Introduction
In rodents, spermatozoa undergo a series of modifications to gain motility and maturity, and to acquire their capacity to fertilize ova during transit through the epididymis. The epididymis is a convoluted reproductive organ divided into four unique anatomical regions, including initial segment (IS), caput (CAP), corpus (COR), and cauda (CAU) (Cosentino and Cockett Reference Cosentino and Cockett1986; Elbashir et al., Reference Elbashir, Magdi, Rashed, Henkel and Agarwal2021; Zhou et al., Reference Zhou, De Iuliis, Dun and Nixon2018). Each region consists of distinct cell types that synthesize and secrete specific proteins and possess distinct transcriptome that plays different physiological functions in the maturation, concentration, and storage of sperm (Dube et al., Reference Dube, Chan, Hermo and Cyr2007; Johnston et al., Reference Johnston, Jelinsky, Bang, DiCandeloro, Wilson, Kopf and Turner2005; Thimon et al., Reference Thimon, Koukoui, Calvo and Sullivan2007). The epididymal epithelium is mainly comprised of principal, basal, apical, clear, halo, and narrow cells found only in the IS (Joseph et al., Reference Joseph, Shur and Hess2011; Martinez-Garcia et al., Reference Martinez-Garcia, Regadera, Cobo, Palacios, Paniagua and Nistal1995; Sullivan et al., Reference Sullivan, Legare, Lamontagne-Proulx, Breton and Soulet2019). These distinct cell types constitute a region-specific luminal microenvironment needed for sperm transport, concentration, and maturation (Cornwall Reference Cornwall2009; James et al., Reference James, Carrell, Aston, Jenkins, Yeste and Salas-Huetos2020).
The IS has a wide luminal diameter lined by elongated epithelial cells containing stereocilia. Reportedly, the epithelial cells of IS absorb ∼90% of the fluid to concentrate the sperm during transition (James et al., Reference James, Carrell, Aston, Jenkins, Yeste and Salas-Huetos2020). According to previous studies, defects in IS formation and differentiation adversely affect sperm maturation and male fertility (Krutskikh et al., Reference Krutskikh, De Gendt, Sharp, Verhoeven, Poutanen and Huhtaniemi2011; O’Hara et al., Reference O’Hara, Welsh, Saunders and Smith2011; Sipila et al., Reference Sipila, Cooper, Yeung, Mustonen, Penttinen, Drevet, Huhtaniemi and Poutanen2002; Sonnenberg-Riethmacher et al., Reference Sonnenberg-Riethmacher, Walter, Riethmacher, Godecke and Birchmeier1996). Thus, investigating the IS functions during spermatozoa transition and maturation in the epididymis will provide molecular insights into the mechanisms underlying male fertility in mammals.
EXOSC10, an important ribonuclease associated with the RNA exosome complex, degrades and processes a wide range of nuclear transcripts through its enzymatic activities (Knight et al., Reference Knight, Bastide, Peretti, Roobol, Roobol, Mallucci, Smales and Willis2016; Pefanis et al., Reference Pefanis, Wang, Rothschild, Lim, Kazadi, Sun, Federation, Chao, Elliott, Liu, Economides, Bradner, Rabadan and Basu2015; van Dijk et al., Reference van Dijk, Schilders and Pruijn2007). Mutations in RNA exosome-related genes are related to various diseases, including myeloma, diarrhea, and neurodegenerative disorders (Hartley et al., Reference Hartley, Zachos, Dawood, Donowitz, Forman, Pollitt, Morgan, Tee, Gissen, Kahr, Knisely, Watson, Chitayat, Booth, Protheroe, Murphy, de Vries, Kelly and Maher2010; Rudnik-Schöneborn et al., Reference Rudnik-Schöneborn, Senderek, Jen, Houge, Seeman, Puchmajerová, Graul-Neumann, Seidel, Korinthenberg, Kirschner, Seeger, Ryan, Muntoni, Steinlin, Sztriha, Colomer, Hübner, Brockmann, Van Maldergem, Schiff, Holzinger, Barth, Reardon, Yourshaw, Nelson, Eggermann and Zerres2013; Weißbach et al., Reference Weißbach, Langer, Puppe, Nedeva, Bach, Kull, Bargou, Einsele, Rosenwald, Knop and Leich2015). Recently, EXOSC10 has been reported to regulate gamete development in mice. Specifically, it promotes the maturation of mouse oocytes by modulating the transcriptome to degrade the growth-phase factors encoding RNAs (Wu and Dean Reference Wu and Dean2020). Additionally, EXOSC10 controls the onset of spermatogenesis in male germ cells (Jamin et al., Reference Jamin, Petit, Kervarrec, Smagulova, Illner, Scherthan and Primig2017); however, the role of EXOSC10 ribonuclease-mediated RNA metabolism within the IS in the regulation of gene expression and sperm maturation remains unclear.
