Mouse dead end1 acts with Nanos2 and Nanos3 to regulate testicular teratoma incidence

Atsuki Imai; Yoshihiko Hagiwara; Yuki Niimi; Toshinobu Tokumoto; Yumiko Saga; Atsushi Suzuki

doi:10.1371/journal.pone.0232047

. 2020 Apr 27;15(4):e0232047. doi: 10.1371/journal.pone.0232047

Mouse dead end1 acts with Nanos2 and Nanos3 to regulate testicular teratoma incidence

Atsuki Imai ¹, Yoshihiko Hagiwara ¹, Yuki Niimi ^1,^¤, Toshinobu Tokumoto ², Yumiko Saga ³, Atsushi Suzuki ^1,^4,^*

Editor: Stefan Schlatt⁵

PMCID: PMC7185693 PMID: 32339196

Abstract

Spontaneous testicular teratomas (STTs) derived from primordial germ cells (PGCs) in the mouse embryonic testes predominantly develop in the 129 family inbred strain. Ter (spontaneous mutation) is a single nucleotide polymorphism that generates a premature stop codon of Dead end1 (Dnd1) and increases the incidence of STTs in the 129 genetic background. We previously found that DND1 interacts with NANOS2 or NANOS3 and that these complexes play a vital role in male embryonic germ cells and adult spermatogonia. However, the following are unclear: (a) whether DND1 works with NANOS2 or NANOS3 to regulate teratoma incidence, and (b) whether Ter simply causes Dnd1 loss or produces a short mutant DND1 protein. In the current study, we newly established a conventional Dnd1-knockout mouse line and found that these mice showed phenotypes similar to those of Ter mutant mice in spermatogenesis, oogenesis, and teratoma incidence, with a slight difference in spermiogenesis. In addition, we found that the amount of DND1 in Dnd1^+/Ter embryos decreased to half of that in wild-type embryos, while the expression of the short mutant DND1 was not detected. We also found that double mutants for Dnd1 and Nanos2 or Nanos3 showed synergistic increase in the incidence of STTs. These data support the idea that Ter causes Dnd1 loss, leading to an increase in STT incidence, and that DND1 acts with NANOS2 and NANOS3 to regulate the development of teratoma from PGCs in the 129 genetic background. Thus, our results clarify the role of Dnd1 in the development of STTs and provide a novel insight into its pathogenic mechanism.

Introduction

Testicular teratomas are tumors that originate from germ cells. A diverse array of cell and tissue types are found differentiating in these tumors: erythrocyte, adipocyte, cartilage, muscle, hair, and glandular tissue, as well as a cluster of stem-like cells from which tumors can be propagated. In mice, spontaneous testicular teratomas (STTs) rarely develop. They predominantly occur in the 129 family of inbred mouse strains; the frequency is 1–7%, depending on the subline [1–3]. In these cases, some primordial germ cells (PGCs) transform to highly proliferative and pluripotent tumor stem cells (embryonal carcinoma [EC] cells) in the embryonic testes at around embryonic day (E) 15.5. Soon after birth, EC cells randomly differentiate into embryonic and adult cells that constitute the tumor in the testes.

In 1973, a spontaneous mutation called Ter was isolated, which increased teratoma incidence to 17% in heterozygotes and 94% in homozygous mutants but did not induce ovarian teratomas in the 129/Sv genetic background [4, 5]. In homozygous for Ter mutant embryos, the number of PGCs drastically decreases during migration and gonad colonization, partly owing to Bax-mediated apoptosis [5–7]. Some of the remaining PGCs in the embryonic testes are thought to give rise to teratomas in the 129/Sv strain. However, in most genetic backgrounds, such as C57BL/6J, LTXBJ, and C3H/HeJ, PGCs disappear until birth and never transform to EC cells, resulting in complete male sterility [8].

In 2005, Ter was mapped to Dead end1 (Dnd1) [9], a mouse homologue of the zebrafish Dead end gene essential for PGC development [10]. Dead end encodes a vertebrate-specific RNA-binding protein possessing two RNA-recognition motifs (RRMs), among which the second RRM does not conserve three aromatic amino acids playing a key role in nucleic acid–binding activity [11, 12]. In the Ter mutation, a single cytosine in the third exon of Dnd1 is changed to thymine, which generates a premature stop codon (S1A and S1C Fig), presumably resulting in a null mutation of Dnd1 by nonsense-mediated mRNA decay [13]. Therefore, the defects observed in Ter mutant mice have been thought to be attributable to loss of Dnd1 expression. We have previously shown that DND1 functions with NANOS2 or NANOS3 in both male embryonic germ cells and adult spermatogonia [14, 15], which raises the question of whether DND1 also acts as a partner of NANOS family proteins to regulate the incidence of testicular teratoma. In addition, it was recently reported that a targeted deletion of Dnd1 did not affect teratoma incidence in the 129S1/SvImJ genetic background; rather, it induced embryonic lethality before E 3.5 [16] (S1B Fig). Thus, these phenotypic differences raised the possibility that the Ter mutation generates a short mutant DND1 protein responsible for the phenotype in mutant mice.

In the current study, we first aimed to elucidate the effect of Dnd1 loss and then to clarify the difference between Dnd1 loss and Ter mutation by comparing our results with previous findings for Ter mutant mice. For this purpose, we newly established a conventional knockout mouse line of Dnd1 and subsequently backcrossed the mutant mice into three different mouse strains: C57BL/6J, MCH(ICR), or 129^+Ter/Sv (hereafter referred to as BL6, MCH, and 129, respectively). We then examined embryonic lethality, spermatogenesis, oogenesis, and incidence of testicular teratoma for the Dnd1 mutant mice in these three mouse strains, especially focusing on the 129 strain, and found phenotypes similar to those of Ter mutant mice. Additionally, we examined genetic interactions between Dnd1 and Nanos2 or Nanos3 in the regulation of STT incidence and showed that double mutants for Dnd1 and Nanos2 or Nanos3 showed increased incidence of STTs in the 129 genetic background.

Materials and methods

Ethics statement

The protocol was approved by the Animal Experiment Committee at Yokohama National University (Project Number: 2019–07; approved on 20 May).

Mice

We had previously established a Dnd1_flox mouse line by using TT2 ES cells and maintained it via intercrosses generating Dnd1^flox/flox mice. To establish the Dnd1 conventional knockout mouse line, we crossed these Dnd1^flox/flox mice with Rosa26^+/CreERT2 mice and obtained Dnd1^+/flox; Rosa26^+/CreERT2 female mice. After administering 75 mg per kg body weight of tamoxifen, we crossed these female mice with wild-type male mice and obtained Dnd1^+/Δ offspring. The Dnd1^+/Δ mice were backcrossed into the BL6, 129, or MCH strains for at least eight generations. All three mouse strains were purchased from CLEA Japan (CLEA Japan, Inc., Japan). The 129^+Ter/SvJcl strain had been established from an STT-high permissive 129/terSv strain [4] by Dr. Noguchi [5] and provided to and maintained at CLEA Japan, whereas the Dnd1^+/Ter mice had been bred in the animal facility at Shizuoka University by Drs. Tokumoto and Noguchi. Genotyping of Dnd1-flox, Δ, and Ter alleles was performed as described previously [9, 14]. The Nanos2^+/LacZ and Nanos3^+/Cre mouse lines were established as previously described [17] and backcrossed into the 129^+Ter/SvJcl strain for at least eight generations.

Histological methods

For histological analysis, the testes and ovaries were fixed with Bouin’s solution and embedded in paraffin. After sectioning (6 μm), the samples were stained with hematoxylin and eosin.

Tumor surveys

Four-week-old male mice were surveyed for testicular teratomas. Teratoma incidence was calculated as the percentage of male mice with at least one testicular teratoma. Histological analysis (hematoxylin and eosin staining) was used to confirm any teratomas that were ambiguous at autopsy.

Sperm count

Mature spermatozoa were isolated from the caudal epididymis, as described previously [18]. Briefly, both epididymides from each mouse were minced and incubated in 1 mL warm phosphate-buffered saline (PBS) for 30 min, following which the sperm suspension was fixed in 10% neutral-buffered formalin. The sperm were counted using a hemocytometer. Data have been shown in terms of mean ± standard error of mean (SEM), and the values were statistically analyzed using a Student’s t-test.

