RESEARCH ARTICLE
Fis1
ablation in the male germline disrupts mitochondrial
morphology and mitophagy, and arrests spermatid maturation
Grigor Varuzhanyan
1
, Mark S. Ladinsky
1
, Shun-ichi Yamashita
2
, Manabu Abe
3
, Kenji Sakimura
3
,
Tomotake Kanki
2
and David C. Chan
1,
*
ABSTRACT
Male germline development involves choreographed changes to
mitochondrial number, morphology and organization. Mitochondrial
reorganization during spermatogenesis was recently shown to
require mitochondrial fusion and fission. Mitophagy, the autophagic
degradation of mitochondria, is another mechanism for controlling
mitochondrial number and physiology, but its role during
spermatogenesis is largely unknown. During post-meiotic spermatid
development, restructuring of the mitochondrial network results
in packing of mitochondria into a tight array in the sperm midpiece
to fuel motility. Here, we show that disruption of mouse
Fis1
in
the male germline results in early spermatid arrest that is associated
with increased mitochondrial content. Mutant spermatids coalesce
into multinucleated giant cells that accumulate mitochondria of
aberrant ultrastructure and numerous mitophagic and autophagic
intermediates, suggesting a defect in mitophagy
.
We conclude that
Fis1
regulates mitochondrial morphology and turnover to promote
spermatid maturation.
KEY WORDS: Autophagy, Mitochondrial dynamics, Mitophagy,
Spermatid, Spermatogenesis, Mouse
INTRODUCTION
Male germline development (spermatogenesis) is one of biology
’
s
most complex developmental programs, transforming spermatogonial
stem cells into highly specialized sperm cells capable of
fertilization. Spermatogenesis requires successive cycles of germ
cell differentiation within the seminiferous epithelium. This tightly
controlled process is regulated by somatic Sertoli cells, which
intercalate with the germ cells and control their microenvironment
(Griswold, 2016). As spermatogonial stem cells differentiate into
sperm cells (spermatozoa), they progressively migrate from the
seminiferous tubule periphery towards the lumen. Spermatozoa are
then released into the lumen and transported to the epididymides
via ATP-dependent tubular contractions (Fleck et al., 2021).
Spermatogenesis is initiated by a pulse of retinoic acid that travels
like a wave along the length of a seminiferous tubule. As a result,
different regions along the longitudinal axis of a tubule are
in distinct developmental phases and display unique cellular
associations, referred to as
‘
stages
’
(Griswold, 2016; Russell
et al., 1993). The stages of the seminiferous epithelium are
defined by the developmental
‘
steps
’
of post-meiotic spermatids.
In mice, spermatid development is divided into 16 steps, which are
defined by the spermatid morphology as well as the extent of
acrosome maturation.
Spermatogenesis is generally divided into three broad categories:
(1) mitotic amplification of spermatogonia before they differentiate
into spermatocytes, (2) meiotic division of spermatocytes to
form haploid spermatids, and (3) maturation of spermatids into
spermatozoa
–
a process termed spermiogenesis. The unique
physiological requirements of these different germ cell types are
regulated by mitochondrial dynamics (fusion and fission)
(Varuzhanyan and Chan, 2020). In undifferentiated spermatogonia,
mitochondria are generally small and fragmented. As spermatogonia
differentiate into spermatocytes and initiate meiosis, their
mitochondria undergo mitofusin-mediated fusion to fuel meiosis
(Chen et al., 2020; Varuzhanyan et al., 2019; Wang et al., 2021;
Zhang et al., 2016). In post-meiotic spermatids, acute mitochondrial
fragmentation mediated by mitochondrial fission factor (MFF)
generates small mitochondrial spheres, which enables their
organization into a spiral array within the midpiece (Varuzhanyan
et al., 2021). At the end of spermatid maturation, excess cellular
components, including mitochondria, are culled into residual bodies
for heterophagic degradation in Sertoli cells (Chemes, 1986). Thus,
dynamic restructuring of mitochondria takes place throughout
spermatogenesis.
Although key roles for mitochondrial fusion and fission have
been demonstrated in spermatogenesis (Varuzhanyan and Chan,
2020), the role of mitophagy
–
the autophagic degradation of
mitochondria (Pickles et al., 2018)
–
is largely unknown (Lv et al.,
2020; Rathje et al., 2019). Because mitophagy counterbalances
mitochondrial biogenesis and can remove dysfunctional
mitochondria, it can control mitochondrial abundance and quality.
