Protein disulfide isomerase is essential for spermatogenesis in mice

found that only PDI deficiency caused the defect in sperm production, uncovering an essential role of PDI in spermatogenesis. The phenotype of sperm production caused by PDI deficiency was confirmed by inducible UBC-Cre –driven whole-body and spermatocyte-specific Stra8-Cre gene–KO strains; in both, PDI was completely deleted without affecting the expression of other members of the PDI family. Furthermore, we found that PDI deficiency induced ER stress and impaired meiosis and apoptosis of spermatocytes, consis-tence with a marked reduction in sperm counts and complete male infertility. The proteomic analysis using 4D-FastDIA indicates the altered profiles of proteins in testes between the littermate control Pdi fl/fl mice and Stra8-Cre/Pdi fl/fl mice, indicating that the critical proteins in meiosis, such as AXDND1, FANCA, HSF5, HSPA4L, MRNIP, RPL10L, SHCBP1L, TMPRSS12, SOX30


Introduction
Spermatogenesis is a highly complex process by which male germline stem cells proliferate, migrate, and differentiate to produce mature haploid spermatozoa (1).There are 3 typical stages: mitosis of spermatogonia, meiosis of spermatocytes, and spermiogenesis of spermatids (2).These processes are subject to complex and precise control at transcription and translation levels, directing the normal expression of specific genes at different stages.Abnormalities at any step may result in meiosis arrest, spermatogenesis failure, and ultimately defective reproductive development.Using genetically modified mouse models, numerous molecules have been identified -such as HSPA4L, SHCBP1L, and DDX4 -that play crucial roles in spermatogenesis (3)(4)(5).However, how the synthesis of these proteins is regulated remains unknown.
The endoplasmic reticulum (ER) is the organelle responsible for protein synthesis and maturation.The protein disulfide isomerase (PDI) family is a group of multifunctional ER enzymes that catalyze the formation of disulfide bonds and is responsible for the quality control of nascent proteins to maintain proteostasis (6,7).Given the rapid proliferation and differentiation of spermatogonia into spermatocytes, the ER-related mechanism is tightly involved in proper and efficient protein synthesis for spermatogenesis.Recent structural and functional studies have suggested that PDI family enzymes are involved in spermatogenesis.PDIA3 (also known as ERp57) was found in the cytoplasm of human testicular spermatogenic cells and the acrosome and flagella of human sperm (8).Another study confirmed that PDIA3 protein exists in cells at all stages of rat spermatogenesis as well as in Leydig cells, and Pdia3 mRNA is similarly transcribed in cells at almost all stages of rat spermatogenesis (9).Moreover, PDIA3 was expressed on the sperm surface and was a component of sperm-egg zona pellucida binding complexes from the cell membrane of capacitive sperm.Functional blockade of PDIA3 with antibodies or inhibitors reduced the surface mercaptan content and zona pellucida binding ability of human sperm (10).In addition, PDIA6 (also known as ERp5), Spermatogenesis requires precise posttranslational control in the endoplasmic reticulum (ER), but the mechanism remains largely unknown.The protein disulfide isomerase (PDI) family is a group of thiol oxidoreductases responsible for catalyzing the disulfide bond formation of nascent proteins.In this study, we generated 14 strains of KO mice lacking the PDI family enzymes and found that only PDI deficiency caused spermatogenesis defects.Both inducible whole-body PDI-KO (UBC-Cre/Pdi fl/fl ) mice and premeiotic PDI-KO (Stra8-Cre/Pdi fl/fl ) mice experienced a significant decrease in germ cells, testicular atrophy, oligospermia, and complete male infertility.Stra8-Cre/ Pdi fl/fl spermatocytes had significantly upregulated ER stress-related proteins (GRP78 and XBP1) and apoptosis-related proteins (Cleaved caspase-3 and BAX), together with cell apoptosis.PDI deletion led to delayed DNA double-strand break repair and improper crossover at the pachytene spermatocytes.Quantitative mass spectrometry indicated that PDI deficiency downregulated vital proteins in spermatogenesis such as HSPA4L, SHCBP1L, and DDX4, consistent with the proteins' physical association with PDI in normal testes tissue.Furthermore, PDI served as a thiol oxidase for disulfide bond formation of SHCBP1L.Thus, PDI plays an essential role in protein quality control for spermatogenesis in mice.

