AF-2364 is a prospective spermicide candidate

Inhibition of sperm motility has recently become a promising target for male contraceptive development. AF- 2364, an analogue of Lonidamine （LND）, had a contraceptive effect when orally administered to adult SpragueDawley rats. LND can also target mitochondria to inhibit oxygen consumption and block energy metabolism in tumour cells. However, there are no reports of the effects of AF-2364 on human sperm function. Herein we describe the action of AF-2364 on human sperm in vitro, as well as the mechanisms involved. AF-2364 specifically blocked human sperm motility in vitro. Further experiments revealed that AF-2364 can target sperm mitochondrial permeability transition （MPT） pores to induce the loss of sperm mitochondrial membrane potential （AqUm） and decrease ATP generation; however, no significant changes in the cytoskeletal network or the human sperm proteome were detected after exposure to AF-2364. Incubation of AF-2364 with other human or mouse cell lines indicated that the spermicidal effect at the lower concentration was specific. In summary, the spermicidal effect olAF-2364 involves direct action on sperm MPT pores, and this compound should be further investigated as a new spermicide candidate.

Anti-VDAC3 recombinant antibody decreased human sperm motility and membrane integrity: A potential spermicide for contraception

Objective:To express recombinant protein that comprises an important fragment of human sperm specific voltage dependent anion channel 3 (VDAC3) protein as a potential molecule for generation of antibody, which can affect sperm function, aiming at spermicide development. Methods: The produce of VDAC3 recombinant protein encoded by cDNA sequence of human VDAC3 exon 5-8, based on experimental design of VDAC3 knock-out mice study. And after the purification of various human sperm VDAC3 recombinant proteins, epitope has been predicted in our recombinant protein determined by ElliPro program. Polyclonal antibody was produced for 14 wk. Then anti-VDAC3-exon 5-8 recombinant antiserum was inoculated to human sperm. After the process, antibody VDAC3 protein in human sperm was incubation with anti-VDAC3 recombinant antibody. Finally evaluation the effect of VDAC3 antiserum to human sperm motility and plasma membrane integrity was proceeded.Results: Human VDAC3 recombinant protein was successfully over-expressed in Escherichia coli and purified by affinity chromatography method. Purified human sperm VDAC3 recombinant protein could stimulate immune response in rabbit producing an antibody against VDAC3. Anti-VDAC3 recombinant antibody recognized VDAC3 antigen in human sperm could decrease human sperm motility and membrane integrity significantly.Conclusions:Anti-VDAC3 recombinant polyclonal antibody that we produced in rabbit by ourselves could decrease sperm motility and sperm membrane integrity. The authors suggest this polyclonal antibody could be used as a candidate agent for male contraception in the future. Furthermore, the authors intend to explore the effect of this antibody into sperm function aiming at male contraceptive vaccine development.

n-Butanol Extract of Rhynchosia volubilis Lour: A Potent Spermicidal Agent In Vitro

Summary： Rhynchosia volubilis Lout has been a major drug in a folk prescription for contraception in China, whereas its mechanism remains unknown. Its antifertility effects on male mice and antimicrobial activities on sexually transmitted infection （STI） pathogens were previously reported. This study was undertaken to develop the n-Butanol extract of Rhynchosia volubilis Lour （BERVL） as a spermicidal agent with STI prevention. The spermicidal activities of BERVL with different doses were assessed using selected high-motile sperms of normal human semen samples, and their inhibitory effects on Lactobacillus acidophilus were determined. The mechanism of the spermicidal activity was explored by aqueous Eosin Y and Hoechst 33342/PI staining. The results showed spermicidal activities and inhibi- tory effects of BERVL on Lactobacillus acidophilus were dose-dependent. Dose of 90 mg/mL BERVL terminated all progressive sperm motility within 2 min, and had slight inhibitory effect on Lactobacillus acidophilus, suggesting it was an effective and safe dose for contraception use. About 80% sperms exposed to BERVL displayed changes consistent with high permeability of head membrane. It is concluded that BERVL as spermicide has advantages over N-9 with strong ability to instantaneously kill human sperm and possesses light inhibitory effect on Lactobacillus acidophilus.

THE CONTRACEPTIVE EFFECT OF MORRHUIC ACID SUPPOSITORY

The morrhuic acid suppository had been used in 1746 women with a total of 16073 women-months cycles. 1354 cases continued to use the suppository for 12 months, 367 cases completed 24 months and 15 cases 25-34 months. The cumulative twelve month life table pregnancy rate was 10.1 per 100 women and the method pergnancy rate was 4.8 per 100 women corresponding to an effective ness rate of 95.2% per 100 women. The gross twelve months cumulative termination rate per 100 women was 27.3 giving a continuation rate of 72.7%. Cervical smears were taken in 499 women showing no carcinomatous changes. It did not interfere with the menstrual cycle and flow. The only adverse side effect was the burning or itching sensation of the vulva which accounts for a 1.2% discontinuation rate. This suppository is sultable for contraception of all women in reproductive age.