In vitro fertilization (IVF)

Three-week-old female B6C3F1 mice were purchased from CLEA Japan and superovulated using intraperitoneal injections of 100 μL of CARD HyperOva (Kyudo Co., Ltd., Japan) followed by intraperitoneal injection of 100 μL of 50 units/mL human chorionic gonadotropin (hCG; Aska Pharmaceutical Co., Ltd., Japan) 48 h later. Eggs were recovered 16 to 17 h after hCG injection and placed in a 200 μL CARD MEDIUM (Kyudo Co., Ltd.) drop covered with liquid paraffin (Nacalai Tesque, Inc., Japan) and then used for the experiments 0.5–1 h after preparation. The spermatozoa were collected from the cauda epididymis of more than 12-week-old wild-type or Dnd1^+/Δ male mice of the 129 strain, suspended in 100 μL CARD FERTIUP (Kyudo Co., Ltd.), and incubated for 1 h for capacitation. Capacitated sperm (3 μL) were added to the drop containing eggs. The eggs were washed in an 80 μL mHTF (Kyudo Co., Ltd.) drop three times after 3 h of coincubation and were transferred to 100 μL KSOM (Kyudo Co., Ltd.) 6 h after insemination. The blastocysts were counted 4 days later.

Comparison of the PGC number in male gonads at E11.5

Embryonic stage was determined as E11.5 on the basis of the number of somites, while the sex of the embryos was determined by polymerase chain reaction (PCR) for the Sry gene using the following primer pairs: Sry-F: 5′-ggttgcaatcataattcttcc-3′ and Sry-R: 5′-cactcctctgtgacactttag-3′.

For section immunostaining, embryonic gonads were fixed with 4% paraformaldehyde overnight at 4°C and then embedded in paraffin. Whole gonads were sectioned (6 μm) and autoclaved with Antigen Unmasking Solution (Vector Laboratories, Inc., USA). After the samples were subjected to blocking with 5% skim milk in PBS, they were incubated overnight at 4°C with primary antibodies against deleted in azoospermia-like (DAZL; 1:1,800) [14] and NANOG (1:5,000; IHC-00205, Bethyl Laboratories, Inc., USA) in Can Get Signal immunostain (NKB-501; Toyobo Co., Ltd., Japan). After the samples were washed, they were incubated with Alexa 488- or Alexa 594-conjugated IgG antibodies at 25°C. The sections were enclosed in Gel/Mount (Biomeda Corp., USA) and observed using fluorescence microscopy (Axio Imager M2, Carl Zeiss, Germany). The number of germ cells in each genital ridge section was assessed by Image J (version 1.50i).

Western blotting analyses

The 3×Flag expression vectors for Dnd1 and Dnd1^Ter were constructed using pcDNA^TM3.1(+) (Thermo Fisher Scientific, USA). HeLa cells were then transfected with these constructs as previously described [19]. After 48 hours, cellular proteins were extracted with 1×sample buffer (100 mM Tris, pH8.3, 2% SDS, 200 mM DTT, 10% glycerol, 1 mM EDTA, 0.05% bromophenol blue), and then resolved on a 12% sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) gel and electroblotted onto nitrocellulose membrane (BioTrace NT, Pall Corporation, USA). The membranes were incubated with primary antibodies; anti-FLAG antibody (1:8,000, F3165, Sigma-Aldrich), or anti-DND1 antibodies (1:1,000) generated from rabbit and guinea pig [20]. These were followed by goat anti-mouse IgG conjugated with alkaline phosphatase (AP; 1:2,000, 69266, Novagen) for anti-FLAG antibody, swine anti-rabbit IgG conjugated with AP (1:2,000, D0306, DAKO) for rabbit anti-DND1 antibody, or goat anti-guinea pig IgG conjugated with AP (1:2,000, sc-2930, Santa Cruz Biotechnology) for guinea pig anti-DND1 antibody. The detection of immunoreactivity was performed using a BCIP/NBT Phosphatase substrate kit (50-81-00, SeraCare Life Sciences, Inc., USA) according to the manufacturer’s instructions.

For western blotting analyses of proteins from E15.5 male gonads, embryonic gonads were excised from E15.5 male embryos from pregnant Dnd1^+/Ter female mice crossed with wild-type male mice and then sonicated with an ultrasonic disruptor (Handy Sonic UR-20P; Tomy Seiko Co., Ltd., Japan.) in 1×sample buffer. Extracts were resolved on 12.5% SDS-PAGE gels and electroblotted onto nitrocellulose membranes (BioTrace NT, Pall Corporation). The membranes were incubated with primary antibodies: anti-DND1 antibody generated by guinea pig #2 (1:1,000) [14] or anti-DAZL antibody generated from rabbits (1:1,000) [20], followed by goat anti-guinea pig IgG conjugated with horseradish peroxidase (HRP; 1:10,000; ab7139, Abcam plc, UK) or goat anti-rabbit IgG conjugated with HRP (1:10,000, sc-2054, Santa Cruz Biotechnology, USA)). Immunoreactivities were visualized as chemiluminescence by using Western BLoT Chemiluminescence HRP Substrate (Takara Bio Inc., Japan) and a lumino-image analyzer (ImageQuant LAS-4000mini, GE Healthcare, England) and then quantitated according to the manufacturer’s instructions.

All antibodies used in western blotting analysis were diluted by Can Get Signal Immunoreaction Enhancer Solution (NKB-101; Toyobo Co., Ltd.).

Flow cytometry

Testis cell suspensions were generated by sequential digestion of dissected and minced seminiferous tubules with 1 mg/mL collagenase (Wako, Japan), followed by 0.25% trypsin (27250018; Thermo Fisher Scientific) containing 1 mM EDTA. Cells were passed through a 40-μm strainer to remove clumps. Harvested cells were resuspended in HBSS containing 5% fetal bovine serum (FBS) and 0.1% bovine serum albumin (BSA). Subsequently, a FoxP3 Transcription Factor Staining Buffer Kit (A25866A; Life Technologies, USA) was used with an antibody against promyelocytic leukemia zinc-finger (PLZF; 1:200; H-300; Santa Cruz Biotechnology) according to the manufacturer’s instructions. DNA was labelled with 4′,6-diamidino-2-phenylindole (DAPI) to gate the 2N and 4N fractions. Stained cells were analyzed with a cell sorter (MoFlo Astrios; Beckman Coulter, USA), and data analysis was performed using KALUZA (version 1.2, Beckman Coulter).

Statistical analysis

The statistical significance of differences in the ratios of testis weight per body weight, ratios of defective tubules, sperm count, and relative DND1 expression were assessed using a two-tailed t-test, whereas the data for genotype distribution in the intercrosses of 129 Dnd1^+/Δ heterozygotes and tumor surveys were analyzed with χ² analysis and Fisher’s exact test, respectively. A P-value of <0.05 was considered to represent statistical significance. Statistical analysis was performed using Microsoft Excel for Mac (version 16.33) or R (version 3.6.0).

Results

Homozygous for Dnd1-Δ mutant mice were born at Mendelian ratios and developed testicular teratoma in the 129 genetic background

To clarify the effect of Dnd1 loss, we aimed to establish a new conventional Dnd1-knockout mouse line. We had previously generated a Dnd1-flox mouse line to conditionally remove DND1 by Cre recombinase [14]. In the current study, a conventional knockout (Dnd1-Δ) mouse line was generated from the Dnd1-flox mice by Cre-mediated excision of exons 2 and 3, which included the two RRMs of DND1 (S1D and S1E Fig), and was subsequently backcrossed into three different mouse strains: BL6, MCH, or 129. Intercrosses of Dnd1^+/Δ heterozygotes within each strain produced the Dnd1^Δ/Δ homozygotes in accordance with a Mendelian expectation of 1:2:1 in all three mouse strains (Table 1), showing no evidence for embryonic lethality of the homozygous mutants even in the 129 genetic background.

Table 1. Genotype distribution of offspring from Dnd1^+/Δ intercrosses in BL6, MCH, and 129 strains.

	Genotype of Dnd1			Total no. examined	x²	P
	+/+	+/Δ	Δ/Δ	Total no. examined	x²	P
BL6	46	82	43	171	0.39	0.82	ns
MCH	93	171	97	361	1.09	0.58	ns
129	102	178	95	375	1.22	0.54	ns

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The number of Dnd1^Δ/Δ progeny derived from Dnd1^+/Δ crosses were compared among BL6, MCH, and 129 strains. Adult offspring of both sexes were genotyped. *ns: not significnat.

