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Article
Rotational motion and rheotaxis of human sperm
do not require functional CatSper channels and
transmembrane Ca
2+
signaling
Christian Schiffer
1
, Steffen Rieger
2
, Christoph Brenker
1
, Samuel Young
1
, Hussein Hamzeh
3
,
Dagmar Wachten
4,5
, Frank Tüttelmann
6
, Albrecht Röpke
6
, U Benjamin Kaupp
3
, Tao Wang
1,7
,
Alice Wagner
1,6
, Claudia Krallmann
1
, Sabine Kliesch
1
, Carsten Fallnich
2,8,*
& Timo Strünker
1,8,**
Abstract
Navigation of sperm in fluid flow, called rheotaxis, provides long-
range guidance in the mammalian oviduct. The rotation of sperm
around their longitudinal axis (rolling) promotes rheotaxis.
Whether sperm rolling and rheotaxis require calcium (Ca
2+
) influx
via the sperm-specific Ca
2+
channel CatSper, or rather represent
passive biomechanical and hydrodynamic processes, has remained
controversial. Here, we study the swimming behavior of sperm
from healthy donors and from infertile patients that lack func-
tional CatSper channels, using dark-field microscopy, optical
tweezers, and microfluidics. We demonstrate that rolling and
rheotaxis persist in CatSper-deficient human sperm. Furthermore,
human sperm undergo rolling and rheotaxis even when Ca
2+
influx
is prevented. Finally, we show that rolling and rheotaxis also
persist in mouse sperm deficient in both CatSper and flagellar
Ca
2+
-signaling domains. Our results strongly support the concept
that passive biomechanical and hydrodynamic processes enable
sperm rolling and rheotaxis, rather than calcium signaling medi-
ated by CatSper or other mechanisms controlling transmembrane
Ca
2+
flux.
Keywords Ca
2+
signaling; CatSper; rheotaxis; rolling; human sperm
Subject Categories Cell Adhesion, Polarity & Cytoskeleton; Membranes &
Trafficking; Signal Transduction
DOI 10.15252/embj.2019102363 | Received 30 April 2019 | Revised 30 October
2019 | Accepted 6December 2019
The EMBO Journal (2020)e102363
Introduction
In fluid flow, mammalian sperm realign their swimming path and
move upstream—a mechanism called rheotaxis (Miki & Clapham,
2013; El-Sherry et al, 2014; Kantsler et al, 2014; Tung et al, 2014,
2015; Bukatin et al, 2015). In the oviduct, long-range navigation via
rheotaxis directs sperm to the site of fertilization (Miki & Clapham,
2013). An important ingredient of rheotaxis is the rotation of sperm
around their longitudinal axis, called rolling (e.g., Miki & Clapham,
2013; Kantsler et al, 2014; Bukatin et al, 2015), resulting in a cone-
shaped beating envelope. Through this mechanism, vertical shear
flow, e.g., near boundary surfaces, exerts a torque that aligns the
longitudinal axis of sperm against the flow direction (Miki &
Clapham, 2013; Kantsler et al, 2014; Bukatin et al, 2015). However,
whether sperm rolling involves full 360°or incomplete rotations of
alternating direction is debated (Miki & Clapham, 2013; Muschol
et al, 2018).
The intracellular Ca
2+
concentration ([Ca
2+
]
i
) controls the flagel-
lar beat and swimming behavior of sperm (Kaupp et al, 2003; Publi-
cover et al, 2008; Fechner et al, 2015). In most sperm species,
[Ca
2+
]
i
is set by the voltage- and alkaline-activated CatSper Ca
2+
channel (Quill et al, 2001; Ren et al, 2001; Kirichok et al, 2006;
Lishko et al, 2010; Lishko et al, 2011; Stru
¨nker et al, 2011; Loux
et al, 2013; Seifert et al, 2015). Mammalian CatSper comprises four
homologous pore-forming subunits (CatSper 1–4) (e.g., Navarro
et al, 2008) and at least six auxiliary subunits (CatSper b,c,d,ɛ,f,
and Efcab9) (Liu et al, 2007; Wang et al, 2009; Chung et al, 2011,
2017; Hwang et al, 2019). The CatSper-channel complex is orga-
nized as quadrilateral threads along the flagellum; the CatSper
threads encompass several other proteins, including
1Centre of Reproductive Medicine and Andrology, University Hospital Münster, University of Münster, Münster, Germany
2Optical Technologies Group, Institute of Applied Physics, University of Münster, Münster, Germany
3Molecular Sensory Systems, Center of Advanced European Studies and Research, Bonn, Germany
4Minerva Max Planck Research Group, Molecular Physiology, Center of Advanced European Studies and Research, Bonn, Germany
5Institute of Innate Immunity, University Hospital, University of Bonn, Bonn, Germany
6Institute of Human Genetics, University of Münster, Münster, Germany
7Institute of Life Science and School of Life Science, Nanchang University, Nanchang, China
8Cells-in-Motion Cluster of Excellence (EXC1003-CiM), Münster, Germany
*Corresponding author. Tel: +49 251 83 36160; E-mail: fallnich@uni-muenster.de
**Corresponding author. Tel: +49 251 83 58238; E-mail: timo.struenker@ukmuenster.de
ª2020 The Authors. Published under the terms of the CC BY NC ND 4.0license The EMBO Journal e102363 |2020 1of 15
Ca
2+
-binding proteins and protein kinases, forming local
Ca
2+
-signaling domains near the membrane surface (Chung et al,
2014, 2017).
In Catsper1
/
mouse sperm, longitudinal rolling and rheotaxis
were abolished (Miki & Clapham, 2013), suggesting that control of
[Ca
2+
]
i
by CatSper is required for rolling and rheotaxis of
mammalian sperm. For human sperm, it was specifically proposed
that rolling is created by asymmetrical Ca
2+
influx via CatSper
channels, stimulated by local pH
i
signaling (Miller et al, 2018).
The H
+
channel Hv1 is organized along the flagellum of human
sperm in two threads near two of the four CatSper threads (Miller
et al, 2018). It was proposed that H
+
efflux via Hv1 organizes
localized Ca
2+
signaling that, ultimately, creates an asymmetry in
calcium-dependent inhibition of dynein-powered microtubule slid-
ing (Miller et al, 2018). However, the concept that rolling and
rheotaxis are enabled by Ca
2+
influx cannot be reconciled with
the finding that rolling of mouse sperm does not require extracel-
lular Ca
2+
(Babcock et al, 2014; Muschol et al, 2018), and that
exposure of human sperm to gradients of flow velocities does not
evoke measurable changes in [Ca
2+
]
i
(Zhang et al, 2016). More-
over, the inventory and regulation of signaling molecules are dif-
ferent among mammalian sperm (Kaupp & Stru
¨nker, 2017). For
example, mouse sperm lack Hv1 channels (Lishko et al, 2010;
Berger et al, 2017), and human CatSper is activated by nanomolar
concentrations of prostaglandins and progesterone (Lishko et al,
2011; Stru
¨nker et al, 2011) that do not activate mouse CatSper
(Lishko et al, 2011). Thus, if the quadrilateral arrangement of
CatSper and its control by pH
i
were key to rolling and rheotaxis of
mouse and human sperm, the underlying mechanisms ought to be
vastly different.
