REPRODUCTIONRESEARCH

ARX/Arx is expressed in germ cells during spermatogenesis in both marsupial and mouse

Hongshi Yu1,2, Andrew J Pask1,2,3, Yanqiu Hu1,2, Geoff Shaw1,2 and Marilyn B Renfree1,2 1ARC Centre of Excellence for Kangaroo Genomics and 2Department of Zoology, The University of Melbourne, Melbourne, Victoria 3010, Australia and 3Department of Molecular and Biology, The University of Connecticut, Storrs, Connecticut 06269, USA Correspondence should be addressed to M B Renfree at Department of Zoology, The University of Melbourne; Email: [email protected]

Abstract

The X-linked aristaless gene, ARX, is essential for the development of the , forebrain, olfactory bulb, , and skeletal muscle in mice and humans. Mutations cause neurological diseases, often accompanied by ambiguous genitalia. There are a disproportionately high number of testis and brain genes on the human and mouse X chromosomes. It is still unknown whether the X chromosome accrued these genes during its evolution or whether genes that find themselves on the X chromosome evolve such roles. ARX was originally autosomal in mammals and remains so in marsupials, whereas in eutherian mammals it translocated to the X chromosome. In this study, we examined autosomal ARX in tammars and compared it with the X-linked Arx in mice. We detected ARX mRNA in the neural cells of the forebrain, midbrain and hindbrain, and olfactory bulbs in developing tammars, consistent with the expression in mice. ARX was detected by RT-PCR and mRNA in situ hybridization in the developing tammar wallaby gonads of both sexes, suggestive of a role in sexual development as in mice. We also detected ARX/Arx mRNA in the adult testis in both tammars and mice, suggesting a potential novel role for ARX/Arx in spermiogenesis. ARX transcripts were predominantly observed in round spermatids. Arx mRNA localization distributions in the mouse adult testis suggest that it escaped meiotic sex chromosome inactivation during spermatogenesis. Our findings suggest that ARX in the therian mammal ancestor already played a role in male reproduction before it was recruited to the X chromosome in eutherians. Reproduction (2014) 147 279–289

Introduction (Gecz et al. 2006). ARX mutations in humans lead to multiple neurological disorders including X-linked The X-linked aristaless homeobox gene, ARX, belongs lissencephaly with or without ambiguous genitalia to the family of paired-type homeodomain transcription (XLAG syndrome), epilepsy, and mental retardation. factors that have essential roles in the development of the ARX plays crucial roles in the development and forebrain, olfactory bulb, pancreas, skeletal muscle, and maintenance of the CNS in embryogenesis and adulthood gonads in mice and humans (Kitamura et al. 2002, Heller et al. 2005, Yoshihara et al. 2005, Biressi et al. 2008). (Miura et al. 1997, Kitamura et al.2002, El-Hodiri et al. ARX was initially identified as an important regulator of 2003, Colombo et al. 2004, Poirier et al.2004, Melkman & forebrain development in mice and zebrafish (Schneitz Sengupta 2005, Yoshihara et al.2005, Friocourt et al. et al. 1993, Miura et al. 1997). Subsequently, ARX 2006). ARX is expressed in the forebrain, predominantly in orthologs were detected in the forebrain of chickens telencephalic structures, of zebrafish, mice, and humans (Cobos et al. 2005), Xenopus (El-Hodiri et al. 2003, Kelly throughout fetal development. The distribution of ARX et al. 2005, Seufert et al. 2005), and Caenorhabditis mRNA in human fetal brain suggests that ARX is involved elegans (Melkman & Sengupta 2005). The ARX in the differentiation and maintenance of specific neuronal protein contains a Q (50) paired-type homeodomain, a cell types in the CNS (Ohira et al. 2002). Arx mutations C-terminal aristaless domain, and an N-terminal octa- lead to the development of a small brain with reduced peptide and acts as a nuclear transcriptional regulator to olfactory bulbs, confirming that the Arx gene is necessary activate or inhibit gene expression (Seufert et al. 2005, for normal brain development (Kitamura et al. 2002, McKenzie et al. 2007, Biressi et al. 2008, Fullenkamp & Yoshihara et al. 2005). El-Hodiri 2008, Colasante et al. 2009). The ARX gene Arx is expressed at all stages of pancreatic develop- encodes a 562-amino acid protein (Ohira et al. 2002, ment in the proliferating epithelium, differentiating Stromme et al. 2002) with four characteristic polyalanine pancreatic precursors, and islets of Langerhans in mice (PolyA) tracts where most of the ARX mutations occur (Collombat et al. 2003). Deficiency of Arx results in a

