Ginsenoside Rg3 protects mouse leydig cells against triptolide by downregulation of miR-26a.

BACKGROUND: Ginsenoside Rg3 has been reported to exert protection function on germ cells. However, the mechanisms by which Rg3 regulates apoptosis in mouse Leydig cells remain unclear. In addition, triptolide (TP) has been reported to induce infertility in male rats. Thus, this study aimed to investigate the protective effect of Rg3 against TP-induced toxicity in MLTC-1 cells. METHODS: CCK-8, immunofluorescence assay, Western blotting and flow cytometry were used to detect cell proliferation and cell apoptosis, respectively. In addition, the dual luciferase reporter system assay was used to detect the interaction between miR-26a and GSK3ß in MLTC-1 cells. RESULTS: TP significantly inhibited the proliferation of MLTC-1 cells, while the inhibitory effect of TP was reversed by Rg3. In addition, TP markedly induced apoptosis in MLTC-1 cells via increasing the expressions of Bax, active caspase 3, Cyto c and active caspase 9, and decreasing the level of Bcl-2. However, Rg3 alleviated TP-induced apoptosis of MLTC-1 cells. Moreover, the level of miR-26a was obviously downregulated by Rg3 treatment. The protective effect of Rg3 against TP-induced toxicity in MLTC-1 cells was abolished by miR-26a upregulation. Meanwhile, dual-luciferase assay showed GSK3ß was the direct target of miR-26a in MLTC-1 cells. Overexpression of miR-26a markedly decreased the level of GSK3ß. As expected, upregulation of miR-26a could abrogate the protective effects of Rg3 against TP-induced cytotoxicity via inhibiting the expression of GSK3ß. CONCLUSION: These results indicated that Rg3 could protect MLTC-1 against TP by downregulation of miR-26a. Therefore, Rg3 might serve as a potential agent for the treatment of male hypogonadism.

Fibroblast growth factor 16 stimulates proliferation but blocks differentiation of rat stem Leydig cells during regeneration.

OBJECTIVES: We aim to investigate the effects of fibroblast growth factor 16 (FGF16) on Leydig cell regeneration in ethane dimethane sulphonate (EDS)-treated rat testis. METHODS: We intraperitoneally inject 75 mg/kg EDS to adult male Sprague Dawley rats and then intratesticularly inject FGF16 (0, 10 and 100 ng/testis/day) from post-EDS day 14 for 14 days. We investigate serum hormone levels, Leydig cell number, gene and protein expression in vivo. We also explore the effects of FGF16 treatment on stem Leydig cell proliferation in vitro. RESULTS: FGF16 lowers serum testosterone levels (21.6% of the control at a dose of 100 ng/testis) without affecting the levels of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) on post-EDS day 28 in vivo. FGF16 increases Leydig cell number at doses of 10 and 100 ng/mg without affecting Sertoli cell number, increases the percentage of PCNA-positive Leydig cells, and down-regulates the expression of Leydig cell genes (Lhcgr, Scarb1, Star, Cyp11a1, Cyp17a1 and Hsd17b3) and Sertoli cell genes (Fshr, Dhh and Sox9) and their proteins in vivo. FGF16 increases phosphorylation of AKT1 and AKT2 as well as EKR1/2 in vivo, indicating that it possibly acts via AKT1/ATK2 and ERK1/2 pathways. FGF16 also lowers medium testosterone levels and down-regulates the expression of Leydig cell genes (Lhcgr, Scarb1, Star, Cyp11a1, Cyp17a1 and Hsd17b3) but increases EdU incorporation into stem Leydig cells in vitro. CONCLUSIONS: These data suggest that FGF16 stimulates stem and progenitor Leydig cell proliferation but blocks their differentiation, thus lowering testosterone biosynthesis.

In vitro study evaluating the instantaneous treatment of ozonised olive oil on human sperm.

OBJECTIVE: The aim of our study was to evaluate the effects of ozonised olive oil (OOO) on human sperm in vitro. METHODS: Human sperm was incubated with OOO for 20 s in vitro. The lowest concentration that completely immobilised all the sperm in 20 s without subsequent recovery of motility was recorded as the minimum effective concentration (MEC). The effects of OOO at MEC on human sperm viability, mitochondrial and acrosomal status, DNA integrity and transmission electron microscopy were observed. RESULTS: Our findings demonstrate that OOO dose-dependently inhibits sperm motility. The MEC of OOO for 100% sperm immobilisation in 20 s was 6 µg/ml. Further experiments showed that sperm ultrastructure, function and DNA integrity were significantly affected after treatment with 6 µg/ml OOO in vitro. CONCLUSIONS: OOO has spermicidal potential and may be explored as a promising vaginal contraceptive agent.

