New insights into CFTR modulation in reproduction: testicular microenvironment imbalance leads to over-activated caspase signalling in spermatogenesis and adversely affects fertility

Cystic fibrosis transmembrane conductance regulator (CFTR) is a prominent chloride channel that governs mucous secretion in multiple organs, including the reproductive tract. According to earlier reports, defective CFTR results in infertility due to congenital bilateral absence of the vas deferens (CBAVD). However, obstruction in the vas deferens is not the only reason CFTR deficiency causes male infertility. The mechanism underlying the loss of mature sperm owing to CFTR deficiency remains elusive. This study aimed to assess the role of CFTR in spermatogenesis, for which 6- and 8-week-old male mice with Cftr+/+, Cftr+/-, and Cftr-/- genotypes were chosen. Furthermore, we assessed the correlation between CFTR deficiency and delayed development of the reproductive system, anomalous apoptosis activation in spermatogenesis, and ionic alterations of the testis lumen. The results demonstrated that the growth of Cftr-/- mice were delayed, with underweight reproductive organs and mild hypospermatogenesis. CFTR depletion destabilizes spermatogenesis by producing abnormal sperm and triggers activation of the Bax/Bcl-2 ratio in Cftr-/- and Cftr+/-mice, causing caspase-mediated irreversible intrinsic apoptosis. Stage-specific apoptosis in germ cells targeted the sexually mature mice, and the testis microenvironment affirmed that ion concentrations influence sperm capacitation. The blood pH determines apoptosis induction, as CFTR is a bicarbonate transporter. In conclusion, Cftr-/- mice were infertile because CFTR deficiency generated an ionic imbalance in the testis lumen, leading to Bax expression and Bcl-2 blockage, which triggered caspases or further activation of voltage-dependent anion-selective channel 1 (VDAC1). Cumulatively, cytochrome C was released due to altered mitochondrial membrane potential. Eventually, anomalous up-regulated apoptosis activation affected spermatogenesis, thus rendering the Cftr-/- male mice infertile. The results supplied new insights into CFTR modulation in reproduction: an imbalanced testicular microenvironment due to CFTR deficiency affects spermatogenesis and fertility in mice through the overactivation of spermatocyte caspase signalling, thus driving us to focus on updated treatments for CFTR deficiency-caused infertility.

destabilizes spermatogenesis by producing abnormal sperm and triggers activation of the Bax/Bcl-2 48 ratio in Cftr -/and Cftr +/mice, causing caspase-mediated irreversible intrinsic apoptosis. Stage-49 specific apoptosis in germ cells targeted the sexually mature mice, and the testis microenvironment 50 affirmed that ion concentrations influence sperm capacitation. The blood pH determines apoptosis 51 induction, as CFTR is a bicarbonate transporter. In conclusion, Cftr -/mice were infertile because 52 CFTR deficiency generated an ionic imbalance in the testis lumen, leading to Bax expression and 53 Introduction passed through the testis, epididymis, and vas deferens were calculated. 142 143

Micro-liquid ion measurement 175
For each mouse, two samples were analyzed for ion concentration: the testis and a blood 176 sample. A homogenate was prepared by mashing a quarter of the rat testis in 200 µL of ddH 2 O and 177 centrifuging it at 15000 rpm for 10 min. From the homogenate, 70 µL of supernatant was placed in 178 an ABL80 flex blood gas analyzer (Åkandevej, Brønshøj, Denmark) to measure the concentration 179 and pH of each ion in the lumen. Another quarter of the testis was mashed to prepare a homogenate 180 which was placed in a shaker for 30 min to evenly distribute the ions in the liquid. Subsequently, 70 181 µL of the supernatant was used in the ABL80 flex blood gas analyzer to measure the testicular ion 182 concentration and blood pH value. These data indicated changes in the microenvironment of the 183 testis lumen. Blood samples were collected from the heart or abdominal aorta, and 70 µL of the 184 blood sample was analyzed. 185

CFTR deficiency affects the early development of body size and fertility 194
