Phosphorylation of the Anaphase Promoting Complex activator FZR1/CDH1 is required for Meiosis II entry in mouse male germ cell

Tanno, Nobuhiro; Kuninaka, Shinji; Fujimura, Sayoko; Takemoto, Kazumasa; Okamura, Kaho; Takeda, Naoki; Araki, Kimi; Araki, Masatake; Saya, Hideyuki; Ishiguro, Kei-ichiro

doi:10.1038/s41598-020-67116-0

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Published: 22 June 2020

Phosphorylation of the Anaphase Promoting Complex activator FZR1/CDH1 is required for Meiosis II entry in mouse male germ cell

Nobuhiro Tanno^1,2,
Shinji Kuninaka²,
Sayoko Fujimura³,
Kazumasa Takemoto¹,
Kaho Okamura¹,
Naoki Takeda⁴,
Kimi Araki^4,5,
Masatake Araki⁴,
Hideyuki Saya² &
…
Kei-ichiro Ishiguro¹

Scientific Reports volume 10, Article number: 10094 (2020) Cite this article

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Abstract

FZR1/CDH1 is an activator of Anaphase promoting complex/Cyclosome (APC/C), best known for its role as E3 ubiquitin ligase that drives the cell cycle. APC/C activity is regulated by CDK-mediated phosphorylation of FZR1 during mitotic cell cycle. Although the critical role of FZR1 phosphorylation has been shown mainly in yeast and in vitro cell culture studies, its biological significance in mammalian tissues in vivo remained elusive. Here, we examined the in vivo role of FZR1 phosphorylation using a mouse model, in which non-phosphorylatable substitutions were introduced in the putative CDK-phosphorylation sites of FZR1. Although ablation of FZR1 phosphorylation did not show substantial consequences in mouse somatic tissues, it led to severe testicular defects resulting in male infertility. In the absence of FZR1 phosphorylation, male juvenile germ cells entered meiosis normally but failed to enter meiosis II or form differentiated spermatids. In aged testis, male mutant germ cells were overall abolished, showing Sertoli cell-only phenotype. In contrast, female mutants showed apparently normal progression of meiosis. The present study demonstrated that phosphorylation of FZR1 is required for temporal regulation of APC/C activity at meiosis II entry, and for maintenance of spermatogonia, which raised an insight into the sexual dimorphism of FZR1-regulation in germ cells.

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Introduction

Anaphase promoting complex/Cyclosome (APC/C) controls timely transitions of mitotic cell cycle phases by promoting ubiquitylation and degradation of many key cell cycle regulators¹. APC/C activity is regulated by either of two co-activators CDC20 and FZR1(CDH1), which determine the substrate specificity of ubiquitylation for each cell cycle phase^2,3,4,5. APC/C^CDC20 activity appears in metaphase-to-anaphase transition, when it has an essential function in promoting chromosome segregation by mediating cyclin B1 and securin degradation. APC/C ^FZR1 is thought to regulate a wide range of cell cycle events, in which significant number of proteins have been identified as APC/C^FZR1 substrates^6,7.

While CDC20 plays a role in APC/C activity at metaphase, when the cyclin-dependent kinase 1 (CDK1) activity is high, FZR1 contributes to APC/C activity when CDK1 activity is sustained at a low level^4,8. APC/C^FZR1 activity is negatively regulated by CDK-mediated phosphorylation of FZR1 during mitotic cell cycle. At mitotic exit, reduction of CDK1 activity leads to phosphatase-mediated dephosphorylation of FZR1 which then binds and activates APC/C until late G1 phase⁹. In yeast mitotic cell cycle, FZR1 is phosphorylated by increased level of CDK activity after late G1/S onward, which subsequently leads to dissociation of FZR1 from APC/C and its inactivation^{10,11,12,13,14,15}. In mammals, mitotic cells transiently transfected with mutant FZR1 (CDH1) that lacked potential CDK1 phosphorylation sites, resulted in premature reduction of cyclin A and cyclin B with decrease in G2/M phase-cells^16,17. Thus, CDK-mediated phosphorylation of FZR1 plays a crucial role in temporal regulation of APC/C activity during mitotic cell cycle. However, the physiological significance of FZR1 phosphorylation is yet to be examined in vertebrate mitotic tissues in vivo.

The meiotic cell cycle consists of one round of DNA replication followed by two rounds of chromosome segregation, producing haploid gametes from diploid cells. Although study in budding yeast elucidated that the role of APC/C in meiosis relies on the meiosis-specific co-activator AMA1^18,19, its homolog or counterpart does not exist in mammals. Instead, CDC20 and FZR1 contribute to the regulation of APC/C activity in mammalian meiosis. In mice, it was demonstrated that loss of FZR1 led to abnormal spermatogonial proliferation and defects in progression of meiotic prophase I in both male and female germ cells²⁰. In female meiosis I, FZR1 plays crucial roles in sustaining dictyate arrest of germinal vesicle (GV) oocytes^21,22,23 and in regulating chromosome segregation^{24,25,26,27,28}. These studies indicate that FZR1 is required for mammalian meiosis, but it remains elusive how APC/C activity regulated by phosphorylation of FZR1 is involved in meiotic cell cycle.