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JCI Insight 2024;9(12):e177743 https://doi.org/10.1172/jci.insight.177743another member of the PDI family, was associated with heat shock protein A2 (HSPA2) in membrane raft microdomains surrounding the acrosome of the sperm head, suggesting that PDIA6 might contribute to fertilization cascade (11).More interestingly, the deletion of PDIA7 (also known as PDILT) in the testis caused infertility in mice, due to the decreased expression of a disintegrin and metalloproteinase 3 (ADAM3), which is a sperm membrane protein that is essential for the migration of sperm from the uterus to the oviduct; the role of PDIA7 is associated with disulfide bond formation of ADAM3 for its expression on sperm surface (12).A more recent study indicated that, in the sperm of a Chinese mitten crab (Eriocheir sinensis), PDI was expressed in the cytoplasm, cell membrane, and extracellular matrix in the spermatogonia, spermatocytes, and stage I and stage II spermatids and was mainly localized in the nuclei of stage III spermatids and sperm (13).Taken together, these previous observations suggest that the PDI family plays a key role in spermatogenesis; however, their exact functions have never been carefully characterized using genetically modified models.As a consequence, the mechanism of proteostasis necessary for spermatogenesis remains unknown.
In this study, we generated 14 strains of PDI family member gene-KO mice to identify their role in spermatogenesis in mice.We found that the deletion of PDI in premeiotic germ cells disrupted homologous recombination in the meiosis of spermatocytes causing complete male infertility, although the gene deletion of other members did not show an obvious phenotype.Our data provide the first genetic evidence to our knowledge demonstrating that PDI is selectively essential for spermatogenesis.
Premeiotic germ cells PDI deficiency results in male infertility.To further confirm the role of PDI family members in spermatogenesis, Pdi fl/fl mice with Stra8-Cre (Stra8-Cre/Pdi fl/fl ) (Figure 2, A and B) and mice lacking spermatocyte PDIA3 (Stra8-Cre/Pdia3 fl/fl ) and PDIA6 (Stra8-Cre/Pdia6 fl/fl ) were mated (Supplemental Figure 2).The Stra8-Cre gene induced the expression of Cre enzyme in the type A1 spermatogonium cells 3 days after birth and reached the peak expression at 7 days (20).With the initiation of the Cre enzyme, PDI was not expressed in germ cells, and this was confirmed by immunofluorescence and Western blotting in testicular tissue (Figure 2, C and D).The male Stra8-Cre/Pdi fl/+ mice had normal fertility (Supplemental Figure 3).Stra8-Cre/Pdi fl/fl mouse survived normally after birth, with normal body size, appearance, and sex ratio.With increasing weekly age, the testicular size of Stra8-Cre/Pdi fl/fl male mice was gradually decreased, and the testicular body weight ratio was significantly reduced (Figure 2E).Male Stra8-Cre/Pdi fl/fl mice were mated with Pdi fl/fl female mice at a ratio of 1:2, and the litter birth of female mice was observed and recorded.Pdi fl/fl female mice appeared to have vaginal plugs but mated continuously for 3 months without offspring (Figure 2F), suggesting that testicle-specific PDI-KO leads to infertility in male mice.The H&E staining of testes showed that, in both Stra8-Cre/Pdia3 fl/fl and Stra8-Cre/Pdia6 fl/fl mice, spermatogenic tubule lumens were filled with spermatocytes, round spermatozoa, and elongated spermatozoa, without vacuole phenomena (Supplemental Figure 2A).The Stra8-Cre/Pdia3 fl/fl and Stra8-Cre/Pdia6 fl/fl mice likely had normal sperm counts in epididymis, shown by the H&E staining (Supplemental Figure 2B).These data suggest that PDI is selectively required for spermatogenesis.
