Caesar has created global headlines with this study: Endocrine disrupting chemicals impair human sperm function and might be involved in fertility disorders seen with increasing frequency in the Western world.
For some time now, it has been observed with concern that an increasing number of couples who are trying to produce a baby require medical help from reproductive clinics. The causes of this are complex. It is suspected that disorders of the hormonal (also known as endocrine) system play a role. The endocrine system keeps our organism in equilibrium: prominent examples include blood sugar level regulation by the hormones insulin and glucagon, as well as the coordination of puberty by testosterone and the estrogens.
Sperm are also controlled by hormones: Progesterone – a female sex hormone released by cells surrounding the egg – acts on the socalled CatSper (Cation channel of Sperm) calcium channels in the membrane of the sperm’s tail - the flagellum. Progesterone opens CatSper, evoking an influx of calcium into the sperm. This calcium increase changes the swimming behavior. It has been proposed that sperm follow the progesterone trail in the oviduct to find their way to the egg: in other words, progesterone could serve as an attractant for human sperm. In addition to its attracting effect, progesterone serves other functions. In the vicinity of the egg, it triggers sperm “hyperactivation”. Hyperactive sperm beat their tails wildly: the hormone puts the sperm into “turbo mode”. This helps them to penetrate the protective egg vestments. In addition, progesterone evokes the acrosome reaction, in which a cocktail of digestive enzymes is released from a vesicle in the spermheads, rendering the egg vestments easier to penetrate.
In our daily lives, we are exposed to numerous chemicals that can influence the hormonal system. These chemicals are referred to as endocrine disrupting chemicals (EDCs). They are omnipresent in foods, plastics, textiles, household products, cosmetics, and toys. It has long been suspected that EDCs interfere with our sex hormone system and have an adverse effect on both unborn babies and on children undergoing puberty. In addition, EDCs seem to impair male fertility, for example by promoting testicular cancer, retaining of testicles, and imparing sperm production. The harmful effect of these chemicals on humans and human fertility has been difficult to prove, because few suitable test systems exist.
We wondered whether EDCs interfere with the hormonal regulation of sperm, and, thereby, with the “chemistry of fertilization”. To this end, we tested around
100 of the most widespread EDCs for their action on intracellular calcium levels in human sperm . Many chemicals, such as the plasticizer bisphenol A, did not affect calcium levels (Figure 1a); however, about one-third of the tested EDCs, for example 4-methylbenzylidene camphor (4-MBC), a UV blocker used in cosmetics, did trigger calcium responses. The calcium responses resembled those evoked by progesterone (Figure 1b) – a truly alarming result.
Figure 1: Do EDCs trigger calcium responses in sperm? A calcium-indicator dye was introduced into sperm to monitor changes in their intracellular calcium concentration. Once inside the sperm, the dye emits fluorescence light. The intensity of the fluorescence depends on the calcium concentration. A fluorescence increase indicates an increase in calcium. a. Bisphenol A (BPA), unlike progesterone (= 2 ?M), does not trigger a calcium response. b. 4-Methylbenzylidene camphor (4-MBC) triggers calcium responses at 10, 1 and 0.1 ?M. The amplitude of the calcium response at 10 ?M 4-MBC is similar to calcium responses evoked by progesterone. c. Analysis of screening trials of about 100 EDCs. The points on the graph represent the mean signal amplitudes of the calcium responses following addition of the EDCs at concentrations of 0.1, 1 and 10 ?M (blue) compared to progesterone (2 ?M; red) and a buffer solution (black). Addition of buffer acts as a negative control: Any EDCs that trigger calcium responses greater than those seen with the buffer (i.e., those in which the fluorescence intensity lies above the gray-hatched area) are defined as “active”.
The “active” chemicals, in addition to 4-MBC, included other UV blockers such as homosalate, benzophenone-3 and padimate O, as well as the plasticizer di-n- butyl phthalate, the pesticide DDT, a growth promoter used to fatten farm animals (?-zearalenol), plus the substances triclosan and n-nonylparaben, which are found in various cosmetics. We characterized the mechanism of action of a selection of active, chemically diverse EDCs (Figure 2).
Figure 2: Chemical structures of EDCs that evoke calcium responses in human sperm.
First of all, we investigated whether EDCs – like progesterone – open CatSper and, thereby, evoke an influx of calcium. To this end, we measured the minute ionic currents flowing through CatSper channels, using the patch-clamp technique. In a patch-clamp experiment, a smallglass electrode is attached to the sperm membrane – a bit like using a stethoscope in medicine – to monitor the opening and closing of the CatSper channels (Figure 3a). Figure 3b illustrates the opening of CatSper by progesterone: the current amplitudes are considerably larger in the presence of progesterone. We performed this experiment also with the UV blocker 4-MBC (Figure 3b, bottom): 4-MBC potentiates the CatSper currents, demonstrating that EDCs imitate the progesterone action on CatSper.
