of 18
Mitochondrial fission factor (
Mff
) is required for organization of
the mitochondrial sheath in spermatids
Grigor Varuzhanyan
1
,
Hsiuchen Chen
1
,
Rebecca Rojansky
1
,
Mark S Ladinsky
1
,
J. Michael
McCaffery
2
,
David C. Chan
1,*
1
Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA
91125, USA.
2
Integrated Imaging Center, Department of Biology, Johns Hopkins University, Baltimore, MD
21218.
Abstract
Background:
Mitochondrial fission counterbalances fusion to maintain organelle morphology,
but its role during development remains poorly characterized. Mammalian spermatogenesis is a
complex developmental process involving several drastic changes to mitochondrial shape and
organization. Mitochondria are generally small and spherical in spermatogonia, elongate during
meiosis, and fragment in haploid round spermatids. Near the end of spermatid maturation, small
mitochondrial spheres line the axoneme, elongate, and tightly wrap around the midpiece to form
the mitochondrial sheath, which is critical for fueling flagellar movements. It remains unclear how
these changes in mitochondrial morphology are regulated and how they affect sperm development.
Methods:
We used genetic ablation of
Mff
(mitochondrial fission factor) in mice to investigate
the role of mitochondrial fission during mammalian spermatogenesis.
Results:
Our analysis indicates that
Mff
is required for mitochondrial fragmentation in haploid
round spermatids and for organizing mitochondria in the midpiece in elongating spermatids. In
Mff
mutant mice, round spermatids have aberrantly elongated mitochondria that often show
central constrictions, suggestive of failed fission events. In elongating spermatids and
spermatozoa, mitochondrial sheaths are disjointed, containing swollen mitochondria with large
gaps between organelles. These mitochondrial abnormalities in
Mff
mutant sperm are associated
*
Correspondence: dchan@caltech.edu.
Credit author statement
Grigor Varuzhanyan: Conceptualization, Investigation, Writing – original draft
Hsiuchen Chen: Investigation, Writing – Review & Editing
Rebecca Rojansky: Investigation
Mark S Ladinsky: Investigation
J. Michael McCaffery: Investigation
David C. Chan: Conceptualization, Supervision, Writing – Review & Editing, Funding acquisition
Competing interests
The authors declare no competing interests.
Publisher's Disclaimer:
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our
customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of
the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered
which could affect the content, and all legal disclaimers that apply to the journal pertain.
HHS Public Access
Author manuscript
Biochim Biophys Acta Gen Subj
. Author manuscript; available in PMC 2022 May 01.
Published in final edited form as:
Biochim Biophys Acta Gen Subj
. 2021 May ; 1865(5): 129845. doi:10.1016/j.bbagen.2021.129845.
Author Manuscript
Author Manuscript
Author Manuscript
Author Manuscript
with reduced respiratory chain Complex IV activity, aberrant sperm morphology and motility, and
reduced fertility.
Conclusions:
Mff
is required for organization of the mitochondrial sheath in mouse sperm.
General Significance:
Mitochondrial fission plays an important role in regulating
mitochondrial organization during a complex developmental process.
Keywords
mitochondrial fission; spermatogenesis; mitochondrial sheath
Introduction
The balance between mitochondrial fusion and fission regulates mitochondrial morphology
and cell metabolism [
1
,
2
]. For some cell type, this balance maintains mitochondrial size,
shape, and number in accordance with cell physiology. In the long processes of neurons, for
example, mitochondria are maintained at a small size compatible with efficient transport
along the long distances from the cell body to the nerve terminal [
3
]. However, the role of
mitochondrial dynamics in regulating mitochondrial organization and distribution during
development is less well understood.
Mitochondrial fission is a multistep process involving several cellular factors. In the initial
phase, the endoplasmic reticulum (ER) constricts the mitochondrion with help from actin
filaments [
4
,
5
]. Next, receptors on the mitochondrial outer membrane recruit DRP1
(Dynamin-related protein 1), which oligomerizes into a ring-like structure on the
mitochondrial surface to further constrict and sever the mitochondrion. In mammals, DRP1
can be recruited by four different receptors: MFF (Mitochondrial fission factor), MID49
(Mitochondrial dynamics protein of 49 kDa), MID51 (Mitochondrial dynamics protein of 51
kDa), and FIS1 (Mitochondrial fission 1 protein), with the latter having only a minor role
[
6
8
]. DNM2 was reported to mediate the final step in fission following DRP1 constriction
[
9
], but this notion has been challenged [
10
,
11
]. Additionally, recent reports showed that
mitochondrial contacts with lysosomes [
12
] and Golgi-derived vesicles [
13
] facilitate
mitochondrial fission.
The molecular mechanisms that orchestrate mitochondrial fission have been mostly
deciphered using cultured cells and the role of fission during development has remained
largely unexplored. To this end, we explored the role of mitochondrial fission during the
development of the male germline (spermatogenesis) in mice. Spermatogenesis is a highly
complex differentiation process associated with several mitochondrial transformations [
14
].