Lonidamine-ethyl ester-mediated remodelling of the Sertoli cell cytoskeleton induces phosphorylation of plakoglobin and promotes its interaction with α-catenin at the blood-testis barrier.

Several compounds affect male fertility by disrupting the adhesion of germ cells to Sertoli cells, which results in the release of undeveloped germ cells into the seminiferous tubule lumen that are incapable of fertilising the ovum. Indazole carboxylic acids are one class of compounds exhibiting such effects and they have been investigated as non-hormonal contraceptives for potential human use. The aims of this study were to investigate the effects of lonidamine-ethyl ester, an indazole carboxylic acid, on spermatogenesis and cell junctions, in particular, desmosomes. We found two doses of lonidamine-ethyl ester at 50mg kg-1 to disrupt Sertoli-germ cell adhesion. By light and fluorescent microscopy, pronounced changes were observed in the distribution of actin microfilaments and intermediate filaments, as well as in the localisation of plakoglobin, a protein with structural and signalling roles at the desmosome and adherens junction at the blood-testis barrier. Furthermore, immunoblotting and immunoprecipitation experiments using testis lysates revealed a significant upregulation (P<0.01) of plakoglobin and Tyr-phosphorylated plakoglobin. Co-immunoprecipitation experiments showed an increase in the interaction between plakoglobin and fyn proto-oncogene, an Src family non-receptor tyrosine kinase, after treatment, as well as an increase in the interaction between plakoglobin and α-catenin. Taken collectively, these data indicate that a disruption of Sertoli cell and spermatocyte-spermatid adhesion in the seminiferous epithelium by lonidamine-ethyl ester results in the phosphorylation of plakoglobin, thereby promoting its interaction with α-catenin at the blood-testis barrier.

The Adverse Effects of Triptolide on the Reproductive System of Caenorhabditis elegans: Oogenesis Impairment and Decreased Oocyte Quality.

Previous studies have revealed that Triptolide damages female reproductive capacity, but the mechanism is unclear. In this study, we used Caenorhabditis elegans to investigate the effects of Triptolide on the germline and explore its possible mechanisms. Our data show that exposure for 4 h to 50 and 100 mg/L Triptolide reduced C. elegans fertility, led to depletion and inactivation of spermatids with the changes in the expression levels of related genes, and increased the number of unfertilized oocytes through damaging chromosomes and DNA damage repair mechanisms. After 24 and 48 h of the 4 h exposure to 50 and 100 mg/L Triptolide, we observed shrink in distal tip cells, an increase in the number of apoptotic cells, a decrease in the number of mitotic germ cells and oocytes in diakinesis stage, and chromatin aggregates in -1 oocytes. Moreover, expression patterns of the genes associated with mitotic germ cell proliferation, apoptosis, and oocyte quality were altered after Triptolide exposure. Therefore, Triptolide may damage fertility of nematodes by hampering the development of oocytes at different developmental stages. Alterations in the expression patterns of genes involved in oocyte development may explain the corresponding changes in oocyte development in nematodes exposed to Triptolide.

Reversible Anti-Spermatogenic Effect of Piperine on Epididymis and Seminal Vesicles of Albino Rats.

BACKGROUND: We have recently proved the interactions of piperine with androgen receptor and androgen binding protein. The present study was aimed to evaluate the antifertility effect of piperine on male albino rats after the treatment period i. e., after 60 days and withdrawal period i. e., after 120 days. MATERIALS AND METHODS: Adult male rats were divided into 4 groups (n=12). Group I: CONTROL: Rats were given vehicle p.o i. e., 0.5% carboxy methyl cellulose (CMC) in normal saline daily for 60 days, Group II: Rats were treated with piperine suspended in 0.5% CMC at a dose of 10 mg/kg daily/60 days. Group III: Rats were treated with piperine suspended in 0.5% CMC at a dose of 10 mg/kg on every 4(th) day for 60 days. Group IV: Rats were treated with piperine suspended in 0.5% CMC at a dose of 10 mg/kg on every 7(th) day for 60 days. RESULTS: Piperine significantly altered the epididymal sperm count, motility, viability, weight of the epididymis, cauda, caput, corpus and seminal vesicles. It also exhibited negative impact on biochemical markers via decreasing epididymal sialic acid levels, seminal fructose content, epididymal anti-oxidant enzyme activities of super oxide dismutase (SOD), catalase (CAT) and by increasing the malondialdehyde content after the treatment period. Histopathological observations also supported the above findings. All the altered values were reinforced after the withdrawal period. CONCLUSION: From the results of this study, we can conclude that piperine has the potential to become a good lead for the reversible male oral contraceptive research.