In this study, we established an Exosc10 conditional knockout mouse (Exosc10 cKO) model by crossing Exosc10 F/F mice with Lcn9-Cre mice that expressed the recombinase in the principal cells of IS as early as post-natal day 17 (P17) (Gong et al., Reference Gong, Wang, Dou, Zhang, Liu, Gao and Sun2021). Histological analysis revealed that Exosc10 cKO males had normal spermatogenesis and development of the IS of the epididymis. Moreover, cauda epididymis of Exosc10 cKO males showed normal sperm abundance, morphology, motility, and spontaneous acrosome reaction frequencies comparable with those of control mice. Thus, the fertility test showed that Exosc10 cKO males had normal fertility. Collectively, these results demonstrate that EXOSC10 deficiency in the IS of the epididymis does not affect sperm development, maturation, motility, or male fertility.
Materials and methods
Animals
All the animal procedures were approved by the Ethics Committee for Animal Research of the School of Life Sciences, Shandong University, China, and were performed according to the guidelines for the care and use of laboratory animals. Exosc10 F/F mice were purchased from Cyagen. The Lcn9-Cre mouse line was provided by Professor Xiao-Yang Sun (Gong et al., Reference Gong, Wang, Dou, Zhang, Liu, Gao and Sun2021).
Mouse genotyping
Mouse tails were lysed in DirectPCR Lysis Buffer with proteinase K at 56 °C overnight, followed by incubation at 85 °C for 1 h to inactivate proteinase K. High-Fidelity PCR Mix (RiboBio Co., LTD) and primers were used to amplify specific DNA fragments. PCR reaction employed an annealing temperature of 58 °C for 35 cycles using Mastercycler Pro (Eppendorf). Genotyping primers are listed in Supplementary Table S1.
Fertility assay
To assess male fertility, control or Exosc10 cKO males were co-caged with wild-type female mice for at least 3 months. The average number of pups per litter was recorded and at least 3 mating cages were set up for each genotype.
Histological analysis
Mouse testes and epididymides were fixed in Bouin’s solution and 4% paraformaldehyde (PFA) overnight at 4 °C for histological analysis and immunostaining, respectively. Samples were embedded in paraffin and cut into 5 μm thick sections, followed by staining with hematoxylin and eosin (H&E). The sperm from cauda epididymis were spread onto slides, air-dried overnight, and fixed with 4% PFA in phosphate-buffered saline (PBS) for 30 min, followed by staining with H&E.
Immunofluorescence
After de-waxing, rehydration, and antigen retrieval with 0.01% sodium citrate buffer (pH 6.0), sections were immersed in blocking buffer containing 0.05% Tween-20 at RT for 1 h and incubated with primary antibodies (Supplementary Table S2) overnight at 4 °C. Specific secondary antibodies (Supplementary Table S2) were used to detect the antigen, and DNA was stained with Hoechst 33342. Brightfield and fluorescent images were captured with a fluorescent microscope (Nexcope NE950).