Here, we show that human sperm undergo continuous full 360°
rotations rather than incomplete rotations of alternating directions.
Moreover, to scrutinize the role of CatSper and Ca
2+
in rolling and
rheotaxis of human sperm, we studied sperm of healthy donors and
patients who suffer from the deafness-infertility syndrome (DIS).
DIS patients lack the CATSPER2 gene (Zhang et al, 2007; Hilde-
brand et al, 2010). We show by 3D-STORM that, in the absence of
CatSper 2, other pore-forming CatSper subunits still assemble into
quadrilateral threads of non-functional CatSper complexes. We
demonstrate that rolling and rheotaxis persist in CatSper-deficient
sperm from DIS patients. Furthermore, we show that rolling and
rheotaxis of human sperm are preserved even when Ca
2+
influx is
completely abolished. Finally, we demonstrate that rolling and
rheotaxis are also preserved in Catsper1
/
mouse sperm, which
lack the CatSper complex and the quadrilateral threads altogether.
We conclude that in mouse and human sperm, neither Ca
2+
influx
via CatSper nor the quadrilateral Ca
2+
-signaling threads organized
by CatSper are required for rolling and rheotaxis.
Results
The expression of pore-forming CatSper subunits is not
strictly interdependent
We examined sperm from five infertile patients suffering from a
homozygous deletion of contiguous genes on chromosome 15,
including the CATSPER2 gene (Fig EV1). This deletion at 15q15.3 is
the hallmark of DIS (Zhang et al, 2007; Hildebrand et al, 2010).
Motile sperm isolated from patients’ ejaculates by the swim-up
procedure were morphologically normal, but lacked CatSper-
mediated Ca
2+
influx (Fig 1A) and CatSper currents (Fig 1B and C),
confirming that the deletion of the CATSPER2 gene abrogates the
expression of functional CatSper channels (Smith et al, 2013;
Brenker et al, 2018a). Antibodies directed against CatSper 3 and
CatSper 4 stained the principal piece of sperm from healthy donors
and DIS patients (Fig 1D and E). 3D-STORM analysis revealed that
the quadrilateral arrangement of CatSper 3 and CatSper 4 along the
flagellum was preserved in DIS patients (Fig 1F and G). Thus, in the
absence of CatSper 2, CatSper 3 and CatSper 4 subunits still assem-
ble into non-functional protein complexes, whose sub-cellular
arrangement is similar to that of the functional CatSper-channel
complex (Chung et al, 2014, 2017).
Human sperm do not require functional CatSper channels
for longitudinal rolling
We examined whether longitudinal rolling is impaired or even
abolished in CatSper-deficient human sperm. Under dim dark-field
illumination, we monitored rolling of sperm in population via
periodic changes in brightness (blinking) of the sperm heads
(Fig 2A–C; Movie EV1). Semi-automated analysis of blinking
events revealed the rotation frequency of each sperm cell in the
field of view. In non-capacitated and capacitated control sperm
from healthy donors, the rotation frequency was normally distrib-
uted (Fig 2D) with a mean value of 4.8 1.5 Hz (n=1,455) and
7.0 2.2 Hz (n=1,097), respectively (Fig 2E) (Rigler & Thyberg,
1984; Aitken et al, 1985; Miller et al, 2018). Bicarbonate (25 mM)
used for capacitation stimulates cAMP synthesis (Carlson et al,
2007; Tresguerres et al, 2011; Brenker et al, 2012) and, thereby,
accelerates the flagellar beat (Esposito et al, 2004; Xie et al,
2006) and rotation frequency (Miki & Clapham, 2013). The rota-
tion frequency decreased with increasing viscosity (Fig 2F), in
line with previous results (e.g., Nosrati et al, 2015; Gallagher
et al, 2019). To study rolling of single sperm cells with high time
resolution and for long recording times, we combined bright-field
microscopy with an optical tweezer (Ashkin et al, 1986) (Fig 2G).
Sperm were trapped perpendicular to the optical axis (Fig 2H,
Movie EV2), and the periodic intensity fluctuations of the laser
light, which was back-scattered from the cell into the microscope
objective, provided a measure of the rotation frequency (Fig 2I).
For optically trapped control sperm from healthy donors, the
rotation frequency was constant for several tens of seconds
(Fig 2I). The frequency distribution and mean frequency of
trapped sperm (6.0 2.1 Hz, n=32) and freely moving sperm
(7.0 2.2 Hz, n=1,097) were similar (compare Fig 2J and D).
Trapping of sperm parallel to the optical axis allowed a frontal
view onto the tip of the sperm head; this view reveals that
human sperm display continuous full 360°rotations (Fig 2K,
Movie EV3), in contrast to incomplete rotations of alternating
directions that have been reported for mouse sperm (Babcock
et al, 2014; Muschol et al, 2018). Remarkably, also CatSper-
deficient human sperm displayed longitudinal rolling (Movie
EV4): In freely moving CatSper-deficient sperm incubated under
non-capacitating or capacitating conditions, the rotation frequency
was normally distributed around a mean value of 6.0 2.6 Hz
2of 15 The EMBO Journal e102363 |2020 ª2020 The Authors
The EMBO Journal Christian Schiffer et al
(n=1,009) and 6.8 3.1 Hz (n=946), respectively (Fig 2L and
M). The CatSper-deficient human sperm swam progressively also
in highly viscous media (Movie EV5), and, like in control sperm,
the rotation frequency of CatSper-deficient sperm decreased with
increasing viscosity (Fig 2N). When optically trapped, the
CatSper-deficient sperm clearly displayed continuous full 360°
rotations (Fig 2O and P, Movie EV6), and the rotation frequency
remained constant over several tens of seconds (Fig 2Q). In
conclusion, human sperm do not require functional CatSper for
longitudinal rolling. If anything, the longitudinal rolling of
CatSper-deficient sperm might be slightly enhanced in the
absence of bicarbonate.
A
B
D
F
E
C
G
Figure 1.
ª2020 The Authors The EMBO Journal e102363 |2020 3of 15
Christian Schiffer et al The EMBO Journal
Longitudinal rolling of human sperm does not require an influx
of Ca
2+
We further examined whether Ca
2+
is required for rolling of human
sperm, using both dark-field microscopy of sperm populations and
optical trapping of single sperm cells. Control sperm from healthy
donors held by the optical tweezer were dragged between parallel
laminar flows of three different solutions (Figs 3A and EV2). This
setup allows monitoring of the rotation frequency upon rapid
switching of solutions. A stimulus buffer (stimulus stream) and
sperm in control buffer (control stream) were separated by a barrier
stream containing fluorescein in control buffer; the buffers were fed
into a capillary via three inlets. The transfer from one to the other
stream was monitored by changes in the fluorescence of fluorescein:
When entering the barrier stream, the fluorescence rose and
resumed basal values when sperm reached the stimulus stream
(Fig 3B). Dragging of control sperm from healthy donors across the
barrier stream was completed within ≤10 s (Movie EV7). Dragging
itself did not affect rolling (Fig 3B and C): The mean rotation
frequency before and after dragging between control buffers was
6.7 2.8 Hz and 6.5 2.8 Hz (n=14), respectively. After drag-
ging from bicarbonate-free to bicarbonate-containing buffer, the
rotation frequency increased from 6.6 2.9 to 11.3 2.5 Hz
(Fig 3D and E, n=5). Next, the rotation frequency of trapped
sperm cells before and after transition from 2 mM to ˂20 nM extra-
cellular Ca
2+
was studied. The rotation frequency was similar in the
absence and presence of Ca
2+
(5.5 3.6 Hz versus 5.9 3.1 Hz,
n=5; Fig 3F and G). Although in dark-field microscopy of sperm
populations, the fraction of motile sperm decreased in Ca
2+
-free
buffer with a time constant (s) of 5.3 min (Fig 3H) (Aaberg et al,
1989; Jin et al, 2007; Torres-Flores et al, 2011), at any time-point
during the decay, motile sperm were also rolling (Movie EV8). The
◀Figure 1. Characterization of CATSPER2-deficient human sperm.
