| Contraception-on-demand, as opposed to traditional technologies, such as the IUD or pill, refers to contraceptive methods that are only employed when needed, such as barrier (condom), postcoital methods, and spermicide. As an important part of contraception-on-demand, spermicides should be developed with a high priority especially on the 15- to 25-year-old demographic since the following two reasons. Firstly, spermicides do provide some protections against pregnancy and the risk of sexually transmitted disease (STD) infection; secondly, the spermicide is women-centered contraception which is the bridge of male and female fertility control.In theory, the perfect spermicide would provide simultaneous protection against both pregnancy and microbicidal infection with good tolerance and less side effects. However, it is not absolutely necessary for the spermicidal and microbicidal activities of a new topical contraceptive agent to reside in the same molecular now. The problem in this area clearly lies not so much with the development of an appropriate two-function molecular but in producing an effective, selective spermicide capable of immobilizing 100×106–200×106 sperm cells in a matter of seconds without local or systemic toxicity.Recently, several investigators have focused on inhibition of sperm motility as a promising target for male contraceptive development. The mitochondrial sheath located in the mid-piece of flagellum can produce ATP through tricarboxylic acid cycle (TCA) which is the major sources for sperm activated motility. Current evidences suggest that the role of activated motility is to aid in propelling the sperm in female reproductive tract to the oviduct. Therefore, the compound targets on the mitochondria might be the candidate of spermicide.Lonidamine (LND) is an antitumoral drug which can target mitochondria permeability transition (PT) pore (also called PT pore complex, PTPC) and cause the loss of mitochondrial membrane potential (ΔΨm) then induces the apoptosis of tumor cells. LND was used to be a strong inhibitor of spermatogenesis; but was aborted for its severe kidney and liver damage. AF-2364, also 1-(2,4-dichlorobenzyl)-1H- indazole-3-carbohydrazide, an analogue of LND, is less toxic but functionally similar to LND. Treatment regimens by gavage, such as 2–5 doses of AF-2364 (50 mg/kg bw q1 week) effectively induced reversible infertility in male rats. Further research revealed that AF-2364 induces adherens junction (AJ) disruption between Sertoli cells and round and elongated spermatids in the adult rat and can be used as a potential oral contraceptive. The other related work are the further description of the above effective pathway or using the AF-2364 induced spermatogenic cell depletion as an in-vivo model to study the dynamic change between the sertoli cells and germ cells. Thus, the present available results focused on the testis but not in sperm functions. Further, there was not report about AF-2364 in human beings. Whether AF-2364 can target the human sperm mitochondrial to influence the sperm function and further develop to be a spermicidal candidate is not reported? In this work, we firstly incubated AF-2364 with human sperm together and explore the possibility of AF-2364 as a spermicide in-vitro; then we studied the molecular mechanism underling the sperm motility inhibition; thirdly, the possible clinical application of AF-2364 in the field as a spermicidal candidate is also tested in the mice model and the result is promising.When human sperm was incubated with 72μM AF-2364 in-vitro, the percentage for total sperm viability and A type sperm decreased dramatically and this sperm motility blocking phenomena was concentration and time dependent. Further experiments focus on the mechanism of action of AF-2364 on sperm mitochondrial. After 15 mins incubation with 72μM AF-2364, the percentage for JC-1 positive sperms and their mean electronic intensities decreased 10.3% and 23.1% relative to the control group, respectively; the loss of spermΔΨm was further severe according to the incubation time. The addition of CsA (a widely used PT pore inhibitor) partial recovered the AF-2364 induced sperm motility blocking and the recovery effects were more notable when the CsA concentration was increased. As the consequence, we observed a significant down regulation of ATP after the time point of 30mins in AF-2364 treated sperm. We also performed the eosin test to confirm that the ATP change after incubation with AF-2364 was not the sequence of sperm death. When incubated with 72μM AF-2364, the sperm survival rates did not show statistically significant differences; while we detected a huge decrease of survival rate (down to less than 30%) when sperm was treated with 96μM AF-2364. Taken together, the above results indicated that AF-2364 induced sperm motility blocking was through the direct action to sperm mitochondrial PT pore and then impaired mitochondrial functions so as to decrease the respiratory ATP generation. With higher concentration, AF-2364 might induce sperm apoptosis through the mitochondrial pathway and finally contribute the sperm death.We also checked the reported AF-2364 regulating cytoskeleton network and explored the other AF-2364 induced molecular pathways by 2-dimensional electrophoresis (2-DE) after treatment of 72μM AF-2364 and found that only the mitochondrial is the main target of AF-2364. Besides, we had observed the similar sperm motility blocking phenomenon of AF-2364 on BALB/c mice and found that the pregnant rate was decreased with AF-2364 vaginal gel.In summary, it was found in this work: 1) AF-2364 can inhibit human sperm motility and can be developed to a candidate spermicide; 2) further work revealed that the AF-2364 interfered with energy production by inhibiting sperm mitochondrial potential (ΔΨm) like LND on the other tumor cells and then impaired the sperm motility; 3) the primary mating result on BALB/c mice with AF-2364 gel indicates the prospective contraceptive effect of AF-2364 and it might be developed to be a new vaginal gel; 4) during the research, we constructed and revised the sperm JC-1 staining and the detection with the flow cytometry methodology. This is one of the up-growing molecular diagnostic projects in male infertility. With the strong background of our laboratory in infertility and sterility diagnosis, we believe that it can be applied clinically soon. |
PDF Full Text Request