Expression of Genes Implicated in Stem Cell Function

Manuscript 2003-09-09571B

Supplementary Information

Expression of genes implicated in stem cell function

In Drosophila, several genes have been identified through loss-of-function and transgenic studies as being central to the maintenance of GSC in the ovary, with the best studied being the RNA binding proteins encoded by piwi and pumilio (Lin, H. The tao of stem cells in the germline. Annu. Rev. Genet. 31, 455-491 (1997); Spradling, A.H., Drummond-Barbosa, D. & and Kai, T. Stem cells find their niche. Nature414, 98-104 (2001); Lin, H. The stem-cell niche theory: lessons from flies. Nature Rev. Genet. 3, 931-940 (2002)). In C. elegans, loss of function of either piwi orthologs (prg-1 and prg-2) or pumilio orthologs (fbf-1 and fbf-2) depletes GSC (Cox, D. N. et al. A novel class of evolutionarily conserved genes defined by piwi are essential for stem cell self-renewal. Genes Dev.12, 3715-3727 (1998); Crittenden, S. L. et al. A conserved RNA-binding protein controls germline stem cells in Caenorhabditis elegans. Nature417, 660-663 (2002)), and mammalian orthologs of piwi (miwi/hiwi and mili) and pumilio (pumilio-1 and pumilio-2) are known to exist (Kuramochi-Miyagawa, S. et al. Two mouse piwi-related genes: miwi and mili. Mech. Dev.108, 121-133 (2001); Spassov, D. S. & Jurecic, R. Cloning and comparative sequence analysis of PUM1 and PUM2 genes, human members of the Pumilio family of RNA-binding proteins. Gene299, 195-204 (2002)). Analysis of ovaries collected from mice at various times during neonatal, juvenile and adult life revealed expression of mili, pumilio-1 and pumilio-2, with mili showing an age-related decline in its levels of expression (Fig. S4). In addition, expression of nucleostemin, a gene recently implicated in stem cell renewal in mammals (Tsai, R. Y. L. & and McKay, R. D. G. A nucleolar mechanism controlling cell proliferation in stem cells and cancer cells. Genes Dev.16, 2991-3003 (2002)), was also identified in the mouse ovary during neonatal, juvenile and adult life (Fig. S4). Although these findings indicate that several genes implicated in stem cell development and function in other organisms or cell lineages are expressed in the postnatal mouse ovary, the relevance of any of these genes to mammalian female GSC remains to be established.

Materials and Methods

Histomorphometrics (Oocyte and Follicle Counts)

Ovaries were fixed (0.34 N glacial acetic acid, 10% formalin, 28% ethanol), paraffin embedded, serially sectioned (8 m), aligned in order on glass microscope slides, and stained with hematoxylin and picric methyl blue. The number of non-atretic primordial, primary and preantral follicles in every fifth section with random start was then determined as detailed previously (Perez, G. I. et al. Prolongation of ovarian lifespan into advanced chronological age by Bax-deficiency. Nature Genet.21, 200-203 (1999); Morita, Y., Perez, G. I., Maravei, D. V., Tilly, K.I. & Tilly, J. L. Targeted expression of Bcl-2 in mouse oocytes inhibits ovarian follicle atresia and prevents spontaneous and chemotherapy-induced oocyte apoptosis in vitro. Mol. Endocrinol.13, 841-850 (1999)). Briefly, primordial follicles were identified as having a compact oocyte surrounded by a single layer of flattened granulosa cells, while primary follicles were identified as having an enlarged oocyte surrounded by a single layer of cuboidal granulosa cells. Intermediate-stage follicles (compact or enlarged oocyte with a single layer of mixed flattened and cuboidal granulosa cells) were scored as primary. Preantral follicles were identified as having an enlarged oocyte surrounded by at least a partial or complete second layer of cuboidal granulosa cells but no more than four complete layers of cuboidal granulosa cells. Only those follicles containing an oocyte with a clearly visible nucleus were scored. Follicles at the primordial, primary and preantral (immature) stages of development were scored as atretic if the oocyte was degenerating (convoluted, condensed) or fragmented. Grossly atretic follicles lacking oocyte remnants were not included in the analyses. Given that this procedure samples one-fifth of the entire ovarian volume, the total number of follicles per ovary (healthy or atretic) was then estimated by multiplying the cumulative counts for each ovary by a correction factor of five (Zuckerman, S. The number of oocytes in the mature ovary. Recent Prog. Horm. Res.6, 63-108 (1951); Tilly, J. L. Ovarian follicle counts – not as simple as 1, 2, 3. Reprod. Biol. Endocrinol.1, 11 (2003)). All counts were conducted by a single trained ovarian histologist in a blinded fashion, and two other members of the group periodically evaluated random samples to verify accuracy and reproducibility of the data.

