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Biochimica et Biophysica Acta 1813 (2011) 1668–1688

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Biochimica et Biophysica Acta

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Expression of nucleocytoplasmic transport machinery: Clues to regulation of spermatogenic development☆

Andrew T. Major a,c, Penny A.F. Whiley b,c, Kate L. Loveland a,b,c,⁎

a Department of Anatomy and Developmental Biology, Monash University, Australia b Department of Biochemistry and Molecular Biology, Monash University, Australia c The ARC Centre of Excellence in Biotechnology and Development, Australia

article info abstract

Article history: Spermatogenesis is one example of a developmental process which requires tight control of gene expression Received 27 August 2010 to achieve normal growth and sustain function. This review is based on the principle that events in Received in revised form 22 February 2011 spermatogenesis are controlled by changes in the distribution of proteins between the nuclear and Accepted 11 March 2011 cytoplasmic compartments. Through analysis of the regulated production of nucleocytoplasmic transport Available online 17 March 2011 machinery in mammalian spermatogenesis, this review addresses the concept that access to the nucleus is tightly controlled to enable and prevent differentiation. A broad review of nuclear transport components is Keywords: Spermatogenesis presented, outlining the different categories of machinery required for import, export and non-nuclear Testis functions. In addition, the complexity of nomenclature is addressed by the provision of a concise yet Importin comprehensive listing of information that will aid in comparative studies of different transport proteins and Exportin the genes which encode them. We review a suite of existing transcriptional analyses which identify common Karyopherin and distinct patterns of transport machinery expression, showing how these can be linked with key events in Nuclear transport spermatogenic development. The additional importance of this for human fertility is considered, in light of data that identify which importin and nuclear transport machinery components are present in testicular cancer specimens, while also providing an indication of how their presence (and absence) may be considered as potential mediators of oncogenesis. This article is part of a Special Issue entitled: Regulation of Signaling and Cellular Fate through Modulation of Nuclear Protein Import. © 2011 Published by Elsevier B.V.

1. Introduction: Nucleocytoplasmic transport in the context affect transcription or perform other nuclear functions, they must of spermatogenesis utilise importin-mediated nuclear import pathways.

This review examines the machinery that controls nucleocyto- 1.1. What is an importin/exportin? plasmic transport in the context of mammalian spermatogenesis. Spermatogenesis is the process by which mature spermatozoa form Central to nucleocytoplasmic transport are the karyopherin family of from spermatogonial stem cells within the testis. Its successful proteins. These ferry cargoes in and out of the eukaryotic cell nucleus completion is dependent on successive division and differentiation and consists of two fundamentally distinct groups, karyopherin αsand steps which require many changes in gene expression; these are karyopherin βs [3–6]. Further categorisation reflects their functions in coordinated by a plethora of transcription and other factors expressed nuclear import (termed importins) and export (exportins). Because the in the testis [1,2]. For these proteins to access the nucleus, and thereby karyopherin αs are all importins, they are often termed importin αs, and the two terms may be used interchangeably. In contrast, the karyopherin βs have roles in nuclear import and export, and this functional distinction usually forms the basis for naming them as Abbreviations: NE, Nuclear envelope; NPC, Nuclear pore complex; NUPs, Nucleo- importin βs and exportins, respectively [3–5]. This separation may be porins; NLS, Nuclear localisation signal; cNLS, Classical nuclear localisation signal; NES, β Nuclear export signal; IBB, Importin beta binding; RAN-GTP, RAN, in its GTP bound inappropriate, as certain karyopherin s are now understood to bind form; RAN-GDP, RAN, in its GDP bound form; SEM, Standard error of the mean; GEO, distinct cargo sets for both import and export [3,7,8]. Gene expression omnibus; TGFβ, Transforming growth factor beta; miRNAs, Micro Each of the more than twenty karyopherin proteins encoded within RNAs; SRY, Sex determining region on the Y chromosome; LMB, Leptomycin B the mouse and human genomes binds a different subset of cargo proteins ☆ This article is part of a Special Issue entitled: Regulation of Signaling and Cellular which contain nuclear localisation signal(s) (NLS) or nuclear export Fate through Modulation of Nuclear Protein Import. ⁎ Corresponding author at: Building 77, Wellington Road, Monash University, signal(s) (NES) [9,10]. Some cargoes can be transported by several Clayton, Victoria, Australia 3800, Tel.: +61 3 99029337 Fax: +61 3 99029500. karyopherins, while others appear to require one specifickaryopherinfor

0167-4889/$ – see front matter © 2011 Published by Elsevier B.V. doi:10.1016/j.bbamcr.2011.03.008 A.T. Major et al. / Biochimica et Biophysica Acta 1813 (2011) 1668–1688 1669 nuclear import or export. Regulated nuclear transport of key factors can facilitated and is typically mediated by a transport carrier (e.g. dramatically affect signalling outcomes and cell fate [11],consistentwith karyopherin). The NPC is comprised of around 30 different nucleo- the concept that the progressive increase in karyopherin numbers with porins (NUPs). Some NUPs contribute to the eight spoke symmetry of evolutionary complexity may enable greater diversity in signalling the NPC nuclear “basket” structure and to the filaments extending into outcomes, required for complex organ development [6,12]. the cytoplasm (Fig. 1) [21]. The FG-NUPs which localise along the inner surfaces of the NPC channel feature hydrophobic repeat 1.2. The contribution of regulated nuclear transport to development sequences, termed FG-repeats, which fill the channel [22]. These FG- NUPs form the barrier that blocks the passage of larger molecules and An early demonstration that regulated importin expression simultaneously provide binding sites for the proteins that mediate controls nuclear protein access and thereby mediates changes in active nuclear transport [22]. function relates to the Drosophila heat shock transcription factor Facilitated transport into the nucleus by importins requires a (HSF). In the early Drosophila embryo, HSF is abundant, but it is unable nuclear localisation signal (NLS) on the cargo protein. The principal to enter the nucleus and the embryo has no heat shock response [13]. feature of the classical NLS (cNLS) is a single stretch of basic amino This has been suggested to enable rapid growth even in the presence acids (monopartite cNLS) or two closely spaced stretches of basic of an environmental insult during early embryogenesis, and after amino acids (bipartite cNLS)[3]). These cNLSs are recognised by cycle 13 the synthesis of the Drosophila homologue of Kpna4 (an importin α(s), while the NLSs that importin β(s) recognise contain importin α) delivers HSF to the nucleus and the heat shock response is much greater variation [10,23,24]. Cargoes of exportin 1 (XPO1) subsequently acquired [13]. contain a nuclear export signal (NES) consisting of hydrophobic Thus, the core concept examined in this review is that to trigger residues, predominantly leucine [25], while different exportins the appropriate developmental changes throughout spermatogenesis, mediate nuclear export in a similar manner (discussed further in correct spatiotemporal production of both nuclear factors and Section 2.4) but may also recognise and export cargoes that do not appropriate importins/exportins is required [14,15]. It builds on contain this motif [26–30]. previous knowledge that importin α family members are differen- tially expressed in certain developmental processes and in specific 2.2. Nuclear import tissue types [16,17], particularly within the testis and brain [16]. This review describes what is currently known about expression of In general terms, active nuclear import is mediated in two different karyopherins and other nucleocytoplasmic transport machinery in ways by importins. During importin β-mediated nuclear import relation to functional changes required through the progressive (Fig. 1A), the NLS-containing cargo forms a transport complex when it events of spermatogenesis in humans and mice. binds importin β in the cytoplasm. Transient interactions between importin β and FG-NUPs mediate NPC docking and translocation of 1.3. The karyopherin calamity: a table of clarity the complex into the nucleus [22,31]. Within the nucleus, the guanine nucleotide binding protein RAN in its GTP bound form (RAN-GTP) The large number of karyopherins identified within the mouse and binds to importin β [4], dissociating the complex and releasing the human genomes, variations in assigned gene names and differences in cargo to perform nuclear action(s) (discussed further in Section 2.4) the number of genes present between these and other species all have [32]. led to some uncertainty regarding to which karyopherin(s) individual In contrast to the importin βs which mediate NPC translocation, publications are referring. To address this issue, Table 1 lists the genes importin α proteins conventionally serve as adaptors that link cargoes encoding karyopherins identified within the human and mouse to the importin β1 protein (IPO1), thereby enabling a diversity of genomes. The abbreviations in the left hand column of Table 1 are cargoes to be selectively carried into the nucleus. Importin α- used throughout this review. Basic gene and protein information, mediated nuclear import is considered the classical nuclear import including ID numbers gathered from NCBI and UniProt websites, is pathway and occurs in a manner similar to importin β-mediated included. Other proteins that form part of the nuclear transport transport (Fig. 1B). Importin α proteins contain distinct importin β machinery, such as RAN and RAN binding proteins (discussed in binding (IBB) and NLS binding domains. As in importin β-mediated Section 2), are included in this list due to their potential to influence nuclear import, nuclear translocation occurs via IPO1 interactions nucleocytoplasmic transport. with the FG-NUPs [22,31]; similarly, RAN-GTP binding to IPO1 triggers importin α/β complex dissociation in the nucleus. 2. Nuclear transport: the basics 2.3. Nuclear export Because nucleocytoplasmic transport is of fundamental impor- tance to cellular physiology, major research efforts have been directed Nuclear export (Fig. 1C) mediated by exportins involves cargoes at understanding nuclear transport functions at the molecular level containing NESs (Section 2.1). RAN–GTP in the nucleus enables (see [4–6,10,18–20] for reviews). We provide here a simplified exportin binding to cargo proteins, thereby forming a trimeric nuclear overview of the nuclear transport system as a framework for export complex. As karyopherin β family members, exportins also subsequent discussion of the functional significance of regulated transiently bind the FG-NUPs of the NPC, thereby facilitating nuclear expression of nuclear transport machinery in spermatogenesis. export complex translocation from the nucleus into the cytoplasm [30]. Once passage is complete, hydrolysis of RAN–GTP into RAN–GDP 2.1. Nucleus, NPCs, NLSs and NESs is mediated at the cytoplasmic face, leading to dissociation of the transport complex (discussed further in Section 2.4). Genomic DNA is contained within the eukaryotic cell nucleus which is bound by the double membrane of the nuclear envelope 2.4. Maintaining nucleocytoplasmic transport: The RAN cycle (NE). Except when NE components are dispersed during cell division, the only passage through the NE is via the nuclear pore complex Active nucleocytoplasmic transport requires the presence of GTP- (NPC). Spanning from cytoplasm to nucleus, the NPC functions as a bound RAN (RAN–GTP) in the nucleus and GDP-bound RAN (RAN– molecular sieve allowing diffusion of proteins and macromolecules of GDP) in the cytoplasm (Fig. 1D) [4,33,34]. Nuclear RAN–GTP is b45 kDa. While molecules b45 kDa can also use facilitated mecha- maintained by the nuclear RAN guanine nucleotide exchange factor, nisms, the transport of larger molecules through the NPC must be RCC1, which catalyses the conversion of RAN–GDP into RAN–GTP. The 1670

Table 1 Major nuclear transport machinery genes and proteins in the mouse and human.