Here, we examined whether phosphorylation of FZR1 is required for the regulation of APC/C activity during mitotic and meiotic cell cycle in vivo, using knockin mice that carries non-phosphorylatable mutations of FZR1. Our study demonstrates that phosphorylation of FZR1 is required for maintenance of spermatogonia for a long period of time. Furthermore, we show that FZR1 phosphorylation has a crucial role in the regulation of APC/C activity during meiosis I-meiosis II transition in juvenile male but not in female. Sexual dimorphism in the requirement of FZR1 phosphorylation raised an insight into different modes of meiosis I-to-meiosis II progression in spermatocytes and oocytes.

Results

Generation of Fzr1 ^9A/9A knockin mice

It has been shown that human FZR1 is phosphorylated during mitotic cell cycle. Ectopic expression of non-phosphorylatable FZR1 mutant in HeLa cells resulted in formation of constitutively active APC/C^FZR1 and a reduction of G2/M phase¹⁷. To analyze the physiological role of FZR1-phosphorylation, we generated a mouse model, Fzr1^9A/9A knockin (Fzr1^9A/9A KI) mice, in which the nine putative CDK-phosphorylation sites of Ser/Thr residues in Fzr1 were substituted with alanine amino acids (Fig. 1A–C). Fzr1^9A/9A KI allele was generated by Cre-mediated site-specific recombination of full length cDNA encoding mutant Fzr1^9A into the endogenous Fzr1 locus using an exchangeable gene-trap (GT) line^29,30. For the control mouse line, full length cDNA cassette encoding wild type (WT) FZR1 was inserted into the targeted locus in the same manner, generating Fzr1^{Gt wt/Gt wt} KI mice (Fig. 1A). To distinguish Fzr1^{Gt wt} KI allele from natural WT Fzr1 allele, hereafter we refer to natural WT Fzr1 allele as Fzr1⁺. The expression levels of FZR1-Gt wt and FZR1–9A proteins in the corresponding KI testes were comparable to the FZR1 expression level in natural WT Fzr1^+/+ testes at postnatal day18 (P18) (Fig. 1D). Further, CDC20, APC/C subunits (CDC27/APC3) and the canonical substrates of APC/C (Cyclin B1, PLK1) overall showed similar expression levels in natural WT Fzr1^+/+, Fzr1^9A/9A KI and Fzr1 ^{Gt wt/Gt wt} KI testes (Fig. 1D). We also confirmed Fzr1^{Gt wt/Gt wt} KI mice showed normal fertility as natural WT Fzr1^+/+, indicating Fzr1^{Gt wt} KI allele functions in a manner indistinguishable from natural WT Fzr1⁺ allele. Immunoprecipitation of CDC27/APC3, a core subunit of APC/C, demonstrated that the non-phoshorylatable FZR1–9A and control FZR1-Gt wt proteins expressed from the corresponding KI alleles were indeed incorporated in APC/C of Fzr1^9A/9A and Fzr1^{Gt wt/Gt wt} KI testes, respectively (Fig. 1E). We noticed that the level of CDC20 incorporated in APC/C in Fzr1^9A/9A testis was less than that in natural WT Fzr1^+/+ and Fzr1^{Gt wt/Gt wt} KI testes. This implies that persistent inclusion of FZR1–9A in APC/C impedes the association of CDC20 to APC/C.

Introduction of non-phosphorylatable mutations of FZR1 leads to male infertility

Inter-crossing of heterozygous (Fzr1^9A/+) males and females produced average 7.285 litter size with normal number of litters of Fzr1^+/+, Fzr1^+/9A and Fzr1^9A/9A genotypes expected by mendelian ratio (total Fzr1^+/+:11 pups, Fzr1^+/9A: 28 pups and Fzr1^9A/9A: 12 pups from 7 pairs of intercrossing. p = 0.9557 by chi-square test). The homozygous Fzr1^9A/9A KI mice developed normally without any overt phenotype in adult somatic tissues, suggesting that FZR1–9A showed little impact on mouse development and mitotic cell cycle of mouse somatic tissues.

Notably, although testes in juvenile Fzr1^9A/9A KI males did not show apparent difference from those of control WT Fzr1^+/+ or Fzr1^+/9A, defects in male reproductive organs were evident with smaller-than-normal testes at adulthood (Fig. 2A). Remarkably, testes in the Fzr1^9A/9A KI males at 3 week or older were significantly smaller compared to the age-matched Fzr1^+/+ males (Fig. 2B). Since it is known that the first wave of spermatogenesis completes meiotic prophase and reaches to meiotic divisions generating haploid spermatids at earliest P18, the observation in Fzr1^9A/9A KI males suggests that the primary defect of Fzr1^9A/9A KI males emerges during or immediately after meiotic prophase.

