MC4R mutant mice develop ovarian teratomas

Teratomas in mice, composed of different tissue types, are derived from primordial germ cells (PGCs) in the foetal gonads. The strongest candidate gene in the testicular teratoma locus (Ter) responsible for testicular teratoma formation was identified as mutation in Dnd1, Dnd1R178*. However, the phenotype of mice with a mutated Dnd1 gene was germ cell loss. This suggests that other genes are involved in teratoma formation. Testicular teratomas can also be induced experimentally (experimentally testicular teratomas: ETTs) in 129/Sv mice by transplanting E12.5 foetal testes into adult testes. Previously, we mapped the ett1 locus, which is the locus responsible for ETT formation on chromosome 18. By exome sequence analysis of the 129 and LTXBJ (LT) strains, we identified a missense mutation in the melanocortin 4 receptor (MC4R) gene among 8 genes in the ett1 region. The missense mutation causes a substitution of glycine 25 by serine. Thus, this gene is a candidate for ETT formation. We established the LT-ett1 congenic strain, which introduced the locus responsible for ETT formation genetically into the genomes of a testicular teratoma non-susceptible strain. In this study, we crossed LT-ett1 and a previously established LT-Ter strain to establish the double congenic strain LT-Ter-ett1. Also, we established a strain with a point mutation in the MC4R gene of the LT strain by genome editing, LT-MC4RG25S. Furthermore, double genetically modified strain LT-Ter-MC4RG25S was established to address the relation between Ter and MC4R. Surprisingly, highly developed ovarian teratomas (OTs), instead of testicular teratomas, appeared not only in the LT-Ter-MC4RG25S and LT-MC4RG25S strains but also in the LT-ett1 and LT-Ter-ett1 strains. The incidence of OT formation was high in double genetically modified strains. The results demonstrated that MC4R is one of the genes responsible for OT formation. It was suggested that the effect of the missense mutation in MC4R on teratoma formation was promoted by abnormal germ cell formation by the mutation in DND1.

Ovarian teratomas (OTs) develop from female germ cells 12,13 . Contrary to STTs, the molecular mechanisms of OTs are largely unknown. An OT response region was reported on chromosome 6, named Ots1, but responsible genes have not yet been identified 14 . However, recent molecular genetic approaches have demonstrated that OTs formation can be induced by various manipulations of genes regulating the activities of follicular cells and OPEN 1 Integrated Bioscience Section, Graduate School of Science and Technology, National University Corporation Shizuoka University, Ohya 836, Suruga-ku, Shizuoka 422-8529, Japan. 2 15 . Follicular cells and oocytes are under the control of many factors, and changes in the quality and quantity of these factors affect the regulation of meiotic arrest and the apoptosis of oocytes or the activity of follicular cells. From the series of results, it is expected that mutations in many of these regulatory genes could cause OTs. Previously, we conducted linkage analysis using F2 inter-crossed foetuses derived from the F1 of mice susceptible to ETTs, which include 129-Ter LT/LT mice and those not susceptible to ETTs, LT mouse hybrids. Fine mapping on chromosomes 18 and 19 of the locus associated with generating ETTs demonstrated the locus responsible for ETT formation as "experimental testicular teratoma 1 (ett1)" on chromosome 18 16 .
Also we established the LT-ett1 congenic strain, the ett1 region of the LT is replaced with the corresponding region of the 129 strain, whereas all other chromosomes are derived from the LT strain. It is confirmed by transplantation experiments using congenic males homozygous for the ett1 loci that ett1 locus contains the gene responsible for ETTs. Eight genes are found in the ett1 locus which span 1.1 Mb (66.3-67.4 Mb) on chromosome 18. By exome sequencing analysis, we identified a SNP that introduces an amino acid substitution in melanocortin 4 receptor (MC4R) among these 8 genes 17 .
In this study, we established a gene-edited strain, LT-MC4R G25S by using a gene transfer method by electroporation (the TAKE method) 18,19 . To introduce single-nucleotide exchange (G25S) into the ett1 candidate MC4R of the LT strain, we designed a set of guide RNAs for CRISPR/Cas9 and a single-stranded oligodeoxynucleotides (ssODN).
As a result, we succeeded in establishing the desired G25S knock-in mouse, LT-MC4R G25S , in addition to various other mutations in MC4R. To explore the roles of MC4R G25S in ETT and STT formation, we established LT-Ter-MC4R G25S by crossing the LT-MC4R G25S and LT-Ter strains. We also established LT-Ter-ett1 by crossing the LT-ett1 and LT-Ter strains. Surprisingly, highly developed ovarian teratomas (OTs) were formed in the females of these strains. The results demonstrated that MC4R is one of the genes responsible for ovarian teratoma formation.