Immunohistochemistry

After fixation in 4% neutral-buffered paraformaldehyde and embedding in paraffin, 6-m tissue sections were cut from ovaries and mounted on slides. The sections were de-waxed in xylenes, re-hydrated, and boiled for 5 min in 10 mM sodium citrate using a microwave. For colorimetric MVH detection, sections were blocked for 30 min in TNK solution (100 mM Tris HCl, pH 7.6; 0.55 M NaCl; 10 mM KCl; 0.02% bovine serum albumin, 1% normal goat serum), followed by incubation with a 1:1,200 dilution of rabbit anti-MVH antibody (Fujiwara, Y. et al. Isolation of a DEAD-family protein gene that encodes a murine ortholog of Drosophila vasa and its specific expression in germ cell lineage. Proc. Natl. Acad. Sci. USA91, 12258-12262 (1994)) in TNK solution for 1 h at room temperature. After three 10-min washes in 1X-PBS, a biotinylated anti-rabbit IgG/streptavidin-peroxidase conjugate system, with diaminobenzidine as the colorimetric substrate, was used as per the manufacturer’s instructions (Vectastain ABC Elite Kit; Vector Labs) for antigen detection. Sections were lightly counterstained with hematoxylin prior to sealing with coverslips. Fluorescence-based immunostaining for MVH was conducted as described above, with the exception that a 1:400 dilution of Alexa Fluor 488-conjugated goat anti-rabbit IgG (Molecular Probes) was used in place of the biotinylated anti-rabbit IgG/streptavidin-peroxidase conjugate system for antigen detection.

Dual fluorescence immunostaining for MVH and BrdU was performed as described above, with the following modifications. A biotinylated anti-BrdU antibody (Zymed) was added to the sections as per the manufacturer’s instructions followed by three 10-min washes in 1X-PBS. Sections were then incubated with MVH antiserum (see above), followed by three additional 10-min washes in 1X-PBS. Alexa Fluor 546-conjugated streptavidin (Molecular Probes) and Alexa Fluor 488-conjugated goat anti-rabbit IgG, each at a 1:400 dilution in TNK solution, were then applied to the sections. After washing, sections were mounted in anti-fade medium (Vectashield, Vector Labs) and visualized using appropriate excitation and detection wavelengths.

Immunostaining for SCP3 was performed essentially as described for the colorimetric detection of MVH, with the following modifications. Normal donkey serum was used in place of normal goat serum in the TNK solution for blocking, and a 1:300 dilution of a goat anti-SCP3 antibody (Walpita, D., Plug, A. W., Neff, N. F., German, J. & Ashley T. Bloom's syndrome protein, BLM, colocalizes with replication protein A in meiotic prophase nuclei of mammalian spermatocytes. Proc. Natl Acad. Sci. USA96, 5622-5627 (1999); Russell, L. B., Hunsicker, P. R., Hack, A. M. & Ashley, T. Effect of the topoisomerase-II inhibitor etoposide on meiotic recombination in male mice. Mutat. Res.464, 201-212 (2000)) was applied to the sections followed by a biotinylated donkey anti-goat IgG (Santa Cruz Biotechnology) for detection using the streptavidin-peroxidase conjugate system with diaminobenzidine as the colorimetric substrate, as described above. To prevent masking of the immunoreaction signal with vital dyes, photomicrographs of the sections were taken under Hoffman optics without prior counterstaining.