To determine whether the introduction of non-phosphorylatable mutations in FZR1 affected male fertility, 8-week-old Fzr1^9A/9A KI males, control WT Fzr1^+/+ males and Fzr1^{Gt wt/Gt wt} KI males were mated with C57BL6 wild-type or corresponding control females over a period of 4 months. While the control WT Fzr1^+/+ and Fzr1^{Gt wt/Gt wt} KI males produced normal size of litters over this period, the Fzr1^9A/9A KI males showed infertility over a period of 4 months (Fig. 2C). Since the control Fzr1^{Gt wt/Gt wt} KI mice showed normal fertility, it is tenable that the infertility in the Fzr1^9A/9A KI males was attributed to the introduction of non-phosphorylatable mutations in FZR1. Since heterozygous Fzr1^9A/+ KI males showed normal fertility, the phenotype exhibited by the Fzr1^9A KI allele was recessive rather than dominant negative in terms of male fertility (Fig. 2B).

Histological analysis revealed that obvious difference was not observed between Fzr1^9A/9A KI and the control seminiferous tubules at P18 (Fig. 2D), suggesting that the first wave of spermatogenesis normally progressed through meiotic prophase in Fzr1^9A/9A KI testis. However, at 5 week-old onward, spermatozoa were absent in the Fzr1^9A/9A KI seminiferous tubules, whereas spermatogonia, spermatocytes and round spermatid-like cells appeared. This observation was in sharp contrast to those of the age-matched natural WT Fzr1^+/+ and the control Fzr1^{Gt wt/Gt wt} KI males (Fig. 2D). Thus, spermatogenesis was severely impaired in the Fzr1^9A/9A KI seminiferous tubules. Accordingly, spermatozoa were completely absent in Fzr1^9A/9A KI epididymis (Fig. 2E), corroborating the infertility of the Fzr1^9A/9A KI males. These observations suggested that although the germ cells had undergone spermatogonial differentiation and meiosis, they failed to progress spermiogenesis beyond round spermatids in the Fzr1^9A/9A KI seminiferous tubules at younger age. Therefore, we reasoned that spermatogenesis defects in Fzr1^9A/9A KI male could be attributed to the failure of differentiation from spermatocyte to round spermatids or in later stage during the juvenile age. Altogether, phosphorylation of FZR1 is required for normal progression of spermatogenesis.

In contrast to the male phenotype, Fzr1^9A/9A KI females were fertile when mated with heterozygous male, and showed apparently no overt histological defects in adult ovaries (Supplementary Fig. S1A,B), although we could not exclude the possibility that more subtle defects might have occurred in the ovaries besides fertility. Thus the infertility caused by non-phosphorylatable mutations in FZR1 was male specific.

Phosphorylation of FZR1 is required for meiosis I-meiosis II transition in male

Given that spermatogenesis defects in Fzr1^9A/9A KI male might arise during or after meiosis in the juvenile age, we further investigated at which stage the primary defect appeared in juvenile Fzr1^9A/9A KI. We analyzed the progression of spermatogenesis in juvenile Fzr1^9A/9A KI males by immunostaining using stage specific markers. Consistent with above observations, immunostaining analysis with antibodies against SYCP3 (a component of meiotic chromosome axis) along with γH2AX (a marker of double strand break for meiotic recombination) or SYCP1 (a marker of homologous chromosome synapsis) demonstrated that spermatocytes underwent meiotic recombination and homologous chromosome synapsis in juvenile Fzr1^9A/9A KI males, which were comparable to age-matched control (Fig. 3A,B). The Fzr1^9A/9A KI spermatocytes reached mid to late pachytene stage as indicated by testis-specific histone H1t, which were also comparable to age-matched WT (Fig. 3C). Thus, spermatocytes progressed the meiotic prophase normally in juvenile Fzr1^9A/9A KI males. We further investigated the meiotic processes before and after the first meiotic division. Spread nuclei from the testes were immunostained with antibodies against SYCP3 along with MEIKIN, a kinetochore marker of late pachytene-metaphase I³¹. In Fzr1^9A/9A KI, spermatogenesis reached metaphase I (centromeric SYCP3 + /MEIKIN + ) and interkinesis (bar-like SYCP3 + /MEIKIN-)(Fig. 4A), an interphase-like stage between meiosis I and meiosis II³², suggesting that at least meiosis I was complete in Fzr1^9A/9A KI spermatocytes. Notably, among the SYCP3 + spermatocytes, interkinesis spermatocytes were accumulated in Fzr1^9A/9A KI compared to WT (p < 0.0001, chi-square test) (Fig. 4B). Intriguingly, although Fzr1^9A/9A KI spermatocytes reached interkinesis at the most advanced stage, we noticed that atypical secondary spermatocyte-like cells appeared in the Fzr1^9A/9A KI (Fig. 4C). Whereas WT round spermatids mostly exhibited single chromocenters (the clusters of centromeric heterochromatin), Fzr1^9A/9A KI secondary spermatocyte-like cells showed multiple number of chromocenters and exhibited round spermatid-like morphology (Fig. 4D). We also observed a small fraction of elongated spermatid-like cells with aberrant shape in Fzr1^9A/9A KI testis (Fig. 4E). Notably, since Fzr1^9A/9A KI secondary spermatocyte-like cells and spermatid-like cells were PNA lectin (a marker of spermatid) positive (Fig. 4D,E), spermiogenesis program at least in part progressed in Fzr1^9A/9A KI atypical spermatid-like cells. Crucially, flow cytometry analysis of testicular cells from Fzr1^9A/9A KI testes (d28) demonstrated complete absence of the 1 C population, despite the existence of spermatid-like cells (Fig. 4F). Therefore, we reasoned that the observed primary defect in Fzr1^9A/9A KI spermatogenesis derived from a process during or at the entry into meiosis II rather than a failure in post-meiotic differentiation into elongated spermatid. Because we did not observe any population with 1 C DNA content in Fzr1^9A/9A KI, secondary spermatocyte-like cells or spermatid-like cells were produced after normal meiosis I chromosome segregation exhibiting 2 C DNA contents. It is plausible that secondary spermatocytes failed to progress from the interkinesis stage to meiosis II, although spermatid program might have been turned on in Fzr1^9A/9A KI testes. Furthermore, a subpopulation of those secondary spermatocyte-like cells was TUNEL positive in Fzr1^9A/9A KI seminiferous tubules, suggesting that those aberrant secondary spermatocyte-like cells and spermatid-like cells were eliminated at least in part by apoptosis (Fig. 4G).

