OBJECTIVE: To comprehensively evaluate the effect of icariin in alleviating reproductive function damage (RFD) in male mice via in vitro and in vivo experiments. METHODS: We isolated Leydig cells from 60 KM male mice in vitro, and examined the toxic effect of icariin on the Leydig cells using Cell Counting Kit-8 (CCK-8). We equally randomized the mice into six groups: normal control, RFD model control (made by intraperitoneal injection of busulfan at 10 mg/kg combined with cyclophosphamide (CP) at 120 mg/kg), positive control, and low-, medium- and high-dose icariin. After modeling, we treated the mice in the positive control group with Wuziyanzong Pills and those in the low-, medium- and high-dose icariin groups by intragastrical administration of icariin at 20, 40 and 80 mg/kg-1, respectively, for 30 successive days. Then we obtained the weight and visceral coefficients of the reproductive organs, calculated the sperm count, observed the pathological changes in the testis tissue by HE staining, measured the serum testosterone (T) level by ELISA, determined the indexes of testicular oxidative stress and nitric oxide (NO) signaling pathway by colorimetric assay, and detected the expression levels of the pro-apoptotic genes Fas and Bax by qRT-PCR. RESULTS: CCK-8 assay confirmed that icariin had no toxic effect on the isolated Leydig cells of the mice, and could effectively reduce busulfan- and CP-induced cytotoxicity and promote the secretion of serum T. Icariin at 80 mg/kg significantly increased the visceral coefficient of the testis and promoted spermatogenesis (P<0.05), but had little effect on the visceral coefficient of the epididymis in the RFD model mice. Testicular histomorphometric observation revealed significantly improved testis structure, intact boundary membrane of seminiferous tubules and increased numbers of various types of spermatogenic cells of the model mice after treated with icariin. Compared with the mice in the model control group, those treated with high-dose icariin showed a significantly reduced content of malondialdehyde (MDA) (by 35.3%, P<0.01), elevated total antioxidant capacity (TAOC) and superoxide dismutase (T-SOD) activity (P<0.05), and decreased NO content and nitric oxide synthase (NOS) activity in the testis tissue (P<0.01). In addition, icariin exhibited an evident inhibitory effect on the expressions of the pro-apoptotic genes Bax and Fas. CONCLUSION Icariin can ameliorate oxidative stress-induced damage to the testicular function and protect spermatogenesis of male mice by elevating TAOC, decreasing NOS activity, inhibiting the NO level in the testis, and suppressing busulfan- and CP-induced apoptosis of testicular cells.
Animals ; Male ; Cyclophosphamide/adverse effects* ; Mice ; Busulfan/adverse effects* ; Flavonoids/pharmacology* ; Leydig Cells/drug effects* ; Oxidative Stress/drug effects* ; Testis/drug effects* ; Apoptosis/drug effects* ; Testosterone/blood*

OBJECTIVETo investigate the effects of hydro-alcoholic extract of Launaea acanthodes, a blood glucose lowering plant in folk medicine of Iran, on the structure of seminiferous tubules and serum gonadotropin and testosterone levels in hyperglycemic rats.

METHODSTwenty-four Wistar rats were randomly allocated into 4 groups (n=6): control, streptozotocin (STZ), STZ + insulin [STZ + Ins, 5 IU/(kg•day)], and STZ + Launaea acanthodes extract [STZ + Ext, 150 mg/(kg•day)]. Blood samples were collected at the 2nd and 4th weeks for detection of testosterone, follicle stimulating hormone (FSH) and luteinizing hormone (LH) with enzyme-linked immuno sorbent assay (ELISA), and the right testes of rats were removed at the 7th week for the evaluation of diameter and wall thickness of seminiferous tubules and number of Leydig cells using unbiased stereological techniques.

RESULTSIn comparison with the control group, at the 2nd week FSH (0.45 vs 0.03, 0.02, 0.02 IU/L in STZ, STZ + Ins and STZ + Ext groups, respectively) and LH (1.02 vs 0.37, 0.2, 0.29 IU/L) showed significant decreases (all P<0.05) and testosterone (4.2 vs 8.37, 7.78, 11.8 ng/mL) showed a remarkable increase (all P<0.05). The levels of these hormones became closer in the STZ + Ext and the STZ + Ins groups to the control at the 4th week. A significant decrease in diameter and wall thickness of seminiferous tubules and number of Leydig cells were observed in the STZ group as compared with the control (P<0.01).