Abbreviation Species Gene (NCBI) Protein (UniProtKB/Swiss-Prot) Subtype (used here) ID-symbol Reference sequence(s) ID, name, aa, mass Alternate names

Official full name Also known as

A: Importin α (karyopherin α) family genes/proteins in the mouse and human Kpna1 Mouse 16646—Kpna1 NM_008465 Q60960 Karyopherin subunit alpha-1, SRP1-beta, α (S) karyopherin (importin) alpha 1 NPI1, Rch2, IPOA5, mSRP1, AW494490 Importin subunit alpha-1 RAG cohort protein 2, NPI-1, Importin alpha-S1 538aa, 60.2 kDa Human 3836—KPNA1 TrnSpt1→NM_002264 P52294 Karyopherin subunit alpha-1, SRP1-beta,

karyopherin alpha 1 TrnSpt2→NR_026698 Importin subunit alpha-1 RAG cohort protein 2, NPI-1 1668 (2011) 1813 Acta Biophysica et Biochimica / al. et Major A.T. (importin alpha 5) RCH2, SRP1, IPOA5, NPI-1 538aa, 60.2 kDa

Kpna2 Mouse 16647—Kpna2 NM_010655 P52293 Karyopherin subunit alpha-2, SRP1-alpha, RAG cohort α (P) karyopherin (importin) alpha 2 Rch1, IPOA1, MGC91246, 2410044B12Rik Importin subunit alpha-2 protein 1, Pendulin, PTAC58, Importin alpha P1 529aa, 57.9 kDa Human 3838—KPNA2 NM_002266 P52292 Karyopherin subunit alpha-2, SRP1-alpha, RAG cohort karyopherin alpha 2 RCH1, IPOA1, SRP1alpha Importin subunit alpha-2 protein 1 (RAG cohort 1, importin alpha 1) 529aa, 57.9 kDa

Kpna3 Mouse 16648—Kpna3 NM_008466 O35344 Karyopherin subunit alpha-3, Qip2, Importin alpha Q2 α (Q) karyopherin (importin) alpha 3 IPOA4 Importin subunit alpha-3 521aa, 57.8 kDa Human 3839—KPNA3 NM_002267 O00505 Karyopherin subunit alpha-3, SRP1-gamma, Qip2 karyopherin alpha 3 SRP1, SRP4, IPOA4, hSRP1, SRP1gamma Importin subunit alpha-3 (importin alpha 4) 521aa, 57.8 kDa

Kpna4 Mouse 16649—Kpna4 NM_008467 O35343 Karyopherin subunit alpha-4, Qip1, Importin alpha Q1 α (Q) karyopherin (importin) alpha 4 IPOA3, 1110058D08Rik Importin subunit alpha-4 521aa, 57.9 kDa Human 3840—KPNA4 NM_002268 O00629 Karyopherin subunit alpha-4, Qip1 karyopherin alpha 4 QIP1, SRP3, IPOA3, MGC12217, MGC26703 Importin subunit alpha-4 (importin alpha 3) 521aa, 57.9 kDa

Kpna5 Mouse –– –– α (S) – 1688 Human 3841—KPNA5 NM_002269 O15131 Karyopherin subunit alpha-5 karyopherin alpha 5 SRP6, IPOA6 Importin subunit alpha-6 (importin alpha 6) 536aa, 60.4 kDa

Kpna6 Mouse 16650—Kpna6 NM_008468 O35345 Karyopherin subunit alpha-6, α (S) karyopherin (importin) alpha 6 IPOA7, Kpna5, NPI-2 Importin subunit alpha-7 Importin alpha-S2 536aa, 60.0 kDa Human 23633—KPNA6 NM_012316 O60684 Karyopherin subunit alpha-6 karyopherin alpha 6 IPOA7, KPNA7, FLJ11249, MGC17918 Importin subunit alpha-7 (importin alpha 7) 536aa, 60.0 kDa

Kpna7 Mouse 381686—Kpna7 NM_001013774 Iso1→C0LLJ0 Iso1→Kpna2-like protein transcript variant 1 α (P) karyopherin alpha 7 Gm1055, AW146299 Iso2→C0LLJ1 Iso2→Kpna2-like protein transcript variant 2 (importin alpha 8) Expressed sequence AW146299 Iso1→499aa, 55.5 kDa Iso2→520aa, 57.8 kDa Human 402569—KPNA7 NM_001145715 A9QM74 Karyopherin subunit alpha-7 karyopherin alpha 7 – Importin subunit alpha-8 (importin alpha 8) 516aa, 56.9 kDa B: Importin β (karyopherin βs→nuclear import) family genes/proteins in the mouse and human. Ipo1 Mouse 16211—Kpnb1 NM_008379 P70168 Karyopherin subunit beta-1, Nuclear factor p97, β (Importin) karyopherin (importin) beta 1 IPOB, Impnb, MGC8315, AA409963 Importin subunit beta-1 PTAC97, SCG 876aa, 97.2 kDa Human 3837—KPNB1 NM_002265 Q14974 Karyopherin subunit beta-1, Nuclear factor p97, karyopherin (importin) beta 1 IMB1, IPO1, IPOB, Impnb, NTF97, MGC2155, Importin subunit beta-1 PTAC97, Importin-90 MGC2156, MGC2157 876aa, 97.2 kDa

Ipo2 Mouse 238799—Tnpo1 TrnSpt 1→NM_178716 Q8BFY9 Importin beta-2, Karyopherin beta-2 β (Importin) transportin 1 TrnSpt2 →NM_001048267 Transportin-1 MIP, TRN, IPO2, MIP1, Kpnb2, AU021749, Iso1→898aa, 102.4 kDa D13Ertd688e Iso2→890aa, 101.3 kDa Human 3842—TNPO1 TrnSpt1 →NM_002270 Q92973 Importin beta-2, Karyopherin beta-2, MIP transportin 1 TrnSpt2 →NM_153188 Transportin-1 MIP, TRN, IPO2, MIP1, KPNB2 Iso1→898aa, 102.4 kDa Iso2→890aa, 101.3 kDa Iso3→848aa, 96.9 kDa — → β

Ipo3 Mouse 212999 Tnpo2 TrnSpt1 NM_001122843 Q99LG2 Karyopherin beta-2b (Importin) 1668 (2011) 1813 Acta Biophysica et Biochimica / al. et Major A.T. transportin 2 TrnSpt2 →NM_145390 Transportin-2 (importin 3, karyopherin beta IPO3, TRN2, Knpb2b, Kpnb2b, MGC6380, AA414969, 887aa, 100.5 kDa 2b) AI464345, AI852433, 1110034O24Rik Human 30000—TNPO2 TrnSpt1 →NM_001136196 O14787 Karyopherin beta-2b transportin 2 TrnSpt2 →NM_013433 Transportin-2 TrnSpt3 →NM_001136195 Iso1→897aa, 101.4 kDa IPO3, TRN2, KPNB2B, FLJ12155 Iso2→887aa, 100.4 kDa

Ipo4 Mouse 75751—Ipo4 NM_024267 Q8VI75 Imp4a, RanBP4 β (Importin) importin 4 Imp4a, RanBP4, AA409693, MGC113723, Importin-4 (Imp4) 8430408O15Rik 1082aa, 119.3 kDa Human 79711—IPO4 NM_024658 Q8TEX9 Imp4b, RanBP4 importin 4 Imp4, FLJ23338, MGC131665 Importin-4 (Imp4) Iso1→1081aa, 118.7 kDa Iso2→1083aa, 118.9 kDa

Ipo5 Mouse 70572—Ipo5 NM_023579 Q8BKC5 Importin subunit beta-3, Karyopherin beta-3, β (Importin) importin 5 IMB3, Kpnb3, C76941, Ranbp5, AA409333, Importin-5 (Imp5) RanBP5 1110011C18Rik, 5730478E03Rik Iso1→1097aa, 123.6 kDa Iso2→1037aa, 116.9 kDa Human 3843—IPO5 NM_002271 O00410 Importin subunit beta-3, Karyopherin beta-3, importin 5 IMB3, KPNB3, RANBP5, MGC2068, FLJ43041, DKFZp686O1576 Importin-5 (Imp5) RanBP5 Iso1→1097aa, 123.6 kDa – Iso2→1037aa, 117.0 kDa 1688 Iso3→1115aa, 125.6 kDa

Ipo7 Mouse 233726—Ipo7 NM_181517 Q9EPL8 RanBP7 β (Importin) importin 7 Imp7, Ranbp7, C330016G14, A330055O14Rik Importin-7 (Imp7) 1038aa, 119.5 kDa Human 10527—IPO7 NM_006391 O95373 RanBP7 importin 7 Imp7, RANBP7, FLJ14581, MGC138673 Importin-7 (Imp7) 1038aa, 119.5 kDa

