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The Journal of Reproduction and Development logoLink to The Journal of Reproduction and Development
. 2017 Jul 9;63(5):481–488. doi: 10.1262/jrd.2017-036

Geminin deletion in pre-meiotic DNA replication stage causes spermatogenesis defect and infertility

Yue YUAN 1,2,*, Xue-Shan MA 1,3,*, Qiu-Xia LIANG 1, Zhao-Yang XU 3, Lin HUANG 1, Tie-Gang MENG 1,2, Fei LIN 1, Heide SCHATTEN 4, Zhen-Bo WANG 1,2, Qing-Yuan SUN 1,2
PMCID: PMC5649097  PMID: 28690291

Abstract

Geminin plays a critical role in cell cycle regulation by regulating DNA replication and serves as a transcriptional molecular switch that directs cell fate decisions. Spermatogonia lacking Geminin disappear during the initial wave of mitotic proliferation, while geminin is not required for meiotic progression of spermatocytes. It is unclear whether geminin plays a role in pre-meiotic DNA replication in later-stage spermatogonia and their subsequent differentiation. Here, we selectively disrupted Geminin in the male germline using the Stra8-Cre/loxP conditional knockout system. Geminin-deficient mice showed atrophic testes and infertility, concomitant with impaired spermatogenesis and reduced sperm motility. The number of undifferentiated spermatogonia and spermatocytes was significantly reduced; the pachytene stage was impaired most severely. Expression of cell proliferation-associated genes was reduced in Gmnnfl/Δ; Stra8-Cre testes compared to in controls. Increased DNA damage, decreased Cdt1, and increased phosphorylation of Chk1/Chk2 were observed in Geminin-deficient germ cells. These results suggest that geminin plays important roles in pre-meiotic DNA replication and subsequent spermatogenesis.

Keywords: DNA replication, Geminin, Meiosis, Spermatogenesis

Geminin (Gmnn) was originally identified in Xenopus embryos as a neutralizing molecule that functions during gastrulation and inhibits DNA replication; it is degraded during mitosis [1, 2]. Geminin has dual roles of safeguarding DNA replication and regulating differentiation [3,4,5]. As an unstable regulator, geminin prevents re-licensing in the G2 phase by preventing loading of minichromosome maintenance (MCM) complexes onto replication origins, a reaction mediated by Cdt1 [6,7,8]. As cells exit mitosis, geminin is directly ubiquitinated by the anaphase-promoting complex, permitting replication in the succeeding cell cycle [2]. Geminin is also thought to influence the function of the histone acetylase HBO1, which has the essential function of maintaining chromatin in an acetylated state in MCM recruitment [9]. As a transcriptional molecular switch directing cell fate decisions, geminin acts on genes that are targets of specific epigenetic regulators, such as the SWI/SNF chromatin-remodeling complex and members of the repressive Polycomb group [10,11,12].

The role of geminin has been examined in various developmental stages. Transient deletion of Geminin causes loss of stem cell identity and trophoblast differentiation, which is dependent on intact Brg1 activity [13]. A lack of Geminin also leads to preimplantation mortality, concomitant with morphological abnormalities that are responsible for the arrested development of embryos [14]. In the developing neural tube, ablation of Geminin during the time window between E8.5 and E10.5, when neural plate patterning and neural tube closure occurs, results in neural tube defects, including decreased differentiation of ventral motor neurons [15]. Our recent study showed that deletion of Geminin in mouse oocytes resulted in developmental delay of zygotes [16], with no defects detected in oocyte development, meiotic maturation, ovulation, or fertilization.

Spermatogenesis is a complex developmental process by which male germline stem cells divide and differentiate to produce mature spermatozoa. In mammalian testes, this process occurs within seminiferous tubules and consists of three phases [17, 18]. First, in the proliferative phase, spermatogonia undergo a series of DNA replication cycles as well as mitoses and then differentiate into primary spermatocytes [19]. During the second phase, primary spermatocytes undergo two meiotic divisions to produce haploid spermatids [20]. This phase is subdivided into leptotene, zygotene, pachytene, diplotene, and diakinesis. During the final process of spermatogenesis, which is termed as spermiogenesis, spermatids differentiate into spermatozoa and are released into the lumen of the tubule [21].