This lengthy process can be divided into three major developmental programs: 1) mitotic
amplification of spermatogonia, 2) meiotic division of spermatocytes to form haploid
spermatids, and 3) morphological transformation of round spermatids into mature sperm, a
process termed spermiogenesis [
15
]. During these developmental transitions, mitochondria
undergo dramatic changes in morphology, distribution, and number [
16
]. Mitochondria are
generally small and fragmented in spermatogonia, elongate and cluster around the nuage
during meiosis, and fragment again in post-meiotic spermatids. Near the end of spermatid
Varuzhanyan et al.
Page 2
Biochim Biophys Acta Gen Subj
. Author manuscript; available in PMC 2022 May 01.
Author Manuscript
Author Manuscript
Author Manuscript
Author Manuscript
maturation, small spherical mitochondria line up longitudinally on the axoneme of the
midpiece[
17
]. These mitochondria elongate and tightly wrap around the axoneme in a
coordinated manner to organize into a compact mitochondrial sheath that fuels sperm
motility [
17
]. The molecular factors that drive these coordinated mitochondrial
rearrangements are unknown.
Because mammalian spermatogenesis involves drastic changes to mitochondrial morphology
and organization, it is a promising model for studying the role of developmentally regulated
alterations to mitochondrial dynamics. Indeed, studies of mitofusins-deficient mice indicate
that mitochondrial fusion is important for maintaining OXPHOS activity in differentiating
spermatogonia and meiotic spermatocytes [
18
,
19
]. However, the role of mitochondrial
fission during spermatogenesis remained largely unknown.
Drp1
knockout mice are
embryonic lethal [
20
], and to our knowledge, a male germline-specific deletion of
Drp1
has
not yet been made. We previously reported that mice homozygous for a gene-trap allele of
Mff
(
Mff
gt
) have reduced fertility and sperm count [
21
]. Therefore,
Mff
gt
mice provide a
model system to decipher the role of mitochondrial fission during male germline
development. Our analysis suggests that mitochondrial fission is required during
spermiogenesis for proper formation of the mitochondrial sheath.
Results and Discussion
Mff
gt
sperm have disjointed mitochondrial sheaths.
To visualize male germ cells within the seminiferous epithelium, we performed Periodic–
acid Schiff staining in wild type (WT) and
Mff
gt
testis sections (Figure 1).
Mff
gt
testes do
not exhibit any obvious degeneration of seminiferous tubules, and all major germ cell types
—spermatogonia (SG), spermatocytes (SC), spermatids (ST), and spermatozoa (SZ)—are
present, indicating that loss of
Mff
does not impair generation of any one cell type. To
examine the ultrastructure of
Mff
gt
sperm, we isolated sperm from caudal epididymides and
subjected them to scanning electron microscopy (SEM).
Mff
gt
sperm often had
morphological abnormalities in the midpiece (Figure 2A, white arrows) and kinking in or
near the midpiece (Figure 2A, yellow arrow). Because the mitochondrial sheath is a major
component of the midpiece, we utilized a mitochondrially targeted Dendra2 (Dn) fluorescent
protein [
22
] to examine mitochondrial structure in sperm (Figures 2B and 2C). WT sperm
have abundant mitochondria tightly packed in the sperm midpiece with little or no gaps
between adjacent organelles. In contrast,
Mff
gt
sperm have disjointed mitochondrial sheaths
with gaps between adjacent organelles. Total mito-Dendra2 fluorescence is greatly reduced
in
Mff
gt
sperm (Figure 2C). In addition, imaging of sperm by differential interference
microscopy (DIC) showed prominent defects in overall sperm morphology (Figures 2D and
2E). Over 60% of
Mff
gt
sperm contain kinks, which are found in the midpiece (>15%), the
principal piece (>40%), or the neck (>5%).
Mitochondria in
Mff
gt
sperm are sparse and swollen.
To visualize the ultrastructure of sperm mitochondria, we performed electron tomography of
epididymal sections to generate three-dimensional renderings of mitochondrial sheaths
(Figures 3A and 3B, and Videos 1–4). When mitochondrial sheaths are visualized in
Varuzhanyan et al.
Page 3
Biochim Biophys Acta Gen Subj
. Author manuscript; available in PMC 2022 May 01.
Author Manuscript
Author Manuscript
Author Manuscript
Author Manuscript
longitudinal sections of WT sperm, thin, rod-like mitochondria can be seen tightly and
uniformly wrapping around the sperm axoneme (Figure 3A and Videos 1–2). In contrast,
mutant sperm have disjointed mitochondrial sheaths with great variation in mitochondrial
morphology. Mutant mitochondria appear highly swollen, have increased transverse
diameters, and are too sparse to pack into a uniform sheath. In transverse sections of sperm
midpieces, mutant mitochondria are also slightly elongated compared to control (Figure 3B
and Videos 3–4). These data suggest that in the absence of fission, aberrantly enlarged
mitochondria are poorly recruited to the sperm midpiece and/or fail to properly wrap around
the axoneme, resulting in disjointed mitochondrial sheaths. TEM analysis of spermatozoa in
testis sections showed similar defects (Figure S1), indicating that spermatozoa contain
defective mitochondria prior to their release into the epididymides.