Ipo8 Mouse 320727—IPO8 NM_001081113 Q7TMY7 RanBP8 β (Importin) importin 8 Om1, MRP7, OM-1, Abcc10, Ranbp8, 6230418K12Rik, Importin-8 (Imp8) C130009K11Rik Iso1→1010aa, 117.1 kDa Iso2→966aa, 111.8 kDa

(continued on next page) 1671 1672

Table 1 (continued) Abbreviation Species Gene (NCBI) Protein (UniProtKB/Swiss-Prot) Subtype (used here) ID-symbol Reference sequence(s) ID, name, aa, mass Alternate names

Official full name Also known as

Human 10526—IPO8 NM_006390 O15397 RanBP8 importin 8 RANBP8, FLJ26580 Importin-8 (Imp8) 1037aa, 119.9 kDa

Ipo9 Mouse 226432—Ipo9 NM_153774 Q91YE6 Imp9a, Imp9b, RanBP9 β (Importin) importin 9 Imp9, C78347, Ranbp9, AI845704, mKIAA1192, 0710008K06Rik Importin-9 (Imp9) 1041aa, 116.1 kDa Human 55705—IPO9 NM_018085 Q96P70 RanBP9 importin 9 Imp9, FLJ10402, DKFZp761M1547 Importin-9 (Imp9) 1041aa, 116.0 kDa — β Ipo11 Mouse 76582 Ipo11 NM_029665 Q8K2V6 RanBP11 (Importin) 1668 (2011) 1813 Acta Biophysica et Biochimica / al. et Major A.T. importin 11 Ranbp11, AI314624, AW555235, 1700081H05Rik, Importin-11 (Imp11) 2510001A17Rik, E330021B14Rik Iso1→975aa, 112.4 kDa Iso2→984aa, 113.3 kDa Human 51194—IPO11 TrnSpt1 →NM_001134779 Q9UI26 RanBP11 importin 11 TrnSpt2 →NM_016338 Importin-11 (Imp11) RanBP11 Iso1→975aa, 112.5 kDa

Ipo12 Mouse 320938—Tnpo3 NM_177296 Q6P2B1 – β (Importin) transportin 3 C81142, Trn-SR, KIAA4133, MGC90049, mKIAA4133, Transportin-3 D6Ertd313e, 5730544L10Rik, C430013M08Rik Iso1→923aa, 104.2 kDa Iso2→929aa, 104.9 kDa Iso3→267aa, 29.9 kDa Human 23534—TNPO3 NM_012470 Q9Y5L0 TRN-SR, Imp12 transportin 3 IPO12, TRNSR, MTR10A, TRN-SR, TRN-SR2 Transportin-3 Iso1→957aa, 108.0 kDa Iso2→923aa, 104.2 kDa Iso3→909aa, 102.5 kDa

Ipo13 Mouse 230673—Ipo13 NM_146152 Q8K0C1 - β (Importin) importin 13 Imp13, Kap13, Ranbp13, MGC18698 Importin-13 (Imp13) Iso1→963aa, 108.2 kDa Iso2→964aa, 108.4 kDa —

Human 9670 IPO13 NM_014652 O94829 RanBP13, Kap13 – importin 13 LGL2, IMP13, KAP13, RANBP13, KIAA0724 Importin-13 (Imp13) 1688 963aa, 108.2 kDa

C: Exportins (karyopherin βs→nuclear export) family genes/proteins in the mouse and human. Xpo1 Mouse 103573—Xpo1 TrnSpt1 →NM_001035226 Q6P5F9 Chromosome region maintenance 1 protein β (Exportin) exportin 1, CRM1 homolog TrnSpt2 →NM_134014 Exportin-1 (Exp1) homolog (yeast) Crm1, Exp1, AA420417 1071aa, 123.1 kDa Human 7514—XPO1 NM_003400 O14980 Chromosome region maintenance 1 protein exportin 1 (CRM1 homolog, CRM1, DKFZp686B1823 Exportin-1 (Exp1) homolog yeast) 1071aa, 123.4 kDa

Xpo2 Mouse 110750—Cse1l NM_023565 Q9ERK4 Importin-alpha re-exporter, Chromosome β (Exportin) chromosome segregation 1-like Cas, Xpo2, Capts, AA407533, 2610100P18Rik Exportin-2 (Exp2) segregation 1-like protein (S. cerevisiae) 971aa, 110.5 kDa Human 1434—CSE1L NM_001316 P55060 Importin-alpha re-exporter, Chromosome CSE1 chromosome segregation 1- CAS, CSE1, XPO2, MGC117283, MGC130036, Exportin-2 (Exp2) segregation 1-like protein, Cellular apoptosis like (yeast) MGC130037 Iso1→971aa, 110.4 kDa susceptibility protein Iso2→195aa, 22.7 kDa Iso3→945aa, 107.8 kDa Xpo3 Mouse 73192—Xpot NM_001081056 Q9CRT8 tRNA exportin, Exportin(tRNA) β (Exportin) exportin, tRNA (nuclear export C79645, AI452076, EXPORTIN-T, 1110004L07Rik, Exportin-T receptor for tRNAs) 3110065H13Rik 963aa, 109.7 kDa Human 11260—XPOT NM_007235 O43592 tRNA exportin, Exportin(tRNA) exportin, tRNA (nuclear export XPO3 Exportin-T receptor for tRNAs) 962aa, 110.0 kDa

Xpo4 Mouse 57258—Xpo4 NM_020506 Q9ESJ0 - β (Exportin) exportin 4 mKIAA1721, B430309A01Rik Exportin-4 (Exp4) 1151aa, 130.0 kDa Human 64328—XPO4 NM_022459 Q9C0E2 - exportin 4 FLJ13046, KIAA1721 Exportin-4 (Exp4) 1151aa, 130.1 kDa

Xpo5 Mouse 72322—Xpo5 NM_028198 Q924C1 Ran-binding protein 21 β (Exportin) exportin 5 Exp5, RanBp21, AI648907, AW549301, Exportin-5 (Exp5) mKIAA1291, 2410004H11Rik, 2700038C24Rik Iso1→1204aa, 137.0 kDa →

Iso2 496aa, 56.1 kDa 1668 (2011) 1813 Acta Biophysica et Biochimica / al. et Major A.T. Iso3→208aa, 24.7 kDa Human 57510—XPO5 NM_020750 Q9HAV4 Ran-binding protein 21 exportin 5 FLJ14239, FLJ32057, FLJ45606, KIAA1291 Exportin-5 (Exp5) 1204aa, 136.3 kDa

Xpo6 Mouse 74204—Xpo6 NM_028816 Q924Z6 Ran-binding protein 20 β (Exportin) exportin 6 R75304, Ranbp20, AI854665, AL022631, mKIAA0370, Exportin-6 (Exp6) 2610005L19Rik, C230091E20Rik Iso1→1125aa, 128.7 kDa Iso2→1124aa, 128.5 kDa Iso3→246aa, 28.7 kDa Human 23214—XPO6 NM_015171 Q96QU8 Ran-binding protein 20 exportin 6 EXP6, RANBP20, FLJ22519, KIAA0370 Exportin-6 (Exp6) 1125aa, 128.9 kDa

Xpo7 Mouse 65246—Xpo7 NM_023045 Q9EPK7 Ran-binding protein 16 β (Exportin) exportin 7 Ranbp16, BB164534, mKIAA0745, 4930506C02Rik Exportin-7 (Exp7) Iso1→1087aa, 123.8 kDa Iso2→1057aa, 120.4 kDa Human 23039—XPO7 TrnSpt1→NM_001100161 Q9UIA9 Ran-binding protein 16 exportin 7 TrnSpt2→NM_015024 Exportin-7 (Exp7) TrnSpt3→NM_001100162 1087aa, 123.9 kDa RANBP16, KIAA0745 –

D: Ran, Ran binding and other major nuclear transport related genes/proteins in the mouse and human (all others). 1688 Ran Mouse 19384—Ran NM_009391 P62827 GTPase Ran, Ras-related nuclear protein, Ran RAN, member RAS oncogene – GTP-binding nuclear protein Ras-like protein TC4 family Ran 216aa, 24.4 kDa Human 5901—RAN NM_006325 P62826 GTPase Ran, Ras-related nuclear protein, Ras-like RAN, member RAS oncogene TC4, Gsp1, ARA24 GTP-binding nuclear protein protein TC4, Androgen receptor-associated protein 24 family Ran 216aa, 24.4 kDa

RanGAP1 Mouse 19387—Rangap1 TrnSpt1→NM_001146174 P46061 Fug1 Ran-GTP Gradient RAN GTPase activating protein 1 TrnSpt2→NM_011241 Ran GTPase-activating Maintenance Fug1, C79654, mKIAA1835 protein 1 (RanGAP1) 589aa, 63.6 kDa Human 5905—RANGAP1 NM_002883 P46060 - Ran GTPase activating protein 1 SD, Fug1, KIAA1835, MGC20266 Ran GTPase-activating protein 1 (RanGAP1) 587aa, 63.5 kDa

(continued on next page) 1673 1674 Table 1 (continued) Abbreviation Species Gene (NCBI) Protein (UniProtKB/Swiss-Prot) Subtype (used here) ID-symbol Reference sequence(s) ID, name, aa, mass Alternate names

Official full name Also known as

Rcc1 Mouse 100088—Rcc1 NM_133878 Q8VE37 Chromosome condensation protein 1 Ran-GTP Gradient regulator of chromosome Chc1, AI326872, 4931417M11Rik Regulator of chromosome Maintenance condensation 1 condensation 421aa, 44.9 kDa Human 1104—RCC1 TrnSpt1→NM_001048194 P18754 Chromosome condensation protein 1, Cell cycle regulator of chromosome TrnSpt2→NM_001048195 Regulator of chromosome regulatory protein condensation 1 TrnSpt3→NM_001269 condensation CHC1, RCC1-I 421aa, 45.0 kDa