PDI is the prototype member of this family and plays a vital role in various physiological conditions (21).We detected high expression of PDI in the liver, testes, and epididymis (Supplemental Figure 4A).At 2, 3, and 7 weeks, PDI expression was relatively high in all cell types, as shown by immunofluorescence staining (Supplemental Figure 4B).Moreover, in the testes of mice aged from 1 to 6 weeks, there was a rapid increase in the expression levels of PDI from 2 weeks until they reached sexual maturity (Supplemental Figure 4C), consistent with its involvement in male fertility.
To understand the cause of infertility in Stra8-Cre/Pdi fl/fl male, we next examined the first wave of spermatogenesis at P10, P12, and P14 corresponding to leptotene, zygotene, and pachytene spermatocytes, respectively (22).The testes of the control Pdi fl/fl mice were filled with various stages of spermatocytes, but the seminiferous tubules of Stra8-Cre/Pdi fl/fl mice contained only a few spermatocytes without an obvious change in number (Figure 3A), suggesting that PDI deficiency affected the prophase of meiosis at the first wave of

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JCI Insight 2024;9(12):e177743 https://doi.org/10.1172/jci.insight.177743spermatogenesis in mice.In the adult mouse testes, the cells of Stra8-Cre/Pdi fl/fl at all levels were poorly developed, and many vacuoles appeared in the seminiferous tubules (Figure 3B).We analyzed early meiotic cells in testes sections after staining with synaptonemal complex protein 3 (SYCP3), typically present in meiotic prophase I spermatocytes, and we found that the number of prophase I spermatocytes was significantly reduced in Stra8-Cre/Pdi fl/fl testes (Figure 3C).FITC-labeled peanut agglutinin (PNA) was used to evaluate the acrosome development (23,24).In the spermatogenic tubules of Pdi fl/fl mice, typically punctated Golgi bodies (stages I-III), capped acrosomes (stages IV-VI), or crescent-shaped acrosomes (stages VII-VIII) were observed in round spermatid acrosomes, and elongated spermatid acrosomes were located on top of the nucleus (stages IX-XII).However, there was no positive signal of FITC-PNA in Stra8-Cre/Pdi fl/fl mice and almost no spermatid in the testes (Figure 3D).The H&E staining of epididymis showed there was a markedly decreased sperm count in Stra8-Cre/Pdi fl/fl mice.The sperm count of Pdi fl/fl male mice was 11.47 × 10 6 ± 1.74 × 10 6 /mL, while that of Stra8-Cre/Pdi fl/fl male mice was only 0.32 × 10 6 ± 0.15 × 10 6 /mL (Figure 3E).Therefore, testicle-specific PDI deficiency leads to infertility in male mice due to the defect in the progress of the first wave of spermatogenesis, following oligospermia.
PDI deficiency disrupts the meiosis of spermatocytes.To further examine the role of PDI in meiosis, the prophase of meiotic passage by chromosomal spreads from the testes of mice was assessed and coimmunostained with SYCP3 and γH2AX (Figure 4A).All stages from leptotene to diplotene were observed in spermatocytes in both control and Stra8-Cre/Pdi fl/fl mice.When DNA double-strand breaks (DSBs) occurred, γH2AX signals were observed throughout the nucleus in leptotene and zygotene.When the autosomal breaks were repaired, γH2AX signals were restricted to the sex chromosome in pachytene and diplotene in control PDI fl/fl mice.Nevertheless, unrepaired DSBs with γH2AX signals partially retained on autosomes occupied 42.44% ± 4.23% of Stra8-Cre/Pdi fl/fl pachytene, which was markedly higher than 11.68% ± 1.80% of the control.In general, meiosis recombination defect is an important cause of pachytene retardation (25).Meiosis recombination depends on the successful repair of the program DSBs.Proteins were involved in the formation and recombination of meiotic-specific programmed DSBs, such as SYCP1, MRE11, RAD51, DMC1, and MLH1.With the help of RAD51 and DMC1 recombinases, the annealed DSB terminal develops into an extended D-loop (26), and the DSB is then repaired (27,28).MLH1 represents the crossover during meiosis recombination (29).The different stages of DSBs processing in chromosome spreads of pachytene spermatocytes were further observed by coimmunostaining SYCP3 with MLH1 and DMC1.The positive signals of spermatocytes were counted in the pachytene stage.We found that the number of DMC1 loci in spermatocytes of Stra8-Cre/Pdi fl/fl mice was significantly increased compared with control Pdi fl/fl mice (Figure 4B), suggesting that the PDI deletion results in an increased incidence of unrepaired DSBs in spermatocytes during the pachytene stage.In addition, Stra8-Cre/Pdi fl/fl mice had significantly fewer MLH1 loci in spermatocytes than control Pdi fl/fl mice (Figure 4C), suggesting that cross-formation is also impaired.Thus, PDI is indispensable in meiosis and plays a pivotal role in DSB repair and synapsis in spermatocytes.