The entire process of spermatogenesis is highly coordinated to protect germ cells against high rates of mutation to maintain genome integrity and involves numerous proteins. Geminin is required for the mitotic self-renewal of spermatogonia but does not regulate spermatocyte meiosis or spermiogenesis, which was evaluated previously by using Vasa-Cre and HspA2-Cre [22]. However, because of complete germ cell loss during the first wave of spermatogenesis by P4 and because HspA2-Cre-mediated recombination begins at the leptotene-zygotene stage [23], the functions of geminin during differentiation from spermatogonia to primary spermatocytes remain unclear.

In the present study, we used the Stra8-Cre/loxP system to investigate the functions of geminin in postnatal, premeiotic male germ cells, whose expression begins in the early stage of spermatogonia at P3 and is detected in pre-leptotene spermatocytes [24]. We found that deletion of Geminin led to infertility and germ cell defects in both undifferentiated spermatogonia and spermatocytes. Impaired proliferation, increased H2AX phosphorylation, and elevated apoptosis were detected. We also observed decreased Cdt1 and increased Chk1/Chk2 phosphorylation. These results indicate that geminin is required not only for the mitotic proliferation of spermatogonia, but also for pre-meiotic DNA replication and thus spermatocyte meiosis during spermatogenesis.

Materials and Methods

Mice

Geminin flox/flox (Gmnnfl/fl) mice were obtained from Jackson Laboratory (016913; Jackson Laboratory, Bar Harbor, ME, USA) [25] and maintained in a 129S4/SvJae; C57BL/6J mixed background. Stra8-Cre mice were maintained in a genomic background of C57BL/6J [24]. To improve knockout efficiency, mutant mice were heterozygous for Stra8-Cre (Gmnnfl/Δ; Stra8-Cre) and control mice were homozygous for Geminin floxed allele and Stra8-Cre-negative (Gmnnfl/fl). Mice were housed with 12-h/12-h light/dark cycles and free access to water and food. All animal operations conformed to the Animal Research Committee of the Institute of Zoology, Chinese Academy of Sciences. DNA extraction from mouse tails was conducted to genotype the Gmnnfl and GmnnΔ alleles as well as Stra8-Cre. The identification primer pair for Gmnnfl was: 1) 5′-GCTCAGAGGTTTCAGGG-3′, 2) 5′-CATCAGGTGTTCTCTCAAGTGTCTG-3′ and 3) 5′-GCTACTTCCATTTGTCACGTCC-3′. The primer pair for GmnnΔ was: 4) 5′-CTAGCCACAGATGTTGAGCTTG-3′ and 5) 5′-CTAGATGGGATGTATTGTATGAGAG-3′. The primer pair for Stra8-Cre was: 6) 5′-GTGCAAGCTGAACAACAGGA-3′ and 7) 5′-AGGGACACAGCATTGGAGTC-3′.

Fertility analysis

Fertile Gmnnfl/fl females were mated with 6-week-old Gmnnfl/Δ; Stra8-Cre and Gmnnfl/fl siblings. Two females were housed together with one male. The number and size of the litters were recorded for a 6-month period.

Sperm motility analysis

Spermatozoa were extruded from the cauda epididymis and incubated for 15 min in 500 μl Human Tubal Fluid culture medium at 37ºC and 5% CO2. For sperm motility analysis, a CASA system (Version. 12 CEROS, Hamilton Thorne Research, Beverly, MA, USA) was used.

