Developing an antibiotic-free wound dressing with effective hemostasis and antibacterial and antioxidant capacity is highly desirable. In this work, a three-dimensional (3D) chitosan/polyvinyl alcohol-tannic acid porous nanofiber sponge (3D-TA) was prepared via electrospinning. Compared with two-dimensional (2D) fiber membrane, the unique fluffy 3D-TA nanofiber sponge had high porosity, water absorption and retention ability, hemostatic capacity. Furthermore, the 3D sponge functionalized by tannic acid (TA) endow the sponge with high antibacterial and antioxidant capacity without loading antibiotics. In addition, 3D-TA composite sponges have shown highly biocompatibility against L929 cells. The in vivo experiment shows the 3D-TA is enable to accelerate wound healing. This newly 3D-TA sponges hold great potential as wound dressings for future clinical application.The online version contains supplementary material available at 10.1186/s12951-023-01922-2. Wound dressings cover damaged skin thereby supplying a temporary healing environment to treat skin wounds. The key factors of these important and widely used medical materials include effective wound sealing to stop bleeding, promotion of blood clotting, prevention of further traumas as well as strong antibacterial and anti-infective capabilities , 2. Many2O2), hydroxyl radical (·OH) and superoxide anion radical [Bacterial infection will trigger body immune response leading to inflammation, tissue damage, and can be potentially life-threatening . Hence,  radical , 16. The radical , 18. TheNatural products of plants are considered to be a source of effective antibacterial agents because of their low toxicities . Tannic 4 foaming technology involve gas generation in aqueous solution [However, the fabrication process of 3D nanofiber sponges will lead to the drugs releasing or inactivated if the drugs are encapsulated in the nanofibers or added before the homogenization treatment . Former solution , thus isvia a large number of reversible hydrogen bonds, making the sponge with good antibacterial and antioxidant performance. So far, owing to the limitation of the fabrication process, a 3D nanofiber sponge with non-antibiotic single agent having both antibacterial and antioxidant capabilities has rarely been reported.Chitosan (CS), a natural polysaccharide biopolymer with good biocompatibility and low toxicity is widely used in the biomedical arena , 33. Howvia the hydrogen bonding between the PVA and TA endows the 3D-TA sponge with both antibacterial and antioxidant properties. The antibacterial activity, clotting activity and in vitro biocompatibility of the 3D-TA nanofiber sponge were evaluated by plate counting, measuring the hemoglobin content, CCK-8 staining and live/dead assay. The results show that the 3D-TA nanofiber sponges exhibit excellent antibacterial, antioxidant activity and biocompatibility. In addition, in vivo animal experiments show that the 3D-TA sponge could accelerate the healing of full-layer skin defect wounds in mice.Herein, a 3D CS/PVA-TA (3D-TA) nanofiber sponge with high porosity, absorptivity, excellent water retention capacity and compression/resilience property was prepared. The grafting of non-antibiotic TA CS, PVA, tert-butylalcohol and acetic acid (36%) were purchased from Aladdin Reagent Co., Ltd. TA was purchased from Macklin Biochemical Technology Co., Ltd. Anhydrous ethanol was purchased from National Pharmaceutical Co., Ltd. Hemoglobin test solution (C021) and 1,1-Diphenyl-2-picrylhydrazyl radical (DPPH) were obtained from Nanjing JianCheng and glutaraldehyde (2.5%) was purchased from Labcoms. The count assay Kit-8 (CCK-8) was received from Soleibao Technology Co., Ltd. All the reagents and materials were used as received without further treatment.Preparing 2D nanofiber membrane: PVA powder (0.77 g) was added into the deionized (DI) water (7 mL) and stirred at 90 °C until the powder was dissolved. Then, CS (0.09 g) was dissolved in acetic acid solution  and stirred for 4 h for dissolving completely. Finally, the cooled PVA solution is mixed with the CS solution under stirring until both the solutions were mixed completely. For electrospinning, the mixture was encapsulated in a syringe (5 mL) with a needle of 21 gauge. The applied voltage was set as 9 kV with a solution flow rate of 0.04 mm/min. The collecting distance was set as approximately 15 cm from the needle to the collector. The 2D nanofiber membrane was dried for at least 24 h at room temperature (RT) to remove residual solvents.Preparing 3D nanofiber sponges: 2D nanofibrous mats were cut into fragments (1 cm × 1 cm) and crushed by a homogenizer  in tert-butylalcohol. The well-dispersed nanofiber mixture was poured into molds froze in the refrigerator for 12 h. The 3D nanofiber sponges were collected after freeze-drying for 24 h. Finally, the 3D samples were cross-linked in 2.5% (v/v) glutaraldehyde vapor for 12 h at RT.0.5 and 3D-TA1.0) were frozen for 12 h and then freeze-dried for 24 h until the prepared sponges were completely dried.3D nanofiber sponge grafted with TA: The cross-linked 3D samples were immersed in TA solution with concentration of 0.5 mg/mL and 1 mg/mL for 12 h. After washing 3 times with DI water, the 3D sponges  with an applied voltage of 15 kV. The samples were sputter-coated with a layer of gold nanoparticles to avoid the surface charge. The diameter of nanofibers was counted by randomly measuring 100 fibers with a Nano Measurer software.− 1.The chemical groups of 3D nanofiber sponges before and after the grafting of TA were characterized by Attenuated Total Reflection Fourier Transform Infrared Spectroscopy . The samples were placed on the sample holder. The spectrum was recorded over the range of 500–4000 cmww. Then the dry samples were immersed in anhydrous ethanol for 2 h and weighed as wd. The porosity of different scaffolds is calculated according to Eq. ρ and v0 are the density of ethanol and the volume of dry samples, respectively.The porosity of different scaffolds was evaluated by ethanol displacement method . Briefly0.5, 3D-TA1.0) were weighed and placed in DI water for soaking for 1 h at RT. Then the water absorbing rate (ARwater) of different samples was evaluated by gravimetric method. The cyclic water absorption performances of 3D nanofiber sponges were designed to compress the cylindrical water-saturated 3D sponges with 80% strain to extrude most of the absorbed water first. After removing the external force, the deformed sponges were then immersed in DI water again. After 20 cycles of absorption and desorption, the ARwater of 3D nanofiber sponges was obtained by Eq. m0 and m1 are the sample weight before and after absorbing the water.Different samples  of different supports was calculated according to Eq. wm is the weight of the sample after it is put into the incubator, while w0 and w1 are the weight of original dry and hydrated samples, respectively.The water retention of Control, 2D, 3D, 3D-TADR) of different samples via Eq. h0 is the original height of samples while hn is the spring back height of samples after compression.The compressibility of 3D nanofiber sponges was characterized by the following steps. Firstly, the original height of each sample was measured. Secondly, the 3D samples were compressed to 80% height of themselves by universal testing machine . Thirdly, the rebound behavior of 3D nanofiber sponges was recorded after the compression force was removed. Repeating the compression for 20 times to calculate the deformation rate (0.5 and 3D-TA1.0) was put into a centrifuge tube (5 mL) and immersed in 3 mL DPPH solution. Then the samples were placed in a dark environment for 1 h. Changes in absorbance at 517 nm were measured using ultraviolet spectrophotometer . The DPPH radical scavenging activity (SADPPH) was calculated according to Eq. Ablank and Asample are the absorbance of DPPH solution and samples mixed with DPPH solution, respectively. Three parallel samples were used for each sample.DPPH radical scavenging method was utilized to assess the antioxidant activity of 3D-TA. The DPPH solution 39.5 mg/L) was prepared by dissolving DPPH in anhydrous ethanol. Then each sample  were scissored into discs (8 mm) and then placed in the middle of separated petri dishes. The blood (50 µL containing sodium citrate) was instilled onto different sample surfaces and incubated in an incubator at 37 ℃ for 20 min. After taking out the samples from the incubator, the DI water (25 mL) was further added into the petri dish. Then the coagulation of various dressings was recorded.The control group (gauze) and the experimental group  of each sample was obtained according to Eq. OD and OD0 are the absorbance of each sample and the blank, respectively.The blood clots on different sample surfaces were measured quantitatively by dissolving clots in hemoglobin test solution. The ultraviolet spectrophotometer was used to determine the corresponding absorbance at λThe interaction between samples and blood cells was investigated by SEM. Briefly, the samples were soaked in PBS. Then the blood (50 µL) was instilled onto the sample surface. After the samples were placed in a 37 ℃ incubator for 20 min and rinsed with PBS, the blood cells on the samples were fixed with 2.5% glutaraldehyde for 2 h and further dehydration for SEM observation.ACblood) was determined by gravimetry. The samples  were weighed and then immersed into the blood for 30 min. After the excessive blood on different sample surfaces was removed with filter paper, and the swollen samples were weighed again. The ACblood of different samples were calculated according to Eq. M0 and M1 are the weight of the dry sample and the sample with blood, respectively.The blood absorption capacity (E. coli) and Staphylococcus aureus (S. aureus) strains were diluted to 1 × 106 CFU/mL. Then 300 µL of bacterial suspension was instilled to plates containing Control, 2D, 3D, 3D-TA0.5 and 3D-TA1.0 samples, respectively, with a diameter of 8 mm and incubated at 37 ℃ for 24 h. Next, the samples were delivered to a new plate and incubated for 2 h with CCK-8 assay solution. After removing the CCK-8 detection solution, the corresponding OD value of 100 µL solution was obtained.CCK-8 assay: First, Escherichia coli (E. coli and S. aureus suspensions (1 × 106 CFU/mL) at 37 ℃ for 24 h. Then the two bacterial solutions were diluted 1 × 104 times and 1 × 102 times and smeared evenly in the solid medium. The number of colonies was recorded accordingly after incubation for 24 h.Co-culture investigation: Briefly, various samples were co-cultured with E. coli and S. aureus were observed by SEM. Briefly, the E. coli and S. aureus (106 CFU/mL) were incubated with various nanofiber dressings at 37 ℃ for 24 h. After rinsing with PBS, the obtained samples were fixed at 4℃ for 6 h with 2.5% glutaraldehyde. The samples were then dehydrated with a series of ethanol with different concentrations . Finally, the interaction between the bacteria and various samples were characterized by SEM.SEM observation: The influence of different dressings on the antibacterial effects against 2/mL. Firstly 2D, 3D, 3D-TA0.5, and 3D-TA1.0 were sterilized on both sides by UV for 30 min. Secondly, the sterilized samples were placed in high glucose DMEM medium  containing 1% penicillin/streptomycin  extracting for 24 h. Thirdly, 10% fetal bovine serum (FBS)  was added to prepare the material extract for cytotoxicity testing. The fresh cell culture medium (100 µL) containing 5 × 103 L929 cells was added to 96-well plates. After the cells were attached, the extracts of different samples were dropped into 96-well plates with attached cells. Control group was placed in DMEM medium without extract. After 72 h of culture , the cells were stained by Calcein-AM/PI double staining kit for live/dead fluorescence imaging.In order to explore the biocompatibility of different samples, the co-culture of the extract from different samples and cells for 72 h was performed. The extract was prepared according to the national extraction standard of China (ISO 10993-12:2021) when the ratio of material surface area to extraction medium is 3 cm3) were incubated with the extraction in an, incubator at 37 ℃ with 5% CO2 for 1, 3 and 5 days. The cell viability of various samples was assessed by CCK-8 method.CCK-8 method: the extraction preparation was the same as that of co-culture method. The L929 cells  were randomly divided into 4 groups before surgery. The mice were anesthetized by intraperitoneal injection and then the dorsal region of the mice was shaved with a razor and cleaned with alcohol. A circular full-thickness wound with a diameter of 8 mm was generated on the dorsal area of each mice. Control, 2D, 3D, and 3D-TAAfter the mice were sacrificed at the 15th day, the wound tissues of each group were excised and fixed with formaldehyde (4%) in PBS buffer for 24 h. Then the biopsies were embedded by paraffin and bisected afterwards. After staining with hematoxylin-eosin (H&E) and Masson trichrome, the slices were investigated by an optical microscope.The quantified results are exhibited as mean ± standard deviation. Student’s t-test is utilized to evaluate differences between groups, where p < 0.05 indicates statistical significance.The preparation of 3D-TA is shown in Fig. 0.5 and 3D-TA1.0) did not affect the hierarchical structure of 3D nanofiber scaffold but slightly reduced the porosity and specific surface area. That may be due to the fact that with the increase of TA content on the surface and within pores of nanofiber sponge, more hydrogen bonds are formed between fibers and TA, resulting in adhesion between fibers, thus reducing the porosity and specific surface area of the sponge [0.5 and 3D-TA1.0 nanofiber sponge is still higher than that of 2D nanofiber mat with the porosity of 86% and 80%, respectively.The macrostructure of bulk 3D nanofiber sponges is shown in Additional file e sponge . However1.0 nanofiber sponge possesses the characteristic peak of carboxylic acid (C = O) vibration at 1719 cm− 1, and the benzene ring C = C vibration absorption peak at 1606 cm− 1 and benzene ring vibration substitution characteristic peaks at 1219 cm− 1. These characteristic peaks were consistent with that of TA indicating the successful grafting of TA onto the 3D nanofiber sponge. Compared with the hydroxyl characteristic absorption peak of 3D (3437 cm− 1), the hydroxyl characteristic peak of 3D-TA1.0 shifted to the left (3471 cm− 1), which attributed to the intermolecular interaction between PVA and TA resulting in the hydrogen bonding [Whether the TA was successfully grafted onto the 3D nanofiber sponges was verified by FTIR Fig. f. The re bonding . FTIR sp0.5 and 3D-TA1.0 are shown in Fig. ARwater values of 551.4% ± 105.7%, 774.2% ± 209.5%, 1848.4% ± 111.6%, 1533.6% ± 77.0% and 1410.0% ± 64.2%, respectively. It is apparent that the absorption ability of 3D sponge is optimal among all the samples (1848.4% ± 111.6%), much higher than that of Control (551.4% ± 105.7%) and 2D nanofiber membrane (774.2% ± 209.5%). That is because the microscale layered porous structure of 3D sponge enhances the water entry and penetration capacity. Moreover, the hierarchical network structure and high porosity of 3D sponge (96%) provide great water storage capacity, making it a good water absorbent. The water absorption of 3D-TA0.5 and 3D-TA1.0 sponges decreased to 1533.6% ± 77.0% and 1410.0% ± 64.2%, because the grafting of TA decreased the porosity of the 3D sponge (86% and 80%), thus the water absorption of 3D-TA0.5 and 3D-TA1.0 sponges decreased. However, the hierarchical network structure was not affected after the grafting of TA, therefore the water absorption of 3D-TA0.5 and 3D-TA1.0 scaffolds was still much higher than that of Control and 2D nanofiber membranes.Wound dressings should possess good absorption capacity of wound exudates to prevent infection . The wat1.0 were verified by a cyclic experiment  was considered as Control. The higher HC of the tested solution indicates its better coagulation effect. The results show the HCs of Control, 2D, 3D, 3D-TA0.5 and 3D-TA1.0 samples were 26.2 ± 10.3 g/L, 14.4 ± 3.4 g/L, 203.2 ± 2.0 g/L, 273. 0 ± 29.1 g/L, 294.4 ± 11.2 g/L, respectively. It is apparent that the HCs of 3D, 3D-TA0.5 and 3D-TA1.0 were much higher than that of Control and 2D samples. Because the abundant porous structure of 3D, 3D-TA0.5 and 3D-TA1.0 could rapidly soak up blood and inhibit the loss of red blood cells and platelets from the wound, thus significantly enhancing their hemostatic capacity. Besides, the grafting of TA (3D-TA0.5 and 3D-TA1.0) further improved the clotting ability of 3D fiber sponges. That is because polyphenols can form precipitable complexes with proteins in the blood in a non-specific manner to promote blood clotting [To quantify the hemostatic capacity of various samples, the red Fig. b. The HCclotting .ACblood was also evaluated as shown in Fig. ACblood value of 424.8% ± 53.9% and 502.6% ± 74.7%, while the 3D, 3D-TA0.5 and 3D-TA1.0 had excellent blood absorption capacity with the ACblood value of 1211.5% ± 38.9%, 1140.6% ± 52.7% and 1089.6% ± 102.9%, respectively. Again, the highly porous structure of the 3D nanofiber sponges accelerated the blood absorption. The addition of TA could affect the porosity of the 3D nanofiber scaffolds, thus would further affect the blood-sucking ability of the 3D nanofiber sponges. However, the difference is not significant.In addition, the 0.5 and 3D-TA1.0) and blood cells was characterized by SEM  on agar plate after being treated with different samples. The corresponding colony count result is shown in Fig. 0.5 and 3D-TA1.0) have excellent antibacterial performance compared with the Control, 2D and 3D samples. Moreover, the bacteriostatic effect enhances with the increase of TA concentration because TA can inhibit the expression of toxin genes in bacteria inducing cell membrane damage, thus leading to the death of bacterial[S. aureus is higher than that of E. coli. Because E. coli has an additional lipopolysaccharide cell wall, which is an effective barrier against antibacterial agents and is more resistant to plant extracts [E. coli  after co-culturing for 72 h. The fluorescence images were consistent with those of CCK-8 results, which demonstrated the good biocompatibility of 3D-TA1.0.In addition, the live-dead cell experiment was also implemented to verify the good biocompatibility of different samples. Figure 1.0) at various day intervals . It is demonstrated that the wound size of all groups decreased with time after treatment. Especially in the 3D-TA1.0 group, the reduction of the wound area was the fastest with an almost completely healing at the 15th day while the other three groups covered by Control, 2D and 3D were much larger than that treated with 3D-TA1.0. The quantitative analysis of the percentage of wound size over the same time period from the photographs , the wound area covered by 3D-TA1.0 was reduced to 34.55% ± 1.88% while that covered by Control, 2D and 3D was 85.09% ± 3.48%, 43.59% ± 1.06% and 45.33% ± 2.91%, respectively. On the 9th day, the wound area of Control, 2D, 3D and 3D-TA1.0 groups were 23.06% ± 2.79%, 21.95% ± 3.86%, 19.05% ± 2.18% and 12.64% ± 2.74%, respectively. It is also indicated that the wound closure ability of 3D group was better than that of Control and 2D group. At 15 days, the wound in 3D group was basically healed and the wound in 3D-TA1.0 group was completely healed without extensive scar and epidermal tissue defect, whereas the obvious wounds were still existed in the other experimental groups. The quantitative results were consistent with the macroscopic observation results, indicating the effective improvement of skin tissue regeneration and promoted cell proliferation capacities of 3D-TA1.0.To verify the cellular results in vitro, the in vivo wound closure efficiency was evaluated by treating full-thickness wound on the dorsal region of mice with different dressings. The medical gauze was used as Control. Figure phs Fig. a is show1.0 underwent more epidermal tissue regeneration (indicated by black arrows) and the presence of fewer inflammation cells (indicated by brown arrows) than that treated with Control, 2D and 3D samples. Moreover, the 3D-TA1.0 treated wound exhibited more angiogenesis (indicated by red arrows) and hair follicle cells (indicated by green arrows). The angiogenesis could promote the migration of fibroblasts to the injured site and maintain low levels of