PDI deficiency induces ER stress and apoptosis of spermatocytes.PDI is critical for correcting protein folding of nascent peptides by catalyzing the formation of disulfide bonds.The decrease in its expression often causes the accumulation of misfolded proteins beyond a specific limit leading to ER stress and unfolded protein response (UPR).In contrast, excessive ER stress may lead to apoptosis if the dysfunction of PDI cannot be compensated (30).Compared with the control group, the level of Bip (also known as GRP78) in Stra8-Cre/Pdi fl/fl testes was significantly increased (Figure 5A), demonstrating the presence of severe ER stress.In addition, Stra8-Cre/PDI fl/fl testes had a significant increase in Cleaved caspase-3 and key proapoptotic protein BAX (Figure 5B), suggesting the activation of the apoptosis signaling pathway.Consistent with the fact that the apoptosis signaling pathway is activated by high levels of intracellular reactive oxygen species (ROS) causing damage to proteins, nucleic acids, lipids, membranes, and organelles (31), Stra8-Cre/Pdi fl/fl spermatogenic cells had a significant increase in ROS (Figure 5C).JC-1 is an ideal fluorescent probe for detecting mitochondrial membrane potential (MMP) and is often used to evaluate apoptosis.The MMP of Stra8-Cre/Pdi fl/fl spermatogenic cells was decreased compared with that of Pdi fl/fl mice, suggesting the apoptosis of cells (Figure 5D).TUNEL staining of the testicular sections showed a significant increase in apoptotic cells in seminiferous tubules of Stra8-Cre/Pdi fl/fl mice (Figure 5E).The above data indicate that the absence of PDI induces irreversible ER stress and cells apoptotic in spermatocytes.

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JCI Insight 2024;9(12):e177743 https://doi.org/10.1172/jci.insight.177743HSPA4L (3), MRNIP (35), RPL10L (36), SHCBP1L (4), TMPRSS12 (37), SOX30 (38), and DDX4 (5) (Figure 6, A-C).Western blotting was used to verify the expression levels of these proteins, showing that the expression of HSPA4L, SHCBP1L, and DDX4, at least, were decreased in Stra8-Cre/Pdi fl/fl testes.However, the expression of PROK2 and RPL10L showed no difference (Figure 7A).Because PDIA3, PDIA4, PDIA5, PDIA6, PDIA9, PDIA14, PDIA15, and PDIA16 were expressed in testes, as shown in Figure 1A, we measured the expression of HSPA4L, SHCBP1L, and DDX4 in these gene deficient mice.Western blotting showed that the expression of HSPA4L, SHCBP1L, and DDX4 in testes of Stra8-Cre/Pdia3 fl/fl , Pdia4 -/-, Pdia5 -/-, Stra8-Cre/Pdia6 fl/fl , Pdia9 -/-, Pdia14 -/-, Pdia15 -/-, and CAG-Cre/Pdia16 fl/fl mice were comparable with their control mice (Supplemental Figure 5, A-H).All of these KO mice had comparable PDI  expression levels in the testicular lysate compared with the control mice, especially the mice lacking PDIA3 and PDIA6 whose structure and amino acid homology are close to PDI.The expression of HSPA4L, SHCBP1L, and DDX4 was selectively decreased by PDI deficiency, consistent with the observations of immunofluorescence staining showing the decreased expression of HSPA4L, SHCBP1L, and DDX4 in Stra8-Cre/Pdi fl/fl testes (Figure 7B).Moreover, in testicular lysate, the physical association of PDI with HSPA4L, SHCBP1L, and DDX4 was detected by coimmunoprecipitation (Figure 7C).These data suggest the specific crucial role of PDI in expression of these proteins, and it likely underlies the phenotypes of PDI deficiency.The most important function of the PDI family is to help the correct folding of the new protein disulfide bonds in the ER, to maintain the proteostasis.To assess the potential role of PDI, free thiols of proteins in the lysate of testes from Pdi fl/fl and Stra8-Cre/Pdi