Immunohistochemistry analysis

Testes were fixed in 4% paraformaldehyde (pH 7.4) at 4ºC overnight, dehydrated in ethanol, and embedded in paraffin. Paraffin-embedded testes were sectioned at a thickness of 5 μm. One or both testes from more than three mice of each genotype were analyzed. After deparaffinization and dehydration, sections were transferred into a sodium citrate solution (0.01 M) in a water bath at 95ºC for 15 min, followed by natural cooling for antigen repair and inactivation of non-specific antigens with 3% H2O2 for 10 min. The samples were washed three times with PBS and blocked with 10% donkey serum for 30 min (serum diluted with PBST, PBST: PBS + 0.1% Tween). These sections were incubated with primary antibodies (Sall4, 1:400, Santa Cruz Biotechnology, Santa Cruz, CA, USA, sc-101147; SCP3, 1:200, Santa Cruz Biotechnology, sc-74569; PCNA, 1:200, Biodragon Immunotechnologies, Beijing, China, B1032) and incubated overnight at 4ºC. The sections were incubated with horseradish peroxidase–conjugated secondary antibodies at 25ºC for 2 h and stained using the DAB chromogenic kit. After staining, the sections were examined with a Nikon microscope and images were captured with a Nikon CCD camera (Nikon, Tokyo, Japan).

Periodic acid Schiff (PAS) staining

Testes were fixed in Bouin’s fixative overnight at room temperature, dehydrated in ethanol, and embedded in paraffin. Paraffin-embedded testes were then sectioned at a thickness of 5 μm. After deparaffinization and dehydration, sections were stained with hematoxylin and Periodic acid Schiff for histological analysis. Images were captured with a Leica Aperio Versa 8 (Leica, Wetzlar, Germany). To count the number of spermatocytes, 3 mice were chosen and germ cells were counted in at least 200 seminiferous tubules observed in cross-sections.

Western blot

Each sample containing 200 mg testicular tissue was added to the tissue lysate containing protease and phosphatase inhibitor, followed by 4ºC lysis for 30 min and centrifugation at 12000 rpm for 20 min at 4ºC. The supernatant was mixed with SDS-PAGE buffer and boiled at 100ºC for 5 min. Western blotting was performed as previously described [26] using an antibody dilution of anti-Geminin (Bioworld Technology, MN, USA, BS7535) at 1:100; β-actin (Zhongshan Golden Bridge Biotechnology, Beijing, China, TA-09) at 1:1000; anti-Cdt1 (Millipore, Billerica, MA, USA, 07-1383) at 1:1000; anti–Pi-Chk1S345 (Cell Signaling Technology, Danvers, MA, USA, 2348) at 1:1000; anti-Pi-Chk2T68 (Bioworld, BS4043) at 1:500; and anti–Pi-H2AXS139 (Cell Signaling Technology, 9718) at 1:1000. The membranes were subsequently incubated with horseradish peroxidase–conjugated secondary antibodies (1:2000; ZB2301 and ZB2305; Zhongshan Golden Bridge Biotechnology) for 1 h at 37ºC. Protein bands were detected using the Thermo Supersignal West Pico chemiluminescent substrate (Waltham, MA, USA).

Immunofluorescence analysis

After deparaffinization and dehydration, sections used for staining were transferred into a sodium citrate solution (0.01 M) water bath at 95ºC for 15 min, followed by natural cooling for antigen repair. The membranes were blocked with 10% donkey serum for 30 min (serum diluted with PBST, PBST: PBS + 0.3% Triton X-100), and then reacted with primary antibodies (MVH at 1:500, Abcam, Cambridge, UK, ab13840; Geminin at 1:50, Santa Cruz Biotechnology; Pi-H2AXS139 at 1:200, Cell Signaling Technology) and incubated overnight at 4ºC. Finally, the membranes were incubated for 1 h at 25ºC with a secondary Alexa Fluor 488-conjugated antibody (1:1000, A11008 and A11055, Life Technologies). After three washes, nuclei were stained with DAPI and the sections were mounted. Fluorescence was examined using a laser-scanning confocal microscope (Zeiss LSM 780, Jena, Germany).