Results
Genome editing to introduce the identified single-nucleotide substitution in MC4R of the LT strain to the 129 strain was performed by using the CRISPR/Cas9 system. Two gRNAs (gRNA1, gRNA2) flanking the target site were selected by CRISPRdirect (https ://crisp r.dbcls .jp). A ssODN was designed to have a 40 bp homologous sequence at each end of the target site (Fig. 1A).
TAKE (Technique for Animal Knockout system by Electroporation) was used as the introduction method 18,19 . This method does not require the injection of a reagent into a fertilized egg and enables easy genome editing.
We crossed LT-MC4R G25S/G25S and LT-Ter mice to address whether the substitution of amino acids in MC4R resulted in the formation of STTs. However, we found that some LT-Ter-MC4R G25S/G25S females had swollen abdomens at approximately 3 months old or older. We found highly developed OTs after dissection in LT-Ter LT/129 -MC4R G25S/G25S mice ( Fig. 2A-C). These results suggested that Dnd1R178* mutation in Ter region and MC4RG25S mutation cooperatively induce OTs.
We also crossed LT-Ter and LT-ett1 mice to establish an LT-Ter-ett1 double congenic strain to address genetic changes in the ett1 region in combination with Ter mutation, resulting in the formation of STTs.
After establishing both the LT-Ter-MC4R G25S and the LT-Ter-ett1 strains, we started to check the ovaries and testes of all the strains for the formation of OTs and STTs. Morphologies of teratomas were photographed and are shown in Supplementary Figs. S2 and S3. The incidents of ovarian teratoma in each genotype are summarized in Fig. 4.
LT-MC4R G25S/G25S and LT-ett1 129/129 homozygous mutant mice developed OTs, but heterozygous MC4R LT/G25S and ett1 LT/129 mice did not. Although only two survivors of LT-Ter 129/129 -MC4R G25S/G25S were obtained, OT was found in both of them (100%). No double-congenic mouse, LT-Ter 129/129 -ett1 129/129 , was obtained. The incidence of OTs was significantly higher in LT-ett1 129/129 mice (75%) than in LT-MC4R G25S/G25S mice (48.7%). The result suggests that the ett1 region contains genes other than MC4R that contribute to OT formation. The incidence of OTs was significantly increased to 66.7% when the Ter region was introduced in heterozygous to MC4R G25S/ G25S mice. In the case of LT-ett1 129/129 , the incidence of OTs was not changed by the introduction of Ter but was lowered in LT-Ter LT*/ LT* -ett1 129/129 mice. Also the incidence of OT formation was decreased in LT-Ter LT*/ LT* -MC4R G25S/G25S mice compared with LT-MC4R G25S/G25S mice. As indicated in Supplementary Table S1, the small region of chromosome 18 in 129 strain identified by the D18Mit84 marker was introduced into LT. Thus, a small fragment of the 129 strain was inserted in LT-Ter LT*/LT* . There is a possibility that other genes that prevent    20,21 . Thus, we checked for this by observing sections prepared from mice that were expected to start to produce teratomas. We prepared sections of ovaries from one-month-old mice. As expected, oocytes divided into 2 cells in the small follicle were found in LT-MC4R G25S/G25S mice (Fig. 5). One to three 2 cells found in whole ovaries before forming OT (1.6% of primary follicles in three ovaries from three different females, 2 ± 0.6 2 cells/ 127.7 ± 7.5 primary follicles).
It is suggested that these 2 cells are produced by the parthenogenesis of the oocyte. It was demonstrated by immunohistochemical analysis that MC4R proteins were expressed in oocytes of LT-MC4R G25S/G25S , but not in oocytes of LT-MC4R +2/+2 mice (Fig. 6).