Mff
gt
is required for developmentally regulated mitochondrial fragmentation in spermatids.
We next visualized mitochondrial morphology in early stage spermatids before they form
mitochondrial sheaths (Figure 4). Whereas round and elongating spermatids of WT mice
almost invariably contain fragmented mitochondria, the vast majority of mutant round and
elongating spermatids contain tubular mitochondria (Figures 4A–4C). To visualize
mitochondrial ultrastructure, we performed transmission electron microscopy (TEM) of
testis sections in WT and
Mff
gt
mice (Figure 4D–4F). Consistent with observations made
using light microscopy, we find that most round spermatid mitochondria are small and
fragmented. In contrast, more than 20% of mutant mitochondria exhibit an elongated and
highly constricted morphology, suggestive of a fission defect. Almost 80% of round
spermatids (31 out of 39) contained one or more constricted mitochondria (Figure 4F).
Mff
gt
sperm have reduced respiratory chain Complex IV activity, motility, and fertility.
To examine sperm mitochondrial function, we examined respiratory chain activity in isolated
sperm by histochemical analysis of respiratory chain Complex IV (cytochrome c oxidase;
COX) and succinate dehydrogenase (SDH) enzyme activity (Figures 5A and 5B). COX
activity is visualized by oxidation of 3,3’-Diaminobenzidine (DAB) by cytochrome
c
into a
brown product that can be visualized by light microscopy. SDH activity is visualized by
oxidation of succinate by SDH followed by reduction of Nitro blue tetrazolium chloride
(NBT), which forms a dark blue precipitate. In WT sperm midpieces, strong and uniform
COX and SDH staining indicated normal OXPHOS activity (Figure 5A; top and middle
panels). When COX and SDH stains are done simultaneously, only COX activity can be seen
because the DAB reaction product saturates the cell (Figure 5A; bottom panel). Midpiece
mitochondria in mutant sperm have a greater than 20% reduction in COX staining and an
almost 20% increase in NBT staining (Figures 5A and 5B). When COX and SDH are
monitored simultaneously in mutant sperm, blue coloring was evident due to decreased COX
staining and increased SDH staining. These results indicate reduced Complex IV activity in
Mff
gt
sperm. In other cell types with low COX/high SDH activity, such as mtDNA-deficient
skeletal muscle, increased SDH activity is correlated with increased mitochondrial mass.
However,
Mff
gt
sperm have reduced mitochondria mass (Figures 2B and 2C). Thus, the
cause of the increased NBT staining in
Mff
gt
sperm is unclear, but previous studies have
correlated increased NBT staining in spermatozoa with increased reactive oxygen species
Varuzhanyan et al.
Page 4
Biochim Biophys Acta Gen Subj
. Author manuscript; available in PMC 2022 May 01.
Author Manuscript
Author Manuscript
Author Manuscript
Author Manuscript
(ROS) [
23
,
24
]. Therefore, future studies should examine whether
Mff
gt
sperm have
increased ROS production.
Because reduced respiratory chain activity has been associated with reduced sperm motility
[
25
], we used mito-Dendra2 to track sperm motility by time-lapse microscopy (Videos 5–6).
Mutant sperm were significantly less motile compared to WT controls. Because
Mff
gt
mice
are runted and exhibit severe cardiomyopathy [
21
], it is important to distinguish whether
their reduced fertility is due to an inherent defect in sperm function or secondary to other
health problems. To this end, we performed
in vitro
fertilization using sperm from WT/Dn
and
Mff
gt
/Dn males with oocytes from WT females and tracked fertilization success by
progression to the 2-cell stage (Figures 5C and 5D). WT sperm successfully fertilized 64.1%
(21.5 out of 33.5) of WT oocytes. In contrast, mutant sperm failed to fertilize any oocytes (0
out of 35.75).
Conclusions
Recent studies have uncovered the importance of mitochondrial dynamics for male fertility
[
14
,
18
,
19
,
21
]. Mitochondria in stem and progenitor spermatogonia are sparse, small, and
spherical. However, as germ cells enter meiosis, mitochondria increase their numbers,
cluster, and undergo fusion, which promotes OXPHOS [
18
]. The increase in OXPHOS at
this time is likely necessary to drive the ATP-dependent processes associated with Meiotic
Prophase I [
26
]. Following meiosis, mitochondria return to a fragmented state, presumably
to facilitate transport and reorganization onto the sperm midpiece during spermatid
maturation. In mature spermatids, small mitochondrial spheres are arranged around the
sperm axoneme, elongate, and tightly wrap around the axoneme to form the mitochondrial
sheath [
17
], which enables sperm motility by fueling dynein motors [
27
].