q 2014 Society for Reproduction and Fertility DOI: 10.1530/REP-13-0361 ISSN 1470–1626 (paper) 1741–7899 (online) Online version via www.reproduction-online.org Downloaded from Bioscientifica.com at 09/25/2021 09:26:15PM via free access 280 H Yu and others loss of mature endocrine a-cells with a concomitant As marsupials occupy an important position in increase in the numbers of b- and d-cells (Collombat et al. X chromosome evolution in mammals, this study exa- 2003), suggesting that Arx is required for the specification mined the expression of autosomal ARX in the tammar and differentiation of a-cells (Collombat et al.2003, wallaby and compared this with the expression of its 2005). Arx is strongly expressed in differentiating X-linked homologue Arx in the mouse. We demonstrate embryonic muscle and is progressively decreased during that autosomal ARX was already dynamically expressed myogenesis, acting as a positive regulator of the during testis development and spermatogenesis before differentiation and specification of skeletal muscle fibers it was recruited to the X chromosome in eutherian (Biressi et al.2008). mammals. ARX is also important for mammalian gonadal development. In mice, Arx is strongly expressed in the fetal testis, principally in the interstitial cells, including Materials and methods peritubular myoid cells, tunica albuginea, endothelial Animals cells, and interstitial fibroblast-like cells. However, Arx is barely detectable in developing Sertoli cells, Leydig Tammar wallabies (Macropus eugenii) from Kangaroo Island (South Australia) were maintained in open grassy yards cells, and germ cells (Kitamura et al.2002). Arx- supplemented with fresh vegetables and water ad libitum in knockout neonatal male mice have smaller testes and our breeding colony in Melbourne, Australia. Pouch young seminiferous tubules of a larger diameter, but do not of various ages were removed from their mother’s pouch survive to adulthood (Kitamura et al. 2002). Arx for sampling. The age of pouch young was estimated using functions through a paracrine signaling pathway to head length from published growth curves (Poole et al. 1991). promote the differentiation of Leydig cells. Loss of Arx Samples of mouse (Swiss white) tissue were collected decreases the number of Leydig cells that produce opportunistically from other ethically approved experiments. testosterone to maintain testis development, resulting in Mice were bred in the Department of Zoology. All sampling X-linked lissencephaly with abnormal genitalia (XLAG) techniques and tissue collection procedures conformed to the syndrome (Ogata et al. 2000, Kitamura et al. 2002). Australian National Health and Medical Research Council Surprisingly, there have been no detailed studies of ARX guidelines (2004) and were approved by the University of expression in the adult testes of any mammal. In addition Melbourne Animal Experimentation and Ethics Committees. to its role in testis development, weak expression of Arx is also observed in the developing mouse at the boundary of the mesonephros at E14.5 (Kitamura et al. Tissues 2002), but a specific role for Arx in ovary development Adult tissues from tammars including brain, pituitary, hypo- has not been investigated yet. thalamus, olfactory bulb, , muscle, spleen, mammary The X and Y chromosomes have evolved from gland, heart, lung, liver, kidney, and adrenal were collected autosomes in the common mammalian ancestor from three adult females (along with uterus and ovary) and two (Ohno 1967, Skaletsky et al. 2003, Ross et al. 2005, adult males (along with testes). Fetal gonads 1 day before birth Graves 2006). The human X chromosome comprises (day 25 of gestation, nZ3), pouch young gonads after birth Z Z Z an X conserved region (XCR) present in all therian (dpp days post partum), d2pp (n 3), d7pp (n 3), d10pp Z Z Z mammals and recently added regions (XAR) present on (n 2), d15pp (n 2), and d24pp (n 3), and mature gonads Z Z the X chromosome in eutherians but located on an (n 2), as well as adult testes from mice (n 4) for molecular autosome in the common therian ancestor and in extant analysis were snap–frozen in liquid nitrogen and stored at K 8 marsupials (Alsop et al. 2005). Comparative mapping in 80 C until use. Olfactory bulbs were collected from both sexes of pouch young aged between d14 and d89pp. All tissues tammars, platypuses, chickens, and fish indicates that were collected under RNase-free conditions. Tissues for in situ the XAR was translocated to the human X chromosome hybridization were fixed overnight in 4% paraformaldehyde, in a block of genes, after the divergence of marsupials washed several times with 1! PBS, and stored in 70% ethanol from eutherian mammals (Graves 2008, Veyrunes et al. before paraffin embedding and sectioning at 7 mm. 2008, Delbridge et al. 2009). ARX maps to the XAR in tammars and so is X-linked in eutherian mammals while being autosomal (on chromosome 5p) in marsu- Cloning of tammar ARX gene and gene structure pials (Delbridge et al. 2008) and other non-mammalian determination vertebrates. Since the X chromosome is hemizygous in Cross-species primers (ARXFw1 and ARXRv1) spanning the males, any genes that confer a reproductive advantage conserved regions of ARX were designed to partially clone ARX can be directly selected for. As a result, the eutherian in tammars. Tammar ARX gene-specific primers (nested X chromosome is thought to have become enriched primers) were designed from the above PCR fragment for with genes that have functions related to reproduction 30 and 50 RACE to isolate the full gene transcript (the sequences (Saifi & Chandra 1999, Wang et al. 2001, Vallender & of all primers are listed in Table 1). cDNA template was Lahn 2004, Ropers & Hamel 2005, Delbridge & Graves derived from reverse-transcribed total RNA from the head of a 2007, Mueller et al. 2008). fetal tammar, using the SMART cDNA Library Construction Kit