CONCLUSIONSAdministration of Launaea extract demonstrated a beneficial impact on the protection of testis from pathogenic and degenerative effects of hyperglycemia which may be partly due to its potential antioxidative effects.

Animals ; Asteraceae ; chemistry ; Blood Glucose ; metabolism ; Cell Count ; Cholesterol ; blood ; Ethanol ; chemistry ; Gonadotropins ; blood ; Hyperglycemia ; blood ; drug therapy ; pathology ; Insulin ; blood ; Leydig Cells ; drug effects ; pathology ; Lipoproteins ; blood ; Male ; Plant Extracts ; pharmacology ; therapeutic use ; Rats, Wistar ; Seminiferous Tubules ; drug effects ; pathology ; Testosterone ; blood ; Triglycerides ; blood ; Water ; chemistry

PURPOSE: Foxo3 in female reproduction has been reported to regulate proliferation of granulose cells that form follicles. There are no reports so far that discuss on the role of Foxo3 in males. This study was designed to outline the role of Foxo3 in the testes. MATERIALS AND METHODS: Testes from mice at birth to postpartum week (PPW) 5 were isolated and examined for the expression of Foxo3 using immunostaining. To elucidate role of Foxo3 in Leydig cells, R2C cells were treated with luteinizing hormone (LH) and the phosphorylation of Foxo3. Testosterone and steroidogenic acute regulatory (StAR) protein levels were measured after constitutive active [triple mutant (TM)] human FOXO3 adenovirus was transduced and StAR promoter assay was performed. RESULTS: Foxo3 expression in the testicles started from birth and lasted until PPW 3. After PPW 3, most Foxo3 expression occurred in the nuclei of Leydig cells; however, at PPW 5, Foxo3 was expressed in both the nucleus and cytoplasm. When R2C cells were treated with luteinizing hormone, Foxo3 phosphorylation levels by AKT increased. After blocking the PI3K pathway, LH-induced phosphorylated Foxo3 levels decreased, indicating that LH signaling regulates Foxo3 localization. When active FOXO3-TM adenovirus was introduced into a Leydig tumor cell line, the concentrations of testosterone and StAR protein decreased. When FOXO3 and a StAR promoter vector were co-transfected into HEK293 cells for a reporter assay, FOXO3 inhibited the StAR promoter. CONCLUSION: FOXO3 affects testosterone synthesis by inhibiting the formation of StAR protein. LH hormone, meanwhile, influences Foxo3 localization, mediating its function.
Animals ; Cell Aging/*physiology ; Cell Nucleus/metabolism ; Cytoplasm/metabolism ; Forkhead Transcription Factors/*metabolism ; HEK293 Cells ; Humans ; Leydig Cells/*drug effects/*enzymology/metabolism ; Luteinizing Hormone/blood ; Male ; Mice ; Phosphatidylinositol 3-Kinases ; Phosphoproteins/metabolism ; Phosphorylation ; Signal Transduction/drug effects ; Testosterone/blood/*metabolism

OBJECTIVETo explore the role of the basic fibroblast growth factor (bFGF) in the directional differentiation of bone marrow mesenchymal stem cells (BMSCs) into Leydig cells.

METHODSAfter purification and identification, we inoculated the third-generation BMSCs of SD rats onto a six-orifice board and then randomly divided them into groups A (normal saline control), B (human chorionic gonadotropin [hCG] + platelet-derived growth factor [PDGF] induction), C (hCG + PDGF + 5.0 ng/ml bFGF induction), D (hCG + PDGF + 10.0 ng/ml bFGF induction), and E (hCG + PDGF + 20.0 ng/ml bFGF induction). On the 7th, 14th and 21st day of induction, we observed the morphological changes of the cells and measured the level of testosterone (T) and expression of 3 beta hydroxy steroid dehydrogenase (3β-HSD) in the supernatant by immunofluorescence staining.

RESULTSAfter induction, the BMSCs of groups B, C, D, and E exhibited microscopic features of enlarged size, inter-connection, long-shuttle or irregular shape, adherent growth, and large round nuclei, all characteristic of Leydig cells. With the prolonging of time and enhanced concentration of bFGF, gradual increases were observed in the T level and the count of 3β-HSD-positive BMSCs in the four induction groups, with statistically significant differences between group B and groups C, D, and E (P < 0.05), as well as between group C and groups D and E (P < 0.05), but not between D and E (P > 0.05).

CONCLUSIONThe bFGF has an obvious promoting effect in the in vitro induced differentiation of rat BMSCs into Leydig cells.