Immunoblot assay
Total protein extraction utilized 1x LDS sample buffer and 1x NuPAGE sample reducing agent (Thermo Fisher Scientific). The extracted proteins were separated on 4–12% Bis-Tris gels and transferred to polyvinylidene fluoride membranes. The membranes were blocked with 5% non-fat milk in Tris-buffered saline containing 0.05% Tween-20 (TBST) for 1 h at RT and incubated overnight with primary antibodies (Supplementary Table S2) at 4 °C. Subsequently, the membranes were washed with TBST and incubated with secondary antibodies (Supplementary Table S2) for 1 h at RT. Thereafter, the membranes were washed with TBST and developed using SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific). Finally, the signals were detected with Hyperfilm ECL (GE Healthcare) according to the manufacturer’s instructions.
RNA isolation and quantitative real-time RT-PCR
Total RNA was extracted from the IS of mouse epididymides using AFTSpin Tissue/Cell Fast RNA Extraction Kit for Animal (ABclonal) and cDNA was synthesized with ABScript III RT Master Mix (ABclonal). Quantitative RT-PCR was performed using Universal SYBR Green Fast qPCR Mix (ABclonal) and the QuantStudio 6 Flex Real-Time PCR System (Thermo Fisher Scientific). The primers are listed in Supplementary Table S1. The relative abundance of each transcript was calculated by 2−ΔΔCt and normalized to endogenous β-actin expression (Livak and Schmittgen Reference Livak and Schmittgen2001).
Computer-assisted sperm analysis
Cauda epididymides were isolated from adult mice and cut into pieces in 500 μl M2 medium (Sigma-Aldrich). Sperm were released for 10 or 120 min at 37 °C under 5% CO2. Then, 10 μl of sperm fluid was put into a glass cell chamber and observed through a 20X objective lens. The sperm concentration and motility were analyzed using computer-assisted sperm analysis (CASA). At least 200 sperm per mouse were analyzed and experiments were repeated thrice.
Spontaneous acrosome reaction
Sperm collected from cauda epididymides were incubated in TYH medium at 37 °C under 5% CO2 for 1 h. Both non-capacitated and capacitated sperm were spread onto slides and air-dried, followed by fixation with 4% PFA for 15 min, washing with PBS, and staining with FITC-conjugated Arachis Hypogaea (peanut) agglutinin (PNA, 15 μg/ml) at 37 °C for 1 h. Simultaneously, the sperm nuclei were stained with Hoechst 33342 and images were captured with a fluorescent microscope (Nexcope NE950). Spontaneous acrosome reaction frequency was calculated by the ratio of PNA-negative sperm to Hoechst 33342-positive sperm. At least 200 sperm per mouse were analyzed and repeated thrice.
Statistical analysis
Statistical analysis was performed using GraphPad Prism. The variances of the two groups were compared by the two-tailed Student’s t-test, and significance was defined as ns, no significance, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.
Results
EXOSC10 expression in the mouse epididymis
To investigate the function of EXOSC10 in mouse epididymis, we first analyzed its mRNA expression levels in different tissues of adult mice by RT-qPCR. The results showed that Exosc10 expressed ubiquitously in mice, including epididymis (Figure 1A). As epididymis is further subdivided into IS, CAP, COR, and CAU (Figure 1B), we performed the immunofluorescence staining of the entire epididymis and found that EXOSC10 was localized in the epithelial cells of the different segments of the epididymis (Figure 1C). Together, these results indicated that EXOSC10 was expressed in the mouse epididymis epithelium.
Figure 1. Expression pattern of EXOSC10 in mouse epididymis. (A) RT-qPCR analysis of Exosc10 mRNA expression in various organs of adult mice. Exosc10 is widely expressed in different organs, including the testis and epididymis. The expression level of Exosc10 in the spleen relative to β-actin was set to 1. Data are presented as mean ± SD for n = 3 biologically independent experiments. (B) Schematic diagram of mouse epididymis, including the IS, CAP, COR, and CAU. (C) Immunofluorescence staining of EXOSC10 (red) and DNA (blue) in adult epididymis. Scale bar, 50 μm. Representative of n = 3 independent biological replicates with similar results per condition. IS, initial segment; CAP, Caput; COR, Corpus; CAU, Cauda.
