SLC9A3 Protein Is Critical for Acrosomal Formation in Postmeiotic Male Germ Cells

Solute carrier family 9 isoform 3 (SLC9A3), a Na+/H+ exchanger, regulates the transepithelial absorption of Na+ and water and is primarily expressed on the apical membranes of the intestinal epithelium, renal proximal tubule, epididymis, and vas deferens. Loss of the Slc9a3 allele in mice enhances intestinal fluid and causes diarrhoea as a consequence of diminished Na+ and HCO3− absorption. Hence, the loss also causes male infertility and reveals the abnormal dilated lumen of the rete testis and calcification in efferent ductules. However, whether loss of Slc9a3 alleles also disrupts mammalian spermatogenesis remains unknown. First, through immunoblotting, we determined that SLC9A3 is highly expressed in the murine testis compared with the small intestine, epididymis, and vas deferens. During murine spermatogenesis, SLC9A3 is specifically expressed in the acrosome region of round, elongating, and elongated spermatids through immunostaining. Furthermore, SLC9A3 signals are enriched in the acrosome of mature sperm isolated from the vas deferens. In Slc9a3 knockout (KO) mice, compared with the same-aged controls, the number of spermatids on the testicular section of the mice progressively worsened in mice aged 20, 35, and 60 days. Sperm isolated from the epididymis of Slc9a3 KO mice revealed severe acrosomal defects. Our data indicated that SLC9A3 has a vital role in acrosomal formation during spermiogenesis.


Solute Carrier Family 9 Isoform 3 (SLC9A3)
SLC9A3 is one of nine plasma membrane Na + /H + exchangers (SLC9A1-9) and is expressed on the apical membranes of the intestinal epithelium, renal proximal tubule, epididymis, and vas deferens [1][2][3][4][5][6][7]. The widely known functions of SLC9A3 are ion homeostasis regulation through Na + and water absorption, and it often functionally couples with transepithelial Cl − /HCO 3 − exchangers in the intestine [8]. Loss of the Slc9a3 allele in mice results in increased intestinal fluid and diarrhoea because of decreased absorption of Na + and HCO 3 − [3]. The bioactivity of SLC9A3 at the apical sites of the epithelial membrane is regulated through the addition or removal of its phosphorylations, protein trafficking, and protein-protein interaction [7]. Recent studies have indicated that variations or

Loss of SLC9A3 Allele Causes Obstructive Azoospermia-Like Phenotype
Mutated cystic fibrosis transmembrane conductance regulators (CFTRs) cause cystic fibrosis (CF), and most also result in congenital bilateral absence of the vas deferens (CBAVD) [17][18][19][20]. It is the major pathological cause of obstructive azoospermia [21]. However, CFTR mutations are absent in most Taiwanese patients with CBAVD; this is consistent with the low frequency of CF mutations in Asian populations [22]. Through oligonucleotide array-based comparative genomic hybridization (array-CGH), we identified the loss of one Slc9a3 allele in 28.57% of Taiwanese men with CBAVD [23]. However, loss of SLC9A3 causes obstructive azoospermia and testicular atrophy [6]. Colleagues' and our own studies have indicated that Slc9a3 −/− adult male mice are completely infertile compared with wild-type (WT) and heterozygous mice [6,15]. Slc9a3 −/− male mice possess an abnormally dilated lumen in the rete testis and calcification in the efferent ductules. Additionally, we identified damaged postmeiotic male germ cells in adult mice (>2 months old) [6]. The proposed pathological cause is efferent ductule obstruction. However, whether loss of the Slc9a3 allele in mice also disrupts the spermatogenic process remains unknown. We sought to determine whether SLC9A3 expression is involved in mammalian spermatogenesis. In this study, we investigated the possible localization and functional roles of SLC9A3 during mammalian spermatogenesis through a KO mouse model.