Ntf2 Mouse 68051—Nutf2 NM_026532 P61971 - Ran-GTP Gradient nuclear transport factor 2 Ntf2, Ntf3, PP15, AI115459, AI413596, AW546000, RNA U transporter 1 (NTF-2) Maintenance MGC102015, 2700067I02Rik 127aa, 14.5 kDa Human 10204—NUTF2 NM_005796 P61970 PP15 1668 (2011) 1813 Acta Biophysica et Biochimica / al. et Major A.T. nuclear transport factor 2 NUTF2, PP15 Nuclear transport factor 2 (NTF-2) 127aa, 14.5 kDa

RanBP1 Mouse 19385—Ranbp1 NM_011239 P34022 RANBP1, HpaII tiny fragments locus 9a protein Ran-GTP Gradient RAN binding protein 1 Htf9a Ran-specific GTPase- Maintenance activating protein 203aa, 23.6 kDa Human 5902—RANBP1 NM_002882 P43487 RanBP1 RAN binding protein 1 HTF9A, MGC88701 Ran-specific GTPase- activating protein 201aa, 23.3 kDa

RanBP2 Mouse 19386—Ranbp2 NM_011240 Q9ERU9 RanBP2 Nucleoporin? RAN binding protein 2 NUP358, AI256741, A430087B05Rik E3 SUMO-protein ligase RanBP2 3053aa, 341.1 kDa Human 5903—RANBP2 NM_006267 P49792 RanBP2, Nuclear pore complex protein Nup358, RAN binding protein 2 ANE1, TRP1, TRP2, NUP358 E3 SUMO-protein ligase Nucleoporin Nup358, 358 kDa nucleoporin, p270 RanBP2 3224aa, 358.2 kDa

RanBP3 Mouse 71810—Ranbp3 NM_027933 Q9CT10 -? – RAN binding protein 3 AA408221, 2610024N24Rik Ran-binding protein 3 1688 (RanBP3) 491aa, 52.6 kDa Human 8498—RANBP3 TrnSpt1→NM_003624 Q9H6Z4 - RAN binding protein 3 TrnSpt2→NM_007320 Ran-binding protein 3 TrnSpt3→NM_007322 (RanBP3) DKFZp586I1520 Iso1→567aa, 60.2 kDa Iso2→562aa, 59.7 kDa Iso3→499aa, 53.4 kDa Iso4→397aa, 42.4 kDa

RanBP6 Mouse 240614—Ranbp6 NM_177721 Q8BIV3 – ? RAN binding protein 6 FLJ11120, C630001B19 Ran-binding protein 6 (RanBP6) 1105aa, 124.6 kDa Human 26953—RANBP6 NM_012416 O60518 – RAN binding protein 6 FLJ54311 Ran-binding protein 6 (RanBP6) 1105aa, 124.7 kDa RanBP9 Mouse 56705—Ranbp9 NM_019930 P69566 RanBPM, IBAP-1 ? RAN binding protein 9 Ibap1, IBAP-1, RanBPM Ran-binding protein 9 (RanBP9) Iso1→653aa, 71.0 kDa Iso2→546aa, 60.1 kDa Human 10048—RANBP9 NM_005493 Q96S59 RanBP7, RanBPM, BPM90, BPM-L RAN binding protein 9 BPM-L, BPM90, RANBPM, RanBP7 Ran-binding protein 9 (RanBP9) Iso1→729aa, 77.9 kDa Iso2→388aa, 43.1 kDa

RanBP10 Mouse 74334—Ranbp10 NM_145824 Q6VN19 – ? RAN binding protein 10 4432417N03Rik Ran-binding protein 10 (RanBP10) 648aa, 70.1 kDa Human 57610—RANBP10 NM_020850 Q6VN20 – RAN binding protein 10 FLJ31165, KIAA1464 Ran-binding protein 10 (RanBP10)

620aa, 67.3 kDa 1668 (2011) 1813 Acta Biophysica et Biochimica / al. et Major A.T.

RanBP17 Mouse 66011—Ranbp17 NM_023146 Q99NF8 – Exportin? RAN binding protein 17 4932704E15Rik Ran-binding protein 17 1088aa, 124.1 kDa Human 64901—RANBP17 NM_022897 Q9H2T7 – RAN binding protein 17 FLJ32916 Ran-binding protein 17 1088aa, 124.4 kDa

Snupn Mouse 66069—Snupn NM_178374 Q80W37 RNA U transporter 1 α homologue snurportin 1 Rnut1, Snupn1, 0610031A09Rik Snurportin-1 (Imports snRNPs) 358aa, 41.0 kDa Human 10073—SNUPN TrnSpt1→NM_001042581 O95149 RNA U transporter 1 snurportin 1 TrnSpt2→NM_001042588 Snurportin-1 TrnSpt3→NM_005701 360aa, 41.1 kDa KPNBL, RNUT1, Snurportin1

Gene information for both mouse and human was gathered by searching the “National Center for Biotechnology Information” (NCBI) website. Protein information was sourced from the Swiss-Prot entries of the “Universal Protein Resource Knowledgebase” (UniProtKB) website [123,124]. Where multiple mRNAs/proteins were present within the databases, all of the transcripts/isoforms are listed and numbered (TrnSpt#/Iso#). The mRNA transcript numbers do not correspond directly to the protein isoform numbers, as each set was gathered independently. Nuclear transport machinery components have been separated into four categories; the importin alphas (Table 1A), the karyropherin β family members involved in nuclear import (Importins [Ipo], see Table 1B), the karyropherin βs involved in nuclear export (Exportins [Xpo], see Table 1C) and all the other nuclear transport factors, such as RAN and RAN binding proteins (Table 1D). The molecular weights of all the proteins are shown in kilodaltons (kDa), rounded to one decimal place. – 1688 1675 1676 A.T. Major et al. / Biochimica et Biophysica Acta 1813 (2011) 1668–1688

Cytoplasm Nuclear Envelope Nucleus

NPC NLS GTP A NLS

NLS

NLS B IBB GTP

IBB

NES NES GTP C Xpo Xpo GDP

RanGAP GTP GTP D Rcc1 RanBP1 GDP GDP

E Ntf2 GDP Ntf2 GTP

Xpo GTP GDP NLS NES IBB cargo with NLS cargo with NES importin importin exportin Ran-GTP Ran-GDP

Fig. 1. Conventional nuclear transport pathways. The cytoplasm and nucleus of a cell are separated by the nuclear envelope, which is traversed by the nuclear pore complex (NPC). Nuclear import: during importin β mediated nuclear import, the importin β subtypes bind directly to the NLS of cargo proteins and transport these into the nucleus (A). Classical importin α mediated nuclear import involves importin α protein binding both the cargo NLS and importin β. The α/β/cargo complex translocates through the NPC (B). In both importin β or α/β mediated nuclear transport, the cargo is released in the nucleus following binding of the monomeric guanine nucleotide binding protein RAN in activated GTP bound form (RAN-GTP) to importin β (A, B). Nuclear export: exportins recognise the cargo NES in the nucleus, forming a complex with cargo and RAN-GTP which translocates through the NPC. In the cytoplasm, this complex dissociates following the hydrolysis of RAN-GTP to RAN-GDP (C). The RAN cycle: RAN-GDP is converted to RAN-GTP in the nucleus by RCC1, while in the cytoplasm the hydrolysis of RAN-GTP to RAN-GDP is facilitated by RAN GTPase activating protein (RANGAP1) and RAN binding protein 1 (RANBP1) (D). NTF2 specifically recognises the RAN-GDP form and facilitates its import into the nucleus (E). cytoplasmic RAN GTPase-activating protein, RANGAP1, in conjunction interact directly with NUPs to transit the NPC [19]. For example, β- with RAN binding protein 1 (RANBP1), mediates RAN–GTP hydrolysis catenin can enter and exit the nucleus independently of importins, into RAN–GDP. Finally, RAN–GDP transport into the nucleus by ATP or RAN [38–40]. In addition, importin α can enter the nucleus in nuclear transport factor 2 (NTF2) enables replenishment of nuclear an ATP-dependent manner, independent of importin β or RAN [41]. RAN–GTP to the high levels required to sustain the transport cycle Under conditions of cellular stress, importin α accumulates in the (Fig. 1E) [35]. Maintenance of the transport cycle also requires that nucleus, shutting down classical importin α/β mediated nuclear importins return to the appropriate compartment for additional cargo import [42,43]. These data are consistent with observations that pickup. Recycling of importin α proteins into the cytoplasm relies on importin α and importin β proteins serves roles additional to cargo active nuclear export mediated by exportin 2 (XPO2) [30,36], while protein transport [6] that include mediating responses to cellular karyopherin β subtypes can traverse the NPC unaided [33,37]. stress [41], nuclear envelope assembly [44], regulation of nuclear lamin polymerisation [45] and facilitating spindle formation [46]. 2.5. Non-conventional transport and non-conventional roles for transport proteins 3. Overview of testis formation and spermatogenesis

While the conventional nuclear transport machinery is the focus of 3.1. Forming the fertile testis this review, this pathway is not the sole mediator of nucleocytoplas- mic protein movements. Instead of using the conventional nuclear Adult spermatogenesis in mammals is the end product of an transport pathway machinery described above, some proteins ongoing series of events that begins in early embryogenesis, when A.T. Major et al. / Biochimica et Biophysica Acta 1813 (2011) 1668–1688 1677

