Utrophin Compensates dystrophin Loss during Mouse Spermatogenesis

Chen, Hung-Chih; Chin, Yu-Feng; Lundy, David J.; Liang, Chung-Tiang; Chi, Ya-Hui; Kuo, Paolin; Hsieh, Patrick C. H.

doi:10.1038/s41598-017-05993-8

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Published: 07 August 2017

Utrophin Compensates dystrophin Loss during Mouse Spermatogenesis

Scientific Reports volume 7, Article number: 7372 (2017) Cite this article

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Abstract

Duchenne muscular dystrophy (DMD) is an X-linked genetic disorder resulting from mutations in the dystrophin gene. The mdx/utrn ^−/− mouse, lacking in both dystrophin and its autosomal homologue utrophin, is commonly used to model the clinical symptoms of DMD. Interestingly, these mice are infertile but the mechanisms underlying this phenomenon remain unclear. Using dystrophin deficient mdx mouse and utrophin haplodeficient mdx/utrn ^+/− mouse models, we demonstrate the contribution of Dp427 (full-length dystrophin) and utrophin to testis and epididymis development, as well as spermatogenesis. We show that Dp427 deficiency disturbed the balance between proliferation and apoptosis of germ cells during spermatogenesis, which was further disrupted with utrophin haplodeficiency, deciphering a compensatory role of utrophin for dystrophin in the male reproductive system. In the spermatozoa, we have found a compensatory response of utrophin to dystrophin deficiency - namely the upregulation and relocation of utrophin to the flagellar midpiece. This study demonstrates the contribution of Dp427 and utrophin in male fertility, suggesting a potential pathology in DMD patients.

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Introduction

Lack of the protein dystrophin results in Duchenne muscular dystrophy (DMD), a devastating hereditary childhood disease. DMD is characterized by progressive muscle degeneration, loss of ambulation in adolescence, and cardiopulmonary failure that leads to the death of DMD patients during the third decades of their lives¹. Dystrophin is encoded by the largest known human gene DMD, which spans about 2.4 Mb of Xp21 and comprises 79 exons^{2, 3}. Dystrophin is a rod-shaped cytoskeletal protein linking extracellular laminin and intracellular F-actin in muscles to mediate force transmission and signal transduction^4,5,6. In addition to this mechanical role, dystrophin also regulates asymmetric division of satellite cells by regulating the polarity of a microtubule kinase MARK2⁷. Alternative promoters and splicing produce tissue-specific isoforms of dystrophin, including the full length Dp427 in skeletal and cardiac muscles, Dp260 in retina, Dp140 in brain and kidney, Dp116 in peripheral nerves, and ubiquitous Dp71⁸. Although predominant in skeletal and cardiac muscles, Dp427 mRNA is also detectable in the mouse testes⁹.

Utrophin (product of UTRN), an autosomal homologue of dystrophin, maps to human chromosome 6 (6q24) and mouse chromosome 10¹⁰. Like dystrophin, utrophin contains an N-terminal actin-binding domain and a C-terminal dystroglycan-binding domain¹¹. The nucleic acids and amino acids of utrophin respectively share 65% and 80% identity with dystrophin in human¹⁰. Utrophin is expressed ubiquitously in many fetal and adult tissues, including liver, spleen, skeletal muscles and testes¹². Utrophin localizes in the neuromuscular junction of adult skeletal muscles, whereas it localizes to the sarcolemma in regenerating muscle fibers^{13, 14}. Sarcolemma recruitment and upregulation of utrophin in the skeletal muscle of mdx mice, the most common animal model of DMD, with a nonsense mutation in exon 23, implies that utrophin plays a role to compensate for the loss of dystrophin^14,15,16. A similar compensatory effect of utrophin has been proposed in DMD patients, who also have a higher level of utrophin in their skeletal muscles¹⁷.

Although dystrophin and utrophin are both expressed in the testes, their function in the male reproductive system remains unclear. Up71 (a short isoform of utrophin) and Dp71 are expressed in the postacrosomal region of the spermatozoa, with the expression of Dp71 extending to the midpiece of the spermatozoal flagella. The absence of Dp71 results in abnormal flagella and reduced fertility in mdx ^3cv; another mouse model of DMD whose AT to A transversion in exon 66 of Dmd causes frameshift of the encoded mRNA¹⁸. Although no altered fertility in mice lacking Dp427 (mdx) or utrophin (utrn ^−/−) has been reported, mdx/utrn ^−/− mice null for both dystrophin and utrophin are infertile^{19, 20}. In this study, we used mdx and mdx/utrn ^+/− mice to unravel the role played by dystrophin and utrophin in the male reproductive system.

Results

The mdx/utrn ^+/− mice have impaired reproductive system

The mdx/utrn ^−/− (dystrophin- and utrophin-deficient) mice show premature death¹⁹. Therefore, we used mdx and mdx/utrn ^+/− (dystrophin-deficient and utrophin-haplodeficient) mice to elucidate the role of dystrophin and utrophin in male mouse reproductive system (Fig. 1a). The C57BL/10 and mdx mice gave birth to similarly-sized litters; however, the average number of pups per litter from mdx/utrn ^+/− mice was only half of C57BL/10 (Fig. 1b). Further examination of the male reproductive organs showed that the relative weights of testis and epididymis of mdx mice were reduced by 24% and 18%, respectively, compared to C57BL/10; nevertheless, utrophin haplodeficiency did not further reduce the size of testis or epididymis in mdx/utrn ^+/− mice (Fig. 1c–e). In addition, the litter size of the utrophin homozygous null mice (utrnko) was also comparable to C57BL/10 and mdx (Fig. 1a,b). Although the results suggest the involvement of dystrophin and utrophin during the development of male reproductive organs, the contribution of utrophin in mdx/utrn ^+/− mouse infertility needs further investigation.

