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doi: 10.1242/10.1242/dev.00221

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DNA repair gene Ercc1 is essential for normal spermatogenesis and oogenesis and for functional integrity of germ cell DNA in the mouse

Kan-Tai Hsia^1,*, Michael R. Millar², Sasha King², Jim Selfridge¹, Nicola J. Redhead³, David W. Melton^3, and Philippa T. K. Saunders²

¹ Institute of Cell and Molecular Biology, University of Edinburgh, King's Buildings, Mayfield Road, Edinburgh EH9 3JR, UK
² MRC Human Reproductive Sciences Unit, Centre for Reproductive Biology, The Chancellor's Building, University of Edinburgh, 49 Little France Crescent, Edinburgh, EH16 4SB, UK
³ Sir Alastair Currie Cancer Research UK Laboratories, Molecular Medicine Centre, University of Edinburgh, Western General Hospital, Edinburgh EH4 2XU, UK
^* Present address: Faculty of Dentistry, National Yang-Ming University, Taiwan, ROC

View larger version (44K): [in a new window]   Fig. 1. Ercc1 expression in mouse testis. (A) Pattern of Ercc1 expression in mouse tissues. Upper panel: total RNA (30 µg) from a range of tissues was analysed by northern blotting using an Ercc1 cDNA probe [Probe a in Selfridge et al. (Selfridge et al., 2001)]. Lower panel: total protein (80 µg) was analysed by western blotting using an antibody raised against a fragment of mouse Ercc1. (B) Developmental pattern of Ercc1 mRNA in testis. Total RNA (30 µg) extracted from testes of mice (ranging from 8-42 days post partum) was analysed by northern blotting using an Ercc1 cDNA probe. The filter was then reprobed for actin mRNA using a mouse -actin cDNA probe (Minty et al., 1981). The Ercc1/actin mRNA ratio was determined by phosphorimagery and is expressed relative to the p8 sample. (C) Developmental pattern of Ercc1 protein in testis. Total protein (80 µg) extracted from testes of mice (ranging from 8-35 days post partum) was analysed by western blotting using the antibody against Ercc1. Wild type (Wt) and Ercc1 null liver samples were used to demonstrate the specificity of the antibody. The filter was then reprobed with an antibody against mitochondrial core protein II (CP II). The Ercc1/CP II protein ratio was determined by densitometry and is expressed relative to the p8 sample. The ratios shown are the means of two separate determinations on two independent samples at each age. (D) Analysis of Ercc1 transgene expression. Upper panel: total RNA (30 µg) extracted from a range of tissues from transgene-positive Ercc1-deficient mice was analysed by northern blotting using a probe from the 5' end of the mouse transthyretin (Ttr) gene [Probe b in Selfridge et al. (Selfridge et al., 2001)]. mRNA from the endogenous Ttr gene and from the transgene (TG) is indicated. Lower panel: total protein (80 µg) was analysed by western blotting using the antibody against Ercc1.

View larger version (19K): [in a new window]   Fig. 2. Testis weights. Testis weights from 6-week old wild-type and transgene-positive Ercc1 nulls are expressed as a percentage of body weight. 6 animals of each genotype were used. The weights of individual testes from the same animal are displayed together.

View larger version (154K): [in a new window]   Fig. 3. Testes from control and Ercc1-null mice immunostained for the germ cell-specific marker Dazl. (A) 3-day wild type, (B) 3-day Ercc1 null, (C) 12-day wild type, (D) 12-day Ercc1 null, (E) 22-day wild type, (F) 22-day Ercc1 null. The number of germ cells in the Ercc1 nulls was reduced compared with wild-type littermates at all ages examined. The number of germ cells in individual tubules in the nulls was highly variable even within the same testis. Tubules devoid of germ cells (*) and germ cells with abnormal morphology (arrows) in the nulls were seen at all ages.