The data presented here indicate that mitochondrial fission is acutely activated in post-
meiotic round spermatids, which have more fragmented mitochondria compared to earlier
meiotic spermatocytes. However, in
Mff
gt
round spermatids, mitochondria fail to fragment,
forming long, tubular mitochondria with striking constrictions that may represent trapped
fission intermediates. Based on the disordered structure of mitochondrial sheaths in
Mff
gt
sperm, we suggest that fission is important for generating small mitochondrial fragments
that can more easily be organized around the sperm midpiece to form the mitochondrial
sheath. In
Mff
gt
mutants, it is likely that the aberrant organization of the mitochondrial
sheath, along with reduced respiratory chain Complex IV activity, contribute to their reduced
fertility.
Materials and methods
Generation of mice
All mouse experiments were approved by the California Institute of Technology Institutional
Animal Care and Use Committee. WT/Dn (
Mff
+/+
;
Rosa26
PhAM(+/excised)
) and
Mff
gt
/Dn
mice (
Mff
gt
;
Rosa26
PhAM(+/excised)
) were generating by crossing
Mff
+/+
and
Mff
gt
mice with
Rosa26
PhAM(excised/excised)
mice.
Mff
gt
mice, described previously [
21
], were maintained on
a 129P2/OlaHsd and C57Bl/6J background and are available at the Mutant Mouse Resource
Varuzhanyan et al.
Page 5
Biochim Biophys Acta Gen Subj
. Author manuscript; available in PMC 2022 May 01.
Author Manuscript
Author Manuscript
Author Manuscript
Author Manuscript
& Research Center (RRID: MMRRC_066700-UCD).
Rosa26
PhAM(excised/excised)
mice were
described previously [
22
] and are available at the Jackson Laboratory (#018397).
Periodic–acid Schiff (PAS) staining
Testes were fixed in Bouin’s fixative overnight at 4°C, dehydrated in a 30–90% ethanol
series, cleared in Xylenes, and embedded in paraffin. Tissue blocks were sectioned at 7 μm,
deparaffinized, and rehydrated before staining. Slides were incubated with 1% periodic acid
(Electron Microscopy Sciences (EMS); 19324–10) for 30 min at RT, washed in running
water for 5 min, then rinsed in deionized water. Slides were incubated with Schiff’s reagent
(EMS; 260582–05) for 30 min at RT and washed as described above before counterstaining
with Gill 2 Hematoxylin for 30 s at RT. Slides were washed in running water for 1 min,
dehydrated with ethanol, cleared with xylene, then mounted using Cytoseal XYL mounting
media (Thermo Fisher Scientific; 22–050-262).
Scanning electron microscopy (SEM)
Epididymides were minced in PBS and incubated at 37 ̊C for 15 minutes for sperm to swim
out. Sperm cells were pelleted with gentle centrifugation at 400
g
for 10 min at RT and
washed once in PBS. The samples were resuspended in sodium cacodylate and fixed with
3.0% formaldehyde and 1.5% glutaraldehyde in sodium cacodylate buffer (0.1M sodium
cacodylate containing 5 mM Ca
2+
and 2.5% Sucrose at pH 7.4). Samples were washed 3X in
sodium cacodylate buffer before proceeding to SEM. Cellulose-nitrate filter circles were
overlaid/activated with 2% glutaraldehyde for 30’ and subsequently washed three times with
ddH
2
O. Sperm were overlaid onto the cellulose-nitrate activated filters for 20’. Filters were
washed gently 1X in 100 mM cacodylate buffer and fixed in Palade’s OsO4 for 1 hr at 4°C
by immersion in the fixative. Filters were then washed in ddH
2
O and dehydrated through a
graded series of ethanol to 100%, followed by 3X washes of 100% ethanol. Filters were then
critical point dried in a Tousimis 795 critical point dryer, or washed 2X with
hexamethyldisilazane and allowed to dry slowly at room temperature under the hood. Filters
were mounted onto aluminum specimen stubs using carbon transfer tabs, grounded to the
stub with silver paste, and sputtered with Pt for one minute in an Anatech Hummer 6.2
sputter coater. Stubs were then observed in an FEI Quanta 200 ESEM at a 10KeV under
high vacuum.
Light microscopy and image processing
Confocal fluorescence images, videos, and differential interference contrast (DIC) images
were acquired using an inverted Zeiss LSM 710 confocal microscope with a 60X Plan-
Apochromat objective. For live-cell motility videos, cells were maintained at 37°C and 5%
CO
2
. Bright-field images for histology were acquired using an upright Nikon Eclipse Ni-E
fluorescence microscope equipped with a Ds-Ri2 camera and CFI Plan Apochromat Lambda
objectives. Z stacks were acquired, and all-in-focus images were created using the NIS
Elements Extended Depth of Focus plugin. All images were processed using ImageJ. All
image modifications were performed on entire images (no masking was used) and were
performed identically between genotypes.
Varuzhanyan et al.
Page 6
Biochim Biophys Acta Gen Subj
. Author manuscript; available in PMC 2022 May 01.