inflammation, which eventually leads to a regenerated upper cortex on the wound [1.0 and the arrangement of collagen fibers was more densely and orderly, similar to normal skin [To confirm the therapeutic effect of various dressings on wounds, the wound tissues dissected after 15 days of treatment were subjected to hematoxylin and eosin (H&E) and Masson’s trichrome staining Fig. . The H&Ehe wound , promotihe wound . The Masmal skin .1.0 exhibit excellent wound healing ability, which is attributed to the unique hierarchical porous structure of 3D nanofibers sponge facilitating nutrient transport, increasing the air permeability, promoting fibroblast proliferation during wound healing. Moreover, the TA-functionalized 3D sponge endows the 3D-TA sponge with excellent antioxidant and anti-inflammatory properties. The decrease of ROS can reduce inflammatory response, reduce the secretion of pro-inflammatory cytokines, increase the number of macrophages, and trigger the generation of collagen around the wound [Overall, the 3D-TAhe wound , 52. In 1.0 nanofiber sponge was prepared via electrospinning, which had high porosity, water absorption and retention ability, hemostatic capacity and keep the wound microenvironment moist simultaneously. Moreover, the 3D-TA1.0 exhibited excellent antioxidant properties and antibacterial ability. The cellulous experiments showed that 3D-TA1.0 had good cell compatibility, which could promote cell adhesion and proliferation. In addition, the promoted wound healing ability of 3D-TA1.0 was verified by in vivo experiments. This study indicates that the prepared 3D-TA1.0 nanofiber sponge could be an ideal alternative to conventional dressings for future clinical applications.In conclusion, a 3D-TAAdditional file 1: Fig. S1 Macroscopic morphology of 3D nanofiber sponge.a Comparison of 3D nanofiber sponge and 2D nanofiber membrane. The photographsof 3D sponges with different b heights and c morphologies prepared via different molds. Fig. S2 Compression/resilience process. The compression/resilience process of 3D and 3D-TA1.0 nanofibersponges. Fig. S3 Cell Live-dead staining. Live-dead staining florescent images of L929 co-cultured with differentsample extracts for 3 days.


RAD21 subunits of cohesin. SGO1 shares this binding interface with CTCF, indicating that these distinct chromosomal regulators control cohesin through a universal principle. This interaction is essential for the localization of SGO1 to centromeres and protects centromeric cohesin against WAPL-mediated cohesin release. SGO1–cohesin binding is maintained until the formation of microtubule–kinetochore attachments and is required for faithful chromosome segregation and the maintenance of a stable karyotype.In the early stages of mitosis, cohesin is released from chromosome arms but not from centromeres. The protection of centromeric cohesin by SGO1 maintains the sister chromatid cohesion that resists the pulling forces of microtubules until all chromosomes are attached in a bipolar manner to the mitotic spindle. Here we present the X-ray crystal structure of a segment of human SGO1 bound to a conserved surface of the cohesin complex. SGO1 binds to a composite interface formed by the SA2 and SCC1 Here, the authors determine the structural basis of how SGO1 protects centromeric cohesin from WAPL-mediated release until the kinetochores and the microtubules have formed functional attachments. In addition, they show that SGO1 and CTCF bind cohesin in a similar manner, suggesting a potential universal mechanism of cohesin control. Cohesin entraps DNA inside its ring-shaped structure4, which at its core consists of SMC1, SMC3 and SCC1 . SCC1 is bound by either of two paralogous HEAT repeat-containing proteins, SA1 or SA2 5.During mitosis, the duplicated genome needs to be accurately distributed over the two daughter cells. The cohesin protein complex holds together the sister DNAs from replication until mitosis6. During mitosis, cohesin is removed from chromosomes in two waves. First, cohesin is removed from chromosome arms in a WAPL-dependent manner through a process known as the prophase pathway10. Cohesion at centromeres is protected by Shugoshin (SGO1)14, giving rise to the typical X-shaped structure of human chromosomes. SGO1 protects centromeric cohesin by recruiting PP2A to counteract cohesin phosphorylation by mitotic kinases, and SGO1 also directly competes with WAPL for cohesin binding18. Centromeric cohesion is maintained until proper attachment of microtubules to the kinetochores, upon which the remaining cohesin is cleaved by separase to trigger anaphase onset19. By protecting centromeric cohesion, SGO1 thus ensures faithful chromosome segregation.Cohesin complexes have a dynamic mode of DNA binding that involves DNA entrapment and release. From DNA replication until mitosis, the cohesin complexes that hold together the sister DNAs are locked on DNA to render cohesin resistant to cohesin’s release factor WAPL20. We showed recently that CTCF binds to cohesin through a conserved YxF motif in the amino terminus of CTCF. This CTCF segment interacts directly with a composite binding interface formed by the SA2 and SCC1 subunits of cohesin21. The SA2 interface is conserved from fungi to mammals and is known as the conserved essential surface (CES)22. For simplicity, we refer to the composite SA2–SCC1 binding pocket as the CES. The direct interaction of the YxF motif of CTCF with the CES is required for formation of CTCF-anchored loops at TAD boundaries21. It has also been suggested that the CES region interacts directly with both SGO1 and WAPL18. The interaction of SGO1 with cohesin is promoted by phosphorylation of SGO1 T346. However, this phosphorylation does not control the direct interaction between the CES and SGO1 (refs. 18). SGO1 contains a YxF motif close to this phosphorylation site that could be key to this interaction. In agreement, previous biochemical experiments have shown that an SGO1 fragment containing the YxF motif can directly compete with CTCF for binding to the CES21. If so, cohesin complexes may be controlled through a shared mechanism, irrespective of whether these complexes build DNA loops or rather hold together the sister DNAs.Cohesin has a dual role, as it not only holds together sister DNAs but also builds the DNA loops that shape the interphase genome. To control this latter function, cohesin is bound by the architectural factor CTCFWe report here the X-ray crystal structure and AlphaFold model of the cohesin subcomplex SA2–SCC1 bound to a fragment of SGO1 Fig. . We demo18. The interacting region contains a YxF motif that is conserved in vertebrate SGO1 proteins , enhances the interaction with SA2–SCC1 but is not essential for binding18. We were able to obtain crystals with a SGO1 peptide spanning amino acids 331–341 containing the YxF motif but not with an extended peptide spanning amino acids 331–349 containing pT346. We determined the X-ray structure by molecular replacement to a minimum Bragg spacing of 3.2 Å  experiments showed that the T346-phosphorylated SGO1 fragment spanning amino acids 331–349 bound SA2–SCC1 with a lower equilibrium dissociation constant (2.3 ± 0.4 μM) compared with a nonphosphorylated SGO1 peptide spanning amino acids 331–341 (13.5 μM ± 1.4) Fig. . Using gY335A F337A as their sole copy of SGO1 . As expected, this SGO1 depletion yielded a massive cohesion defect, but this defect was no stronger than the defect observed in the SGO1Y335A F337A cells. SGO1 depletion in SGO1Y335A F337A cells also barely if at all worsened the cohesion defect of these cells -tagged SGO1 that was either wild type or harbored the SGO1Y335A F337A mutation. We then scored for SGO1 localization by immunofluorescence chromosome spreads, comparing the absence versus the presence of microtubule–kinetochore attachments, using nocodazole- or MG132-treated cells, respectively. In nocodazole-treated cells, wild-type SGO1–GFP localized to the inner centromere as expected. The SGO1Y335A F337A mutant, however, did not localize to the inner centromere and was primarily found at the kinetochores . Here, SGO1 through CES binding protects centromeric cohesin from the WAPL-dependent prophase pathway. Upon establishment of bipolar microtubule attachment, SGO1 then relocates towards kinetochores25 , STAG1W337A (5′-AGTACTGAGACAAACATAACTTCCATCAAAGCTTAGAACAG AGTAACTTACCCTGTCGTGAAGAGTAGCGCCAACATATTTTAGGTAACTGTCATTTAGGAAGGCATCACTATACATTTTCATC-3′) and STAG2W334A (5′-CTTAATGACAGTTATTTAAAATATGTTGGTGCGACTATGCATGATAAGGTAAGATGTGCCCTTCAGACTGCTTCTTTCTATACATCGGCGTGGCTGTCTGCACCTCTCATTCATGAG-3′). We cotransfected pBabePuro at a ratio of 1:10 to the pX330 plasmid. Cells were treated with 2 μg μl−1 puromycin for 2 days for selection. Colonies were picked, genomic DNA of clones was isolated and mutations were validated by Sanger sequencing.HAP1 cells were cultured in Iscove’s modified Dulbecco’s medium (Invitrogen), containing 10% fetal bovine serum (Clontech), 1% UltraGlutamin (Lonza) and 1% penicillin–streptomycin (Invitrogen). Mutant cells were generated by CRISPR–Cas9 technology. Guide RNAs targeting exon 6 of SGO1 , exon 10 of STAG1 (5′- TTGGCTGGACTCTTCATGAC-3′) and exon 11 of STAG2 (5′-GACAGTTATTTAAAATATGT-3′) were annealed into pX330. To mutate the locus of interest, we cotransfected a 100–120 base pair repair oligonucleotide with the desired mutation as well as a silent mutation: for SGO1YAll siRNAs were manufactured by Dharmacon (ON-TARGETplus). For SGO1 and luciferase we used SMARTpools, and for WAPL we used the following sequence: 5′-CAACAGUGAAUCGAGUAAUU-3′. Transfection was performed with 20 μM per siRNA final concentration, using Invitrogen RNAiMAX (Life Technologies), following the manufacturer’s instructions.31. Images were randomized by a homemade ImageJ macro and then visually assigned their corresponding phenotype. A parametric two-tailed t test was used to compare the scoring of cohesion phenotypes.Cells were transfected with the corresponding siRNAs, and after 2 days the cells were treated with nocodazole as described previously31. For immunofluorescence spreads, cells were treated with the corresponding siRNA. After 24 h, cells were transfected using FuGENE transfection reagent (Promega) with 0.8 μg SGO1–GFP plasmid (kindly