Dystrophin and utrophin are expressed in the testes

Next, we analyzed the expression of dystrophin and utrophin in the testes. Quantitative real-time PCR (qPCR) revealed the expression of utrophin in the testes of C57BL/10, mdx and mdx/utrn ^+/− mice (Fig. 2a). Interestingly, the expression of utrophin rose 1.5 fold in mdx testes (Fig. 2a). Western blotting confirmed the absence of dystrophin protein in mdx and mdx/utrn ^+/− testes and a 1.5 fold increase of utrophin protein in the mdx testes (Fig. 2b and Supplementary Fig. S1a,b), suggesting a compensatory expression of utrophin in response to dystrophin deficiency. Immunofluorescent labeling showed the presence of full-length dystrophin (dystrophin with the molecular weight of 427 kDa and hereinafter referred to as Dp427) in the germ cells in C57BL/10 testes but not in mdx or mdx/utrn ^+/− testes (Fig. 2c). Similar to dystrophin in C57BL/10 testes, utrophin was also observed in the germ cells of C57BL/10, mdx and mdx/utrn ^+/− with more intense labeling for utrophin in mdx (Fig. 2d). These results indicate that dystrophin and utrophin may participate in the germ cell function.

Spermatozoa from mdx and mdx/utrn ^+/− have impaired mobility and morphology

We isolated spermatozoa from the cauda epididymidis of C57BL/10, mdx and mdx/utrn ^+/− mice in order to examine the influence of dystrophin/utrophin on spermatozoa (Fig. 3a). The motility rate of spermatozoa was determined as the percentage of spermatozoa with normally beating flagella. Nearly 50% of C57BL/10 spermatozoa were found to be motile; whereas the mobility rate of the spermatozoa from mdx or mdx/utrn ^+/− mice was only 30% (Fig. 3b and Supplementary Fig. S2a). Furthermore, the utrnko spermatozoa exhibited comparable motility rate as the mdx and mdx/utrn ^+/− spermatozoa (Fig. 3b). Aberrant spermatozoal morphologies such as folding or double tails influence their mobility, thus we next explored the spermatozoal morphology (Fig. 3c). By contrast to C57BL/10 spermatozoa, which show 30% of abnormality, both mdx and mdx/utrn ^+/− had up to 56% of the spermatozoon with abnormal morphology (Fig. 3d). Despite a comparable percentage of morphological abnormality in the spermatozoa of mdx and mdx/utrn ^+/− mice, the mdx spermatozoa had similar percentages of abnormality in the tail and the head (26.8 ± 5.9% vs. 30.1 ± 6.3%; p = 0.71) while the mdx/utrn ^+/− spermatozoa had a higher percentage of abnormality in the head over the tail (35.2 ± 3.8% vs. 21.7 ± 2.6%; p = 0.015) (Fig. 3d,e). Collectively, these data suggest both Dp427 and utrophin may be required for normal production of spermatozoa.

Dp71 is the shortest and the predominant dystrophin isoform in the spermatozoa, whose expression affects the flagellum function and thus fertility¹⁸. To evaluate whether absence of the Dp427 affects Dp71 in spermatozoa, we immunofluorescently labeled Dp427, Dp71 and utrophin in spermatozoa isolated from 10-week-old C57BL/10, mdx and mdx/utrn ^+/−. Dp427 localized in the spermatozoon head and the flagellar midpiece in C57BL/10 and, as expected, was absent from the spermatozoa from mdx and mdx/utrn ^+/− (Fig. 4a). Consistent with the previous report, Dp71 localized in the head of the spermatozoa and the midpiece of the flagella in C57BL/10 (Fig. 4b)¹⁸. Moreover, we observed no altered localization of Dp71in spermatozoa from mdx and mdx/utrn ^+/− compared to C57BL/10 (Fig. 4b), suggesting that Dp427 exerts no effect on the expression and localization of Dp71 in spermatozoa. Interestingly, utrophin was also located in the flagellar midpiece of the spermatozoa from C57BL/10, mdx and mdx/utrn ^+/− mice and was upregulated in the mdx spermatozoa (Fig. 4c), arguing partial functional redundancy of utrophin and dystrophin in the spermatozoal flagella. Collectively, these data indicate that Dp427 and utrophin may affect the spermatozoal mobility.

The mdx and mdx/utrn ^+/− testes have degenerated seminiferous tubules

The testes are occupied by the seminiferous tubules within which the germ cells lie and undergo spermatogenesis. Using hematoxylin-eosin staining, we investigated the effects of dystrophin/utrophin deficiency on testicular morphology. The majority of the C57BL/10 seminiferous tubules were composed of layers of spermatogonia, preleptotene spermatocytes, pachytene spermatocytes, round spermatids and elongated spermatids which are characteristic of spermatogenesis at stage VII-VIII (Supplementary Fig. S3a,b)^{21, 22}. The elongated spermatids in the lumen of seminiferous tubules were fewer in the mdx testes than in the C57BL/10 testes (Supplementary Fig. S3a,b,d,e). Similarly, the epididymisal lumen also showed a reduction of spermatozoa in mdx mice (Supplementary Fig. S3c, compared with Fig. S3f). In addition, the seminiferous tubules of the mdx testes exhibited mild focal vacuolation, suggestive of moderate degeneration of seminiferous tubules (Supplementary Fig. S3e; arrow). Interestingly, utrophin haplodeficiency worsened the degeneration of testicular seminiferous tubules in mdx/utrn ^+/−; the mdx/utrn ^+/− testes showed not only prominent vacuolation in most tubules (Supplementary Fig. S3g; arrows), but also multinucleated giant cells (Supplementary Fig S3g; arrowheads), which may be attributed to degeneration and widening of intercellular bridges between germ cells²³. There was also a severe loss of elongated spermatids and spermatozoa in the tubule lumen of mdx/utrn ^+/− testes and epididymidis respectively (Supplementary Fig. S3h,i). These data indicate the deficiency of dystrophin and utrophin may lead to degeneration of seminiferous tubules.