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Table 1 Primers designed for the analysis of ARX expression by PCR. Primers Sequence (50–30) Application ARXFw1 CAGTTTCAGCTGC(A/T)GGA(A/G)CTGG Cross-species cloning and RT-PCR ARXRv1 TTCCC(A/C)GG(C/G)AGGAT(A/G)TTGA Cross-species cloning and RT-PCR ARXFw2 ACTCACCCTCCAGGTTTGC 30 RACE ARXRv2 CTGGGTGATGAGGAGGGA 50 RACE SMART IV AAGCAGTGGTATCAACGCAGAGTGGCCATTACGGCCGGG 50 RACE 0 CDS III ATTCTAGAGGCCGAGGCGGCCGACATG-d(T)30NK1N 3 RACE Z Z (n A, G, C, or T; NK1 A, G, or C) 50 PCR primers AAGCAGTGGTATCAACGCAGAGT 50 RACE ARXFw3 ACTCACCCTCCAGGTTTGC RT-PCR ARXRv3 TGAGGAGGAAAGGACAGG RT-PCR 18S F GATCCATTGGAGGGCAAGTCT RT-PCR 18S R CCAAGATCCAACTACGAGCTTTTT RT-PCR All the primers are listed in the 50 to 30 orientation; CDSIII, Smart IV, and 50 PCR primers were synthesized by SIGMA (Genosys) according to the SMART cDNA Library Construction Kit (Clontech).

