This cellular defect is observed by light microscopy as LC3 tubulation and aggregation ( Yamano et al., 2014 ). It is generally accepted that spermatogonia depend on glucose for energy production through glycolysis ( Rato et al., 2012 ). Mitochondria in spermatogonia are generally small, spherical and contain 'orthodox' cristae (De Martino et al., 1979;
They elongate during pachytene and then re-fragment into post-meiotic spermatids (De Martino et al., 1979). Our recent study expanded on the role of mitochondrial fusion during spermatogenesis in mice (Varuzhanyan et al., 2019). In Pink1 mutant flies, spermatids have aberrant mitochondria and defects in individualization ( Clark et al., 2006 ).
The acrosome has been classically characterized as a Golgi-derived lysosome-associated organelle (Khawar et al., 2019). For example, male testis germ cell-specific Rab GTPase-activating proteins (MgcRabGap) colocalize with RAB3A at the acrosome ( Lin et al., 2011 ).
Figure Legends
A) (Left panel) Anatomy of the mammalian testis, highlighting the convoluted seminiferous tubules in which spermatogenesis occurs. Right panel) Schematic representation of the seminiferous epithelium, highlighting the close association between somatic Sertoli cells and germ cells. Note that meiotic spermatocytes and post-meiotic spermatids develop on the adluminal side of the blood-testis barrier (BTB).
Spermatogonia in the basal region have direct access to systemic glucose, which they use for glycolysis. Spermatocytes and spermatids in the adluminal region, however, are separated from the vasculature and interstitial space by the blood-testicular barrier and thus rely on Sertoli cells as a carbon source. Sertoli cells take up systemic glucose and glycolytically convert it to pyruvate, which is converted to lactate via pyruvate dehydrogenase (PDH).
After crossing the blood-testis barrier (dashed red line) and entering the adluminal compartment, mitochondria elongate and coalesce around the nuage, also called the intermitochondrial cementum (IMC). Finally, near the end of spermiogenesis, the mitochondria elongate and squeeze tightly around the center of the sperm.
Figures Figure 1.1 Figure 1.1
Mutant spermatozoa also show morphological defects, notably near or mid-twisting (Figures 2.1E and 2.1G) and almost complete loss of motility (Figure 2.1H; Videos 2.1-3). Haploid spermatids were identified using an antibody against SP-10 (Osuru et al., 2014), which labels the spermatid acrosome (Figure 2.2C). PAS-stained testis sections of P56 S8::Dm mice showed greater germ cell depletion and more widespread Sertoli cell vacuolization (Figure 2.4A) compared to P24 mutants.
Quantification from testis sections in WT and S8::Dm mice shows that the vast majority of GFRα1-positive spermatogonia express Stra8-Cre/Dn (Figure 2.4G and Figure 2.S4B). Thus, mitochondrial fusion is required for maintenance of all differentiated germ cell types, but is dispensable for self-renewal of stem-like undifferentiated spermatogonia (Figure 2.4J). Quantification of c-Kit-expressing spermatogonia showed that both mutants have severe depletion of differentiating spermatogonia, close to the level observed in S8::Dm mice (Figure 2.S4E).
Our GO analysis also revealed an upregulation of mitochondrial import proteins in Mfn1/Mfn2-null MEFs (Figure 2.6A). Consistent with the progressive loss of differentiated spermatogonia in S8::Dm mice, there is also mitochondrial heterogeneity in this cell type (Figures 2.S8B and 2.S8C). Consistent with upregulation of OXPHOS during pachytene, others and we have found high expression of the mtDNA-encoded COXI protein (Figure 2.8A, middle panel) (Jiang et al., 2017) and mRNA (Saunders, Millar, West, & Sharpe, 1993) in pachytene spermatocytes.
Diploid, tetraploid and haploid cells were isolated as previously described (Bastos et al., 2005), and verified using germ cell-specific markers (Figure 2.S3A).
Figure legends
Mutant values are plotted relative to control, which is set to 100% and indicated by the gray bar. Adult mutant mice have an earlier defect, in which all differentiating germ cell types are lost, but self-renewal of stem-like undifferentiated spermatogonia remains intact.
Figures Figure 2.1Figure 2.1
Supplemental figure legends Figure 2.S1
Note the loss of germ cells from the midtubules in both S8::Mfn1 and S8::Mfn2 mice. Mitochondria are labeled with the outer mitochondrial membrane marker Tom20 (red). B) Analysis of mitochondrial heterogeneity in differentiated and undifferentiated spermatogonia. MTCOXI protein was examined in c-Kit-positive cells (differentiated spermatogonia) and PLZF-positive cells (undifferentiated spermatogonia).
Supplemental figures Figure 2S.1
The acrosomal protein SP-10 (Acrv1) is an ideal marker for staging the cycle of seminiferous epithelium in the mouse. Using genetic ablation in mice, we discover that mitochondrial fission factor (Mff) is required for mitochondrial fragmentation in haploid round spermatids and for organizing mitochondria in the midpiece of elongating spermatids. In the initial phase, the endoplasmic reticulum (ER) constricts the mitochondrion with the help of actin filaments (Friedman et al., 2011; Korobova et al., 2013).
During these developmental transitions, mitochondria undergo dramatic changes in morphology, distribution, and number (De Martino et al., 1979b). Indeed, mitochondrial fusion has been shown to be important for maintaining OXPHOS activity in meiotic spermatocytes (Varuzhanyan et al., 2019b; Zhang et al., 2016b). Furthermore, we previously reported that mice homozygous for a recessive allele of Mff (Mffgt) have reduced fertility and sperm count (Chen et al., 2015b), but the underlying reasons were unclear.