provided by S. Lens) or a SGO1Y335A F337A–GFP mutant plasmid. One day later, cells were treated with nocodazole for 1.5 h or with MG132 for 2 h, and mitotic cells were collected by shake-off. Cells were washed once in phosphate-buffered saline, followed by a quick spin onto microscope slides with a Shandon Cytospin centrifuge. Cells were extracted with PBS containing 0.3% Triton-X for 5 min and fixed in 4% paraformaldehyde for 15 min. The coverslips were washed three times with PBS containing 0.1% Triton-X before being incubated with antibodies at a 1:1000 dilution in PBS containing 3% BSA and 0.1% Triton-X overnight at 4 °C. Secondary antibody incubations were performed by incubation at room temperature for 1 h with DAPI in PBS containing 3% BSA and 0.1% Triton-X. Coverslips were mounted in Prolong Gold (Invitrogen).For immunofluorescence, cells were treated with nocodazole, fixed and stained as described previouslyImages were obtained using a DeltaVision deconvolution microscope (Applied Precision), and images were acquired using Softworx (Applied Precision) and ImageJ. To establish levels of SGO1 in prometaphase cells, we used an ImageJ macro that allowed us to calculate the level of SGO1 relative to CENPA. To identify the location of SGO1–GFP in mitotic cells, we first blinded the channel corresponding to GFP to prevent bias towards a phenotype. Next, we drew a straight line on four random chromosomes that showed two distinct centromeres and obtained the plot profile of both CENPA and GFP for each location.Cells were grown on glass-bottomed dishes (LabTek). To visualize the DNA, 2 h before imaging, a SiR-DNA probe  was added. Images were taken using a DeltaVision deconvolution microscope (Applied Precision). Cells were imaged every 5 min using a ×40 air objective with 4 × 2.5 μm Z stacks. Images were acquired using Softworx (Applied Precision) and ImageJ.Prometaphase samples cells were obtained as described above. Fixed cells were dropped on cover slides and then dried. We added probes against the centromere of chromosome 8  and shielded the cells with a coverslip and rubber cement. The slides were incubated for 2 min at 75 °C, followed by overnight incubation at 37 °C. The cells were washed with 0.4× SSC at 72 °C for 2 min, followed by washing at room temperature with 2× SSC, 0.05% Tween-20, for 30 s. The slides were washed with water and stained with DAPI, followed by mounting with Prolong Gold (Invitrogen).G2 samples were collected by treating the cells for 18 h with RO-3306. We verified that the cells were synchronized in G2 by incubation in Nicoletti buffer followed by flow cytometry (BD LSRFortessa). Plots were generated with FlowJo (v.10). G2-synchronized cells were spun down and resuspended with fixative solution , followed by the same protocol as described above.32.Images were taken using a DeltaVision deconvolution microscope (Applied Precision), and images were acquired using Softworx (Applied Precision) and ImageJ. The fluorescence signal was categorized as singlet  or doublet , as described previously33.Immunoblot and coimmunoprecipitation were performed as previously describedThe following antibodies were used as primary antibodies for immunofluorescence microscopy: SGO1 , GFP  and CENPA . For immunoblotting, the following primary antibodies were used: SA1 , SA2 , SMC1 , SCC1 , WAPL , Sororin , HSP90 (sc-13119(F-8), Santa Cruz) and α-tubulin . All primary antibodies were used at a 1:1000 dilution with the exception of HSP90 and α-tubulin (1:10000). For coimmunoprecipitation, we used 4.5 μg of SMC1  or IgG  per sample. Secondary antibodies were used at a 1:1000 dilution. For immunofluorescence microscopy we used: Alexa Fluor 488 goat anti-mouse , Alexa Fluor 568 goat anti-mouse , Alexa Fluor 488 goat anti-rabbit  and Alexa Fluor 568 goat anti-rabbit . For western blots, we used the following secondary antibodies: anti-goat-PO , anti-rabbit-PO  and anti-mouse-PO .21. Expression and purification were done as described previously21. SGO1 constructs were cloned into the BamHI and NotI sites of pGEX-6P1. Mutagenesis was done using a Q5 Site-Directed Mutagenesis Kit (New England Biolabs). All proteins were expressed in Escherichia coli BL21 (DE3) by autoinduction, and purification was done as described previously21.SA2 amino acid residues 80–1060 were expressed as a GST fusion protein and SCC1 amino acid residues 281–420 as an N-terminally 6×His-tagged fragment as described previously21. Crystals were soaked for 7 days with a 500 μM peptide solution including SGO1 amino acid residues 331–341 SNDAYNFNLEE. Crystals were cryoprotected as described previously21. Diffraction data were collected at 100 K at an X-ray wavelength of 0.9687 Å at beamline ID30A-1/MASSIF-1 (ref. 34) of the European Synchrotron Radiation Facility, with a Pilatus3 2M detector, using automatic protocols for the location and optimal centering of crystals35.Crystallization of the SA2–SCC1 complex was done as described previously36 and imported into CCP4 format using AIMLESS37. The structure was determined by molecular replacement using Phaser (Phenix 1.14-3260)38. A final model was produced by iterative rounds of manual model-building in Coot (COOT 0.8.0-3)39 and refinement using PHENIX (1.14-3260)40. The SA2–SCC1–SGO1 model was refined to a resolution of 3.2 Å with Rwork and Rfree values of 25% and 28%, respectively . The computational model shown in Fig. 42. The computational model shown in Fig. Data were processed with XDSr Phenix .14-3260321 with small modifications. Briefly, 50 μM GST-tagged SGO1 constructs were mixed in 50 μl buffer 1  containing 25 μl of a 50% slurry of GST Sepharose beads (Cytiva) per reaction. GST beads were incubated for 1 h at 4 °C, followed by four washes with 500 μl of buffer 1. Then, 2.5 μM of SA2–SCC1 was added, followed by overnight incubation at 4 °C. A 25-μl volume of the reaction was withdrawn as the reaction input, and the remainder was washed five times with 500 μl of buffer 1. Samples were boiled in 1× sodium dodecyl sulfate (SDS) sample loading buffer (New England Biolabs) for 5 min to obtain the bound fraction, followed by SDS polyacrylamide gel electrophoresis analysis. ITC was performed as described previously21. ITC data were analyzed with Origin 7.0.GST pulldowns were done as described previouslyt test analyses, with a probability threshold of P = 0.05. GST pulldowns were repeated at least three times with consistency.No statistical method was used to predetermine the sample size. No data were excluded from the analyses. All experiments with phenotype calling were randomized, and the SGO1 signal was blinded in all experiments for SGO1 localization with respect to the centromere. Data were visualized with Prism 9. For all pairwise comparisons, we performed Further information on research design is available in the Any methods, additional references, Nature Portfolio reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at 10.1038/s41594-023-00968-y.Supplementary InformationSupplementary Fig. 1 FACS gating strategy.Reporting Summary


A complex network of signaling proteins participates in this reaction. Here, we identify key players that regulate intracellular pH (pHi) and HCO3− in human sperm by quantitative mass spectrometry (MS) and kinetic patch-clamp fluorometry. The resting pHi is set by amiloride-sensitive Na+/H+ exchange. The sperm-specific putative Na+/H+ exchanger SLC9C1, unlike its sea urchin homologue, is not gated by voltage or cAMP. Transporters and channels implied in HCO3− transport are not detected, and may be present at copy numbers < 10 molecules/sperm cell. Instead, HCO3− is produced by diffusion of CO2 into cells and readjustment of the CO2/HCO3−/H+ equilibrium. The proton channel Hv1 may serve as a unidirectional valve that blunts the acidification ensuing from HCO3− synthesis. This work provides a new framework for the study of male infertility.The reaction of CO 3−) is critical in sperm for stimulation of cAMP synthesis during fertilization, though there is dispute over how  HCO3− is transported into sperm. Here the authors use limit-of-detection LC/MS to characterize sperm protein expression and show that HCO3− is produced from CO2 diffusion into sperm rather than active transport.Bicarbonate (HCO Arguably, the most important cellular reaction that controls motility and capacitation of sperm is the CO2 + H2O ⇄ HCO3− + H+ equilibrium (CO2/HCO3−/H+). HCO3− and H+ feed into a sprawling network of signaling proteins that translate the state of this equilibrium into changes in cAMP and Ca2+, the intracellular messengers . The CO2/HCO3−/H+ reaction is catalyzed by carbonic anhydrases (CA). The extra- and intracellular CO2/HCO3−/H+ equilibria are coupled across membranes not only in sperm but also surrounding epithelia cells by CO2 diffusion, and HCO3− and H+ transport via ion channels and solute carriers (SLC).Fertilization is a complex multi-stage process. After ejaculation, a maturation process, called capacitation, sets in, whereby sperm become motile and acquire the ability to locate and fertilize the egg. During capacitation, sperm adopt a whiplash-like flagellar beat pattern, called hyperactivation, that is required to penetrate the protective egg investment3− concentration that stimulates cAMP synthesis by a HCO3−-activated soluble adenylate cyclase (sAC)9, which initiates protein kinase A (PKA)-mediated phosphorylation that, among other events, enhances flagellar beat frequency11. A second hallmark of capacitation is an increase of intracellular pH (pHi), which activates the exquisitely pH-sensitive, sperm-specific Ca2+ channel CatSper12. The Ca2+ influx is important for sperm hyperactivation13, although the extent of this regulation varies among mammals. Moreover, Ca2+ and alkaline pHi activate the sperm-specific Slo3 K+ channel in human and mouse, respectively15. Finally, hyperpolarization of the membrane potential reportedly is key for capacitation2. The physiological consequences of the hyperpolarization are not precisely known.Three key events reportedly are required for capacitation: first, an increase of intracellular HCO4. HCO3− has been proposed to enter sperm via transporters of the solute carrier families SLC4 (ref. 17) and SLC26 (ref. 22). HCO3− could also enter