Phosphorylation of FZR1 is required for the entry into meiosis II in male meiosis

Given that Fzr1^9A/9A KI spermatocytes undergo meiosis I but do not produce 1 C DNA content-spermatids, the secondary spermatocyte-like cells may be a consequence of the failure in meiosis II. Homologous chromosomes are segregated in meiosis I, whereas sister chromatids are separated in meiosis II. In order to examine the chromosome composition in Fzr1^9A/9A KI secondary spermatocyte-like cells, we performed immuno-FISH assays using a probe that detects a specific DNA sequence in the mid-arm region of chromosomes 3. In both WT and Fzr1^9A/9A KI mice, interkinesis spermatocytes showed a pair of FISH signals (Fig. 5A), indicating that homologous chromosomes or sister chromatids were disjoined after meiosis I. In contrast, while WT round spermatids showed a single FISH signal, most of Fzr1^9A/9A KI secondary spermatocyte-like cells showed a pair of FISH signals (Fig. 5B), suggesting that in Fzr1^9A/9A KI interkinesis spermatocytes failed to enter meiosis II. Moreover, immuno-FISH assays using probes that detect a centromere proximal region of X chromosome and whole Y chromosome showed that Fzr1^9A/9A KI secondary spermatocyte-like cells have either X or Y chromosome, but not both (Fig. 5C). This suggests that homologous chromosomes rather than sister chromatids were disjoined in meiosis I in Fzr1^9A/9A KI as normal spermatocytes. Thus, in Fzr1^9A/9A KI male, homologous chromosomes underwent disjunction at meiosis I, but failure in meiosis II entry consequently led to non-disjunction of sister chromatids. These results suggested that phosphorylation of FZR1 is required for the progression from interkinesis to meiosis II in male meiosis. In mitotic cell cycle, constitutively active APC/C^FZR1 reduces cyclin A and cyclin B levels prematurely with decrease in G2/M phase cells¹⁷. Our observations in Fzr1^9A/9A KI male implies that without phosphorylation of FZR1, APC/C^FZR1 substrates might undergo premature degradation, which impairs the entry into meiosis II in male.

Phosphorylation of FZR1 is required for maintaining spermatogonia population

We noticed that the loss of germ cells was more severe in aged Fzr1^9A/9A KI seminiferous tubules, with residual spermatogonia, spermatocytes and Sertoli cells remaining along the basement compartment of the tubules (Fig. 6A), which was accompanied by complete absence of spermatozoa in the epididymis (Fig. 6B). Immunostaining of the aged Fzr1^9A/9A KI seminiferous tubules revealed that TRA98 positive cells were lost while SOX9 positive cells remained in a subpopulation of the tubules, suggesting that germ cell populations were depleted (Fig. 6C). This trend was further augmented at older age in Fzr1^9A/9A KI seminiferous tubules (Fig. 6D). The aged Fzr1^9A/9A KI seminiferous tubules showed a severe loss of male germ cells, which was similar to Sertoli cell-only phenotype. This implied spermatogonia was depleted over a long period of time in Fzr1^9A/9A KI testis. Therefore, it is plausible that phosphorylation of FZR1 is required also for maintaining the spermatogonia population.

Discussion

Little impact of Fzr1^9A/9A KI allele on somatic cells

Contrary to previous observation in human cell culture study^16,17, Fzr1^9A/9A KI mice showed no overt phenotype in mouse somatic tissues. This suggests that somatic cells undergo normal mitotic cell cycle in Fzr1^9A/9A KI mice, despite the constitutive activity of APC/C^FZR1. It is possible that premature degradation of APC/C^FZR1 specific substrates, such as cyclin A, cyclin B, DNA replication regulators (CDC6 and Geminin) and mitosis regulators (Aurora A, Aurora B, PLK1, CDC20 and SGO1) might be compensated by robust balance of cell cycle regulators in somatic tissues in the mutant mice.