A Representative Ca
2+
signals in sperm from a patient with deafness-infertility syndrome lacking functional CatSper channels (CATSPER2
/
; red) and a healthy donor
(black), evoked by progesterone, PGE1,NH
4
Cl, or ionomycin. NH
4
Cl increases the intracellular pH. Bar graph: Amplitudes (n=4; mean SD) of Ca
2+
signals in
CATSPER2
/
sperm.
B Representative monovalent CatSper currents in CATSPER2
/
sperm (blue, green, orange, purple, brown) and in sperm from a healthy donor (black), and
corresponding current-voltage relationship (right). The membrane voltage was stepped from 100 to +100 mV in increments of 10 mV from a holding potential of
80 mV.
C Outward and inward current amplitudes (mean SD) at + 100 mV and -100 mV, respectively, in CATSPER2
/
sperm (color code: panel B) and sperm from
healthy donors (black).
D, E Representative immunocytochemical staining of control sperm from healthy donors and CATSPER2
/
sperm from DIS patients using antibodies directed against
CatSper 3(D) or CatSper 4(E); DNA was labeled with DAPI (blue). Scale bars represent 10 lm.
F3D-STORM image in xy projection of sperm from a healthy donor labeled with the anti-CatSper 3antibody (left). Axial projection of the boxed region (right). Scale
bars represent 5lminxy projections and 200 nm in axial projections.
G3D-STORM images in xy projection of CATSPER2
/
sperm (left) labeled with the anti-CatSper 3(upper panel) or anti-CatSper 4(lower panel) antibody. Axial
projection of the boxed regions (right). Scale bars represent 5lminxy projections and 200 nm in axial projections.
▸
Figure 2. Analysis of longitudinal rolling of human sperm.
A Experimental setup for population analysis by dark-field microscopy.
B Dark-field microscopy of a single sperm cell; shown are single frames obtained at t=0,48 ,96, and 136 ms. Scale bar = 25 lm.
C Dark-field imaging of a sperm population; left: single frames at t=185,363,540, and 718 ms. Sperm selected for analysis are highlighted (1–4). Right: temporal
change in the brightness (blinking) of sperm heads. The blue lines correspond to the time-points of the single frames. Scale bar = 25 lm.
D Representative distribution of rotation frequencies of freely swimming sperm incubated under non-capacitating (0mM bicarbonate; black; n=218) and capacitating
(25 mM bicarbonate; red; n=232) conditions determined by dark-field imaging.
E Rolling frequency (mean SD) of sperm incubated under non-capacitating (0mM bicarbonate, n=1,455; three experiments) and capacitating (25 mM bicarbonate,
n=1,097, eight experiments) conditions.
F Rolling frequency (mean SD) of freely swimming sperm in 0(n=1,175; five experiments), 0.2(n=832; three experiments), and 1%(n=599; three experiments)
methyl cellulose (w/v).
G Experimental setup for the laser-based optical tweezer.
H Bright-field images of an optically trapped sperm cell obtained at t=0,55,155, and 185 ms. Scale bar represents 10 lm.
I Representative time-course of the rotation frequency of trapped sperm (each sperm cell is represented by a different color). Error bars indicate the full width at half
prominence of the frequency peaks determined by the fast Fourier analysis.
J Distribution of rotation frequencies in trapped sperm (n=32; mean frequency SD 6.02.1Hz).
K Image series of a sperm cell trapped parallel to the optical axis; images were obtained at t=0,95,165, and 205 ms. The red bar indicates the 360°rotation of the tip
of the head. Scale bar represents 10 lm.
L Representative distribution of rotation frequencies of freely swimming CatSper-deficient sperm incubated under non-capacitating (0mM bicarbonate; black; n=73)
and capacitating conditions (25 mM bicarbonate; red; n=272).
M Rolling frequency (mean SD) of freely swimming CATSPER2
/
sperm incubated under non-capacitating (0mM bicarbonate, n=1,009; four experiments) and
capacitating (25 mM bicarbonate, n=946; seven experiments) conditions.
N Rolling frequency (mean SD) of freely swimming CATSPER2
/
sperm in 0(n=457; four experiments), 0.2(n=389; two experiments), and 1%(n=187; two
experiments) methyl cellulose (w/v).
O Image series of a CATSPER2
/
sperm cell optically trapped perpendicular to the optical axis; images were obtained at t=0,75,150, and 225 ms. Scale bar
represents 10 lm.
P Image series of a CATSPER2
/
sperm cell optically trapped parallel to the optical axis; images were obtained at t=0,60,195, and 320 ms. The red bar indicates the
360°rotation of the tip of the head. Scale bar represents 10 lm.
Q Representative time courses of the rotation frequencies of optically trapped CATSPER2
/
sperm (each sperm is represented in a different color). Error bars indicate
the full width at half prominence of the frequency peaks determined by the fast Fourier analysis.
4of 15 The EMBO Journal e102363 |2020 ª2020 The Authors
The EMBO Journal Christian Schiffer et al
mean rotation frequency and the rotation frequency-histogram
(determined at ≤5 min in Ca
2+
-free buffer) were similar to those
under control conditions (6.3 1.9 Hz, n=224; Fig 3I). These
results show that Ca
2+
influx is not required for rolling of human
sperm.
CatSper-deficient human sperm display rheotaxis
Next, we studied the swimming behavior of human sperm in a glass
capillary with and without fluid flow. Sperm were tracked in the field
of view, and the starting point of each track was shifted to the origin
AC
B
D
I
L
OP Q
MN
JK
EF G H
Figure 2.
ª2020 The Authors The EMBO Journal e102363 |2020 5of 15
Christian Schiffer et al The EMBO Journal
of a coordinate system (Fig 4A, C, E, G, I, K). To quantify the rheo-
tactic behavior, we determined the angular swimming directions and
plotted the mean relative frequency of sperm swimming with
angular directions of 45°–135°, 135°–225°, 225°–315°, and 315°–45°
in a spider plot. Under no-flow conditions, control sperm swam
randomly without any preferred directional angle (Fig 4A and B). In
contrast, under flow conditions, a large fraction of sperm aligned
their swimming path against the flow direction (Fig 4C and D, angu-
lar direction of the flow =0°). The fraction of sperm swimming with
directional angles between 135°and 225°was 25.7 1.7% (n=7,
1,301 sperm) and 44.3 8.6% (n=7, 1,083 sperm) in the absence
and presence of a flow, respectively. For CatSper-deficient sperm
under no-flow conditions, the angular swimming directions were
random (Fig 4E and F; Movie EV9). Under flow conditions, like in
control sperm, a large fraction of the CatSper-deficient sperm aligned
their swimming path against the flow direction (Fig 4G and H; Movie
EV10). The fraction of CatSper-deficient sperm swimming with direc-
tional angles between 135°and 225°was 27.6 1.8% (no-flow;
n=6, 1,068 sperm) versus 47.8 3.4% (flow; n=7, 1,068 sperm).