Counting Sall4-positive cells

To count the number of Sall4-positive cells, 2 testis sections were scored per animal and at least 3 mice were used. Germ cells were counted in at least 100 seminiferous tubules observed in cross-sections. Tests sections were scored randomly to avoid experimental bias.

TUNEL assay

A TUNEL assay was conducted using the In Situ Cell Death Detection Kit, Fluorescein (Promega BioSciences, Madison, WI, USA) as recommended by the manufacturer. Images were captured using a laser-scanning confocal microscope (Zeiss LSM 780).

Statistical analysis

All experiments were repeated three times. The data were analyzed with SPSS 17.0 software (SPSS, Chicago, IL, USA) by independent sample t-test. The data in the graphs are presented as the mean ± standard error (S.E.M.) and were considered significant when P < 0.05 (*), 0.01 (**), or 0.001 (***).

Results

Deletion of Geminin gene from male germline

Given that Geminin-null mice are early embryonic lethal [14, 27], we crossed Gmnnfl mice in which exons 5–7 were flanked by two loxP sites with transgenic mice expressing Stra8 promotor-driven Cre recombinase. In Stra8-Cre mice, Cre recombinase was only activated in males at day 3 after birth in early-stage spermatogonia and detected in preleptotene spermatocytes [24]. To examine the deletion efficiency of Geminin, geminin expression in the testes was examined by western blotting and immunofluorescence. Western blot analysis showed that geminin was notably depleted at the protein level (Fig. 1A). As shown in Fig. 1B, geminin was highly expressed in spermatocytes of control testes, whereas little geminin was detected in those from Gmnnfl/Δ; Stra8-Cre mice. These data indicate that geminin was efficiently deleted in Gmnnfl/Δ; Stra8-Cre mouse testes.

Fig. 1.

Fig. 1.

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Deletion efficiency of geminin. A: The protein level of geminin was markedly reduced in Gmnnfl/Δ; Stra8-Cre mice. B: Germ cell staining to show deletion of geminin. Geminin (green) in testes; nuclei were stained with DAPI (blue).

Selective deletion of Geminin in pre-meiotic germ cells results in germ cell loss and infertility

Gmnnfl/Δ; Stra8-Cre mice developed normally with no developmental defects observed until at least 8 months of age. However, adult Gmnnfl/Δ; Stra8-Cre males exhibited markedly smaller testes compared to littermate controls (7-week-old, Fig. 2A), and the testis weight of Gmnnfl/Δ; Stra8-Cre mice was significantly reduced (Fig. 2B, P < 0.001). To investigate the reason for the dramatically decreased testis weight, we performed histological analysis of testes employing immunofluorescence staining. Atrophic tubules and greatly fewer MVH-positive germ cells were observed in Gmnnfl/Δ; Stra8-Cre testes (Fig. 2C), indicating that Geminin plays a critical role in spermatogenesis and that deletion of this gene results in germ cell loss.

Fig. 2.

Fig. 2.

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Deletion of geminin in the testes by Stra8-Cre resulted in massive germ cell loss. A, B: The size and the weight of testes from adult Gmnnfl/Δ; Stra8-Cre mice were significantly smaller than those of control littermates. Testis weight/body weight: Gmnnfl/fl, 0.39 ± 0.03; Gmnnfl/Δ; Stra8-Cre, 0.14 ± 0.02, P < 0.001. C: Germ cells were labeled with anti-MVH antibody (green) and DAPI (blue). Markedly fewer MVH-positive germ cells were observed in the seminiferous tubules from Gmnnfl/Δ; Stra8-Cre testis (white asterisks). D: Histological analysis of the caudal epididymides of Gmnnfl/fl and Gmnnfl/Δ; Stra8-Cre mice. E: The percentage of progressive spermatozoa (S.E.M., N = 3, P < 0.001) and sperm motion variables including average path velocity, straight line velocity, curvilinear velocity, and lateral amplitude were assessed (S.E.M., N = 3, P < 0.05). Gmnnfl/Δ; Stra8-Cre mice showed significantly impaired sperm motility.