Animals ; Bone Marrow Cells ; cytology ; drug effects ; metabolism ; Cell Differentiation ; Cells, Cultured ; Chorionic Gonadotropin ; metabolism ; Fibroblast Growth Factor 2 ; pharmacology ; Humans ; Leydig Cells ; cytology ; Male ; Mesenchymal Stromal Cells ; cytology ; drug effects ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Testosterone ; metabolism

OBJECTIVETo investigate the effects and mechanisms of diethylhexylphthalate (DEHP) on morphology and function of progenitor Leydig cells (PLC) in rats.

METHODSTwenty pregnant SD rats were randomly divided into 4 groups ( n = 5): normal control group, DEHP low dose group , middle dose group, and high dose group, which were treated from postnatal day (PND) 1 to PND 21 of the pubs with DEHP at the doses of 0, 10, 100, 750 mg/(kg · d) in 0.5 ml of corn oil by gavage respectively. At the end of the treatment, the male pups were killed and blood samples were collected for determination of serum testosterone concentration by chemiluminescence method. The body weight, testis weight and anogenital distance (AGD) were measured. The morphology of PLC was observed by light and transmission electron microscopy. The protein expression of steroidogenic acute regulatory protein(StAR) in PLC was determined by immunohistochemistry. The mRNA expression of insulin-like growth factor-I (IGF-I) in the testis was assayed by real-time PCR.

RESULTSCompared with normal control group, the serum testosterone and AGD of male pubs from the middle and high dose groups were declined significantly (P < 0.01), the testis weight and body weight from high dose group were decreased significantly (P < 0.01), while the testis weight increased in the low dose group (P < 0.05). Under light microscope, PLC showed hyperplasia and cluster aggregation in the low dose group and focal hyperplasia in the middle and high dose group. The spermatogenic cells in seminiferous tubules showed decrease, apoptosis and unfix in the high dose group. Under transmission electron microscope, the PLC showed decreased lipid droplets, smooth endoplasmic reticulum and mitochondriae in the treated group. The mRNA expression of IGF-I increased in the low dose group, and the protein expression of StAR decreased in the middle and high dose group.

CONCLUSIONLactating exposure to DEHP may interfere with the synthesis of testosterone of PLC in male pubs, the decrease of StAR and the damage of PLC may be involved in it.

Animals ; Body Weight ; Diethylhexyl Phthalate ; adverse effects ; Female ; Germ Cells ; drug effects ; Insulin-Like Growth Factor I ; metabolism ; Lactation ; Leydig Cells ; cytology ; drug effects ; Male ; Organ Size ; Phosphoproteins ; metabolism ; Pregnancy ; Rats ; Rats, Sprague-Dawley ; Stem Cells ; cytology ; drug effects ; Testis ; Testosterone ; blood

OBJECTIVETo study the impact of the water extract from Codonopsis thalictrifolia Wall (CTW) on the reproductive

METHODSWe divided 32 male SD infant rats into four groups of equal number to be treated intragastrical-system of male infant rats. ly with distilled water (control) and CTW at 10 g/kg (low dose) , 20 g/kg (medium dose), and 40 g/kg (high dose), respectively, twice a day for 2 weeks. Then we killed the rats, measured the levels of testosterone (T), luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in the serum, obtained the testis weight, body weight, testis visceral coefficient and sperm concentration, and detected sperm viability, sperm motility and the level of cyclic adenosine monophosphate (cAMP) in the Leydig cells, followed by

RESULTSCompared with the control group, the low-dose, me-analysis of differences among different groups using the SPSS software. Medium-dose and high-dose CTW groups showed significant decreases in the serum T level ([3.09 +/-0.42] vs [1.22 +/-0. 32] , [1.06 +/- 0.29] and [0.57 +/-0.18] nmol/L, P<0.01), testis weight ([1.40 +/-0.16] vs [0.96 +/-0.09], [0.92 +/-0.11] and [0.91 +/- 0.08] g, P <0.01), and sperm concentration ([1.03 +/-0.16] vs [0.19 +/-0.07], [0.17 +/-0.08] and [0.16 +/-0.07] x 10(6)/ml, P <0.01), but a dramatic elevation in the testis visceral coefficient ([42.22 +/- 3.02] vs [51.39 +/- 3.09], [52.28 +/- 4.86] and [54.13 +/-6.06] mg/10 g, P <0.01); the medium- and high-dose CTW groups exhibited remarkable increases in the levels of serum LH ([13.62+/-0.89] vs [14.69 +/-0.12] and [14.93 +/-0.28] ng/L, P<0.01) and FSH ([4.32 +/-0.18] vs [4.77 +/-0.23] and [4.89 +/-0. 38] IU/L, P <0.05); all the three CTW groups showed markedly inhibited serum T secretion ([1.85 +/- 0.18] vs [1.42 +/-0.15], [1.12+/-0.18] and [0.88 +/-0.21] nmol/L, P<0.01) and intracellular cAMP ([5.51 +/-0.12] vs [4.39+/-0.06], [4.28 +/-0.07] and [4.11 +/- 0.10] nmol/L, P <0.01) in the Leydig cells.

