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Regulation of The Sperm Calcium Channel CatSper by Endogenous Steroids and Plant Triterpenoids PDF
Regulation of The Sperm Calcium Channel CatSper by Endogenous Steroids and Plant Triterpenoids PDF
Edited by David E. Clapham, Howard Hughes Medical Institute, Boston Children’s Hospital, Boston, MA, and approved April 20, 2017 (received for review
January 10, 2017)
The calcium channel of sperm (CatSper) is essential for sperm CatSper in a manner similar to P4 and explore the possibility of
hyperactivated motility and fertility. The steroid hormone pro- PregS binding to the same sperm receptor as progesterone.
gesterone activates CatSper of human sperm via binding to the As spermatozoa travel through the male and female repro-
serine hydrolase ABHD2. However, steroid specificity of ABHD2 ductive tract, they are exposed to a variety of steroid hormones,
has not been evaluated. Here, we explored whether steroid such as testosterone and estrogen. The rising levels of hydro-
hormones to which human spermatozoa are exposed in the male cortisone (HC) in the body as a result of stress can impact fer-
and female genital tract influence CatSper activation via modula- tility (16) by interfering with spermatogenesis and/or sperm
tion of ABHD2. The results show that testosterone, estrogen, and functions. Therefore, we have also explored what influence tes-
hydrocortisone did not alter basal CatSper currents, whereas the tosterone, estrogen, and HC have on CatSper activation. The
neurosteroid pregnenolone sulfate exerted similar effects as pro- structural precursor of all steroid hormones in animals is the
gesterone, likely binding to the same site. However, physiological triterpenoid lanosterol, which is formed upon cyclization of
concentrations of testosterone and hydrocortisone inhibited CatSper the linear triterpene squalene. Structurally related plant tri-
activation by progesterone. Additionally, testosterone antago- terpenoids have been shown to exhibit antifertility properties in
PHYSIOLOGY
nized the effect of pregnenolone sulfate. We have also explored mice and rats. The most extensively studied plant triterpenoid is
whether steroid-like molecules, such as the plant triterpenoids lupeol, which is found in mangoes, grapes, and olives. It has been
pristimerin and lupeol, affect sperm fertility. Interestingly, both shown to possess antifertility properties when orally adminis-
compounds competed with progesterone and pregnenolone sul- tered to rats, resulting in a significantly reduced number of
fate and significantly reduced CatSper activation by either steroid. motile sperm (17), although the underlying mechanisms are not
Furthermore, pristimerin and lupeol considerably diminished hyper- understood. A plausible explanation is that lupeol blocks CatSper,
activation of capacitated spermatozoa. These results indicate that which is indispensable for sperm motility (4). Another plant tri-
(i) pregnenolone sulfate together with progesterone are the main terpenoid, pristimerin, which is found in Tripterygium wilfordii (also
steroids that activate CatSper and (ii) pristimerin and lupeol can act known as “Thunder God Vine”) and Celastrus regelii, acts as a
as contraceptive compounds by averting sperm hyperactivation, monoacylglycerol lipase (MAGL) inhibitor. It inhibits the hydro-
thus preventing fertilization. lysis of 2-arachidonoylglyceol (2-AG) to arachidonic acid (AA) and
glycerol (18). In sperm, 2-AG acts as an endogenous CatSper in-
CatSper | steroids | lupeol | triterpenoids | pristimerin hibitor, and 2-AG degradation is mediated by the α/β hydrolase
domain-containing protein 2 (ABHD2) in a P4-dependent man-
ner (7). Because it is likely that pristimerin, lupeol, as well as
S teroid hormones control fundamental organism functions
such as development, metabolism, inflammation, ion ho-
meostasis, and reproduction. According to the conventional Significance
model, steroid hormones signal through a corresponding geno-
mic receptor, which upon binding to a steroid hormone initiates The calcium channel of sperm—CatSper—is vital for male fer-
the translocation of the hormone–receptor complex to the nu- tility. CatSper is activated by the hormone progesterone,
cleus. There it acts as a transcription factor by altering gene but its pharmacological profile is not well studied. By exploring
expression, and such a process can take hours (1). However, steroid selectivity of CatSper activation, we found one addi-
there is another, much faster pathway that is initiated by steroid tional agonist—pregnenolone sulfate—and the two plant-derived
hormone binding to its membrane receptor on the extracellular inhibitors pristimerin and lupeol. By averting sperm hyper-
side of the cell. The latter signaling involves second messengers activation, both inhibitors can prevent fertilization, thus acting
and signal-transduction cascades, which often result in the acti- as contraceptive agents. Additionally, by exploring CatSper reg-
vation of ion channels (1). The female hormone progesterone ulation by endogenous steroids, we explain why CatSper is silent
(P4) activates the principal calcium channel of sperm (CatSper) within the male reproductive tract and is only activated in close
(2–5) and blocks the potassium channel KSper (6) via these proximity to the egg. Interestingly, both testosterone and hy-
nongenomic pathways (7). The modulation of ion channel drocortisone antagonize the action of progesterone at physio-
functions by steroid hormones has been reported in the heart (8, logical concentrations, which may explain why elevated levels of
9), neurons (10–12), smooth muscle (13), and pancreatic beta these steroids in the female organism affect fertility.