fl/fl mice were labeled with 3-(N-maleimide-propionyl)-biocytin (MPB), and the MPB-labeled proteins were pulled down by streptavidin beads.The immunoblotting results revealed more abundant free thiols of SHCBP1L in PDI-deficient testes lysate than that of the control, although the total SHCBP1L protein was much less in PDI-deficient testes lysate (Figure 7D).The ratio of normalized levels of MPB-labeled SHCBP1L/total SHCBP1L shows that PDI deficiency increased free thiols in SHCBP1L, suggesting that PDI serves as a thiol oxidase that is responsible for the disulfide bond formation of SHCBP1L.However, the free thiols of SHCBP1L in the lysate of testes were comparable between Pdia3 fl/fl mice and Stra8-Cre/Pdia3 fl/fl mice (data not shown).Although PDI deficiency did not change free thiols in HSPA4L and DDX4 proteins, we have to admit the shortage/limitation of this MPB labeling technique, as it could not identify the disulfide isomerization reaction.Taken together, our data imply that PDI plays an essential role in spermatogenesis, at least in part, through the regulation of HSPA4L, SHCBP1L, and DDX4 expressions in spermatocytes.

Discussion
In the present study, using a group of gene-KO mice deficient in 14 members of the PDI family, we found that only PDI deficiency caused the defect in sperm production, uncovering an essential role of PDI in spermatogenesis.The phenotype of sperm production caused by PDI deficiency was confirmed by inducible UBC-Cre-driven whole-body and spermatocyte-specific Stra8-Cre gene-KO strains; in both, PDI was completely deleted without affecting the expression of other members of the PDI family.Furthermore, we found that PDI deficiency induced ER stress and impaired meiosis and apoptosis of spermatocytes, consistence with a marked reduction in sperm counts and complete male infertility.The proteomic analysis using 4D-FastDIA indicates the altered profiles of proteins in testes between the littermate control Pdi fl/fl mice and Stra8-Cre/Pdi fl/fl mice, indicating that the critical proteins in meiosis, such as AXDND1, FANCA, HSF5, HSPA4L, MRNIP, RPL10L, SHCBP1L, TMPRSS12, SOX30, and DDX4, were significantly downregulated.Taken together, our study demonstrates an essential role for PDI in the proteostasis of spermatocytes to support meiosis.
PDI is one of the most abundant and critical protein-folding catalysts in the ER of eukaryotic cells (30).Its a and a′ domains have catalytic activity, while the b and b′ domains have substrate binding activity (7).Although PDI has more than a single function in a variety of cellular processes and is responsible for the folding and synthesis of microsomal triglyceride transfer protein, insulin, and P4H, which are critical for lipid and glucose metabolism and bone formation (39)(40)(41), its role in spermatogenesis has never been studied, to our knowledge, using gene-modified models.A previous study has shown that mice lacking PDIA7 had a defect in male fertility due to loss of sperm migration from the uterus into the oviduct because PDIA7 deletion reduced the expression of ADAM3.However, PDIA7 deficiency did not affect spermatogenesis, and sperm produced were morphologically normal (12).Our data indicate that PDI and other PDI family members PDIA3, PDIA6, and PDIA7 were highly expressed in testes (Figure 1A), consistent with previous studies.PDIA3 and PDIA6 have similar structures to PDI and both contain 2 CGHC active domains.However, the deficiency of PDIA3 and PDIA6 as well as the other 11 members of this family did not cause the defect in spermatogenesis (Supplemental Figures 1 and 2), suggesting that PDI is selectively required for this process and its function is indispensable.
