Team:Montpellier/Demonstrate
Objective of this analysis
Antimicrobial peptides (AMPs) have a great affinity for spermatozoa membranes. They are composed of anionic phospholipids such as phosphatidylglycerol and phosphatidylserine in the plasma membrane. Therefore AMPs have to ability to inhibit motility of spermatozoa and prevent the fertilization [1].
Antisperm antibodies are antibodies that can fix on specific parts of spermatozoa (YLP12 antigen) and inhibit their motility. The exact mechanism for this motility inhibition is not known yet [2]. We quantified the action of the AMPs and antibodies on the motility of spermatozoa. This is our proof of concept to test the anticonceptional efficiency of the molecules produced by our bacteria.
Use of mice sperm
The animals that we are using are males Mus musculus, 8 weeks old. They are used by the team of Thomas Robert, at the IGF of Montpellier, that is working on the genetic factors that control the integrity of the meiotic cell division. For our study, the epididymis was taken to collect the sperm of the animal. This tissue is not used by the team of Thomas Robert. The epididymis is squeezed to take the sperm out of it. Following extraction, the sperm is stored in BWW buffer for the experiments. We are using the spermatozoa from the mice’s epididymis cauda (Figure 1) because the maturation of spermatozoa is more advanced in this region of the epididymis and thus represent in the best way fully mature spermatozoa while still being easy to extract.
For our experiments there were two cases. Experiments could be done right after the dissection and extraction of the sperm, else we did the experiment one or two days after the dissection. In the first case we put the sperm in Biggers-Whitten-Whittingham medium (BWW). Otherwise we used a conservation method, which allows to conserve spermatozoa and their motility during 72 hours (preservation protocol).
Moreover for our observations we needed to have the most representative samples possible. Knowing that immotile spermatozoa would more go down the sample than motile ones, we had to take this into account for the samples put on the slides. We calculated the volume of sample required so that the height would let the spermatozoa move without having them at different heights. The head of mice sperm measuring 10µm we calculated, as we do not want to have multiple spermatozoa on the same X and Y dimensions but we don’t want the sperm to be crushed either. For this we approximated the height needed at 20µm. The coverslips measured 24mm for each side. The calculation was the following 20.10-3*24² = 11.5 mm3 = 11.5 µL. We chose to use 20 µL for the sample volume to be sure that sperm are able to move in the liquid as this is a more important constraint than the movement in two dimensions.
The use of mice sperm is discussed in our ethics page.
Experiments
We carried out different types of measurements, including their controls. Negative controls allow us to quantify the motility of spermatozoa alone, while positive controls measure the efficiency and the concentration needed to completely stop the motility of spermatozoa of nisin.
For each experiments the work was the following. After the dissection of the animal the sperm is preserved in BWW. Then some of the sperm is diluted to the 100th in BWW. Nisin can be added at this step. Right after 20 µL of the sample is put on an observation slide. The sample is then recorded by a camera through a microscope (magnificence 20x). The videos recorded are 10 seconds long and the location on the sample is randomly selected.
You can find more details on the protocol on the protocols page.
Negative control
Negative controls were done for each experiment. There was done because the motility can vary depending on the mouse so it is not possible to have only one negative control for all experiments. The negative control consisted of spermatozoa in the BWW buffer. This control permitted to know the normal motility of sperm.
Positive control
Our positive control was the use of a peptide whose activity against sperm motility was demonstrated [3]: nisin. Nisin is an antimicrobial peptide secreted by Lactococcus lactis and is a member of the Class I AMPs. Nisin is specially active against Gram-positive bacteria but not against Lactobacilli in the vaginal flora which makes it a good candidate for contraceptive uses. According to our bibliography 400µg/mL of nisin was enough to inhibit motility of 100% of spermatozoa. Our first positive control was to put sperm in BWW containing different concentrations of nisin. Nisin was prepared with commercial Sigma nisin peptide and according to nisin elution protocol.
Here you can see two videos of spermatozoa. The first one is the negative control, there are only spermatozoa in the media BWW (Figure 2). The second one is the positive control, spermatozoa with nisin at a 400µg/mL concentration (Figure 3).
To analyse this videos, we used our tracking software to construct differents histogramms. In addition we measured the motility for different concentrations of Nisin, to see if there is a dose effect (Figure 4)
The control for this experiments was the analysis of sperm motility in BWW alone. As our experiment for the nisin took one hour to be completed we decided to have as a negative control the motility of sperm during an hour (Figure 5).