It was demonstrated that while maternal store of FZR1 was not essential for completion of oocyte meiosis II following fertilization, loss of function of both maternal and paternal Fzr1 alleles led to developmental defects in early embryo, where aberrant two independent spindles were formed following pronuclear fusion and the chromosomes failed to mix in 1-cell zygotes³³. Thus, FZR1 is essential for development of early embryo. In contrast, since homozygous Fzr1^9A/9A KI animals were normally produced with expected mendelian ratio, it is plausible that maternal store of constitutive active APC/C ^FZR1–9A had little impact on oocyte meiosis or development of early embryo.

Sexual dimorphisms of Fzr1^9A/9A KI phenotype

In male germ cells, it was shown that loss of function of FZR1 led to failure of spermatocytes to progress beyond zygotene and raised higher Cyclin B1 levels, indicating that APC/C ^FZR1 activity is essential for progression of meiotic prophase²⁰. Crucially, constitutively active APC/C ^FZR1–9A showed little impact on the progression of meiotic prophase (Fig. 3) but resulted in failure of spermatocytes to enter meiosis II, consequently producing 2 C DNA-content secondary spermatocyte-like cells (Fig. 7). This phenotype is reminiscent of the observation that large round spermatid-like cells were accumulated in Meikin KO testes, where meiosis II was blocked as a result of failure in reductional chromosome segregation during meiosis I³¹. The presence of PNA positive 2 C DNA-content cells (Figs. 4 and 5) suggested that the Fzr1^9A/9A KI spermatocytes were not simply arrested before meiosis II. At least, the Fzr1^9A/9A KI spermatocytes reached interkinesis but failed to enter meiosis II, which is similar to a phenomenon “mitosis skip” caused by premature activation of APC/C ^FZR1³⁴. FZR1 should be once released from APC/C by CDK-mediated phosphorylation after meiosis I to allow critical substrates accumulate for meiosis II entry. Otherwise, such substrates will be destroyed precociously by constitutively active APC/C ^FZR1. It should be mentioned that despite failure in meiosis II entry, the Fzr1^9A/9A KI spermatocytes still showed an ability to undergo spermiogenesis. This implies that meiotic cell cycle program is genetically separable from spermiogenesis.

In female, loss of function of FZR1 led to impairment in progression of meiotic prophase, and consequently reduced the number of primordial follicle pool²⁰. Furthermore, loss of function of FZR1 accelerated meiotic resumption of GV oocytes^21,22,27, and elevated chromosome missegregation at meiosis I due to failure in removal of SGOL2 from the chromosome arms²⁴. Maternal store of FZR1 is not essential for completion of oocyte meiosis II following fertilization³³.

In female, non-phosphorylatable mutation of FZR1 did not exhibit apparently overt impact on female fertility, showing sexual dimorphism of the Fzr1^9A/9A allele. However, we still do not officially exclude the possibility that constitutively active APC/C ^FZR1–9A may give rise to more subtle effect on meiotic resumption of GV oocytes or chromosome missegregation at meiosis I, which will be a motive for future investigation using in vitro oocyte culture. The observed difference of Fzr1^9A/9A KI phenotypes in male and female might be attributed to the sexual difference in the mode of meiosis I-meiosis II transition. In male, spermatocytes pass through a transient interphase-like state³² with chromosome de-condensation and reassembly of nuclear membrane after completion of meiosis I. In female, chromosomes are persistently condensed without nuclear compartmentalization after completion of meiosis I until metaphase II. Since CDK-mediated phosphorylation of FZR1 inactivates APC/C ^FZR1 through preventing its association with APC/C^{10,11,12,16,17}, it is possible that APC/C ^FZR1-mediated degradation of a critical regulator for male meiosis II entry is prevented by CDK-mediated phosphorylation, which requires further investigations. Alternatively, constitutive binding of FZR1–9A may compete with another activator CDC20 for the association with APC/C before meiosis II, which in turn raises a situation that APC/C ^CDC20 specific substrates fail to undergo destruction in meiosis II.

Impact of Fzr1^9A/9A KI allele on spermatogonial population in aged male

We have also shown that the Fzr1^9A/9A KI testes show Sertoli-cell only phenotype at older age (Fig. 6). Paradoxically, in the absence of APC/C ^FZR1 activity, spermatogonial population was progressively lost at adulthood showing Sertoli-cell only phenotype²⁰. Although the impact on APC/C ^FZR1 activity should be opposite in Fzr1 KO and Fzr1^9A/9A KI testes, the consequent phenotypes were apparently similar between those testes (Fig. 6). Because APC/C ^FZR1 activity prevents mitotic cell cycle from premature S-phase onset and from spontaneously entering quiescence^35,36, it is assumed that spermatogonia undergo precocious quiescence in Fzr1 KO testis²⁰. On the other hand, depletion of germ cells observed in adult Fzr1^9A/9A KI testes (Fig. 6) may imply that constitutive APC/C ^FZR1–9A activity enforces persistent proliferation of spermatogonia, prematurely exhausting spermatogonial population. Thus, CDK-mediated phosphorylation of FZR1 is required for sustaining spermatogonia over a long period of time.

