Rationalising drug delivery using nanoparticles: a combined simulation and immunology study of GnRH adsorbed to silica nanoparticles.

Silica nanoparticles (SiNPs) have been shown to have significant potential for drug delivery and as adjuvants for vaccines. We have simulated the adsorption of GnRH-I (gonadotrophin releasing hormone I) and a cysteine-tagged modification (cys-GnRH-I) to model silica surfaces, as well as its conjugation to the widely-used carrier protein bovine serum albumin (BSA). Our subsequent immunological studies revealed no significant antibody production was caused by the peptide-SiNP systems, indicating that the treatment was not effective. However, the testosterone response with the native peptide-SiNPs indicated a drug effect not found with cys-GnRH-I-SiNPs; this behaviour is explained by the specific orientation of the peptides at the silica surface found in the simulations. With the BSA systems, we found significant testosterone reduction, particularly for the BSA-native conjugates, and an antibody response that was notably higher with the SiNPs acting as an adjuvant; this behaviour again correlates well with the epitope presentation predicted by the simulations. The range of immunological and hormone response can therefore be interpreted and understood by the simulation results and the presentation of the peptides to solution, paving the way for the future rational design of drug delivery and vaccine systems guided by biomolecular simulation.

Preliminary study on antifertility activity of Enicostemma axillare leaves and Urena lobata root used in Indian traditional folk medicine.

OBJECTIVE: To evaluate the possible antifertility activity of Enicostemma axillare (E. axillare) leaves and Urena lobata (U. lobata) root in adult male Wistar albino rats. METHODS: Six groups of rats were treated with ethanolic (70%v/v) extracts of E. axillare (375 and 750 mg/kg body weight) and U. lobata root (300 and 600 mg/kg body weight) once daily for 55 days. Control groups received the distilled water and vehicle. All the treated rats had corresponding recovery groups. At the end of each treatment periods, animals were killed and organ weights, sperm characteristics, testicular and epididymal biochemicals as well as testicular enzymes were assessed. RESULTS: The E. axillare and U. lobata at tested doses did not decrease body weight, whereas the weight of testes, epididymides and seminal vesicles were significantly (P<0.01) reduced. Significantly (P<0.01) more reductions in the sperm motility, viability and counts, epididymal and testicular protein contents were noted in the rats treated with higher dose of both the plants. Both the plants at the higher dose caused a marked increase (P<0.01) in sperm morphological abnormalities, testicular cholesterol and ascorbic acid contents were remarkably increased (P<0.01), while, the activities of testicular glucose-6-phosphate dehydrogenase (G-6-PDH) and Δ(5)-3ß-hydroxy steroid dehydrogenase (Δ(5)-3ß-HSD) were significantly reduced (P<0.01). However, reversal of these changes occurred after 55 days of treatment withdrawal. CONCLUSIONS: This study suggests that the E. axillare leaves and U. lobata root reversibly inhibited spermatogenesis and steroidogenesis indicating reversible antifertility activity which could partially support the traditional of these plants as male contraceptives.

Safety evaluation of long term oral treatment of methanol sub-fraction of the seeds of Carica papaya as a male contraceptive in albino rats.

ETHNOPHARMACOLOGICAL RELEVANCE: The manuscript is one of the series of attempts in authenticating scientific documentation of the seeds of Carica papaya being traditionally used for contraception. AIMS OF THE STUDY: To establish safety of the methanol sub-fraction (MSF) of the seeds of Carica papaya as a male contraceptive following long term oral treatment. MATERIALS AND METHODS: MSF was administered orally to albino rats at multiples of contraceptive dose (CD) at 50 (1x), 100 (2x), 250 (5x) and 500 (10x)mg/kg body weight daily for 52 weeks. Body weight, organs weight, morbidity, mortality, clinical chemistry, sperm analysis, histopathology and serum testosterone were evaluated to assess the safety and contraceptive efficacy. RESULTS: MSF treatment at various dose regimens, daily for 52 weeks did not show significant changes in body weight, organs weight, food and water intake and pre-terminal deaths compared to those of control animals. Sperm count and viability in 50mg/kg body weight treated animals and the weight of epididymis, seminal vesicle and prostate of all the treated animals showed significant reduction compared to control. Cauda epididymal spermatozoa of 50mg/kg body weight treated animals were immotile. Azoospermia was observed in 100, 250 and 500 mg/kg body weight treated animals. Serum clinical parameters, serum testosterone and histopathology of vital organs were comparable to those of control animals. Histology of testis revealed adverse effects on the process of spermatogenesis, while the histology of epididymis, seminal vesicles and ventral prostate showed no changes compared to control. CONCLUSION: The long term daily oral administration of MSF affects sperm parameters without adverse side effects and is clinically safe as a male contraceptive.