Spermicidal Effect of Propranolol and Its Isomers onHuman Sperm in vitro

Fourly semen samples were collected from healthy volunteers by masturbation. The samples were then divided into four groups according to the quality and quantity of the sperm. The propranolol (D,L-P) and its isomers including the Dextko-propranolol (D-P) and LevoAnpropranolol (L-P) solution of various concentrations were tested. SPermicidal e/fect at various intervals was observed by microscopy, and then compared to the Nonoxynol (NP-9) as a reference spermicide.The results showed that the mean values of lowest effective spermicidal concentration of D,L-P, D-P and L-P within twenty seconds were 1.00±0.63,1.10±0.62 and 0.91 ±0.54 (mg/ml) respectively, there was no signijicant difference among them (P>0.05), but they were signilicantly different from that of NP-9 (P<0.001). It is well known that the D- P has no significant beta- adrenoceptor blocking activity, so the D-P might be a new promising vaginal contraceptive drug.

Human sperm immobilization effect of Carica papaya seed extracts: an in vitro study

Aim: To examine if the seed extracts of Carica papaya, which showed anfispermatogenic/sperm immobilizationproperties in animal models, could cause human sperm immobilization in vitro. Methods: Chloroform extract, ben-zene chromatographic fraction of the chloroform extract, its methanol and ethyl acetate sub-fractions and the isolatedcompounds from the sub-fractions i. e., ECP 1 & 2 and MCP 1 & 2, of the seeds of Carica papaya were used at con-centrations of 0.1%, 0.5%, 1% and 2%. Sperm motility was assessed immediately after addition of extracts and ev-ery 5 minutes thereafter for 30 minutes. Results: There were dose-dependent spermicidal effects showing an instantfall in the sperm motility to less than 20% at 2% concentration. Isolated compounds ECP 1 & 2 were more effective in-ducing a motility of less than 10%. Many of the spermatozoa became vibratory on the spot. Total inhibition of motilitywas observed within 20-25 rain at all concentrations of all products. Scanning and transmission electron microscopyrevealed deleterious changes in the plasma membrane of the head and mid-piece of spermatozoa. Sperm viability testand the number of abnormal spermatozoa after completion of incubation suggested that the spermatozoa were infertile.The effects were spermicidal but not spermiostatic as revealed by the sperm revival test. Conclusion: The results re-veal spermicidal activity in vitro of the seed extracts of Carica papaya.

Sperm immobilization activity of Allium sativum L. and other plant extracts

<abstract>Aim: To identify possible spermicidal agents through screening a number of edible medicinal plants with antimicrobial activity. Methods: Initial screening was made on the basis of ram cauda epididymal sperm immobilization immediately after addition of extracts. The most potent extract was selected and was evaluated on both ram and human spermatozoa. To unravel its mode of action several sperm functional tests were carried out, namely viability of cells, hypo-osmotic swelling test for membrane integrity and assays of membrane-bound enzyme 5'-nucleotidase and acrosomal marker enzyme acrosin. Results: The crude aqueous extract of the bulb of Allium sativum L. showed the most promising results by instant immobilization of the ram epididymal sperm at 0.25 g/mL and human ejaculated sperm at 0.5 g/mL. Sperm immobilizing effects were irreversible and the factor of the extract responsible for immobilization was thermostable up to 90 癈. On boiling at 100 癈 for 10 minutes, this activity was markedly reduced. Moreover, this extract was able to cause aggregation of ram sperms into small clusters after 30 minutes of incubation at 37 癈. However this property was not found in human spermatozoa. More than 50 % reduction in sperm viability and hypo-osmotic swelling occurred in treated sperm as compared with the controls, indicating the possibility of plasma membrane disintegration which was further supported by the significant reduction in the activity of membrane bound 5'-nucleotidase and acrosomal acrosin. Conclusion: The crude aqueous extract of A. sativum bulb possesses spermicidal activity in vitro.

Human Spermicidal Activity of Passiflora edulis Extract

Objective To evaluate the spermicidal activity of Passiflora edulis extract on human spermatozoa. Methods Human spermatozoa were incubated with P. edulis extracts and their motility and viability were evaluated; additionally, the cytotoxic effect of the extracts was evaluated by the tetrazolium bromide （MTT） reduction assay. Results The motility and viability were decreased immediately after treatment with a 21% dilution of the supernatant of the extract of P. edulis （P〈0.01）. No cytatoxic effect of the extracts studied was found on proliferation of MDBK and VERO cells. Conclusion These results may open the way for the use of P. edulis as a spermicidal product with less adverse effects.