cells (14). In marine invertebrates, the sulfated steroid choles-
Author contributions: N.M. and P.V.L. designed research; N.M., M.R.M., and P.V.L. per-
tane acts as a chemoattractant for sea squirt sperm, which also
formed research; N.M. contributed new reagents/analytic tools; M.R.M. helped with pilot
happens via the nongenomic pathway (15). The latter represents experiments; P.V.L. led the research study; N.M. and P.V.L. analyzed data; and N.M. and
an unconventional chemoattractant for ascidian sperm, as such P.V.L. wrote the paper.
factors are usually proteins or peptides and not steroids. In hu- Conflict of interest statement: P.V.L. and N.M. are inventors on a patent application filed
mans, pregnenolone sulfate (PregS), a sulfated steroid hormone by University of California, Berkeley related to the work presented in this paper.
similar in structure to P4, is a nongenomic activator of the This article is a PNAS Direct Submission.
transient receptor potential cation channel subfamily M member 3 Freely available online through the PNAS open access option.
(TRPM3) channel in pancreatic beta cells (14). However, the 1
To whom correspondence should be addressed. Email: lishko@berkeley.edu.
precise mechanism of this activation is unknown. Here, we This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
report that PregS also activates the sperm calcium channel 1073/pnas.1700367114/-/DCSupplemental.
A B C D E
progesterone (P4) testosterone (T) 17beta-estradiol (E2) hydrocortisone (HC) pregnenolone sulfate (PregS)
+80 mV
0 mV
200 pA/pF
1 µM P4 DVF -80 mV 1 µM E2 + 1 µM HC +
1 µM T + 1 µM P4 1 µM P4 1 µM PregS
DVF 1 µM P4 1 µM E2 DVF
1 µM T DVF 1 µM HC
200 ms HS HS HS DVF HS
HS
ICatSper (-pA/pF) at -80 mV
****
400 (7) 400 400 400 400
ICatSper (-pA/pF)
ICatSper (-pA/pF)
ICatSper (-pA/pF)
ICatSper (-pA/pF)
300 300 300 300 300
*** ** ****
200 200 200 NS (4) 200 (5) 200 (13)
NS NS NS
100 (7) 100 (8) 100 (4) 100 (5) 100
(8) (8) (4) (5) (13)
0 0 0 0 0
1 µM P4 - + 1 µM T - + + 1 µM E2 - + + 1 µM HC - + + 1 µM PregS - +
1 µM P4 - - + 1 µM P4 - - + 1 µM P4 - - +
F 1.0
(5)
G 1.0 H 1.0 I IC50 (nM) slope
dose- (4) factor
I/Imax at -80 mV
I/Imax at -80 mV
I/Imax at -80 mV
PHYSIOLOGY
serum concentration of PregS in adults reaches levels of 130 nM centrations of P4 and PregS (Fig. 3 A and C, red trace and Table
(women) and 140 nM (men) (14). It is also abundant in testes, S3). The experiments were repeated in the reverse order (Fig. 3
in which the total PregS concentrations can reach 2.8 μmol/kg B and C and Table S3). The increase of CatSper currents by
(3.8 μM), whereas it drops to 0.76 μmol/kg (190 nM) in the ep- PregS was not further potentiated upon addition of P4. The same
ididymis (14). Such high PregS concentrations may result in result was obtained when sperm cells were first stimulated with
premature CatSper activation, while spermatozoa are still in the P4 and then with a combination of P4 and PregS. These experi-
male reproductive tract, which would be detrimental for sperm ments suggest that P4 and PregS likely compete for the same
fertility. Because testosterone is also abundant in the male re- ABHD2 binding site and that CatSper activation by PregS follows
productive tract, we have explored whether it can block CatSper the same mechanism as CatSper activation by P4.