SLC9A3 is Specifically Expressed in Postmeiotic Male Germ Cells
The expressional patterns of SLC9A3 are restricted to several tissues (e.g., intestines, kidneys, epididymides, and vas deferentia) in rodents and humans [11]. To determine whether SLC9A3 expresses in testicular tissues, murine testicular tissues were evaluated through Western blotting. SLC9A3 is expressed in the murine intestine, epididymis, vas deferens, and testis in WT mice ( Figure 1A Figure S2). Furthermore, testicular sections from adult mice were used to determine the localization. We determined that SLC9A3 is principally expressed in the postmeiotic male germ cells ( Figure 1B and Supplementary Materials Figure S3). Through costaining with acrosomal marker (Lectin), we determined that SLC9A3 is specifically localized in the acrosomal regions of postmeiotic male germ cells. testicular sections and separated male germ cell populations were subject to immunofluorescence analysis. Figure 2 illustrates SLC9A3 expression in the acrosomal region at stages VI-VII of murine spermatogenesis ( Figure 2E-H). Figure 2H depicts the specific granules vesicles of the acrosomal region. At stages X-XII, SLC9A3 covers the upper region of the sperm heads of the elongating spermatids ( Figure 2I-L). Figure 1. Solute carrier family 9 isoform 3 (SLC9A3) expression in the murine testis (A) SLC9A3 expression in the intestines, epididymides, vas deferentia, and testes of adult wild-type mice was compared with that in the intestines of Slc9a3 −/− mice through western blotting. The upper image shows SLC9A3 expression (arrow), and the lower image shows Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression, which was used as a loading control; (B) Testis of WT mouse was detected through immunofluorescence staining with anti-SLC9A3 primary antibodies (SLC9A3 signals; green) (a). (b) Lectin (acrosome marker; red) and (c) 4′,6-diamidino-2-phenylindole (DAPI) (nucleus marker; blue) were costained and are displayed as a merged image (d). Scale bar = 50 μm. Figure 1. Solute carrier family 9 isoform 3 (SLC9A3) expression in the murine testis (A) SLC9A3 expression in the intestines, epididymides, vas deferentia, and testes of adult wild-type mice was compared with that in the intestines of Slc9a3 −/− mice through western blotting. The upper image shows SLC9A3 expression (arrow), and the lower image shows Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression, which was used as a loading control; (B) Testis of WT mouse was detected through immunofluorescence staining with anti-SLC9A3 primary antibodies (SLC9A3 signals; green) (a). (b) Lectin (acrosome marker; red) and (c) 4 ,6-diamidino-2-phenylindole (DAPI) (nucleus marker; blue) were costained and are displayed as a merged image (d). Scale bar = 50 µm.

SLC9A3 is Involved in Acrosomal Formations
To determine the precise expressional stages of SLC9A3 during murine spermiogenesis, testicular sections and separated male germ cell populations were subject to immunofluorescence analysis. Figure 2 illustrates SLC9A3 expression in the acrosomal region at stages VI-VII of murine spermatogenesis ( Figure 2E-H). Figure 2H   To evaluate SLC9A3 expressed in the murine spermatocytes and the first waves of early-round spermatids, testicular sections from 20-day-old mice were used. The 20-day-old mice lacked SLC9A3 signals of testicular sections, which comprised spermatogonia, spermatocyte, and early-round spermatids, whereas 35-day-old mice exhibited strong expression of the testicular tissues, which had completed the first spermatogenic waves ( Figure 3). During sperm-head formation, SLC9A3 covered the acrosomal regions of elongating ( Figure 4A-C) and elongated spermatids ( Figure 4D-F) isolated from the testicular tissue. These results indicated that SLC9A3 was specifically expressed for acrosomal formation in elongating and elongated spermatids.