Phytoestrogen treatment induces testis alterations in dogs. Potential use in population control.

Dog overpopulation is considered a human health risk; they are the terrestrial vector of rabies and reservoirs for other human diseases. Surgical neutering and intratesticular injections have been used in male dogs. Physiological and morphological alterations in reproductive organs can be induced by phytoestrogens. Our goal was to evaluate the effect of oral coumestrol on dog ejaculates and testis histology. Two groups of 5 healthy adult dogs were used. One coumestrolcontaining biscuit was given once a week for a 4 week period to the experimental group. Ejaculates were obtained and evaluated. After treatment, testis were obtained and processed for histology. Compared to controls, treated dogs have reduced tubules (462 +/- 1.4 vs 336 +/- 2 micron(2)), spermatogenic epithelium (49.1 +/- 0.01 vs 13.3 +/- 0.01 micron(2)), and lumen opening (891 +/- 1.4 vs 530 +/- 26.9 micron). Ejaculates from treated animals have increased numbers of abnormal spermatozoa and reduced sperm concentration.

Reversible changes in the antifertility induced by Aegle marmelos in male albino rats.

To study the effects of Aegle marmelos on the testicular reproductive system, a 50% ethanolic extract of Aegle marmelos leaves (AMLEt) was fed orally to male albino rats at the dose levels of 200 and 300 mg/kg body wt./day for 60 days. Recovery was assessed for an additional 120 days. Oral administration of AMLEt did not cause body weight loss. The motility and sperm concentration were significantly reduced along with complete inhibition of fertility at a dose of 300 mg/kg. The level of serum testosterone also declined and spermatogenesis was impaired. The number of normal tubules and the height of epithelial cells of the caput and cauda were reduced significantly. The cross sectional surface area of Sertoli cells and mature Leydig cells was reduced along with a dose dependent reduction of preleptotene and pachytene spermatocytes. Thus the antifertility effects of Aegle marmelos seemed to be mediated by disturbances in structure and function in testicular somatic cells including Leydig and Sertoli cells resulting in an alteration in physio-morphological events of spermatogenesis. However, complete recovery was observed after a 120 day withdrawal.

Tropaeolum tuberosum (Mashua) reduces testicular function: effect of different treatment times.

Tropaeolum tuberosum Ruiz & Pavon, along with other several species, is an edible-tuber crop that grows in the Andean region. Folk medicine describes the use of mashua to reduce reproductive function in men. The present study aimed to evaluate the effect of mashua (1 g kg(-1)) on sperm production in rats during 7, 12, 21 and 42 days of treatment. The following parameters were assessed: reproductive organ weights, spermatid count and daily sperm production (DSP), sperm count in epididymis and sperm transit and serum testosterone levels. Freeze-dried extract of mashua had 3.7 g 100 g(-1) of benzyl glucosinolate. Mashua-treated rats showed a reduction in testicular spermatid number and DSP from day 12 to day 42; meanwhile, the effect of mashua was noted in epididymal sperm count after 12 and 42 days of treatment. In addition, epididymal sperm transit time was delayed at day 7 and it was accelerated on days 12 and 21 of treatment. No differences in serum testosterone levels were found between rats treated with vehicle and mashua after 42 days of treatment. Finally, mashua reduces testicular function after one spermatogenic cycle by reducing spermatid and sperm number, DSP and epididymal sperm transit time.

The development of a testosterone stimulation test in the Virginia opossum (Didelphis virginiana) and its use in evaluating deslorelin contraception.

