Researchers locate novel targets for development of male contraceptive

Researchers at São Paulo State University (UNESP) in Brazil have studied a protein in sperm and discovered two new targets that can be used in combination to develop male contraceptives. The study also demonstrates the feasibility of using mice as a model for in vivo trials during the drug development process. Scientists interested in the protein have so far used primates in their experiments, making the trials more complex, time-consuming and costly.

The focus for the project is epididymal protease inhibitor (EPPIN), a protein whose main function is modulating sperm motility – their ability to move through the female reproductive tract and reach the egg. Scientists and the pharmaceutical industry aim to develop male contraceptives that inhibit sperm motility because it is very difficult to develop a drug that prevents production of sperm. 

“Sperm production is far more complex than egg production. Spermatogenesis takes about two months and is continuous. A male contraceptive designed to block sperm production wouldn’t take effect for three to four months,” said Erick José Ramo da Silva, a professor in the Department of Biophysics and Pharmacology at the Botucatu Institute of Biosciences (IBB-UNESP), and last author of the article on the research published in the journal Molecular Human Reproduction.  

Authorization to swim

An outline of the human fertilization process will help explain how EPPIN works. When a man ejaculates, semen is forcibly expelled from the epididymis, where it is stored, and through the urethra, along the way receiving fluids from the seminal vesicles, prostate gland, and other glands. 

In the case of mammals, especially primates, recently ejaculated semen is gelatinous and viscous, containing several proteins that form the so-called semen coagulum. One of these is semenogelin, with which EPPIN interacts to block sperm motility. “Until ejaculation, sperm don’t swim, although they have the machinery to do so,” Ramo da Silva explained. 

In order for sperm to continue on their voyage to the egg, they must be released from the coagulum, which is similar to a gel owing to the presence of proteins secreted by the seminal vesicles. Here a key role is played by prostate-specific antigen (PSA), well-known as a diagnostic marker for prostate cancer. The protease cleaves the proteins that form the coagulum, including semenogelin, to produce semen liquefaction. 

“This cleaving by PSA releases the sperm in a process we call progressive motility. They are then able to penetrate the outer layers of the egg in a movement known as hyperactivated motility,” he said. 

PSA-mediated cleavage takes place in the female reproductive tract between five and ten minutes after ejaculation. “Up until the moment of PSA-mediated cleavage, the only mechanism that moves sperm to the woman’s reproductive tract is ejaculation. Sperm motility isn’t needed before that. They save energy for the rest of the journey to the uterus and then the egg,” he said. 

In previous research, monkeys were vaccinated with human recombinant EPPIN [produced in the laboratory by genetically modified microorganisms] and developed antibodies that bound to the protein, causing infertility by blocking semenogelin cleavage and delaying semen liquefaction. This proved that semenogelin plays a role in controlling sperm motility. 

Given the differences between rodents and primates, the scientists first set out to work with the latter, but the study by Ramo da Silva’s group opted for a murine model because mice have a protein called SVS2 that plays the same role as semenogelin in humans, binding to EPPIN and blocking sperm motility. 

Results

In the study conducted at UNESP, mice received three types of antibodies to see if they would bind to EPPIN, in which case they would show which domains of the protease inhibitor could be targeted to reduce or block sperm motility. 

“The antibodies act against a specific piece of the EPPIN, and don’t bind to other parts,” Ramo da Silva said. The antibodies that inhibited sperm motility bound to an initial region called the C-terminal extension of the Kunitz domain, as expected, but other bodies, which bound to the N-terminal end of the WFDC domain, were also capable of inhibiting sperm motility, and this was a novelty for the scientists. In addition, both antibodies against the C-terminal and N-terminal regions inhibited the in vitro fertilization rate, confirming that their connection to EPPIN affects the sperm’s potential fertility.

Sperm motility was measured and found to have decreased, proving that protease inhibitors regulated sperm motility in both regions and could be targets for novel drugs. In other words, the study showed that it is possible to design molecules that bind not just to C-terminal but to both domains, blocking sperm motility. 

The study showed which sequences in the EPPIN chain of 133 amino acids should be targeted by a male contraceptive based on sperm motility inhibition. Lastly, it proved that mice can be used as models for in vivo trials, promising to make preclinical studies simpler, faster and cheaper. 

The lead authors of the Molecular Human Reproduction article reporting these results are Alan Andrew dos Santos Silva and Tamiris Rocha Fanti Raimundo, both of whom conducted the research while studying for a master’s degree at IBB-UNESP. Their co-authors are affiliated with the Federal University of São Paulo (UNIFESP) and Argentina’s Institute of Biology and Experimental Medicine (IByME), as well as IBB-UNESP. 

The study was supported by FAPESP via three projects (2015/08227-0, 2017/11363-8, and 2019/13661-1).

In next steps, a team led by Ramo da Silva will test small amino acid sequences designed to bind to EPPIN in a similar manner to semenogelin to see if they interrupt sperm motility. A partnership with researchers in Portugal and the United Kingdom has been arranged for this purpose. 

The tests will be performed with mice in Brazil and human semen in Portugal, where procedures to obtain human biological material are faster. “We now want to study the action mechanism, how inhibition works, and what stages happen for motility to be interrupted. We may then be able to identify other targets – specific proteins in sperm that are involved in this process,” Ramos da Silva said.

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