(Clontech). 50 RACE was first carried out with primer 50 PCR (ND-1000 Spectrophotometer, Wilmington, DE, USA). Total and ARXRv1 and then 50 PCR and nested primer ARXRv2 were RNA of 2 mg was treated with DNaseI (Ambion, Inc.) for 30 min used to carry out 50 RACE. 30 RACE was carried out using the at 37 8C. Total RNA of 1 mg was reverse-transcribed using the ARXFw1 and CDS III primers. Nested PCR was carried out SuperScript III kit (Invitrogen). using the ARXFw2 and CDS III primers. PCR cycling conditions To check the expression pattern of ARX in developing tissues were as follows: 35 cycles of 30 s, 95 8C; 60 s, 50 8C; and 90 s, and adult tissues, PCR was carried out in a 25 ml reaction 72 8C, in a 25 ml reaction mixture with GoTaq Green Master mixture with GoTaq Green Master Mix and 0.4 mmol of each Mix (Promega) and 0.4 mM of each primer. primer (ARXFw3 and ARXRv3). Amplification conditions were Comparative analysis was carried out to determine the gene as follows: 95 8C, 30 s; 53.5 8C, 60 s; and 72 8C, 120 s for 35 structure of ARX in tammars using the gene structure of cycles (ARX) or 25 cycles (18S). Samples were analyzed on a humans, mice, rats, South American grey short-tailed opossum 1.2 and 2% agarose gel for ARX and 18S respectively. (Monodelphis domestica), and the platypus (Ornithorhynchus anatinus)(http://www.ensembl.org/). Partial genomic sequence of M. eugenii was obtained from the trace archives at NCBI RNA isolation and northern blotting hybridization (http://www.ncbi.nlm.nih.gov/) to confirm the number of exons Total RNA was isolated from snap-frozen adult tissues with and their length and define the position of introns. RNAwiz (Ambion, Inc.). The concentration and quality of total RNA were examined as described above. Equivalent amounts of RNA (about 12 mg) were subjected to electrophoresis in a 1% Sequence alignment analysis agarose gel containing formaldehyde (Sambrook & Russell The ARX protein sequences were retrieved from Ensembl 2001). Northern blotting was carried out according to standard (http://www.ensembl.org/) or NCBI (http://www.ncbi.nlm.nih. methods. Hybridization was carried out at 42 8C in ULTRAhyb gov/protein/): human – AAK93901; macaque – ENSMMUP solution (Ambion, Inc.) with [a-32P]dCTP-labeled cDNA probe 00000024253; mouse – NP_031518; rat – NP_001093644; from exon 2 to the end of 30 UTR. dog – NP_001108138; opossum – ENSMODP00000009070; tammar – GU369938; Xenopus – NP_001079329; zebrafish – BAA21764; nematode – NP_509860; and fruit fly – AAF51505. ARX mRNA in situ hybridization Sequence identity was calculated by BLAST2 (http://blast.ncbi. Antisense and sense RNA probes were prepared separately in Z nlm.nih.gov/Blast.cgi?PROGRAM blastp&BLAST_PROGR- the exon 2 region and were about 680 bp in length. Probes Z Z AMS blastp&PAGE_TYPE BlastSearch&SHOW_DEFAUL- were synthesized and labeled with digoxigenin-UTP using SP6 Z Z Z TS on&BLAST_SPEC blast2seq&LINK_LOC blasttab& or T7 RNA polymerase. Tissues were fixed in 4% paraformal- Z Z LAST_PAGE blastn&BLAST_INIT blast2seq). ARX protein dehyde overnight at 4 8C, rinsed several times with 1! PBS, sequences were aligned with Clustal-X (http://www.molecular- embedded in paraffin, and sectioned onto polylysine slides evolution.org/software/alignment/clustal) and viewed and (Menzel-Gla¨ser, Braunschweig, Germany). After dewaxing, the edited by GeneDoc (http://www.nrbsc.org/gfx/genedoc/). sections were washed several times with 1! PBS, glycine, Triton X-100, and triethanolamine buffer and were then immediately hybridized with probes at 42 8C. Hybridization Non-quantitative RT-PCR signals were detected using anti-Dig alkaline phosphatase- Total RNA was isolated using RNAwiz (Ambion, Inc., Austin, conjugated antibody and visualized with NBT/BCIP chromo- TX, USA) according to the manufacturer’s instructions. The gen, according to the manufacturer’s instructions (Roche quality and quantity of total RNA were verified by gel GmbH). The sections were counterstained with 0.1% Fast electrophoresis and optical density reading with a Nanodrop Red (Aldrich Chemical Corp., Milwaukee, WI, USA). www.reproduction-online.org Reproduction (2014) 147 279–289

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Results The tammar ARX gene encodes a protein of 552 amino acids, highly conserved with eutherian ARX (Fig. 1B). We Tammar ARX gene is highly conserved among mammals compared the ARX proteins of humans, mice, tammars, The tammar ARX gene was cloned using degenerate and Xenopus to determine which regions were under primers and RACE PCR (GenBank accession number strongest selection (Fig. 1C). ARX is especially conserved GU369938). ARX mRNA has 2747 bp, consisting of five within the functional domains including the N-terminal exons (Fig. 1A). The sequence was confirmed from the octapeptide, known to repress transcriptional activity and trace archives of the tammar (http://blast.ncbi.nlm.nih. act as a nuclear localization signals recognized by gov/Blast.cgi) and compared with the human ARX gene importin, the homeodomain with its characteristic structure, in which the conserved homeobox region helix-turn-helix DNA-interacting motif and the spans exon 2 to exon 4. C-terminal aristaless transcription activation domain.

(A)

(B)

(C)

Figure 1 Characterization of tammar ARX. (A) The tammar ARX gene has five exons (black/white boxes) with a total open reading frame length of 2719 bp. The white portion of boxes indicates the UTRs and black portions indicate protein-coding sequences (CDS). The length of each exon is labeled above the box. The broken line between exons represents introns of unknown size, while intron 4 (the solid line between exon 4 and exon 5) has a length of 2621 bp. (B) Cross-species ARX protein sequence comparison. The highest identity (93%) was observed between tammars and opossums and the lowest identity was observed between mammals and invertebrates as expected. (C) ARX protein sequence alignment between humans, mice, tammars, and Xenopus. The octapeptide (dashed box), nuclear localization signals (NLS, black underline), homeodomain (dashed box), fourth polyalanine (p(A)4 dashed box), and aristaless domain (dashed box) are highly conserved in all the species. The first and third p(A) are highly conserved in humans, mice, and tammars. The amino acids shaded black are identical across all the species.

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There are three conserved nuclear localization signals (NLS) localized in the N-terminal region and both sides of the homeodomain in humans, mice, tammars, and Xenopus. Interestingly, three of the four PolyA tracts important for brain development in humans and mice are found only in therian mammals. However, the fourth tract is conserved in all the four species (Fig. 1C).