To understand whether CFTR affected the overall development and fertility of mice, an initial 195 morphological assessment was performed to evaluate the growth of the 6-8-week-old mice with 196 variable genotypes. The morphological comparison between 6-and 8-week-old mice is displayed in 197 body sizes of the 6-and 8-week-old Cftr -/mice were smaller than those of the Cftr +/+ and Cftr +/-199 mice (Figures 1-A and B). Cftr -/mice had a significantly lower body weight than age-matched 200 Cftr +/+ and Cftr +/mice (Figures 1-C and D). 201 Bodyweight (gram) of C. 6-week and D. 8-week-old Cftr +/+ , Cftr +/-, and Cftr -/mice. Statistics 213 from 6-week-old Cftr +/+ (n=13), Cftr +/-(n=8), and Cftr -/-(n=6) mice, and 8-week-old Cftr +/+ (n=10), 214 From 6 weeks after birth, male mice of different genotypes were caged with Cftr +/+ female mice for 217 2 weeks. We found that male Cftr -/mice were infertile. Male Cftr +/mice exhibited a poorer 218 breeding rate than the male Cftr +/+ mice. While there was a trend of marginally lower litter numbers 219 in the Cftr +/group compared to Cftr +/+ mice, it did not reach statistical significance (Table 1) Cftr +/-, and Cftr -/mice that were bred with female Cftr +/+ mice. The breeding rates were calculated 224 as "the number of litters with offspring" divided by "the total number of breeding litters." 225 ***P<0.001 when compared to the male Cftr +/+ mice group. 226

CFTR deficiency reduces reproductive organ weight 228
To determine the impact of CFTR deficiency on the male reproductive ducts in mice, 229 morphological development and structural deformities of the male reproductive organs were 230 assessed in 6-and 8-week-old mice with variable genotypes. abnormal secretory products in the lumen in 6-week-old Cftr -/mice (Figure 3-A). The lumen of the 277 vas deferens in 8-week-old Cftr -/mice became significantly smaller and displayed partial 278 morphological obstruction (Figure 3-B). In addition, the sperm count in the vas deferens of Cftr -/-279 mice was significantly lower than that in the Cftr +/+ mice (Figures 4-A and B); however, the 280 morphological structure of spermatozoa of all genotypes was normal (Figures 4-C and D). Cftr +/+ , Cftr +/-, and Cftr -/mice. Scale bar = 100 μm. The assay was duplicated for cross-verification. 344

CFTR deficiency activates the apoptotic pathway in mice testis
To determine the cause of CFTR-induced hypospermatogenesis, the apoptotic pathway was 350 analyzed. The Bax to Bcl-2 ratio indicated the tendency to inhibit apoptosis. Bcl-2 was considered 351 an anti-apoptotic factor that was expressed less in the testes of Cftr -/mice. Bax was a pro-apoptotic 352 factor which counteracted the Bcl-2 function in Cftr -/mice. The Bax to Bcl-2 ratio was higher in 353 the Cftr -/mice than in the Cftr +/+ mice. Therefore, the pro-apoptotic tendency in the Cftr -/mouse 354 testis might have been higher than that in the Cftr +/+ control mouse. When Bcl-2 does not inhibit 355 Bax, it interacts with VDAC1. These events may induce alterations in the mitochondrial membrane 356 potential, which triggers the release of cytochrome C and caspase activation. Hence, we determined 357 the caspase signalling changes in the Cftr -/mouse. Caspase-9, an initiator caspase, was activated 358 more in the Cftr -/mouse than in the other two genotypes in both 6-and 8-week-old mice. Caspase-3, 359 an effector caspase, shared the same results as Caspase-9. However, cleaved (activated) Caspase-7 360 showed no difference between the genotypes (Figure 7). cleaved Caspase-7, (pro-) Caspase-9, cleaved Caspase-9, and β-actin of the testis. The statistical 384 results of B. 6-and C. 8-week-old Cftr +/+ , Cftr +/-, and Cftr -/mice. The ratio of Bax to Bcl-2 was 385 quantified to show the tendency of apoptosis. The ratio of cleaved Caspase-3, 7, and 9 to β-actin 386 were respectively quantified to show the caspase cascade activation. β-actin was one of the 387 housekeeping genes which can be used to standardize the cellular protein quantity. *P<0.05 388 Western blotting and quantification were performed in triplicate. 389 390  391 Caspase-9 and 3 might share some features in common; the observed results showed that both 392 exhibit an increased expression of Cftr -/-, less expression of Cftr +/-, and almost no expression of 393 Cftr +/+ in testicular seminiferous tubules (Figures 8-A and B). In the 6-week-old Cftr -/mouse testis, 394

CFTR deficiency shows a stage-specific-pattern of Caspase signalling in spermatogenesis