Fig. 2. Spermatogenesis. Cellular differentiation of the male germline is presented as (A) foetal and (B) postnatal stages of development. Both phases feature a range of tightly regulated, dynamic cell cycle activities, as well as extensive chromatin remodelling. The relevant ages are listed for events in the mouse (E=embryonic day; dpp=days post partum; from [73]). cells are set aside during gastrulation to form the pluripotent germline Leydig cells (the steroidogenic lineage), fibroblasts, immune cells and lineage; it is first completed after puberty and is sustained throughout both endothelial and lymphatic vessels. Important transformations in adulthood [47,48]. These cells, termed primordial germ cells, will testicular somatic cell populations in juvenile life are required to sustain subsequently multiply and migrate to the indifferent foetal gonads and, full spermatogenesis in adequate numbers to achieve fertility. A crucial under the influence of somatic-derived factors in either the ovary or step is the transition of Sertoli cells in juvenile life from an immature, testis, become committed to their fate of forming either female or male proliferative state into the post-mitotic, highly polar mature cells linked gametes, respectively [49,50] (Fig. 2A). Retinoic acid exposure in the by tight junctions which form the tubular epithelium required to foetal ovary triggers germ cell entry into meiosis [51,52], thereby support adult sperm production [57]. It is within this epithelium that commencing the terminal differentiation pathway of oogenesis. In spermatogonial stem cells are triggered from puberty onwards to males, somatic cell cues initiated by expression of SRY in pre-Sertoli cells undergo the amplification and developmental changes of spermato- direct the transformation of primordial germ cells into gonocytes [53]; genesis (Fig. 2B). Key cues driving spermatogenesis are also understood unlike their female counterparts, cells in the male germline retain the to be mediated from the vasculature and other cells outside of the capacity for pluripotent differentiation. Gonocytes in the rodent foetal seminiferous tubule, but the identity and control of such signals are not testis have a period of quiescence [54], then re-enter the cell cycle and fully understood. However, all germ cells are continuously exposed, migrate to the cord basement membrane as they transform into directly or indirectly, to both hormones and locally-produced signals spermatogonia, generating the stem cell population required to sustain which are mediated by the regulated transmission of resulting signals to sperm production through adult life [55,56]. Normally, gonocytes which the nucleus [58]. Recent reviews highlight the specific examples which do not undergo this transformation die by apoptosis. These events occur underpin this perspective and illustrate the intense interest in relatively early in foetal life in human males (weeks 6–8 of gestation) understanding how such signals are integrated [14,59]. but commence immediately after birth in rodents. Events which impede The progressive stages of spermatogenesis, therefore, require that this change or enable gonocytes to survive inappropriately are believed a particular sequence of gene expression changes occurs [58] (Fig. 2B). to underlie human testicular cancer. Typically diagnosed after puberty, This mediates unique chromatin remodelling events required for in this disease a gonocyte-like cell in a post-pubertal human testis maintenance of pluripotency, repression of somatic genes and nuclear (termed carcinoma in situ [CIS] of the testis) is triggered to differentiate DNA repackaging during the latter stages as mature spermatozoa are into either a seminoma or a non-seminoma by the hormonal changes of formed. In juveniles, the onset of the first wave of spermatogenesis puberty [55]. The mechanisms by which pluripotent germ cells are occurs when a subset of undifferentiated spermatogonia first sustained into adulthood and then driven to malignancy into the testis commence a series of mitotic divisions and simultaneously change are not understood. transcriptional activity, losing the capacity to return to a pluripotent state. These differentiating spermatogonia become responsive to a 3.2. Spermatogenesis in adulthood new set of signalling cues which ultimately enable them to enter, transit and complete meiosis as spermatocytes. Two rounds of nuclear Survival, proliferation and maturation of spermatogenic cells are meiotic divisions form the initially round haploid spermatids in which driven by dynamic somatic cell cues. The male germline develops the last transcriptional activity occurs. The last stage of spermato- throughout life embedded in the seminiferous epithelium formed by genesis, termed spermiogenesis, requires many days of morphological Sertoli cells. Comprised of cords (in the immature testis) or tubules remodelling in the absence of gene transcription, and a key feature of (formed at puberty when Sertoli cells adopt a polarised, secretory round spermatids is the cytoplasmic accumulation of a large mass of phenotype) and surrounded by contractile peritubular myoid cells, the stored mRNAs. Their subsequent transformation into the elongating spermatogenic epithelia are loosely joined by an interstitium containing spermatid form involves growth of the sperm tail around the 9+2 1678 A.T. Major et al. / Biochimica et Biophysica Acta 1813 (2011) 1668–1688

α microtubule doublet of the axoneme, development of the acrosome as Importin Expression in the Embryonic Mouse Testis a flattened, enzyme-bearing vesicle along the nuclear apex, and 15000 Kpna1 reshaping of the spermatid cytoplasm to be discarded as a single Kpna2 droplet when sperm are released into the tubule lumen. Nuclear DNA Kpna3 is compacted through the large-scale removal of histones which are 10000 Kpna4 replaced first with transition proteins and then with protamines. Kpna6 Coordination of these processes within the seminiferous epithelium is rigidly managed by the Sertoli and other somatic cells which contain 5000 receptors for the hormones required to sustain full spermatogenesis.

The germ cells influence the pace of these events, however, as is Relative Expression evident from xenografts of spermatogonial stem cells into host testes 0 lacking endogenous spermatogenesis [60]. 2 E11 E1 E14 E16 E18 2dpp 0.5dpp 4. Regulated synthesis of nucleocytoplasmic transport fi components during spermatogenesis Fig. 4. Transcript pro ling example using the gene expression omnibus (GEO). The expression profiles shown are from the GEO dataset GDS2098. This was generated by Gaido and Lehmann (no linked publication) from normal whole mouse testes from As described above (Section 3), spermatogenesis is governed by embryonic day (E) 11 through to 2 days post partum (dpp). The magnitude of the signal complex and dynamic interacting signals from somatic cells. The for Kpna2 is far greater than that of the other importin α genes, suggesting it may be the manner in which these inputs are coordinated to produce adequate key mediator of nucleocytoplasmic transport in the embryonic testis. Note that subtle changes in the other importin αs are poorly visualised, highlighting the necessity of sperm numbers for fertility, and the factors contributing to sub/ splitting probesets into the higher and lower groups as in Figs. 5–7 (see Fig. 5A for a infertility or testis cancer, are of intense interest. The nucleus, as the direct comparison). site where transcription occurs, is the cellular compartment in which these signals are ultimately integrated. Thus, developmentally regulated nucleocytoplasmic transport has been proposed to influ- hosted at the NCBI website [65–71], for the genes listed in Table 1.In ence spermatogenesis and male fertility [57]. the next sections, these data will be used to describe the expression profiles of major nucleocytoplasmic transport protein family mem- 4.1. The knowledge gap and how to fill it bers throughout testis development and spermatogenesis, whilst comparing these to published expression data. These data are While it has been known for some time that importin α family presented in temporal sequence for the mouse, beginning with the members are produced at different levels in the testis [16], a limited foetal testis (Figs. 4 and 5), then progressing through the first wave of number of publications report expression of the importins and other spermatogenesis and including cell types isolated from the adult testis nucleocytoplasmic transport components in specific spermatogenic (Figs. 6 and 7). Additional information from an analysis of human cells. In fact, few studies have investigated importin synthesis during testis cancer specimens is also presented (Fig. 8). development of any biological system, with a small number In many GEO datasets, each gene listed in Table 1 is represented by comprehensively documenting expression patterns of importin α more than one oligonucleotide probeset. In such cases, the expression subtypes [16,61,62] and three importin β proteins, IPO1 [62,63], IPO5 profiles for each probeset was compared and a single set chosen to [62,63] and IPO13 [64]. Fig. 3 summarises the cell-specific synthesis represent the gene in this review. As an example, Fig. 4 presents data and subcellular distribution changes we have documented for for the importin α genes as recorded in the foetal mouse testis importins during rodent spermatogenesis in the adult testis. Beyond between the time of sex determination (E11) and shortly after birth this, the stages at which other importin family members or other (2 dpp). The magnitude of the signal for Kpna2 is obviously far greater factors required for nucleocytoplasmic transport have not been than that of the other importin α genes, which suggests it is the key extensively investigated within the testis. Such information could mediator of nucleocytoplasmic transport in the testis. In subsequent potentially identify important points of control and mechanisms that Figures, graphs of all transcript groupings are separated into the control in the unique cascade of cellular differentiation events, should higher and lower level probesets to enable developmentally regulated they be temporally linked with specific stages of spermatogenesis. changes to be visualised for each mRNA. In selecting data to include, To address this potential, we undertook the approach of collating preference was given to probesets that displayed higher levels of information available through the Gene Expression Omnibus (GEO), detection, displayed less variation between replicates and/or better represented the general trend displayed by all probesets of a given gene. These basic rules of selection were also applied where conflicting probeset outcomes were found and some of these conflicts are mentioned within the descriptive text. A full list of GEO datasets and the oligonucleotide probeset IDs used for Figs. 4–8 is available within the supplementary data (S1A-D). Where individual data replicates produced a “Detection Call=ABSENT,” the data were deemed uninformative from a quantitation perspective and excluded from consideration. Data presented in Figs. 4–8 were prepared using GraphPad Prism v5.03 software.