Generation of Exosc10 cKO mice
To elucidate the role of EXOSC10 during epithelial cell differentiation in the IS, we established an Exosc10 cKO model by crossing Exosc10 F/F mice with Lcn9-Cre knock-in mice which expressed CRE recombinase, primarily in the principal cells of the IS as early as at post-natal day 17 (P17). Exosc10 exons 4, 5, and 6 flanked with LoxP sites were excised after crossing with Lcn9-Cre mice (Figure 2A). After three generations of breeding (Figure 2B), we obtained control and Exosc10 cKO mice and performed genomic PCR for genotypic analysis (Figure 2C). The Exosc10 mRNA and EXOSC10 levels were significantly reduced in the IS of Exosc10 cKO mice compared with those in the control mice as estimated by RT-qPCR and immunoblot assay, respectively (Figure 2D–F). Moreover, the results of the immunofluorescence staining demonstrated that EXOSC10 was absent in the principal cells of the IS but present in other cell types of Exosc10 cKO mice (Figure 2G). Collectively, these results indicated that EXOSC10 was successfully ablated in the principal cells of the IS in Exosc10 cKO mice.
Figure 2. Generation of Exosc10 cKO mice with Lcn9-Cre. (A) Schematic diagram of targeting strategy for generation of Exosc10 cKO mice. Exons 4, 5, and 6 were deleted upon CRE-mediated recombination. (B) The schematic diagram of breeding strategies to obtain Exosc10 cKO mice. (C) Genomic PCR genotyping to detect LoxP band (222 bp) and Cre band (252 bp). (D) Exosc10 mRNA expression levels in the initial segment of adult control and Exosc10 cKO mice. The expression level of Exosc10 in control relative to β-actin was set to 1. Data are presented as mean ± SD for n = 3 biologically independent experiments. ***P < 0.001. (E) Western blotting depicting the levels of EXOSC10 in the initial segment of control and Exosc10 cKO mice. Representative of n = 3 independent biological replicates with similar results per condition. (F) Quantification of EXOSC10 protein levels in the initial segment of control and Exosc10 cKO mice. *P < 0.05 (G) Immunofluorescence staining of EXOSC10 in the initial segment of control and Exosc10 cKO mice. DNA was stained with Hoechst 33342. Scale bar, 50 μm. Representative of n = 3 independent biological replicates with similar results per condition.
Morphology of testis and epididymis in Exosc10 cKO mice
The effects of EXOSC10 on the testis and epididymis were examined by analyzing their morphology in Exosc10 cKO mice. The results revealed no significant differences in the size and weight of the testis and epididymis in 3-month-old Exosc10 cKO mice compared with the controls (Figure 3A–D). Moreover, the H&E staining of the testis and epididymis showed no obvious anomalies with normal spermatogenesis even in the absence of EXOSC10 (Figure 3E). In addition, epithelial cells of the IS and other segments, including CAP, COR, and CAU displayed normal histology in Exosc10 cKO mice (Figure 3F). Besides, the numbers of spermatozoa in CAP, COR, and CAU were comparable between control and Exosc10 cKO mice (Figure 3F). These results indicated that Exosc10 deficiency in the IS did not affect spermatogenesis and epididymal development.
Figure 3. Histological analysis of testis and epididymis in adult Exosc10 cKO mice. (A, B) Morphology of 3-month-old testis and epididymis of control and Exosc10 cKO mice. (C, D) Histograms of testis and epididymis weights from control and Exosc10 cKO mice. Mean ± SD for n = 3 biologically independent samples from 3 different animals. ns, no significance. (E) H&E staining of testes from control and Exosc10 cKO mice. Scale bar, 50 μm. (F) H&E staining of different segments of epididymis from control and Exosc10 cKO mice, including initial segment (IS), Caput (CAP), Corpus (COR), and Cauda (CAU). Scale bar, 50 μm. Representative of n = 3 (E, F) independent biological replicates with similar results per condition.