Overall, temporal regulation of FZR1 by phosphorylation is essential for the maintenance of spermatogonia population and meiosis II entry in male germ cells.

Materials and Methods

Animal experiments

Fzr1^9A/9A KI and Fzr1 ^{Gt wt/ Gt wt} KI mice were 129/C57BL6 mixed genetic background. Whenever possible, each knockin animal was compared to littermates or age-matched non-littermates from the same colony, unless otherwise described. Since Fzr1^+/+, Fzr1^{Gt wt/Gt wt} KI, Fzr1^+/9A KI mice showed indistinguishable phenotype in fertility and testes morphology, they were used as controls when compared to Fzr1^9A/9A KI mice. All the animal methods were carried out in accordance with relevant guidelines and regulations. Animal experiments were approved by the Institutional Animal Ethics Committees of Keio University (approval 16037-1) and Kumamoto University (approval F28-078, A2020-006, A30-001, A28-026).

Generation of Fzr1 knock-in mouse and genotyping

Fzr1^+/GT allele was generated in TT2 embryonic stem (ES) cell line³⁷ by integration of the pU-17 exchangeable GT vector²⁹ into the Fzr1 locus³⁰. Characterization of the vector insertion site was performed by 5′ rapid amplification of cDNA ends (5′ RACE) and plasmid rescue experiments. Genotyping of the mutant mice was performed using a PCR protocol based on the primers Gs4 (5′ -CCTCCACTACAGCAGCACG-3′), Gas7 (5′-CTCCAAGGCCTTTGTGAGGC-3′), and SA6as (5′-CCGGCTAAAACTTGAGACCTTC-3′). For detection of the Fzr1–β-geo fusion mRNA, oligo(dT)-primed cDNAs derived from mutant mice were subjected to PCR using the primers 5NC-s (5′-TGTTCCTGGGACCGGCGGGAAC-3′) and LZUS-3 (5′-CGCATCGTAACCGTGCATCT-3′). The amplification product was cloned into the TA cloning vector and sequenced.

To produce ES cells in which the β-geo gene cassette of Fzr1^+/GT cells was replaced with cDNA encoding mouse wild type Fzr1 (Fzr1^{Gt wt}) or Fzr1-9A (Fzr1^9A), we introduced the P17/FZR1 replacement vector together with pCAGGS-Cre³⁸ into Fzr1^+/GT ES cells using electroporation. The P17/FZR1 replacement vector was designed to insert full length cDNA of mouse Fzr1^{Gt wt} or Fzr1^9A between 5′ lox 66 and 3′ lox P. Point mutations that substituted Thr/Ser codons with Ala codons were introduced into Fzr1 cDNA (Eurofins), generating the P17/FZR1-9A replacement vector. After electroporation, ES cells were cultured in medium containing puromycin for 1 day to isolate cell lines that had undergone Cre-mediated recombination. Puromycin selection was performed twice at a 2-day interval. To detect the expression from the Fzr1^wt and Fzr1^9A knock-in (KI) alleles in the recombinant ES cell lines, we performed reverse transcription-PCR (RT-PCR) analysis using the primers 5NC-s2 (5′-TCGAACAGGCGCGGCGTGTT-3′) and mFzr as2 (5′-ATAGTCCTGGTCCATGGTG GAG-3′). The PCR product was cloned into the pGEM-T easy vector (Promega) and sequences were verified. Chimera mice were generated by morula aggregation (host ICR) of recombinant ES cells. Chimeric males were mated to C57BL/6N females and the progenies were genotyped by PCR using the following primers. Gs4 (5′-CCTCCACTACAGCAGCACG-3′) and Gas7 (5′-CTCCAAGGCCTTTGTGAGGC-3′) for the wild-type allele (0.4 kb).

Gs4 and SA6as (5 -CCGGCTAAAACTTGAGACCTTC-3′) for the knock-in allele (0.7 kb).

C57BL-mFzr1-9A GT (Fzr1^9A/9A) and CB-Fzr1-wt GT (Fzr1^{Gt wt/ Gt wt}) knockin mouse lines generated in this study have been deposited to Center for Animal Resources and Development (CARD) with ID3000 and ID1748, respectively.

Preparation of testis extracts and immunoprecipitation

Testis extracts were prepared as described previously³⁹. Briefly, testicular cells were suspended in low salt extraction buffer (20 mM Tris-HCl [pH 7.5], 100 mM KCl, 0.4 mM EDTA, 0.1% TritonX100, 10% glycerol, 1 mM β-mercaptoethanol) supplemented with Complete Protease Inhibitor (Roche). After homogenization, the soluble chromatin-unbound fraction was separated after centrifugation at 100,000 g for 30 min. The solubilized chromatin fraction was collected after centrifugation at 100,000 g for 30 min at 4 °C.