The major function of PDI is to catalyze the disulfide bond formation of native proteins in ER, and this is necessary for normal protein conformation to maintain their function.If PDI is deleted or has low activity, a large number of misfolded proteins accumulate in the ER, leading to ER stress, increased intracellular ROS, and decreased protein synthesis.In this study, we found that ER stress marker Bip was highly upregulated together with the activation of UPR activation pathways involving XBP1 (Figure 5A).These results suggest that PDI deficiency causes severe ER stress and UPR.However, the deleterious effect of PDI deficiency cannot be compensated for by any other PDI enzymes, leading to persistent intracellular ROS elevation and triggering proapoptotic pathways (Figure 5, B-D).Therefore, PDI is a critical component of proteostasis in spermatocytes.It is estimated that approximately 10%-20% of couples cannot conceive a child in the world (42).Male factor infertility is estimated at 30%-50% of infertility cases (43).Azoospermia, or severe oligospermia, is a more serious condition of male infertility caused by multiple factors such as genetic, epigenetic, and environmental factors.Genetic factors may account for about 15%-30% of cases of male infertility, with over 2,000 genes devoted to spermatogenesis (44).Our finding on the essential role of spermatogenesis will boost further investigation to understand whether the decreased expression and enzymatic activity of PDI is involved in male infertility.
Using a high-throughput technique, we found that PDI deficiency caused a significant decrease of 341 proteins with a ≥1.5-fold change, among which AXDND1, FANCA, HSF5, HSPA4L, MRNIP, RPL10L, SHCBP1L, TMPRSS12, SOX30, and DDX4 have been shown having a role in spermatogenesis.Using Western blotting, we have so far confirmed the decreased expression of HSPA4L, SHCBP1L, and DDX4 (Figure 7A).As found in a previous study, in Hspa4l -/-mice, spermatogenic tubules underwent apoptosis and their mature sperm number was significantly reduced (3).Shcbp1l -/-mice exhibited an increase in metaphase or anaphase-arrested spermatocytes, as well as impaired fertility in male mice (4).DDX4 KO also resulted in male infertility and their proliferative activity of primordial germ cells was reduced; DDX4-null spermatogonia failed to reach the fertilized egg stage and undergo apoptosis (5).These proteins are necessary for spermatogenesis and fertility.The decreased expression of all of these proteins may underlie the phenotypes of PDI deficiency and suggest a role of PDI in the proteostasis of spermatocyte ER that is crucial for correct folding and synthesis of these critical proteins.Presumably, PDI is required for the formation of structural disulfide bonds of HSPA4L, SHCBP1L, and DDX4; the deficiency of PDI might cause the unfolded or misfolded structure of these proteins, leading to their degradation.To understand the specific role of PDI in regulation of these proteins, it will be helpful to identify the cysteines/disulfides targeted by PDI in these proteins.Mouse and human HSPA4L have 15 and 18 Cys residues, respectively (45).Both mouse and human SHCBP1L have 13 Cys residues.There are 15 Cys residues in mouse and human DDX4.However, the crystal structure of these proteins has not been characterized so far, their disulfide pairings are not known.In this case, we studied the oxidation of protein by PDI using the thiol labeling technique.We found the association of PDI deficiency with the increase in free thiols of these proteins, suggesting that PDI catalyzes the disulfide oxidation of these proteins.Our observations point out the need to characterize the disulfide pairing of these proteins so that we will be able to map the targeting cysteines of PDI using new techniques, such as differential thiol labeling and mass spectrometry (MS).
In summary, our data provide genetic evidence demonstrating that PDI plays an essential role in spermatogenesis and male fertility and that its function in meiosis is indispensable.Our findings highlight the importance of spermatocyte proteostasis and an ER quality control system for spermatogenesis as well as the distinct requirement of different PDI family members in this process.In the future, it will be necessary to characterize the disulfide pairing of the key molecules in spermatogenesis for a better understanding of the thiol-based redox network driven by PDI in the physiological and pathophysiological settings.