These results demonstrate that functional CatSper channels are
dispensable not only for rolling, but also for rheotaxis of human
sperm.
A
DE
F
HI
G
BC
Dragging
Barrier stream
Barrier stream
Stimulus stream
Control stream
Barrier stream
Barrier stream
Figure 3. The action of bicarbonate and Ca
2+
on longitudinal rolling of human sperm.
A Experimental setup to subject optically trapped sperm to different conditions in a three-channel microfluidic capillary.
B Rotation frequency of a trapped sperm cell before and after dragging across the barrier stream. The green trace indicates the fluorescence of fluorescein included in
the barrier stream. Error bars indicate the full width at half prominence of the frequency peaks determined by the fast Fourier analysis.
C Paired plot of rotation frequencies of individual sperm cells before and after dragging across the barrier stream.
D Rotation frequency of a trapped sperm cell before and after dragging from the control stream containing 0mM bicarbonate into the stimulus stream containing
25 mM bicarbonate. Error bars indicate the full width at half prominence of the frequency peaks determined by the fast Fourier analysis.
E Paired plot of rotation frequencies of individual sperm at 0and 25 mM bicarbonate.
F Rotation frequency of a trapped sperm cell in the presence and absence of extracellular Ca
2+
. Error bars indicate the full width at half prominence of the frequency
peaks determined by the fast Fourier analysis.
G Paired plot of rotation frequencies in the presence and absence of extracellular Ca
2+
.
H Fraction of motile sperm (mean SD) in a sperm population incubated in the presence (black) and absence (at t=0) of extracellular Ca
2+
(red; n≥5).
I Distribution of rotation frequencies in populations of freely swimming sperm in the presence (black, n=335) and absence (red, n=224) of extracellular Ca
2+
.
6of 15 The EMBO Journal e102363 |2020 ª2020 The Authors
The EMBO Journal Christian Schiffer et al
Rheotaxis of human sperm does not require Ca
2+
influx
Finally, we studied the trajectories of CatSper-deficient sperm in
Ca
2+
-free buffer ([Ca
2+
]˂20 nM). Under no-flow conditions,
the angular swimming directions were random (Fig 4I and J).
Under flow conditions, like in the presence of extracellular Ca
2+
,
a large fraction of the CatSper-deficient sperm aligned their
swimming path against the flow direction (Fig 4K and L); in
Ca
2+
-free buffer, the fraction of CatSper-deficient sperm swim-
ming with directional angles between 135°and 225°was
28.2 2.7% (no-flow; n=4, 442 sperm) versus 43.3 3.6%
(flow; n=4, 620 sperm). These results demonstrate that Ca
2+
influx in general is dispensable for both rolling and rheotaxis of
human sperm and that rheotaxis of CatSper-deficient human
sperm is similar in the absence and presence of extracellular
Ca
2+
.
ABCD
EFGH
IJKL
Figure 4.
ª2020 The Authors The EMBO Journal e102363 |2020 7of 15
Christian Schiffer et al The EMBO Journal
Rolling and rheotaxis persist in CatSper-deficient mouse sperm
Although both devoid of functional CatSper channels, rolling and
rheotaxis are largely unaffected in human CATSPER2
/
sperm, but
seem to be abolished in mouse Catsper1
/
sperm (Miki & Clapham,
2013). A major difference is that the quadrilateral Ca
2+
-signaling
threads are disrupted in Catsper1
/
mouse sperm (Chung et al,
2014), but not in CATSPER2
/
human sperm (Fig 1G). This finding
suggests that the supramolecular CatSper organization might be
required for rolling and rheotaxis. To test for this possibility, we
re-examined rolling and rheotaxis in mouse Catsper1
/
sperm.
Surprisingly, not only wild-type (Fig 5A, Movie EV11) but also
Catsper1
/
sperm (Fig 5B, Movie EV12) clearly displayed longitudi-
nal rolling. The mean rotational frequency of wild-type and
Catsper1
/
sperm was 2.9 1.3 Hz (n=24) and 2.6 0.7 Hz
(n=24), respectively (Fig 5A and B). We studied the swimming
behavior of wild-type and Catsper1
/
mouse sperm in a glass capil-
lary with and without fluid flow. We tracked sperm in the field of
view, and the starting point of each track was shifted to the origin of
a coordinate system (Fig 5C, E, G, I). To quantify the rheotactic
behavior, we determined the angular swimming directions and
plotted the mean relative frequencies of sperm swimming with an
angular direction of 45°–135°, 135°–225°, 225°–315°, and 315°–45°
in a spider plot. Under no-flow conditions, wild-type mouse sperm
swam randomly without any preferred directional angle (Fig 5C and
D). In contrast, under flow conditions, a large fraction of sperm
aligned their swimming path against the flow direction (Fig 5E and F;
angular direction of the flow =0°). The fraction of sperm swimming
with directional angles between 135°and 225°was 25.6 5.9%
(n=3, 127 sperm) and 53.2 6.7% (n=3, 175 sperm) in the
absence and presence of flow, respectively (see also Movies EV13
and EV14). For Catsper1
/
mouse sperm under no-flow condi-
tions, the angular swimming directions were random (Fig 5G and
H, Movie EV15). Under flow conditions, like in wild-type sperm, a
large fraction of Catsper1
/
sperm aligned their swimming path
against the flow direction (Fig 5I and J, Movie EV16). The fraction
of Catsper1
/
sperm swimming with directional angles between
135°and 225°was 26.0 1.5% (no-flow; n=4, 297 sperm)
versus 44.2 10.9% (flow; n=4, 261 sperm), respectively. These
results demonstrate that also rolling and rheotaxis of mouse sperm
◀Figure 4. Rheotaxis of human sperm.
A Trajectories of human sperm in the absence of a fluid flow. Sperm were tracked for 1.86 s. The starting point of each trajectory was centered to the origin of a
coordinate system, represented by the intersection of the dotted lines in the center of the circle.
B Spider-web plot of the mean (SD) relative frequencies of sperm swimming with an angular direction of 315°–45°,45°–135°,135°–225°, and 225°–315°(n=7;1,301
sperm) in the absence of a fluid flow.
C Representative trajectories of human sperm in the presence of a fluid flow.
D Spider-web plot of the mean (SD) relative frequencies of angular swimming directions (n=7;1,083 sperm) in the presence of a fluid flow. The red arrow indicates
the flow direction.
E Representative trajectories of CATSPER2
/
sperm in the absence of a fluid flow.
F Spider-web plot of the mean (SD) relative frequencies of angular swimming directions of CATSPER2
/
sperm in the absence of a fluid flow (n=6;1,068 sperm).