Author Manuscript
Author Manuscript
Author Manuscript
Author Manuscript
Transmission electron microscopy (TEM) of testis sections
Testes were dissected, cut at the poles and fixed for one hour at RT with 3% formaldehyde
and 1.5% glutaraldehyde in sodium cacodylate buffer (0.1M sodium cacodylate containing 5
mM Ca
2+
and 2.5% Sucrose at pH 7.4). Samples were washed 3X in sodium cacodylate
buffer. Samples were then postfixed in Palade’s OsO4, stained in Kellenberger uranyl
acetate, dehydrated through a graded series of EtOH, and flat embedded in EMBED 812
(EMS). 80-nm testis sections were prepared on an ultramicrotome (UCT; Leica), collected
onto 400 mesh high-transmission nickel grids, and poststained with lead citrate and uranyl
acetate. Images were collected with a transmission electron microscope (Tecnai 12; FEI)
operating at 100 kV and equipped with an Olympus Soft Imaging System (OSIS) digital
camera (Megaview III; Olympus).
3D Electron tomography of epididymal sperm
Caudal epididymides were dissected and immediately fixed with cold 3% glutaraldehyde,
1% paraformaldehyde, 5% sucrose in 0.1 M sodium cacodylate trihydrate. Pre-fixed pieces
of tissue were rinsed with fresh cacodylate buffer and placed into brass planchettes (Type A;
Ted Pella, Inc, Redding, CA) prefilled with 10% Ficoll in cacodylate buffer. Samples were
covered with the flat side of a Type-B brass planchette and rapidly frozen with a HPM-010
high-pressure freezing machine (Leica Microsystems, Vienna Austria). The frozen samples
were transferred under liquid nitrogen to cryotubes (Nunc) containing a frozen solution of
2.5% osmium tetroxide, 0.05% uranyl acetate in acetone. Tubes were loaded into an AFS-2
freeze-substitution machine (Leica Microsystems) and processed at −90°C for 72 hr,
warmed over 12 hr to −20°C, held at that temperature for 8 hr, then warmed to 4°C for 2 hr.
The fixative was removed, and the samples were rinsed 4x with cold acetone, and then were
infiltrated with Epon-Araldite resin (Electron Microscopy Sciences, Port Washington PA)
over 48 hr. Samples were flat-embedded between two Teflon-coated glass microscope slides,
and the resin polymerized at 60°C for 24–48 hr.
Flat-embedded epididymis samples were observed with a stereo dissecting microscope, and
appropriate regions were extracted with a microsurgical scalpel and glued to the tips of
plastic sectioning stubs. Semi-thick (400 nm) serial sections were cut with a UC6
ultramicrotome (Leica Microsystems) using a diamond knife (Diatome, Ltd. Switzerland).
Sections were placed on Formvar-coated copper-rhodium slot grids (Electron Microscopy
Sciences) and stained with 3% uranyl acetate and lead citrate. Gold beads (10 nm) were
placed on both surfaces of the grid to serve as fiducial markers for subsequent image
alignment. Grids were placed in a dual-axis tomography holder (Model 2040, E.A.
Fischione Instruments, Export PA) and imaged with a Tecnai TF30ST-FEG transmission
electron microscope (300 KeV) equipped with a 2k × 2 k CCD camera (XP1000; Gatan, Inc
Pleasanton CA). Tomographic tilt-series and large-area montaged overviews were acquired
automatically using the SerialEM software package (Mastronarde, 2005). For tomography,
samples were tilted + /− 64° and images collected at 1° intervals. The grid was then rotated
90° and a similar series taken about the orthogonal axis. Tomographic data was calculated,
analyzed and modeled using the IMOD software package (Kremer et al., 1996; Mastronarde,
2008) on MacPro computers (Apple, Inc, Cupertino, CA).
Varuzhanyan et al.
Page 7
Biochim Biophys Acta Gen Subj
. Author manuscript; available in PMC 2022 May 01.
Author Manuscript
Author Manuscript
Author Manuscript
Author Manuscript
COX/SDH Enzyme Histochemistry
COX/SDH labeling was performed as described previously [
28
], with minor modifications.
Briefly, sperm cells were isolated from the caudal epididymis as described above, placed
onto glass slides, and dried under a ventilated hood. Slides were stained with COX buffer for
25 min at RT in the dark, washed twice with dH
2
0 for 5 min, then stained with SDH buffer
at 37°C for 45 min in the dark. Slides were washed two more times with dH
2
0 and destained
using a 30%–90%–30% acetone gradient. After two additional washes in dH
2
0, slides were
mounted using Fluorogel.
Sperm motility
Cauda epididymides of
Mff
gt
and WT male littermates were dissected into 0.5 mL modified
Tris-buffered medium (mTBM) pre-warmed to 37°C and 5% CO
2
. mTBM was composed of
113.1 mM NaCl, 3 mM KCl, 7.5 mM CaCl
2
, 5 mM sodium pyruvate, 11 mM glucose, 1 mM
caffeine, and 20 mM Tris., as described before [
29
]. Sperm were released by mincing the
tissue with a 26-gauge needle and incubated at 37°C and 5% CO
2
for 15 minutes to allow
for swim out. After incubation, the tissue was removed, and the fluid was mixed gently and
collected into 1 mL Eppendorf tubes. Sperm were washed twice by gentle centrifugation at
833
g
for 5 minutes, resuspended in 0.5 mL mTBM, then incubated at 37°C and 5% CO
2
for
30 minutes for capacitation. Sperm were plated at 5×10
5
sperm/mL in Nunc Lab-Tek II
Chambered Coverglass slides (154852, Thermo), and videos were acquired as described
above.