through a Cl− channel, the cystic fibrosis transmembrane regulator (CFTR)27. Alternatively, CFTR might balance Cl− fluxes associated with Cl−/HCO3− exchange, and reportedly affects HCO3− transport by interactions with SLC4 and SLC26 transporters28 and with the epithelial sodium channel (ENaC)27. Finally, HCO3− could be synthesized from CO2 that enters by diffusion across the cell membrane. The mechanism by which HCO3− rises is crucial because it determines whether sperm alkalize or acidify.Many proteins and mechanisms have been implied in the execution and coordination of these three signaling events Fig. 2,4. HCO3i could be accomplished by several different mechanisms: H+ efflux via the voltage-gated proton channel Hv1 (HVCN1)30 or two different types of sodium/proton (Na+/H+) exchangers—sperm-specific sNHE (SLC9C1)33 and the Na+/H+ antiporters NHA (SLC9B1 and SLC9B2)35. Alternatively, by feeding into and readjusting the CO2/HCO3−/H+ equilibrium, which is catalyzed by carbonic anhydrases CA2 and CA4 (ref. 37), HCO3− influx could alkalize sperm indirectly. Finally, hyperpolarization could be brought about by activation of Slo3 K+ channels, HCO3− or Cl− influx via CFTR channels, blockage of Na+ inward currents via ENaC channels, or H+ efflux via Hv1 channels. Because membrane potential Vm is set by the balance of inward and outward currents, any one of these various channels may participate in producing the hyperpolarization  In summary, the literature abounds with competing hypotheses how the increase of HCO3−, alkalization, and hyperpolarization is generated. This is not surprising, considering that many of the chemical reactions and molecular components are coupled and cannot be varied independently from each other.The increase of pH6. Hv1 channels exist in human and boar but not mouse sperm39. The K+ channel Slo3 is controlled by pHi in mouse41 but primarily by Ca2+ in humans14. The Ca2+ channel CatSper is activated by the female sex hormone progesterone in human43 but not mouse42.The complexity is all the more perplexing as both the properties of these signaling molecules and the protein inventory of sperm differ markedly among mammalian speciesi/HCO3− network, and the generalization of concepts across phyla must be viewed with due caution6.Considering the diversity of sperm-specific molecules and the uncertainty of their actions, it is a daunting task to unravel this perplexing cellular pH47. Moreover, fertilization phenotypes may result from defects in spermiogenesis, and targeted proteins that exist in precursor cells may be absent from mature sperm.Our present knowledge about the presence of these signaling proteins in human sperm is largely based on immunodetection, the pharmacology of signaling components, and on concepts derived from knockout-mouse lines or other mammalian species. However, Fc binding sites on sperm can confound immuno-detection, and some drugs display multiple off-target actions+ and Na+ fluxes in single sperm cells using fluorescence microscopy and patch-clamp fluorometry. We show that CO2/HCO3− acidifies rather than alkalizes sperm, and that synthesis of HCO3− from CO2 dominates HCO3− entry via HCO3− solute carriers. Finally, we propose that H+ efflux by the Hv1 proton channel keeps the acidification resulting from HCO3− synthesis at bay and, thereby, sustains high HCO3− levels inside sperm.Here we examine the presence or absence of signaling proteins in human sperm using quantitative liquid chromatography (LC)-coupled mass spectrometry (MS). We determine the copy number of several signaling proteins and, using limit-of-detection (LOD) values, we estimate the maximal copy number for those proteins, which we could not detect. The large dynamic range between copy numbers and LOD values seriously constrains hypotheses about the physiological function of molecules that are either absent or of low abundance. We corroborate the MS data by functional measurements of H51 that differ markedly in size and in key signaling components (Supplementary Table 50). In addition, targeted MS studies of human sperm have also been performed52. Here, we determined the proteome from three different donors by LC-MS/MS ; the Slo3 channel and its auxiliary subunit LRRC5214; the Na+/H+ exchangers SLC9B1 and 2 (ref. 34), carbonic anhydrases CA2 and CA4 (refs. 37), and the Hv1 channel39. Regarding sNHE, we detected two isoforms—SLC9C1 and SLC9C2; SLC9C2 is an orphan protein that was discovered genomically but has not yet been detected or characterized in any cell type57. Most of these components are integral membrane proteins that are notoriously more difficult to detect than soluble proteins. Moreover, all enzymes and targets involving cAMP signaling were detected, including the soluble adenylyl cyclase sAC9, the phosphodiesterase PDE10 (ref. 58), all catalytic and regulatory subunits of protein kinase A (PKA), the PKA-scaffolding proteins AKAP3 and 4, and the sperm-specific phosphatase PPP1CC59.We searched our data notably for those proteins that had been implied in transport of HCOa2+ Fig. . All sig50. With very few exceptions, our MS data agree with a previously published proteome of similar size51  speaks to the sensitivity, robustness, and reliability of our approach. In previous proteomic studies only CatSper1, 2, 4, B, and D were identified60 are PKA subunits, the anchor proteins AKAP3 and 4, carbonic anhydrase CA2 and 4, the Na+/K+-ATPase subunits ATP1A4 and ATP1B3, the Ca2+-transporting ATPase ATP2B4, the phosphatase PPP1CC, and the proton channel HVCN1  or its auxiliary ß and γ subunits. Proteins that had been commonly implied in HCO3−/pHi/Cl− sperm signaling but were not detected by LC-MS/MS in all three samples are assembled in a ‘negative’ list  and, except one protein, have also not been detected by another comprehensive MS study of human sperm51. Furthermore, we ascertained that protein extraction was complete using harsh extraction conditions followed by protein aggregation capture (PAC)63, and deep proteomic profiling was ensured by off-line basic reverse-phase chromatography as an additional separation step prior to LC-MS/MS.Unexpectedly, we could not detect many proteins implied by previous studies either directly or indirectly in HCO3− transport  for CA2; 23,100 ± 15,400 (n = 4) for SLC26A3; 2500 ± 1800 (n = 4) for CatSper 1; 7100 ± 2500 (n = 4) for CatSper 2; 5000 ± 2600 (n = 4) for CatSper 3; 2700 ± 1300 (n = 4) for CatSper 4; 5400 ± 1000 (n = 4) for CatSper B; and 5300 ± 2500 (n = 3) for Slo3.Next, we explored the dynamic range between minimum copy numbers defined by LOD values of proteins that were not detected and copy numbers of signaling proteins that were detected. Indeed, the absolute abundance of any protein from the positive list has not yet been reported. To initiate benchmarks, we determined the copy numbers of a few selected proteins by titration with standard peptides including CA2, SLC26A3, CatSper1-4, CatSperB, and Slo3  for sperm from two distant species—sea urchin and humans—overall the copy numbers of CatSper subunits are of similar scale (between 2000 and 8000 molecules/cell) . Alternatively, the CatSper density probed by tomography may not be uniform along the flagellum. Whatever the reason for this difference might be, both studies report a high density of CatSper channels in the flagellum. Additionally, the copy numbers of two different solute carriers, SLC26A3 in human and SLC9C1 in sea urchin sperm, are also similar . In conclusion, the copy numbers of three different types of proteins—ion channels, solute carriers, and a CA enzyme—are one to four orders of magnitude higher than the typical range of LOD values .The precision of PRM analysis using standard peptides primarily depends on the nature of the protein (soluble or membrane-spanning), the choice of suitable standard peptides, the digestion conditions, and the modification of endogenous peptides3− transport or regulation of the CO2/HCO3−/H+ equilibrium. We recognize the iconoclastic nature of these results and, therefore, discuss here potential pitfalls and the relative weight of each result. Our conclusions rest on several rationales.These results question the presence of several proteins that have been reported to be key players either directly or indirectly in HCO3− or Cl− that operate in the millimolar range.First, low copy numbers derived from peptide-specific LOD values are difficult to reconcile with a physiological function. For example, transport capacity should be able to significantly change substrate concentrations within a few seconds. SLC transporters feature transport rates of a few hundred substrate molecules/s; thus, even if present, copy numbers in the range of <10-20 molecules/cell could not substantially change concentrations of substrates such as HCO3− transporters of the SLC4 and SLC26A families have been proposed to physically interact with CFTR and with each other to form heterodimers, and that HCO3− enters sperm via these complexes68. Our data seriously constrain hypotheses about such potential interactions. A case in point is the proposed interaction between SLC4A4 and SLC26A318: the LOD value of SLC4A4 ( < 10 copies/cell) is ~2,000-times lower than the copy number of SLC26A3 . Thus, even if SLC4A4 were present, SLC26A3 would be the physiologically dominant HCO3− transporter present in human sperm, unless its transport number is several orders of magnitude lower than that of SLC4A4. The large mismatch between LOD values of SLC4A4 and high copy numbers for SLC26A3 also shows that hetero-dimers either do not exist or are too rare to be physiologically relevant in human sperm. The same argument holds for potential interactions between CFTR/ENaC channels and the SLC26A3 transporter. This comparison underscores that the physiological significance of these low-abundance proteins either on their own or in complex with other abundant proteins needs to be revisited.Second, the wide dynamic range between such low LOD values vs. the high copy numbers of proteins that serve similar functions precludes any prominent role of the low-copy-number proteins. Moreover, several known or potential HCO3− transport and hyperpolarization of mammalian sperm. It is disputable as to how CFTR or ENaC channels can affect