The mdx and mdx/utrn ^+/− mice have perturbed gene expression for spermatogenesis

To establish whether the absence of dystrophin/utrophin influences Sertoli cells and germ cells of different stages, we evaluated the expression of genes specific to these cells²⁴. Compared to C57BL/10, the expression of Gdnf was slightly increased in both mdx and mdx/utrn ^+/− Sertoli cells (Fig. 5a). Although the expression of Sertoli cell marker Rhox1 in mdx/utrn ^+/− was only half that in C57Bl/10, the expression of another Sertoli cell marker Rhox8 was constant in all three strains (Fig. 5a). The expression of the self-renewal related spermatogonium markers Cdh1, Pou5f1 (also known as Oct4) and Zbtb16 (also known as Plzf) was not significantly different in all three strains (Fig. 5b); however, Stra8, which is required for spermatogonium differentiation, was down-regulated in both mdx and mdx/utrn ^+/− (Fig. 5b). Except for Bmp8b, whose expression was not altered in spermatocytes of all three mouse strains, expression of the other spermatocyte markers Clgn, Dmc1, Id2 and Sycp3 decreased drastically in mdx/utrn ^+/− mice (Fig. 5c). With regard to the spermatids, there was a marked recession of Camk4, Crem, Odf1, Pgk2, Prm2 and Tnp1 in mdx/utrn ^+/− compared to C57BL/10 (Fig. 5d). Similarly, the expression of Camk4, Crem, Odf1 and Prm2 was also decreased in mdx in comparison with C57BL/10 (Fig. 5d). The results argue that inadequacy of dystrophin/utrophin may contribute to perturbation of the progression through spermatogenesis in the testes.

The number of spermatogenic cells diminishes in the mdx and mdx/utrn ^+/− testes

Spermatogenesis, a process involving differentiation of spermatogonia to spermatocytes and eventually the spermatids, initiates within the seminiferous tubules of the testis and requires the support of Sertoli and Leydig cells (Fig. 6a). To establish whether the deficiency of dystrophin/utrophin has an influence on germ cells, we focused on the changes of germ cell number. The spermatozoa concentration of the mdx and utrnko cauda epididymidis were less than that of C57BL/10 by 28% and 21% respectively though not statistically significant; moreover, the spermatozoa concentration further reduced by 58% in the mdx/utrn ^+/− cauda epididymis compared to C57BL/10 (Fig. 6b). In addition, there was a dramatic decrease of viable spermatozoal concentration determined with computer-assisted sperm assay in the mdx cauda epididymis (1.27 ± 0.12 million/ml) compared to C57BL/10 (8.17 ± 0.42 million/ml); the viable spermatozoal concentration further decreased in the mdx/utrn ^+/− epididymis (Supplementary Fig. 2b), reflecting that both dystrophin and utrophin play a role during spermatogenesis. We then investigated the average number of germ cells per seminiferous tubules at spermatogenic stages VII-VIII to determine the effects of dystrophin/utrophin insufficiency on spermatogenesis (Fig. 6c). The number of Sertoli cells per seminiferous tubule did not differ among C57BL/10, mdx and mdx/utrn ^+/− testes (Fig. 6c,d). However, the number of spermatogonia per seminiferous tubule decreased from 33.08 ± 2.24 in C57BL/10 to 20.78 ± 2.52 in mdx and further to 13.80 ± 1.696 in mdx/utrn ^+/− (Fig. 6c,e). In addition, the number of pachytene spermatocytes per seminiferous tubule in mdx (20.78 ± 2.52) and mdx/utrn ^+/− (13.80 ± 1.70) exhibited 37% and 58% reductions, respectively, compared to C57BL/10 (33.08 ± 2.24) (Fig. 6c,f). The number of round and elongated spermatids per seminiferous tubule reduced by 32% and 37% respectively in mdx by contrast to C57BL/10 (99.30 ± 11.72 for round spermatids and 103.50 ± 4.13 for elongated spermatids) (Fig. 6c,g,h). Moreover, deficiency of dystrophin/utrophin further declined the number of round and elongated spermatids per seminiferous tubules in mdx/utrn ^+/− by 54% and 68% respectively in comparison to C57BL/10 (Fig. 6c,g,h). These results demonstrate that dystrophin deficiency compromises spermatogenesis and concomitant utrophin haplodeficiency further exacerbates germ cell loss.

The mdx and mdx/utrn ^+/− mice have increased apoptosis and decreased proliferation of spermatogenic cells

The reduction of spermatogenic cells in mdx and mdx/utrn ^+/− could be attributed to increment in apoptosis and/or decline in proliferation. Loss of dystrophin in satellite cells results in downregulation of the serine-threonine kinase MARK2 (a cell polarity regulator), leading to defects in asymmetric division and prolonged cell cycle of satellite cells⁷. In the normal testes, Sertoli cells extend from the basal to the luminal compartment of the seminiferous tubules to support, nourish and organize spermatogenic cells^{25, 26}. We first stained microtubules in the testes to identify the morphological changes of Sertoli cells. Protrusion of Sertoli cells was less in mdx and nearly loss in mdx/utrn ^+/− by contrast to that in C57BL/10 (Fig. 7a), which may lead to disorganization and loss of germ cells. We also observed significant decline of MARK2 positive germ cells in mdx and mdx/utrn ^+/−, especially the mdx/utrn ^+/− (Fig. 7b), indicating impaired polarity of spermatogenic cells undergoing cell division.