Tammar ARX revealed highly conserved and novel sites of expression ARX plays critical roles in development of the forebrain, olfactory bulb, and gonads during embryogenesis in mice. To examine tammar ARX gene expression, we carried out RT-PCR using various tissues. Tammar ARX was widely expressed during embryo- Figure 3 Northern blotting and RT-PCR in tammar adult tissues. genesis (data not shown) and was cloned from the Northern blot (top panel) identified a single transcript of 2.8 kb that was developing brain. We detected ARX expression through- predominant in the testis, but not detected in other tissues consistent out gonadal development (Fig. 2). ARX was detected in the with the RT-PCR. RT-PCR (middle panel) revealed that ARX was strongly expressed in the adult testis and weakly expressed in the gonads before sexual differentiation (2 days before birth), olfactory bulb, adrenal, ovary, and muscle. 18S (bottom panel) was during the time that testicular cords begin to form in males the positive control for the RT-PCR. (around day 2pp) andwhen the cortex and medulla form in the ovary in females (around day 7–8pp), and later during germ cell proliferation in the ovary (on days 2–20pp) and Tammar ARX maintains a conserved localization in when the ovarian germ cells begin to enter meiosis the developing brain and gonads (around day 25pp) (Renfree et al.1996). There was no ARX plays an important role in brain development obvious difference in ARX expression between male and in humans, mice, and zebrafish. To examine ARX female samples at any time point (Fig. 2), suggesting that it expression and localization within the developing may be important in sexual differentiation of both sexes. brain in marsupials, we examined the head on the day Using RT-PCR and northern blotting, we examined of birth, a stage that is equivalent to early intra-uterine ARX expression in various adult tissues and confirmed development period of most eutherian mammals the broad expression pattern in the brain observed in (Fig. 4A and B). a previous report on the tammar (Delbridge et al. 2008). ARX mRNA was detected in the epidermis (Fig. 4C) A northern blot confirmed that there was only one surrounding the head, and strong expression was transcript of w2.8 kb in adult testis (Fig. 3), in contrast to detected in the tongue and palate epithelium (Fig. 4D the three isoforms detected in human skeletal muscle and E), as well as in the nasal epithelium. ARX mRNA (Stromme et al. 2002). staining was also detected in the olfactory bulb (Fig. 4F) Interestingly, ARX was most strongly expressed in and the vomeronasal organ (VNO) (Fig. 4G) and, using the adult testis, but, by comparison, it was only weakly RT-PCR, strongly in the developing olfactory bulb (data expressed in the olfactory bulb, ovary, and skeletal not shown). Tammar ARX was expressed in the neuronal muscle (Fig. 3). This finding suggests a previously cells at the site of the trigeminal ganglion (Fig. 4H), unreported potential role for ARX in the adult testis. telencephalon (Fig. 4I), mesencephalon, metencephalon Therefore, we compared ARX mRNA localization and (Fig. 4J), and myelencephalon (Fig. 4K) in a pattern protein distribution in the adult testes of a marsupial and similar to that observed in mice and zebrafish. a eutherian mammal. We examined ARX mRNA distribution in the devel- oping gonads of tammar wallabies from before birth to 26 days after birth with mRNA in situ hybridization (Fig. 5). Germ cells in the tammar wallaby testis are still actively mitotically proliferating in the postnatal testis, peaking in number on day 25pp. At this stage, the germ cells are developing gonocytes and are not yet Figure 2 Tammar ARX gene expression in developing gonads. The spermatogonia (Tyndale-Biscoe & Renfree 1987). Before tammar ARX gene was expressed in the developing ovary and testis at sex determination, tammar ARX was localized in the all stages examined from before birth (fetus: at 25 days gestation) to day 45 post partum throughout the period of gonadal sex determination and cytoplasm of germ cells (Fig. 5A and B). Two days after differentiation. 18S rRNA was the positive control, and the last lane is birth, during the formation of testicular cords, ARX was the negative control where template DNA was omitted from the PCR. detected in the cytoplasm of somatic cells and germ cells www.reproduction-online.org Reproduction (2014) 147 279–289

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Figure 4 ARX has a conserved mRNA localization in the developing brain. In situ hybridization of the tammar head with antisense ARX. mRNA distribution is indicated by dark blue staining (A). The sense probe hybridization control is shown in the inset box. The tissue is also shown with hematoxylin and eosin staining (B). mRNA was localized in the spinosum and granulosa cells of epidermis (C) surrounding the head and in the tongue epithelium (D) and palate epithelium (E). ARX staining was also found in the olfactory bulb (F) and vomeronasal organ (G), both regions important for smell and signal detection in mammals. ARX transcripts were found in the conserved regions of the developing brain in the neural cells of trigeminal ganglion (H), telencephalon (I), metencephalon (J), and myelencephalon (K).