We then examined whether the Dnd1-Δ allele affects the occurrence of testicular teratoma. For this purpose, testes from 4-week-old Dnd1^Δ/Δ mice, Dnd1^+/Δ mice, and their Dnd1^+/+ wild-type control littermates of all three strains were harvested and surveyed for teratomas (Table 2 and S2 Fig). In the 129 strain, the Dnd1-Δ allele significantly increased teratoma occurrence in male mice from a baseline of 4.2% to 28.8% in Dnd1^+/Δ heterozygotes and 92.6% in Dnd1^Δ/Δ homozygotes, which are ratios similar to those for Ter mutant mice [4, 5, 16, 21]. In contrast, teratomas developed in the testes of approximately 10% of the MCH Dnd1^Δ/Δ male mice, indicating that the MCH strain has low sensitivity to testicular teratoma, while teratomas were not observed in the testes of all three genotypes of the BL6 strain. Collectively, the results indicate that the Dnd1-Δ allele is inherited in accordance with Mendelian ratios, similar to the findings for the Dnd1-Ter allele, and that the ratios of testicular teratoma incidence are similar between the Δ and Ter alleles.

Table 2. Genotype of Dnd1 and the incidence of male mice affected with testicular teratomas in the BL6, MCH, and 129 strains.

BL6	% of affected males
Genotype	total^*	unilateral (L)	unilateral (R)	bilateral
+/+	0% (0/32)	-	-	-
+/Δ	0% (0/36)	-	-	-
Δ/Δ	0% (0/20)	-	-	-
MCH	% of affected males
Genotype	total^*	unilateral (L)	unilateral (R)	bilateral
+/+	0% (0/47)	-	-	-
+/Δ	0% (0/85)	-	-	-
Δ/Δ	10.2% (6/59)	5.1% (3/59)	1.7% (1/59)	3.4% (2/59)
129	% of affected males
Genotype	total^*	unilateral (L)	unilateral (R)	bilateral
+/+	4.2% (4/97)	3.1% (3/97)	1.0% (1/97)	-
+/Δ	28.8% (42/146)	14.4% (21/146)	7.5% (11/146)	6.8% (10/146)
Δ/Δ	92.6% (50/54)	9.3% (5/54)	20.4% (11/54)	63.0% (34/54)

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Dnd1^+/Δ female mice were crossed with Dnd1^+/Δ or Dnd1^+/+ male mice among the BL6, MCH, and 129 strains to test the incidence of male offspring with at least one testicular teratoma.

*Fisher's exact test: MCH-Dnd1^+/Δ versus MCH-Dnd1^Δ/Δ, P = 0.00404; 129-Dnd1^+/+ versus 129-Dnd1^+/Δ, P = 5.28E-07; 129-Dnd1^+/Δ versus 129-Dnd1^Δ/Δ, P = 2.2E-16

Dnd1 loss caused defects in both spermatogenesis and oogenesis

We also examined the effect of Dnd1 loss on germ cell development. For this purpose, we first compared testes from 4-week-old Dnd1^+/+ wild-type, Dnd1^+/Δ, and Dnd1^Δ/Δ male mice that did not have testicular teratomas. We found that even testes of Dnd1^+/Δ mice appeared to be smaller than those of wild-type male mice, in addition to a clear decrease in testis size noted in Dnd1^Δ/Δ male mice in all three strains (Fig 1A–1C). The ratios of testis weight per body weight of Dnd1^+/Δ male mice were significantly lower than those of wild-type male mice in all three strains, while the ratios of Dnd1^Δ/Δ male mice showed a drastic decrease compared with those of wild-type and Dnd1^+/Δ male mice (Fig 1D). Histological analyses showed some seminiferous tubules with impaired spermatogenesis in Dnd1^+/Δ testes, while no germ cells were observed in Dnd1^Δ/Δ testes (Fig 1E–1M). We counted the tubules with impaired spermatogenesis in Dnd1^+/Δ testes and found significant increases in the ratio of defective tubules as compared with those of wild-type testes in all three strains, especially in the 129 strain (Fig 1N–1P). These data indicate that Dnd1 loss results in disappearance of germ cells in adult testes and that spermatogenesis is impaired in a part of the seminiferous tubules, even in Dnd1^+/Δ heterozygous male mice.

Fig 1 — (A–C) Comparison of the testis size of 4-week-old littermates of wild-type, *Dnd1*^+/Δ, and *Dnd1*^Δ/Δ mice of the BL6 (A), MCH (B), and 129 (C) strains. Scale bar: 5 mm in A for A–C. (D) Box plots (median [horizontal line], 25th and 75th percentiles [box], and maximum and minimum [error bars]) indicate the testis weight per body weight of male mice not affected with teratomas. ***P < 0.001 (Student’s t-test). (E–M) Testis sections of 4-week-old littermates of wild-type (E, H, K), *Dnd1*^+/Δ (F, I, L), and *Dnd1*^Δ/Δ (G, J, M) mice of the BL6 (E, F, G), MCH (H, I, J), and 129 (K, L, M) strains were stained with hematoxylin and eosin. Asterisks indicate tubules with defective spermatogenesis in *Dnd1*^+/Δ testes (F, I, L). Insets show enlarged views to better visualize tubules. Scale bar: 200 μm in E for E–M. (N–P) Comparison of percentages of seminiferous tubules with defective spermatogenesis among wild-type and *Dnd1*^+/Δ mice of the BL6 (N), MCH (O), and 129 (P) strains; more than 180 tubules from three independent cross-sections of the testes were scored (n = 3). *P < 0.01, **P < 0.005, ***P < 0.001 (Student’s t-test).

We then compared the ovaries of 4-week-old female mice of each genotype among the three strains (Fig 2A–2C) and found that the Dnd1^+/Δ ovaries were as large as the wild-type ovaries in all the three strains, in contrast to the findings for testes. Only the Dnd1^Δ/Δ ovaries from the 129 female mice appeared to be slightly larger than those of the other two strains, although the ovaries from Dnd1^Δ/Δ female mice were smaller than those of wild-type and Dnd1^+/Δ female mice in all three strains. Histological analyses showed that a considerable number of oocytes were developing in the ovaries from 129 Dnd1^Δ/Δ female mice, whereas the other two strains had few oocytes in Dnd1^Δ/Δ ovaries (Fig 2D–2L). We then crossed these female mice at 6 weeks of age with MCH male mice and counted the number of offspring to examine their fertility until they reached 30 weeks of age (Fig 2M). These analyses showed no significant difference between wild-type and Dnd1^+/Δ female mice in each strain; no offspring were born from Dnd1^Δ/Δ female mice in both BL6 and MCH strains. However, the 129 female mice delivered offspring even in the case of Dnd1^Δ/Δ mice, but the number was fewer than those of wild-type and Dnd1^+/Δ female mice, indicating that 129 Dnd1^Δ/Δ female mice were subfertile.

Collectively, the data indicate that the spermatogenic and oogenic phenotypes observed in Dnd1-Δ mutant mice were very similar to those previously reported for Ter mutant mice [5].

Dnd1^+/Δ male mice progressively lost fertility because of sperm count decrease and impaired sperm function in the 129 strain

We next checked whether the Dnd1-Δ mutant mice exhibited phenotypes other than those mentioned above. We noted that 129 Dnd1^+/Δ male mice produced fewer offspring than BL6 and MCH strains in their intercrosses. For more in-depth analysis of the phenotype, we crossed 10-week-old Dnd1^+/Δ male mice of each strain with female mice of the MCH strain and determined the litter size. BL6 or MCH Dnd1^+/Δ male mice continuously impregnated the female mice and produced offspring until they were 20–24 weeks old (Fig 3A and 3B). In contrast, female mice that were crossed with 129 Dnd1^+/Δ male mice produced offspring until the male mice reached 10–12 weeks of age, but the number of offspring drastically decreased after 12 weeks of age (Fig 3C). Subsequently, they stopped giving birth when the male mice were at 20–24 weeks of age; however, copulatory plugs were continuously found in the female mice. Since wild-type 129 male mice could impregnate the female mice even after they were 12 weeks old (Fig 3D), this phenotype was caused by the heterozygous Dnd1-Δ mutant allele only in the 129 genetic background.