The aims of the present study were to examine the variability of testosterone secretion in the Virginia Opossum over a 24 h period and to develop a testosterone stimulation test that would provide an index of the prevailing testosterone biosynthetic capacity of the testes; the latter was used to clinically evaluate the efficacy of a gonadotrophin-releasing hormone agonist contraceptive. Sexually-mature captive opossums (n = 12) located in Africam Safari (Mexico) sampled every 12 h over 24 h consistently showed basal (<0.21 ng mL(-1)) blood testosterone concentrations. Intra-muscular injection of buserelin (2 microg mL(-1)) and human chorionic gonadotrophin (hCG; 1000 IU) resulted in an increase (P < 0.05) of plasma testosterone concentrations with maximal concentrations (3.9 ng mL(-1) and 5.8 ng mL(-1) respectively) occurring 120 min after injection. Plasma testosterone declined relatively rapidly to basal concentrations after 240 min with hCG but remained elevated after the same period of time with buserelin. Male opossums treated with (n = 6) and without (n = 6) a controlled-release deslorelin implant (Suprelorin; 4.7 mg deslorelin) were evaluated over a 10-week period for changes in testosterone secretion (hCG stimulation test) and sperm production (spermatorrhea). At the end of this period, the animals were hemi-castrated and their relative testicular quantitative histology compared. Testosterone concentration decreased over the course of the study in both treated and control animals (P < 0.0001) but there was no apparent effect of deslorelin on testosterone secretion, testicular histology (relative proportions of testicular cell types and seminiferous tubule diameter), or sperm production (presence of sperm in the cauda epididymis or urine).

Development of l-CDB-4022 as a nonsteroidal male oral contraceptive: induction and recovery from severe oligospermia in the adult male cynomolgus monkey (Macaca fascicularis).

The present study was undertaken to examine the antispermatogenic effect of l-CDB-4022 in the adult male cynomolgus monkey. Monkeys (four per group) were dosed via nasogastric tube for 7 d with l-CDB-4022 at 12.5 mg/kg.d or vehicle (d 0=first day of dosing). Plasma levels of l-CDB-4022 and its deesterified metabolite were nondetectable prior to treatment and in all vehicle-treated monkeys. Peak levels of l-CDB-4022 and its metabolite were observed at 4 h after dosing with steady-state levels apparent around d 4. Sperm concentration and total sperm per ejaculate were decreased to levels below 1x10(6) sperm/ml or sperm/ejaculate in l-CDB-4022-treated monkeys by d 17 and remained suppressed through wk 6. Sperm motility also declined to 0% for 6 wk. Testicular volume was reduced in l-CDB-4022-treated monkeys through d 21. The left testis and epididymis were removed from all monkeys on d 24. At this time, the most mature germ cells in the seminiferous tubules of testes from l-CDB-4022-treated monkeys were either spermatocytes or round spermatids. Immature germ cells, but not mature sperm, were found in the efferent ducts and collapsed epididymal lumen of l-CDB-4022-treated monkeys. A steady recovery in sperm motility, concentration, and total sperm per ejaculate was observed in l-CDB-4022-treated monkeys such that these parameters were not different from those of vehicle-treated monkeys by wk 16. Volume of the remaining testis increased in vehicle- and l-CDB-4022-treated monkeys after hemicastration; however, the increase in l-CDB-4022-treated monkeys was delayed compared with that observed in the vehicle-treated monkeys. The morphology of the remaining testis and epididymis, which were removed on wk 17, was normal. Serum inhibin B levels were increased in l-CDB-4022-treated monkeys during the dosing interval; thereafter serum inhibin B levels declined such that there was no difference between the groups by wk 3. l-CDB-4022 treatment did not affect circulating levels of testosterone, LH, FSH, or estradiol. In conclusion, these data indicate that in the cynomolgus monkey, a representative higher primate, l-CDB-4022 exerts a selective antispermatogenic action, which was reversible under the conditions of this study and thus has potential as a nonhormonal oral male contraceptive.

Effect of isolated fractions of Barleria prionitis root methanolic extract on reproductive function of male rats: preliminary study.

Male rats treated with 100 mg/kg for 60 days of isolated fractions of the Barleria prionitis root methanolic extract (Fr. I and Fr. II) showed a significant reduction on spermatogenesis without affecting general body metabolism. Sperm motility as well density in cauda epididymides was reduced significantly. The fertility was decreased by 33.4% in Fr. I and 100% in Fr. II treated rats. The blood parameters were within the normal range. Total protein, glycogen and sialic acid contents of testes were reduced after the plant fractions treatment. Seminal vesicular fructose was decreased significantly after the treatment. The population of various spermatogenic cells such as primary spermatocytes, secondary spermatocytes and round spermatids were declined significantly in Fr. II treatment groups whereas in Fr. I treated animals preleptotene spermatocyte and spermatid number was decreased. There was no significant change in the number of Sertoli cells and spermatogonia in any of the treatment group.

A combined regimen of gossypol plus methyltestosterone and ethinylestradiol as a contraceptive induces germ cell apoptosis and expression of its related genes in rats.