(Fig. 5C and D). With testicular cord formation, ARX was Dynamic regulation of tammar ARX during expressed in the cytoplasm of some developing Sertoli spermatogenesis in the adult testis cells and in germ cells (Fig. 5E). During ovarian differentiation (Fig. 5F), it was also cytoplasmic in the ARX mRNA was abundant in spermatogenic cells at germ cells and some somatic cells. Around 25 days after specific stages and in some somatic cells (Fig. 6). birth, when male germ cells began to enter mitotic arrest, Interestingly, unlike in the developing gonocytes, there ARX was similarly detected in the cytoplasm of some was no expression in the mitotic spermatogonia and Sertoli cells and germ cells (Fig. 5G). When female germ expression was only observed in these cells after the cells began to enter meiosis about day 25 after birth, ARX initiation of meiosis (Fig. 6A, B, C, D, and E). ARX mRNA was observed predominantly in the cytoplasm of germ was especially abundant in spermatocytes (Fig. 6A). cells as well as somatic cells (Fig. 5H). As meiosis proceeded, it became restricted to the newly

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spermatids. No mRNA expression was detected in Sertoli cells at any stage (Fig. 6B).

Dynamic regulation of Arx during spermatogenesis in the adult mouse testis This study, to our knowledge, is the first report of ARX expression in any adult mammalian testis. To determine whether our findings in the tammar represent a con- served role of ARX in the adult testis, we examined Arx expression at the mRNA level in the adult mouse testis. The Arx mRNA expression pattern in the mouse testis was similar to that in the tammar testis, especially in spermatogenic cells. There was no detectable staining in the spermatogonia and somatic cells (Fig. 7A, B, C, and D), but strong expression was detected in the primary and secondary spermatocytes as well as round spermatids (Fig. 7A, B, C, and D). At later stages of the spermatogenic cycle, there was very weak staining in the cytoplasm of elongated spermatids (Fig. 7A), but no mRNA staining was detected in the mature spermatozoa (Fig. 7B).

Discussion

Figure 5 mRNA in situ showing ARX localization in tammar developing Comparative analysis of ARX/Arx in tammars and mice gonads. ARX mRNA localization in the developing gonads of tammars. suggests that it has a highly conserved expression pattern mRNA distribution is indicated by dark blue staining and tissue is during spermatogenesis, despite its different chromo- counterstained with 0.1% Nuclear Fast Red. On day 25 of gestation, somal location in these two species (X-linked in mice ARX was detected in the cytoplasm of germ cells (A and B). On day 2 vs autosomal in marsupials). This study is the first to after birth during the formation of testicular cords, ARX mRNA was demonstrate that ARX may have a highly conserved role detected in the cytoplasm of some Sertoli cells and germ cells (C), and no difference in ARX mRNA was found in the equivalently staged ovary in spermatogenesis in addition to its established roles (D). On day 8 after birth, there was strong staining in the cytoplasm of in neurogenesis. germ cells and some Sertoli cells in the testis (E), and during ovarian differentiation, the same pattern of ARX staining was observed in the cytoplasm of germ cells and some somatic cells in the ovary (F). When Evolution and conservation of ARX male germ cells began to enter mitotic arrest on day 25 after birth, ARX ARX has a conserved five-exon gene structure across transcripts were strongly expressed in the cytoplasm of germ cells and some Sertoli cells (G). When female germ cells began to enter meiosis eutherian and marsupial mammals and fruit flies. ARX is on day 16 after birth, ARX staining was observed in the cytoplasm of highly conserved at the protein level, with up to 93% both germ cells and somatic cells (H). A, C, E, and G show developing identity between tammars and opossums, 85% with testes, while B, D, F, and H show developing . Black dotted box primates, and 56% with zebrafish (Fig. 1B). Conservation represents gonads; blue dotted box represents seminiferous tubules; is even higher across the functional domains, such as and blue dotted line in H separates the cortex and medulla in the ovary. the octapeptide domain, nuclear localization domain, Scale barZ100 mm. homeodomain, and aristaless domain from the amphi- bian Xenopus to eutherian mammals. As in the mouse, formed round spermatids, but there was no staining in the tammar ARX has three nuclear localization signals the spermatocytes (Fig. 6B). As round spermatids (NLS) that are identical to those of other species, developed, ARX staining became weaker in the cyto- consistent with their conserved role in directing and plasm, but it was still strong in the round spermatids importing ARX into the nucleus, possibly mediated by (Fig. 6C). No mRNA staining was detected in the importin (Lin et al. 2009, Shoubridge et al. 2010). Thus, elongated spermatids and spermatozoa (Fig. 6D and E), ARX has been highly conserved despite its physical but there was still strong expression in the cytoplasm of relocation in the genome from an autosome to the XAR round spermatids at these stages (Fig. 6D and E). ARX of the eutherian X chromosome (Delbridge et al. 2008). mRNA was also observed in some Leydig cells (Fig. 6A) ARX has conserved roles in early development. The and peritubular myoid cells at early stages of the strong mRNA staining of neural cells in the forebrain, spermatogenic cycle (Fig. 6A, B, C, D, and E) before midbrain, and hindbrain suggests that ARX plays a round spermatids were transformed into elongated pivotal role in CNS development in tammars as it does in www.reproduction-online.org Reproduction (2014) 147 279–289