Fig 3 — (A–D) Litter size analysis of *Dnd1*^+/Δ male mice of the BL6, MCH, and 129 strains and wild-type male mice of the 129 strain. A 10-week-old male mouse was crossed with three wild-type female MCH mice until the male mouse reached 24 weeks of age. Three male mice (Mouse 1, Mouse 2, and Mouse 3) were analyzed per strain or genotype. (E–G) Comparison of testis weight per body weight ratios between 4-week-old and 12-week-old wild-type and *Dnd1*^+/Δ mice from the BL6, MCH, and 129 strains. Error bars represent mean ± SD; three mice were analyzed per genotype and strain. *P < 0.01, **P < 0.005 (Student’s t-test). (H–J) Sperm count analysis of 12-week-old wild-type and *Dnd1*^+/Δ male mice of the BL6, MCH, and 129 strains. Error bars represent mean ± SD; three mice were analyzed per genotype and strain. *P < 0.01, **P < 0.005, ***P < 0.001 (Student’s t-test). (K) IVF analysis using sperm from more than 12-week-old wild-type and *Dnd1*^+/Δ male mice of the 129 strain. Error bars represent mean ± SD; three mice were analyzed per genotype. ***P < 0.001 (Student’s t-test).

To identify the cause of infertility in 129 Dnd1^+/Δ male mice, we compared the ratios of testis weight per body weight of 4-week-old wild-type and Dnd1^+/Δ male mice with those of 12-week-old male mice in each of the three strains. The ratios of Dnd1^+/Δ male mice were lower than those of wild-type male mice at both time points and in all three strains (Fig 3E–3G), consistent with the results shown in Fig 1D. Furthermore, we found that the ratio significantly decreased from 4 to 12 weeks of age in only 129 Dnd1^+/Δ male mice, whereas in the other two strains, the ratio increased even in Dnd1^+/Δ male mice, suggesting testicular growth retardation due to impaired spermatogenesis. We therefore determined the sperm count of 12-week-old wild-type and Dnd1^+/Δ male mice in each strain and found that the count had significantly decreased in Dnd1^+/Δ male mice of all the strains. However, the sperm counts of 129 Dnd1^+/Δ male mice decreased to approximately one-fifth of those of wild-type male mice (Fig 3J), whereas the decrease was only approximately one-half in the other two strains (Fig 3H, 3I), suggesting that such a large reduction in sperm count might lead to fertility loss in 129 Dnd1^+/Δ male mice. However, since the sperm count reduction does not necessarily cause fertility loss [22], we further examined whether the sperm generated in 129 Dnd1^+/Δ male mice were functional by performing IVF with sperm from more than 12-week-old 129 wild-type or Dnd1^+/Δ male mice (Fig 3K). Nearly 90% of oocytes developed to blastocysts when sperm from wild-type male mice were used. However, in the case of sperm from Dnd1^+/Δ male mice, the developmental rate drastically decreased to 3.9%, indicating that the sperm were not fully functional in 12-week-old 129 Dnd1^+/Δ male mice. Collectively, the findings indicate that the 129 Dnd1^+/Δ male mice lost fertility, presumably because of a significant decrease in sperm count and impaired function of sperm after 12 weeks of age.

To determine why a large decrease in sperm count and impaired sperm function occurred only in 129 Dnd1^+/Δ male mice after 12 weeks of age, we speculated that the impaired spermatogenesis causing these phenotypes might be attributable to some defect in spermatogonia because Dnd1 is expressed in spermatogonia and is required for the maintenance of these cells [15]. To examine this hypothesis, cells in the testes from 12-week-old wild-type and Dnd1^+/Δ male mice of all three strains were subjected to flow cytometric analysis performed using antibodies against promyelocytic leukemia zinc-finger (PLZF) because we have previously shown that PLZF is expressed in all populations of DND1-positive spermatogonia [15]. These analyses revealed that the number of PLZF-positive spermatogonia significantly decreased in both 129 and BL6 Dnd1^+/Δ male mice (S3A–S3F Fig). However, although BL6 Dnd1^+/Δ male mice showed a slightly greater decrease in the number of PLZF-positive spermatogonia than 129 Dnd1^+/Δ male mice did (S3C and S3F Fig), the decrease in sperm count in BL6 Dnd1^+/Δ male mice was much lesser than that in 129 Dnd1^+/Δ male mice (3H and J). Furthermore, although the number of spermatogonia was unchanged between wild-type and Dnd1^+/Δ male mice in the MCH strain (S3G–S3I Fig), the sperm count significantly decreased in Dnd1^+/Δ male mice (Fig 3I). These data indicate no correlation between the decrease in spermatogonia and the decrease in sperm count in Dnd1^+/Δ male mice.

Progressive loss of fertility did not occur in 129 Dnd1^+/Ter male mice

In relation to testicular teratoma incidence, spermatogenesis, and oogenesis, the phenotypes of Dnd1-Δ mutant mice were similar to those of Ter mutant mice [5]. However, the progressive loss of fertility observed in 129 Dnd1^+/Δ male mice has not been previously reported in 129 Dnd1^+/Ter male mice. To determine whether the Ter mutant allele leads to the same phenotype for fertility as the Dnd1-Δ mutant allele in the 129 strain, we compared the ratio of testis weight per body weight between 12-week-old wild-type and Dnd1^+/Ter male mice in the 129 strain and found a significant decrease in the ratio to 70% of that of wild-type male mice in the case of Dnd1^+/Ter male mice (Fig 4A). However, the reduction rate was lesser than that of Dnd1^+/Δ male mice since the Dnd1-Δ mutant allele decreased the ratio by almost half (Fig 3G). We therefore compared the sperm count of 12-week-old 129 Dnd1^+/Ter male mice with those of wild-type male mice and found that the number tended to decrease but was not significantly changed as compared with that of wild-type male mice (Fig 4B). We then crossed 129 Dnd1^+/Ter male mice with female mice of the MCH strain and determined the litter size, as mentioned in Fig 3, to check whether the Ter mutant allele caused progressive loss of fertility, as is the case with 129 Dnd1^+/Δ male mice (Fig 3C). These analyses revealed that 129 Dnd1^+/Ter male mice continuously impregnated the female mice and produced offspring until they were 20–24 weeks old, unlike the 129 Dnd1^+/Δ male mice (Fig 4C). These data thus showed a clear phenotypic difference between Dnd1^+/Δ and Dnd1^+/Ter male mice in the 129 strain, suggesting that either the Dnd1-Δ or Ter mutant allele caused some gene expression change, other than Dnd1 loss, and that such changes generated these phenotypic differences.

Fig 4 — (A) Testis weight per body weight ratios of 12-week-old wild-type and *Dnd1*^+/Ter mice of the 129 strain were measured. Error bars represent mean ± SD; three mice were analyzed per genotype. **P < 0.005 (Student’s t-test). (B) Sperm count analysis of 12-week-old wild-type and *Dnd1*^+/Ter male mice of the 129 strain. Error bars represent mean ± SD; three mice were analyzed per genotype. (C) Litter size analysis of *Dnd1*^+/Ter male mice of the 129 strain. A 10-week-old *Dnd1*^+/Ter male mouse was crossed with three wild-type female MCH mice until the male mouse was 24 weeks of age. Three male mice (Mouse 1, Mouse 2, and Mouse 3) were analyzed. (D) Western blotting analyses of Flag-tagged wild-type DND1 and DND1^Ter in HeLa cells transfected with FLAG-tagged *Dnd1*, *Dnd1*^Ter, or control vector by using anti-DND1 antibodies generated by rabbit #1, guinea pig #1 (gp #1), guinea pig #2 (gp #2), and guinea pig #3 (gp #3). (E) Western blotting analyses of proteins from E15.5 male gonads of wild-type and *Dnd1*^+/Ter embryos, performed using anti-DND1 antibodies generated by guinea pig #2 (gp #2). DAZL is a loading control. Note that no specific bands were observed in all five *Dnd1*^+/Ter male gonads at approximately 27 kDa. (F) Comparison of the DND1 relative expression level in wild-type and *Dnd1*^+/Ter E15.5 male gonads. The signal intensities of DND1 in (E) were normalized by those of DAZL. Error bars represent mean ± SD; **P < 0.005 (Student’s t-test).