G Trajectories of CATSPER2
/
sperm in the presence of a fluid flow.
H Spider-web plot of the mean (SD) relative frequencies of angular swimming directions of CATSPER2
/
sperm in the presence of a fluid flow (n=7;1,068 sperm).
The red arrow indicates the fluid flow direction.
I Representative trajectories of CATSPER2
/
sperm in Ca
2+
-free buffer in the absence of a fluid flow.
J Spider-web plot of the mean (SD) relative frequencies of angular swimming directions (n=4;442 sperm) of CATSPER2
/
sperm in Ca
2+
-free buffer in the absence
of a fluid flow.
K Representative trajectories of CATSPER2
/
sperm in Ca
2+
-free buffer in the presence of a fluid flow.
L Spider-web plot of the mean (SD) relative frequencies of angular swimming directions (n=4;620 sperm) of CATSPER2
/
sperm in Ca
2+
-free buffer in the presence
of a fluid flow. The red arrow indicates the flow direction.
▸
Figure 5. Rolling behavior and rheotaxis of mouse sperm.
A Left: Bright-field image series of a freely swimming wild-type sperm cell at t=0,151, and 303 ms. Scale bar represents 10 lm. Right: rotation frequency
(mean SD) of freely swimming wild-type sperm (n=24, three experiments).
B Left: Bright-field image series of a freely swimming Catsper1
/
sperm cell at t=0,151, and 294 ms. Scale bar represents 10 lm. Right: rotation frequency
(mean SD) of freely swimming Catsper1
/
sperm (n=24, three experiments).
C Representative trajectories of wild-type sperm in the absence of a fluid flow. The starting point of each trajectory was centered to the origin of a coordinate system,
represented by the intersection of the dotted lines in the center of the circle. Each color represents one trajectory.
D Spider-web plot of the mean (SD) relative frequencies of sperm swimming with an angular direction of (binning: 315°–45°,45°–135°,135°–225°, and 225°–315°
(n=3;127 sperm) in the absence of a fluid flow.
E Representative trajectories of wild-type sperm in the presence of a fluid flow.
F Spider-web plot of the mean (SD) relative frequencies of angular swimming directions (n=3;175 sperm) of wild-type sperm in the presence of a fluid flow. The
red arrow indicates the flow direction.
G Representative trajectories of Catsper1
/
sperm in the absence of a fluid flow. The starting point of each trajectory was centered to the origin of a coordinate
system, represented by the intersection of the dotted lines in the center of the circle. Trajectories are magnified by a factor of 2.05 with respect to the plots C and E
to compensate for the reduced swimming speed of the Catsper1
/
sperm.
H Spider-web plot of the mean (SD) relative frequencies of angular swimming directions of Catsper1
/
sperm (n=4;297 sperm) in the absence of a fluid flow.
I Representative trajectories of Catsper1
/
sperm in the presence of a fluid flow; trajectories are magnified by a factor of 2.05 with respect to the plots C and E to
compensate for the reduced swimming speed of the Catsper1
/
sperm, and two trajectories were truncated (indicated by two parallel lines).
J Spider-web plot of the mean (SD) relative frequencies of angular swimming directions (n=4;261 sperm) of Catsper1
/
sperm in the presence of a fluid flow. The
red arrow indicates the flow direction.
8of 15 The EMBO Journal e102363 |2020 ª2020 The Authors
The EMBO Journal Christian Schiffer et al
do not require functional CatSper channels. For the time being, we
cannot reconcile ours with previous results (Miki & Clapham,
2013). We suggest that other laboratories examine rolling and
rheotaxis of CatSper-deficient mouse sperm independently.
Discussion
The Ca
2+
channel CatSper has been implicated in rolling and
rheotactic steering of mammalian sperm (Miki & Clapham, 2013;
A
B
CD EF
GH I J
Figure 5.
ª2020 The Authors The EMBO Journal e102363 |2020 9of 15
Christian Schiffer et al The EMBO Journal
Miller et al, 2018). We show that, in fact, rolling and rheotaxis of
both human and mouse sperm do not require Ca
2+
influx via
CatSper. This conclusion agrees with other reports: Rolling of mouse
sperm does not require extracellular Ca
2+
(Babcock et al, 2014;
Muschol et al, 2018), and exposure of human sperm to gradients of
flow velocities does not evoke measurable changes in [Ca
2+
]
i
(Zhang et al, 2016). Furthermore, it has been proposed that the
quadrilateral organization of CatSper and associated signaling
components provides the flagellar ultrastructure required for rolling
and rheotaxis (Miller et al, 2018). The CatSper complex and the
quadrilateral Ca
2+
-signaling domains are abolished in Catsper1
/
mouse sperm (Chung et al, 2014), but not in human CATSPER2
/
sperm. Yet, both sperm species display rheotaxis and undergo
rolling, suggesting that also the quadrilateral flagellar architecture
along with potential asymmetric cytosolic Ca
2+
gradients estab-
lished by this structure is dispensable. Furthermore, mouse sperm
lack Hv1 channels (Lishko et al, 2010; Berger et al, 2017), indicating
that asymmetrical, spatially confined pH gradients established by
Hv1 are not required for rolling and rheotaxis. Altogether, our
results strongly support the concept that passive biomechanical and
hydrodynamic processes enable rolling and rheotaxis rather than
active spatio-temporally confined Ca
2+
and H
+
signaling.
On a broader perspective, rolling is not a unique feature of sperm
that undergo rheotaxis: Sperm from marine external fertilizers also
exhibit longitudinal rolling (Jikeli et al, 2015), although in their
aquatic habitat, no gradients of fluid velocity exist and chemotaxis
rather than rheotaxis is employed for navigation.
Of note, even in a viscous medium, rheotaxis of CatSper-deficient
and healthy human sperm was rather similar (Fig 6A and B). Thus,
at the particular flow velocity and viscosity that we used, and with
respect to the parameters that we analyzed, the lack of functional
CatSper does not seem to affect rheotaxis in more viscous fluid.
However, human sperm display rheotaxis within a broad range of
physiological flow velocities and viscosities (Kantsler et al, 2014).
The relation of upstream versus shear velocity is bell-shaped
(Kantsler et al, 2014), demonstrating that the rheotactic perfor-
mance peaks at a particular shear profile. Furthermore, the trajecto-
ries of human sperm swimming against a flow feature a transverse
component, and in cylindrical tubes, sperm swim on spiral-shaped
trajectories along the walls, thereby exploring the tube’s surface
(Kantsler et al, 2014). The transversal component is positively and
negatively related to the shear velocity and viscosity, respectively.
Thus, subtle differences in the 3D beat of CatSper-deficient sperm
might compromise rheotactic performance under some conditions
encountered in the oviduct. For example, rheotaxis might be
compromised at certain shear velocities and/or fluid viscosities, the
relationship of upstream versus shear velocity might be shifted,
and/or the transversal component might be altered. The beat
envelope is a critical determinant of the rheotactic performance:
Mouse sperm that lack CatSper fsuffer from a rather stiff proximal
flagellum, altering the 3D flagellar envelope and hamper the reorien-
tation in fluid flow (Chung et al, 2017). Future studies need to
quantify the 3D flagellar beating pattern of control versus CatSper-
deficient human sperm as well as their rheotactic performance over
a broad range of fluid flows and viscosities.
