture Techniques ; Radioimmunoassay ; Sertoli Cells ; cytology ; Spermatogonia ; cytology ; Testis ; Testosterone ; chemistry ; Vimentin ; metabolism

Congenital lipoid adrenal hyperplasia (lipoid CAH) is the most fatal form of CAH, as it disrupts adrenal and gonadal steroidogenesis. Most cases of lipoid CAH are caused by recessive mutations in the gene encoding steroidogenic acute regulatory protein (StAR). Affected patients typically present with signs of severe adrenal failure in early infancy and 46,XY genetic males are phenotypic females due to disrupted testicular androgen secretion. The StAR p.Q258X mutation accounts for about 70% of affected alleles in most patients of Japanese and Korean ancestry. However, it is more prevalent (92.3%) in the Korean population. Recently, some patients have been showed that they had late and mild clinical findings. These cases and studies constitute a new entity of 'nonclassic lipoid CAH'. The cholesterol side-chain cleavage enzyme, P450scc (CYP11A1), plays an essential role converting cholesterol to pregnenolone. Although progesterone production from the fetally derived placenta is necessary to maintain a pregnancy to term, some patients with P450scc mutations have recently been reported. P450scc mutations can also cause lipoid CAH and establish a recently recognized human endocrine disorder.
Alleles ; Asian Continental Ancestry Group ; Cholesterol ; Cholesterol Side-Chain Cleavage Enzyme ; Female ; Gonads ; Humans ; Hyperplasia* ; Male ; Placenta ; Pregnancy ; Pregnenolone ; Progesterone

OBJECTIVETo explore the effects of di-butyl phthalate (DBP) on the reproductive system of adolescent male rats.

METHODSSprague-Dawley (SD) rats aged 5 weeks were assigned to receive corn oil (vehicle control) or DBP orally at 10, 100 and 500 mg/(kg x d) for 30 days. After the exposure, the testis, epididymis, liver and pituitary of the rats were weighted and their ratios to the body weight obtained. Histopathological changes of the testis and epididymis were examined by Hematoxylin-eosin staining, the levels of testosterone (T), luteinizing hormone (LH) and follicle stimulating hormone (FSH) in the serum were measured by radioimmunoassay, and the relative mRNA expressions of the steroidogenesis acute regulatory protein (StAR), proliferating cell nuclear antigen (PCNA), cytochrome P450 cholesterol side chain cleavage enzyme (P450scc) and scavenger receptor (SR) were detected by real-time quantitative RT-PCR.

RESULTSDBP induced significant histopathological changes in the testicular tissue at 100 and 500 mg/(kg x d), and decreased the testicular and epididymal weights, inhibited the mRNA expressions of StAR and PCNA, reduced the levels of T and LH, and elevated the level of FSH at 500 mg/(kg x d). At the dose of 10 mg/(kg x d), DBP increased serum LH and FSH and the mRNA expression of P450scc. While the SR mRNA expression showed no significant changes in all the groups.

CONCLUSIONHigh level of DBP has apparent toxic effect on reproductive system of male rats. Low - dose DBP can increase the level of serum gonadotropin LH and affect the mRNA expression of P450scc in the testis.

Animals ; Cholesterol Side-Chain Cleavage Enzyme ; metabolism ; Dibutyl Phthalate ; administration & dosage ; toxicity ; Follicle Stimulating Hormone ; blood ; Luteinizing Hormone ; blood ; Male ; Phosphoproteins ; metabolism ; Proliferating Cell Nuclear Antigen ; metabolism ; RNA, Messenger ; genetics ; Rats ; Rats, Sprague-Dawley ; Receptors, Scavenger ; metabolism ; Testis ; drug effects ; metabolism

AIMTo study the effect and mechanism of gonadotrophin-releasing hormone (GnRH) on murine Leydig cell steroidogenesis.

METHODSPurified murine Leydig cells were treated with GnRH-I and -II agonists, and testosterone production and steroidogenic enzyme expressions were determined.