The current state of male hormonal contraception.

World population continues to grow at an unprecedented rate, doubling in a mere 50years to surpass the 7-billion milestone in 2011. This steep population growth exerts enormous pressure on the global environment. Despite the availability of numerous contraceptive choices for women, approximately half of all pregnancies are unintended and at least half of those are unwanted. Such statistics suggest that there is still a gap in contraceptive options for couples, particularly effective reversible contraceptives for men, who have few contraceptive choices. Male hormonal contraception has been an active area of research for almost 50years. The fundamental concept involves the use of exogenous hormones to suppress endogenous production of gonadotropins, testosterone, and downstream spermatogenesis. Testosterone-alone regimens are effective in many men but high dosing requirements and sub-optimal gonadotropin suppression in 10-30% of men limit their use. A number of novel combinations of testosterone and progestins have been shown to be more efficacious but still require further refinement in delivery systems and a clearer understanding of the potential short- and long-term side effects. Recently, synthetic androgens with both androgenic and progestogenic activity have been developed. These agents have the potential to be single-agent male hormonal contraceptives. Early studies of these compounds are encouraging and there is reason for optimism that these may provide safe, reversible, and reliable contraception for men in the near future.

Development of water-soluble polyanionic carbosilane dendrimers as novel and highly potent topical anti-HIV-2 microbicides.

The development of topical microbicide formulations for vaginal delivery to prevent HIV-2 sexual transmission is urgently needed. Second- and third-generation polyanionic carbosilane dendrimers with a silicon atom core and 16 sulfonate (G2-S16), napthylsulfonate (G2-NS16) and sulphate (G3-Sh16) end-groups have shown potent and broad-spectrum anti-HIV-1 activity. However, their antiviral activity against HIV-2 and mode of action have not been probed. Cytotoxicity, anti-HIV-2, anti-sperm and antimicrobial activities of dendrimers were determined. Analysis of combined effects of triple combinations with tenofovir and raltegravir was performed by using CalcuSyn software. We also assessed the mode of antiviral action on the inhibition of HIV-2 infection through a panel of different in vitro antiviral assays: attachment, internalization in PBMCs, inactivation and cell-based fusion. Vaginal irritation and histological analysis in female BALB/c mice were evaluated. Our results suggest that G2-S16, G2-NS16 and G3-Sh16 exert anti-HIV-2 activity at an early stage of viral replication inactivating the virus, inhibiting cell-to-cell HIV-2 transmission, and blocking the binding of gp120 to CD4, and the HIV-2 entry. Triple combinations with tenofovir and raltegravir increased the anti-HIV-2 activity, consistent with synergistic interactions (CIwt: 0.33-0.66). No vaginal irritation was detected in BALB/c mice after two consecutive applications for 2 days with 3% G2-S16. Our results have clearly shown that G2-S16, G2-NS16 and G3-Sh16 have high potency against HIV-2 infection. The modes of action confirm their multifactorial and non-specific ability, suggesting that these dendrimers deserve further studies as potential candidate microbicides to prevent vaginal/rectal HIV-1/HIV-2 transmission in humans.

Alterations of gene profiles in Leydig-cell-regenerating adult rat testis after ethane dimethane sulfonate-treatment.