Furthermore, in human sperm, CatSper translates stimulation
with oviductal hormones, like steroids and prostaglandins (Lishko
et al, 2011; Stru
¨nker et al, 2011; Brenker et al, 2012, 2018a; Miller
et al, 2016; Mannowetz et al, 2017), into Ca
2+
and motility
responses that are important for human sperm chemotaxis and
hyperactivation (Schaefer et al, 1998; Harper et al, 2003; Oren-
Benaroya et al, 2008; Publicover et al, 2008; Baldi et al, 2009; Kilic
et al, 2009; Alasmari et al, 2013; Schiffer et al, 2014; Tamburrino
et al, 2014, 2015; Rennhack et al, 2018). In the absence and
ABC D
Figure 6. Rheotaxis of human sperm in viscous media and in the presence of progesterone.
A Spider-web plot of the mean (SD) relative frequencies of sperm swimming with an angular direction of 315°–45°,45°–135°,135°–225°, and 225°–315°(n=3;403
sperm) of human sperm swimming in buffer fortified with 0.2% (w/v) methyl cellulose in the presence of a fluid flow.
B Spider-web plot of the mean (SD) relative frequencies of angular swimming directions (n=3;520 sperm) of human CATSPER2
/
sperm swimming in buffer
fortified with 0.2% (w/v) methyl cellulose in the presence of a fluid flow.
C Spider-web plot of the mean (SD) relative frequencies of sperm swimming with an angular direction of 315°–45°,45°–135°,135°–225°, and 225°–315°(n=3;454
sperm) of human sperm swimming in buffer fortified with 100 nM progesterone in the presence of a fluid flow.
D Spider-web plot of the mean (SD) relative frequencies of angular swimming directions (n=3;372 sperm) of human CATSPER2
/
sperm swimming in buffer
fortified with 100 nM progesterone in the presence of a fluid flow. Data information: The red arrow indicates the flow direction.
10 of 15 The EMBO Journal e102363 |2020 ª2020 The Authors
The EMBO Journal Christian Schiffer et al
presence of progesterone (100 nM), rheotaxis of control and
CatSper-deficient human sperm was rather similar (Fig 6C and D),
indicating that under our conditions, progesterone activation of
CatSper does not affect rheotaxis. However, to decipher how the
ligand control of CatSper and, thereby, [Ca
2+
]
i
are intertwined with
rheotaxis, it is required to quantify rheotaxis of control and CatSper-
deficient human sperm in the absence and presence of pico- to
micromolar progesterone concentrations over a broad range of flow
velocities and viscosities that emulate in vitro the complex physico-
chemical landscape of the oviduct.
On a final note, it is unknown whether sub- or infertility in men
correlates with the failure of sperm to undergo rheotaxis. Studying
rheotaxis is technically demanding. Therefore, we propose to assess
longitudinal rolling as a surrogate biomarker for infertility. The
population analysis introduced in this study can be readily incorpo-
rated into existing computer-assisted sperm analysis setups for
clinical diagnostics.
Materials and Methods
Reagents
Reagents were obtained from Sigma-Aldrich (USA) unless otherwise
indicated.
Sperm preparation and buffer conditions
The studies were performed in accordance with the standards set by
the Declaration of Helsinki. Samples of human semen were obtained
from healthy volunteers and DIS patients with their prior written
consent, under approval of the institutional ethical committees of
the medical association Westfalen-Lippe and the medical faculty of
the University of Mu
¨nster (4INie). Ejaculates were allowed to
liquefy at 37°C for 30–60 min. Motile sperm were purified by a
“swim-up” procedure: Liquefied semen (0.5–1 ml) was layered in a
50-ml Falcon tube below 4 ml of human tubal fluid (HTF) medium
containing (in mM): 93.8 NaCl, 4.69 KCl, 0.2 MgSO
4
, 0.37 KH
2
PO
4
,
2.04 CaCl
2
, 0.33 Na-pyruvate, 21.4 lactic acid, 2.78 glucose, 4
NaHCO
3
, and 21 HEPES, pH 7.35 (adjusted with NaOH). Alterna-
tively, the semen was diluted 1:10 with HTF and sperm, somatic
cells, and cell debris were pelleted by centrifugation at 700 gfor
20 min (37°C). The pellet was resuspended in the same volume
HTF, 50-ml Falcon tubes were filled with 5 ml of the suspension,
and cells were pelleted by centrifugation at 700 gfor 5 min (37°C).
In either case, motile sperm were allowed to swim up into HTF for
60–90 min at 37°C. After swim-up, sperm were washed twice
(700 g, 20 min) with HTF, the sperm concentration was adjusted,
and HTF was supplemented with 3 mg/ml human serum albumin
(HSA, Scientific Irvine, USA; referred to as HTF
+
); under these
conditions, sperm are non-capacitated. For capacitation, sperm were
resuspended after the second wash in HTF
++
medium, containing
(in mM): 72.8 NaCl, 4.69 KCl, 0.2 MgSO
4
, 0.37 KH
2
PO
4
, 2.04 CaCl
2
,
0.33 Na-pyruvate, 21.4 lactic acid, 2.78 glucose, 25 NaHCO
3
, and 21
HEPES, pH 7.35 (adjusted with NaOH), and supplemented with
3 mg/ml HSA. Sperm were capacitated in HTF
++
for at least 3 h.
Alternatively, swim-up and washing were directly performed in
HTF
++
. To study non-capacitated sperm in the absence of
bicarbonate and the motility response evoked by a step increase in
bicarbonate, swim-up and washing were performed in bicarbonate-
free HTF containing (in mM): 97.8 NaCl, 4.69 KCl, 0.2 MgSO
4
, 0.37
KH
2
PO
4
, 2.04 CaCl
2
, 0.33 Na-pyruvate, 21.4 lactic acid, 2.78
glucose, and 21 HEPES, pH 7.35 (adjusted with NaOH). HSA (3 mg/
ml) was added prior to the experiment to prevent attaching of sperm
to the surface of the recording chamber. C57BL/6 wild-type and
Catsper1
/
mice were handled and sacrificed in accordance with
the German Animal Welfare Act and the district veterinary office
under approval by the LANUV (84-02.05.20.13.115). Mouse epidi-
dymis was obtained from at least 21-week-old male mice that were
anaesthetized with CO
2
or isoflurane (AbbVie Deutschland, Ludwig-
shafen, Germany) and sacrificed by cervical dislocation. Mouse
sperm were isolated by incision of the cauda epididymis in modified
TYH medium containing (in mM): 138 NaCl, 4.8 KCl, 2 CaCl
2
, 1.2
KH
2
PO
4
, 1 MgSO
4
, 5.6 glucose, 0.5 sodium pyruvate, 10 sodium
DL-lactate, and 10 HEPES, pH 7.4. Sperm were allowed to swim out
for 30 min at 30°C and 10% CO
2
.Catsper1
/
mice (Ren et al,
2001) were generously provided by David Clapham (Janelia
Research Campus, USA).