There is evidence that the autophagy pathway functions to eliminate
excess cellular material in spermatids during their transformation
into highly compacted sperm cells. Deletion of the core autophagy
gene
Atg7
has been shown to diminish autophagic flux in
spermatids, cause acrosome fragmentation (Wang et al., 2014),
and prevent spermatid polarization and cytoplasmic removal (Shang
et al., 2016). However, it remains unknown whether spermatids use
mitophagy to control mitochondrial density and remodeling.
The mitochondrial dynamics gene
Fis1
has been shown to
mediate mitochondrial fission in the budding yeast
Saccharomyces
cerevisiae
(Fekkes et al., 2000; Kraus et al., 2021; Mozdy et al.,
2000; Tieu and Nunnari, 2000). However, mammalian
Fis1
has little
(Losón et al., 2013) or no (Osellame et al., 2016; Otera et al., 2010)
Handling Editor: Swathi Arur
Received 6 April 2021; Accepted 13 July 2021
1
Division of Biology and Biological Engineering, California Institute of Technology,
Pasadena CA 91125, USA.
2
Department of Cellular Physiology, Niigata University
Graduate School of Medical and Dental Sciences, Niigata 951-8510, Japan.
3
Department of Animal Model Development, Brain Research Institute, Niigata
University, Niigata 951-8585, Japan.
*Author for correspondence (dchan@caltech.edu)
T.K., 0000-0001-9646-5379; D.C.C., 0000-0002-0191-2154
This is an Open Access article distributed under the terms of the Creative Commons Attribution
License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use,
distribution and reproduction in any medium provided that the original work is properly attributed.
1
© 2021. Published by The Company of Biologists Ltd
|
Development (2021) 148, dev199686. doi:10.1242/dev.199686
DEVELOPMENT
role in mitochondrial fission. Instead,
Fis1
has been implicated in
mitophagy in multiple species and in a variety of cell types, for
example cultured cells (Rojansky et al., 2016; Shen et al., 2014; Xian
et al., 2019; Yamano et al., 2014, 2018), nematodes (Shen et al.,
2014), early mouse embryos (Rojansky et al., 2016), mouse skeletal
muscles (Zhang et al., 2019), and leukemia stem cells (Pei et al.,
2018). Furthermore,
Fis1
was recently implicated in an asymmetric
type of mitochondrial fission that is associated with mitophagy
(Kleele et al., 2021). Some evidence indicates that
Fis1
may also
have a more general function during nonselective autophagy.
Fis1
-
deficient worms treated with mitochondrial toxins accumulate large
autophagic structures (Shen et al., 2014). Furthermore,
Fis1
can
regulate mitochondrion-lysosome contacts via the
Tbc1d15/Rab7
pathway (Wong et al., 2018; Yu et al., 2020), and can affect
lysosomal function (Joshi et al., 2019; Kim et al., 2016). Finally,
Fis1
genetically interacts with the amyotrophic lateral sclerosis gene
C9orf72
(Chai et al., 2020), which is involved in membrane
trafficking and autophagy (Nassif et al., 2017). Thus,
Fis1
is
implicated in mitophagy and may have a more general role in
regulating nonselective autophagy.
Here, we investigate the role of
Fis1
during mouse
spermatogenesis, a process that is highly sensitive to perturbations
in mitochondrial dynamics and autophagy. To this end, we
generated and characterized male germ cell-specific
Fis1
knockout
mice and male germ cell-specific mitophagy reporter mice. Our
analysis indicates that
Fis1
is required for the development of the
male germline by regulating mitochondrial morphology, mitophagy
and autophagy during spermatid maturation.
RESULTS
Fis1
is required for spermatogenesis
To study the role of
Fis1
during male germline development,
we generated mice with a conditional
Fis1
allele (Fig. S1A).
To remove
Fis1
from the male germline, we crossed conditional
Fis1
mice to the
Stra8-Cre
driver (Sadate-Ngatchou et al., 2008). We refer
to the mutants as S8/
Fis1
, and their littermate controls as S8/Control.
Stra8-Cre
expression begins at postnatal day (P) 3 (Sadate-Ngatchou
et al., 2008) in the majority of stem-like GFRA1-positive
spermatogonia (Hobbs et al., 2015; Varuzhanyan et al., 2019).
Therefore, all male germ cell types should be depleted of
Fis1
.We
confirmed gene knockout by genotype analysis of tail DNA
(Fig. S1B) and immunostaining of testis sections with an antibody
against FIS1 (Fig. S1C). In control mice,
Fis1
is expressed in the
mitochondria of both germ and Sertoli cells. In mutant mice,
Fis1
expression is eliminated selectively from germ cells.