The endogenous APC/C was immunoprecipitated from chromatin-unbound fraction of Fzr1^+/+, Fzr1^{Gt wt/Gt wt}, Fzr1^9A/9A mice testes, using 2 µg of mouse anti-CDC27 antibody and 50 µl of protein A-Dynabeads (Thermo-Fisher). The beads were washed with low salt extraction buffer. The bead-bound proteins were eluted with 40 µl of elution buffer (100 mM Glycine-HCl [pH 2.5], 150 mM NaCl), and then neutralized with 4 µl of 1 M Tris-HCl [pH 8.0].The immunoprecipitated proteins were run on 4-12% NuPAGE (Thermo-Fisher) in MOPS-SDS buffer and immunoblotted. For the immunoblot of testes extracts, whole lysates were prepared in RIPA buffer and run on 8% Laemmli SDS-PAGE in Tris-Glycine-SDS buffer. Immunoblots were detected by secondary antibody VeriblotBlot for IP detection reagent (HRP) (ab131366, abcam, 1:3000 dilution), ECL rabbit IgG HRP-linked F(ab’)2 fragment, ECL mouse (NA9340, GE, 1:3000 dilution) or IgG HRP-linked F(ab’)2 fragment (NA9310, GE, 1:3000 dilution). Immunoblot image was developed using ECL prime (GE healthcare) and captured by FUSION Solo (VILBER).

Antibodies

The following antibodies were used for immunoblot (IB) and immunofluorescence (IF) studies: rabbit anti-Actin (IB, 1:1000, Sigma A2066), mouse anti-CDC27 (IB, 1:1000, Abcam: ab10538), rabbit anti-FZR1 (IB, IF, 1:1000, Abcam: ab118939), rabbit anti-CDC20 (IB, 1:1000, Bethyl:A301-180A), rabbit anti-SOX9 (IF, 1:1000, Milipore: Ab5535), rabbit anti-CyclinB1(IB, 1:1000, Santa Cruz: sc-595), mouse anti-PLK1(IB, 1:1000, Abcam ab17056), rabbit anti-SYCP1 (IF, 1:1000, Abcam ab15090), rabbit anti-γH2AX (IF, 1:1000, Abcam ab2893), rat anti-TRA98 (IF, 1:1000, ab82527), rabbit anti-CENPC³⁹, rabbit anti-MEIKIN (IF, 1:1000)³¹, rat anti-SYCP3⁴⁰, guinea pig anti-H1t (IF, 1:2000, kindly provided by Marry Ann Handel).

Histological analysis

For hematoxylin and eosin staining, testes, epididymis and ovaries were fixed in 10% formalin or Bouin solution, and embedded in paraffin. Sections were prepared on CREST-coated slides (Matsunami) at 6 μm thickness. The slides were dehydrated and stained with hematoxylin and eosin.

For Immunofluorescence staining, testes were embedded in Tissue-Tek O.C.T. compound (Sakura Finetek) and frozen. Cryosections were prepared on the CREST-coated slides (Matsunami) at 8 μm thickness, and then air-dried and fixed in 4% paraformaldehyde in PBS at pH 7.4. The serial sections of frozen testes were fixed in 4% PFA for 5 min at room temperature and permeabilized in 0.1% TritonX100 in PBS for 10 min. The sections were blocked in 3% BSA/PBS, and incubated at room temperature with the primary antibodies in a blocking solution. After three washes in PBS, the sections were incubated for 1 h at room temperature with Alexa-dye-conjugated secondary antibodies (1:1000; Invitrogen) in a blocking solution. For paraffin embedded section, sections were deparaffinized, rinsed in distilled water and incubated with PBS for 30 minutes at 37°C. Then, sections were incubated in PBS containing for Proteinase K for 30 minutes at 37°C and rinsed in distilled water. After treatement, immunofluorescence staining were performed with same procedure of frozen tissue sections.

TUNEL assay was performed using MEBSTAIN Apoptosis TUNEL Kit Direct (MBL 8445). DNA was counterstained with Vectashield mounting medium containing DAPI (Vector Laboratory). Lectin staining was done using Lectin from Arachis hypogaea FITC Conjugate (IF, 1:1000, Sigma: L7381).

Immunostaining of spermatocytes

Spread nuclei from spermatocytes were prepared as described⁴¹. Briefly testicular cells were suspended in PBS, then dropped onto a slide glass together with an equal volume of 2% PFA, 0.2% (v/v) Triton X-100 in PBS, and incubated at room temperature in humidified chamber. The sides were then air-dried and washed with PBS containing 0.1% Triton-X100 or frozen for longer storage at −80 °C. The serial sections of frozen testes were fixed in 4% PFA for 5 min at room temperature and permeabilized in 0.1% TritonX100 in PBS for 10 min. The slides were blocked in 3% BSA/PBS, and incubated at room temperature with the primary antibodies in a blocking solution. After three washes in PBS, the sections were incubated for 1 h at room temperature with Alexa-dye-conjugated secondary antibodies (1:1000; Invitrogen) in a blocking solution. DNA was counterstained with Vectashield mounting medium containing DAPI (Vector Laboratory).