View larger version (121K): [in a new window]   Fig. 4. Testes from control and transgene-positive Ercc1-deficient mice. (A) 3-week wild type, immunostained for Dazl, containing a normal complement of germ cells including pachytene spermatocytes (P) and round spermatids (R). (B) 3-week transgene-positive Ercc1 null, immunostained for Dazl; note that germ cells are present up to and including pachytene spermatocytes (P), but many tubules are SCO (Sertoli cell only, *). Although germ cell numbers are reduced compared with wild type (A) they are substantially increased compared to age-matched null animals (Fig. 3F). (C) 6-week wild type, Haematoxylin and Eosin stained; full spermatogenesis is present with germ cells at different stages of development arranged in characteristic associations (stages), mature spermatozoa (s) are seen in the centre of the tubule at stage VIII. (D) 7-week transgene-positive Ercc1 null, Haematoxylin and Eosin stained; note that the germ cell complement is variable with some SCO tubules (*), whilst in others, although significant germ cell loss has occurred (arrows point to gaps within the epithelium), germ cells including spermatocytes (arrowheads) and elongate spermatids () are present. (E) 10-week wild-type, plastic section; note very occasional small lipid droplets close to the basement membrane (arrows). (F) 10-week transgene-positive Ercc1 null, plastic section; note reduced diameter of the seminiferous tubules due to reduced germ cell numbers (double-headed arrow) compared with age-matched wild type (E), and accumulation of numerous lipid droplets within the Sertoli cells (arrows). Positive lipid staining was noted in interstitial Leydig cells in both wild-type and Ercc1 null testes (L).

View larger version (73K): [in a new window]   Fig. 5. (A,B) Apoptosis in Ercc1-deficient testes and (B,C) pattern of expression of the Ercc1 protein. (A) 7-week wild type and (B) 7-week transgene-positive Ercc1 deficient testes. Note that apoptosis, visualised by the Apotag assay, was detected in only a few cells in each sample. Apoptotic cells in the wild type (arrows) were stage dependent (stages I and XII), whereas in Ercc1-deficient testis some apoptotic cells were observed at all ages and often occurred in clusters (arrows in B). (C) 9-week wild-type and (D) 9-week transgene-positive Ercc1-deficient testes. Non-specific staining of sperm tails was seen in both samples. Weak immunopositive staining was detected in cell nuclei of Leydig and Sertoli cells from wild-type testis. Examination of specific staining of wild-type germ cells (C) revealed that some immunopositive reaction was present in pre-meiotic germ cells and that the most intense immunopositive reaction was localised to late pachytene spermatocytes (stages IX-XI) and round spermatids (stages I-VII, arrowheads). Very faint specific nuclear staining was detected in a few round spermatids from transgene-positive Ercc1-deficient testis (arrowheads in D) (*, SCO). (E) A summary diagram based on the stages of the spermatogenic cycle (adapted from Oakberg, 1956) showing germ-cell-specific staining for Ercc1. As germ cells proceed through meiosis from pre-meiotic spermatogonia (bottom left) to mature spermatozoa (top right) they are arranged vertically in the seminiferous tubules in characteristic associations (numbered stages) as indicated. The intensity of the immunopositive staining observed is indicated by the intensity of shading: the heaviest shading denotes the intense immunopositive reaction from late pachytene spermatocytes (stages IX-XI) to round spermatids (stages I-VII); the lighter shading denotes the faint staining in pre-meiotic germ cells through to mid pachytene spermatocytes.

View larger version (29K): [in a new window]   Fig. 6. Morphology and comet assay on sperm isolated from 7-week wild-type and Ercc1-deficient mice. Morphology of (A) wild-type and (C) transgene-positive Ercc1-deficient sperm, note abnormal shapes of sperm heads () and presence of tail abnormalities (*). Comet assay of (B) wild-type and (D) transgene-positive Ercc1-deficient sperm. Note increased size of comet tails compared with B. (E) Summary of comet assays. At least 200 sperm from four animals of each genotype were scored. The median value for the percentage of the DNA in the comet tail is shown for each sample, along with the median value (black diamond) for each genotype (+/+, wild type; +/-, Ercc1 heterozygote; -/-, transgene-positive Ercc1 deficient). DNA damage was significantly higher (P<0.05 by Mann-Whitney U test) in transgene-positive Ercc1-deficient than wild-type sperm.

View larger version (169K): [in a new window]   Fig. 7. Ovaries from control and Ercc1-deficient mice. (A) 8-day wild type, (B) 8-day Ercc1 null, (C) 14-day wild type, (D) 14-day Ercc1 null, (E) 6-week wild type, (F) 6-week transgene-positive Ercc1 null mice. Note that numerous primary follicles (arrows) are present in the wild-type samples (A,C,E), but appear to be absent from the ovaries in both types of Ercc1-deficient sample (B,D,F). Organisation of granulosa cells (gc) around oocytes occurred in both wild-type and Ercc1-deficient ovaries. By day 14 some follicles had matured sufficiently to form an antrum (A) in both wild-type and Ercc1-null ovaries (C,D). The development of the most mature follicles appeared less advanced in the null animals on day 14 and in the adult transgene-positive females compared with their wild-type littermates. Oocytes with an abnormal appearance were frequently observed in both types of Ercc1-deficient ovary (*). Scale bar, 100 µm.