Caspase-9 expression showed a mild increase in spermatocytes and spermatids at stages I-IV and 395 V-VIII (Figure 8-A). In 8-week-old Cftr -/mouse testis, Caspase-9 expression was significantly 396 increased in spermatocytes and spermatids at stages I-IV and V-VIII. Caspase-9 was not expressed 397 during stages IX-XII. (Figure 8-A). However, the expression pattern of Caspase-3 differed slightly 398 from that of Caspase 9. It is worth noting that Caspase-3 was found to increase spermatocytes at stages V-VIII in 6-week-old Cftr +/mouse testes. In 6-week-old Cftr -/mice testis, Caspase-3 was 400 expressed in spermatocytes at all stages. Concurrently, after 8 weeks, Cftr +/testes were similar to 401 that of 6-week-old Cftr +/testes. Intriguingly, Caspase-3 was highly expressed in stages I-IV 402 spermatocytes, even in spermatogonia, and less expressed in stages V-VIII and IX-XII 403 spermatocytes (Figure 8-B). CFTR is a chloride ion channel located on the apical epithelial membrane that mediates 428 transepithelial chloride ion and fluid movement. We aimed to identify if CFTR deficiency causes 429 overactivation of the apoptotic pathway during spermatogenesis. First, we evaluated the changes in 430 testicular ion concentration during normal reproductive developmental processes. We found that the 431 concentration of all testicular ions (including chloride, sodium, and calcium ions) in 6-week-old 432 wild-type mice was significantly higher than that in the 8-week-old wild-type mice (Figure 9). In 433 sexually immature 6-week-old mice, a higher concentration of chloride, sodium, and calcium was 434 observed in the seminiferous tubule. In contrast, sexually mature 8-week-old mice displayed a 435 lower concentration of these ions than 6-week-old mice. Herein, ion concentration might have acted 436 as a potent indicator of the developmental condition of the testis. This result confirmed that ion 437 changes play an important role in reproductive development and testicular maturation. 438 The experimental results indicated that the concentrations of chloride, sodium, and calcium ions in 439 Cftr -/mice were lower than that in 6-week-old Cftr +/+ mice but higher than that in 8-week-old 440 Cftr +/+ mice. In particular, we found that there was no significant difference between the ion 441 concentrations in the testes of 6-and 8-week-old CFTR-deficient mice, and both violated the 442 normal physiological ion level requirements in testicular development. Therefore, CFTR-deficient 443 mice have a significantly decreased ability to regulate the testicular ionic environment normal 444 development. After confirming that CFTR plays a vital role in testis growth, we analyzed the 445 performance of Cftr +/mice. With only one Cftr allele being knocked out, the chloride, sodium, and 446 calcium ion distribution showed a pattern between Cftr +/+ and Cftr -/-. The ion concentration gap 447 between 6-and 8-week-old mice was the largest in the Cftr +/+ group, was lower in Cftr +/group, and 448 was the smallest in Cftr -/group (Figure 10).

Association between CFTR depletion, maintenance of blood ions, and alteration in pH 469
We wondered whether testicular environment dysregulation caused by CFTR deficiency was 470 mediated through the systemic circulation system. In 6-week-old mice, we found that the blood pH 471 value was lower in the Cftr -/mouse than in the Cftr +/+ and Cftr +/mice (Figure 11-A). In contrast, 472 CFTR is an apical membrane chloride channel, and its depletion causes male infertility. Our 487 study found that Cftr -/mice were infertile, and their reproductive system suffered from delayed 488 maturation. CFTR depletion leads to abnormal chloride distribution in the seminiferous tubules, 489 which disrupts the balance of other testicular ions. An ion concentration imbalance alters membrane 490 potential and activates apoptosis via the Bax/Cytochrome C/caspase-9/caspase-3 pathway. 491 Consequently, abnormal spermatogenesis leads to fewer spermatozoa, and male Cftr-/-mice become 492 infertile. 493 The male Cftr -/mice with disrupted CFTR showed delayed growth and were downsized and 494 underweight compared to the Cftr +/+ and Cftr +/mice. Generally, low body weight and retarded 495 growth are typical features of CFTR depletion; Cftr -/mice suffer from a severe intestinal disease, 496 primarily obstruction, and require a liquid diet to decrease the frequency of this lethal complication.