Sperm morphology and motility of Exosc10 cKO mice
IS of epididymis synthesizes and secretes a set of proteins to provide a unique luminal environment required for sperm maturation. First, no significant difference was observed in the sperm counts in the cauda epididymis of control and Exosc10 cKO mice (Figure 4A). Next, results of H&E staining revealed a normal morphology of most sperm (∼80%) in both control and Exosc10 cKO mice and only ∼20% abnormal sperm in Exosc10 cKO mice relative to the control males (Figure 4B, C). This indicated that Exosc10 cKO did not affect sperm concentration and morphology. Furthermore, sperm motility was analyzed using CASA and the results demonstrated no significant differences in cauda sperm motility and progressive motility (Figure 4D, E). Nevertheless, sperm parameters, including straight line velocity (VSL), curvilinear velocity (VCL), average path velocity (VAP), and amplitude of lateral head displacement (ALH) after 10 min (or 120 min) of incubation remained unaffected in the absence of EXOSC10 (Figure 4F–I). These data suggested that sperm motility was not affected after EXOSC10 ablation in the IS.
Figure 4. Sperm morphology and motility in control and Exosc10 cKO mice. (A) Sperm counts of cauda epididymis from control and Exosc10 cKO mice. (B) H&E staining of sperm from cauda epididymis of control and Exosc10 cKO mice. Scale bar, 10 μm. (C) The percentage of normal and abnormal sperm from cauda epididymis of control and Exosc10 cKO mice. CASA analysis of (D) sperm motility, (E) progressive motility, (F) VSL, straight-line velocity, (G) VCL, curvilinear velocity, (H) VAP, average path velocity, and (I) ALH, amplitude of lateral head displacement. Data are presented as mean ± SD for n = 3 biologically independent experiments. ns, no significance.
Sperm acrosome reaction in Exosc10 cKO mice
Acrosome exocytosis during fertilization releases lytic enzymes to facilitate sperm passage through the cumulus cells and zona pellucida of the eggs; therefore, the integrity of the acrosome is an important indicator of sperm quality. The frequency of acrosome exocytosis was estimated by staining cauda sperm with PNA and calculating the ratio of spontaneous acrosome reaction. The results revealed that the rates of spontaneous acrosome reaction in non-capacitated and capacitated conditions were comparable between control and Exosc10 cKO mice (Figure 5A–C), indicating a normal sperm acrosome reaction frequency in Exosc10 cKO mice.
Figure 5. Frequency of sperm acrosome reaction in control and Exosc10 cKO mice. (A, B) Representative images of sperm acrosome reaction in control and Exosc10 cKO mice in non-capacitated (A) and capacitated (B) conditions. Sperm acrosome was labeled by PNA. DNA was stained with Hoechst 33342. White arrowheads, incomplete acrosomes. Scale bar, 50 μm. Representative of n = 3 independent biological replicates with similar results per condition. (C) The frequency of spontaneous acrosome reaction in non-capacitated or capacitated sperm. Data are presented as mean ± SD for n = 3 biologically independent experiments. ns, no significance.
Alterations of IS differentiation-related genes in Exosc10 cKO mice
To determine the effect of EXOSC10 unavailability on the transcriptome of IS, a set of genes associated with IS differentiation was analyzed by RT-qPCR. The results indicated that most of the gene expression levels remained unaffected, including Pten, Ros1, Mst1, Src, and Esr1. While the expression levels of Mst2 and Ar were slightly increased, those of Dicer1 and Esr2 were somewhat decreased (Figure 6A–I). Although the levels were statistically different for all four genes, the change was less than 1.5-fold. These data suggested that the loss of EXOSC10 had no significant influence on the IS transcriptome in Exosc10 cKO mice.
Figure 6. Relative expression levels of differentiation-related genes in the initial segment. RT-qPCR analysis of (A) Pten, (B) Ros1, (C) Mst1, (D) Mst2, (E) Dicer1, (F) Ar, (G) Src, (H) Esr1, and (I) Esr2 in control and Exosc10 cKO mice. The expression levels of genes in control relative to β-actin were set to 1. Data are presented as mean ± SD for n = 3 biologically independent experiments; *P < 0.05, **P < 0.01, ***P < 0.001, ns, no significance.