A B
ICatsper density(-pA/pF)
-80 mV 200
DVF
1 µM PregS +
1 µM NNC 100 (4)
HS (4)
0 Fig. 2. PregS activates CatSper through a molecular
1 µM PregS - + +
mechanism, which is similar to the P4-mediated
- - +
5 µM 1 µM NNC CatSper activation. (A) Representative ICatSper traces
1 µM in response to stimulation with 1 μM PregS (blue) or
C 10 nM D 1.0 (13/4)
1 μM PregS + 1 μM NNC 55–0396 (red). The same
I/Imax at -80 mV
100 nM 0.8
1 nM (4) voltage protocol was used for all traces. (B) Averaged
0 nM (3) inward ICatSper densities of control and test conditions.
0.6
HS (C) Representative ICatSper with different concentra-
0.4 (4) (3) PregS tions of PregS (1 nM, light blue; 10 nM, dark blue;
(5) 100 nM, magenta; 1 μM, red; 5 μM, green). (D) Dose–
0.2 PregS + 1µM T
response of ICatSper activation with PregS alone (black
(nM)
1 µM PregS 0.0 trace) or with various concentrations of PregS in the
10-1 101 103 105 107 presence of 1 μM T (red trace). The data were fitted
E F PregS
with a Hill-based equation. For PregS alone, the EC50
**
DVF was 15.2 ± 5.0 nM with a slope factor of 0.6 ± 0.1.
ICatsper density (-pA/pF)
ICatSper (-pA/pF)
1 µM PregS pristimerin and lupeol inhibit it, we explored whether VCL val-
at -80 mV
-80 mV 1 µM PregS + NS
1 µM P4 200 ues of human sperm were affected in the presence of steroid
(3) (3)
DVF hormones or plant triterpenoids. As expected, neither of the
HS
100 compounds changed VCL values of noncapacitated sperm cells
(6) (Fig. S3, Bottom panels and Table S6). In capacitated sperma-
1 µM P4
1 µM P4 + tozoa, P4 stimulation increased VCL by 12%, comparable to
0
200 pA/pF
1 µM PregS 1 µM PregS - + + - + numbers reported earlier (7, 21), but VCL values remained un-
B DVF 1 µM P4 - - + + +
changed in the presence of PregS, pristimerin, or lupeol alone
0.2 s HS (Fig. S3A, Top and Inset and Table S6). When spermatozoa were
stimulated with P4 + pristimerin or P4 + lupeol, VCL values
were reduced by 39% and 48%, respectively, in comparison with
VCL values obtained in the presence of P4, and the reduced
VCL values were comparable to those of noncapacitated cells
Fig. 3. PregS and P4 compete for the ABHD2 binding site. (A) Represen- (Fig. S3B and Table S6). Stimulating capacitated sperm with
tative ICatSper in response to the indicated voltage ramp in the presence of PregS in combination with pristimerin or lupeol decreased VCL
1 μM PregS (blue) or 1 μM PregS + 1 μM P4 (red). (B) ICatSper recording with by 18% and 9%, respectively, compared with sperm, which were
1 μM P4 (blue) followed by 1 μM P4 + 1 μM PregS (red). (C) Averaged ICatSper treated with PregS alone (Fig. S3C, Top and Table S6). VCL
densities obtained at –80 mV and presented as mean ± SEM; n indicates values of noncapacitated sperm remained unchanged under such
number of individual cells analyzed. conditions (Fig. S3C, Bottom and Table S6). To explore whether
pristimerin and lupeol also affect basal sperm motility, we
The Two Plant Triterpenoids Pristimerin and Lupeol Significantly assessed the effect of these compounds on motility of capacitated
Reduce the Activation of CatSper by Either P4 or PregS. 2-AG hy- and noncapacitated sperm cells. The number of both capacitated
drolysis by monoacyl lipases, such as ABHD2, is required for and noncapacitated motile spermatozoa remained unchanged
CatSper activation. The plant triterpenoid pristimerin was reported when stimulated with either compounds (Fig. S4A and Table S7).