2002). In addition, a study of CFTR expression in the mouse liver showed that CFTR had a close 499
interaction with the selective induction of sulfotransferase (SULT) 1E1 in mouse hepatocytes. The 500 interactions between insulin-like growth factor-1 (IGF-1) and Cftr -/mouse livers were positively 501 correlated with body weight and negatively correlated with SULT1E1 activity. 502 Growth hormone (GH) is vital in regulating IGF-1 expression, suggesting that β-estradiol 503 levels are implicated in GH-signaling in hepatocytes. In addition, it is also evident that estrogen 504 plays a significant role in regulating body weight (Li et al., 2009). Hence, Cftr -/mice exhibit abrupt 505 stunted growth and are underweight. CFTR expression has been observed during several germ cell 506 developmental phases. As an ion channel, it plays many dynamic roles in embryonic development. 507 Nevertheless, CFTR regulation is typically observed in the most primitive type of germ cells, the 508 primordial germ cells (PGCs) (Liao et al., 2018). Therefore, CFTR-depleted mice are more likely to 509 have a reduced male fertility phenotype. Our observations confirmed that the male Cftr -/mice were 510 infertile, and the male Cftr +/mice had a lower breeding rate than Cftr +/+ mice. The results obtained 511 in this study are substantiated by the findings in previous studies. To avoid the influence of female 512 infertility, Cftr +/+ female mice were used for breeding in this study. 513 Apart from the impeded morphology, male Cftr -/mice were observed to have downsized and 514 underweight reproductive organs, which indicated that their reproductive capacity might be 515 decreased. The comparative statistical data between body and organ weight also indicated a lower 516 weight of the male Cftr -/mice than the other genotypes. Although the structure of Cftr -/-517 spermatozoa was normal, the results indicated that the male Cftr -/mice had less sperm in the vas 518 deferens and were less active than the Cftr +/+ spermatozoa. This scenario proved that 519 oligozoospermia (count) and asthenozoospermia (motility) were significant reasons for infertility in 520 male Cftr -/mice. Recent reports have validated these results and stated that asthenozoospermia and and CFTR expression in sperm (Fok et al., 2014). In addition, decreased sperm motility and poor 523 capacitation are reportedly distinctive features of CFTR mice (Yefimova et al., 2019). 524 The thinner testicular layer in Cftr -/mice implied that CFTR disruption influences 525 spermatogenesis and induces reproductive disorders. The Cftr -/mouse testes were filled with sperm 526 during our observations. These findings led to further morphological studies of the head (caput) and 527 tail (cauda) of the epididymis, which exhibited a normal state in all mice genotypes. Furthermore, 528 morphological observation of the sperm in Cftr -/caput showed a less normal state, and the sperm in 529 Cftr -/cauda and vas deferens were significantly lower. Collectively, these results led us to conclude 530 that spermatogenesis in the testis or sperm maturation in the epididymis might be responsible for 531 oligozoospermia in Cftr -/mice. Defects in spermatogenesis lead to infertility, including 532 azoospermia, oligospermia, and teratozoospermia. In affirming the current findings, histological 533 evidence observed in CF males with CBAVD showed severely diminished spermatogenesis with 534 abnormal sperm forms and a reduced sperm count (Chen et al., 2012). We observed that the 535 epididymal epithelium was normal in Cftr -/mice; however, the epithelial layer of Cftr -/testis was 536 thinner than that of the wild-type control. In addition, the intact vas deferens with an unimpeded 537 lumen indicated that Cftr -/mice might not mimic humans with CBAVD. A similar result was 538 previously observed that substantiated the present findings; the Cftr -/mice had intact vas deferens 539 (Reyneart et al., 2000). Furthermore, an updated study confirmed that the heterozygous mutations 540 of CFTR increased the risk of male infertility, except the CBAVD (Chen et al., 2012). Our findings 541 also revealed that the diameters of vas deferens from all genotypes were similar. This result strongly 542 suggests