Only occupying about 1%-5% of total testicular cells, the adult Leydig cell (ALC) is a unique endocrine cell that produces androgens. Rat Leydig cells regenerate after these cells in the testis are eliminated with ethane dimethane sulfonate (EDS). In this study, we have characterized Leydig cell regeneration and messenger ribonucleic acids (mRNA) profiles of EDS treated rat testes. Serum testosterone, testicular gene profiling and some steroidogenesis-related proteins were analyzed at 7, 21, 35 and 90 days after EDS treatment. Testicular testosterone levels declined to undetectable levels until 7 days after treatment and then started to recover. Seven days after treatment, 81 mRNAs were down-regulated greater than or equal to two-fold, with 48 becoming undetectable. These genes increased their expression 21 days and completely returned to normal levels 90 days after treatment. The undetectable genes include steroidogenic pathway proteins: steroidogenic acute regulatory protein, Scarb1, Cyp11a1, Cyp17a1, Hsd3b1, Cyp1b1 and Cyp2a1. Seven days after treatment, there were 89 mRNAs up-regulated two-fold or more including Pkib. These up-regulated mRNAs returned to normal 90 days after treatment. Cyp2a1 did not start to recover until 35 days after treatment, indicating that this gene is only expressed in ALCs not in the precursor cells. Quantitative polymerase chain reaction, western blotting and semi-quantitative immunohistochemical staining using tissue array confirmed the changes of several randomly picked genes and their proteins.

Effect of aqueous leaf extract of Dalbergia sissoo Roxb. on spermatogenesis and fertility in male mice.

OBJECTIVES: Antifertility effects of Dalbergia sissoo in male mice were investigated. METHODS: Adult Parkes strain male mice were orally administered aqueous leaf extract of Dalbergia sissoo (50 and 100 mg/kg body weight/day) or distilled water or no treatment (controls) for 35 days (n = 5/group). Motility, viability and number of spermatozoa in the cauda epididymidis; testis histology; serum level of testosterone; and toxicological parameters were evaluated. To assess reversibility, more mice were treated with 100 mg/kg body weight of Dalbergia sissoo or distilled water (n = 5/group) for 35 days and sacrificed 56 days later. Fertility was also assessed separately. RESULTS: Histologically, testes of Dalbergia-treated mice showed dissimilar degenerative changes in the seminiferous tubules. Significant reductions were noted (i) in epididymal sperm motility, viability and number, and (ii) in serum level of testosterone in Dalbergia-treated mice compared to controls. However, serum levels of alanine aminotransferase, aspartate aminotransferase and creatinine, and haematological parameters were not affected. Also libido of Dalbergia-treated males showed no change, but their fertility was markedly suppressed. By 56 days of treatment withdrawal, alterations induced in the above parameters returned to control levels. CONCLUSIONS: Dalbergia sissoo treatment caused reversible suppression of spermatogenesis and fertility in P mice, without eliciting detectable toxic effects.

Evaluation of atorvastatin efficacy and toxicity on spermatozoa, accessory glands and gonadal hormones of healthy men: a pilot prospective clinical trial.

BACKGROUND: Recommendations for cardiovascular disease prevention advocate lowering both cholesterol and low-density lipoprotein cholesterol systemic levels, notably by statin intake. However, statins are the subject of questions concerning their impact on male fertility. This study aimed to evaluate, by a prospective pilot assay, the efficacy and the toxicity of a decrease of cholesterol blood levels, induced by atorvastatin on semen quality and sexual hormone levels of healthy, normocholesterolaemic and normozoospermic men. METHODS: Atorvastatin (10 mg daily) was administrated orally during 5 months to 17 men with normal plasma lipid and standard semen parameters. Spermatozoa parameters, accessory gland markers, semen lipid levels and blood levels of gonadal hormones were assayed before statin intake, during the treatment, and 3 months after its withdrawal. RESULTS: Atorvastatin treatment significantly decreased circulating low-density lipoprotein cholesterol (LDL-C) and total cholesterol concentrations by 42% and 24% (p<0.0001) respectively, and reached the efficacy objective of the protocol. During atorvastatin therapy and/or 3 months after its withdrawal numerous semen parameters were significantly modified, such as total number of spermatozoa (-31%, p<0.05), vitality (-9.5%, p<0.05), total motility (+7.5%, p<0.05), morphology (head, neck and midpiece abnormalities, p<0.05), and the kinetics of acrosome reaction (p<0.05). Seminal concentrations of acid phosphatases (p<0.01), α-glucosidase (p<0.05) and L-carnitine (p<0.05) were also decreased during the therapy, indicating an alteration of prostatic and epididymal functions. Moreover, we measured at least one altered semen parameter in 35% of the subjects during atorvastatin treatment, and in 65% of the subjects after withdrawal, which led us to consider that atorvastatin is unsafe in the context of our study. CONCLUSIONS: Our results show for the first time that atorvastatin significantly affects the sperm parameters and the seminal fluid composition of healthy men.