to inhibit the activity of the ABHD-related enzyme mono- However, when P4 was coapplied with pristimerin or lupeol, the
acylglycerol lipase (MAGL) (18). If pristimerin can also inhibit percentage of motile capacitated sperm decreased by 19%, whereas
ABHD2, then sperm exposure to this compound should prevent the values of noncapacitated cells did not change (Fig. S4B and
CatSper activation by P4 or PregS. To test this hypothesis, human Table S7). Costimulation with PregS and pristimerin or lupeol,
spermatozoa were stimulated with pristimerin, followed by expo- however, did not result in a significant reduction of motile
sure to either a mixture of pristimerin and P4 or to a mixture of capacitated or noncapacitated sperm (Fig. S4C and Table S7).
pristimerin and PregS. The mere presence of pristimerin did not These results demonstrate that both pristimerin and lupeol have
affect basal ICatSper (Fig. 4A, Fig. S2 A–D, and Table S4), which the capacity to significantly reduce sperm hyperactivation by
indicates that pristimerin did not target the channel directly. blocking the P4-mediated activation of CatSper but also have a
However, coapplication of pristimerin with P4 or PregS signifi- minor effect on basal sperm motility of capacitated cells.
cantly reduced CatSper activation. Compared with stimulation with
Discussion
P4 or PregS alone, ICatSper stimulation was reduced by 63% (pris-
timerin and P4) and by 48% (pristimerin and PregS; Fig. 4A, Fig. Our results demonstrate that testosterone, estrogen, and hy-
S2 A–D, and Table S4). To determine the IC50 value of pristi- drocortisone alone did not activate CatSper but that they reduce
merin’s inhibitory effect on CatSper activation by P4, human sperm or prevent CatSper activation by progesterone (P4). In the case
cells were first preexposed to various concentrations of pristimerin. of testosterone and hydrocortisone, the effects were the stron-
Then, cells were exposed to the respective pristimerin concentra- gest with a significant inhibition of P4-mediated CatSper stim-
tion of the first stimulus plus 1 μM P4. The IC50 of pristimerin in ulation under physiological concentrations of either steroid. One
the presence of 1 μM P4 was 116 ± 77 nM (Fig. 4B and Table S4). possibility for this effect could be that testosterone binds with a
Another pharmacologically active plant triterpenoid, which
affects sperm functions when orally administered to rats, is lupeol
(17). Comparable to pristimerin, lupeol alone did not affect basal
ICatSper (Fig. 5A, Fig. S2 E and F, and Table S5). However, ad- A ****
B
ICatSper (-pA/pF) at -80 mV
400
ministration of lupeol with P4 or PregS led to an even stronger (7) 1.0
(3)
inhibition of ICatSper than pristimerin. Compared with CatSper 300 0.8
I/Imax at -80 mV
300 (4)
PHYSIOLOGY
Testosterone blocked CatSper activation even in the presence of
and P4 act synergistically or independently to activate CatSper.