To examine Gmnnfl/Δ; Stra8-Cre mouse fertility, adult males were mated with wild-type females for 6 months, while Gmnnfl/fl males were used as a control group. Breeding assays showed that Gmnnfl/Δ; Stra8-Cre mice were completely infertile (Table 1). Most Geminin-deficient males had few spermatozoa in the cauda epididymis. However, a small number of elongating spermatids was observed in both seminiferous tubules (not shown) and the epididymal lumens of Gmnnfl/Δ; Stra8-Cre males (Fig. 2D). We therefore used computer-aided sperm analysis (CASA) to investigate the sperm motility characteristics after sperm incubation for 15 min at 37ºC. The percentage of progressive sperm was substantially reduced in Gmnnfl/Δ; Stra8-Cre mice compared to in the control group. Sperm motion variables including average path velocity, straight line velocity, curvilinear velocity, and lateral amplitude were also assessed, which showed consistent results (Fig. 2E). These results suggest that selective deletion of Geminin from pre-meiotic spermatogonia leads to infertility in mice.

Table 1. Breeding assay of Gmnnfl/Δ; Stra8-Cre males.

Group Number Litters/male in 6 months
(mean ± S.E.M.)		 Pups/litter
(mean ± S.E.M.)
Gmnnfl/fl 7 9.29 ± 0.63 7.14 ± 0.75
Gmnnfl/Δ; Stra8-Cre 6 0 0
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Undifferentiated spermatogonia and spermatocytes are reduced in Geminin-deficient males

To precisely determine which stage of spermatogenesis was affected by Geminin knockout, the expression of undifferentiated spermatogonia marker Sall4 [28] and meiotic marker SCP3 [29] was analyzed. Sall4-positive germ cells were detected in the seminiferous tubules of both control and Geminin-deficient testes (Fig. 3A), but the number of Sall4-positive germ cells was significantly reduced in Gmnnfl/Δ; Stra8-Cre tubules compared to in controls (Fig. 3B, P < 0.01). SCP3-positive germ cells were detected in most seminiferous tubules of control testes and were evenly arranged in the seminiferous epithelium layer. In contrast, the number of SCP3-positive germ cells was dramatically reduced in Gmnnfl/Δ; Stra8-Cre testes based on weaker immunohistochemistry staining (Fig. 3C). Western blot analyses also confirmed that SCP3 was markedly decreased in Gmnnfl/Δ; Stra8-Cre testes (Fig. 3D). To further explore which stage of germ cells was affected by Geminin deletion, PAS and hematoxylin staining were conducted [30]. As shown in Fig. 3E and 3F, the numbers of preleptotene, leptotene, zygotene, pachytene, and diplotene spermatocytes were dramatically reduced. Among these stages, the number of pachytene spermatocytes from Geminin-deficient testes was most severely reduced. These findings demonstrate that specific deletion of Geminin results reduced the number of both undifferentiated spermatogonia and spermatocytes, and that the pachytene stage was the most severely impaired.

Fig. 3.

Fig. 3.

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Undifferentiated spermatogonia and spermatocytes were reduced in Geminin-deficient males. A: Sall4 immunohistochemical analysis of germ cells in Gmnnfl/fl and Gmnnfl/Δ; Stra8-Cre mice. B: Quantification of Sall4-positive germ cells in the seminiferous tubules of Gmnnfl/fl and Gmnnfl/Δ; Stra8-Cre mice. Gmnnfl/fl: 14.09 ± 1.09, Gmnnfl/Δ; Stra8-Cre: 10.31 ± 0.74, N = 100, P < 0.01. C, D: SCP3 immunohistochemical analysis and western blotting indicated a decrease in SCP3 protein level. E: PAS staining analysis of testis sections in Gmnnfl/fl and Gmnnfl/Δ; Stra8-Cre mice. PreL-preleptotene spermatocytes; L-leptotene spermatocytes; Z-zygotene spermatocytes; P-pachytene spermatocytes; D-diplotene spermatocytes; M-meiotic divisions; Rst-round spermatids; Est-elongated spermatids. Scale bar: 50 μm. F: Quantification of preleptotene, leptotene, zygotene, pachytene, diplotene spermatocytes in the seminiferous tubules of in Gmnnfl/fl and Gmnnfl/Δ; Stra8-Cre mice.