We then evaluated whether deficiency on dystrophin/utrophin affects the rates of apoptosis and proliferation by TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) assay and Ki67 (a nuclear protein associated with cell proliferation) staining respectively. The seminiferous tubules examined were at the stage VII-VIII of spermatogenesis. The number of TUNEL-positive spermatogonia and pachytene spermatocytes per seminiferous tubule were 3.6 fold and 6.25 fold more in mdx testes than in C57BL/10 respectively (Fig. 7c,d,e). Furthermore, 13.2 fold more TUNEL-positive spermatogonia and 23.6 fold more TUNEL-positive spermatocytes were observed in mdx/utrn ^+/− testes than in C57BL/10 respectively (Fig. 7c,d,e), suggesting that utrophin haplodeficiency further exacerbates germ cell apoptosis in the absence of dystrophin. On the other hand, the number of Ki67-spermatogonia and spermatocytes per seminiferous tubule in the mdx testes were only 67% and 50% that in C57BL/10 (46.08 ± 2.56 for spermatogonia and 58.98 ± 4.87) respectively (Fig. 7f,g,h). In addition, a further reduction in the number of Ki67-positive spermatogonia and spermatocytes per seminiferous tubule was observed in the mdx/utrn ^+/− testes which were only 38% and 19% that in C57BL/10 (Fig. 7f,g,h), indicating that dystrophin/utrophin deficiency also depleted proliferating germ cells. The results argue that both dystrophin and utrophin are required for spermatogenesis and that utrophin-haplodeficiency further exacerbates the effects of dystrophin deficiency on apoptosis and proliferation of germ cells.

Discussion

In addition to the skeletal and cardiac muscles, dystrophin and utrophin are also expressed in the adult testes, in which their function remains unclear^{9, 12}. Our goal was to investigate the role of dystrophin and utrophin in male reproductive system. In DMD patients, the level of utrophin negatively correlates with the disease severity^{11, 17}. In the mdx mice, transgenic expression of both full-length and truncated utrophin lacking most spectrin repeats restores the components of dystrophin-glycoprotein complex to the sarcolemma to ameliorate the degeneration of myofibers and improves the muscle function^27,28,29. All of these reports suggest a compensatory role of utrophin for dystrophin in skeletal muscles. In this report, we have demonstrated that dystrophin deficiency in the testes also up-regulates utrophin in a similar manner to the skeletal muscle (Fig. 2)^{15, 17}, which appears to play a compensatory role. Interestingly, we found that dystrophin deficiency and utrophin haplodeficiency compromise fertility of male mdx/utrn ^+/− mice (Fig. 1). Consequently, we demonstrated perturbed spermatogenesis in male mdx/utrn ^+/− mice.

Previously, Hernandez-Gonzalez et al. showed the expression of Dp71 in the spermatozoal head and the midpiece of flagellum where it tightly regulates the level and localization of voltage-dependent Na⁺ (μ1), K⁺ (Kv1.1) channels and neural nitric oxide synthase (nNOS), and therefore the flagellar movement¹⁸. In this study, we demonstrated that Dp427 also participates in modulating the flagella function of spermatozoa (Figs 3 and 4). However, whether dystrophin performs its function in the flagella of spermatozoa via association with nNOS and voltage-dependent Na⁺ (μ1) and K⁺ (Kv1.1) channels has yet to be determined. One intriguing finding is that utrophin is not only located in the midpiece of the flagella but its expression is also upregulated in the absence of Dp427 (Fig. 4c), indicating that both utrophin and dystrophin play roles in the spermatozoal flagella. Although the motility rate of utrnko spermatozoa also decreased to a similar degree as mdx spermatozoa, neither the spermatozoal morphology nor the spermatozoal motility was further deteriorated in mdx/utrn ^+/− (Fig. 3 and Supplementary Fig. S2a) which suggest that dystrophin and utrophin may affect the spermatozoal motility through different mechanisms. One possible explanation why the spermatozoal motility was neither further exacerbated in mad/utrn ^+/− and nor restored in mdx may be due to the distinct binding properties of dystrophin and utrophin to the cytoskeleton. In vitro, binding of utrophin to actin filaments renders more restriction to the dynamics of actin filaments than the dystrophin does³⁰. Furthermore, utrophin fails to restore the microtubule disorganization resulted from dystrophin deficiency in the mdx skeletal muscle³¹.

Another interesting discovery is that impaired spermatogenesis would be due to the dysfunction of Sertoli cells and the direct effect of dystrophin/utrophin deficiency on germ cells. Rhox1 expression in Sertoli cells correlates with the proliferation of both Sertoli cells and spermatogonia³². We observed downregulation of Rhox1 (Fig. 5a) and severe impairment of microtubule-mediated expansion of Sertoli cells in mdx/utrn ^+/− (Fig. 7a), suggesting that lack of dystrophin and utrophin leads to the failure of Sertoli cells to support proliferation and survival of germ cells. This is consistent with the previous reports showing that dysregulation of microtubules in the Sertoli cells accounts for the loss of germ cells^{25, 26}. Moreover, downregulation of the undifferentiated spermatogonia marker Cdh1 and differentiating spermatogonia marker Stra8 in mdx/utrn ^+/− (Fig. 5b) correlates with the decrease in the number of spermatogonia in the mdx/utrn ^+/− seminiferous tubules (Fig. 6e)^{33, 34}. Likewise, downregulation of the markers for spermatocytes (Clgn, Dmc1, Id2 and Sycp3) and spermatids (Camk4, Crem, Odf1, Pgk2, Prm2 and Tnp1) in mdx/utrn ^+/− (Fig. 5c,d) is also coincident with the decrease in the number of spermatocytes and spermatids in the mdx/utrn ^+/− seminiferous tubules (Fig. 6f–h). Although most of the germ cell markers did not alter in mdx (Fig. 5) the number of spermatogonia and spermatids decreased significantly in mdx compared to C57BL/10 (Fig. 6e,g,h), suggesting that dystrophin also involves in the progress of spermatogenesis. One thing noteworthy is that the number of germ cells declined further in mdx/utrn ^+/−, indicating that up-regulation of utrophin in mdx may partially compensate the role of dystrophin during spermatogenesis.

