Tesmin, Metallothionein-Like 5, is Required for Spermatogenesis in Mice†

Abstract In mammals, more than 2000 genes are specifically or abundantly expressed in testis, but gene knockout studies revealed several are not individually essential for male fertility. Tesmin (Metallothionein-like 5; Mtl5) was originally reported as a testis-specific transcript that encodes a member of the cysteine-rich motif containing metallothionein family. Later studies showed that Tesmin has two splicing variants and both are specifically expressed in male and female germ cells. Herein, we clarified that the long (Tesmin-L) and short (Tesmin-S) transcript forms start expressing from spermatogonia and the spermatocyte stage, respectively, in testis. Furthermore, while Tesmin-deficient female mice are fertile, male mice are infertile due to arrested spermatogenesis at the pachytene stage. We were able to rescue the infertility with a Tesmin-L transgene, where we concluded that TESMIN-L is critical for meiotic completion in spermatogenesis and indispensable for male fertility.


Introduction
Spermatogenesis is a complicated process involving mitotically dividing spermatogonial cells, spermatocytes dividing meiotically, and spermatid maturation into spermatozoa. While spermatogonial stem cells reside at the basement of the seminiferous tubules and continuously produce progeny cells to maintain the stem cell pool, some of the progeny cells start differentiation into primary spermatocyte (4C). The primary spermatocytes undergo meiosis I to form secondary spermatocytes (2C) and meiosis II to form spermatids (1C). The spermatids elongate their cilia and drastically change their morphology to form mature spermatozoa, which are released into the lumen of seminiferous tubules. Each of these events is supported by stage-and cell-specific gene expression.
In mammals, gene-expression analysis estimates that more than 2300 genes are expressed predominantly in the male germ line [1], and these testis-enriched genes are believed to play critical roles for male fertility. While knockout (KO) mouse studies have revealed essential genes for male fertility, recent CRISPR/Cas9-mediated KO studies suggested that many of the testis-enriched genes are not individually essential [2]. One can posit that paralogous genes may complement the testis-specific gene functions. However, it should be noted that many genes shown essential for male fertility are simultaneously expressed with their paralogues during spermatogenesis (e.g., Ccna2/Ccna1 [3], Hspa1a/Hspa2 [4]). Thus, the KO approach remains suitable to determine whether the target gene has an essential role in vivo.
Metallothioneins (MTs) belong to a group of low-molecularweight cysteine-rich proteins, which bind to heavy metal ions via their cysteine-rich (CXC) motif [5,6]. MTs fall into four subgroups (MT1-4), Mt1 and Mt2 are ubiquitously expressed in mice, Mt3 is expressed mostly in the brain, and Mt4 was detected in stratified squamous epithelial cells. All are located on a single chromosome (chromosome 8 in mice and chromosome 11 in human) [7]. Additionally, Tesmin was identified as a fifth member, which is strongly expressed in testis [8]. It was discovered that Tesmin encodes two variants with the longer variant having an additional 180 amino acids at the N-terminus [9]. We refer to the long form as TESMIN-L and the short form as TESMIN-S, respectively. Both TESMIN-variant proteins contain the CXC-domain like cysteinerich domains and a hinge region as described in previous reports [9,10]. Since TESMIN-L had been reported to change its localization in response to metal stress in vivo and in vitro [9,11], TESMIN proteins are assumed to chelate metal ions like other MTs. However, TESMIN-L and TESMIN-S have some differences compared with other members of the MT family. For example, TESMIN proteins are relatively larger than other family members: TESMIN-L consists of 475 amino acids and TESMIN-S consists of 295 amino acids, while MT1-4 consists of 61, 61, 68, and 62 amino acids, respectively. Moreover, Tesmin is located on chromosome 19 in mice, while the other Mt1-4 genes cluster on chromosome 8.
TESMIN-L has also been reported to translocate from the cytoplasm to the nucleus before meiotic division even without metal ion stress [9,11]. Furthermore, as described in a previous study, meiosis-stage-specific expression of Tesmin suggests a germ-cellspecific function independent of the metal ion stress response [12]. Despite these findings, it remains unclear whether TESMIN proteins play critical roles in meiosis and which isoform (TESMIN-L and TESMIN-S) works in testis. In the present study, we generated Tesmin KO mice and demonstrated its essential role in the completion of meiosis in male germ cells.