Interaction of oligomeric breast cancer resistant protein (BCRP) with adjudin: a male contraceptive with anti-cancer activity.

Breast cancer resistant protein (BCRP, ABCG2) is an ATP-binding cassette (ABC) transporter, which together with two other ABC efflux drug pumps, namely P-glycoprotein (P-gp, ABCB1) and multidrug resistance-related protein 1 (MRP1, ABCC1) is the most important multidrug resistance protein foun d in eukaryotic cells including cells in the testis. However, unlike P-gp and MRP1, which are components of the Sertoli cell blood-testis barrier (BTB), BCRP is not expressed at the BTB in rodents and human testes. Instead, BCRP is expressed by peritubular myoid cells and endothelial cells of the lymphatic vessel in the tunica propria, residing outside the BTB. As such, the testis is equipped with two levels of defense against xenobiotics or drugs, preventing these harmful substances from entering the adluminal compartment to perturb meiosis and post-meiotic spermatid development: one at the level of the BTB conferred by P-gp and MRP1 and one at the tunica propria conferred by BCRP. The presence of drug transporters at the tunica propria as well as at the Sertoli cell BTB thus poses significant obstacles in developing non-hormonal contraceptives if these drugs (e.g., adjudin) exert their effects in germ cells behind the BTB, such as in the adluminal (apical) compartment of the seminiferous epithelium. Herein, we summarize recent findings pertinent to adjudin, a non-hormonal male contraceptive, and molecular interactions of adjudin with BCRP so that this information can be helpful to devise delivery strategies to evade BCRP in the tunica propria to improve its bioavailability in the testis.

Potential of triptolide in reproductive management of the house rat, Rattus rattus.

Mature and healthy male house rats, Rattus rattus (n= 160) were fed on bait (cracked wheat: powdered sugar, 98:2) containing different concentrations of triptolide (0.1, 0.05, 0.025 and 0%) for 7 and 14 days in no-choice and bi-choice feeding tests in the laboratory. The objective of the study was to record the antifertility affects of triptolide after 30 and 60 days of termination of treatment. Results revealed no significant effect of triptolide treatment on weights of testis, epididymis, seminal vesicles and prostate gland of rats. Overall, sperm motility, live sperm count, sperm density and sperm morphology in the cauda epididymal fluid were found to differ significantly (P≤ 0.05) between untreated and treated groups of rats. The major effect of triptolide on sperm morphology was in the form of sperm head tail separation, which was up to 56.0% in rats treated for 14 days in no-choice and autopsied after 30 days. A significant effect (P≤ 0.05) of triptolide treatment was observed on the histomorphology of the testis, which included a dose-dependent decrease in diameter of seminiferous tubules, thickness of germinal epithelium and numbers of various spermatogenic cells. Cell associations in the seminiferous epithelial cycle were poorly developed in rats ingesting medium (4.7-5.1 mg/100 g bw) and high doses (6.9-7.2 mg/100 g bw) of triptolide than rats ingesting low doses (1.8-2.3 mg/100 g bw) and untreated rats. The cell stages affected had not recovered fully within the 60 day period following triptolide withdrawal. The present study suggests the potential of triptolide in reproductive management of Rattus rattus.

Reversible antispermatogenic and antisteroidogenic activities of Feronia limonia fruit pulp in adult male rats.

OBJECTIVE: To explore the antispermatogenic and testicular antisteroidogenic activities of Feronia limonia fruit pulp southern India. METHODS: Fourty Wistar male albino rats (Rattus norvegicus) were equally divided into four groups. Experimental groups were administered with the ethanolic extract of Feronia limonia (F. limoni) fruit pulp at doses of 250 and 500 mg/kg body weight once daily for 55 days. All treated rats had corresponding recovery groups. At the end of each treatment periods, various spermatological indices, tissue biochemicals and testicular enzymes levels were analysed. Blood profiles were also estimated. RESULTS: Compared with the control, the F. limonia fruit pulp at both dose levels did not decrease body weight, which were associated with decline in epididymal sperm count, motility, viability and increased percent of abnormal sperm. Further, F. limonia fruit pulp at 500 mg/kg body weight markedly reduced the epididymal and testicular protein content by 24.58% and 29.86%, respectively, as well as the glucose-6-phosphate dehydrogenase and Δ(5)-3ß-hydroxy steroid dehydrogenase) levels by 42.82% and 38.08%, respectively, while a significant elevation was observed in testicular cholesterol and ascorbic acid content. A gradual recovery of all parameters was observed after 55 days of treatment withdrawal. No significant alterations in haematological indices were observed. CONCLUSIONS: The present findings indicate that F. limonia fruit pulp may have reversible antispermatogenic and antisteroidogenic properties, and could partially support the traditional use as male contraceptive.