In this context, we focused on the Ter mutant allele because it was previously suggested that Ter was not a mutation causing loss of Dnd1 expression, but instead generated a short mutant DND1 protein, called DND1^Ter, consisting of Dnd1 exons 1–2 and a part of exon 3 (S1C Fig) [16, 23]. To examine DND1^Ter expression, we first tested whether antibodies against DND1, which we previously generated from a rabbit and three guinea pigs [14], could detect DND1^Ter. For this purpose, wild-type DND1 or DND1^Ter were forcibly expressed in HeLa cells by transfection of 3×Flag-tagged Dnd1 or Dnd1^Ter and then subjected to western blot using four different antibodies against DND1. Our analyses showed that the antibodies generated from guinea pigs #1, #2, and #3 could detect Flag-tagged DND1^Ter at the same level as the anti-FLAG antibody, while the antibody generated from rabbit #1 barely detected it (Fig 4D). We then analyzed the in vivo expression of DND1^Ter in the E15.5 male gonads of 129 Dnd1^+/Ter embryos by using the antibody against DND1 generated from guinea pig #2. However, western blot analyses revealed that DND1^Ter was undetectable in all five Dnd1^+/Ter embryos (Fig 4E), whereas the DND1 levels decreased to approximately half of those in wild-type embryos (Fig 4F). These results support the possibility that Ter is a loss of Dnd1 mutation, which in turn suggests that the Dnd1-Δ mutant allele causes some defect leading to sperm count decrease and sperm malfunction independently of Dnd1 loss.

Dnd1 genetically interacted with Nanos2 and Nanos3 for suppression of testicular teratomas

We have previously shown that both NANOS2 and NANOS3 interact with DND1 and that these complexes are essential for germ cell development [14, 15], leading us to speculate that both NANOS2 and NANOS3 play a vital role with DND1 even in the regulation of testicular teratoma incidence. To examine this hypothesis, we crossed Dnd1-Δ mutant mice with both Nanos2 or Nanos3 mutant mice to establish male mice that were double mutants for Dnd1 and Nanos2 or Nanos3 and subsequently analyzed these mice for testicular teratomas in the 129 genetic background.

In the case of crossing with Nanos2 mutant mice (Figs 5A and S4A), the ratio of affected male mice slightly increased from 2.9% in wild-type male mice to 8.8% in Nanos2^+/LacZ male mice and 11.8% in Nanos2^LacZ/LacZ male mice, indicating that Nanos2 is one of the genes responsible for testicular teratomas, as previously reported [24]. In addition, introducing the heterozygous Dnd1-Δ mutant allele into both Nanos2^+/LacZ and Nanos2^LacZ/LacZ male mice increased the ratio to 38.8% and 80.0%, respectively, indicating a synergistic effect of combining mutations for Dnd1 and Nanos2 since only 28.5% (Table 2) of Dnd1^+/Δ male mice were affected. In the case of crossing with Nanos3 mutant mice (Figs 5B and S4B), the ratio of affected male mice increased from 4.4% in wild-type male mice to 24.2% in Nanos3^+/Cre male mice, indicating that Nanos3 is one of the genes responsible for testicular teratomas, as previously reported [25]. In addition, the male mice that were double mutants for Dnd1^+/Δ; Nanos3^+/Cre showed a drastic increase in the ratio to 84.3%, indicating a synergistic effect of combining heterozygous mutations for Dnd1 and Nanos3. Based on these data, we conclude that both Nanos2 and Nanos3 interact with Dnd1 in the regulation of testicular teratoma incidence.

Fig 5 — (A, B) Incidence of testicular teratomas in 129 male mice carrying the *Dnd1*-Δ allele and *Nanos2*-LacZ (A) or *Nanos3*-Cre (B) allele. The numbers of male mice examined were as follows: *wild-type* (N = 35), *Nanos2*^+/LacZ (N = 57), *Nanos2*^LacZ/LacZ (N = 17), *Dnd1*^+/Δ; *Nanos2*^+/LacZ (N = 49), and *Dnd1*^+/Δ; *Nanos2* ^LacZ/LacZ (N = 10) in (A), and *wild-type* (N = 45), *Nanos3*^+/Cre (N = 62), *Dnd1*^+/Δ; *Nanos3*^+/Cre (N = 51), *Nanos3*^Cre/Cre (N = 30), and *Dnd1*^+/Δ; *Nanos3*^Cre/Cre (N = 20) in (B). Blue, orange, and gray boxes in (A) and (B) indicate the percentages of cases where left, right, and both testes, respectively, were affected. (C, D) Comparison of the PGC number among wild-type, *Nanos3*^+/Cre, and *Nanos3*^Cre/Cre (C) or wild-type, *Dnd1*^+/Δ, and *Dnd1*^Δ/Δ (D) male embryos at E11.5.

Testicular teratomas were not observed in Nanos3^Cre/Cre male mice, even when these mice had a heterozygous Dnd1-Δ mutant allele. To determine why Nanos3^Cre/Cre male mice were not affected, we counted the number of PGCs in Nanos3-Cre mutant male embryos at E11.5 by section immunostaining of gonads with antibodies against DAZL and NANOG and then compared the numbers with those in Dnd1-Δ mutant male embryos. These analyses revealed that PGCs were almost completely absent in Nanos3^Cre/Cre embryos at E11.5 (Fig 5C), which is before the transformation to EC cells. This might be because the PGCs undergo apoptotic cell death [26] or transdifferentiate into somatic cells as observed in other species [27, 28] during migration stages in the absence of Nanos3. In contrast, a small number of PGCs were still observed in the male gonads of Dnd1^Δ/Δ embryos (Fig 5D), suggesting that some of these survivor cells eventually acquire pluripotency at the late stages of embryogenesis and then develop to form teratomas after birth.

Discussion

In the current study, we newly generated Dnd1-Δ, a conventional knockout allele of Dnd1 (S1E Fig), and found that the Dnd1-Δ mutant mice showed defects similar to those of Ter mutant mice in spermatogenesis, oogenesis, and incidence of testicular teratomas, with a slight difference in spermiogenesis. In addition, a short mutant DND1 protein, called DND1^Ter, was not detected in E15.5 male gonads from Dnd1^+/Ter embryos in our western blotting analysis (Fig 4D and 4E), whereas the amounts of full-length DND1 in Dnd1^+/Ter embryos decreased to about half of those in wild-type embryos (Fig 4F). On the basis of these data, we suggest that the Ter mutation simply causes loss of Dnd1 expression, as previously reported [9]. However, we cannot rule out the possibility that DND1^Ter is highly expressed in germ cells other than at E15.5 or that DND1^Ter is translated but is undetectable because its expression level is too low. The expression level of DND1^Ter was lesser than that of wild-type DND1 when both proteins were force-expressed in cultured cells (Fig 4D), which was consistent with a previous study suggesting that DND1^Ter was unstable [23]. In either case, DND1^Ter might be involved in the development of testicular teratomas. Alternatively, given that the defects in Dnd1-Ter mutant mice were milder than those in Dnd1-Δ mutant mice (Figs 3J and 4B), it is also possible that DND1^Ter alleviates the phenotypes of Dnd1 loss, thereby leading to the phenotypic differences in sperm count between Dnd1-Δ and Ter mutant mice.

The 129 Dnd1^+/Δ male mice lost fertility after 12 weeks because of sperm count decrease and impaired sperm function, which was not observed in 129 Dnd1^+/Ter male mice (Figs 3 and 4C). If Ter is a loss of Dnd1 mutation as suggested, these phenotypic differences between Dnd1^+/Δ and Dnd1^+/Ter might be caused by sequence deletion in the Dnd1-Δ allele. Generating the Dnd1-Δ allele might collaterally remove regulatory sequence(s) located between two loxP sequences controlling the expression of gene(s) around Dnd1, which might cause changes in the expression of other gene(s), rather than loss of Dnd1. This is because Dnd1 is located in a genomic region where 16 genes are closely placed within 150 kilobase pairs, thereby resulting in sperm count decrease and impaired sperm function. However, it is unclear which gene expression level is affected by the targeted deletion and which of the cells are affected by these gene expression changes occurring with the Dnd1-Δ mutant allele because no study has reported the physiological role of the 15 neighboring genes in spermatogenesis (S5 Fig). Since no correlation was observed between the decrease in spermatogonia and the decrease in sperm count in Dnd1^+/Δ male mice (S3 Fig), the sperm count decrease and impaired sperm function could be attributed to a defect in cells other than spermatogonia.