Attempts to develop gossypol and steroidal hormones alone as a male contraceptive have been tested for many years; however, both caused undesirable side effects that have prevented their acceptance. In this study, we formulated a regimen of combined gossypol at a low dose of 12 mg/kg or a high dose of 50 mg/kg plus methyltestosterone 20 mg/kg and ethinylestradiol 100 g/kg daily (12 mg G+H and 50 mg G+H) administered for 6 weeks in adult rats. The possible roles of germ cell apoptosis and related genes expression were studied by techniques of TdT-mediated dUTP nick end-labeling (TUNEL), agarose gel electrophoresis of low-molecular-weight DNA, in situ hybridization and reverse transcription-polymerase chain reaction detection. Results showed that germ cell apoptosis and related genes expression were significantly induced after combined drug administration. The apoptosis index increased 3.86- and 9.65-fold in the 12-mg and 50-mg G+H-treated groups, respectively, as compared to the control group. DNA ladder formation on the agarose gel further validated the findings of TUNEL-stained apoptotic cells. The apoptosis-related genes fas mRNA expression levels increased 0.44- and 1.39-fold, bax mRNA 0.74- and 2.56-fold, caspase-3 mRNA 0.60- and 1.29-fold, and caspase-9 mRNA 2.50- and 4.08-fold, respectively, in the 12-mg and 50-mg G+H-treated groups vs. the control group. These results indicated that our drug regimen applied as a contraceptive could induce rat germ cell apoptosis. The apoptotic process involved fas system, bax and caspase family genes and the apoptotic extent and cell types were gossypol dose-dependent.

Effects of plumieride, an iridoid on spermatogenesis in male albino rats.

UNLABELLED: Oral feeding of male rats with plumieride (15 mg/rat/day) for the period of 60 days did not cause any significant change in the body weight of treated rats. However, the weights of testes, epididymides, seminal vesicle and ventral prostate were significantly reduced when compared to control values. The production of step-19 spermatids was reduced by 87.26% in plumieride treated rats. The population of preleptotene and pachytene spermatocytes were decreased by 64.26% and 55.13% respectively. Spermatogonia and sertoli cell population was also affected. Plumieride treatment resulted in an arrest of spermatogenesis without any systemic side effect. Sperm motility as well as sperm density was reduced significantly. The number of mature Leydig cells was decreased and complete suppression of fertility was observed. A significant fall in the protein and sialic acid contents of the testes, epididymides, seminal vesicle and ventral prostate as well as glycogen content of testes was also noticed. Fructose in seminal vesicle was lowered whereas testicular cholesterol was elevated. There was no significant change in RBC and WBC count, haemoglobin, haematocrit and sugar in the whole blood and total protein, cholesterol, phospholipid and triglycerides in the serum. CONCLUSION: Plumieride administration arrests spermatogenesis in male rats without noticeable side effects. For the clinical use more experiments should be carried out in a phased programme.

Male rat infertility induction/spermatozoa and epididymal plasma abnormalities after oral administration of Kalanchoe gastonis bonnieri natural juice.

Natural aqueous crude extracts (NACE) of several Crassulaceae family plants have been applied as a vaginal contraceptive by the populace. The aim of this work was to evaluate the inhibition of fertility in male Wistar rats and some physiological and biochemical changes in spermatozoa and epididymal plasma induced by NACE from Kalanchoe gastonis bonnieri (K. g. b.) (Crassulaceae). The NACE was obtained by mechanic pressure on grinding fresh plant leaves. Sublethal doses (150-300 mg/kg body weight) of NACE were orally administered to adult and fertile male rats daily for 30 days in a search for a contraceptive effect, and physiological and biochemical modifications on sperm cells and cauda epididymal plasma. The toxicity studies revealed that the lethal dose (LD(50)) calculated was 11 g/kg body weight. Sublethal doses induced 50%-100% fertility inhibition, with 100% recovery of fertility 30 days after stopping the treatment. The sperm motility, viability and spermatic density were also significantly decreased (p < 0.001). The outstanding biochemical change observed in the cauda epididymal plasma was a decrease of carnitine concentration. The NACE of K. gastonis contains one substance active on fertility by affecting spermatozoa motility, viability and sperm density with a significantly decreased carnitine and sialic acid (p < 0.001) in the caudal epididymal plasma.

Antifertility effect of Tinospora cordifolia (Willd.) stem extract in male rats.