These results show that nisin has an effect on the sperm motility that is significantly higher than the one of time alone. For the same amount of time where the sperm were out of the mouse, nisin decreased the motility to 0% as opposed to 40% with the effect of time alone. Moreover this experiment confirms that 400µg/mL of nisin is the concentration that inhibit 100% of sperm motility. This experiments confirms that our experimental procedure is efficient to measure the activity of spermicidal molecules.
Supernatant
The goal of this experiment is to show the effect of molecules present in the supernatant (secreted and from dead cells) on the mice sperm motility. The control for this experiment is to put the sperm in contact with only the culture media and no bacteria. This permits to have information about the effect of the media on sperm motility. The other control needed is the presence of wild non transformed bacteria culture media After doing both controls we can measure and quantify the activity of bacteria supernatant on mice sperm.
For the medium control we put a half of BWW buffer with sperm diluted to 1/100 and half of media (LB and MRS) [4].
Sperm motility was largely inhibited by the media (Figure 7). Also we can see in the video some sperm were able to move their tail but it seemed that the media was blocking them. The same results were found for MRS media. We decided to reduce the concentration to 1/10 (Figure 8).
Once again the sperm motility was inhibited by the medium even with a lower concentration. The same results were found with LB.
We concluded that it was not possible to have supernatant of bacteria as an experiment as the media themselves inhibited sperm motility with the concentrations used. We were not able to continue those experiments to find an alternative. Also this experiments showed the non reproducibility of the method used for the publication we based this experimental procedure on [4]. The use of different concentrations or of a different protocol could prove the usefulness of such an experiment.
Tracking
Our script for tracking spermatozoa is based on Trackpy v0.3.2 Package. Trackpy is able to track any biological samples because it performs a band pass and threshold to locate any particle. Different parameters for the characterization of the tracked object like its size and its intensity (mass) are to take into account to allow good tracking [5]. All of those are specified in the protocol for the tracking (Tracking). We also use tolls for filtering out spurious features, and also to filter the data by their appearance to eliminate undesirable data that has been tracked. Our script track the spermatozoa over time and then give us the X and Y positions for each frame. This allows us to determine each spermatozoa trajectory and velocity. Therefore, we can calculate the mean velocity to analyze the motility of sperm samples depending on different conditions. You can find more information on this page Software We are sharing the fully commented script so everyone can freely understand it and use it as they want : tutorial
Proof of concept
These experiments proved that it was possible, using our experimental procedure, to analyze the effect of molecules on sperm motility. Moreover, we proved that the nisin peptide has an inhibitory effect on the mice sperm motility, which confirms that the use of spermicidal peptides is a good way for a new mean of contraception. This peptide was one of our candidate for our project. Even though we were not able to transform our constructs into L. jensenii and to test their own activity we were able to clone LL-37 and antisperm antibodies inserts and to transform them into our gram positive model bacterium, Bacillus subtilis. These two aspects together show that the way of using bacteria to produce antisperm molecules is possible and could be in the future a new non hormonal contraception.
What should be done next
First, experiments with lysed cells should be conducted to measure the efficiency of the produced molecules by the bacteria. Spermatozoa should be in presence of cell lysate of transformed cells (and their control) to measure the inhibition of motility achieved by the produced intracellular LL-37. Moreover, the experiments with the supernatant should be conducted again with another protocol to measure the secretion of LL-37 by the bacteria. Finally, these experiments should be done with transformed L. jensenii. Once a good protocol to transform L. jensenii is found and proved to be the best, plasmids coding LL-37 (and other molecules) should be transformed into the bacteria to test its activity.
| References | |
|---|---|
| [1] | K V R Reddy, C Aranha, S M Gupta and R D Yedery . (2004). Evaluation of antimicrobial peptide nisin as a safe vaginal contraceptive agent in rabbits: in vitro and in vivo studies. Reproduction Volume 128. Page(s): 117–126. |
| [2] | Samuel, A. S., & Naz, R. K. (2008). Isolation of human single chain variable fragment antibodies against specific sperm antigens for immunocontraceptive development. Human reproduction, 23(6), 1324-1337. |
| [3] | Rajesh K. Naz. (2004). Contraceptive efficacy of antimicrobial peptide Nisin : in vitro and in vivo studies. Contraception Vol 69:333-8. |
| [4] | Bhandari, P., & Prabha, V. (2015). Evaluation of profertility effect of probiotic Lactobacillus plantarum 2621 in a murine model. The Indian journal of medical research, 142(1), 79. |
| [5] | Goodson SG et al., (2011). Classification of Mouse Sperm Motility Patterns Using an Automated Multiclass Support Vector Machines Mode. Biology of Reproduction Vol 84: 1207-15. |