Rolling analysis in sperm populations
The longitudinal rolling of human sperm was recorded in glass
chambers (depth of ~100 lm) under an inverted microscope
(IX73; Olympus, Germany), equipped with a condenser (IX2-
LWUCD; Olympus, Germany) with a custom-made dark-field filter,
a10×objective (UPLFLN10X2PH1; Olympus, Germany), and addi-
tional 1.6×magnification lenses (16×final magnification). The
samples were illuminated with a red light-emitting diode (LED;
M660D2; Thorlabs, Germany) and a custom-made power supply.
To study sperm rolling in bicarbonate-free HTF or HTF
++
, sperm
were diluted 1:9 in the respective buffer 5–30 min prior to the
experiment. To study rolling in the absence of extracellular Ca
2+
([Ca
2+
]
o
˂20 nM), sperm in HTF
++
were diluted 1:9 only prior
to the experiment into a Ca
2+
-free HTF medium (HTF
0Ca
), contain-
ing (in mM): 69.8 NaCl, 4.69 KCl, 0.2 MgSO
4
, 0.37 KH
2
PO
4
,5
EGTA, 0.33 Na-pyruvate, 21.4 lactic acid, 2.78 glucose, 25
NaHCO
3
, and 21 HEPES, pH 7.35 (adjusted with NaOH), and
supplemented with 3 mg/ml HSA. To study sperm rolling in
viscous medium, sperm in HTF
+
were diluted in HTF
+
fortified
with methyl cellulose. Over 4–10 min, short movies (~725 ms) of
sperm in different fields of view in the observation chamber were
recorded at 124 Hz with a high-speed sCMOS camera (Zyla, 4.2
plus, Andor, UK). Longitudinal rolling was assessed with a
custom-made program written in the ImageJ macro language (Ras-
band, 1997–2016). In brief, moving sperm heads were tracked, and
rotation was monitored by an oscillating change in head bright-
ness. The rotation frequency was computed from the average
temporal distance between two intensity peaks. The sperm head is
approximately plane symmetrical with planes intersecting the
length axis and, therefore, lights up twice per 360°rotation. Thus,
the rotation frequency (F
Rot
) is given by F
Rot
=F
Blink
×0.5. Sperm
that displayed less than three relative maxima within the observa-
tion time were excluded from the analysis, yielding a cut-off for
F
Rot
of about 1.5 Hz; immotile sperm were excluded from the anal-
ysis. The rolling of mouse sperm in TYH was studied in observa-
tion chambers (depth of ~400 lm; Ibidi, Germany) under an
ª2020 The Authors The EMBO Journal e102363 |2020 11 of 15
Christian Schiffer et al The EMBO Journal
inverted microscope (IX73; Olympus, Germany) equipped with a
condenser (IX2-LWUCD; Olympus, Germany), a 10×objective
(UPLFLN10X2PH1, Olympus, Germany), and, optionally, an addi-
tional 1.6×magnification lens. The sample was illuminated by a
red LED (M660D2; Thorlabs, Germany). Movies of sperm were
recorded with a high-speed sCMOS camera (Zyla, 4.2 plus; Andor,
UK). The rolling frequency of sperm was determined by visual
frame-by-frame analysis.
Optical trapping of sperm
Unless otherwise indicated, we used capacitated sperm in HTF
++
for optical-trapping experiments. The trapping of sperm cells was
achieved with an optical tweezer (Ashkin et al, 1986; Fig EV2),
using a continuous-wave (cw) diode laser (Lumics LU0975M500,
Germany) at a wavelength of 976 nm (red beam in Fig EV2). The
laser beam was expanded by a telescope setup of two lenses (L) to a
diameter of 1.7 mm (full width at half maximum) and directed into
a 100×oil immersion objective (MO, Plan-Apochromatic 100×/1.40
Oil DIC M27, Zeiss, Germany) with a numerical aperture (NA) of
1.4, allowing for tight focusing of the laser beam onto the head of a
sperm swimming inside an observation capillary. To assess the rota-
tion frequency of trapped sperm, the laser light reflected by the
sperm head into the objective was directed onto a photomultiplier
tube (PMT, H10721-20, Hamamatsu, Japan) by a beam splitter (re-
flectivity ~4%). A long-pass filter in front of the PMT blocked both
the bright-field illumination and ambient light. The PMT signal was
sampled with a frequency of 2 kHz, and every 10 points were aver-
aged. The rotation frequency was determined by a fast Fourier
transformation in a moving time window of 1.5 s. In parallel to the
quantification of the back-reflected laser light, we recorded the
trapped sperm with a bright-field microscope. A blue LED, equipped
with a 450-nm short-pass filter (SP) and a collimator lens, served as
a light source (blue beam in Fig EV2). The bright-field image was
reflected by a dichroic mirror (DM), projected by a lens onto the
chip of a charge-coupled device (CCD) camera (UI-3140CP-M-GL,
IDS, Germany) and recorded with a frame rate of 200 Hz. To
measure the rotation frequency of a sperm at different conditions,
we trapped sperm inside a microfluidic capillary (dimensions
[height ×width]: 0.4 ×0.33 lm; l-Slide III 3in1; Ibidi, Germany)
with three separate inlets to establish a continuous, parallel laminar
flow of three solutions ((i) control stream with sperm, (ii) barrier
stream, and (iii) stimulus stream) with a flow speed of 65 lm/s.
The barrier stream was supplemented with fluorescein (1 lM).
Fluorescein was excited with the blue LED; fluorescence light was
collected through the microscope objective and reflected by two
DMs through a long-pass filter onto a second PMT (H10721-210;
Hamamatsu, Japan). Using a custom-built mechanical scanning
table, the microfluidic capillary was moved in the horizontal plane
orthogonal to the flow direction, dragging a trapped sperm within
6.8 s from the control stream through the barrier stream into the
stimulus stream. The fluorescein fluorescence, recorded synchro-
nously to the back-reflected laser light and the bright-field images,
provided a readout of the position of the trapped sperm inside the
flow profile. For control experiments, the control stream with
sperm, the barrier stream, and the stimulus stream consisted of
HTF
++
. To study the action of Ca
2+
, the stimulus stream consisted
of HTF
0Ca
. To study the action of bicarbonate, the control stream
with sperm consisted of bicarbonate-free HTF. For paired-plot analy-
sis, the change in frequency was determined after reaching a stable
value.
Rheotaxis assay
Human sperm in HTF
+
, in HTF
+
containing 100 nM progesterone,
or in HTF
+
containing 0.2% methylcellulose, or mouse sperm in
TYH were observed in shallow microfluidic channels with rectangu-
lar cross section of 0.4 ×3.8 mm (Ibidi, Germany) under an
inverted microscope (IX73; Olympus, Germany) equipped with a
condenser (IX2-LWUCD; Olympus, Germany) and a 10×objective
(UPLFLN1X2PH; Olympus, Germany). The sample was illuminated
by a red LED (M660D2; Thorlabs, Germany). Images were collected
at ~80 to ~125 Hz using a sCMOS camera (Zyla 4.2 Plus; Andor,
UK). Sperm were exposed to a buffer flow of ~13.5 ll/min (human)
or ~10 ll/min (mouse), respectively, using a syringe pump (World
Precision Instruments, USA). Individual human sperm were tracked
over 1.86 s in a semi-automatic fashion using a custom-made track-
ing tool based on the Mtrack2 plugin for ImageJ (NHI, Bethesda,
USA). The angle of each track was defined by its start and end point
in a two-dimensional Cartesian coordinate system with the flow
direction pointing to an angle of 0°and a trajectory straight against
the flow pointing to an angle of 180°. Immotile and surface-attached
sperm were excluded from analysis. For each experiment, computed
trajectory angles were binned into angle intervals of 90°and
expressed as fractions of the sperm population (e.g., Fig 4B). Indi-
vidual mouse sperm were tracked manually for 2 s; the angle of
each track was defined by its start and end point in a two-dimen-
sional Cartesian coordinate system with the flow direction pointing
to an angle of 0°and a trajectory straight against the flow pointing
to an angle of 180°. Like for human sperm, immotile and surface-
attached sperm were excluded from analysis. For each experiment,
computed trajectory angles were binned into angle intervals of 90°
and expressed as fractions of the sperm population (e.g., Fig 4D).