Deletion of Geminin results in impaired proliferation, increased H2AX phosphorylation, and apoptosis

To further examine the functions of Geminin in spermatogenesis, immunostaining for the cell proliferation marker proliferating cell nuclear antigen (PCNA) [31] was performed in 8-week-old testis sections. PCNA-positive staining was prominent in control testes, whereas a markedly decreased number of PCNA-positive germ cells was observed in Gmnnfl/Δ; Stra8-Cre mice (Fig. 4A).

Fig. 4.

Fig. 4.

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Deletion of geminin resulted in impaired proliferation, increased H2AX phosphorylation, and elevated apoptosis. A: The number of PCNA-positive germ cells was dramatically reduced. B, C: The expression of γH2AX of control and Gmnnfl/Δ; Stra8-Cre testes was examined by immunofluorescence and western blotting. The level of γH2AX (B, green, white arrowheads) was dramatically increased (B, white asterisks). D: The number of apoptotic cells was markedly increased in Gmnnfl/Δ; Stra8-Cre testes (white arrowheads). Nuclei were stained with DAPI (blue).

It has been reported that Geminin deletion induces DNA damage and elevates apoptosis in oocytes and early embryos [14, 16, 32]. Therefore, the expression of phosphorylated H2AX (γH2AX) was examined by immunofluorescence and western blotting in Gmnnfl/Δ; Stra8-Cre testes. The level of γH2AX was dramatically increased compared to that seen in the control group, showing a stronger fluorescence signal (Fig. 4B). Consistent results were obtained by western blot analysis (Fig. 4C). In addition, numerous TUNEL-positive cells were detected in the seminiferous tubules of Gmnnfl/Δ; Stra8-Cre mice, but not in control testes (Fig. 4D).

Depletion of Geminin decreases Cdt1 but increases phosphorylation of Chk1 and Chk2 in germ cells

The absence of geminin, an inhibitor of Cdt1, results in accumulation and proteolysis of Cdt1, leading to DNA re-replication and triggering the Chk1/Chk2-dependent checkpoint [32,33,34,35]. Western blot analysis of testes showed that Cdt1 was decreased, while the levels of Chk1 and Chk2 phosphorylation were increased (Fig. 5). These results suggest that ablation of Geminin from the male germline induces DNA re-replication and activates a cell cycle checkpoint.

Fig. 5.

Fig. 5.

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Deletion of geminin resulted in decreased Cdt1 and increased phosphorylation of Chk1 and Chk2 in germ cells. Western blot analysis of testes showed that Cdt1 protein was decreased, whereas the levels of Chk1 and Chk2 phosphorylation were increased.

Discussion

We found that specific deletion of Geminin from pre-meiotic spermatogonia resulted in testicular developmental defects and abnormal spermatogenesis. Gmnnfl/Δ; Stra8-Cre males showed complete infertility. Fewer Sall4-positive and SCP3-positive germ cells were observed in Gmnnfl/Δ; Stra8-Cre testes compared to in control males, indicating that both undifferentiated spermatogonia and spermatocytes were affected by ablation of Geminin. The decreased number of PCNA-positive germ cells in Gmnnfl/Δ; Stra8-Cre testes compared to in control males indicates that geminin is required for germline cell proliferation. Changes in the levels of γH2AX, Cdt1 protein, and Chk1/Chk2 phosphorylation suggest that depletion of geminin in germ cells causes DNA damage and damage-induced apoptosis as a result of DNA re-replication, which causes meiotic defects in spermatocytes.