Spermicidal Effects of Methanolic Extract of Cestrum parqui Leaves on Human Spermatozoa:A View through DNA Breakage and Disruption of Membrane Ultrastructure

Objective To focus on the possible mechanism about the spermicidal effect of methanolic extract of leaves of Cestrum parqui （Solanacea） on human spermatozoa. Methods Sperm motility and viability were noted according to the guideline of WHO. The morphological changes in chromatin materials and in plasma membrane at the head part of the spermatozoa were assessed under transmission electron microscopy （TEM）. DNA fragmentation index （DFI） of spermatozoa in percentage was assessed by terminal deoxynucleotidyl transferase mediated dUTP nick end labelling （TUNEL） using kits. For confirmation of DNA breakage, gel electrophoresis of DNA was conducted using 1.2 % agarose gel. Results Sperm viability and motility were both decreased in dose-dependent manner from 50 to 300 lzg/ml of methanolic extract in respect to the control. Loss of viability and motility both were noted 100% at the dose of 300 #g/ml within 5 min. From the microphotography it has been revealed that chromatin condensation at the dose of 200 lzg/ml is more than the control without any noticeable alteration in plasma membrane. In contrast, chromatin decondensation has been noticed at the dose of 300 I^g/ml along with a remarkable disruption in plasma membrane. Analysis of DNA damage by TUNEL revealed a significant elevation （P〈0.01） in the level of% DFI at the dose of 200 μg/ml （moderate dose） after 17 h in respect to the control. But at the dose of 300 lzg/ml, no significant variation was observed in this parameter upto 24 h of exposure though a significant variation （P〈0.01） was noted after 48 h of exposure compared with the control. Gel electrophoresis of DNA also followed the same type of results i.e. 200 lzg/ml affected DNA integrity but other doses were ineffective in this concern. Conclusion The extract of Cestrum parqui at moderate dose exhibited spermicidal activity by inducing apoptotic pathway but at the high dose it caused necrosis.

Reversible Antifertility Activity of Hydroalcoholic Extract of Ficus Racemosa L. in Male Mice

Objective To evaluate male antifertility activity of hydroalcoholic extract of Ficus racemosa bark. Methods Swiss male mice were orally administered hydroalcoholic extract of Ficus racemosa bark （50 mg/kg for 30 d and 100 mg/kg body weight for next 30 d）, and the effect of the treatment on body weight, reproductive organs weight, sperm, biochemical profile （sialic acid in epididymis and fructose in seminal vesicle）, fertility and vaginal contraceptive efficacy was investigated. Recovery studies were also performed. Results Extract reduced fertility to 70% within 60 d. Suppression of cauda epididymis sperm count, motility, viability and abnormal morphology was observed. Marked reduction was noted in the weight of reproductive organs and the level of sialic acid in epididymis and fructose in seminal vesicle. Vaginal application of bark extract exhibited 80% vaginal contraceptive efficacy. After cessation of plant extract treatment, the altered parameters recovered after 60 d. Conclusion Clinical assessment of male antifertility agents should include acceptability, safety and efficacy during and after the treatment. The above results revealed the potential, reversible male antifertility effect of hydroalcoholic extract F. racemosa bark.

Effects of the crude extract of Polygala tenuifolia Willd on human sperm in vitro

The aim of the present study is to analyze sperm membrane changes and the spermicidal effect in treatment with the crude extract from Polygala tenuifo/ia Willd （PTW） in vitro. The root of PTW was extracted in distilled water. Normal human spermatozoa were used to assess the spermicidal activity （Sander-Cramer assay） of the extract from the PTW root. The hypo-osmotic swelling （HOS） test and the eosin Y （EY） staining were used to detect the integrity of sperm membrane and vitality. The sperm chromatin dispersion （SCD） test was performed to determine sperm DNA integrity. N-9 was used as a reference standard and semen added to physiological saline was used as the control. Semen samples were donated by 42 healthy fertile men. The crude extract from the root of PTW could immobilize and kill 100% spermatozoa within 20 s in vitro at the concentrations of 20.0 and 10.0 mg/ml; at the concentration of 5.0 mg/ml, spermatozoa were immobilized in （39.5±3.2） s. In the groups of the crude extract from the root of PTW and N-9 solution the rate of the normal HOS （tails swollen） and the white head （unstained） was 0%, and the rate of the abnormal HOS （tails unswollen） and red head （stained） was 100%. Sperm DNA fragmentation showed no change in exposure to the crude extract from the root of PTW and N-9 solution. The sperm revival test did not show any spermatozoa that recovered their motilities. The rapid spermicidal activity of the crude extract from the root of PTW in vitro may occur by the disruption of the sperm membrane integrity.