The decrease in the germ cell number may be attributed to the decreased proliferation and/or increased apoptosis of the germ cells. In the present study, we demonstrated that deficiency of dystrophin and utrophin results in arrest of germ cell proliferation by showing a decrease in Ki67-positive and microtubule associated protein MARK2-positive spermatogenic cells (Fig. 7b,f–h), considering that MARK2 associates with dystrophin and utrophin and loss of dystrophin hinders the polarity of MARK2 and consequent catastrophe of asymmetric division of satellite cells⁷. In addition, we also showed the contribution of increased apoptosis in germ cells to the reduction of germ cells in mdx and mdx/utrn ^+/− (Fig. 7c–e). Collectively, our data demonstrated the requirement of dystrophin and utrophin during spermatogenesis through regulating the balance between proliferation and apoptosis.

In conclusion, we demonstrated in this report that (1) Dp427 affects the spermatozoal motility; (2) germ cell survival and proliferation require dystrophin and utrophin and (3) utrophin compensates for dystrophin deficiency during spermatogenesis but not on spermatozoal motility.

Methods

Animals

C57BL/10SnJ (C57BL/10), C57BL/10ScSn-Dmd ^mdx/J (mdx mice), and Utrn^tm1JrsDmd^mdx/J (mdx/utrn ^+/− mice) mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). The C57BL/10 mice were first crossbred with mdx/utrn ^+/− mice to obtain the utrn ^+/− (utrophin heterozygous knockout) mice, and the F1 utrn ^+/− mice were interbred to obtain the homozygous utrophin null mice, the utrnko mice. All experiments were in accordance with the Guides for the Use and Care of Laboratory Animals (ARRIVE guidelines), and the animal protocols have been approved by Experimental Animal Committee, Academia Sinica, Taiwan. The mice were housed in individually ventilated cages (IVCs) system in Academia Sinica SPF Animal Facility. The average litter size was determined by mating three pairs of each strain.

Mouse testes and epididymides size determination

The body weights of the male mice were measured on a Toledo Precision Balance (Mettler Toledo, Switzerland). Sedentary male mice were sacrificed and the testes and epididymides were dissected at the age of 10 weeks. The weights of the testes and epididymides were measured on a Toledo Analytical Balance (Mettler Toledo, Switzerland). The sizes of the testes and epididymides were presented as the ratio to the body weight (%).

Mouse sperm preparation

To evaluate the cauda epididymal spermatozoal concentration and motility, fresh cauda epididymidis were dissected from 10-week old male mice, placed into 2 ml of Whitten’s HEPES medium (100 mM NaCl, 4.4 mM KCl, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄, 5.4 mM glucose, 0.8 mM pyruvate, 4.8 mM lactic acid and 20 mM HEPES) and kept in 5% CO₂ at 37 °C. Each epididymis was minced to allow the spermatozoa to disperse in the medium. After 30 minutes, the spermatozoal suspension was collected and diluted for quantitative assessment. The number of motile and total sperms were determined using a Hemocytometer (Hausser Scientific, USA).

Spermatozoal morphology analysis

The spermatozoa from 10-week-old C57BL/10, mdx and mdx/utrn ^+/− were fixed with 4% paraformaldehyde (Sigma-Aldrich, 158127) for 15 minutes, washed with 100 mM ammonium acetate (Merck, Darmstadt, DE) and spread on slides (Marienfeld-Superior, Lauda-Königshofen, DE). The spermatozoal spread was stained with 0.22% Coomassie blue (Sigma-Aldrich, B0770) in 50% Methanol (9070; Avantor Performance Materials, Center Valley, PA, USA) and 10% Acetic Acid (9375; Avantor Performance Materials, Center Valley, PA, USA) for 30 minutes and the morphology were examined under microscope. 6 mice of reach strain were examined and more than 500 spermatozoa from each mouse were scored. The spermatozoal morphology was classified according to the criteria used by Wyrobek et al.³⁵ and Smith et al.³⁶ as normal, folded, double-tailed, pin head, amorphous head or head with a wrong-angled hook, very short hook or no hook^{35, 36}.

Histology and immunofluorescent labeling

Tissues were fixed overnight in Bouin’s solution (Sigma-Aldrich, HT10132), paraffin-embedded and sectioned at 3 μm. Sections were subjected to hematoxylin-eosin stain and periodic acid–Schiff stain (Merck Millipore, Billerica, MA, USA) following manufacturer’s instructions. For immunofluorescent labeling, sections were first antigen retrieved in boiling 10 mM citrate buffer (pH = 6.0) for 12 minutes. The sections and the spermatozoal spreads were blocked with 10% goat serum (Gibco, Grand Island, NY, USA) and 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA). The following primary antibodies were used for detecting full length dystrophin Dp427 (MANDYS1 3B7; 1:500; Wolfson CIND, OSWESTRY, UK), Dp71 (MANDRA1 7A10; 1:100; Wolfson CIND, OSWESTRY, UK), utrophin (MANCHO3 8A4; 1:100; Wolfson CIND, OSWESTRY, UK), α-tubulin (1:1000; Sigma-Aldrich, T9026), ki67 (1:500; GTX16667; GeneTex, Irvine, CA, USA) and MARK2 (1:100; GTX111783; GeneTex, Irvine, CA, USA). The spermatozoal spreads were also immunofluorescently labeled with MitoTracker® Green (200 nM; Invitrogen, Carlsbad, CA, USA). The following second antibodies were used to provide fluorescent signals: anti-mouse IgG-Alexa Flour 488 (1:1,000; Invitrogen, Carlsbad, CA, USA), anti-rabbit Igs-Alexa Flour 568 (1:1,000; Invitrogen, Carlsbad, CA, USA).