4.2. Expression during embryonic testis development

Fig. 3. Importins in adult spermatogenesis. Published [61,63] and unpublished data There is a remarkable paucity of data regarding the synthesis or from our laboratory have identified the steps in spermatogenesis where each importin function of nucleocytoplasmic transport machinery in the foetal testis, α mRNA (solid bars) and/or protein (hatched lines) is readily detected by in situ despite the absolute dependence of human male sex determination hybridisation or immunohistochemistry, respectively. Note that KPNA3 and KPNA4 are detected in different subcellular compartments, despite being most closely related to on nuclear transport of the sex-determining switch protein, SRY, a each other based on structural characteristics. phenomenon discussed in Section 4.5. A.T. Major et al. / Biochimica et Biophysica Acta 1813 (2011) 1668–1688 1679

A (High) Importin α Subtypes (Low) 15000 2500 Kpna2 Kpna1 Kpna3 2000 Kpna4 10000 Kpna6 1500

1000 5000 500 Relative Expression Relative Expression

0 0 6 p E11 E12 E14 E1 E18 E11 E12 E14 E16 E18 2dpp 2dpp 0.5dp 0.5dpp B (High) Importin β Subtypes (Low) 6000 2000 Ipo1 Ipo2 Ipo4 Ipo3 Ipo5 1500 Ipo7 4000 Ipo9 Ipo8 Ipo11 1000 Ipo12 Ipo13 2000 500 Relative Expression Relative Expression

0 0

E11 E12 E14 E16 E18 E11 E12 E14 E16 E18 2dpp 2dpp 0.5dpp 0.5dpp C (High) Exportin Subtypes (Low) 8000 2500 Xpo1 Xpo3 Xpo2 Xpo4 2000 6000 Xpo5 Xpo6 1500 Xpo7 4000 1000

2000 500 Relative Expression Relative Expression

0 0 8 p E11 E12 E14 E16 E1 E11 E12 E14 E16 E18 2dpp 2dpp 0.5dp 0.5dpp D (High) Ran Binding & Other Nuclear Transport Proteins (Low) 6000 Ntf2 2000 Ran RanBP2 RanGAP1 RanBP9 Rcc1 1500 4000 RanBP1 RanBP3 1000 RanBP6 RanBP10 2000 RanBP17 500 Snupn Relative Expression Relative Expression

0 0 1 2 8 E11 E12 E14 E16 E18 E1 E1 E14 E16 E1 2dpp 2dpp 0.5dpp 0.5dpp

Fig. 5. Expression of the nuclear transport machinery during embryonic testis development. The expression profiles shown are from the GEO dataset GDS2098. This was generated by Gaido and Lehmann (no linked publication) from normal whole mouse testes from embryonic day (E) 11 through to 2 days post partum (dpp). Data are separated into lower/higher detection levels for visualisation and into four groups; the importin α subtypes (A), the importin β subtypes involved in nuclear import (B), the exportin subtypes involved in nuclear export (C) and other proteins involved in nuclear transport (D). Error bars represent the standard error of the mean (SEM), where multiple values were present. 1680 A.T. Major et al. / Biochimica et Biophysica Acta 1813 (2011) 1668–1688

To examine levels of transcripts encoding the karyopherins in days 4–6, Type B spermatogonia on day 8, pachytene spermatocytes on foetal and newborn mouse testes, we used the GEO dataset GDS2098, day 16, round spermatids on day 20, elongating spermatids on day 30 derived from the GEO series GSE4818 (Gaido and Lehmann, no and spermatozoa on day 42 (see [73] for overview). Another GEO publication). This dataset was selected as it had triplicate samples, dataset, GDS2390 (derived from GSE4193), contained an analysis of each one representing the testes of an individual animal, with purified germ cell subtypes, type A spermatogonia, type B spermato- measurements at embryonic days (E) 11, 12, 14, 16, 18 and 2 days gonia, pachytene spermatocytes and round spermatids [74].These post partum (dpp). Key time points for germline transition are E11.5 spermatogonia were isolated from day 8 mice, and thus may exhibit for germline gender assignment in males [53], E14.5 for complete some transcripts absent from spermatogonia in the adult testis. The cessation of gonocyte proliferation [54] and day 1 for the onset of spermatocytes and spermatids were purified from adult animals. proliferation (Fig. 2). The platform used for this dataset also had the Because most of the transcripts from specific germ cell types should highest number of karyopherins present, compared to other GEO be consistently higher at the ages immediately following when they first datasets. A minor limitation of this particular dataset is inclusion of emerge during testis development, this information has been presented only one 0.5 dpp sample. together in Figs. 6 and 7 to facilitate this comparison. The extracted data relating to importin expression in the embryonic testis are presented in Fig. 5. The left hand and right 4.3.1. Importin alphas—spermatogenesis hand panels illustrate the transcripts present at higher and lower Importin α subtype expression throughout spermatogenesis is levels, respectively, for each of the following groups which are plotted particularly interesting, as each importin α shows a distinct separately to reflect their functional characterisation and for clarity of expression pattern, and there are published data with which to data visualisation: importin α, importin β (import function), importin compare the GEO datasets shown here (compare Fig. 3 and Fig. 6A). β (export function) and RAN binding plus other nucleocytoplasmic All importin α transcripts show expression profiles that match the transport functions. expression patterns described by Hogarth et al. using in situ During embryonic testis development, the importin α that hybridisation and RT-PCR methods [61], however these datasets displayed the most dynamic expression pattern was Kpna2, exhibiting offer additional insights, based on results such as the magnitude of the a very high signal (~6–12 times higher than other importin α signal for Kpna2 relative to the other transcripts. For example, Kpna1 transcripts) and a distinct peak around E12, suggesting it plays a was detected at lower, but consistent levels throughout spermato- major role in nucleocytoplasmic transport and serves important genesis. The Kpna2 value was consistently much higher than all others functions at the time of sexual differentiation. Kpna3 and Kpna4 and was highly elevated within spermatocytes and spermatids, which transcript levels were higher at the beginning of the series (E11) and appear from approximately day 15 onwards. Kpna3 displayed an early slowly declined. Both Kpna1 and Kpna6 exhibited lower but generally peak in expression, consistent with the published finding that it is consistent levels of expression throughout testis development. predominantly found within spermatogonia [61]. Kpna4 shows high In the importin β (import function) subgroup, Ipo4, Ipo5 and Ipo9 levels predominantly in spermatocyctes, with a peak between days 14 had the highest signals at all ages. Ipo11 and Ipo12 had two of the most and 35 in the total testis samples, also consistent with previous data distinctive expression profiles, showing higher signals at E11 and E12 [59]. Kpna6 was not detected (Detection Call=ABSENT) until day 8, that dropped markedly by E14 (~2×) to a level that was sustained and its signal did not peak until around day 30, when the population until after birth. Although the Ipo7 signal was lower, it also displayed a of round spermatids greatly increases. This is also consistent with peak at E12, similar to Kpna2. Ipo1, Ipo2 and Ipo9 all had higher published data detecting Kpna6 mRNA only in round spermatids by in expression levels at the beginning of the embryonic testis age series situ hybridisation, as illustrated in Fig. 3 [61]. Despite their structural which progressively diminished until birth. Both Ipo8 and Ipo4 similarities, the importin α family members clearly have distinct roles displayed the highest expression value at E14. and individual expression profiles throughout spermatogenesis Of the exportin subtypes, Xpo1 and Xpo2 had the highest signal [16,61,75]. A newly defined importin, Kpna7 [76–78], consistently values, while Xpo3 showed its highest value at E12, as did RanBP1. registers ABSENT detection calls in embryonic and postnatal testis These findings collectively indicate there is a high level of nucleocy- datasets and so has not been reported here. toplasmic trafficking in the testis concordant with sex determination, a proposal which is supported by the high level of Ntf2 in the earlier 4.3.2. Importin betas—spermatogenesis ages of this series. Several importin β subtypes also show peaks of transcript expression Amongst these transport components, the expression of KPNA2, associated with specific stages of spermatogenesis (Fig. 6B), with Ipo4 KPNA3, IPO1 and IPO5 proteins in the foetal testis has been and Ipo5 particularly striking. The Ipo5 signal rises after birth to peak at documented using immunohistochemistry. KPNA2, KPNA3 and IPO1 day 3, at the onset of spermatogenesis, as gonocytes complete the proteins all appear cytoplasmic in foetal germ cells with IPO1 transition into spermatogonia. The transcript level drops 2-fold to day exhibiting an unusual perinuclear localisation in gonocytes at E16.5, 10, then increases N3-fold to peak by day 18, an age when pachytene when these cells are quiescent [62,63]. While the Ipo5 mRNA level spermatocytes predominate in the postnatal testis. The Ipo5 transcript does not vary remarkably in foetal life (Fig. 5B), the protein appears to was first identified by differential display targeting genes expressed undergo gender-specific movement between nucleus and cytoplasm speci fically within germ cells of the testis [79]. In situ hybridisation data in the mouse gonad [62,63]. The functional implications of these in this study identified Ipo5 mRNA within pachytene spermatocytes and changes remain to be discerned. round spermatids [79]. In our comprehensive study of regulated IPO1 and IPO5 synthesis during rodent spermatogenesis [63], we reported 4.3. Expression throughout spermatogenesis the expression profiles of IPO1 and IPO5 in the foetal gonad, during the first wave of spermatogenesis and in the adult testis using PCR, in situ We explored several GEO datasets to identify karyopherin family hybridisation and immunohistochemistry. Consistent with the GEO expression profiles in the postnatal mouse testis. The GEO datasets, profiles databases, Ipo5 mRNA was identified predominantly within GDS605, GDS606 and GDS607 (all derived from the GEO series GSE926) round spermatids by in situ hybridisation, while the IPO5 protein was represent a testis age series ranging from birth (day 0) to adulthood [72]. detected in elongating spermatids [63], demonstrating a translational The power and value of this series is that it follows the first wave of delay for IPO5 that is a common feature of transcripts encoding proteins murine spermatogenesis, in which progressively maturing germ cell required for the period in spermiogenesis when the male germ cell is types first appear at or around a specific postnatal day: undifferentiated transcriptionally inactive [1]. Thus synthesis of other karyopherin Type A spermatogonia on day 1, differentiated Type A spermatogonia on proteins identified here may similarly exhibit translational delay, A.T. Major et al. / Biochimica et Biophysica Acta 1813 (2011) 1668–1688 1681 A (High) Importin α Subtypes – Age Series (Low) 2000 800 Kpna2 Kpna1 Kpna6 Kpna3 1500 600 Kpna4

1000 400

500 200 Relative Expression Relative Expression 0 0

day 0day 3day 6day 8 day 0day 3 day 6day 8 day 10day 14day 18day 20day 30day 35day 56 day 10day 14day 18day 20day 30day 35day 56 (High) Importin α Subtypes – Isolated Cells (Low) 4000 500 Kpna2 Kpna1 Kpna4 Kpna3 400 3000 Kpna6

300 2000 200

1000 100 Relative Expression Relative Expression 0 0

A SpG B SpG A SpG B SpG Pch SpC Rnd SpT Pch SpC Rnd SpT B (High) Importin β Subtypes – Age Series (Low) 1500 400 Ipo1 Ipo5 Ipo2 Ipo7 Ipo3 300 Ipo8 1000 Ipo4 Ipo11 Ipo9 Ipo12 200