Discussion
We introduced the Ter gene, which is one of the causative genes for testicular teratoma formation found in 129 strains that develop STTs and ETTs, into LT strains that do not develop STTs and ETTs. A single-nucleotide substitution was introduced into the MC4R gene to introduce the same amino acid substitution as in the 129 strain. The introduction of the Ter gene resulted in a germ-cell-loss phenotype in both males and females, as in the case of the Ter-gene-introduced lines produced from other lines 9 . Then, it was revealed that the Ter gene product DND1 is essential for germ cell formation and that other genes are also necessary for the onset of teratomas. We mapped the ett1 region, which is a candidate region for ETTs, and found a SNP in the MC4R gene that causes an amino acid change between the 129 strains and the LT strains among the 8 genes existing in the ett1 region 17 . Thus, the single amino acid change in the MC4R gene is supposed to be a candidate for ett1. Then, to identify the ett1 gene, a knock-in strain, LT-MC4R G25S/G25S , in which this single base substitution was introduced into the LT strain, was created by genome editing. By crossing this knock-in line with the LT-Ter line, we tried to investigate the possibility of developing STTs or ETTs due to the combined effect of Ter and MC4R G25S . Surprisingly, highly developed OTs developed at an extremely high rate (48.7%). The original LT strain was established as a susceptible strain for OTs, but at the end of 30 years of passage, the current strain has changed so that OTs do not develop at all. At least one and a half years after starting the establishment of the double-gene-modified strain of LT-Ter-MC4R G25S mice, no case of OTs was found in the original strain LT during the observations over four generations. Thus, we introduced two candidate teratoma-causing genes into a line that does not develop OTs and analysed their combined effects. From many years of research so far, the mechanism of ovarian teratoma development is that some of the germ cells begin parthenogenesis for some reason in the process of germ cell apoptosis 13 . Our results suggest that MC4R is involved in the regulation of germ cell apoptosis and that changes in the MC4R-mediated signal transduction system cause germ cell parthenogenesis.
The MC4R gene is involved in energy intake and energy expenditure 22 . MC4R coupled with stimulating G protein (Gs) promotes protein kinase A (PKA) activation and induces cAMP production. The signal transduction pathway through MC4R suppresses c-Jun N-terminal kinase (JNK) 23 . From the results of MC4R gene targeting, it is well known that MC4R-gene-deficient mice become obese, and the MC4R gene is deeply involved in energy control 24 . Melanocyte-stimulating hormone (MSH), agouti and agouti-related protein (AGRP) are known as   25 . 129-Sv-Ay (Ay/+) male mice are known to have a tenfold lower rate of testicular teratoma formation than wild-type mice 26 . Ay is a dominant mutation in the agouti gene and causes ectopic expression of agouti protein and the inhibition of MC1R and MC4R chronic signal transduction systems [27][28][29] . As an agonist of MC4R, agouti-related (AGRP) has been found to be a related gene of agouti, and it has been reported that AGRP has a 100-fold higher binding affinity to MC4R than agouti 30 . According to the docking model of MC4R and its ligand, the binding of AGRP and MC4R involves the N-termini of MC4R K33, Y35 and D37, and the binding of MSH and MC4R does not involve the N-terminus of MC4R 31 . From this, it can be speculated that the ligand of MC4R in the development of ovarian teratomas in this double-gene-modified line is AGRP. Additionally, the G25S missense mutation of MC4R inhibits the binding to AGRP, which suppresses the action of the MC4R antiapoptotic signal. As indicated in this study, MC4R is expressed in mouse oocytes. It is hypothesized that the modification of the antiapoptotic pathway of oocytes is relatively enhanced to increase the rate of parthenogenesis and cause ovarian teratoma formation. Detailed analysis of the function of DND1 identified as the Ter gene is underway. Yamaji et al. indicated that DND1-dependent mRNA destabilization is required for the survival of mouse PGCs and spermatogonial stem cells by suppressing apoptosis 32 . By contrast, there is a report that showed that DND1 protects and maintains germ cell fate 33 . DND1-deficient germ cells transdifferentiate into somatic cells. The translation of nanos1, The OT-related locus was reported previously; however, a mutated gene in the Ots1 locus has not yet been identified 14 . However, the genes suggested to be responsible for OTs were found with gene knockout approaches. As a result of the generation of many genetically modified mice so far, it has been reported that ovarian teratomas also develop by different mechanisms. The onset of OTs due to parthenogenesis has been reported in mice in which the Mos gene has been knocked out, which has the function of meiotic arrest 21 . Yang et al. showed that conditional knockout mice of retinoblastoma protein 1 (Rb1) developed ovarian teratomas and that abnormalities of somatic follicular cells also cause teratoma formation 15 . Similarly, the overexpression of Bcl-2, constitutive active mutation in the FSH receptor, a missense mutation in Foxo3a and the Tgkd transgene insertion mutation in the Inpp4b gene are other examples of teratoma formation caused by somatic abnormalities and the genetic background of mouse strains [37][38][39][40] . Although the mechanism of teratoma formation is unknown, global knockdown of the Gata4 gene by siRNA also induced the formation of OTs 41 . In this way, it has become clear that there is an onset mechanism caused by germ-cell-derived teratomas and somatic cells. Many gene mutations can cause teratomas because the formation and divisional control of germ cells are performed under complicated control of the follicular cells covering them and growth factors in the ovary. To elucidate the whole picture of the mechanism of teratoma formation, it is necessary to analyse it at the whole-genome level.