TUNEL assay

The testes of the ten-week-old mice were fixed in Bouin’s Solution, paraffin-embedded and sectioned at 3 μm. Levels of germ cell apoptosis were measured by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay using ApopTag® Plus In Situ Apoptosis Fluorescein Detection Kit (Merck Millipore, Billerica, MA, USA). DNA breaks were labeled with fluorescein and visualized with Rhodamine.

Separation of the testicular germ cell population

Separation of spermatogenic cells was based on the centrifugal elutriation system³⁷. The testes of 10-week-old C57BL/10, mdx and mdx/utrn ^+/− mice were excised, decapsulated and cut into small pieces in dishes. Testes were incubated in 50 ml DMEM/F12 (Invitrogen, Carlsbad, CA, USA) with 1 mg/ml trypsin (Invitrogen, Carlsbad, CA, USA), 0.75 mg/ml collagenase (Invitrogen, Carlsbad, CA, USA), protein inhibitor (Sigma-Aldrich), DNase I (Sigma-Aldrich; 10104159001) and 1% penicillin/streptomycin (Sigma-Aldrich; P4333) and shacked at 180 rpm/minute for 90 minutes. The medium was loaded on 40 um filters (BD Bioscience, Sparks, MD, USA) and washed with DMEM/F12 medium. The cell suspensions were subjected to centrifugal elutriation to collect suspensions containing germs cells of different stages in the following sequence: 700 × g for 1 minute for spermatogonia, 400 × g for 1 minutes for round spermatids, 200 × g for 1 mins for secondary spermatocytes, 100 × g for 1 minutes for primary spermatocytes. The collected suspensions were centrifuged at max 3,000 × g for 10 minutes and the pellets were subjected to RNA purification using TRIZOAL reagent (Invitrogen, Carlsbad, CA, USA).

Quantitative Real-Time PCR (qPCR)

Testes and epididymides were homogenized by MagNA lyser system (Roche, Basel, CH) and total RNA was extracted by TRIZOL reagent following manufacturer’s RNA extraction protocol. RNA was reversely transcribed by SuperScript III First-Strand Synthesis system (Invitrogen, Carlsbad, CA, USA) to generate cDNA in accordance with manufacturer’s instruction. In each qPCR reaction, the cDNA equivalent to 25 ng of RNA was used. Amplification of cDNA was monitored on an ABI 7500 real-time PCR system (Applied Biosystems, Foster City, CA, USA) with OmicsGreen qPCR Master Mix (Omics Bio, Taipei, Taiwan) and the forward and reverse primer each at a final concentration of 0.25 µM. The amplification protocol consisted of 2 minutes 50 °C and 10 minutes 95 °C followed by 50 cycles of 15 minutes 95 °C and 1 minute 60 °C; and completed with a standard melting curve protocol. Results were normalized to the expression of Csnk2a2. The mRNA primer sequences are detailed in Supplementary Table S1.

Western blotting

The testes and epididymides of C57BL/10, mdx and mdx/utrn ^+/− were homogenized in RIPA buffer (50 mM HEPES, pH7.5; 140 mM NaCl; 1 mM EDTA; 1% Triton X-100 and 0.1% SDS) supplemented with protease inhibitor cocktail (Sigma-Aldrich) by MagNA lyser system. The lysate was then centrifuged at 15,800 × g for 10 minutes at 4 °C. 50 μg of protein lysate was denatured and separated using SDS-polyacrylamide electrophoresis following Dr. Thomas Krag’s protocol for separating dystrophin and utrophin³⁸. Proteins were loaded on 5% polyacrylamide resolving gel directly and separated at 40 mA/gel in running buffer (192 mM glycine, 25 mM Tris base and 0.1% SDS) for 3–4 hours. Proteins were transferred to the PVDF membrane (PerkinElmer, Richmond, CA, USA) in transfer buffer (192 mM glycine, 25 mM Tris base, 15% methanol and 0.1% SDS) at 300 mA overnight. Dystrophin and utrophin were detected with anti-dystrophin (MANDYS1 3B7; 1:500; Wolfson CIND, OSWESTRY, UK) and anti-utrophin (MANCHO3 8A4; 1:100; Wolfson CIND, OSWESTRY, UK) respectively.

Statistical analysis

Statistical analysis with GraphPad Prism 5 software (GraphPad Software, San Diego, CA, USA) was performed using the two-tailed unpaired Student’s t-test or one-way analysis of variance (ANOVA) followed by post hoc Tukey HSD (Honestly Significant Difference) multiple comparison tests. The results were recognized as statistically significant at P < 0.05. Asterisks and hashtags indicate the level of statistical significance compared with C57BL/10 and mdx respectively: *P < 0.05; **P < 0.01; ***P < 0.001; ^# P < 0.05; ^## P < 0.01; ^### P < 0.001; ns indicates not significant. The data were presented as means mean ± SEM.