In the context of phenotypic difference, Zechel et al. reported that, in the 129S1/SvImJ genetic background, homozygous deletion from Dnd1 exon 1 to most of exon 3 (S1B Fig) induced embryonic lethality before E3.5 [16]. In addition, they showed that the incidence of testicular teratomas did not increase in the 129S1/SvImJ Dnd1^+/KO heterozygous mutant male mice although Dnd1^+/Ter male mice showed increased incidence in the same genetic background. These phenotypes are different from those of both Dnd1-Δ and Ter mutant mice. As mentioned above, the deletion from Dnd1 exon 1 to most of exon 3 might collaterally remove regulatory sequence(s) that control the expression of genes placed close to Dnd1, leading to phenotypes different from those in Dnd1-Δ and Ter mutant mice. Alternatively, the phenotypic difference might be caused by the neomycin selector unit because it is widely known that insertion of a pgk-neo cassette or reporter gene into a specific locus might affect the expression of the neighboring genes [29, 30]. It is also possible that a de novo mutation occurred during establishment of the Dnd1-KO mouse line and caused changes in the expression of the neighboring genes, resulting in the phenotypic differences. The expression profile and phenotypes of mice with knockout of the genes around Dnd1 would yield valuable information for further understanding the phenotypic differences among Dnd1-Δ, Ter, and Dnd1-KO mutant mice.

We have also shown synergistic increase in testicular teratoma incidence in the male mice that were double mutants for Dnd1 and Nanos2 or Nanos3 (Fig 5A and 5B). In addition to these genetic interactions, given that both NANOS2 and NANOS3 associate with DND1, it is highly plausible that both DND1-NANOS2 and DND1-NANOS3 complexes regulate teratoma incidence in male embryonic germ cells. We have previously shown that DND1 plays a role in loading RNAs onto the NANOS2-CNOT deadenylase complex, leading to degradation of the target RNAs [15]. In addition, in the absence of NANOS2, a part of male embryonic germ cells continuously proliferate and cannot undergo cell-cycle arrest [31], which increases the incidence of transformation of germ cells to EC cells in the 129 strain [24]. These results suggest that the DND1-NANOS2 complex suppresses target RNAs that induce cell-cycle arrest via CNOT deadenylase–mediated RNA degradation, thereby suppressing the transformation of germ cells to EC cells. NANOS3 is highly expressed in migrating and proliferative PGCs but its expression gradually disappears after colonization to male gonads, in accordance with the increase in NANOS2 expression [17, 32]. Therefore, the regulatory mechanisms of the DND1-NANOS3 complex in the case of testicular teratoma incidence are still unknown and may differ from those of NANOS2. Comprehensive identification of target RNAs for DND1-NANOS2 and DND1-NANOS3 complexes is required to elucidate the transformation of germ cells to EC cells.

Supporting information

S1 Fig. Comparison of Dnd1 genome structure.

Structures of the Dnd1 (A-E) wild-type allele (A), KO allele generated by Zechel et al. [16] (B), Ter allele [9] (C), floxed allele generated by Suzuki et al. [14] (D), and Δ allele (E).

(PDF)

Click here for additional data file.^{(473.2KB, pdf)}

S2 Fig. Testicular teratomas in the Dnd1-Δ mutant mice of the 129 strain.

Comparison of the testes from 4-week-old Dnd1^+/Δ and Dnd1^Δ/Δ mice of the 129 strain. Note that testicular teratomas developed only in the right testis in the Dnd1^+/Δ mice, whereas both testes have testicular teratoma in the Dnd1^Δ/Δ mice. Scale bar: 5 mm.

(PDF)

Click here for additional data file.^{(889.6KB, pdf)}

S3 Fig. Reduction in spermatogonial number is not related to sperm count decrease and impaired sperm function.

Representative flow cytometric analyses of testis cells from 12-week-old wild-type (A, D, G) and Dnd1^+/Δ (B, E, H) mice of the 129 (A–C), BL6 (D–F), and MCH (G–I) strains for PLZF. Percentages of cells within each PLZF-positive gate (A, B, D, E, F, G) were normalized by the ratio of PLZF-positive cells from wild-type mice and indicated (C, F, I). Error bars represent mean ± SD; three mice were analyzed per genotype and strain. *P < 0.05 (Student’s t-test).

(PDF)

Click here for additional data file.^{(1.1MB, pdf)}

S4 Fig. Testicular teratomas in mice that were double mutants for Dnd1 and Nanos2 or Nanos3 in the 129 strain.

Comparison of the testes from 4-week-old Dnd1^+/Δ; Nanos2^+/LacZ and Dnd1^+/Δ; Nanos2^LacZ/LacZ mice (A), or wild-type and Dnd1^+/Δ; Nanos3^+/Cre mice of the 129 strain (B). Scale bars: 5 mm in (A) and (B).

(PDF)

Click here for additional data file.^{(1.5MB, pdf)}

S5 Fig. The 15 genes neighboring Dnd1 and their knockout mouse lines.

The 15 genes neighboring Dnd1 are listed in order of their location on chromosome 18. Their knockout mouse (KO) lines and phenotypes are also mentioned in the list. IMPC: The International Mouse Phenotyping Consortium.

(PDF)

Click here for additional data file.^{(764.5KB, pdf)}

S1 Raw images

(JPG)

Click here for additional data file.^{(777.4KB, jpg)}

Acknowledgments

We thank H. Nishimura for her technical assistance with the tumor survey.

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

This work was supported by the Japan Society for the Promotion of Science (KAKENHI) grants 16H01252 and 17H05046 to AS. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0232047.r001

Decision Letter 0

Stefan Schlatt

26 Feb 2020

PONE-D-20-03327

Dead end1 acts with Nanos2 and Nanos3 to regulate testicular teratoma incidence

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1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: General comments:

In this study, Imai and Colleagues followed on their previous work on the mechanisms of action of Dnd1 in germ cells of 129/Sv mice, which frequently develop spontaneous testicular teratomas (STT). In the current study they examined the consequences of Dnd1 loss and looked at interactions between DND1 and NANOS2 and NANOS3.

The authors established a dnd1-KO mouse line and back-crossed it to three different mouse strains characterised by very different frequencies of SST, including a highly permissive 129/terSv and a resistant C57BL/6J strain. Furthermore, they crossed the 120+ter/SvJcl strain with either Nanos2- or Nanos3- manipulated strains. The detailed analysis of the phenotypes comprised incidence of teratomas, fertility, and embryonic and postnatal testis and ovary histology, with germ cell quantification, including analysis of flow-sorted PLZF-positive germ cells. Germ cell recognition / analysis of gene expression was done by IHC (DAZL, NANOG) and quantitative analysis of studied proteins (DND1; DAZL) by western blotting. The authors reported a significant rise in the incidence of SST only in 129/Dnd1- mice, with rates similar to Ter mice. Cross-breeding experiments with Nanos2- or Nanos3- showed that both these genes are involved in the origin of teratomas in mice. In all strains they found decreased adult testis size, significant germ cell loss in dnd1-heterozygotes and a complete germ cell loss in the homozygotes. The sperm quality decreased with age, but not in the 129ter. Oogenesis and fertility were affected in all dnd1- strains, with the mildest phenotype in 129dnd1- strain.

The results concerning the phenotypic differences between 129ter and 129dnd1- as well as the cross-breeding experiments with Nanos2- or Nanos3- are novel and shed light on the mechanisms of STT pathogenesis. Although the conclusion that ter mutation causes dndl1 loss confirms previous studies, this is the most rigorous confirmation of this hypothesis. The work is very comprehensive and technically very well done. The figures are very nice and informative. The findings are discussed carefully, without over-interpreting the data.

I congratulate the authors for their excellent work and think that the paper is ready for publication essentially as is.

Minor specific comments:

1. The studied species (mouse) should be mentioned in the title.

2. The description of figures is included in the text of the Results and there are no separate figure legends. In my opinion this avoids redundant data repeating but the editors can decide whether or not this is OK.