To this end, we analyzed the mitochondrial defects present in the male germ cells of Mffgt mice. Because the mitochondrial sheath is a major component of the midpiece, we used a mitochondrial-targeted Dendra2 (Dn) fluorescent protein (Pham et al., 2012) to examine mitochondrial structure in sperm (Figures 3.2B and 3.2C). WT sperm had abundant mitochondria tightly packed in the sperm midpiece with little or no gaps between adjacent organelles.
These data suggest that in the absence of fission, aberrantly enlarged mitochondria are poorly recruited to the midpiece of the sperm, resulting in detached mitochondrial envelopes. Because reduced respiratory chain activity has been associated with reduced sperm motility (Ruiz-Pesini et al., 1998), we examined sperm motility by time-lapse microscopy (Video 3.5-6). Recent studies have revealed the importance of mitochondrial dynamics for male fertility (Chen et al., 2015b; Varuzhanyan et al., 2019b; Zhang et al., 2016b).
Mffgt mice, previously described ( Chen et al., 2015b ), were maintained on a 129P2/OlaHsd and C57Bl/6J background and are available from the Mutant Mouse Resource & Research Center (RRID: MMRRC_066700-UCD). Rosa26PhAM (cut/excised) mice have been described previously ( Pham et al., 2012 ) and are available from The Jackson Laboratory (#018397). Tomographic data were calculated, analyzed, and modeled using the IMOD software package (Kremer et al., 1996; Mastronarde, 2008) on MacPro computers (Apple, Inc, Cupertino, CA).
Figure Legends
Note that Mffgt sperm have wider mitochondria, and large empty spaces where mitochondria should be present. For more information on the 3D versions, see Videos 3.1-2. B) 3D electron tomography of cross-section of epididymal sperm. At least 20 seminiferous tubule cross-sections from each of two WT and three Mffgt mice were assessed.
A total of 36 cells from two WT mice and 39 cells from one Mffgt mouse were quantified.
Figures Figure 3.1Figure 3.1
Supplementary figure Figure 3S.1
To better understand the spermatogenic defect in S8/Fis1 mice, we performed periodic Acid-Schiff (PAS) staining of adult testis sections (Figure 4.2A). To determine the stage of spermatogenic arrest in S8/Fis1 mice, we performed PAS staining in juvenile P28 mice, which are undergoing the first round of spermatogenesis. To determine the stage of spermatogenic arrest in S8/Fis1 mice, we examined younger mice (P23) whose acrosomes formed first (Figure 4.3E).
Strikingly, no Step-9 elongating spermatids could be found in juvenile or mature S8/Fis1 testes (Figure 4.3F). Taken together, these data indicate that spermatogenesis in S8/Fis1 mice arrests between Stage V and Stage VIII. We noted a marked increase in mitochondrial Dendra2 fluorescence intensity in S8/Fis1 GCs compared to WT spermatids (Figure 4.4A), indicating mitochondrial accumulation.
Using this system, we found an increase in red-only signals in S8/Fis1 GCs, which was much greater compared to control (Figures 4.4B and 4.4C). S8/Fis1 GCs showed increased COX/SDH staining, indicating increased respiratory chain complex IV and II activity (Figures 4.6E and 4.6F). Relative protein quantification shows an upregulation of the nuclear DNA damage response in S8/Fis1 germ cells.
As described above, Fis1-null spermatids have a pronounced increase in mito-Dendra2 fluorescence intensity, indicating mitochondrial accumulation (Figure 4.4A). We have shown above that the mitochondrial accumulation in S8/Fis1 GCs is associated with a block in autophagic flux, manifesting as a build-up of autophagic intermediates (Figure 4.5). These observations are consistent with the aforementioned mitochondrial accumulation (Figure 4.4A) and increased respiratory chain complex IV and II activity (Figures 4.6E and 4.6F) in Fis1-null GCs.
Acrosomes also fragment in Atg7-deficient germ cells (Wang et al., 2014), providing further support for a defect in autophagic flux in S8/Fis1 mice. Several lines of evidence point to possible mechanisms by which DNA damage may contribute to spermatogenic arrest in S8/Fis1 mice. Our proteomics analysis indicated that the DNA damage response protein, ESCO2, is the most downregulated protein in the S8/Fis1 testes (Figure 4.7B).
Figure legends
The seminiferous epithelium stage is indicated by Roman numerals, and the spermatid stage is indicated by Arabic numerals. The stage of seminiferous epithelium is indicated by Roman numerals and the spermatid stage is indicated by Arabic numerals. The stages of spermatid differentiation are indicated and correspond to the stage of the cycle of the seminiferous epithelium.
Note the localization of ATG9A on the acrosome (white arrows) and the accumulation of ATG9A in mutant spermatids. The control photomicrograph is from a round spermatid, and the mutant photomicrographs are from spermatid giant cells (GCs). Quantification showing the percentage of GC-containing tubules in which at least 1 GC showed strong γH2AX staining.
Figures Figure 4.1Figure 4.1
Supplementary figure legends Figure 4.S1: Related to Figure 4.1
The TUNEL assay was performed in P35 testis sections co-stained with an antibody against γH2AX and DAPI.
Supplementary figures Figure 4.S1Figure 4.S1