S8/
Fis1
mice were healthy, displaying no changes in body weight
compared with controls (Fig. 1A). However, their testes were
smaller and weighed substantially less than those of age-matched
controls (Fig. 1B). Mutant epididymides were completely devoid of
spermatozoa (Fig. 1C,D), indicating an essential role for
Fis1
during
spermatogenesis. To examine apoptosis, we performed terminal
deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) of
testis sections. S8/
Fis1
testes had a greater than four-fold increase in
TUNEL-positive tubules, indicating increased cell death by
apoptosis (Fig. 1E,F). In cultured cells, downregulation of
Fis1
has been shown to inhibit apoptosis (Lee et al., 2004); therefore,
these results indicate that the effect of
Fis1
depletion on apoptosis is
highly context dependent.
A basal level of apoptosis occurs during normal germ cell
development, and the affected germ cells are phagocytosed by
Sertoli cells (Braun, 1998; Elliott et al., 2010). To address whether
the large numbers of apoptotic germ cells in S8/
Fis1
mutants were
similarly phagocytosed, we performed the TUNEL assay and
co-stained for RAB7A, a small GTPase associated with phagophore
maturation and subsequent fusion with lysosomes (Zhang et al.,
2009) (Fig. 1G,H). In control testes, about one-third of TUNEL-
positive cells were enclosed by ring-like structures decorated with
RAB7A, suggesting that they are on the pathway to phagocytic
degradation. S8/
Fis1
sections had a five-fold increase in the number
of RAB7A-positive phagosomes. Thus, depletion of
Fis1
causes
apoptotic loss of germ cells, which are then likely eliminated by
Sertoli cell phagocytosis.
Germ cell
Fis1
deletion results in multinucleated spermatid
giant cells
To gain a better understanding of the spermatogenic defect in S8/
Fis1
mice, we performed periodic acid-Schiff (PAS) staining of
adult testis sections (Fig. 2A). Control seminiferous tubules
exhibited classical germ cell organization and their lumens were
filled with spermatozoa. In stark contrast, S8/
Fis1
tubules were
devoid of spermatozoa and were filled with structures that resemble
previously described multinucleated giant cells (GCs) (MacGregor
et al., 1990). To verify that these structures are multinucleated, we
stained testis sections with DAPI and visualized germ cell
boundaries with the plasma membrane marker sodium/potassium-
transporting ATPase subunit alpha-1 (Na/K-ATPase) (Fig. S2). GCs
did indeed contain multiple nuclei that were not compartmentalized
by plasma membrane. The nuclear morphology of the GCs
indicated that they are comprised primarily of spermatids. To
verify this, we immunostained testis sections with the spermatid-
specific acrosome marker SP-10 (Acrv1) (Osuru et al., 2014). In
control testis sections, SP-10 expression was restricted to round and
elongating spermatids, with the most intense staining highlighting
the crescent-shaped acrosome (Fig. 2B). In S8/
Fis1
sections, GCs
stained intensely for SP-10, which was present diffusely throughout
the GC cytosol (Fig. 2B,C). Finally, the majority of S8/
Fis1
tubules
contained GCs (Fig. 2D), indicating that they are a prominent
pathological feature in mutant testes.
Fis1
giant cells have ectopic
γ
H2AX expression
Because S8/
Fis1
mice exhibit arrest in spermatid development,
we next checked whether spermatid precursor cells, the meiotic
spermatocytes, displayed any abnormalities. We visualized
spermatocytes by staining testis sections with the double-strand
break (DSB) repair protein
γ
H2AX, which differentially labels
spermatocytes in different stages of meiosis.
γ
H2AX is first observed
in early prophase I spermatocytes, when programmed DSBs are
generated to enable homologous recombination (Hamer et al., 2003;
Mah et al., 2010). During pachytene, these DSBs are resolved as
homologous recombination takes place, but
γ
H2AX persists on the
XY body, which is thought to be associated with silencing of the
unsynapsed sex chromosomes (Baarends et al., 2005; Fernandez-
Capetillo et al., 2003; Turner, 2007). Our analysis indicated that
pachytene spermatocytes formed normally in S8/
Fis1
mice (Fig. 3A).
By the end of meiosis I, the
γ
H2AX signal is completely resolved
and does not reappear until the histone to protamine transition in
steps 10-12 spermatids (Jha et al., 2017; Meistrich et al., 2003).
Consistent with these epigenome dynamics, control round
spermatids lacked
γ
H2AX staining (Fig. 3A,B). Unexpectedly, we
found that 30% of S8/
Fis1
GCs have ectopic
γ
H2AX expression.
We checked whether cells with ectopic
γ
H2AX expression are
apoptotic. However, TUNEL staining showed that
γ
H2AX-positive
cells in mutant samples were not associated with TUNEL labeling
(Fig. S3).
2
RESEARCH ARTICLE
Development (2021) 148, dev199686. doi:10.1242/dev.199686
DEVELOPMENT