Loss of Slc9a3 Allele Disturbs the Spermiogenic Process of 35-Day-Old Mice
To determine the early progressive effects of SLC9A3 deficiency, we evaluated the testicular sections of WT and Slc9a3 −/− mice at 20, 35, and 60 days old. The arrangements of the seminiferous tubules and interstitial tissue in the 20-, 35-, and 60-day-old WT mouse testes were effectively organized, and the male germ cells exhibited complete development ( Figure 5A-F). The male germ cell population and numbers in seminiferous tubules of 20-day-old WT and Slc9a3 −/− mice were comparable ( Figure 5G,H). First, in 35-day-old testes, spermatogenesis was moderately decreased and fewer spermatids were observed in the lumen compared with WT testes ( Figure 5C,D,I,J; Arrowheads). Second, in 60-day-old mice, most testicular lumens in Slc9a3 −/− males lacked postmeiotic male germ cells and exhibited an inferior spermiogenic process ( Figure 5K-L). The affected testicular sections of Slc9a3 −/− mice and the similar duration for SLC9A3 expression on acrosomal formation indicated that SLC9A3 was involved in acrosomal formation during the spermiogenic process.

SLC9A3 is Essential for Acrosome Integrity
To determine whether loss of SLC9A3 disrupts the terminal development and maturation of male germ cells, we evaluated the sperm counts and spermatozoa integrations from the vas deferens and epididymis. First, sperm was collected from the vas deferentia of WT (>60 days old, n = 10) and Slc9a3 −/− mice (>60 days old, n = 16). Figure 6 shows that the sperm of Slc9a3 −/− mice were absent, in To evaluate SLC9A3 expressed in the murine spermatocytes and the first waves of early-round spermatids, testicular sections from 20-day-old mice were used. The 20-day-old mice lacked SLC9A3 signals of testicular sections, which comprised spermatogonia, spermatocyte, and early-round spermatids, whereas 35-day-old mice exhibited strong expression of the testicular tissues, which had completed the first spermatogenic waves (Figure 3). During sperm-head formation, SLC9A3 covered the acrosomal regions of elongating ( Figure 4A-C) and elongated spermatids ( Figure 4D-F) isolated from the testicular tissue. These results indicated that SLC9A3 was specifically expressed for acrosomal formation in elongating and elongated spermatids.

Loss of Slc9a3 Allele Disturbs the Spermiogenic Process of 35-Day-Old Mice
To determine the early progressive effects of SLC9A3 deficiency, we evaluated the testicular sections of WT and Slc9a3 −/− mice at 20, 35, and 60 days old. The arrangements of the seminiferous tubules and interstitial tissue in the 20-, 35-, and 60-day-old WT mouse testes were effectively organized, and the male germ cells exhibited complete development ( Figure 5A-F). The male germ cell population and numbers in seminiferous tubules of 20-day-old WT and Slc9a3 −/− mice were comparable ( Figure 5G,H). First, in 35-day-old testes, spermatogenesis was moderately decreased and fewer spermatids were observed in the lumen compared with WT testes ( Figure 5C,D,I,J; Arrowheads). Second, in 60-day-old mice, most testicular lumens in Slc9a3 −/− males lacked postmeiotic male germ cells and exhibited an inferior spermiogenic process ( Figure 5K-L). The affected testicular sections of Slc9a3 −/− mice and the similar duration for SLC9A3 expression on acrosomal formation indicated that SLC9A3 was involved in acrosomal formation during the spermiogenic process.