CONCLUSIONThe water extract from CTW may reduce the synthesis of testosterone in the serum of male infant rats through the PKA pathway and consequently inhibit their testicular development and sperm production and affect the development of their reproductive system.

Animals ; Codonopsis ; chemistry ; Cyclic AMP ; metabolism ; Leydig Cells ; metabolism ; Male ; Plant Extracts ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Testosterone ; blood ; Urogenital System ; drug effects

OBJECTIVETo investigate the effects of total flavonoids of Litsea Coreana (TFLC) on the gap junction (GJ) intercellular communication in TM3 testicular Leydig cells and whether TFLC can reduce the cytotoxicity of oxaliplatin (OHP) in vitro.

METHODSWe detected the effect of TFLC on the dye spread of the in vitro cultured TM3 cells by parachute assay, observed changes in the expression of connexin 43 (Cx43) total protein in the TFLC-treated TM3 cells by Western blot, and determined the effects of TFLC on the expression of Cx43 on the membrane of the TM3 cells by immunofluorescence assay and on the cytotoxicity of OHP by MTT assay.

RESULTSTFLC obviously enhanced the GJ function with the increasing of the TFLC concentration in the TM3 cells. Western blot and immunofluorescence assay confirmed that TFLC significantly enhanced the expression of Cx43 total protein and Cx43 expression on the membrane of the TM3 cells. MTT assay showed that at a high cell density (confluent with GJ formation), 20 microg/ml TFLC enhanced the GJ function of the TM3 cells and reduced the cytotoxicity of OHP (P < 0.05), while at a low density (preconfluent with no GJ formation), TFLC exhibited no effect on the cytotoxicity of OHP (P > 0.05).

CONCLUSIONTFLC increases the Cx43 expression and GJ function in normal TM3 Leydig cells, and the enhancement of GJ function reduces the cytotoxicity of OHP.

Antineoplastic Agents ; toxicity ; Cell Communication ; drug effects ; physiology ; Cell Count ; Connexin 43 ; metabolism ; Flavonoids ; pharmacology ; Gap Junctions ; drug effects ; Humans ; In Vitro Techniques ; Leydig Cells ; drug effects ; ultrastructure ; Litsea ; chemistry ; Male ; Organoplatinum Compounds ; antagonists & inhibitors ; toxicity ; Proteins ; metabolism

OBJECTIVETo explore the repair of Xiangsha Liujunzi Decoction (XSLJZD) on interstitial cells of Cajal (ICC) and gap junction (GJ) in the gastric muscular layer of rats of Pi-qi deficiency syn- drome (PQDS).

METHODSPQDS was established using purgative method with bitter and cold drugs in 30 healthy Wistar rats. After successful modeling they were randomly divided into the treatment group and the model group, 15 in each group. Another 15 healthy Wistar rats were recruited as the healthy control group. Rats in the treatment group were gastric administered with XSLJZD at 2 mL/100 g body weight, once daily for 14 successive days. Equal volume of normal saline was gastrically administered to those in the healthy control group and the model group. The gastric muscle tissues were taken out before modeling, before intervention, and after intervention, respectively. Ultrastructural changes of ICC and GJ were observed using transmission electron microscope (TEM). The number and distribution of Connexin43 (Cx43) were detected using immunohistochemistry.

RESULTSResults of TEM indicated that compared with the healthy control group, both ICC and GJ in the model group showed obvious injury. ICC and GJ were apparently repaired after intervention in the treatment group. Compared with the same group before modeling, the integrated optical density (IOD) of the Cx43 expression significantly decreased in the model group before and after intervention (P <0.05). Compared with before intervention, the IOD of the Cx43 expression significantly increased in the treatment group (P <0.05). Compared with the healthy control group, the IOD of the Cx43 expression significantly decreased in the model group before and after intervention (P <0.05). Compared with the model group, the IOD of the Cx43 expression significantly increased in the treatment group (P <0.05).