Reviewer #2: Imai et al. introduce a new Dnd1Δ/Δ mouse line with a loss of function phenotype and show that double mutants of Dnd1 with Nanos2 and Nanos3 increase the testicular teratoma incidence. By comparing their knockout phenotype in different genetic backgrounds to the well-described Dnd1+/Ter mice, they show similarities in the phenotypes supporting the hypothesis that the Ter mutation leads to a loss of functional protein. By using different genetic backgrounds, they address the known influence of the genetic background on the DND1 mutations on the tumor incidence.

The authors present a well designed study. They showed appropriate controls and their conclusions are supported by statistical analyses. All methods are described in sufficient detail.

Minor points for improvement prior to publication:

- Figure presentation: The labeling of the graphs is relatively small and may be very hard to read when considering the usual figure size in the formatted manuscript.

- Line 105: What is the genetic background of the DND1flox/flox used to produce the Dnd1Δ/Δ mouse line? The cited publication (Suzuki et al. 2016) does not describe this specific line in detail.

- Line 526: In your discussion, you mention the observed differences to the study of Zechel et al. While I appreciate the given explanations, I was wondering how you ensured that the backcrosses still share the 100% of the genetics and that no other (de novo) mutations occurred explaining the striking differences between Dnd1Δ/Δ and Dnd1KO/KO mice?

- Line 540: You mention genes close to DND1, that may be regulated by deleted regions and potentially explain observed differences. Your point may be strengthened by giving specific examples, for example if neighboring genes play a known role in developmental processes affected or if the genetic organization (TADs, known enhancers) in this region are described in the literature and do confirm your hypothesis.

Reviewer #3: The paper “Dead end1 acts with Nanos2 and Nanos3 to regulate testicular teratoma incidence” by Imai et al present functional analysis of the Dead end protein in mouse.

The authors present the phenotype of the “full knockout” of the gene and compare it with that observed in the Ter mutation where the protein is truncated.

The authors suggest only the development of the germline is affect in the mutant, which is different from the conclusion of a previous paper where Dead end was considered to be essential for embryonic development. The authors provide evidence that from the point of view of teratoma development the complete null and the Ter allele are similar. The authors also present the phenotype Dead end loss of function has on ovary development, which is a poorly studied topic. Last, the authors present a series of experiments should genetic interactions between Dead end and Nanos proteins, thereby contributing to the understanding of the molecular mechanisms of Dead end function.

Overall, the paper provides new data that is of importance for researchers in the field of reproductive biology and the work should definitely be published in the journal after addressing the following points.

1. The paper would benefit a lot from English editing. In many cases the message is understood only after reading certain sentences several times.

2. “In the BL6 strain, teratomas were not observed in the testes of all three genotypes, whereas teratomas developed in the testes of approximately 10% of the MCH Dnd1Δ/Δ male mice, indicating that the MCH strain has low sensitivity to testicular teratoma.” – reading this sentence one should would define the MCH strain as having HIGH sensitivity, since the comparison is to the BL6 strain that does not form teratomas. The authors should modify / correct this sentence.

3. In Figure 1A- C the authors present examples for the testes phenotypes. The least severe difference between the wild-type and the mutant is observed in Figure 1C (for the 129 strain). From Figure 1D, the 129 strain shows the strongest effect. The authors should present in 1A-C examples that fit the average phenotype presented in Figure 1D.

4. In Figure 1E-M, show magnified boxes in all cases not only in panels F, I and L. This way the phenotypes can be better appreciated.

5. “Only the Dnd1Δ/Δ ovaries from the 129 female mice appeared to be slightly larger than those of the other two strains…”. If this data exists, provide quantitative information rather than “slightly”.

6. In Figures 2D-L, provide magnification boxes and marked specific cell types / structures such that the information is more accessible for a broader readership.

7. In Figure 3A, B, mark the mice as Nr 1 , Nr 2 and Nr 3 rather than No 1, No 2 and No 3. As it is, it looks like a gene name and not a mouse number. The best would be to use Mouse 1 , Mouse 2 and Mouse 3 as there is enough space for that.

8. The authors should try to reduce the text in the figure legends such that the information is not redundant with what is provided in the figure itself. For example in the legend of Figure 3- “The vertical axis represents the average number of offspring per copulatory plug, while the horizontal axis represents the age of the male mice.” The same information is provided in the Figure itself.

9. “…were subjected to flow cytometric analysis performed using antibodies against PLZF, because… “ define PLZF in the text the first time it is mentioned and not only in the methods section.

10. The authors start a section with the sentence “We next checked whether the Dnd1-Δ mutant mice exhibited phenotypes other than those of Ter mutant mice.”. In this section the Ter mutant is not directly referred to. The authors should rephrase it.

11. “These results support the idea that Ter is a loss of Dnd1 mutation, which in turn suggests that the Dnd1-Δ mutant allele causes some defect leading to sperm count decrease and sperm malfunction independently of Dnd1 loss.” This statement is very strong and is not proven by the data provided. The assumption that Ter is a complete loss of function, which questions the specificity of the knockout phenotype relies on Western blots. As very low amounts of the shorter version of Dead end are not necessarily detected, it could in principle be that the milder phenotype of Ter results from low amounts of the truncated version of the protein. In addition, it could be that the level of the Ter protein is higher at stages that differ from those examined in the Western blots. This point should be addressed and the statement the authors put forward should either be deleted, or presented as one of several options.

12. “In addition, introducing the heterozygous Dnd1-Δ mutant allele into both Nanos2+/LacZ and Nanos2LacZ/LacZ male mice increased the ratio to 38.8% and 80.0% (Fig. S4A)..” – the information is not. Provided in Figure S4A. Perhaps the authors mean Figure 5a here.

13. In Figure 5 the lack of germ cells in homozygous nanos3 mutants is determined by expression of daz and nanog. The authors conclude that the cells underwent apoptosis, but do not show it directly. The loss of cells could for example be transfating etc. The sentence should just be rephrased or the apoptosis claim deleted.

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6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes: Erez Raz and Kim Westerich

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2020 Apr 27;15(4):e0232047. doi: 10.1371/journal.pone.0232047.r002

Author response to Decision Letter 0

26 Mar 2020

Responses to reviewers’ comments

Reviewer #1: General comments:

I congratulate the authors for their excellent work and think that the paper is ready for publication essentially as is.

Minor specific comments:

1. The studied species (mouse) should be mentioned in the title.

Response: Thank you for the suggestion. As per the reviewer’s suggestion, we have indicated the species studied by adding the word “Mouse” in the title.

Response: As per the journal’s formatting guidelines, we had placed the figure caption along with the corresponding legend directly after the paragraph in which the corresponding figure had been first cited.

The authors present a well designed study. They showed appropriate controls and their conclusions are supported by statistical analyses. All methods are described in sufficient detail.

Minor points for improvement prior to publication:

- Figure presentation: The labeling of the graphs is relatively small and may be very hard to read when considering the usual figure size in the formatted manuscript.

Response: We have increased the font size of the labels used for the graphs in all the Figures.

- Line 105: What is the genetic background of the DND1flox/flox used to produce the Dnd1Δ/Δ mouse line? The cited publication (Suzuki et al. 2016) does not describe this specific line in detail.

Response: Thank you for highlighting this point. We had previously established a Dnd1_flox mouse line by using TT2 ES cells and maintained it via interbreeding to generate Dnd1flox/flox mice. We used these Dnd1flox/flox mice to produce the Dnd1_Δ mouse line. We have added this information in the “Mice” subsection of the Materials and methods section (Lines 104–111).

Response: We understand the reviewer’s concern. To address this point in the manuscript, we have now mentioned the possibility that a de novo mutation could have occurred during the establishment of the Dnd1-KO mouse line, resulting in the observed striking differences (Lines 544–546).

Response: To the best of our knowledge, no study has reported the physiological role of the neighboring genes in spermatogenesis. We have mentioned this in the Discussion (Lines 527 and 528). In addition, we have listed the genes neighboring Dnd1 and the phenotypes of their knockout mouse lines in Figure S5.

Reviewer #3: The paper “Dead end1 acts with Nanos2 and Nanos3 to regulate testicular teratoma incidence” by Imai et al present functional analysis of the Dead end protein in mouse.

The authors present the phenotype of the “full knockout” of the gene and compare it with that observed in the Ter mutation where the protein is truncated.

1. The paper would benefit a lot from English editing. In many cases the message is understood only after reading certain sentences several times.