Measurement of changes in [Ca
2+
]
i
Changes in [Ca
2+
]
i
were measured in sperm (in HTF
+
) loaded with
the fluorescent Ca
2+
indicator Fluo-4-AM at 30°C in 384 multi-well
plates in a fluorescence plate reader (Fluostar Omega, BMG
Labtech, Ortenberg, Germany) at 30°C as described before (Schiffer
et al, 2014; Brenker et al, 2018b). Briefly, sperm were loaded with
Fluo-4-AM (5 lM, 20 min) at 37°C in the presence of Pluronic F-
127 (0.05% w/v). After incubation, excess dye was removed by
centrifugation (700 g, 5 min, room temperature). Sperm were resus-
pended in HTF at a density of 5 ×10
6
cells/ml. The wells were
filled with 54 ll of the sperm suspension; fluorescence was excited
at 480 nm (Fluo-4), and fluorescence emission was recorded at
520 nm. Changes in Fluo-4 fluorescence are depicted as DF/F
0
(%),
that is, the change in fluorescence (DF) relative to the mean basal
fluorescence (F
0
) before application of buffer or stimuli (6 ll).
Electrophysiology
We recorded from sperm in the whole-cell configuration as
described before (Stru
¨nker et al, 2011). Seals between pipette and
sperm were formed either at the cytoplasmic droplet or in the neck
12 of 15 The EMBO Journal e102363 |2020 ª2020 The Authors
The EMBO Journal Christian Schiffer et al
region in standard extracellular solution (HS) containing (in mM):
135 NaCl, 5 KCl, 1 MgSO
4
, 2 CaCl
2
, 5 glucose, 1 Na-pyruvate, 10
lactic acid, and 20 HEPES, adjusted to pH 7.4 with NaOH. Monova-
lent currents were recorded in a sodium-based divalent-free solution
(NaDVF) containing (in mM): 140 NaCl, 40 HEPES, and 1 EGTA,
adjusted to pH 7.4 with NaOH; the pipette (10–15 MΩ) solution
contained (in mM): 130 Cs-aspartate, 5 CsCl, 50 HEPES, and 5
EGTA, adjusted to pH 7.3 with CsOH. Data were not corrected for
liquid junction potentials.
Immunocytochemistry
Sperm were immobilized on microscope slides and fixed for 10 min
with paraformaldehyde in PBS (4%; PBS containing [in mM]: 137
NaCl, 2.7 KCl, 10 Na
2
HPO
4
, 1.8 KH
2
PO
4
, pH 7.4–7.5). To block
unspecific binding sites, sperm were incubated for 1 h with blocking
buffer (0.5% Triton-X 100 and 5% ChemiBLOCKER [Millipore,
USA] in 0.1 M PBS, pH 7.4). Primary antibodies (anti-CatSper 4,
ACC-304, Alomone Labs, Israel; polyclonal antibody raised in
rabbits, directed against amino acids 384–402 of CatSper 3) were
diluted in blocking buffer and incubated overnight. Fluorescent
secondary antibodies were diluted in blocking buffer containing
0.5 mg/ml DAPI (Invitrogen, USA), and pictures were taken with a
confocal microscope (FV1000; Olympus, Japan).
3D-STORM microscopy
Experiments were performed with a Ti-E microscope (Nikon, Japan)
in an imaging buffer (50 mM Tris, pH 8, 10 mM NaCl) with an
oxygen scavenging system (0.5 mg/ml glucose oxidase, 40 lg/ml
catalase [Roche Applied Science, Germany or Sigma-Aldrich], and
10% [w/v] glucose), and 10 mM 2-aminoethanethiol. Images were
acquired with an iXON 897 EMCCD camera (Andor, UK). 10,000–
60,000 frames were acquired per data set using a 647-nm excitation
laser at 100 mW at the sample plane, unless mentioned otherwise.
A 405-nm laser was used to maintain an adequate number of local-
izations per frame. For 3D STORM acquisition, a cylindrical lens
was introduced into the detection path; the “perfect focus system”
(Nikon) and a vibration isolation table were used to minimize axial
and lateral drifting, respectively. STORM movies were analyzed as
described previously using the Nikon software package based on a
technology developed by Dr. Xiaowei Zhuang (Huang et al, 2008).
Briefly, fluorescence peaks corresponding to individual molecules
were identified in each frame and fit, using least-squares fitting or
maximum-likelihood estimator fitting, with a two-dimensional
Gaussian to determine the (x,y) position of each molecule. For 3D
imaging, the ellipticity of the Gaussian was used to assign the z
coordinate. Drift correction was applied using cross-correlation.
STORM images were rendered with each localization plotted as a
Gaussian whose width is weighted by the inverse square root of the
number of detected photons for that switching event. Images were
filtered to reject molecules with emitted photon number below 500.
Molecules with an aspect ratio higher than 1.5 for 2D and 2.5 for 3D
datasets were rejected. Moreover, molecules that appeared for more
than 10 consecutive frames were rejected. Background noise in
STORM images caused by non-specifically bound antibodies,
appearing as scattered localizations at low local densities, was
removed by a local density filter. Low-density localizations were
filtered out by removing a localization if it was surrounded by fewer
than 10 localizations in the 80 nm ×80 nm region surrounding the
localization.
Array CGH analysis
Patients were analyzed by array comparative genomic hybridization
(CGH; Agilent platform, Agilent Technologies, Santa Clara, Califor-
nia, USA) using 400k arrays (#G4448A). For details, see Tu
¨ttelmann
et al (2011).
Statistical methods
Unless otherwise indicated, data are displayed as mean SD.
Expanded View for this article is available online.
Acknowledgements
This work was supported by the German Research Foundation (CRU326 to T.S.
and F.T.) and the Cells-in-Motion (CiM) Cluster of Excellence, Münster
(FF-2016-17 to T.S. and C.F.).
Author contributions
CB, CF, and TS conceived the project. CS, SR, CB, SY, HH, DW, FT, AR, UBK, TW,
AW, CK, SK, CF, and TS designed research, performed experiments, acquired,
analyzed, and/or interpreted data. CS and TS wrote the manuscript. All authors
revised the manuscript critically for important intellectual content and
approved the manuscript.
Conflict of interest
C.B., C.S., and T.S. filed a patent entitled “Method for assessing the fertilizing
potential of sperm based on longitudinal axis rotation”EP 19 191 395.3
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