Fertility of Exosc10 cKO male mice
The adult control or Exosc10 cKO male mice mated with the wild-type female mice for at least 3 months and male fertility was assessed. The results showed that Exosc10 cKO males had nearly the same number of pups per litter as control males (Figure 7). This indicated that EXOSC10 in the IS was not essential for male fertility.
Figure 7. Fertility test of Exosc10 cKO male mice. Each male mouse mated with one wild-type female mouse for at least 3 months (n = 3 per genotype). The number of pups per litter was recorded. Data are presented as mean ± SD; ns, no significance.
Discussion
Previous studies have reported that epididymal aging occurs in a segment-dependent manner and the proximal epididymis is particularly vulnerable due to rich blood supply (Huang et al., Reference Huang, Li, Sun, Yao, Gao, Wang, Hu, Wang, Ouyang, Tu, Zou, Liu, Lu, Deng, Yang and Xie2021; Markey and Meyer Reference Markey and Meyer1992). IS is the initial part of the epididymis and its dysfunction may cause sperm maturation defects and male infertility (Kiyozumi et al., Reference Kiyozumi, Noda, Yamaguchi, Tobita, Matsumura, Shimada, Kodani, Kohda, Fujihara, Ozawa, Yu, Miklossy, Bohren, Horie, Okabe, Matzuk and Ikawa2020; Krutskikh et al., Reference Krutskikh, De Gendt, Sharp, Verhoeven, Poutanen and Huhtaniemi2011; Murashima et al., Reference Murashima, Miyagawa, Ogino, Nishida-Fukuda, Araki, Matsumoto, Kaneko, Yoshinaga, Yamamura, Kurita, Kato, Moon and Yamada2011; O’Hara et al., Reference O’Hara, Welsh, Saunders and Smith2011; Sonnenberg-Riethmacher et al., Reference Sonnenberg-Riethmacher, Walter, Riethmacher, Godecke and Birchmeier1996). These findings suggest that IS plays a critical role during sperm transition and maturation in the epididymis. Therefore, our study focused on the development and function of IS in the mouse epididymis.
The effects of various IS genes on sperm maturation and male fertility have been characterized. Ros1 KO males show sperm maturation defects and sterility due to the disruption of IS formation (Sonnenberg-Riethmacher et al., Reference Sonnenberg-Riethmacher, Walter, Riethmacher, Godecke and Birchmeier1996). Lgr4 mutant males are completely infertile with immotile cauda sperm due to defects in post-natal epididymal coiling and IS differentiation (Hoshii et al., Reference Hoshii, Takeo, Nakagata, Takeya, Araki and Yamamura2007; Mendive et al., Reference Mendive, Laurent, Van Schoore, Skarnes, Pochet and Vassart2006). Moreover, androgen receptor (Ar) KO mice display a sperm transition defect and accumulation of spermatozoa in the efferent ducts, which suggests the importance of epididymal androgen signaling in IS formation and principal cell differentiation (Murashima et al., Reference Murashima, Miyagawa, Ogino, Nishida-Fukuda, Araki, Matsumoto, Kaneko, Yoshinaga, Yamamura, Kurita, Kato, Moon and Yamada2011; O’Hara et al., Reference O’Hara, Welsh, Saunders and Smith2011). Similarly, Dicer1 ablation in the epididymis causes dedifferentiation of the epithelium and imbalanced sex steroid signaling (Bjorkgren et al., Reference Bjorkgren, Saastamoinen, Krutskikh, Huhtaniemi, Poutanen and Sipila2012). Besides, OVCH2, a secreted protease, is exquisitely localized to the IS of the epididymis and its absence causes sperm defects, including aberrant ADAM3 processing, inability to bind to the zona pellucida, and passage through the utero-tubal junction (Kiyozumi et al., Reference Kiyozumi, Noda, Yamaguchi, Tobita, Matsumura, Shimada, Kodani, Kohda, Fujihara, Ozawa, Yu, Miklossy, Bohren, Horie, Okabe, Matzuk and Ikawa2020).