Response: Our manuscript had already been proofread, as indicated by the certificate provided on the last page of this file. However, in accordance with the reviewer’s comment, the manuscript was submitted for English language proofreading again after incorporating the changes suggested by the reviewers.

Response: We understand the reviewer’s concern. We have revised this portion in the manuscript to avoid misinterpretation by the readers (Lines 233–239).

Response: Thank you for your suggestion. However, we faced limitations in obtaining 129 Dnd1+/+, Dnd1+/Δ, and Dnd1Δ/Δ male mice in a litter by intercrossing 129 Dnd1+/Δ mice because 129 Dnd1+/Δ male mice show decreased fertility and eventually become sterile by 12 weeks of age, as indicated in Figure 3C. In addition, approximately 95% of Dnd1Δ/Δ male mice have testicular teratomas. Thus, we have a very low chance to obtain 129 Dnd1+/+, Dnd1+/Δ, and Dnd1Δ/Δ testes with no teratomas in a litter. From these reasons, we currently do not have another picture for Figure 1C and it would take much time to obtain new one. As pointed out by the reviewer, Figure 1C does not represent the average phenotype presented in Figure 1D; however, it fits in the data range. Therefore, we think that Figure 1C would not critically hinder the reader’s sound understanding of the information provided.

4. In Figure 1E-M, show magnified boxes in all cases not only in panels F, I and L. This way the phenotypes can be better appreciated.

Response: Thank you for your suggestion. We have now provided magnified insets for the remaining figure panels also in Figure 1E-M.

Response: We do not have quantitative data for this analysis because the Dnd1Δ/Δ ovaries were too tiny for accurate measurement of their weight. However, as the ovaries of the 129 Dnd1Δ/Δ mice were obviously larger than those of the other two strains (Figure 2A-C) and contained a considerable number of oocytes, unlike the ovaries of the other two strains (Figure 2L). We hope that the present data would not hamper readers’ understanding of the data being presented.

6. In Figures 2D-L, provide magnification boxes and marked specific cell types / structures such that the information is more accessible for a broader readership.

Response: We have provided magnification insets for better visualization of the oocytes.

Response: In accordance with the reviewer’s comment, we have changed the labels for the mouse number in Figure 3A–C and 4C.

Response: We have deleted redundant information from the legends of Figures 2, 3, and 4.

9. “…were subjected to flow cytometric analysis performed using antibodies against PLZF, because… “ define PLZF in the text the first time it is mentioned and not only in the methods section.

Response: As per your instructions, we have now defined PLZF at the first instance it is used in the Results section (Lines 370 and 371).

Response: We have rephrased the sentence as “We next checked whether the Dnd1-Δ mutant mice exhibited phenotypes other than those mentioned above.”

Response: To address the point raised by the reviewer, we have revised the sentence “These results support the idea that…” to “These results support the possibility that…”. In addition, we have mentioned the possibility that DND1Ter is still expressed and that this protein alleviates the phenotype of Dnd1 loss causing the phenotypic difference between Δ and Ter in the Discussion section (Lines 505–514).

Response: We are afraid that our figure citations might have led to some confusion. To avoid this confusion, we have now cited Fig. S4A along with Fig. 5A (Line 456). Similarly, we have now cited Fig. S4B along with Fig. 5B (Line 464).

Response: As per the reviewer’s comment, we have rephrased the sentence mentioning the possibility of transdifferentiation to somatic cells (Lines 488–492).

PLoS One. doi: 10.1371/journal.pone.0232047.r003

Decision Letter 1

Stefan Schlatt

7 Apr 2020

Mouse Dead end1 acts with Nanos2 and Nanos3 to regulate testicular teratoma incidence

PONE-D-20-03327R1

Dear Dr. Suzuki,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

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If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

With kind regards,

Stefan Schlatt

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: Yes

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6. Review Comments to the Author

Reviewer #1: Thank you for this excellent and well written paper. I have no additional comments.

The paper is ready for publication.

Reviewer #2: Dear authors.

Thank you for submitting the revised manuscript. My questions were answered approprietly and the suggested corrections were included in the manuscript.

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

PLoS One. doi: 10.1371/journal.pone.0232047.r004

Acceptance letter

Stefan Schlatt

10 Apr 2020

PONE-D-20-03327R1

Mouse Dead end1 acts with Nanos2 and Nanos3 to regulate testicular teratoma incidence

Dear Dr. Suzuki:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

For any other questions or concerns, please email plosone@plos.org.

Thank you for submitting your work to PLOS ONE.

With kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Stefan Schlatt

Academic Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. Comparison of Dnd1 genome structure.

Structures of the Dnd1 (A-E) wild-type allele (A), KO allele generated by Zechel et al. [16] (B), Ter allele [9] (C), floxed allele generated by Suzuki et al. [14] (D), and Δ allele (E).

(PDF)

Click here for additional data file.^{(473.2KB, pdf)}

S2 Fig. Testicular teratomas in the Dnd1-Δ mutant mice of the 129 strain.

(PDF)

Click here for additional data file.^{(889.6KB, pdf)}

S3 Fig. Reduction in spermatogonial number is not related to sperm count decrease and impaired sperm function.

(PDF)

Click here for additional data file.^{(1.1MB, pdf)}

S4 Fig. Testicular teratomas in mice that were double mutants for Dnd1 and Nanos2 or Nanos3 in the 129 strain.

(PDF)

Click here for additional data file.^{(1.5MB, pdf)}

S5 Fig. The 15 genes neighboring Dnd1 and their knockout mouse lines.

(PDF)

Click here for additional data file.^{(764.5KB, pdf)}

S1 Raw images

(JPG)

Click here for additional data file.^{(777.4KB, jpg)}

Data Availability Statement

All relevant data are within the manuscript and its Supporting Information files.

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PERMALINK

Mouse dead end1 acts with Nanos2 and Nanos3 to regulate testicular teratoma incidence

Atsuki Imai

Yoshihiko Hagiwara

Yuki Niimi

Toshinobu Tokumoto

Yumiko Saga

Atsushi Suzuki

Roles

Abstract

Introduction

Materials and methods

Ethics statement

Mice

Histological methods

Tumor surveys

Sperm count

In vitro fertilization (IVF)

Comparison of the PGC number in male gonads at E11.5

Western blotting analyses

Flow cytometry

Statistical analysis

Results

Homozygous for Dnd1-Δ mutant mice were born at Mendelian ratios and developed testicular teratoma in the 129 genetic background

Table 1. Genotype distribution of offspring from Dnd1+/Δ intercrosses in BL6, MCH, and 129 strains.

Table 2. Genotype of Dnd1 and the incidence of male mice affected with testicular teratomas in the BL6, MCH, and 129 strains.

Dnd1 loss caused defects in both spermatogenesis and oogenesis

Fig 1. Comparison of testicular phenotype of Dnd1-Δ mutant male mice among the BL6, MCH, and 129 strains.

Fig 2. Comparison of ovarian phenotype of Dnd1-Δ mutant female mice among the MCH, BL6, and 129 strains.

Dnd1+/Δ male mice progressively lost fertility because of sperm count decrease and impaired sperm function in the 129 strain

Fig 3. 129 Dnd1+/Δ male mice progressively lost fertility because of sperm count decrease and sperm malfunction.

Progressive loss of fertility did not occur in 129 Dnd1+/Ter male mice

Fig 4. Dnd1+/Ter mutant male mice maintained fertility and did not produce a short mutant DND1 protein.

Dnd1 genetically interacted with Nanos2 and Nanos3 for suppression of testicular teratomas

Fig 5. Both Nanos2 and Nanos3 are involved in the regulation of Dnd1-mediated teratoma susceptibility.

Discussion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Stefan Schlatt

Roles

Author response to Decision Letter 0

Decision Letter 1

Stefan Schlatt

Roles

Acceptance letter

Stefan Schlatt

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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Table 1. Genotype distribution of offspring from Dnd1^+/Δ intercrosses in BL6, MCH, and 129 strains.

Dnd1^+/Δ male mice progressively lost fertility because of sperm count decrease and impaired sperm function in the 129 strain

Fig 3. 129 Dnd1^+/Δ male mice progressively lost fertility because of sperm count decrease and sperm malfunction.

Progressive loss of fertility did not occur in 129 Dnd1^+/Ter male mice

Fig 4. Dnd1^+/Ter mutant male mice maintained fertility and did not produce a short mutant DND1 protein.