1 μM P4 with an IC50 of 429 nM, which is within the physiological Our earlier findings identified the acylglycerol lipase ABHD2
range, as testosterone concentrations reach 2 μM in the blood as the P4 binding partner (7). Therefore, we tested whether the
plasma of men (23). CatSper can also be activated by PregS, monoacylglycerol lipase inhibitor pristimerin, a plant triterpe-
which can reach high concentrations in male reproductive tissues. noid (18), can inhibit both the P4- and the PregS-mediated ac-
However, testosterone also inhibits CatSper response to PregS by tivation of CatSper of human sperm. Although basal CatSper
shifting its EC50 10-fold to 172 nM. Therefore, even if sperm are currents were not affected, both P4 and PregS-mediated CatSper
exposed to elevated concentrations of PregS, high concentrations potentiation was significantly reduced. Lupeol, another plant
of testosterone in the male genital tract prevent premature triterpenoid, had similar effects on ICatSper as pristimerin. It is
CatSper activation by P4, or even PregS. It is therefore possible possible that both triterpenoids occupy the steroid binding site of
that testosterone acts as an anticapacitation factor by preventing ABHD2, thus preventing CatSper activation by P4 via a com-
CatSper activation until it is removed in the female reproductive petitive antagonist-type mechanism. Both compounds were also
tract, presumably by chelation with albumin. able to inhibit sperm hyperactivation and slightly reduced basal
Resting serum E2 levels in women are about 110 pM, which motility of capacitated sperm cells, as evident from computer-
peak at ovulation to concentrations of around 403 pM (24). assisted sperm analyses (CASAs). Interestingly, pristimerin and
Therefore, sperm encounter an E2-enriched milieu in the uterus lupeol had no effect on sperm motility of noncapacitated cells,
and the fallopian tube. Our results show that E2 did not alter which indicates their low toxicity effect toward spermatozoa.
resting CatSper currents but that it also did not allow full These results correlate with our electrophysiological data, which
channel activation by P4. We assume that elevated levels of showed a significant reduction of ICatSper with pristimerin + P4
E2 during ovulation render CatSper in its closed state to prevent and lupeol + P4, respectively. Because CatSper is indispensable
premature calcium influx and thus sperm activation. However, for hyperactivated sperm motility (36–40), it is evident that the
the significantly weaker IC50 for E2 of 833 nM indicates that reduction of sperm hyperactivation by both triterpenoids may
E2 acts as a much less potent P4 antagonist. Right after ovula- significantly impair sperm ability to fertilize an egg.
tion, E2 levels decrease, whereas P4 concentrations surge to CASA experiments also revealed that although P4 increased
∼7 nM (24) in the blood. Because cumulus cells surrounding the hyperactivated motility, PregS failed to do so. This could be due
oocyte also secrete P4 (25-28), sperm travel through a P4 gra- to the fact that PregS is a charged molecule, which cannot
dient with maximal concentrations in close proximity to the egg. pass the plasma membrane (41). It is therefore possible that the
It is therefore possible that P4 outcompetes E2, leading to nonpolar P4 can activate additional intracellular pathways, con-
CatSper activation, as P4 is the natural E2 antagonist (29). tributing to a more pronounced activation of CatSper. Indeed, for
Various conditions, such as stress and elevated levels of glu- full sperm hyperactivation in vitro, two events must be met at the
cocorticoids, particularly hydrocortisone, are known to impair same time: (i) CatSper must be relieved from inhibition by 2-AG,
male fertility (16) by either inhibiting spermatogenesis (30) or and (ii) the sperm plasma membrane needs to be depolarized.
reducing sperm counts and sperm motility (31). Stress and ele- Because CatSper is a voltage-dependent channel, it requires at least
vated hydrocortisone levels can also affect female fertility. One +30 mV for half-activation (3). The latter can be achieved via the
study shows that in women who underwent in vitro fertiliza- P4-mediated inhibition of the potassium channel KSper (6), which
tion, baseline urine cortisol levels increased from ∼230 nM to creates membrane depolarization required for full CatSper activa-
∼500 nM (32). According to our results, hydrocortisone blocked tion. If P4 inhibits KSper from the intracellular side, which PregS
CatSper activation by P4 with an IC50 of 153 nM. It is therefore fails to do, as it cannot cross plasma membrane, then P4 is able to
possible that elevated levels of stress hormones in the female cause a more pronounced hyperactivation.
genital tract impair not only early stages but also late stages of In conclusion, our findings show that apart from P4, PregS is
sperm acquisition of their fertilizing potential, thus significantly another steroid hormone that can activate CatSper via ABHD2
contributing to infertility. in human spermatozoa, whereas testosterone, E2, and HC may
Another interesting candidate among the hormones tested in bind to ABHD2 competitively to modulate the response to P4
this study was the sulfated neurosteroid PregS, as it stimulates and PregS. In addition, we describe two plant triterpenoids that
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