RESULTSGnRH-I and -II agonists significantly stimulated murine Leydig cell steroidogenesis 60%-80% in a dose- and time-dependent manner (P < 0.05). The mRNA expressions of steroidogenic acute regulatory (StAR) protein, P450scc, 3beta-hydroxysteroid dehydrogenase (HSD), but not 17alpha-hydroxylase or 17beta-HSD, were significantly stimulated by both GnRH agonists with a 1.5- to 3-fold increase (P < 0.05). However, only 3beta-HSD protein expression was induced by both GnRH agonists, with a 1.6- to 2-fold increase (P < 0.05).

CONCLUSIONGnRH directly stimulated murine Leydig cell steroidogenesis by activating 3b-HSD enzyme expression.

3-Hydroxysteroid Dehydrogenases ; biosynthesis ; genetics ; Animals ; Blotting, Western ; Cell Separation ; Cells, Cultured ; Cholesterol Side-Chain Cleavage Enzyme ; biosynthesis ; Dose-Response Relationship, Drug ; Gonadotropin-Releasing Hormone ; agonists ; pharmacology ; Leydig Cells ; drug effects ; metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Phosphoproteins ; biosynthesis ; genetics ; RNA ; biosynthesis ; isolation & purification ; Reverse Transcriptase Polymerase Chain Reaction ; Sexual Maturation ; physiology ; Steroids ; biosynthesis ; Testosterone ; biosynthesis

OBJECTIVETo investigate the effects of Ginkgo biloba extract (EGB) on the testosterone synthesis in the Leydig cells of type 2 diabetic rats.

METHODSThirty male SD rats were equally randomised into a normal control, a type 2 diabetic and an EGB group. Morphological changes of Leydig cells were observed by light microscopy (LM) and transmission electron microscopy (TEM), concentrations of serum luteinizing hormone (LH) and testosterone (T) were determined by enzyme linked immunosorbent assay (ELISA), and the mRNA levels in the steroidogenic acute regulatory protein (StAR), cytochrome P450 side chain cleavage (P450scc), cytochrome P450 17a-hydroxylase (P450c17), 17beta-hydroxysteroid dehydrogenase 3 (17beta-HSD3) and 3beta-hydroxysteroid dehydrogenase (3beta-HSD1) from the Leydig cells were examined by RT-PCR.

RESULTSCompared with the normal control, there was a significant decrease in the number and volume of Leydig cells, the levels of serum LH and T and the expression of mRNA in StAR, P450scc, 17beta-HSD3 and 3beta-HSD1 in the type 2 diabetes group. And the expression of the P450c17 gene showed a tendency of descending, but with no significance. Compared with the type 2 diabetes group, 12 weeks of EGB treatment caused very slight pathological changes in the Leydig cells, significantly increased the concentrations of blood LH and T, markedly elevated the levels of mRNA in StAR and P450scc and induced an ascending tendency of the expressions of P450c17, 17beta-HSD3 and 3beta-HSD1.

CONCLUSIONEGB enhances testosterone synthesis and secretion of Leydig cells by reducing the impairment of the testis in type 2 diabetic rats.

17-Hydroxysteroid Dehydrogenases ; genetics ; Animals ; Cholesterol Side-Chain Cleavage Enzyme ; genetics ; Diabetes Mellitus, Type 2 ; blood ; genetics ; physiopathology ; Enzyme-Linked Immunosorbent Assay ; Gene Expression ; drug effects ; Ginkgo biloba ; chemistry ; Hydroxysteroid Dehydrogenases ; genetics ; Leydig Cells ; drug effects ; metabolism ; ultrastructure ; Luteinizing Hormone ; blood ; Male ; Microscopy, Electron, Transmission ; Phosphoproteins ; genetics ; Plant Extracts ; pharmacology ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction ; Testosterone ; biosynthesis ; blood