the membrane potential in the presence of the Slo3 channel that features a 10-fold larger single-channel conductance (CFTR vs. Slo3 is 7.9 pS vs. 70 pS)14 and is up to a 1000-fold more abundant. In sperm of a CatSper-/- and Slo3-/- double knock-out mouse, no other conductance above the background or leak conductance is detected69, excluding currents carried by CFTR and ENaC channels also in mouse sperm. In agreement with these physiological results in mouse, we could not detect CFTR and ENaC proteins in human sperm, and, according to our LOD values, maximally between 10 and <40 molecules/cell would be present if any. CFTR has been implied in capacitation of human sperm based on the inhibitor CFTRinh-172 (ref. 70) This compound also inhibits the H+/Cl− exchange transporter 3 (CLCN3)71. Indeed, CLCN3 was identified in our proteome analysis, suggesting that the effect of CFTRinh-172 on capacitation might result from its action on CLCN3.For additional reasons, CFTR and ENaC channels may be irrelevant for HCO-/- and SLC26A8-/- mice that suffer from subfertility72. SLC4A4-/- mice do not survive breeding age; therefore, the fertility phenotype is not known. These SLC members are expressed elsewhere in the male reproductive tract, where they serve important roles in spermiogenesis72. The SLC26A family is diverse, features various and often unclear substrate specificities, and it serves broad functions73. Once the substrate specificity and turnover number of SLC26A3 is known, its contribution to HCO3− metabolism relative to CO2 diffusion can be more quantitatively estimated.In support of these conclusions, human mutations or targeted disruption in mice of SLC26 members do not cause fertility phenotypes, except for SLC26A3+/K+- ATPase. Yet, even those proteins use some subunits that are sperm-specific. This almost perfect all-or-none pattern reflects the historical course of fertilization research. Scientists chose signaling systems from somatic cells and hypothesized that related signaling pathways, furnished with similar molecules, operate in sperm. A case in point are concepts of olfactory and taste signaling in the nose and the tongue, respectively that were adopted as a signpost that guide the study of chemosensation in sperm. Only later was it recognized that sperm employ sperm-specific molecules that are not expressed in somatic cells. We have also failed to detect a plethora of those somatic proteins implied in chemotaxis or thermotaxis of sperm that, however, are not considered here. Our LOD approach holds promise to provide definitive answers as to which molecules are involved in these sensing mechanisms and anticipate that other proteins from the negative list . By readjustment of the CO2/HCO3−/H+ equilibrium, influx of HCO3− or intracellular synthesis of HCO3− from CO2, will either alkalize or acidify the cell, respectively. Therefore, we examined changes in pHi that might provide cues for mechanisms of H+ and HCO3− transport. We recorded pHi in intact single human sperm cells using the fluorescent pH-indicator pHrodo Red 74, before sperm returned slowly to resting pHi. Restoration of pHi required extracellular Na+: the acidification persisted when ES/NH4Cl was replaced by ES that contained NMDG+ instead of Na+ (to inactivate Na+/H+ exchange), demonstrating that pHi is adjusted by Na+/H+ exchange . In conclusion, sperm recover from acidification by amiloride-sensitive Na+/H+ exchange. Because SLC9A1 is absent from sperm, SLC9B1, SLC9B2, and SLC9C1/2 are the remaining candidates for Na+/H+ exchange. However, human SLC9B2 and sea urchin SLC9C1 are insensitive to amiloride76, and amiloride sensitivity of SLC9B1 is not known.The MS analysis revealed that components previously implied in HCORed Fig. . The dye+]o Fig. . We expo+]o Fig. . The dyenge Fig. . Acidifi+/H+ exchange. A unique hallmark of sNHE activity in sea urchin sperm, which sets it apart from all other NHEs, is gating by hyperpolarized Vm and modulation by cAMP77. Voltage gating and cAMP sensitivity rest on a channel-like voltage-sensor motif (VSD) and cyclic nucleotide-binding domain (CNBD) that are absent from all other NHEs31. To test for hyperpolarization-triggered Na+/H+ exchange in human sperm, we employed patch-clamp fluorometry (PCF). Sperm were loaded via a patch pipette with the pH dye pHrodo Red  of human sperm79, where sNHE and Hv1 are not active. A step from −30 mV to +70 mV, which activates Hv1, evoked a proton outward current and a concomitant pHi increase 31. However, even in the presence of HCO3−, which elevates cAMP in mammalian sperm80, no hyperpolarization-induced alkalization was detected , Na+/H+ exchange via SLC9C1 in mammals may be mechanistically and pharmacologically different. The lack of voltage gating is not entirely surprising given that three of four key Arg residues in the S4 voltage-sensor motif of sea urchin SLC9C1 are absent in the mouse and human SLC9C1orthologues31. Considering that SLC9C2 is also present in human sperm, we speculate that SLC9C1 and SLC9C2 forms heterodimers that may adopt new properties and mediate amiloride-sensitive Na+/H+ exchange via a classical alternating-access mechanism81 rather than by Vm gating and modulation by cAMP. Alternatively, mammalian SLC9C1 may transport substrates other than Na+ or H+. This conclusion is also plausible because a highly conserved Asn-Asp or Asp-Asp motif, which is key for coordination of Na+ ions in Na+/H+ exchangers76, is lacking in mammalian SLC9C1 (ref. 31) Moreover, SLC9C1-/- sperm do not show any alteration in resting pHi32, something to be expected when Na+/H+ exchange is abolished. Finally, Na+/H+ exchange activity of heterologously expressed SLC9C1 is weak if not absent32. In this scenario, it is probably SLC9B1 that controls Na+/H+ exchange in human sperm. Indeed, SLC9B1 predominantly mediates the zona pellucida-induced pHi increase that is greatly attenuated in SLC9B1-/- mouse sperm35. Definitive answers are awaited from SLC9B1-/-/SLC9B2-/- double KO mouse and functional co-expression of SLC9C1/SLC9C2.sNHE/SLC9C1 is sperm-specific, belongs to the SLC9 family of Na-/- sperm are lacking the full-length form of sAC, and motility and infertility phenotypes can be rescued in vitro by cAMP35. Thus, the primary defect of SLC9C1-/- sperm may be in cAMP rather than pHi regulation. The reciprocal control of Na+/H+ exchange by SLC9C1 and cAMP synthesis by sAC has recently been shown in sea urchin sperm82. Non-mammalian sAC is directly activated at alkaline pHi and not by HCO3−: alkalization of sea urchin sperm by SLC9C1 stimulates cAMP synthesis82, and the rise of cAMP modulates the voltage dependence of SLC9C1 activity31. Although such reciprocal control is straightforward for a sAC that is controlled by pHi, it is unclear how sNHE controls mammalian sAC, which is activated by HCO3− and not pH82. Thus, sAC and sNHE/SLC9C1 in human sperm may have adopted new mechanisms of mutual control that remain to be elucidated.A related intriguing feature that remains to be solved is the functional and even molecular interaction between SLC9C1 and sAC. SLC9C1o)/low-HCO3− of the epididymis and ends in high-pHo/high-HCO3− environs of the oviduct. An increase of intracellular HCO3− and pHi are considered key events during sperm capacitation84, and both have been attributed to HCO3− influx via SLC4 and SLC26 transporters or CFTR/ENaC27 . We compared the kinetics of ΔpHi and intracellular ∆[Na+]i using the Na+-sensitive dye ANG-2. The t½ and time course of ΔpHi (H+ efflux) and Δ[Na+]i (Na+ influx) were not significantly different , whereas cells in the body reside in a quasi-closed system where pCO2 is more than 100-fold higher (pCO2 = 40 mmHg) , the pHi increased slowly from 6.6 ± 0.1 to 7.0 ± 0.1   Fig. . We emul0.1 Fig. . In the ly) Fig. . Thus, io = 7.35 from a HCO3−-free solution (open system) to a CO2/HCO3−-containing solution (from ES to ES/5%CO2/25 mM HCO3−) evoked a rapid and robust acidification   )  to an open system (pCO2 = 0.3 mmHg) also caused acidification albeit at a slower rate   Fig.  with a t42) Fig. . The sol4)) Fig. , suggesters Fig. . Finally2/HCO3−   markedly slowed down acidification kinetics in sperm and HEK293 but not CHO cells  Fig. . The larlls Fig. , suggestlls Fig.  and its lls Fig. . The equ2/HCO3−-evoked pHi changes in mouse sperm, which lack Hv1-mediated proton currents30 but express the sNHE SLC9C133. Both wt and SLC9C1-/- mouse sperm displayed similar behavior: Superfusion with CO2/HCO3− also evoked rapid acidification, whereas de- or hyperpolarizing Vm steps did not evoke proton currents or pHi changes, either before or after HCO3− treatment  limited time resolution, which missed initial acidification; (ii) use of an open CO2/HCO3− system in equilibrium with atmospheric rather than tissue pCO2; during hour-long capacitation times, sperm slowly alkalize due to an increase in pHo by CO2 degassing into the air; and (iii) difficulties with pH control of HCO3− solutions46.Exposure to higher COi = -0.15 reflects the production of 10-30 mM H+ and, thereby, HCO3− (see Methods). This concentration matches the HCO3− sensitivity range of sAC (K1/2 = 14 mM)87. Thus, small changes in pHi reflect physiologically relevant changes in HCO3− concentrations. Cytosolic alkalization has been considered a key step for capacitation However, because HCO3− and pHi are inextricably linked by the CO2/HCO3−/H+ equilibrium, it is difficult to gauge the physiological significance of pHi changes on their own for capacitation. As first physico-chemical principles of CO2 diffusion across membranes are generally valid90, we anticipate that HCO3− regulation in sperm of other mammalian species is governed by similar mechanisms.We estimate that acidification as small as ∆pHv1 contributes to pHi homeostasis by PCF, and how its activity is affected by changes in the CO2/HCO3−/H+ equilibrium. The mechanism of Hv1 activation is unique, as it depends on both Vm and the transmembrane pH difference (ΔpHtm = pHo − pHi)91. When ΔpHtm = 0, the threshold voltage Vthr at which Hv1 activity becomes noticeable is about +30 mV; when ∆pHtm > 0 or <0, the activation curve is shifted to more negative and positive potentials, respectively91  and t½ac = 2.5 ± 1.2 s (ncells = 7))  (ncells = 4) ) Fig. . Of notelux