CONCLUSIONSUltrastructures of ICC and GJ in the gastric muscular layer of rats of PQDS were obviously damaged. XSLJZD could repair the structural damage of ICC and GJ in the gastric muscle tissues of rats of PQDS.

Animals ; Connexin 43 ; Drugs, Chinese Herbal ; pharmacology ; Gap Junctions ; Interstitial Cells of Cajal ; drug effects ; Leydig Cells ; Male ; Muscle, Smooth ; Qi ; Rats, Wistar ; Syndrome

OBJECTIVETo investigate the effects of di-(2-ethylhexyl) phthalate (DEHP) on the expressions of Caspase-3 and Caspase-9 genes in rat Leydig cells and the apoptosis of the cells in vitro.

METHODSLeydig cells were isolated from male SD rats, primarily cultured and treated with DEHP at a low (10 nmol/L), a medium (50 nmol/L) and a high dose (100 nmol/L) for 24 hours. Then the mRNA expressions of Caspase-3 and Caspase-9 genes in the Leydig cells were detected by real time PCR, their protein expressions determined by Western blot, and the apoptosis of the Leydig cells measured by flow cytometry.

RESULTSCompared with the DMSO control group, the low-, medium- and high-dose DEHP groups showed significantly upregulated expressions of Caspase-3 mRNA (1.69 +/- 0.38 vs 3.82 +/- 0.39, 6.91 +/- 0.40 and 15.47 +/- 0.40, P < 0.05), Caspase-3 protein (0.18 +/- 0.0.09 vs 0.32 +/- 0.10, 0.61 +/- 0.08 and 0.89 +/- 0.09, P < 0.05), Caspase-9 mRNA (2.24 +/- 0.41 vs 5.16 +/- 0.43, 9.61 +/- 0.45 and 19.22 +/- 0.43, P < 0.05) and Caspase-9 protein (0.26 +/- 0.07 vs 0.40 +/- 0.08, 0.68 +/- 0.09 and 0.96 +/- 0.08, P < 0.05), as well as increased apoptosis rate of Leydig cells (4.36 +/- 1.11 vs 7.52 +/- 1.09, 12.72 +/- 1.10 and 24.59 +/- 1.11, P < 0.05), all in a dose-dependent manner.

CONCLUSIONDEHP can induce the apoptosis of rat Leydig cells by activating the apoptosis Caspase pathway, and consequently affect the function of Leydig cells.

Animals ; Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Caspase 9 ; metabolism ; Diethylhexyl Phthalate ; toxicity ; Leydig Cells ; drug effects ; metabolism ; Male ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo study the effect of ethane dimethane sulfonate (EDS) injection on the volumes of different histological structures in the seminal vesicles of adult rats.

METHODSTwenty-seven male SD rats aged approximately 90 days were randomly divided into a control group (n = 14) and an EDS group (n = 13) to receive one intraperitoneal injection of normal saline and EDS (75 mg/kg bodyweight), respectively. At 7 and 12 days after treatment, the unilateral seminal vesicles were removed, methacrylate resin-embedded sections prepared and the total volumes of various structures in the seminal vesicles estimated using stereological methods.

RESULTSEDS treatment almost completely destroyed the Leydig cells in the testis, resulting in a drastic testosterone deficiency. The volume of the seminal vesicle (including the coagulating gland attached to the vesicle) was decreased by 53% in the 7 d EDS group (n = 6) in comparison with the 7 d control group (n = 7) ([138.2 +/- 12.9] vs [64.9 +/- 3.6] mm3, P < 0.01), but showed no significant difference between the 7 d and the 12 d EDS (n = 7) groups ([64.9 +/- 3.6] vs [55.4 +/- 7.7] mm3, P > 0.05). The total volumes of the glandular lumen, glandular epithelium, smooth muscular layer and adventitia were decreased by 96.7, 80.3, 57.6 and 67.0%, respectively, in the 12 d EDS group as compared with the 12 d control group (n = 7).

CONCLUSIONEDS induces drastic testosterone deficiency in adult rats, and significantly reduces the total volumes of the seminal vesicle lumen, glandular epithelium, smooth muscular layer and adventitia.

Animals ; Leydig Cells ; drug effects ; Male ; Mesylates ; pharmacology ; Organ Size ; drug effects ; Rats ; Rats, Sprague-Dawley ; Seminal Vesicles ; drug effects ; pathology ; Testis ; cytology ; drug effects ; pathology