Oral administration of 70% methanolic extract of T. cordifolia stem to male rats at the dose level of 100 mg/rat/day for 60 days did not cause body weight loss but decreased the weight of testes, epididymis, seminal vesicle and ventral prostate in a significant manner. Sperm motility as well as sperm density were reduced significantly which resulted in reduction of male fertility by 100%. The stem extract brought about an interference with spermatogenesis. The round spermatids were decreased by 73.12%. However, the population of preleptotene and pachytene spermatocytes were decreased by 47.60% and 52.85% respectively, followed by secondary spermatocytes (48.10%). Leydig cell nuclear area and mature Leydig cell numbers were significantly reduced when compared with controls. Serum testosterone levels showed significant reduction after Tinospora extract feeding. Seminiferous tubule diameter, Leydig cell nuclear area as well as cross sectional surface area of Sertoli cells were reduced significantly when compared to controls. Biochemical parameters i.e. protein, sialic acid, glycogen contents of testes decreased significantly. Seminal vesicular fructose also depleted whereas, testicular cholesterol was elevated significantly followed by a reduction in testosterone levels. These results suggested antifertility effects of the stem extract of T. cordifolia in male rats.

Expression of Hsp70-2 in rhesus monkey testis during germ cell apoptosis induced by testosterone undecanoate.

Hsp70-2 functions as a molecular chaperone that assists other proteins in their folding, transport and assembly into complexes, and is postulated to be linked to the mechanisms that inhibit apoptosis. Here we have determined the association between Hsp70-2 gene and germ cell apoptosis induced by a high dose of testosterone undecanoate (TU). In this study, in situ analysis of cell DNA fragmentation and expression of Hsp70-2 in TU-treated monkey testes were compared with the normal testes. The TUNEL analysis data showed that a large number of germ cell apoptosis occurred in the testes on Day 30 after TU injection. Therefore, we speculate that spermatogenesis failure in TU-treated monkey testis may be a result of the germ cell apoptosis induced by a high dose of TU. As compared with that of normal testes, however, the level of Hsp70-2 mRNA was only slightly decreased while that of Hsp70-2 protein was almost unchanged in the testes from Day 7 to day 30 at the early stage of the germ cell apoptosis after TU treatment, but the levels of both Hsp70-2 mRNA and protein dropped dramatically on Day 60 when a large number of germ cells had undergone apoptosis and were depleted. Therefore, it is suggested that the Hsp70-2 may be not a molecule to prevent germ cell apoptosis induced by injection of TU in the testes at the early stage.

Current status of fertility control methods in India.

Approximately 48.2% of couples of 15 to 49 years of age practice family planning methods in India. Female sterilization accounts for 34.2%, with male sterilization declining from 3.4% in 1992-93 to 1.9% in 1998-99. Use of the condom increased to 3.1% from 2.4%. There is an urgent need for research to develop new contraceptive modalities especially for men and also for women and to make existing methods more safe, affordable and acceptable. Current efforts in India to develop a male contraceptive are mainly directed towards (i) development of antispermatogenic agents to suppress sperm production, (ii) prevention of sperm maturation, (iii) prevention of sperm transport through vas deferens or rendering these sperm infertile and (iv) prevention of sperm deposition. Research work in the field of prevention of sperm transport through vas deferens has made significant advances. Styrene maleic anhydride (SMA) disturbed the electrical charge of spermatozoa leading to acrosome rupture and consequent loss in fertilizing ability of sperm. A multicentre phase-III clinical trial using SMA is continuing and it is hoped that the SMA approach would be available in the near future as an indigenously developed injectable intra-vasal male contraceptive. The safety and efficacy of available oral contraceptives were evaluated. An indigenously developed oral contraceptive 'Centchorman', which is a nonsteroidal, weakly estrogenic but potently antiestrogenic, was found to be safe and effective and is now being marketed in India since 1991 as a 'once a week' pill. Cyclofem and Mesigyna have been recommended as injectable contraceptives with proper counselling and service delivery by Indian studies. It has been recommended that these injectable contraceptives be added to the existing range of contraceptive methods available in the National Family Planning Programme. Based on the Indian studies CuT 200 was also recommended. Studies have indicated the advantage of intrauterine devices (IUD); they are long acting, relatively easily removed and fertility returns rapidly after their removal. Recent studies have recommended CuT 200 for use up to 5 years. The combination of some plant products i.e. Embelia ribes, Borax and Piper longum has been found to be safe and effective as a female contraceptive and the results of phase-I clinical trials are encouraging. Research work is going on in the country in various areas with special reference to hormonal contraceptive - a three monthly injectable contraceptive, immuno-contraceptives, antiprogestins, etc.