Immunofluorescence results showed that geminin was highly expressed in spermatocytes (Fig. 1B), which is consistent with the results of a previous study [22]. In the study, Geminin was selectively eliminated from spermatogonia by using Vasa-Cre, which is expressed from E15, resulting in the complete spermatogonial loss of Gmnnfl/Δ; Vasa-Cre males during the first wave of spermatogenesis by P4. Additionally, geminin is not essential for male germ cell meiosis or spermiogenesis, as revealed by using the HspA2-Cre-mediated knockout system. Because expression of HspA2-Cre begins in spermatocytes in the leptotene-zygotene stage, in which spermatogonia have already differentiated into primary spermatocytes, the functions of geminin in pre-meiotic DNA replication and subsequent spermatogenesis were unclear. Therefore, in this study, Gmnnfl mice were crossed with transgenic mice expressing Stra8-Cre, which is detected in early-stage spermatogonia from P3 and becomes stronger at P7 through preleptotene-stage spermatocytes, the developmental stage that occurs around the onset of meiosis [24].

Breeding assays indicated that the lack of geminin caused infertility, but elongating spermatids were observed in seminiferous tubules and epididymides. This may be because of incomplete Cre excision; as reported previously, Cre is only active in a subpopulation of undifferentiated spermatogonia [24]. In addition, spermatogonial stem cells undergo continuous self-renewal and differentiation throughout the male reproductive life [36]. The short expression time of Stra8 promoter-driven Cre and sustained spermatogenesis led to the production of spermatozoa. Five DNA repair mechanisms [37], such as nucleotide excision repair, have evolved to maintain genomic integrity to compensate for DNA damage in spermatozoa, which may also play a role. As shown in our study, γH2AX accumulation and increased apoptosis were detected. Additionally, notably stronger signals for γH2AX were observed in the elongated spermatids of conditional knockout mice (Fig. 4B, right panel). This suggests that deletion of geminin leads to DNA damage in elongated spermatids and results in motility defects in spermatozoa [38, 39].

Sall4 is a marker for undifferentiated As, Apr, and Aal spermatogonia, which undergo mitosis [28]. Geminin deletion by the Stra8-Cre conditional knockout system begins in Type A spermatogonia [24]. The decreased number of Sall4-positive spermatogonia suggests that geminin is required for the mitotic proliferation of spermatogonia, which we described previously [22]. A reduced number of spermatocytes in all stages was observed in Geminin-deficient testes and that of pachytene spermatocytes was decreased most severely among these stages. The mid-pachytene checkpoint occurs at testis epithelial stage IV and pairing problems of aberrant chromosomes leads to apoptosis of these spermatocytes [40, 41]. Pachytene spermatocytes from Gmnnfl/Δ; Stra8-Cre testes may therefore undergo apoptosis. A small number of pachytene, diplotene spermatocytes, and round and elongated spermatids were observed in geminin-deficient testes, potentially because of insufficient Cre excision.

Previous studies showed that the main role of geminin is to bind to and inhibit Cdt1 protein [6, 33, 42,43,44,45,46], which contributes to genome stability by rigorously controlling genome replication exactly once per cell cycle [47]. Deletion of Geminin activates Cdt1 and induces DNA re-replication [48, 49]. The overreplication of DNA then activates the ATM/ATR pathway, which triggers cell cycle checkpoint arrest [48], resulting in increased Chk1/Chk2 phosphorylation, as shown in our study (Fig. 5). The lower Cdt1 level may be a consequence of Cdt1 proteolysis [50], possibly because of re-replication-induced DNA damage [51].

In summary, Stra8-Cre-driven germline-specific deletion of Geminin results in infertility and germ cell loss. Geminin, which is required for accurate DNA replication, is important for both spermatogonial self-renewal and meiosis progression and is essential for DNA damage repair during spermatogenesis.

Acknowledgments

We thank the Jing-Pian Peng lab and En-kui Duan lab for technical assistance. This study was supported by the National R&D Program of China (2016YFC1000600) and National Natural Science Foundation of China (30930065) to Q-Y S and Z-B W.

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