Declarations

Ethics approval and consent to participate

All experiments were in accordance with the Institute of Animal Care and Use Committee of Academia Sinica, Taiwan. The IACUC reference number is 1109211.

References

Fairclough, R. J., Wood, M. J. & Davies, K. E. Therapy for Duchenne muscular dystrophy: renewed optimism from genetic approaches. Nat Rev Genet 14, 373–378 (2013).
Article CAS PubMed Google Scholar
Den Dunnen, J. T. et al. Topography of the Duchenne muscular dystrophy (DMD) gene: FIGE and cDNA analysis of 194 cases reveals 115 deletions and 13 duplications. Am J Hum Genet 45, 835–847 (1989).
Google Scholar
Koenig, M. et al. Complete cloning of the Duchenne muscular dystrophy (DMD) cDNA and preliminary genomic organization of the DMD gene in normal and affected individuals. Cell 50, 509–517 (1987).
Article CAS PubMed Google Scholar
Koenig, M., Monaco, A. P. & Kunkel, L. M. The complete sequence of dystrophin predicts a rod-shaped cytoskeletal protein. Cell 53, 219–228 (1988).
Article CAS PubMed Google Scholar
Ervasti, J. M. & Campbell, K. P. A role for the dystrophin-glycoprotein complex as a transmembrane linker between laminin and actin. J Cell Biol 122, 809–823 (1993).
Article CAS PubMed Google Scholar
Brenman, J. E., Chao, D. S., Xia, H., Aldape, K. & Bredt, D. S. Nitric oxide synthase complexed with dystrophin and absent from skeletal muscle sarcolemma in Duchenne muscular dystrophy. Cell 82, 743–752 (1995).
Article CAS PubMed Google Scholar
Dumont, N. A. et al. Dystrophin expression in muscle stem cells regulates their polarity and asymmetric division. Nat Med 21, 1455–1463 (2015).
Article CAS PubMed PubMed Central Google Scholar
Muntoni, F., Torelli, S. & Ferlini, A. Dystrophin and mutations: one gene, several proteins, multiple phenotypes. Lancet Neurol 2, 731–740 (2003).
Article CAS PubMed Google Scholar
‘t Hoen, P. A. et al. Generation and characterization of transgenic mice with the full-length human DMD gene. J Biol Chem 283, 5899–5907 (2008).
Article PubMed Google Scholar
Love, D. R. et al. An autosomal transcript in skeletal muscle with homology to dystrophin. Nature 339, 55–58 (1989).
Article ADS CAS PubMed Google Scholar
Tinsley, J. M. et al. Primary structure of dystrophin-related protein. Nature 360, 591–593 (1992).
Article ADS CAS PubMed Google Scholar
Love, D. R. et al. Tissue distribution of the dystrophin-related gene product and expression in the mdx and dy mouse. Proc Natl Acad Sci USA 88, 3243–3247 (1991).
Article ADS CAS PubMed PubMed Central Google Scholar
Ohlendieck, K. et al. Dystrophin-related protein is localized to neuromuscular junctions of adult skeletal muscle. Neuron 7, 499–508 (1991).
Article CAS PubMed Google Scholar
Amenta, A. R. et al. Biglycan recruits utrophin to the sarcolemma and counters dystrophic pathology in mdx mice. Proc Natl Acad Sci USA 108, 762–767 (2011).
Article ADS CAS PubMed Google Scholar
Banks, G. B., Combs, A. C., Odom, G. L., Bloch, R. J. & Chamberlain, J. S. Muscle structure influences utrophin expression in mdx mice. PLoS Genet 10, e1004431 (2014).
Article PubMed PubMed Central Google Scholar
Bulfield, G., Siller, W. G., Wight, P. A. & Moore, K. J. X chromosome-linked muscular dystrophy (mdx) in the mouse. Proc Natl Acad Sci USA 81, 1189–1192 (1984).
Article ADS CAS PubMed PubMed Central Google Scholar
Kleopa, K. A., Drousiotou, A., Mavrikiou, E., Ormiston, A. & Kyriakides, T. Naturally occurring utrophin correlates with disease severity in Duchenne muscular dystrophy. Hum Mol Genet 15, 1623–1628 (2006).
Article CAS PubMed Google Scholar
Hernandez-Gonzalez, E. O., Mornet, D., Rendon, A. & Martinez-Rojas, D. Absence of Dp71 in mdx3cv mouse spermatozoa alters flagellar morphology and the distribution of ion channels and nNOS. J Cell Sci 118, 137–145 (2005).
Article CAS PubMed Google Scholar
Deconinck, A. E. et al. Utrophin-dystrophin-deficient mice as a model for Duchenne muscular dystrophy. Cell 90, 717–727 (1997).
Article CAS PubMed Google Scholar
Grady, R. M. et al. Skeletal and cardiac myopathies in mice lacking utrophin and dystrophin: a model for Duchenne muscular dystrophy. Cell 90, 729–738 (1997).
Article CAS PubMed Google Scholar
Ahmed, E. A. & de Rooij, D. G. Staging of mouse seminiferous tubule cross-sections. Methods Mol Biol 558, 263–277 (2009).
Article PubMed Google Scholar
Meistrich, M. L. & Hess, R. A. Assessment of spermatogenesis through staging of seminiferous tubules. Methods Mol Biol 927, 299–307 (2013).
Article CAS PubMed Google Scholar
Creasy, D. et al. Proliferative and nonproliferative lesions of the rat and mouse male reproductive system. Toxicol Pathol 40, 40S–121S (2012).
Article PubMed Google Scholar
Schwab, K. R., Smith, G. D. & Dressler, G. R. Arrested spermatogenesis and evidence for DNA damage in PTIP mutant testes. Dev Biol 373, 64–71 (2013).
Article CAS PubMed Google Scholar
Tanwar, P. S., Kaneko-Tarui, T., Zhang, L. & Teixeira, J. M. Altered LKB1/AMPK/TSC1/TSC2/mTOR signaling causes disruption of Sertoli cell polarity and spermatogenesis. Hum Mol Genet 21, 4394–4405 (2012).
Article CAS PubMed PubMed Central Google Scholar
Komada, M., McLean, D. J., Griswold, M. D., Russell, L. D. & Soriano, P. E-MAP-115, encoding a microtubule-associated protein, is a retinoic acid-inducible gene required for spermatogenesis. Genes Dev 14, 1332–1342 (2000).
CAS PubMed PubMed Central Google Scholar
Squire, S. et al. Prevention of pathology in mdx mice by expression of utrophin: analysis using an inducible transgenic expression system. Hum Mol Genet 11, 3333–3344 (2002).
Article CAS PubMed Google Scholar
Deconinck, N. et al. Expression of truncated utrophin leads to major functional improvements in dystrophin-deficient muscles of mice. Nat Med 3, 1216–1221 (1997).
Article CAS PubMed Google Scholar
Tinsley, J. M. et al. Amelioration of the dystrophic phenotype of mdx mice using a truncated utrophin transgene. Nature 384, 349–353 (1996).
Article ADS CAS PubMed Google Scholar
Prochniewicz, E., Henderson, D., Ervasti, J. M. & Thomas, D. D. Dystrophin and utrophin have distinct effects on the structural dynamics of actin. Proc Natl Acad Sci USA 106, 7822–7827 (2009).
Article ADS CAS PubMed PubMed Central Google Scholar
Belanto, J. J. et al. Microtubule binding distinguishes dystrophin from utrophin. Proc Natl Acad Sci USA 111, 5723–5728 (2014).
Article ADS CAS PubMed PubMed Central Google Scholar
Maclean, J. A. 2nd et al. Rhox: a new homeobox gene cluster. Cell 120, 369–382 (2005).
Article CAS PubMed Google Scholar
Tokuda, M., Kadokawa, Y., Kurahashi, H. & Marunouchi, T. CDH1 is a specific marker for undifferentiated spermatogonia in mouse testes. Biol Reprod 76, 130–141 (2007).
Article CAS PubMed Google Scholar
Anderson, E. L. et al. Stra8 and its inducer, retinoic acid, regulate meiotic initiation in both spermatogenesis and oogenesis in mice. Proc Natl Acad Sci USA 105, 14976–14980 (2008).
Article ADS CAS PubMed PubMed Central Google Scholar
Wyrobek, A. J. & Bruce, W. R. Chemical induction of sperm abnormalities in mice. Proc Natl Acad Sci USA 72, 4425–4429 (1975).
Article ADS CAS PubMed PubMed Central Google Scholar
Smith, M. A. et al. Anatase titanium dioxide nanoparticles in mice: evidence for induced structural and functional sperm defects after short-, but not long-, term exposure. Asian J Androl 17, 261–268 (2015).
Article CAS PubMed Google Scholar
Yeh, Y. C., Yang, V. C., Huang, S. C. & Lo, N. W. Stage-dependent expression of extra-embryonic tissue-spermatogenesis-homeobox gene 1 (ESX1) protein, a candidate marker for X chromosome-bearing sperm. Reprod Fertil Dev 17, 447–455 (2005).
Article CAS PubMed Google Scholar
Krag, T. O. et al. Heregulin ameliorates the dystrophic phenotype in mdx mice. Proc Natl Acad Sci USA 101, 13856–13860 (2004).
Article ADS CAS PubMed PubMed Central Google Scholar