Fig. , suggestion Fig. . In addi 4) Fig. , suggestv1 (CHO-hHv1) confirmed these results obtained in sperm. Hv1 currents rapidly activated, whereas the ensuing alkalization was delayed, slower, and never saturated during a 3-s Vm pulse 95 together with the high CA2 copy number of 154,000 molecules/cell renders the CO2/HCO3−/H+ equilibrium a powerful buffering system that rapidly responds to changes in pHi and HCO3− concentration . Although the positive Vm values required for Hv1 activation in vitro are unlikely to prevail in sperm97, the presence of proteolytically cleaved Hv1 isoforms in human sperm39 and albumin that binds to Hv1 (ref. 98) shift the V1/2 to more favorable potentials. In light of the substantial H+ production by HCO3− synthesis and Hv1’s unique regulation by both Vm and ∆pH, we suggest that Hv1 curtails strong acidification during HCO3− synthesis from CO2 and, thus, sustains HCO3− synthesis. It is in this sense that Hv1 is required for capacitation rather than promoting alkalization per se. The dependence of Hv1 on both Vm and ∆pHtm (ref. 91) implies that Hv1 opens only at Vm values positive to the Nernst potential for protons, i.e., Hv1 can carry only proton outward currents  for 30–60 min and purified by the “swim-up” procedure43 in human tubular fluid (HTF) medium containing (in mM): NaCl 93.8, sodium-lactate 21.4, HEPES 21, KCl 4.69, sodium bicarbonate (NaHCO3) 4, D-glucose 2.78, CaCl2 2.04, sodium pyruvate 0.33, MgSO4 0.2, and potassium dihydrogen phosphate (KH2PO4) 0.37 (adjusted to pH 7.35 with NaOH) and 5% CO2. After 30-45 min, motile sperm were collected from the supernatant, centrifuged at 700 xg for 10 min and resuspended in fresh HTF. Samples were stored in HTF at 37 °C diluted to 1*107 cells/ml until they were further prepared for recordings.Human semen samples were donated by healthy adult caucasian males (age: 21–56 years) with their prior written consent and the approval of the ethic committee of the University of Bonn (042/17). Handling and experiments with human semen samples were performed in agreement with the standards set by the Declaration of Helsinki. Ejaculates were liquefied at room temperature  and killed by cervical dislocation according to the German law of animal protection and the district veterinary office . sNHE knockout mice were purchased from the Jackson Laboratory . Mice were housed at 21 °C with 55% relative humidity at a 12 h light/dark cycle.2 2.0, KH2PO4 1.2, MgSO4 1.0, sodium-pyruvate 0.5 (adjusted to pH 7.4 with NaOH) for the “swim-up” procedure. After 15 min, sperm were collected from the supernatant, centrifuged at 700 xg for 10 min and resuspended in fresh TYH medium. Sperm were diluted to 1*107 cells/ml in TYH and stored at 37 °C until further processed.For mouse sperm preparation, cauda and corpus epididymides were excised and separated from fat. The tissue was incised several times and transferred to modified TYH medium containing (in mM): NaCl 138, sodium-lactate 10, HEPES 10, glucose 5.6, KCl 4.8, CaClFor single-cell fluorometry, cells were loaded with 3.3 µM pHrodo-Red-acetoxymethyl ester  for 12 min, 2 µM Calbryte520-AM  and 0.05% Pluronic F-127 for 60 min, or 10 µM Asante Natrium Green  and 0.05% Pluronic F-127 for 60 min, at 37 °C for pH, calcium, and sodium measurements, respectively, followed by centrifugation (700 x g for 10 min) and washing.6 sperm were extracted and subjected to in-solution digestion as follows. The sample was diluted two-fold by addition of an equal volume of human sperm lysis buffer (8% (w/v) sodium dodecyl sulfate (SDS), 10 mM ethylenediaminetetraacetic acid (EDTA), 10 mM ethylene glycol-bis(2-aminoethylether)-N, N, N’, N’-tetraacetic acid (EGTA), 20 mM dithiothreitol (DTT), and 100 mM NH4HCO3), and boiled for 10 min at 99 °C. The sample was sonicated in a pre-warmed (15 °C) sonication device  for 10 min with alternating cycles of 30 s ON, followed by 30 s OFF. After sonication, 50 mM NH4HCO3 buffer (pH 8) was added resulting in a final SDS concentration of 1%, and the sample was incubated with 500 units of universal nuclease  in the presence of 1 mM MgCl2 for 30 min at 37 °C. Proteins were reduced with 10 mM DTT for 30 min at 60 °C and then alkylated with 40 mM iodoacetamide (IAA) for 30 min at 37 °C, followed by another incubation step with 20 mM DTT for 5 min at 37 °C. SP3 beads were prepared as described and 3 mg of SP3 beads were added to the reduced and alkylated proteins, followed by addition of 100% ethanol (LiChrom grade) to a final concentration of 50% (v/v) ethanol and incubation for 5 min at 24 °C with shaking at 1000 rpm in a ThermoMixer® C . SP3 beads were washed three times with 80% (v/v) ethanol using an in-house made magnetic rack. After the final washing step, SP3 beads were resuspended in 50 mM NH4HCO3 buffer. Rapigest  was added to a final concentration of 0.1% (w/v) and trypsin  was added in 1/20 (w/w) trypsin to protein ratio for protein digestion overnight at 37 °C. Digestion was stopped by acidifying the sample with 100% formic acid (FA) so that the pH of the sample was between 2 and 3 and incubated for 30 min at 37 °C, followed by centrifugation for 10 min at 4 °C and 17,000 xg. Using a magnetic rack, the peptide containing supernatant was transferred to another reaction tube and 80% (v/v) acetonitrile (ACN)/0.1% (v/v) formic acid (FA) were added to the supernatant yielding a concentration of 4.5% ACN, pH 2-3. The peptide mixture was cleaned up using a C18 spin column  according to the manufacturer’s protocol and dried in a SpeedVac concentrator. For whole-proteome LC-MS/MS analysis, dried peptides were reconstituted in 50 µl (containing peptides derived from 1-5×106 sperm cells) 10 mM NH4OH, pH 10 for further separation by basic reverse-phase chromatography (see below). For PRM-MS analysis (see below), peptide samples derived from sperm were aliquoted in amounts containing peptides derived from 180,000 sperm cells and dried in a SpeedVac concentrator.Proteins from 1–5 × 10To control for complete solubilization and protein recovery, human sperm cells were denatured as described above and then in an additional step centrifuged at 30,000 xg  to separate insoluble material. The residual small pellet was again dissolved in 8% (w/v) SDS, digested, and analyzed by LC-MS. Furthermore, to control for lost peptides during enzymatic digestion, SP3 beads were—after the above-described elution steps of peptides—incubated with 50% (v/v) ACN and 0.1% (v/v) FA for 15 min at 37 °C. The solution containing eventually residual extracted peptides was dried in a SpeedVac concentrator and then analyzed by LC-MS as above.4HCO3 buffer (pH 8), 500 units of universal nuclease and 1 mM MgCl2. Further sample processing was performed exactly as described for sperm sample above. The peptide mixture was stored in 10 µg aliquots at −20 °C.HEK293 cell lysate containing 500 µg of protein was directly subjected to in-solution digestion starting with nucleic acid digestion in the presence of 50 mM NH6 sperm cells were dissolved in 50 μl mobile phase A  and injected. Elution was performed at a flow rate of 60 μl/min using mobile phase A and B  with a Waters XBridge C18 column . The gradient was 5% B for 5 min, then changed to 10% B in 3 min, to 35% B in 34 min, to 50% B in 8 min, to 90% B in 1 min, to 95% in 5 min, back to 5% B in 2 min, then held at 5% B for 6 min . The elution was recorded at 214 nm. 60 µl fractions including the flow-through were collected. Fractions were combined into total 24 fractions, dried in a SpeedVac concentrator, and stored at −20 °C for subsequent LC-MS analysis.Off-line by bRP-HPLC was performed on an Agilent 1100 series HPLC system. Peptides derived from digestion of 1–5 × 102O. 24 individual fractions were analyzed on Orbitrap Exploris™480 mass spectrometer (Thermo Fisher Scientific) or on Q Exactive HF-X Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Fisher Scientific), coupled to a Dionex UltiMate 3000 UHPLC system (Thermo Fisher Scientific) equipped with an in-house-packed C18 column . Mass spectrometers were operated using the software Thermo XCalibur 4.4.16.14 for the Orbitrap Exploris™480 mass spectrometer and Thermo XCalibur 4.0.27.19 for the Q Exactive HF-X Hybrid Quadrupole-Orbitrap Mass Spectrometer. Samples were separated applying the following 118 min gradient: mobile phase A consisted of 0.1% (v/v) FA in H2O, mobile phase B of 80% (v/v) ACN/0.08% FA (v/v) in H2O. The gradient started at 5% B, increasing to 8% B within 10 min, followed by 8–35% of B within 90 min and by 35% to 55% within 7 min. Finally, the mobile phase was set to 90% of B for 4 min. After each gradient, the column was again equilibrated with 5% B for 6 min. The flow rate was 300 nl/min. Eluting peptides were analyzed in positive mode using a data-dependent acquisition method with a time between master scans of 3 s. MS1 spectra were acquired with a resolution of 120,000 full width at half maximum (FWHM) in the Orbitrap covering a mass range of 350–1600 m/z (data type—profile). Injection time was set to 25 ms and automatic gain control (AGC) target to 300% (100% = 1 × 106). Dynamic exclusion covered 25 s and intensity threshold was set to 5 × 103. Precursor ion charge state screening was enabled, all unassigned charge states were rejected and only precursors with a charge state of 2-6 were included. MS2 spectra were recorded with a resolution of 15,000 FWHM in the Orbitrap (data type – centroid), injection time was set to 32 ms, AGC target to 75% (100% = 1 × 105) and the isolation window to 1.4 m/z. Fragmentation was enforced by higher-energy collisional dissociation (HCD) at 28%.Dried peptides in each bRP-HPLC fraction were dissolved in 4% (v/v) ACN/0.1% (v/v) TFA in H2O, the different peptides were mixed at equimolar concentrations of 1 nanomol, split into aliquots of 10 pmol, dried in a SpeedVac concentrator and stored at –20 °C. Standard peptides from JPT were solubilized in 80% (v/v) 100 mM NH4HCO3 buffer and 20% (v/v) ACN in H2O. The JPT peptides required an additional endoproteinase digest to remove the Q-tag. For this, 500 pmol of each peptide were mixed, and 0.5 µg trypsin was added to the peptide mixture. Digestion was performed at 37 °C for 5 h, as instructed by JPT. The reaction was quenched with 5 µl of 