that CBAVD did not cause infertility in male patients with CF. 543 Immunofluorescence demonstrated an elevated level of CFTR expression in the spermatocytes 544 and spermatids in mouse testicular epithelium. The quantification results confirmed that CFTR 545 expression in germ cells was higher than that in other cells. Indeed, increased CFTR expression has 546 been observed in rat Sertoli, germ cells, and epididymis (Chen et al., 2012). However, our result event that significantly regulates germ cell development by diminishing the abnormal or damaged 549 germ cells from the seminiferous tubules and ensuring good sperm quality (Aitken & Baker, 2013); 550 a defect in this process leads to male infertility. CFTR plays a pivotal role in this process, which 551 leads to apoptosis and possibly controls the intracellular reactive oxygen species equilibrium by 552 altering the glutathione concentration (l 'Hoste et al., 2009). It has been observed that CFTR 553 depletion destabilizes spermatogenesis and produces abnormal germ cells. The results showed that 554 Cftr -/mice had fewer spermatozoa in the vas deferens but normal morphology. This scenario 555 enumerated the process of apoptosis, which may have eliminated abnormal spermatozoa and 556 excluded normal spermatozoa. 557 Caspases are cysteine proteases that negatively regulate the activation of Bcl-2 family 558 proteins (TSujimoto, 1998). From the observed results, the Bax to Bcl-2 ratio was high in Cftr -/-, 559 moderate in Cftr +/-, and low in Cftr +/+ mice. The higher Bax to Bcl-2 ratio indicated that the Cftr -/-560 mice testes had an increased tendency to undergo apoptosis. The activated caspases triggered 561 several apoptotic pathways, and we targeted the over-regulated apoptotic pathway in the CFTR-562 depleted condition with few specific caspases. Auto-initiation of cleavage of downstream pro-563 caspase activated downstream caspases (Shi, 2002). Western blotting results revealed that cleaved 564 caspase-9 was much lower in Cftr +/+ and Cftr +/testes than in the Cftr -/testes, confirming that the 565 intrinsic apoptotic pathway was involved in the present study. Cleaved caspase-9 potentially 566 regulates several effector caspases, including caspases-3 and 7. Caspase-7 was almost absent in 567 Cftr -/mice testes; in contrast, cleaved caspase-3 was over-regulated in Cftr -/germ cells but not in 568 those of Cftr +/+ and Cftr +/-. Caspase-3 is a vital executor of apoptosis and is mainly involved in the 569 cleavage of several significant cellular proteins that lead to typical morphological alterations in 570 apoptotic cells (Li et al., 2009). The presence of caspase-3 indicated that the apoptosis process had 571 reached an irreversible phase. 572 Apoptosis often displays a stage-specific pattern during spermatogenesis. In adult testes, it caspase-9 expression in spermatocytes and spermatids. This expression pattern indicated a strong 576 correlation between CFTR deficiency and apoptosis. The apoptosis mechanism comprises multiple 577 checkpoints with specific demarcations in transcription and translation and often exhibits stage-578 specific expression patterns (Plastria et al.,2007); at 6 weeks, Cftr -/mouse testes expressed 579 caspase-3 at all stages and in spermatids at stages V-VIII. At 8 weeks, Cftr -/mouse testis expressed 580 caspases -3 in spermatocytes in stages I-IV, expressed less in stages V-VIII, and expressed least in 581 stages IX-XII. These results suggest that the 6-week-old mice might be immature and in the early 582 stage of sexual development; hence, the elimination mechanism might not be as timely. However, 583 the 8-week-old mice were sexually mature, and the elimination mechanism forced the abnormal 584 germ cells into apoptosis at stages I-IV. In affirming this mechanism, a large number of degraded 585 sperm with a shrunken nucleus in the lumen of some thin tubules were observed in the testes of 586 Cftr -/mice, indicating that over-regulated apoptosis undermined the testicular epithelial structure. 587 Therefore, it was confirmed that the lack of CFTR activity triggered intrinsic apoptosis in germ 588 cells, negatively influencing CFTR -/mouse spermatogenesis. 