The steroidogenic acute regulatory (StAR) protein is an essential component in the regulation of steroid biosynthesis in adrenal and gonadal cells. The StAR protein has a high tissue specificity, located on the mitochondrial membranes of some relative cells. It regulates the transfer of cholesterin from extracellular into intracellular and plays a dominant role in steroidogenic synthesis. Recent studies have also shown that the transcription and expression of StAR are modulated not only through the cAMP-PKA dependent pathway, but also by multiple hormones and cytokines, which contributes to the regulation of cholesterin synthesis.
Animals ; Cholesterol ; metabolism ; Cholesterol Side-Chain Cleavage Enzyme ; metabolism ; Ethisterone ; metabolism ; Gene Expression Regulation ; Humans ; Mice ; Mitochondria ; metabolism ; Phosphoproteins ; genetics ; physiology ; Rats

OBJECTIVETo study the influence of electromagnetic irradiation on cytochrome P450 cholesterol side chain lyase (P450scc) in adult rat testis tissues and to assess the protective effect of the copper shield.

METHODSHealthy male Wistar rats were randomized into a control group, an electromagnetic irradiation group and a wholly shielded group (with the copper shielding net). The electromagnetic irradiation group and the shielded group were set for 4 phases of 3, 6, 24 and 72 hours after irradiation, 15 rats for each phrase. The testosterone contents in the serum of the irradiated rats at 3, 6, 24 and 72 hours and in that of the controls were measured by radioimmunoassay(RIA), and so was the level of the P450scc mRNA in the testis tissues by semi-quantitative RT-PCR. And the effect of the copper shielding net on testosterone and P450scc mRNA was observed.

RESULTSThe contents of testosterone and the P450scc mRNA level in the irradiated group were significantly lower than in the control rats, decreased by 83.9% and 56.9% at 3 hours (P < 0.01), 54.8% and 27.3% at 6 hours (P < 0.01), restored to normal at 24 hours, but again reduced by 60.1% and 56.1% respectively (P < 0.01). While in the shielded group, no significant change was observed either in the testosterone of the serum or in the P450scc mRNA expression in the testis tissues.

CONCLUSIONElectromagnetic irradiation may affect the transcription of P450scc in adult rat Leydig cells and thereby decrease the testosterone synthesis. Whole-body shielding with the copper net may achieve satisfactory effect.

Animals ; Cholesterol Side-Chain Cleavage Enzyme ; biosynthesis ; genetics ; Copper ; Male ; RNA, Messenger ; genetics ; Radiation Protection ; instrumentation ; Radioimmunoassay ; Random Allocation ; Rats ; Rats, Wistar ; Reverse Transcriptase Polymerase Chain Reaction ; Testis ; metabolism ; radiation effects ; Testosterone ; blood

To determine the (tttta)n repeat polymorphisms at the promoter region of CYP11alpha gene, and study its linkage to hyperandrogenism of polycystic ovary syndrome (PCOS) in Chinese women, a case-control study was conducted in the Reproductive Medical Center of the Second Affiliated Hospital of Zhengzhou University (Zhengzhou, China). 96 PCOS patients and 78 healthy control women were included. CYP11alpha (tttta)n repeat-polymorphism genotyping analysis was performed by using polymerase chain reaction (PCR). Serum pituitary hormone and total testosterone levels were measured by ELISA. 4 different CYP11alpha (tttta)n allelles were identified, corresponding to 4-, 6-, 8-, and 9-repeat-unit alleles. The frequency and distribution of these alleles are 0.16, 0.33, 0.38, and 0.13 respectively in PCOS patients, as compared with 0.20, 0.34, 0.35, and 0.11 respectively in healthy controls. There were no significant differences between these two groups. Moreover, no correlation between the polymorphism of CYP11alpha gene and serum testosterone level of patients with PCOS and controls was observed. It is concluded that microsatellite polymorphism (tttta)n of gene CYP11alpha exists in Chinese women and the polymorphism of CYP11alpha gene does not play an important role in the pathogenesis of Chinese patients with PCOS, especially in patients with hyperandrogenism.
Cholesterol Side-Chain Cleavage Enzyme/*genetics ; Hyperandrogenism/complications ; Hyperandrogenism/*genetics ; Microsatellite Repeats ; Polycystic Ovary Syndrome/complications ; Polycystic Ovary Syndrome/*genetics ; *Polymorphism, Genetic/genetics