SLC9A3 is Essential for Acrosome Integrity
To determine whether loss of SLC9A3 disrupts the terminal development and maturation of male germ cells, we evaluated the sperm counts and spermatozoa integrations from the vas deferens and epididymis. First, sperm was collected from the vas deferentia of WT (>60 days old, n = 10) and Slc9a3 −/− mice (>60 days old, n = 16). Figure 6 shows that the sperm of Slc9a3 −/− mice were absent, in contrast to WT mice. To determine the possible ultrastructure effects of SLC9A3 on sperm through transmission electron microscopy, tiny sperm were collected from the epididymis of the Slc9a3 −/− mice (n = 3) for comparison with the sperm of WT mice (n = 3). The acrosome from the sperm of the Slc9a3 −/− mice revealed a fragment-like structure, are similar to the small vesicles, in Figure 7B (indicated by the arrow), compared with the sperm of WT mice. These results indicate that the SLC9A3 function is critical to the integrity of sperm acrosome. contrast to WT mice. To determine the possible ultrastructure effects of SLC9A3 on sperm through transmission electron microscopy, tiny sperm were collected from the epididymis of the Slc9a3 −/− mice (n = 3) for comparison with the sperm of WT mice (n = 3). The acrosome from the sperm of the Slc9a3 −/− mice revealed a fragment-like structure, are similar to the small vesicles, in Figure 7B (indicated by the arrow), compared with the sperm of WT mice. These results indicate that the SLC9A3 function is critical to the integrity of sperm acrosome.    contrast to WT mice. To determine the possible ultrastructure effects of SLC9A3 on sperm through transmission electron microscopy, tiny sperm were collected from the epididymis of the Slc9a3 −/− mice (n = 3) for comparison with the sperm of WT mice (n = 3). The acrosome from the sperm of the Slc9a3 −/− mice revealed a fragment-like structure, are similar to the small vesicles, in Figure 7B (indicated by the arrow), compared with the sperm of WT mice. These results indicate that the SLC9A3 function is critical to the integrity of sperm acrosome.

Immunoblotting
The murine tissues were homogenised in a lysis buffer and total protein extractions were heated for 5 min at 37 °C before SDS-PAGE [24]. The antibodies against SLC9A3 (ab95299; Abcam, Cambridge, MA, USA) and GAPDH (G8795; Sigma-Aldrich, St. Louis, MO, USA) were applied and detected through chemiluminescence [6].

Immunoblotting
The murine tissues were homogenised in a lysis buffer and total protein extractions were heated for 5 min at 37 • C before SDS-PAGE [24]. The antibodies against SLC9A3 (ab95299; Abcam, Cambridge, MA, USA) and GAPDH (G8795; Sigma-Aldrich, St. Louis, MO, USA) were applied and detected through chemiluminescence [6].

Separation of the Murine Male Germ Cell Populations
The male germ cells were isolated from the testicular tissues of adult mice (60-day-old; n = 3). As described in our previous article, male germ cells were separated depending on the density of the various male germ cell types through a centrifugal system [25]. Further, spermatogonia, spermatocyte, and spermatids were separated and dried on slides. Through MitoTracker staining, elongating and elongated spermatids (n > 10) were identified.

Electron Microscopy
Sperm were isolated from the caput epididymides from WT (60-day-old; n = 3) and Slc9a3 −/− mice (60-day-old; n = 3), and were directly fixed with 4% paraformaldehyde and 0.1% glutaraldehyde overnight at 4 • C. Subsequently, the sperm were washed with 0.1 M phosphate buffer (pH 7.2) and were rinsed with 1% osmium tetroxide at 25 • C for 2 h. After being re-treated with phosphate buffer, the samples were progressively dehydrated by raising the ethanol concentration. Subsequently, the sperm were embedded with Spurr's resin kit (cat-14300; Electron Microscopy Sciences, Hatfield, PA, USA) overnight at 25 • C. The embedded samples were sectioned into 75-nm-thick sections using an ultramicrotome (EM UC7; Leica Microsystems, Wetzlar, Germany) and were mounted onto copper grids. Ultramicrographs were acquired using a transmission electron microscope (JEM-1400; JEOL, Tokyo, Japan) at 100 kVA.

Discussion
Studies have indicated that disrupted SLC9A3, a Na + /H + exchanger, results in male sterility because of the disturbed acidification and obstructed male reproductive tract. In this study, we found that SLC9A3 is expressed in the acrosomal region of spermatids. Additionally, loss of SLC9A3 reduced sperm production in the testis and disrupted the acrosome formation in vivo. Our evidence suggests that SLC9A3 not only maintains the pH balance of the male reproductive tract but also facilitates acrosome development during murine spermiogenesis.