Sterility due to inhibition of sperm motility by oral administration of benzene chromatographic fraction of the chloroform extract of the seeds of Carica papaya in rats.

The contraceptive effects of benzene chromatographic fraction of the chloroform extract of the seeds of Carica papaya have been reported in male albino rats at the dose regimens 5 and 10 mg/animal/day; oral for 150 days. The body weight, weight of testis, epididymis, seminal vesicle and ventral prostate remained unaltered during the entire course of the investigation. Total suppression of cauda epididymal sperm motility coincided with a decrease in sperm count, viability and an increase in per cent abnormal spermatozoa during 60-150 days observation period. Minor changes in the germ cell proliferations in the testis and vacuolization and pyknotic nuclei in the few epithelial cells of the cauda epididymis were observed. Histology and biochemical composition of testis and accessory sex organs, haematology and serum clinical biochemistry and serum testosterone levels remained unchanged throughout the course of the investigation. Test for estrogenicity indicated mild estrogenicity. Monthly fertility test showed negative fertility. All the altered parameters returned to normal level following 60 days withdrawal of the treatment. The results suggest that the benzene chromatographic fraction of the chloroform extract of the seeds of Carica papaya exerts antifertility effects in rats without adverse toxicity and that the effects may be directly rendered on the spermatozoa.

The reversible antifertility effect of Piper betle Linn. on Swiss albino male mice.

To study the antifertility effect of an extract (alcoholic) of the leaf-stalk of Piper betle Linn., one set of experiments with two different doses in Swiss male albino mice were evaluated. Initially, 500 mg of the leaf-stalk extractive for 30 days and then 1000 mg for next 30 days/animal/day/kg body weight were administered orally. The extract reduced fertility to 0% within 60 days. Suppression of cauda epididymal sperm count and motility (p <0.05) was observed. Biochemical parameters did not show any marked alterations in testosterone content in serum nor 17beta-hydroxysteroid dehydrogenase (17beta-HSD) activity in testes although fructose content in seminal vesicles was reduced as are the weights of reproductive organs. The cholesterol content in testes increased, although not appreciably. After cessation of drug (plant extract) treatment, the altered parameters recovered. Results suggest that the contraceptive effect of the extract of leaf-stalk of Piper betle Linn. is mainly on the maturation process of spermatozoa in epididymides without influencing hystemic hormonal profiles. Withdrawal of the extract restored all altered parameters including organ weights and fertility after 60 days.

Subcapsular intratesticular assay: a preliminary screening method for putative male antifertility drugs.

The subcapsular intratesticular assay was developed for the preliminary screening of male antifertility agents. In the assay, small amounts of the test sample are injected twice, 1 week apart just beneath the tunica albuginea of the testes of rats. The rats were killed within 3 weeks of treatment to examine epididymal spermatozoa and the histology of the testis and epididymis. There is an excellent agreement between this method and the modified MB-50 assay developed by the World Health Organization. To date, more than 300 samples have been assayed, of which the results of 10 plant-derived compounds are reported here. Not one active sample has been missed as confirmed by the modified MB-50 method, although one compound which was positive in the subcapsular assay did not show antifertility action in the in-vivo assay. Compared with the latter assay, the advantages of this method are: simplicity, time-saving, rat-saving and the need for far smaller amounts of test sample. The absence of false-negative results makes the subcapsular intratesticular assay applicable as a preliminary screening method to test potential male fertility regulating agents.

[Intratesticular subcapsular assay, a simple method for the preliminary screening of male contraceptives].

The MB-50 protocol worked out for long by the World Health Organization has been widely employed for the screening of male contraceptives. With this method, 40-80 rats will be used for 1 sample and the course of study will be about 3 months. In view of the high expenses and long duration of time needed, it seems to be not suitable for a preliminary screening. We have exploited a much simpler method, the intratesticular subcapsular assay. The method is as follows: Puncture the scrotal skin and the tunica albuginea at the equatorial plane of the testis just opposite to the epididymis with an intradermal needle fixed at a tuberculin syringe. Then push on the needle for 3-4 mm along the equator parallel to the tunica albuginea. Inject 100 microliters of solution containing 0.1-5 mg of the sample to each testis. To the controls, only the vehicle is injected. One week later the same procedure is repeated and 2 more weeks later, the animals are sacrificed and the data are observed (see Table 1 and 2). In this paper, 7 samples screened with this method were compared with results of the MB-50 method, no positive sample was missed except one false positive sample. These results indicate the coincidence between these two methods.