Animals
All animal experiments were conducted in accordance with the guidelines of "Animal experiment rules" established by the Research Institute for Microbial Diseases, Osaka University, and were approved by the Animal Care and Use Committee of the Research Institute for Microbial Diseases, Osaka University. B6D2F1 and ICR mice were purchased from CLEA (Tokyo, Japan) and SLC (Shizuoka, Japan). Mouse cDNA was prepared from various tissues of adult ICR,  and testes from 0 to 35 days after birth. RT-PCR was performed using 10 ng of cDNA with the following forward and  reverse primers: 5 -CCAGCAGGGCTAGGGATAGA-3 and 5 -CATGGGGCTGGAGTCTTTCTTCC-3 for Tesmin-S, 5 -ATG-GAGGACGCGCTGCTCGG-3 and 5 -CATGGGGCTGGAGT-CTTTCTTCC-3 forTesmin-L, 5 -AAGTGTGACGTTGACATCCG-3 and 5 -GATCCACATCTGCTGGAAGG-3 for the Actb gene. The amplification conditions were 1 min at 94 • C, followed by 40 cycles (for Tesmin-S) or 35 cycles (for Tesmin-L, Actb) of 94 • C for 30 s, 65 • C for 30 s, and 72 • C for 30 s, with a final 1-min extension at 72 • C.

Cell culture and transfection
Cos7 cells were maintained with Dulbeccos modified Eagles medium, 100 U/ml of penicillin, 0.1 mg/ml of streptomycin sulfate, and 10% fetal bovine serum under 5% CO 2 at 37 • C. For transfection, the cells were transfected at approximately 70% confluence with 1 μg of pCAG-Flag-Tesmin-S or pCAG-Flag-Tesmin-L using Lipofectamine LTX & PLUS technology (Life Technologies, Carlsbad, CA, USA) according to the manufacturer's instructions.

Immunofluorescence staining of Cos7
Transfected Cos7 cells were seeded onto coverslips in 6-well plates. After 2 days, cells were fixed with 2% paraformaldehyde in phosphate-buffered saline (PBS) for 10 min. After washes with PBS, cells were permeabilized with 0.2% Triton X-100 for 7 min and then blocked with 10% goat serum in PBS. Then, coverslips were incubated with anti-FLAG (1:200) antibody for overnight at 4 • C. After incubation with Alexa Fluor 488 conjugated secondary antibody (1:200) (Thermo Fisher Scientific, MA, USA) at room temperature for 1 h, cells were counterstained with Hoechst33342 (Life Technologies) and mounted on slides.

Guide RNA design and cloning
Tesmin-specific guide sequences (sgRNAs) were designed and inserted into the pX330 plasmid (#42230, Addgene, Cambridge, MA, USA). Validation of DNA cleavage activity of these plasmids was performed as described previously [15,16].

Assessment of the fertility of TESMIN deficient mice
Sexually mature male mice of genotypes Tesmin +/em1 and KO were caged with B6D2F1 female mice (at least 2 months old) for 3 months, and the number of pups in each cage was counted within a week of birth.

Immunoblotting
Protein extracts from whole testis were subjected to 10% polyacrylamide gel electrophoresis and blotted onto a polyvinylidene fluoride membrane. Membranes were blocked in 10% skim milk powder in tris-buffered saline with Tween20 and probed with primary (overnight at 4 • C) and secondary (1 h at RT) antibodies in blocking solution. Chemiluminescent signals were detected using ImageQuant LAS 4000 mini (GE Healthcare, Munich, Germany) after incubation of the membrane with ECL prime western blotting detection reagent (GE Healthcare).

Histological analysis
Testes were fixed in 4% paraformaldehyde in PBS and embedded in Technovit 8100 resin (Heraeus-Kulzer, Wehrheim, Germany) according to the manufacturer's instructions. Sections were cut at 5 μm and stained with periodic acid-Schiff (PAS) and then counterstained with Mayers hematoxylin solution (Wako, Japan).

Immunohistochemistry
The spreading procedure of testicular cells was carried out as described [17]. In brief, hypotonic treated cells were spread and fixed on slides with 1% paraformaldehyde and 0.1% Triton X-100. Spread samples were blocked with 10% goat serum in PBS and then incubated with anti-SYCP3 (1:500) and anti-phosphohistone H2A.X (1:500) antibodies overnight at 4 • C in blocking solution. After incubation with Alexa Fluor 488 or Alexa Fluor 546conjugated secondary antibody (1:250) at room temperature for 1 h, samples are counterstained with Hoechst33342, and mounted with Immu-Mount (Thermo Fisher Scientific).