The LT-Ter-MC4R G25S double-gene-modified line we established was used to elucidate the cause of testicular teratomas. Transplantation studies are currently underway to determine whether MC4R amino acid substitution is responsible for ETTs. Additionally, it is currently unknown whether STTs will develop in the double-genemodified strain. These results will be published in the future.

Methods
Mice. All animals were housed under temperature-controlled conditions and had free access to food and water. All animal procedures were performed per the protocols approved by the Institutional Animal Care and Use Committee of Shizuoka University (29A-8, 2018A-19, 2019A-8, 2020A-7). All experiments were performed in accordance with the guideline of the committee. The study was carried out in compliance with the ARRIVE guidelines 42 .
The 129/Sv-Ter and LTXBJ mice 12 , generous gifts from Dr Leroy Stevens (The Jackson Laboratory, Bar Harbor, ME, USA), were bred in the animal facilities at Shizuoka University. The LT males exhibited normal  Table S1). *, **, *** indicate significant differences between the value of LT at the P < 0.05, < 0.001, < 0.0001, respectively.  13 . The LT strain used as the original strain was established as a strain susceptible to ovarian teratomas, but at the end of 30 years of passage, the current strain has changed so that OTs do not develop at all. At least one and a half years  www.nature.com/scientificreports/ after the establishment of the double-gene-modified strain of LT-Ter-MC4R G25S , no OT has been found in the original strain LT during the observations over four generations. The LT-ett1 congenic strain was established previously and maintained 16 . The LT-ett1 strain also exhibited normal spermatogenesis and did not generate STTs but formed ETTs when foetal testes were transplanted into adult testes. In the LT-ett1 strain, the genome region including the ett1 locus (which exists at 62.0-70.2 M bp and includes peak markers D18Mit81 and D18Mit184) and a region identified by D18Mit84 (33.8 M bp) were substituted with the region from 129-Sv-Ter mice 17 .
LT-Ter 129/129 exhibited the germ-cell-loss phenotype in both heterozygous and homozygous mutants. In the LT-Ter 129/129 strain, the genome region including the Ter locus (which exists at 33.8-46.4 Mbp and includes the Dnd1 gene) was substituted with the 129/Sv-Ter region 43 .
The LT-Ter-ett1 strain was established by crossing these two strains and then back-crossing at least 5 more times with LT-ett1 129/129 .
For the sampling of specimens, the mice were sacrificed by cervical dislocation. All efforts were made to minimize animal suffering.
Creation of genome-edited mice. Using the TAKE method based on CRISPR/Cas9 system, a change in a single nucleotide was introduced into the LT strain by using a ssODN 18,19 . As gRNA, two gRNAs sandwiching the target site were selected by CRISPRdirect (https ://crisp r.dbcls .jp) (Fig. 1). An 81-bp ssODN with a 40-bp homologous sequence covering the target region was designed. The synthesis of an ssODN was requested from STAR Oligo (RIKAKEN, Nagoya, Japan). PAGE purification was chosen as the grade of synthesis. Genomeedited mice that could be identified by sequence analysis were back-crossed to LT strain mice. The F1 offspring obtained were also genotyped by sequence analysis and then transferred to sibling mating between the same genotypes.
Genotyping. Genotyping by PCR-Simple sequence length polymorphism (SSLP) and DNA sequencing were done using genomic DNA extracted from the ear of each mouse. MC4R target site was amplified by PCR using a primer set (F: 5′-CCG AAC CCA GAA GAG ACC AA, R: 5 '-GAC CCA TTC GAA ACG CTC AC). DNA sequencing was outsourced to Fasmac Co., Ltd. The DNA sequence analysis was performed using Codon Code Aligner (http://www.codon code.com/align er/) and GENETX-MAC (Ver.14.0.3).
Observation of ovarian teratomas. The morphology of the ovaries of each mouse after three months of age was observed and photographed after dissection. Whether the teratoma was developed on both sides or on which side when developed on only one side was recorded. The weight of the ovaries was measured.
Histological analysis. Tissues were dissected and fixed in Bouin's solution, embedded in paraffin, and serially sectioned at 6-μm thickness. Deparaffinized sections were stained with haematoxylin, eosin and alcian blue (H-E-A) and screened for the presence of OTs under a light microscope.

Statistical analysis.
OT formed mice and none-formed mice of each strain were binarized and the teratoma incidence of each strain was analyzed by one-way analysis of variance (ANOVA) (Tukey's multiple comparisons test). ANOVA was performed using GraphPad Prism (San Diego, CA).