589 The testicular micro-environment in a 6-week-old Cftr +/+ mouse exhibited an increased 590 concentration of chloride, sodium, and calcium ions in the seminiferous tubules due to rapid 591 maturation. In contrast, the 8-week-old, mature Cftr +/+ mouse displayed a decreased ion 592 concentration. Sperm capacitation is a crucial event in the maturation process that is poorly 593 mouse testes with a high ratio of K + (Fraser, 1983). Many studies have pointed out that Ca 2+ , K + , 601 and many other ions have a significant impact on differentiation and maturation processes. We 602 discovered a correlation between mouse ion concentration and sperm maturation; however, the 603 underlying mechanism remains elusive. 604 The variable ion concentrations between the 6-and 8-week-old Cftr -/mice showed that CFTR 605 depletion caused delayed sexual testicular development, and was delayed when compared with that 606 of the Cftr +/+ wild-type control. Since CFTR is a chloride channel, depletion of CFTR is likely to 607 cause abnormal ion balance. The increased chloride concentration was higher in the 8-week-old 608 Cftr -/mice than in the 6-week-old Cftr -/mice, which might have been due to the compensatory 609 effect from other ion channels, which rescued the functional loss caused by CFTR depletion. 610 However, the compensatory effect is likely to involve cations, particularly sodium. Sodium is the 611 richest cation and is often co-transported with anions such as chloride. Furthermore, the 612 accumulation of chloride disrupts the potential gradient across the plasma membrane of germ cells 613 and causes a change in sodium distribution, downregulating sodium transporters, such as SLC9A3. 614 Our previous study has confirmed that SLC9A3 interacts closely with CFTR (Wang et al., 2017). 615 Thus, the concentrations of sodium and chloride in the seminiferous tubule fluid are strongly 616 correlated. Co-expression of SLC26A3, CFTR, and SLC9A3 in the testis and epididymis affects the 617 pathophysiology of male subfertility. Moreover, CFTR and SLC26A3 have been shown to be 618 immunolocalized in elongating spermatids (Hihnala et al., 2006). 619 Ion channels are involved in many physiological mechanisms, including cell proliferation and 620 apoptosis. Ca 2+ is a widely employed intracellular messenger that regulates apoptosis via multiple 621 pathways. The ionic concentration imbalance forces survival stress in germ cells, and an elevated 622 intracellular Ca 2+ concentration indicates the initiation of apoptosis (Kondratskyi et al., 2015). The 623 classified data from our study indicated analogous results. The microdialysis method was used to 624 retrieve fluid from the Cftr -/mouse vas deferens to measure ion concentration, and it exhibited 625 elevated levels of sodium and chloride similar to that in the 8-week-old Cftr -/mouse seminiferous 26 tubule fluid. This study indicated that altering the microenvironment was a survival stress for germ 627 cells. These results collectively suggest that the microenvironment of the Cftr -/seminiferous 628 tubules indicate a delayed pattern in developing growth. 629 The apoptotic machinery is an extremely complicated and well-refined mechanism involving a 630 range of molecular players. CFTR deficiency alters the microenvironment in the testis and causes 631 abnormal spermatogenesis. It has been demonstrated that an imbalanced distribution of Hsp90 632 downregulation triggers the degradation of protein kinase b (AKT) in CFTR-knockdown cells. 633 Although the mechanism is elusive, phosphorylated-AKT levels were significantly decreased in 634 CFTR knockdown cells. AKT participates in the upregulation of Bcl2 expression through the 635 cAMP-response element-binding protein. In CFTR-knockdown cells, Bcl-2 expression is lower 636 than normal via Hsp90-mediated degradation of phosphorylated AKT (Liu et al., 2019). Bcl-2 is an 637 anti-apoptotic protein that inhibits the heterodimeric formation of binding Bax and Bak. Bax and 638 Bak are pro-apoptotic proteins that undergo conformational modifications on the mitochondrial 639 outer membrane when Bcl-2 expression is lower than under normal conditions. Activation of the 640 Bcl-2 protein family triggers the release of cytochrome C from the mitochondrial inner membrane 641 into the cytosol (Chen et al., 2001). Furthermore, Bax interacts with VDAC1, and CFTR inhibition 642 triggers overexpression of VDAC-1, leading to a significantly increased concentration of ATP in 643 CFTR mutant mice than in wild-type controls (Yan et al., 2016). 