Expression of Tesmin isoforms
Tesmin was originally identified as a gene encoding a testis-specific 30-kDa protein in mice [8] but later shown to have another splicing variant [9]. Because Tesmin-L and Tesmin-S transcripts utilize different starting exons but share common exons 2-9, we designed specific primers and performed RT-PCR with various adult tissues (Figure 1a). While the Tesmin-S signal was weak but specifically observed in testis, the Tesmin-L signal was strongly detected in testis with weak signals in spleen, liver, and kidney ( Figure 1b). As shown in Figure 1b, we further detected two bands of Tesmin-S by RT-PCR. Sequencing analysis revealed that the upper band includes an additional 69 bp outside the coding region ( Figure S1a). When we assessed the expression pattern of Tesmin variants during spermatogenesis (Figure 1c), Tesmin-L appears in a 6-day-old testis, which contains only spermatogonia and somatic cells, whereas Tesmin-S appears in a 18-day-old testis in which spermatocytes finish meiosis and haploid spermatids appear.
Next, to confirm the presence of TESMIN protein, we attempted to produce anti-TESMIN antibodies. One TESMIN-L specific antibody worked and detected a band near 50 kD, which is the expected size of TESMIN-L. Western blot analysis also detects TESMIN-L expression in a 14-day-old testis when differentiating germ cells enter the early pachytene stage of meiosis (Figure 1d). Since our anti-TESMIN-L antibody did not work for immunofluorescence in testis, we stained FLAG-tagged TESMIN-L and TESMIN-S transiently transfected into the Cos7 cell line with anti-FLAG antibody to know the localization of each variant ( Figure S1b). Interestingly, TESMIN-L localized to the cytoplasm but TESMIN-S localized in close proximity to the nucleus, suggesting different roles between TESMIN-L and TESMIN-S in cells.

CRISPR/Cas9-mediated generation of Tesmin KO mice
To analyze the physiological function of TESMIN proteins in vivo, we generated Tesmin KO mice using CRISPR/Cas9-mediated genome-editing in mouse zygotes. To disrupt both Tesmin-L and Tesmin-S genes, we designed a single guide RNA (sgRNA) against the common third exon (Figure 2a). We microinjected the sgRNA/Cas9 expressing plasmid into 105 zygotes and obtained eight newborn pups. By PCR amplification of the target region and subsequent sequence analysis, we confirmed three pups carrying mutations. While two of the three mice had indel mutations (+/i3 and +/i5d3), the third had a 316 bp-deletion (hereinafter, referred to as "em1" mutation) that includes the entire exon 3 (Figure 2b). Because exon 3 consists of 121 nucleotides, the lack of exon 3 results in a frameshift mutation in both Tesmin-L and Tesmin-S transcripts. We, therefore, bred this em1 mutant mouse line (B6D2-Mtl5<em1Osb>) to obtain Tesmin KO mice. Tesmin KO mice were born at the expected Mendelian ratio (+/+: 27, +/em1: 62, em1/em1: 29; n = 118), and no overt abnormality was observed in both females and males throughout their life. We confirmed the disappearance of TESMIN-L protein in the Tesmin KO mouse by western blotting (Figure 2c). Since an antibody against TESMIN-S was not available, we performed RT-PCR with Tesmin-S specific primers and confirmed the frameshift mutation (Figure 2d). These results indicate that both the TESMIN-L and TESMIN-S proteins are deleted in the Tesmin KO mice.

Fertility of Tesmin KO male mice
To evaluate the effect of TESMIN deficiency on fecundity, Tesmin KO female (n = 10) and male (n = 3) mice were mated with WT males or females for 3 months, respectively. Whereas the Tesmin KO female mice were fertile (litter size of +/em1: 7.9 ± 2.0, number of litters n = 15, litter size of em1/em1: 7.1 ± 2.7, number of litters n = 15), the Tesmin KO male mice were infertile despite showing normal mating behavior (litter size of 0, Figure 2e). To define the causes of male infertility, we measured testicular weights in Tesmin +/em1 and KO mice. Although the testicular weight of Tesmin KO mice was comparable with that of Tesmin +/em1 10-day-old mice, a significant decrease appeared in testis weight in Tesmin 14-day-old KO mice and remained smaller throughout their life (Figure 2f and g). These results indicated that the lack of TESMIN impairs testis function.  Percentage of spermatocytes from Tesmin +/em1 and KO male mice that are categorized as late zygotene, early pachytene and late pachytene by the feature of SYCP3 and γ H2AX staining. Three mice (over 5 weeks old) are used for each genotype and more than 50 nuclei were observed from one mouse. The numbers in bar graph indicate the ratio (%) of each category. P-values of each categories are; zygotene: 0.0373, early pachytene: 0.0033, and late pachytene/diplotene: 0.0002, respectively. Welch t test was used for statistical analysis.