SLC9 Family Is Implicated in Sperm Formation
Maintaining and regulating intracellular pH (pHi) homeostasis are critical for cell physiology (e.g., regulating cell proliferation and cell survival) [26,27]. The SLC family is one of the major modelling protein families. The SLC9 family, including SLC9A, SLC9B, and SLC9C, are wildly expressed and extremely efficient in pHi regulation (Fuster et al. [28]). SLC9A1, SLC9A2, and SLC9A3 are enriched in the efferent ducts and regions of the epididymis [14]. Furthermore, SLC9A1, SLC9A5, and SLC9C1 (sNHE) are expressed in mature spermatozoa [16]. Three of these genes in the SLC9 family have been disrupted in mice. First, SLC9A1, located at the sperm midpiece and the disrupted Slc9a1 allele in mice revels normal in male reproduction [29]. SLC9C1 is specifically expressed in the sperm principal piece, and loss of Slc9c1 causes dramatic immotility of sperm and reduced male fertility [30]. Third, Slc9a3 KO causes male infertility and reveals the abnormal dilated lumen of the rete testis and efferent ductules [15]. The major cellular function of SLC9A3 is transporting H + in exchange for Na + on the cell membrane, resulting in fluid absorption and acidification of the male reproductive tract [1]. In this study, we determined that the SLC9A3 expressed during acrosome formation and Slc9a3 allele KO disrupted the acrosomal structure in mature sperm. We speculate that the loss of SLC9A3 protein disturbs fluid absorption and Na + homeostasis during acrosome biogenesis, resulting in disintegration of the acrosomal membrane. This is first study to identify the SLC9 family's involvement in acrosome formation and integration.

Oestrogen Receptor α Regulates SLC9A3 Expression in Male Reproduction
Zhou Qing et al. reported that oestrogen receptor α (ERα) may regulate the SLC9A3 mRNA expression in the efferent ductules by determining the SCL9A3 expression in an ERα knockout mice model [15]. Loss of ERα in mice resulted in testis weight reduction, lower sperm count, abnormal dilated lumen of the rete testis, and calcification in the efferent ductules [31,32]. These phenotypes are similar to those of Slc9a3 knockout mice [6,15]. Recently, Joseph et al. revealed the high-frequency spontaneous acrosomal reactions of mature spermatozoa of ERα knockout mice [33]. In the present study, we characterized the SLC9A3 involved in sperm production and acrosomal integration of sperm. The similar results indicated that ERα may not only regulate SLC9A3 expression in the efferent ductules but also acrosomal development and maintenance.

SLC9A3-CFTR Complexes and Male Germ Cell Production
In a colleague's study and our previous study, SLC9A3-CFTR formed a complex and disrupted the Slc9a3 allele in mouse to decrease the CFTR levels in the epididymis and vas deferens [6,34]. In clinical studies, loss of CFTR resulted in CBAVD and obstructive azoospermia [17,18,20]. The CBAVD cases also reduced the success rates of fertilization in intracytoplasmic sperm injections using epididymal sperm [35,36]. Wu et al. found that CFTR is expressed in the human and mouse sperm head [37]. Furthermore, loss of CFTR reduces capacitation, disturbs the levels of pHi and cAMP production, and reduces membrane hyperpolarization of mouse sperm [37]. We speculate that loss of the Slc9a3 allele in sperm production may also reduce CFTR, resulting in a disturbed intracellular pHi in sperm, and concluding in the disintegration of the membrane of the acrosome.

Conclusions
In this study, we determined that SLC9A3 also expresses in sperm and is critical for acrosomal integration. However, whether SLC9A3 affects the physiological functions of sperm (e.g., acrosome reaction and fertilizing capacity) remains to be investigated.