Figure 3: EDC-induced CatSper currents, recorded with the patch-clamp technique. a. A glass electrode is placed carefully on the “neck” of the sperm. The membrane patch below the electrode opening is then disrupted with a short pulse of negative pressure to gain electric access to the sperm’s interior. This technique can be used to measure minute currents of a few hundred picoamperes, flowing through the ion channels in the sperm membrane. b. CatSper currents in human sperm in the absence (left; basal) and presence of progesterone and 4-MBC. The CatSper currents were recorded at membrane potentials of -80 mV to +80 mV (Figure 3a from ).
Next, we investigated whether EDCs stimulated behavioral responses similar to progesterone. To this end, we analyzed flagellar beat under the microscope. The flagellar beat is very regular and symmetric (Figure 4a). However, addition of 4-MBC decreases the beat frequency and the beating pattern becomes asymmetric (Figure 4b) – a characteristic feature of hyperactive swimming. Moreover, we were able to show that EDCs also trigger the acrosome reaction. At first glance, the EDC action on sperm would not appear to represent a threat – after all, CatSper activation, hyperactivation, and the acrosome reaction are all needed anyway for fertilization.In contrast to progesterone, however, EDCs are not present exclusively in the vicinity of the egg, but rather throughout the female reproductive tract. If EDCs already trigger hyperactivation at the entrance of the oviduct, hyperactivated sperm may get left behind and fail to reach the egg.
Figure 4: EDCs change sperm motility and cooperate to trigger calcium responses. Flagellar beating pattern of sperm before (a) and after (b) stimulation with 4-MBC (6.8 ?M). Superimposed single images taken during the beat cycle. Scale bar = 15 ?m. c. Calcium responses of sperm, evoked by individual EDCs at very low concentrations (black) and by a cocktail of EDCs at the same individual concentrations (red).
Once it reaches the egg, each sperm has only one shot at penetrating the egg´s protective vestments. If the enzyme cocktail is not released at the right place and time, it fizzles out ineffectively and the sperm is no longer capable of fertilization. EDCs can “cheat” the sperm into “thinking” that they have reached the egg, thus, triggering the acrosome reaction far too soon. In other words: an EDC-induced hyperactivation and acrosome reaction could interfere considerably with the complex steps of fertilization. It gets even worse: in the presence of EDCs, sperm become “blind” to progesterone. If sperm are bathed in 4-MBC, their sensitivity to progesterone is reduced by 50%.
How far can our research results in the laboratory be applied to “everyday life”? The concentration dependence of the EDC effects on sperm is the key factor in this respect: in many cases, the EDCs are effective at concentrations that are also detectable in the body in real life. For example, when certain sunscreens are applied, the concentration of UV blockers in the blood rises to values which evoke calcium responses in sperm in the laboratory.
In our daily life, however, we are exposed to a variety of EDCs simultaneously. How did we account for this circumstance in our study? We added a number of EDCs to sperm in two separate experimental set-ups, and measured the resultant calcium responses: in one, each individual EDC was added at its threshold concentrations to evoke a calcium response, while in the other, the EDCs were added together at the same concentrations, as an “EDC cocktail” (Figure 4c). As expected, when added alone, the individual EDCs evoked miniscule calcium responses. In the cocktail, however, the miniscule effects of the individulal EDCs added up to one considerable calcium signal. Thus, EDCs cooperate to a certain extent, at least when it comes to interfering with sperm function.
In summary, it may be said that EDCs can impair fertilization in a number of ways: calcium responses control key sperm functions, such as navigation in the female reproductive tract, hyperactivation and the acrosome reaction. Progesterone, which is released by cells surrounding the egg, synchronizes these sperm functions in a precisely coordinated temporal and spatial rhythm. EDCs can lead the sperm astray, and can trigger both hyperactivation and the acrosome reaction at the wrong time and in the wrong place.
Our study, however, could not deliver the final proof that EDCs contribute to the increase in fertility disorders in the West. German law, namely, prohibits studies to determine whether the fertilization of human egg is truly less effective in the presence of EDCs. Since rodents possess different sperm-attractant systems, these experimental animals are not a suitable model for the situation in humans.
On the other hand, our study does show that human sperm are suitable as a test system for the investigation and risk assessment of EDCs. The EU Commission is currently reviewing guidelines for EDC limit values. However, our cocktail experiment shows that limit values for individual EDCs do not offer sufficient protection, since the effects of EDCs can be additive. Thus, our work has provided scientific evidence that could help to establish new guidelines.
 Schiffer, C., Müller, A., Egeberg, D.L., Alvarez, L., Brenker, C., Rehfeld, A., Frederiksen, H., Wäschle, B., Kaupp, U.B., Balbach, M., Wachten, D., Skakkebaek, N.E., Almstrup, K. & Strünker, T. (2014) “Direct action of endocrine disrupting chemicals on human sperm” EMBO Rep., DOI:10.15252/embr.201438869
 Lishko, P.V., Botchkina, I.L., Fedorenko, A., Kirichok, Y. (2010) “Acid extrusion from human spermatozoa is mediated by flagellar voltage-gated proton channel” Cell140, 327-337