100% (v/v) FA. The peptides were divided into 10 pmol aliquots, dried in the SpeedVac and stored at −20 °C.For the absolute quantification of CA2, SLC26A3, CatSper1-4, CatSperB, and Slo3, and the determination of LOD values for SLC4A4, SLC26A6, SCNN1A, and CFTR, isotope-labelled standard peptides from Thermo Fisher Scientific and for analysis of CatSperB, isotope-labelled standard peptides from JPT  were used : 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1.0, 5.0, and 10.0. For quantitative analysis with the JPT standard peptides, the following final concentrations in 7 µl of injected sample were used (in fmol): 0.1, 0.5, 1.0, 5.0, 10.0, and 50.0. To reach the aforementioned absolute amount of standard peptides in 7 µl sample, the biological sample , was dissolved in 14 µl or 21 µl of the respective standard peptide dilutions. By this, 7 µl that were injected into the mass spectrometer contained endogenous peptides derived from either 90,000 or 60,000 sperm cells and the specified amount of standard peptide.For the positive control with peptides derived from HEK293 cells, concentrations of standard peptides were prepared as follows (in fmol): 0.5, 1.0, 5.0, 10.0 and 20.0 of peptide in 7 µl of MS loading buffer. To each aliquot of HEK293 peptides, 70 µl of standard peptide dilution was added yielding 1 µg of peptides in 7 µl.2O gradient and analyzed via an acquisition method involving an MS1 survey scan and targeted MS2 scans. For MS1 scans, the Orbitrap resolution was set to 120,000 FWHM, the scan range (m/z) was adjusted to the m/z range of the peptides to be detected, a standard AGC target was used and data was acquired in positive mode. For the targeted MS2 scan, the isolation window (m/z) was set to 1.4 m/z, a normalized collision energy of 30% and a normalized AGC target of 100% was used, the maximum injection time was adjusted to 100 ms, the Orbitrap resolution was set to 60,000 FWHM, and data was acquired in positive mode. For targeting, the mass and charge state of the peptides as well as the retention time windows were defined in the PRM method settings in the mass spectrometer. The retention time windows were newly determined for each set of measurements.For absolute quantification, 7 µl of injected samples were measured on an Orbitrap Exploris™480 mass spectrometer (Thermo Fisher Scientific) equipped as above. Peptides were separated using a 58 min 0–42% ACN (v/v)/0.1% (v/v) in H102 with its built-in Andromeda peptide search engine103; the following settings were used to identify proteins: trypsin/P was used as digestion enzyme with maximal two missed cleavage sites; MS2 spectra were searched against the UniProt complete Homo sapiens proteome sequence database (downloaded in June 2020); carbamidomethylated cysteines were set as fixed, oxidation of methionine and N-terminal acetylation as variable modifications ; maximum false discovery rate was set to 0.01 both on peptide and protein levels. One peptide with a minimum length of seven amino acids was required for protein identification.Raw files were processed by MaxQuant (MQ) software (version 1.6.5.0)60.To generate a label-free absolute quantification intensity matrix, the “iBAQ” built-in option in the MaxQuant software was selected without further changes of the default settings. iBAQ intensities calculations use the approach taken in the iBAQ method13C, 15N-labelled lysine and arginine were added; in the transition settings section, the instruments and measurement details were provided to the Skyline software. The MS raw files were imported into Skyline, and retention times of the different LC-MS runs were aligned. Transitions of endogenous peptides were compared to signals of labelled peptides; transitions from endogenous peptides were only considered when they displayed the same retention time and fragmentation pattern as the labelled peptides. Only peaks/signals were included in quantification calculations that fulfilled the following criteria: a minimum of three transition ions, minimum of three to five acquired data points. The total integrated peak area was exported from Skyline for both endogenous and standard peptides. The signals from the standard peptides were used to generate calibration curves, which were used to calculate the molar amounts of endogenous peptides. From that, the molecules/cell was calculated. For plotting, data from all peptides corresponding to the same protein were merged , R Studio, version 2022.07.2). Data displayed as box plots represent 25%, 50% (median), and 75% quartiles, with whiskers displaying minimum and maximum data points, except for outliers outside of 1.5-times interquartile range (IQR).The PRM data was analyzed with Skyline . The mass accuracy (delta mass ≤ 10 ppm) of the transitions was used as cut-off filter to reduce the number of false positives. The Skyline program was provided with the peptide sequences; in the peptide settings section, the isotope modifications for 2 1.8, MgCl2 1.0 (adjusted to pH 7.35 with NaOH). For “acid-load” experiments, ES with NH4Cl contained (in mM): NaCl 125, NH4Cl 15, mannitol 10, KCl 5.4, HEPES 5, CaCl2 1.8, MgCl2 1.0 (adjusted to pH 7.35 with NaOH); ES with N-methyl-D-glucamine (NMDG+) contained (in mM): NMDG+ 140, mannitol 10, KCl 5.4, HEPES 5, CaCl2 1.8, MgCl2 1.0 (adjusted to pH 7.35 with HCl). For pH calibration high-K+ calibration solutions contained (in mM): KCl 135, HEPES/MES/Tris 20, CaCl2 1.8, MgCl2 1.0, Nigericin 0.015 . The ES/pH7.35/CO2/HCO3−, which was always used when cells were exposed to CO2/HCO3− unless otherwise mentioned, contained (in mM): NaCl 115, NaHCO3 25, mannitol 10, KCl 5.4, HEPES 5, CaCl2 1.8, MgCl2 1.0 (adjusted to pH 7.35 with NaOH). The pH conditions of the epididymis (pH 6.5) and oviduct (pH 7.5) were mimicked with the following solutions. ES/pH6.5/ CO2/HCO3− contained (in mM): NaCl 137, NaHCO3 3, mannitol 10, KCl 5.4, MES 5, CaCl2 1.8, MgCl2 1.0 (adjusted to pH 6.5 with NaOH); ES/pH 7.5/CO2/HCO3− contained (in mM): NaCl 110, NaHCO3 30, mannitol 10, KCl 5.4, HEPES 5, CaCl2 1.8, MgCl2 1.0 (adjusted to pH 7.5 with NaOH). HCO3−-containing solutions were bubbled with Carbogen  for at least 30 min before the pH was adjusted. HCO3−-containing solutions were continuously bubbled with Carbogen during experiments to maintain constant pCO2 and pH. Prior to recordings, the pH of all solutions was checked with a calibrated pH-meter  and adjusted, if necessary. All extracellular solutions had osmolarities of 290-300 mOsm/l.For fluorometric and electrophysiological recordings, human and mouse sperm were mixed with 3 mg/ml human serum albumin (HSA) or bovine serum albumin (BSA), respectively, and pipetted on poly-L-lysine coated coverslips submerged in extracellular recording solution (ES), containing (in mM): NaCl 140, mannitol 10, KCl 5.4, HEPES 5, CaCl+ 140, Mg-ATP 4.0, NaCl 1.4, EGTA 1.0, MES 1.0 . Prior to recording, 12.5 µM pHrodo-Red-maleimide was added.For patch-clamp recordings of mouse sperm, the pipette solution contained (in mM): aspartic acid 130, Mg-ATP 4, NaCl 1.4, MES 1.0, EGTA 1.0 . For all other cell types, the intracellular solution contained (in mM): NMDGAll chemicals and inhibitors were purchased from Carl Roth , Sigma Aldrich , or Thermo Fisher Scientific  if not stated otherwise.Fluorometry and patch-clamp fluorometry (PCF) experiments were performed on an inverted IX71 microscope  equipped with a 60× water immersion objective and an additional 1.6x magnification lens for PCF. Fluorescent indicators were excited with LED light . Excitation and emission filters used were 543/22 nm and 568LP nm for pHrodo-Red and 472/30 nm and 525/35 nm for CalBryte-520 and ANG-2. An EMCCD camera  controlled by the Andor Solis software was used for imaging.An Axopatch 200B amplifier connected to Digidata 1440 A acquisition board controlled by the software ClampEx  was used for electrophysiological recordings. Patch pipettes were pulled from borosilicate capillaries  using a DMZ puller . All experiments were conducted at RT.104, was excluded from analysis. During the whole experiment, a continuous flow of perfusion was applied and minimized motion of sperm head and midpiece. Depending on the density of the sperm cells on the cover slip, one to several sperm cells could be imaged simultaneously. Number of experiments (nexp) denotes the number of cover slips recorded in imaging experiments. Number of cells (ncells) denotes the total number of cells imaged.For fluorometric imaging, coverslip areas with non-overlapping cells were chosen. Motile sperm with only their head attached to the coverslip were chosen for analysis. ROIs were drawn by hand and restricted to the sperm head, the midpiece, and up to the first quarter of the principal piece Fig. . These pVm = −30 mV. During the break-in process, loading of cells with pHrodo-Red maleimide was observed with the EMCCD camera. The half-time t1/2 of filling sperm flagella with dye was about 50 s  in ClampFit (Molecular Devices). Data displayed as box plots represent 25%, 50% (median), and 75% quartiles, with whiskers displaying minimum and maximum data points, except for outliers outside of 1.5-times interquartile range (IQR). Statistical analysis was performed with Prism 9 . Data sets were tested for normal distribution using the D’Agostino & Pearson test. Based on the results, the following statistical test were chosen: statistical significance was tested with the Mann-Whitney test for data in Figs. t-test . Movies from PCF experiments were motion-corrected with the “MultiStackReg” ImageJ plugin to compensate for slow drifts of the patch pipette (less than 1 μm/min). The mean intensity of the ROIs was calculated with ImageJ and further analyzed with Igor Pro . Fluorescence data are presented as ΔF/F. For some experiments, pH signals were calibrated at the end of the recording in order to quantify changes in pHpHi Fig. . PCF fluest Fig.  or the pest Fig.  were usei were used to calculate the changes in H+ by the following equation ∆H+/∆pH = −2.3ß, wherein ß is the intrinsic buffer capacity. The ß of cells ranges between 40–80 mM107. For mammalian sperm, β was estimated to range between 30–87 mM109.Changes in pHFurther information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Reporting SummarySource Data