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Acknowledgements

We thank Dr. G. Morris for providing anti-dystrophin and anti-utrophin antibodies. We also thank the Confocal Microscopy Core Facility at IBMS, Academia Sinica, Taipei, Taiwan for assisting the image acquisition and processing. We would like to thank the Academia Sinica SPF Animal Facility for helping to maintain the mice. We thank Dr. L.C. Pan and F.C. Hsu for assisting the CASA analysis.

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Authors and Affiliations

Institute of Biomedical Sciences, Academia Sinica, Taipei, 115, Taiwan
Hung-Chih Chen, Yu-Feng Chin, David J. Lundy & Patrick C. H. Hsieh
National Laboratory Animal Center, National Applied Research Laboratories, Taipei, 115, Taiwan
Chung-Tiang Liang
Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Zhunan, Miaoli County, 350, Taiwan
Ya-Hui Chi
Department of Obstetrics & Gynecology, College of Medicine, National Cheng Kung University, Tainan, 701, Taiwan
Paolin Kuo
Institute of Medical Genomics and Proteomics, Institute of Clinical Medicine and Cardiovascular Surgery Division, National Taiwan University and Hospital, Taipei, 100, Taiwan
Patrick C. H. Hsieh

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Hung-Chih Chen
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Contributions

H.C.C. designed and performed experiments, analyzed data, wrote and edited the manuscript. Y.F.C. and C.T.L. performed the experiments. D.J.L., Y.H.C., and P.K. edited the manuscript. P.C.H.H. supervised the project and edited the manuscript. All authors reviewed the manuscript.

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Correspondence to Patrick C. H. Hsieh.

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Chen, HC., Chin, YF., Lundy, D.J. et al. Utrophin Compensates dystrophin Loss during Mouse Spermatogenesis. Sci Rep 7, 7372 (2017). https://doi.org/10.1038/s41598-017-05993-8

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Received: 10 January 2017
Accepted: 07 June 2017
Published: 07 August 2017
DOI: https://doi.org/10.1038/s41598-017-05993-8

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