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Figure 6 mRNA in situ showing ARX localization in tammar adult testis. In situ hybridization of adult tammar testes with antisense ARX. mRNA distribution is indicated by dark blue staining and tissue is counterstained with 0.1% Nuclear Fast Red. Tammar ARX was strongly expressed in the round spermatids (RS) throughout all stages (A, B, C, D, E, F, and G) and there was no expression in the spermatogonia (SG), but there was stage- specific expression in other spermatogenic cells from the spermatocytes (SP) to the elongating spermatids (ES). At early stage I (A), ARX was expressed in the SP and some of the Leydig cells (LC) and peritubular myoid cells (PC) in the seminiferous cord. Slightly later in spermatogenesis at stage I (B), ARX was expressed in the RS and some of the LC, but in no other spermatogenic cells and Sertoli cells (SC). At stage IV (C), ARX was still strongly expressed in the RS, but weakly in the cytoplasm of ES. By stage V/VI (D), ARX was restricted to the RS. With the continuation of spermatogenesis (stage VIII, E), ARX was observed in the SP again besides the RS. At the early stage of spermatogenesis (F) and later stages (G), there was a dynamic expression of ARX in the LC but no expression in the SG in all the stages. The stages of spermatogenesis are described in Tyndale-Biscoe & Renfree (1987). SG, spermatogonia; SP, spermatocytes; RS, round spermatids; ES, elongating/elongated spermatids; SZ, spermatozoa; LC, Leydig cells; PC, peritubular myoid cells; SC, Sertoli cells. Scale barZ100 mm. mice (Kitamura et al. 2002, Seufert et al. 2005). The E14.5 gonads (Kitamura et al. 2002). Tammar ARX similarity in tammar and murine ARX/Arx localization in mRNA had a restricted pattern of expression in adult the developing olfactory bulbs suggests that ARX is tissues with the highest expression in the adult testis and important for the normal development of this organ extremely weak expression in the olfactory bulb, (Yoshihara et al. 2005). The mRNA distribution both in adrenal, ovary, and muscle. By contrast, ARX is highly the olfactory epithelium and in the VNO suggests that expressed in skeletal muscle in humans (Ohira et al. ARX could be important for developing the sense of 2002, Stromme et al. 2002). smell that is so essential for directing newborn wallabies into the pouch to locate the teats (Schneider et al. 2009). Taken together, the conserved expression pattern in the ARX escapes meiotic sex chromosome inactivation during spermatogenesis developing brain of marsupials and mice suggests that ARX is a critical gene for brain development throughout This is the first report of the mRNA expression of ARX in therian mammalian evolution. Strong mRNA staining the adult testis of any mammal during spermatogenesis. was also observed in the epithelium of the tongue and The spermatogenic cycle of the germ cells in the testis is palate and in the underlying muscle. Early tongue coordinated and maintained by the somatic cells development is especially important in the head of the including the Sertoli cells, peritubular myoid cells, and newborn marsupial, which must be able to attach to the Leydig cells that participate in spermatogenesis via their nipple and suck at the very early stages of development response to gonadotrophic signals (LH and FSH) from the (Hughes & Hall 1988). hypothalamic–pituitary–testicular axis (Roser 2008, Yan No obvious differences based on the RT-PCR were et al. 2008, He et al. 2009, Li et al. 2009, Walker 2009, detected between the developing testis and ovary in Cheng et al. 2010). However, most of the genes on the tammar wallabies, similar to mice (Kitamura et al. 2002). sex chromosomes are silenced during the meiotic phase The mRNA cellular location (Fig. 5) is similar to the of spermatogenesis by a process known as meiotic sex pattern shown using whole-mount in situ hybridization chromosome inactivation (MSCI), but are active in the of the mouse E11.5 testes (Colombo et al. 2004) and mitotic stage and early meiosis (Hornecker et al. 2007,