In vitro fertilization
For oocyte and sperm collection and fertilization all media was pre-warmed to 37 ̊C at 95%
humidity, 5% CO
2
, 5% O
2
, and 90% N
2
. Ovarian stimulation was performed as described
previously [
30
]. Briefly, female C57BL/6J mice between 21 and 25 days of age were
injected intraperitoneally with 25 I.U. of PMSG (G-4877 Sigma) on day −2, followed by 5
I.U. of HCG (C-1063, Sigma) 48 hours later on day 0. Sperm were collected from the cauda
epididymides of WT/Dn or
Mff
gt
/Dn mice into 0.5 mL of Fertiup medium (KYD-002–05-
EX, Cosmo Bio USA) at 37 ̊C, counted with a hemocytometer, and capacitated at a
concentration of 2–4 × 10
6
cells/mL for 1.5–2 hr at 37 ̊C, 95% humidity, 5% CO
2
, 5% O
2
,
90% N
2
.
Primed females were anesthetized with Ketamine-Xylazine and oviducts were dissected into
0.5 mL of Cook’s IVF medium (K-RVFE-50, Cooks) to wash, then transferred to 0.15 mL
of Cook’s IVF medium overlaid with mineral oil (M-8410, Sigma) into which the cumulus
mass was released. Capacitated sperm were diluted to a final concentration of 1–2 × 10
5
sperm/mL in 0.2 mL CARD medium (KYD-003-EX, Cosmo Bio USA) overlaid with
mineral oil, into which the cumulus mass was then transferred. The sperm and ova were co-
incubated for 4–6 hours at 37 ̊C, 95% humidity, 5% CO
2
, 5% O
2
, and 90% N
2
, transferred
through four 0.15 mL washes of Cook’s IVF medium overlaid with mineral oil, then
cultured in 0.5 mL Cook’s IVF medium at 37 ̊C, 95% humidity, 5% CO
2
, 5% O
2
, 90% N
2
for 16–18 hr, at which point presumptive embryos were examined for progression to 2-cell
stage.
Varuzhanyan et al.
Page 8
Biochim Biophys Acta Gen Subj
. Author manuscript; available in PMC 2022 May 01.
Author Manuscript
Author Manuscript
Author Manuscript
Author Manuscript
Quantification
Quantification of total Dn fluorescence in sperm midpieces was performed on confocal
images in which the sperm midpiece had been cropped and straightened using ImageJ.
Integrated density (mean times area) values were plotted. Dn intensity plots in Figure 2B are
“column average plots” generated from entire midpieces that were straightened using
ImageJ. Quantification of sperm morphology was performed under the 100X objective of an
upright Nikon Eclipse Ni-E fluorescence microscope. Quantification of mitochondrial
morphology in round and elongating spermatids from WT/Dn and
Mff
gt
/Dn mice was by
done by scoring seminiferous tubule transverse sections that were circular and had an
obvious lumen. Seminiferous tubules in which the majority of round or elongating
spermatids contained tubular mitochondria were scored as “tubular”. All others were scored
as “fragmented”.
Replicates and statistical reporting
Pairwise comparisons were made using the Student’s
t
-test. When multiple pairwise
comparisons were made from the same dataset,
p
-values were adjusted using the Bonferroni
correction. Number of mice and replicates are indicated in figure legends. All outliers were
included in the analysis. All data are represented as mean ± SEM. **** indicates p≤0.0001;
*** indicates p≤0.001; ** indicates p≤0.01; * indicates p≤0.05.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
Acknowledgements
We thank all members of the Chan Lab for helpful discussions. We thank the Caltech Kavli Nanoscience Institute
for maintenance of the TF-30 electron microscope.
Funding
This work was supported by the National Institutes of Health (R35 GM127147). Grigor Varuzhanyan was supported
by a National Science Foundation Graduate Research Fellowship (DGE
1144469) and a National Institutes of
Health Cell and Molecular Biology Training Grant (GM07616T32). Mark S. Ladinsky was supported by the
National Institute of Allergy and Infectious Diseases (NIAID) (2 P50 AI150464) (awarded to Pamela J. Bjorkman,
Caltech).
References
[1]. Mishra P, Chan DC, Metabolic regulation of mitochondrial dynamics, J. Cell Biol. 212 (2016)
379–387. doi:10.1083/jcb.201511036. [PubMed: 26858267]
[2]. Chan DC, Mitochondrial Dynamics and Its Involvement in Disease, Annual Review of Pathology:
Mechanisms of Disease. 15 (2020) null. doi:10.1146/annurev-pathmechdis-012419-032711.