Experimental design and data analysis

All experiments were independently replicated at least 3 times, using different mice to collect the tissues for each experimental replicate. Quantitative data shown, which represent the mean ± S. E. of combined results from replicate experiments, were analyzed by a one-way ANOVA followed by the Tukey-Kramer post-hoc test for statistical differences between mean values. Qualitative data shown are representative of results obtained in replicate experiments.

Table S1 Developmental degeneration and clearance of oocytes in the neonatal mouse ovary.

Table S2. Details regarding the RT-PCR analysis of gene expression.

Dmc1, -d3

1 F, forward primer; R, reverse primer

2 Nucleotide numbering corresponds to the A of the start codon (ATG) being designated as nucleotide 1.

3 The Dmc1-d splice variant has been reported (Habu, T., Taki, T., West, A., Nishimune, Y. & Morita, T. The mouse and human homologs of DMC1, the yeast meiosis-specific homologous recombination gene, have a common unique form of exon-skipped transcript in meiosis. Nucleic Acids Res.24, 470-477 (1996)) but the sequence has not yet been deposited in GenBank.

Supplementary Figure Legends

Figure S1 Grafted wild-type ovarian tissue adheres to transgenic host ovarian tissue and becomes vascularized. a, b, Gross morphology of a representative ovarian graft at 3 weeks post-surgery, prior to (a) and after (b) removal from the bursal cavity. Once removed from the bursa, neovascularization, as well as adhesion of the grafted ovarian tissue (broken white line) to the host ovary, can be easily visualized. c-f, Gross histological appearance of representative ovarian grafts (broken white line) as viewed under light (c, e) and fluorescence (green fluorescent protein, GFP; d, f) microscopy at 3-4 weeks post-surgery. Note that GFP-positive cells appear to infiltrate the grafted tissue by this time, although a low level of autofluorescence was noted in scattered somatic cells of wild-type ovarian tissue prior to grafting (Fig. S2).

Figure S2 Wild-type ovarian tissue exhibits a low level of background autofluorescence. Representative photomicrographs of transgenic (tg) and wild-type (wt) ovarian tissue analyzed by fluorescence microscopy for GFP expression prior to grafting, demonstrating sporadic autofluorescence in scattered somatic cells of the wild-type tissue. However, autofluorescence was never observed in existing oocytes (asterisk) of wild-type ovaries. PI, propidium iodide counterstaining.

Figure S3 Additional example of folliculogenesis in grafted ovarian tissue. These data show two adjacent immature follicles composed of GFP-transgenic oocytes and wild-type granulosa cells present within wild-type (wt) ovarian tissue 4 weeks after grafting into the ovarian bursal cavity of a GFP-transgenic recipient female (PI, propidium iodide counterstaining). Note also the presence of GFP-positive cells within the theca-interstitial cell layers of the follicles in the grafted tissue (see also Fig. 5). Given that the blood supply of developing follicles is restricted to these outer cell layers, these cells are presumed to be host-derived microvascular endothelial cells that orchestrated neovascularization of the graft. In all grafts, GFP-positive somatic cells were also observed in the ovarian stromal cell compartment, indicative of host stromal cell and/or immune cell infiltration into the grafted tissue during the 3-4 week period following transplantation.

Figure S4 Postnatal ovarian expression of stem cell-associated genes. a, RT-PCR analysis of mili, pumilio-1 (pum1), pumilio-2 (pum2) and nucleostemin expression in mouse ovaries collected at the indicated days of age or at 8 months (8m) postpartum. The ribosomal gene, L7, was co-amplified as an internal control; no product was observed in mock-transcribed (Mock) ovarian RNA samples. b, Tissue distribution analysis of the genes indicated above in RNA samples prepared from ovaries, brains, hearts, kidneys, lungs and spleens of female mice at 40-42 days of age postpartum. Additional details on PCR primers and product sequence information are provided in Table S2.