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number of testis and brain genes have accumulated on the human and mouse X chromosomes. It is still unknown whether the X chromosome has accrued testis and brain genes during its evolution or whether genes that find themselves on the X chromosome evolve these roles. We examined how gene expression changed for the ARX gene when it became relocated from an autosome in the therian mammalian ancestor to the X chromosome in eutherians. ARX was originally autosomal in mammals and remains so in marsupials, making it an excellent comparison to the mouse X-linked Arx. Our findings suggest that ARX/Arx already played a role in male reproduction long before it was recruited to the X chromosome in eutherians, adding to the pool of data suggesting that the eutherian X chromosome actively recruits testis genes (Saifi & Figure 7 Arx localization in the adult mouse testis. mRNA in situ Chandra 1999, Wang et al. 2001, Vallender & Lahn 2004, hybridization of adult mouse testes with antisense mouse Arx. mRNA Mueller et al. 2008). localization is indicated by dark blue staining and tissue is counter- Our investigations also uncovered the previously stained with 0.1% Nuclear Fast Red. At stage II (A), Arx mRNA was unreported dynamic expression of ARX/Arx during mainly localized to the cytoplasm of SP and RS and there was no spermatogenesis. However, as yet, there is no direct expression in SG, PC, and LC; at stage VII (B), Arx was also expressed in the cytoplasm of SP and RS, but no expression was detected in SG and evidence to confirm a functional role for ARX/Arx in SZ; at stage X (C), Arx was stained in SP and weakly in ES, but no spermatogenesis. In mice, mutations in Arx cause early staining was found in SG and LC; at stage XI (D), Arx was only detected postnatal death (Kitamura et al. 2002), making it in RS, but not in SG, ES, and SC. The stages of spermatogenesis are impossible to examine the function of Arx in spermato- described in Hess & de Franca (2008). The abbreviations are the same genesis in adults. A conditional knockout approach Z as those in Fig. 6. Scale bar 100 mm. is needed to clarify this question. In non-traditional model species such as marsupials, functional analyses Cloutier & Turner 2010). Tammar ARX is autosomal, but are extremely difficult and it is not possible at present mouse Arx is X-linked. We examined the change in the to generate a transgenic or conditional transgenic expression of ARX/Arx between marsupials and mice tammar. However, X-linked ARX/Arx mRNA is expressed during spermatogenesis to gain insight into how sex and in spermatocytes and round spermatids in both reproduction-related (SRR) genes evolved. While tam- tammars and mice maintaining this profile of expression mar ARX can be expressed at any stage of spermatogen- for over 160 million years of divergent evolution (Fig. 7). esis due to its autosomal localization, mouse Arx would In addition, if mouse Arx is subject to MSCI, it would not be expressed in the meiotic phase of spermatogen- not be expressed at these stages in the germ cells of esis if it is silenced by MSCI. Tammar ARX mRNA was the adult testis. Our study provides strong evidence that predominantly transcribed in the cytoplasm of sperma- Arx must escape MSCI. Thus, we postulate that the tocytes and round spermatids within the seminiferous dynamic expression of Arx/ARX during spermatogenesis tubules, which is similar to that of NANOG expression in in both mice and tammars is highly suggestive of a the adult testis (Kuijk et al. 2010). This requires potential role in spermatogenesis. expression during meiosis when genes on the sex chromosomes are normally silenced. If mouse Arx is subject to MSCI, it would not be expressed at equivalent Conclusions stages in the germ cells of the adult testis. To determine A similar but subtly different expression pattern during whether this is the case, we carried out mRNA in situ spermatogenesis between tammars and mice highlights hybridization in mouse testes and, surprisingly, found an the similarities in the basic processes of gametogenesis expression pattern in the germ line that was almost in marsupial and eutherian mammals. Although ARX identical to that observed in tammar testes. Thus, ARX is located on an autosome in tammars and not on appears to escape MSCI to maintain its role in the X chromosome as in eutherians, its expression does postmeiotic stages of spermatogenesis. not appear to have been altered. This lends support to the theory that the mammalian X chromosome may selectively acquire SRR genes, rather than such Relationship of autosomal ARX in a marsupial with genes evolving SRR functions after relocation to the X-linked Arx in mouse X chromosome. Mouse Arx escapes MSCI during An important aim of this study was to understand the spermatogenesis, suggesting that it has a potentially fundamental question of why a disproportionately high important function in mammalian spermatogenesis that www.reproduction-online.org Reproduction (2014) 147 279–289

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