Meiotic defect in Tesmin KO male mice
To define the cause of the smaller testis in Tesmin KO mice, we observed testicular sections stained with hematoxylin and PAS, which detect nuclei and carbohydrate chains, respectively (Figure 3a). There were no significant differences between Tesmin KO and +/em1 testis cross sections in 10-and 14-day-old testes, respectively (Figure 3a). Sections from 5-week-old Tesmin KO testis showed apparent abnormalities compared with that of Tesmin +/em1 mice. Instead of three layers seen in control sections, only two layers of germ cells were observed at the periphery at 5 weeks in KO seminiferous tubules with a few cells beyond the meiotic stage (Figures 3a and S2). These results indicated that spermatogenesis was predominately arrested at an early stage before the appearance of haploid spermatogenic cells. Due to an apparent defect in meiosis in the KO, we focused on the meiotic events and performed immunofluorescence staining of synaptonemal complex protein 3 (SYCP3) and γ -H2AX, a histone variant (Figure 3b). During meiotic prophase I, SYCP3 protein begins to assemble along each sister-chromatid pair at leptonema to form the axial elements, which appears as short filaments dispersed in the nucleus. Then, during zygotene, these filaments begin to associate in pairs to form thicker filaments, called the lateral elements of synaptonemal complex [18,19]. Meanwhile, γ -H2AX, which is a marker of the response to DNA double-strand breaks, localizes throughout the entire nucleus during leptonema and early zygonema when programmed double-strand breaks are being generated by the Spo11 endonuclease [19]. Thereafter, it accumulates in the chromatin of the XY body from late zygotene until diplotene [18]. We prepared meiotic nuclear spreads from heterozygous and KO males and quantified zygotene, pachytene, and diplotene spermatocytes based on SYCP3 and γ -H2AX staining patterns according to published reports [20,21]. In heterozygous, the major population (∼80%) of selected spermatocytes showed a γ -H2AX foci in the XY body, which is the typical feature of late-pachytene to diplotene spermatocytes (Figure 3b and c). This distribution of spermatocytes among different meiotic stages was consistent with that of WT mice in a previous report [22]. However, in the majority of Tesmin KO spermatocytes, the staining pattern of γ -H2AX was not restricted to the XY body in fully synapsed chromosomes, which is found in zygotene and early-pachytene spermatocytes. These data suggest that meiosis was arrested before late-pachytene stage in the absence of Tesmin.

Transgenic rescue of Tesmin KO mice
To exclude the possibility that the infertility phenotype was caused by an off-target effect from CRISPR/Cas9 cleavage, or an aberrant genetic modification near the Tesmin locus, we performed a rescue experiment by generating transgenic mouse lines expressing Tesmin-L driven by the CAG promoter on a Tesmin KO background (Figure 4a). CAG promoter is a strong ubiquitous promoter frequently used to drive high levels of gene expression [13]. From pronuclear injection of 88 zygotes, we generated five founder lines. Western blotting analysis confirmed the expression of the transgene in these mice (Figure 4b). In Tesmin KO mice carrying the Tesmin-L transgene, the testicular weight (99.3 ± 9.8 mg) and spermatogenesis were comparable with the control Tesmin +/em1 mice (Figure 4c and d). In addition, the infertile phenotype was rescued (litter size of 7.8 ± 2.5, number of litters n = 21). These data that indicate infertility in Tesmin KO mice are due to the absence of TESMIN protein.