[3]. Schwarz TL, Mitochondrial trafficking in neurons, Cold Spring Harb Perspect Biol. 5 (2013).
doi:10.1101/cshperspect.a011304.
[4]. Friedman JR, Lackner LL, West M, DiBenedetto JR, Nunnari J, Voeltz GK, ER Tubules Mark
Sites of Mitochondrial Division, Science. 334 (2011) 358–362. doi:10.1126/science.1207385.
[PubMed: 21885730]
[5]. Korobova F, Ramabhadran V, Higgs HN, An actin-dependent step in mitochondrial fission
mediated by the ER-associated formin INF2, Science. 339 (2013) 464–467. [PubMed: 23349293]
Varuzhanyan et al.
Page 9
Biochim Biophys Acta Gen Subj
. Author manuscript; available in PMC 2022 May 01.
Author Manuscript
Author Manuscript
Author Manuscript
Author Manuscript
[6]. Chan DC, Fusion and Fission: Interlinked Processes Critical for Mitochondrial Health, Annual
Review of Genetics. 46 (2012) 265–287. doi:10.1146/annurev-genet-110410-132529.
[7]. Losón OC, Song Z, Chen H, Chan DC, Fis1, Mff, MiD49, and MiD51 mediate Drp1 recruitment
in mitochondrial fission, Mol. Biol. Cell. 24 (2013) 659–667. doi:10.1091/mbc.E12-10-0721.
[PubMed: 23283981]
[8]. Otera H, Wang C, Cleland MM, Setoguchi K, Yokota S, Youle RJ, Mihara K, Mff is an essential
factor for mitochondrial recruitment of Drp1 during mitochondrial fission in mammalian cellsMff
is essential for mitochondrial recruitment of Drp1, J Cell Biol. 191 (2010) 1141–1158.
doi:10.1083/jcb.201007152. [PubMed: 21149567]
[9]. Lee JE, Westrate LM, Wu H, Page C, Voeltz GK, Multiple dynamin family members collaborate to
drive mitochondrial division, Nature. 540 (2016) 139–143. doi:10.1038/nature20555. [PubMed:
27798601]
[10]. Fonseca TB, Sánchez-Guerrero Á, Milosevic I, Raimundo N, Mitochondrial fission requires
DRP1 but not dynamins, Nature. 570 (2019) E34–E42. doi:10.1038/s41586-019-1296-y.
[PubMed: 31217603]
[11]. Kamerkar SC, Kraus F, Sharpe AJ, Pucadyil TJ, Ryan MT, Dynamin-related protein 1 has
membrane constricting and severing abilities sufficient for mitochondrial and peroxisomal
fission, Nat Commun. 9 (2018) 1–15. doi:10.1038/s41467-018-07543-w. [PubMed: 29317637]
[12]. Wong YC, Ysselstein D, Krainc D, Mitochondria-lysosome contacts regulate mitochondrial
fission via RAB7 GTP hydrolysis, Nature. 554 (2018) 382–386. doi:10.1038/nature25486.
[PubMed: 29364868]
[13]. Nagashima S, Tábara L-C, Tilokani L, Paupe V, Anand H, Pogson JH, Zunino R, McBride HM,
Prudent J, Golgi-derived PI(4)P-containing vesicles drive late steps of mitochondrial division,
Science. 367 (2020) 1366–1371. doi:10.1126/science.aax6089. [PubMed: 32193326]
[14]. Varuzhanyan G, Chan DC, Mitochondrial dynamics during spermatogenesis, J Cell Sci. 133
(2020). doi:10.1242/jcs.235937.