500 100 Relative Expression Relative Expression

0 0

day 0 day 3 day 6 day 8 day 0 day 3 day 6 day 8 day 10day 14day 18day 20day 30day 35day 56 day 10day 14day 18day 20day 30day 35day 56 (High) Importin β Subtypes – Isolated Cells (Low) 5000 1000 Ipo1 Ipo2 Ipo4 Ipo3 4000 800 Ipo5 Ipo7 Ipo8 3000 600 Ipo9 Ipo11 2000 400 Ipo12 Ipo13 1000 200 Relative Expression Relative Expression 0 0

A SpG B SpG A SpG B SpG Pch SpC Rnd SpT Pch SpC Rnd SpT

Fig. 6. Expression of the importin αs and importin βs throughout spermatogenesis. The expression profiles shown for the mouse testis age series are from the GEO datasets GDS605, GDS606 and GDS607 [72]. The isolated mouse germ cell data (GDS2390) contains Type A spermatogonia (A SpG), Type B spermatogonia (B SpG), pachytene spermatocytes (Pch Spc) and round spermatids (Rnd SpT) [74]. Data are separated into lower/higher detection levels for visualisation, and into the importin α subtypes (A) and the importin β subtypes involved in nuclear import (B). Error bars represent the standard error of the mean (SEM), where multiple values were present. particularly if their encoding transcripts are produced during post- the cytoplasm of spermatogonia, spermatocytes and Sertoli cells, with mitotic differentiation. minimal to no signal in round and elongating spermatids [63]. The Using the non-quantitative approaches of in situ hybridisation and transcript levels of Ipo1, Ipo3 and Ipo11 are each moderately higher in immunohistochemistry, Ipo1 mRNA and IPO1 protein were detected in pachytene spermatocyte cells than in purified spermatogonia or 1682 A.T. Major et al. / Biochimica et Biophysica Acta 1813 (2011) 1668–1688 A (High) Exportin Subtypes – Age Series (Low)

Xpo1 600 1000 Xpo2 Xpo6 Xpo3 Xpo5 400 Xpo7

500 200 Relative Expression Relative Expression 0 0

day 0day 3day 6day 8 day 0day 3day 6day 8 day 10day 14day 18day 20day 30day 35day 56 day 10day 14day 18day 20day 30day 35day 56 (High) Exportin Subtypes – Isolated Cells (Low) 2500 400 Xpo1 Xpo4 Xpo2 Xpo5 2000 Xpo3 300 Xpo7 Xpo6 1500 200 1000 100 500 Relative Expression Relative Expression 0 0

A SpG B SpG A SpG B SpG Pch SpC Rnd SpT Pch SpC Rnd SpT B (High) Ran Binding & Nuclear Transport Proteins – Age Series (Low) 1000 6000 Ran RanGAP1 Ntf2 Rcc1 RanBP2 800 RanBP1 Snupn RanBP3 4000 600 RanBP9 RanBP10 400 RanBP17 2000 200 Relative Expression Relative Expression 0 0

day 0day 3day 6day 8 day 0day 3day 6day 8 day 10day 14day 18day 20day 30day 35day 56 day 10day 14day 18day 20day 30day 35day 56 (High) Ran Binding & Nuclear Transport Proteins – Isolated Cells (Low) 1000 Ran RanGAP1 3000 Ntf2 Rcc1 800 RanBP1 RanBP2 RanBP9 RanBP3 2000 600 RanBP6 RanBP10 400 RanBP17 1000 Snupn 200 Relative Expression Relative Expression 0 0

A SpG B SpG A SpG B SpG Pch SpC Rnd SpT Pch SpC Rnd SpT

Fig. 7. Expression of the exportins and other nuclear transport proteins during spermatogenesis. The expression profiles shown for the mouse testis age series are from the GEO datasets GDS605, GDS606 and GDS607 [72]. The isolated germ cell data (GDS2390) contain Type A spermatogonia (A SpG), Type B spermatogonia (B SpG), pachytene spermatocytes (Pch Spc) and round spermatids (Rnd SpT) [74]. Data are separated into lower/higher detection levels for visualisation and into the exportin subtypes involved in nuclear export (A) and other proteins involved in nuclear transport (B). Error bars represent the standard error of the mean (SEM).

spermatid preparations, though this modest peak is not obvious by This correlates with the elevation recorded in the total testis age series inspection of the testis age series. samples visible from day 18 onwards. A function for IPO4 in haploid germ Similar to Ipo5,theIpo4 transcript level is higher in isolated cells has been identified using a combination of binding assays and in pachytene spermatocytes and round spermatids than in spermatogonia. vitro nuclear transport assays that indicated transition protein 2 (TNP2) A.T. Major et al. / Biochimica et Biophysica Acta 1813 (2011) 1668–1688 1683 is imported into the spermatid nucleus by IPO4 [80]. Transition proteins RanGAP1 and Rcc1 (the products of the latter two contribute to RAN– participate in a key step in chromatin remodelling during spermiogen- GTP gradient maintenance) all display higher values in older testes esis, where most of the histones are replaced, firstlybytransition and in the later stages of spermatogenesis. Both RanGAP1 and Rcc1 proteins and then by protamines, to yield the tightly compacted DNA dramatically increased (~4-fold) from day 20 onwards, suggesting a present in mature spermatozoa [58], so the upregulation of IPO4 at this profound increase occurs in the level of global nuclear transport in stage is predicted to mediate this developmental function. post-mitotic germ cells. There were, however, oligonucleotide IPO13 has been identified as a nuclear import receptor for UBC9, probesets within the databases for both that did not show this the E2 conjugating enzyme in the sumoylation pathway [7]. It can also increase. Ran transcript values increased from day 14 onwards and exhibit nuclear export activity towards eIF1A, a translation initiation were higher in spermatocytes and spermatids than in spermatogonia. factor [7]. While there was no informative oligonucleotide probeset RAN functions in spermatogenesis, in addition to nuclear transport, for Ipo13 within the testis age series GEO datasets described here, it may be to participate in microtubule assembly [89]. A second, testis- was readily detected and displayed a 2-fold greater microarray signal specific Ran isoform has been identified, which further indicates RAN in pachytene spermatocytes and round spermatid cell preparations may serve roles specific to spermatogenesis, but whether this isoform compared to both spermatogonial subtypes. This agrees with the functions in nuclear transport or other processes is currently report that Ipo13 within mouse male and female germ cells is most unknown [90]. abundant during the pachytene phase of meiosis [64].During RanBP2 signal levels are highest within the early days of the age spermatogenesis, Ipo13 expression precedes the nuclear accumula- series, then peak at day 14. This peak is not evident in the data from tion of its cargo, UBC9. Using siRNA to reduce IPO13 expression in the isolated germ cell types, which suggests that the highest level of foetal ovaries lowered nuclear accumulation of UBC9 and limited this transcript falls within the early spermatocytes. The implications oocyte progression to the later meiotic stages [64]. A truncated, testis- of changing RANBP2 production levels are discussed in Section 4.4,as specific Ipo13 (TS-Ipo13) transcript was discovered to be the product it relates to nucleoporins in the testis. of an alternate transcriptional start site; it appears slightly later than RanBP3 shows a very defined expression peak around day 20, its full length counterpart and reaches maximal levels in total testis which correlates to the expression values recorded as being highest in lysates from adult mice [64]. The product of this TS-Ipo13 transcript is the pachytene spermatocytes. RANBP3 is understood to act as a co- predicted to serve as a dominant-negative regulator of IPO13 cargo factor for XPO1-mediated nuclear export by stabilising interactions transport, due to its ability to bind IPO13 cargoes while lacking an N- between XPO1 and cargo proteins [91,92]. RANBP3 has also been terminal RAN–GTP binding domain [64,81]. shown to act in an XPO1-independent manner to facilitate nuclear export of β-catenin and SMAD2/SMAD3 [93,94]. This suggests it has 4.3.3. Exportins—spermatogenesis the capacity to terminate or control the duration of Wnt and TGF-β Most exportins displayed distinct expression profiles (Fig. 7A). signalling pathway activation, respectively [93,94], both pathways of Xpo1 and Xpo6 transcript levels are inversely related after day 14. known importance to testis growth and function [95,96]. Xpo1 levels are highest in A and B type spermatogonia as well as in the The RanBP9 signal is low in the early ages of the testis age series first half of the total testis age series, while in contrast Xpo6 rises ~3- and in spermatogonia, then increases from day 14 onwards to reach a fold between days 10 and 20 and is ~3-fold higher in spermatocytes level N6-fold higher in the adult testis and in spermatocytes and and spermatids than in spermatogonia. XPO1 exports Snupn and spermatids. While RANBP9 was first identified as a RAN binding nuclear export cargoes containing a hydrophobic NES (Section 2.1) protein [97], it has not yet been shown to have any direct roles in [25], while XPO6 is reported to predominantly export actin/profilin nuclear transport. Its capacity to bind RAN does, however, indicate its complexes from the nucleus [27]. This suggests that functional potential to influence nuclear transport. While it is beyond the scope requirements for nuclear export differ significantly between mitotic of this review, RANBP9 is of specific interest to spermatogenesis, given and post-mitotic cells. In addition, transcripts encoding exportins that it is predominantly expressed within the chromatoid bodies of XPO2, XPO4 and XPO7 were higher in A and B type spermatogonia testicular germ cells [98], interacts with DEAD box polypeptide 4 than in post-mitotic cells, and the profiles of Xpo2 and Xpo7 in whole (DDX4) which has essential roles in the male germ cells [98] and is testis RNA peaked at ~day 14 (no data available for Xpo4). The only believed to affect the transcriptional activity of both androgen and identified XPO2 cargoes are importin α proteins, which it exports glucocorticoid receptors [99]. back to the cytoplasm to enable additional nuclear import cycles Another gene that displayed higher expression levels in post- [30,36]. In contrast, protein binding assays have demonstrated that mitotic germ cells is RanBP17.NoRanBP17 was recorded within the XPO7 provides a more generalised export pathway with broader cargo early timepoints for this age series, but a signal was detected from day specificity [26]. While only a few cargoes have been identified for 18 onwards. RANBP17 appears to be a homologue of XPO7 [100,101] XPO4 [29], several of these have important nuclear roles in testis and is thus likely to act as an exportin. Northern blot analysis of biology. SMAD3, a transcription factor in the transforming growth human tissue samples has shown RanBP17 to be expressed predom- factor beta (TGFβ) superfamily signalling pathway, mediates signal- inantly within the testis, and the gene appears to yield two transcripts ling by activin and TGFβ [82] and can be exported from the nucleus by [100]. RANBP17 displays a nuclear localisation in HeLa cells [100], XPO4 [83]. In addition, XPO4 can act as a nuclear import receptor for similar to XPO7 and consistent with its predicted function as a nuclear SOX2 and SRY [8]. Thus, similarly to IPO13, XPO4 imports some export receptor [100,101]. In situ hybridisation analysis of mouse cargoes and exports others. The importance of SRY and SOX proteins tissues indicated that RanBP17 is present mainly within primary in the testis are discussed further in Section 4.5. spermatocytes [100], but to date there are no functional data. XPO5 mediates the nuclear export of short hairpin RNAs and pre- miRNAs [28,84–86], of which there are many unique to the testis 4.4. Importins, exportins, nucleoporins and the testis which are implicated in regulation of spermatogenesis [87,88]. The Xpo5 transcript was ~2-fold higher in pachytene spermatocytes and Most karyopherins have been demonstrated to bind, and therefore round spermatids relative to spermatogonia, although the signal transport, a specific subset of cargoes through binding to an NLS(s)/ recorded for the testis age series did not reflect this difference. NES(s). The data reviewed here demonstrate distinct transcript profiles for karyopherins throughout spermatogenesis, suggesting 4.3.4. Other nuclear transport related proteins—spermatogenesis that each has discrete functions relating to particular phases of germ Transcripts encoding RAN, the RAN binding proteins and other cell maturation. With advances in imaging techniques [102,103], proteins involved in nuclear transport are shown in Fig. 7B. Ran, structural/computer modelling [21] and the intuitive collection of 1684 A.T. Major et al. / Biochimica et Biophysica Acta 1813 (2011) 1668–1688