644 VDAC1, a Ca 2+ channel on the outer mitochondrial membrane, plays a critical role in ATP 645 production, which induces oxidative stress that triggers an imbalance in the antioxidant defence 646 system, leading to impairment of the OXPHOS system. VDAC1 enhances the efflux of calcium 647 ions from the mitochondria to the cytosol and alters Ca 2+ hemostasis and the mitochondrial potential. 648 Subsequently, cytochrome C is released from the outer mitochondrial membrane and binds to 649 apoptotic protease activating factor 1 (APAF1) and ATP to form the apoptosome. This apoptosome 650 potentially cleaves the pro-caspase-9 and activates it; activated pro-caspase-9 tends to cleave pro-caspase-3 (Marek, 2013). Cleaved caspase-3 signifies that apoptosis is already at an irreversible 652 step and finally leads to DNA fragmentation (Saha et al., 2016). 653 Abdominal aortic blood was obtained, and ion concentration was estimated for each 654 genotype of the mice allotted for the study. The results revealed that the alteration of the ionic 655 balance in Cftr -/mouse testes were unrelated to the circulatory system. All mice were reared in a 656 specific pathogen-free (SPF) room to avoid lung infection; hence, all were free from CF. The blood 657 of CF mice typically exhibited a lower partial pressure of O 2 (Peotta et al., 2014) combined with a 658 higher partial pressure of CO 2 . The partial pressure of O 2 in our Cftr -/mice was even higher than 659 that in the other two genotypes, which might be due to the smaller body size of the Cftr -/mice. Its 660 survival required more energy density; therefore, the supply of arterial O 2 was higher than that of 661 larger mice. Our results showed that the arterial blood samples of Cftr -/mice did not exhibit lung 662 diseases such as CF; hence, we confirmed that our blood ion data were not affected by lung diseases. 663 In addition, the chloride, sodium, and calcium ions from the blood showed no difference between 664 each genotype, indicating that the circulatory system does not contribute to Na + and Clalteration of 665 Cftr -/seminiferous tubule fluid. Collectively, these results proved that alterations in ion 666 concentration in seminiferous tubule fluid in Cftr -/mouse might be caused by a local deficiency of 667 CFTR in the testis. 668 The 6-week-old Cftr -/mice demonstrated a lower pH than that in the wild-type control. The 669 blood of Cftr -/mice possessed a lower partial pressure of CO 2; hence, the acidic nature of the blood 670 was not caused by the blood gas but probably due to the CFTR deficiency. CFTR depletion 671 destabilizes the transportation of bicarbonate and reduces bicarbonate levels leading to the acidic might be the reason for the alteration of the blood pH value. However, the precise mechanism of 678 how blood pH alteration activates germ cell apoptosis in CFTR requires further investigation. 679 680

Conclusion 681
In the present study, we observed that the Cftr -/mice were infertile and endured delayed 682 reproductive developmental maturation. This state was induced by the upregulation of apoptosis via 683 the caspase-9/caspase-3 intrinsic pathway. CFTR has a high affinity for several ion channels, such 684 as SLC9A3, and CFTR deficiency influences other ion channels, leading to ion imbalance in the 685 testis lumen. Alteration of the testicular microenvironment triggered Bax, impeded Bcl-2, and 686 promoted Bax activation of VDAC1. Subsequently, the mitochondrial membrane potential was 687 altered, and cytochrome C was released, which induced a cascade of caspase actions. As a result, 688 abnormal apoptosis activation affects spermatogenesis, causing sperm reduction in Cftr -/mice and 689 infertility. Cumulatively, these novel findings will provide a better understanding of the importance 690 of CFTR proteins in spermatogenesis. We also consider these findings as an eye-opener for 691 developing new therapeutic strategies for treating CFTR-defective infertile patients.