[15]. Griswold MD, Spermatogenesis: The Commitment to Meiosis, Physiological Reviews. 96 (2016)
1–17. doi:10.1152/physrev.00013.2015. [PubMed: 26537427]
[16]. De Martino C, Floridi A, Marcante ML, Malorni W, Scorza Barcellona P, Bellocci M, Silvestrini
B, Morphological, histochemical and biochemical studies on germ cell mitochondria of normal
rats, Cell and Tissue Research. 196 (1979) 1–22. [PubMed: 421242]
[17]. Ho H-C, Wey S, Three dimensional rendering of the mitochondrial sheath morphogenesis during
mouse spermiogenesis, Microscopy Research and Technique. 70 (2007) 719–723. [PubMed:
17457821]
[18]. Varuzhanyan G, Rojansky R, Sweredoski MJ, Graham RL, Hess S, Ladinsky MS, Chan DC,
Mitochondrial fusion is required for spermatogonial differentiation and meiosis, ELife. 8 (2019)
e51601. doi:10.7554/eLife.51601. [PubMed: 31596236]
[19]. Zhang J, Wang Q, Wang M, Jiang M, Wang Y, Sun Y, Wang J, Xie T, Tang C, Tang N, Song H,
Cui D, Chao R, Ding S, Ni B, Chen X, Wang Y, GASZ and mitofusin-mediated mitochondrial
functions are crucial for spermatogenesis, EMBO Reports. 17 (2016) 220–34. doi:10.15252/
embr.201540846. [PubMed: 26711429]
[20]. Wakabayashi J, Zhang Z, Wakabayashi N, Tamura Y, Fukaya M, Kensler TW, Iijima M, Sesaki
H, The dynamin-related GTPase Drp1 is required for embryonic and brain development in mice,
J. Cell Biol. 186 (2009) 805–816. doi:10.1083/jcb.200903065. [PubMed: 19752021]
[21]. Chen H, Ren S, Clish C, Jain M, Mootha V, McCaffery JM, Chan DC, Titration of mitochondrial
fusion rescues Mff-deficient cardiomyopathyCardiac physiology restored in Mff/Mfn1 mutants, J
Cell Biol. 211 (2015) 795–805. doi:10.1083/jcb.201507035. [PubMed: 26598616]
[22]. Pham AH, McCaffery JM, Chan DC, Mouse lines with photo-activatable mitochondria to study
mitochondrial dynamics, Genesis. 50 (2012) 833–843. doi:10.1002/dvg.22050. [PubMed:
22821887]
[23]. Baehner RL, Boxer LA, Davis J, The biochemical basis of nitroblue tetrazolium reduction in
normal human and chronic granulomatous disease polymorphonuclear leukocytes, (1976).
Varuzhanyan et al.
Page 10
Biochim Biophys Acta Gen Subj
. Author manuscript; available in PMC 2022 May 01.
Author Manuscript
Author Manuscript
Author Manuscript
Author Manuscript
[24]. Tunc O, Thompson J, Tremellen K, Development of the NBT assay as a marker of sperm
oxidative stress, International Journal of Andrology. 33 (2010) 13–21. doi:10.1111/
j.1365-2605.2008.00941.x. [PubMed: 19076253]
[25]. Ruiz-Pesini E, Diez C, Lapeña AC, Pérez-Martos A, Montoya J, Alvarez E, Arenas J, López-
Pérez MJ, Correlation of sperm motility with mitochondrial enzymatic activities, Clin Chem. 44
(1998) 1616–1620. doi:10.1093/clinchem/44.8.1616. [PubMed: 9702947]
[26]. Ranjha L, Howard SM, Cejka P, Main steps in DNA double-strand break repair: an introduction
to homologous recombination and related processes, Chromosoma. 127 (2018) 187–214.
doi:10.1007/s00412-017-0658-1. [PubMed: 29327130]
[27]. Serohijos AWR, Chen Y, Ding F, Elston TC, Dokholyan NV, A structural model reveals energy
transduction in dynein, PNAS. 103 (2006) 18540–18545. doi:10.1073/pnas.0602867103.
[PubMed: 17121997]
[28]. Ross JM, Visualization of mitochondrial respiratory function using cytochrome c oxidase/
succinate dehydrogenase (COX/SDH) double-labeling histochemistry, Journal of Visualized
Experiments : JoVE. (2011) e3266. doi:10.3791/3266. [PubMed: 22143245]
[29]. Park K-W, Niwa K, Bovine Oocytes Can Be Penetrated in Modified Tris-buffered Medium, Asian
Australas. J. Anim. Sci. 22 (2009) 500–506. doi:10.5713/ajas.2009.80431.
[30]. Lois C, Hong EJ, Pease S, Brown EJ, Baltimore D. Germline transmission and tissue-specific
expression of transgenes delivered by lentiviral vectors. Science 295 (2002) 868–872.
doi:10.1126/science.1067081 [PubMed: 11786607]
Varuzhanyan et al.
Page 11
Biochim Biophys Acta Gen Subj
. Author manuscript; available in PMC 2022 May 01.
Author Manuscript
Author Manuscript
Author Manuscript
Author Manuscript
Highlights
Mff
is required for mitochondrial fragmentation in post-meiotic spermatids.
Mff−/−
mitochondria have central constrictions, suggestive of failed fission
events.
Spermatozoa in
Mff
-deficient mice have discontinuous mitochondrial sheaths.
Mutant mitochondria have reduced respiratory chain Complex IV activity.
Mutant spermatozoa have aberrant morphology and reduced motility and
fertility.
Varuzhanyan et al.
Page 12
Biochim Biophys Acta Gen Subj
. Author manuscript; available in PMC 2022 May 01.
Author Manuscript
Author Manuscript
Author Manuscript
Author Manuscript
Figure 1. Histological analysis of seminiferous epithelium of
Mff
gt
mice.
Periodic-acid Schiff staining of Bouin’s-fixed testis sections. Note that all major germ cell
types are present in
Mff
gt
mice. SG, spermatogonium; SC, spermatocyte; ST, spermatid; SZ,
spermatozoa. Scale bars, 50 μm.
Varuzhanyan et al.
Page 13
Biochim Biophys Acta Gen Subj
. Author manuscript; available in PMC 2022 May 01.
Author Manuscript
Author Manuscript
Author Manuscript
Author Manuscript