Toxicological studies of momordica charantia linn seed extracts in male mice / Estudios toxicológicos de los extractos de la semilla de Momordica charantia Linn en ratones macho

An attempt to find out the male contraceptive molecule of plant origin, the extracts of seeds of Momordica charantia were tested in male mice. Petroleum ether, chloroform and ethanolic extracts of Momordica charantia were administered at the dose level of 25mg/100gm body weight to the albino mice for 48 days intraperitoneally. All the extracts showed antispermatogenic effect as the number of spermatogonia, spermatocytes, spermatids and spermatozoa were decreased. The increase in the weight of epididymis, prostate gland, seminal vesicle and vas deferens indicates clearly the androgenic property of these extracts. After subjecting to preliminary phytochemical screening ethanol extract showed positive tests for alkaloids, flavonoids, glycosides, phenols, tannins, oils and fats. Out of the three extracts tested, the ethanol extract seems to be more potent in its contraceptive and androgenic activities.

En un intento por descubrir la molécula de anticoncepción masculina de origen vegetal, fueron probados los extractos de semillas de Momordica charantia en ratones machos. Extractos de éter de petróleo, cloroformo y etanol de Momordica charantia fueron administrados en dosis de 25mg/100g de peso corporal a ratones albinos de 48 días por vía intraperitoneal. Todos los extractos mostraron un efecto antiespermatogénicos, con reducción del número de espermatogonias, espermatocitos, espermátidas y espermatozoides. El aumento de peso del epidídimo, próstata, vesículas seminales y conductos deferentes indica claramente la propiedad androgénica de estos extractos. Después de someter el extracto de etanol a la detección preliminar fitoquímica se observaron resultados positivos para alcaloides, flavonoides, glucósidos, fenoles, taninos, aceites y grasas. De los tres extractos probados, el extracto de etanol parece ser más potente en sus actividades de anticonceptivas y androgénicas.

Palladium(II) and platinum(II) derivatives of benzothiazoline ligands: Synthesis, characterization, antimicrobial and antispermatogenic activity.

A series of Pd(II) and Pt(II) complexes with two N(∩)S donor ligands, 5-chloro-3-(indolin-2-one)benzothiazoline and 6-nitro-3-(indolin-2-one)benzothiazoline, have been synthesized by the reaction of metal chlorides (PdCl2 and PtCl2) with ligands in 1:2 molar ratios. All the synthesized compounds were characterized by elemental analyses, melting point determinations and a combination of electronic, IR, 1H NMR and 13C NMR spectroscopic techniques for structure elucidation. In order to evaluate the effect of metal ions upon chelation, both the ligands and their complexes have been screened for their antimicrobial activity against the various pathogenic bacterial and fungal strains. The metal complexes have shown to be more antimicrobial against the microbial species as compared to free ligands. One of the ligands, 5-chloro-3-(indolin-2-one)benzothiazoline and its corresponding palladium and platinum complexes have been tested for their antifertility activity in male albino rats. The marked reduction in sperm motility and density resulted in infertility by 62-90%. Significant alterations were found in biochemical parameters of reproductive organs in treated animals as compared to control group. It is concluded that all these effects may finally impair the fertility of male rats.

[Anti-fertility effect of Tongbi composition and its reversibility in male rats].

OBJECTIVE: To study the anti-fertility effect of maximum-dose Tongbi Composition and its reversibility in male rats. METHODS: Thirty-six male SD rats were equally randomized into a control group and a medication group, the former given normal saline at 10 ml/(kg x d), while the latter treated with Tongbi Composition at 10 g/(kg x d), both for 60 days. Half the rats of each group were sacrificed randomly at the cessation of treatment, and the rest killed at 72 days after it. The relative testis weight, testis volume, sperm concentration and sperm motility were measured, and the pathological changes in the testicular tissue observed under the optical microscope. RESULTS: After 60 days of treatment, no statistically significant differences were found between the two groups in the relative testis weight, testis volume and sperm concentration (P > 0.05) , and the sperm motility of the medication group dropped to zero, but it was restored to normal at 72 days after drug withdrawal. Almost no lesions were observed in the testis tissue of the medication group. CONCLUSION: The short-term use of Tongbi Composition at the maximum clinical dose has an obvious anti-fertility effect, but it is reversible.