Methods
Sex as a biological variable.Our study used male mice because the phenotype of spermatogenesis is exclusively relevant in males.

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JCI Insight 2024;9(12):e177743 https://doi.org/10.1172/jci.insight.177743KO first (Pdia11 -/-) mice (16), and Pdia3 fl/fl mice (14) were described in our previous studies.Spermatocyte-specific-KO mice deficient in PDI, PDIA3, and PDIA6 were generated by mating floxed mice with Stra8-Cre mice.Genotyping of mice was performed by PCR analysis of tail DNA, and primers used for genotyping are listed in Supplemental Table 1.
Assessment of fertility.Each adult Stra8-Cre/Pdi fl/fl male or littermate control Pdi fl/fl male mouse was mated with 2 adult female Pdi fl/fl mice for fertility tests.The number of pups per litter and the date of delivery were recorded.The fertility test lasted for at least 3 months.
TUNEL assay.Mouse testicular frozen sections were fixed with 4% paraformaldehyde for 30 minutes and incubated with PBS containing proteinase K for 20 minutes.After washing 3 times with PBS, the testes sections were incubated with an equilibration buffer for 20 minutes.The TUNEL assay was performed according to the manufacturer's instructions (Service Bio, G1501).In brief, the sections were incubated with the TUNEL kit including TdT enzyme and dUTP at 37°C for 1 hour.Sections were washed with PBS and then counterstained with DAPI.Slides were imaged under an FV3000 Confocal laser scanning microscopy (Olympus).
Histological and immunofluorescence staining analysis.Histological and immunohistochemical staining were performed as previously described with slight modifications (46).For paraffin sectioning, testes were excised and fixed in 4% paraformaldehyde at 4°C overnight.After the samples were dehydrated with graded ethanol and were paraffin embedded, the specimens were sectioned at 5 μm and mounted on glass slides.The sections were then deparaffinized, hydrated, and stained with H&E.The slides were imaged with a Leica DM2000 microscope.For immunofluorescence staining, testes were fixed in 4% paraformaldehyde at 4°C overnight and then dehydrated with 20% sucrose solution overnight.The testes were dipped in OCT compound and then quickly frozen in liquid nitrogen-frozen sections with a thickness of 10 μm and fixed in precooled acetone for 20 minutes.The slides were blocked with Immunol staining blocking buffer (QuickBlock Blocking Buffer for Immunol Staining, Beyotime, P0260) for 1 hour before being incubated with primary antibodies overnight at 4°C.After washing in PBS 3 times, the sections were incubated with secondary

Figure 6 .
Figure 6.Identification of protein profiles in mouse testes altered by PDI deficiency.(A-C) Reactome, gene ontology, and protein domain analysis of downregulated and upregulated genes in control Pdi fl/fl and Stra8-Cre/Pdi fl/fl testes.

Figure 7 .
Figure 7. PDI is required for the expression of HSPA4L, DDX4, and SHCBP1L in spermatogenic cells.(A) Western blotting analysis of proteins in testes from adult control and Stra8-Cre/Pdi fl/fl mice, normalized to GAPDH (mean ± SEM, n = 3, *P < 0.05, **P < 0.01, Student's t test).(B) Immunofluorescence staining of HSPA4L, DDX4, and SHCBP1L in testes from control and Stra8-Cre/Pdi fl/fl mice at 3 weeks old.Scale bar: 50 μm.(C) Immunoprecipitation of testes lysates of WT mice was performed with anti-PDI antibody.Detection of HSPA4L, DDX4, and SHCBP1L in anti-PDI immunoprecipitants with Western blotting.(D) Pdi fl/fl and Stra8-Cre/Pdi fl/fl testes lysates were labeled with MPB, and the labeled proteins were pulled down by streptavidin beads and analyzed with Western blotting using the indicated antibodies.The relative abundance of reduced protein was calculated by comparing the density of protein before and after the pull-down, and then the value of the PDI fl/fl group was normalized to be 1 (mean ± SEM, n = 4, *P < 0.05, Student's t test).