Discussion
The study of MTs stretches back decades when they were first identified in the late 1950s [23]. MTs belong to the group of intracellular cysteine-rich, metal-binding proteins that have been found in bacteria, plants, invertebrates, and vertebrates [24]. Mammalian MTs are involved in diverse intracellular functions, with their role in detoxification of heavy metals and maintenance of essential metal ion homeostasis having been mostly investigated. However, TESMIN, which was identified as a MT-like protein (also known as Mtl5) and abundantly expressed in testes had not been studied well. In our experiments using KO mice, we demonstrated that TESMIN is an essential protein for meiotic progression in male germ cells.
Previous studies revealed two Tesmin splicing variants, named Tesmin-S and Tesmin-L in this paper [8,9]. In this study, we confirmed that Tesmin-S and Tesmin-L encoding mRNAs that begin expression before and after meiosis, respectively and their localization in Cos7 cells were completely different. These results are consistent with the previous report, which claimed that TESMIN localizes in the cytoplasm of spermatocytes and then begins to localize on the nuclear membrane just under the acrosome vesicle of elongated spermatids [9]. It allows us to speculate that the splicing variants of Tesmin may have distinct roles in spermatogenesis. However, we demonstrated that the Tesmin-L transgene alone could rescue the meiotic defect and male fertility in Tesmin KO mice, suggesting that TESMIN-S is not essential for spermatogenesis.
A previous report showed that TESMIN-L localizes to the cytoplasm until the pachytene stage of meiosis then moves into the nucleus just before meiotic division [9,11]. Our immunofluorescence staining of FLAG-TESMIN-L in Cos7 cells showed TESMIN-L localization in the cytoplasm, which is consistent with the first localization in testis. From immunofluorescence staining of meiotic markers SYCP3 and γ -H2AX in meiotic nuclear spreads, we found spermatogenesis arrests at the early pachytene stage in the majority of spermatocytes in Tesmin KO testis. The timing of the arrest in spermatogenesis corresponds to the change of Tesmin-L localization from the cytoplasm to nucleus, where TESMIN-L is thought to play an important role for meiosis in cytoplasm, and then moves into nucleus.
Previously, a similar phenotype, spermatogenesis arrest at the early pachytene stage with abnormal localization of γ -H2AX, has been reported in several mice lines deficient in proteins related to chromosome-structure during prophase I [25][26][27]. Based on these reports, TESMIN-L may be involved in such specific events in the nucleus at meiosis-I, but it could be indirect because of its cytoplasmic localization. Moreover, although a great majority of Tesmin KO spermatocytes are arrested at the early pachytene stage, a small fraction of cells complete meiosis and cease development at the metaphase/anaphase or haploid stage ( Figure S2). To address these questions, more precise assessment of the arrested stages by immunofluorescence staining with other meiotic markers such as RAD51 and SYCP1 would be beneficial.
Tesmin was originally identified as an MT family-like gene because of its metal-binding, cysteine-rich domain, and early studies showed that metal ion-treated mice translocated TESMIN from the cytoplasm to the nucleus during meiosis [8,11]. These results indicate that TESMIN responds to metal ions, like other MT genes; however, there are some differences between canonical MTs and TESMIN, as discussed previously [9]. TESMIN is ten times larger than other MT family proteins, and its cysteine composition is only 7.8% compared to 30% in other MT proteins ( Figure S3). This suggests that the non-CXC regions of TESMIN might have different roles from other MTs. Furthermore, the metal-responsive element (MRE), a DNA sequence located upstream of MT family genes that are activated by the binding of the metal-responsive transcription factor (MTF-1), is not present upstream of Tesmin [9]. MTF-1 is known to be translocated from the cytoplasm into the nucleus upon heavy metal exposure or stress stimuli and bind to the MRE [28,29]. The translocation of TESMIN in a similar manner to MTF-1 upon metal exposure suggests it may have a similar function during meiosis. Notably, with the absence of an MRE, Tesmin expression would be regulated by meiosis-specific transcription factors rather than in a metal-dependent manner.
The CXC in TESMIN are also present in LIN54, which is an essential cell cycle regulator. The CXC domains form a larger domain within LIN54 called the CXC-hinge-CXC (CHC) domain that is conserved in many LIN54 orthologs in various species, such as plants, invertebrates, and mammals [30][31][32]. Previous reports showed that LIN54 binds to the promoters of mitotic genes, such as cdc2 through the CHC domain and that LIN54 depletion through RNAi arrested the cell cycle [33,34]. In addition, LIN54 has been assumed to change its subcellular localization in a cell-cycle-dependent manner [35]. Moreover, in Drosophila, mutation of a member of the CXCdomain family, tombola (tomb), which is expressed specifically in testis, fails to express Cyclin B and shows meiotic arrest in the male germline [31]. These reports allowed us to anticipate that TESMIN might have a similar function to LIN54 and tomb such as cell-cycle regulation in the nucleus at the end of meiosis during spermatogenesis.
Here, we demonstrated that TESMIN is essential for the completion of meiosis in male germ cells. Further studies on TESMIN will elucidate the mechanism of meiosis in male germ cells and shed light on the study of male infertility. We would expect that Tesmin KO mice would provide a clue to elucidate how TESMIN relates to meiosis in future research.

Supplementary data
Supplementary data is available at BIOLRE online.