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Fig. 8. Expression of nuclear transport machinery in testicular seminomas. The data presented are from GDS2842 and show 40 human samples from both normal tissue and testicular seminoma tumours at various stages of progression (pT1, pT2, pT3) [119]. All values are units of relative expression with dots representing individual samples, while lines represent the mean of each sample and error bars represent the standard error of the mean (SEM). experimental data [22,31,104,105], the processes by which these investigation. There is significant evidence showing that transient transport complexes translocate through the NPC and the molecular interactions between the transport complexes and the FG-NUPs which mechanisms that underpin how the NPC functions are under line the NPC channel mediate passage of transport complexes through A.T. Major et al. / Biochimica et Biophysica Acta 1813 (2011) 1668–1688 1685 the NPC. Because individual FG-NUPs are required for translocation of and export in developing gonads that ultimately drives selection of transport complexes formed by specific importins [31,106–108], the male or female developmental program. regulated synthesis of NUPs could also change the import/export Many karyopherins have recently been linked with human cancers. efficiencies of each importin/cargo subset. While not the focus of this For example, XPO1 has been suggested to contribute to human ovarian review, there is evidence that at least some of the NUPs show cancer, as tumours of this type with the highest levels of XPO1 spatiotemporal regulation throughout developmental processes, correlate with poorpatientoutcomes[118].Toconsiderhow including spermatogenesis. The expression of RanBP2, also known as karyopherins may be involved with disease states of the testis, we Nup358, demonstrates this: its transcript was highest in the isolated examined another GEO dataset within the NCBI database (GDS2842, pachytene spermatocyte sample, with a peak around day 14 of the age derived from GEO series GSE8607) which contains data from normal series (Fig. 7B). Interestingly, there is a testis-specific transcript splice human testis in comparison with testicular seminomas at three stages variant of RanBP2 which encodes a truncated protein variant termed of progression [119] classified (pT1, pT2 and pT3) as proposed by the BS-63 [109,110]. This isoform has been identified within the NPC of “International Union Against Cancer” and the “American Joint spermatids and in the nuclear membrane of human sperm, suggesting Committee on Cancer” [119,120]. The pT1 samples correspond to it has specialised transport roles in the most mature germ cell types tumours that are exclusively within the testis tubule confines, while [109,110]. Similarly NUP50, while not restricted to the testis, has been pT2 and pT3 represent progressive levels of invasion [120]. Presented identified to arise from two splice variants in humans with different in Fig. 8, this GEO dataset revealed differences in karyopherin effects on nuclear transport [111]. Although many details remain to be transcripts between normal testicular tissue and testicular seminoma resolved, regulated synthesis of NUPs also appears to be important to subtypes. At a gross level, this would reflect the distinction between a nuclear transport outcomes, as their presence or absence dictates healthy testis, which is populated by all germ cell types and containing which importin-transport complexes have access or preferential a predominance of post-mitotic germ cells, and the diseased testis access to translocation across the NE. which has a highly restricted compliment of cell types. Overall, the nucleocytoplasmic transport system is highly dynamic, Kpna6, Ipo2 and RanBP9 were each represented by multiple with the transport of specific cargoes being affected at multiple levels: oligonucleotide probesets that did not display the same trend as the (1) at the cargo level, through sequestration or recognition of cargo single dataset presented here. This may reflect detection of transcript NLS(s)/NES(s) which can be modulated through phosphorylation, splicing variants or homologous transcripts. While some transport masking, or additional binding interactions [9,11,112], (2) at the level factors, such as Ipo1 or Xpo3 have relatively uniform transcript levels, of karyopherins through regulated expression of the importins/ others such as Kpna3, Kpna4, Kpna6, Ipo13 and RanBP9 appeared to be exportins which directly define the complement of cargoes that can broadly up- or down-regulated in all seminoma samples when enter/exit the nucleus via facilitated transport, and (3) at the NPC, via compared with normal tissues. All three classes of seminoma samples regulated expression of the NUPs, which can differentially modulate the transport efficiencies of import/export complexes. With so many means of regulating nuclear transport, it seems likely that even subtle changes may compound to impact in ways that could easily alter Table 2 fl Summary of most abundant transcripts encoding nuclear transport machinery during biological thresholds and have dramatic in uences on cell signalling spermatogenesis. and fate. This review has focused on the regulated expression of the fi karyopherins throughout spermatogenesis, because it is undoubtedly Implicated by Identi ed in expression profile experimental one of the most powerful links in the transport chain. An increase or data decrease in the expression of a single karyopherin in developing cells Sex Determination Kpna2 (Fig. 4 and 5A) Xpo1 [117] might quickly grant or restrict nuclear access for many factors (E11 - E12) Ipo7, Ipo11, Ipo12 (Fig. 5B) (cargoes) simultaneously. This would serve as an efficient way to Xpo3 (Fig. 5C) achieve large changes in gene expression in cells undergoing extensive RanBP1 (Fig. 5D) differentiation and remodelling, as occurs during spermatogenesis. Quiescence Ipo8 (Fig. 5B) (E14.5—day 0) Re-entry into cell cycle Ipo9 (Fig. 6B) (day 0–day 1) Xpo3 (Fig. 5C) 4.5. Fertility, health and disease Migration to cord perimeter Xpo3 (Fig. 5C) (day 0–day 2) Nuclear transport defects have been directly linked with failure to Spermatogonial mitosis Xpo1, Xpo2, Xpo7 (Figure 7A) Ipo1 [63] (day 6–day 8) differentiate normally during the early foetal gonad development. In Spermatocyte meiosis Kpna2, Kpna3, Kpna4 (Figure 6A) Kpna2 [61] humans, several mutations within the sex determining region on the (day 14–day 19) Ipo1, Ipo3, Ipo4, Ipo5, Ipo11, Ipo13 Y chromosome (SRY) that specifically cause defective NLS recognition (Figure 6B) Kpna4 [61] by importins and therefore a failure in SRY nuclear accumulation have Xpo1, Xpo2, Xpo6, Xpo7 (Figure 7A) Ipo1 [63] been identified; individuals with any of these mutations exhibit XY Ran, RanBP2, RanBP9, RanBP17, Ipo5 [79] Snupn (Figure 7B) Ipo13 [64] sex reversal [113,114]. SOX9 transcription factor up-regulation is one RanBP17 of the earliest and most important outcomes of SRY nuclear activity, as [100] ectopic SOX9 synthesis is sufficient to induce testis formation in Round spermatids/mRNA Kpna2, Kpna6 (Fig. 6A) Kpna2 [61] female (XX) mice [115]. Similar to SRY, defects in SOX9 nuclear storage and translational Ipo4, Ipo5, Ipo13 (Fig. 6B) Kpna6 [61] arrest (day 20–day 30) Xpo6 (Fig. 7A) Ipo4 [80] import can also cause sex reversal [116]. Embryonic gonad exposure Ran, Rcc1, RanGAP1, RanBP9, Ipo5 [63,79] to the XPO1-mediated nuclear export inhibitor, leptomycin B (LMB) RanBP17 (Fig. 7B) Ipo13 [64] around the time of sex determination provided evidence that nuclear Elongated spermatids/ Kpna2, Kpna6 (Fig. 6A) Ipo4 [80] export in general, including export of SOX9, is also important for spermiogenesis (day 30~) Ipo4 (Fig. 6B) correct gender development in females [117]. Ovaries treated with Xpo6, (Fig. 7A) Ipo5 [63] Ran, Rcc1, RanGAP1, RanBP9, LMB display SOX9 in the nucleus of germ cells and partial formation of RanBP17 (Fig. 7B) testis cord-like structures [117]. 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