Recent studies have shown that EXOSC10 is involved in the regulation of gamete and embryonic development in mice. Exosc10 inactivation in the oocytes using Gdf9-Cre impairs oocyte development and maturation, leading to a depleted ovarian reserve (Demini et al., Reference Demini, Kervarrec, Guillot, Com, Lavigne, Kernanec, Primig, Pineau, Petit and Jamin2023). Moreover, the deletion of Exosc10 in the oocytes using Zp3-Cre causes female subfertility due to delayed germinal vesicle breakdown (Wu and Dean Reference Wu and Dean2020). In addition, conditional disruption of Exosc10 in male germ cells using Ddx4-Cre or Stra8-Cre impairs the growth and development of germ cells (Jamin et al., Reference Jamin, Petit, Kervarrec, Smagulova, Illner, Scherthan and Primig2017). According to another study, Exosc10 (−/−) mutant embryos are arrested at the eight-cell embryo/morula transition stage (Petit et al., Reference Petit, Jamin, Kernanec, Becker, Halet and Primig2022). To further investigate whether EXOSC10 is required for IS differentiation and sperm maturation, we established an Exosc10 cKO mouse model using Lcn9-Cre knock-in mice which expressed CRE enzyme in the principal cells of the IS as early as at P17 (Gong et al., Reference Gong, Wang, Dou, Zhang, Liu, Gao and Sun2021). Unexpectedly, Exosc10 cKO males showed normal spermatogenesis, IS development, sperm morphology, motility, and fertility. Moreover, we examined a set of IS differentiation-related genes and found that Exosc10 deficiency had no significant effect on the transcriptome levels. Only Mst2, Dicer1, Ar, and Esr2 mRNA levels were slightly modulated in the absence of EXOSC10. Consequently, our findings suggest that EXOSC10 is dispensable for IS differentiation.
The Lcn9-Cre-mediated EXOSC10 deletion occurs only in the principal cells of the IS in the entire epididymis. It is speculated that deletion of a large part of the epididymis or other cell types may affect the phenotype and fertility. Some other Cre lines, such as Defb41-Cre and Cyp17a1-Cre, express recombinase in the epithelium of the most proximal part of the epididymis (IS and CAP) and epithelial cells of the epididymis, respectively (Bjorkgren et al., Reference Bjorkgren, Saastamoinen, Krutskikh, Huhtaniemi, Poutanen and Sipila2012; Gannon et al., Reference Gannon, Darbey, Chensee, Lawrence, O’Donnell, Kelso, Reed, Parameswaran, Smith, Smith and Rebourcet2022); therefore, they may be used to explore the potential function of EXOSC10 in the development of epididymis and sperm maturation. In conclusion, our findings suggest that Exosc10 deficiency in the IS is dispensable for epididymal differentiation, sperm maturation, and male fertility.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S0967199424000418
Acknowledgments
We thank Bullet Edits for the English language editing of the manuscript. The authors are grateful to Professor You-Qiang Su from the School of Life Sciences, Shandong University for providing his lab facilities, and Professor Xiao-Yang Sun from the School of Life Sciences, Shandong University for providing Lcn9-Cre mice.
Author contributions
N. Li., and Z. Wang. designed the experiments. M. Zhou., J. Yu., Y. Xu., H. Li., YQ. Feng., X. Wang., and F. Qiu. performed the experiments. M. Zhou., N. Li., and Z. Wang. analyzed the data. M. Zhou., and Z. Wang. wrote the paper.
Competing interests
None.
Funding
This work was supported by grants from the National Natural Science Foundation of China (NSFC 32370904); the Shandong Excellent Young Scientists Fund Program (Overseas), grant number 2022HWYQ-026; the Natural Science Foundation of Shandong Province, grant number ZR2022MC005; the Qilu Scholarship of Shandong University, grant number 61200082163142.
Ethical standards
This study was carried out in the guidelines of the School of Life Sciences, Shandong University, China.