Fluorescence in situ hybridization (FISH) on immunostained nuclei

For immuno-FISH, structurally preserved nuclei (SPN) from spermatocytes were prepared as described⁴¹ with modification. Briefly testicular cells were collected in PBS by mincing seminiferous tubules into small pieces with fine-tipped tweezers and then pipetting. After removal of tissue pieces, the cell suspension was filtered through a Cell strainer (BD Falcon) to remove debris. The cell suspension (~5 μl) was dropped onto a MAS-coated slide glass (Matsunami) and fixed with 10 μl of 2% Paraformaldehyde (PFA)/100 mM sucrose in PBS for 10 min followed by the addition of 1.5 μl of 1.25 M Glycine/PBS, and then air-dried at room temperature. Immediately before they were completely air-dried, the slide glasses were washed with PBS containing 0.1% Triton-X100 or frozen for longer storage at −80 °C.

Immuno-stained samples of SPN were fixed in 4% paraformaldehyde for 5 min, washed with PBS, and subjected to sequential dehydration through 70%, 80%, 90%, 100% ethanol. Immuno-stained SPNs were denatured in 50% formamide, 2x SSC at 72 °C for 10 min. Hybridization was conducted with a fluorescence-labeled point probe in buffer containing 50% formamide, 2x SSC, 20% dextran sulfate at 37 °C for 12-16 h. The slides were washed sequentially at room temperature in 2x SSC for 1 min, 0.4x SSC/0.3% Tween20 solution for 2 min, and 2xSSC at room temperature for 1 min. The mouse point probe derived from BAC clone RP23-6I6 detects the mid region of chromosome 3. A biotinylated point probe that detects X chromosome and a FITC-labeled painting probe that detects whole Y chromosome (MXY-10, Chromosome Science Labo, Hokkaido, Japan) were used. The biotinylated X chromosome point probe was detected by Alexa555-streptavidine (Thermo, S21381).

Imaging

Immunostaining images were captured with DeltaVision (GE Healthcare). The projection of the images was processed with the SoftWorx software program (GE Healthcare). For Fig. S2B, images were captured with Zeiss LSM-710 confocal microscope and processed with ZEN software. All images shown were Z-stacked. Bright field images and immunofluorescent images for counting seminiferous tubules, were captured with BIOREVO BZ-X710 (KEYENCE), and processed with BZ-H3A program.

All methods were carried out in accordance with relevant guidelines and regulations.

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Acknowledgements

The authors thank Tomohiko Wakayama, Yuki Takada (Kumamoto University), Yuzuru Kato (National Institute of Genetics) for technical support and valuable advice. This work was supported in part by KAKENHI grant #16H01257, #16H01221, #17H03634, #18K19304, #19H05245, #19H05743, #20H03265, #JP 16H06276 from MEXT, Japan (to K.I.), the IMEG program of the Joint Usage/Research Center for Developmental Medicine (to H.S.).

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Authors and Affiliations

Department of Chromosome Biology, Institute of Molecular Embryology and Genetics (IMEG), Kumamoto University, Kumamoto, 860-0811, Japan
Nobuhiro Tanno, Kazumasa Takemoto, Kaho Okamura & Kei-ichiro Ishiguro
Division of Gene Regulation, Institute for Advanced Medical Research, Keio University School of Medicine, 160-8582, Tokyo, Japan
Nobuhiro Tanno, Shinji Kuninaka & Hideyuki Saya
Liaison Laboratory Research Promotion Center, IMEG, Kumamoto University, Kumamoto, 860-0811, Japan
Sayoko Fujimura
Institute of Resource Development and Analysis, Kumamoto University, Kumamoto, 860-0811, Japan
Naoki Takeda, Kimi Araki & Masatake Araki
Center for Metabolic Regulation of Healthy Aging, Kumamoto University, Kumamoto, 860-0811, Japan
Kimi Araki

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Nobuhiro Tanno
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Contributions

N.Tanno performed most of the experiments. K.A. and M.A. designed the knockin mice. S.F. performed histological analyses. S.K., K.T., K.O. N.Takeda supported the experiments. H.S. and K.I. supervised experiments and conducted the study. K.I. and N.T. wrote the manuscript.

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Correspondence to Kei-ichiro Ishiguro.

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Tanno, N., Kuninaka, S., Fujimura, S. et al. Phosphorylation of the Anaphase Promoting Complex activator FZR1/CDH1 is required for Meiosis II entry in mouse male germ cell. Sci Rep 10, 10094 (2020). https://doi.org/10.1038/s41598-020-67116-0

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Received: 13 March 2020
Accepted: 03 June 2020
Published: 22 June 2020
DOI: https://doi.org/10.1038/s41598-020-67116-0

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