New frontiers in nonhormonal male contraception.

The world's population is nearing 6.8 billion, and we are in need of a male contraceptive that is safe, effective, reversible and affordable. Hormonal approaches, which employ different formulations of testosterone administered in combination with other hormones, have shown considerable promise in clinical trials, and they are currently at the forefront of research and development. However, the long-term effects of using hormones throughout a male's reproductive life for contraception are unknown, and it may take decades before this information becomes available. Because of this, many investigators are aiming to bring a nonhormonal male contraceptive to the consumer market. Indeed, there are several distinct but feasible avenues in which fertility can be regulated without affecting the hypothalamus-pituitary-testis axis. In this review, we discuss several approaches for fertility control involving the testis that one day may lead to the development of a nonhormonal male contraceptive.

Inhibition of hyaluronidase activity of human and rat spermatozoa in vitro and antispermatogenic activity in rats in vivo by Terminalia chebula, a flavonoid rich plant.

Our interest in development of hyaluronidase inhibitors as male antifertility agents led to identification of Terminalia chebula (T. chebula) plant with hyaluronidase (HAase) inhibitory activity of human spermatozoa ( approximately 93% inhibition) and rat caudal epididymal spermatozoa ( approximately 86% inhibition) in vitro at 30 mg/ml. We further demonstrated inhibition of hyaluronidase activity of testis and epididymal spermatozoa in vivo coincident with antispermatogenic activity and contraceptive efficacy of TC extract administered at 50 and 100mg/kg/day orally for 60 days in male albino rats. The significant decrease in motility, count and increase in morphological abnormalities of epididymal spermatozoa and severe reduction in fertility (-100%) of male rats treated with T. chebula fruit extract at 100mg/kg dose could be attributed to either direct effect on testis or direct or indirect interference with sperm maturation in epididymis, and/or inhibition of testicular and epididymal sperm hyaluronidase enzyme in vivo probably caused by flavonoids like tannins present in T. chebula.

Recrudescence of spermatogenesis in the dog following downregulation using a slow release GnRH agonist implant.

The present study examined the degree to which downregulation with a GnRH agonist impaired spermatogenesis and the time course of morphological and hormonal changes that occurred during recrudescence of spermatogenesis. Using a control group (group 1, n = 5) of dogs, the effect of a removable slow release GnRH-agonist implant was investigated in beagle dogs (group 2, n = 30). The implant was removed after 5 months (week 0) and three to four dogs were castrated at weeks 0, 3, 6, 9, 12, 15, 18, 21 and 24. The degree of downregulation and recrudescence of spermatogenesis was assessed by evaluation of 200 tubular cross-sections, resulting in an assigning of dogs of group 2 to testis developmental groups (DG) according to the most developed germ cell observed: DG A, spermatocytes; DG B, round spermatids; DG C, elongating spermatids and DG D, elongated spermatids. Downregulation led to an arrest of spermatogenesis at the level of spermatogonia/primary spermatocytes. The time course of recrudescence showed high individual variations and the number of dogs falling into DG A, B, C and D was 4, 3, 6 and 17 respectively. Spermatogenesis in group 2, DG D was not different from group 1 (control). In DG A, mean area of Leydig-cell nuclei was lower (p < 0.001) than in the other DG and group 1 and resembled that of juvenile dogs (group 3, n = 3); nuclei of Sertoli cells had changed from more flat/polygonal (group 1, group 2, DG C and D) to round/ovoid and had moved to a more luminal position. As indicated by basal testosterone (T), luteinizing hormone (LH) and follicle stimulating hormone (FSH) concentrations at implant removal, full downregulation had been obtained. Testosterone, LH and FSH concentrations [X(g) (DF), ng/ml] increased (p < 0.05) from implant removal to DG B [T: 0.1 (1.24) vs 2.12 (2.31); LH: 0.2 (2.15) vs 1.11 (1.7); FSH: 0.37 (3.50) vs 6.37 (1.68)] and were more or less constant thereafter indicating that onset of spermatogenesis was related to an increase of plasma T occurring in a very narrow time window. Following GnRH implantation, the size of the testes and the prostate decreased by approximately 55% (p < 0.001), they increased to sizes similar to pre-treatment values following implant removal.