Hedgehog Signaling in Mouse Ovary: Indian Hedgehog and Desert Hedgehog from Granulosa Cells Induce Target Gene Expression in Developing Thec
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内分泌学杂志 2005年第8期
Department of Reproduction and Development, Erasmus University Medical Center, 3000 DR Rotterdam, The Netherlands
Address all correspondence and requests for reprints to: Dr. Mark Wijgerde, Department of Reproduction and Development, Erasmus University Medical Center, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands. E-mail: m.wijgerde@erasmusmc.nl.
Abstract
Follicle development in the mammalian ovary requires interactions among the oocyte, granulosa cells, and theca cells, coordinating gametogenesis and steroidogenesis. Here we show that granulosa cells of growing follicles in mouse ovary act as a source of hedgehog signaling. Expression of Indian hedgehog and desert hedgehog mRNAs initiates in granulosa cells at the primary follicle stage, and we find induced expression of the hedgehog target genes Ptch1 and Gli1, in the surrounding pre-theca cell compartment. Cyclopamine, a highly specific hedgehog signaling antagonist, inhibits this induced expression of target genes in cultured neonatal mouse ovaries. The theca cell compartment remains a target of hedgehog signaling throughout follicle development, showing induced expression of the hedgehog target genes Ptch1, Ptch2, Hip1, and Gli1. In periovulatory follicles, a dynamic synchrony between loss of hedgehog expression and loss of induced target gene expression is observed. Oocytes are unable to respond to hedgehog because they lack expression of the essential signal transducer Smo (smoothened). The present results point to a prominent role of hedgehog signaling in the communication between granulosa cells and developing theca cells.
Introduction
IN THE MAMMALIAN OVARY, developing follicles represent the functional units that carry out and coordinate the two most important ovarian functions, gametogenesis and steroidogenesis (1, 2, 3). During adult reproductive life, recruitment of follicles from the primordial pool is a continuous process that results in formation of primary follicles and sets the basis for subsequent follicle development (4). In a developing follicle, proliferation and differentiation of the somatic granulosa and theca cell compartments, and development of the oocyte, are highly coordinated. This requires intercellular communication between these cell types and compartments (5). When a primordial follicle enters the pool of growing primary follicles, the flattened granulosa cells become cuboidal and change from a quiescent to a proliferative state (6). From this stage, granulosa cells secrete proteins like stem cell factor (or Kit ligand) and anti-Müllerian hormone (Amh), which are implicated in control of follicle development (7, 8, 9). In a primary follicle, the oocyte starts to produce growth differentiation factor (Gdf) 9. As shown by the ovarian phenotype of Gdf9 knockout mice, this factor is important for development of follicles beyond the primary follicle stage and formation of the first theca cells (10). Pre-theca cells originate from mesenchymal precursor cells within the ovarian cortical stroma around the primary follicles but do not emerge as fully differentiated steroidogenic theca cells before the onset of the preantral follicle stage (11). The role of Gdf9 involves action on granulosa cells (12). Little is known about factors from granulosa cells involved in formation and differentiation of pre-theca cells.
Hedgehog proteins form a small and highly conserved family of intercellular signaling molecules, whose first known member was initially described as a protein encoded by a segment polarity gene in Drosophila, and that protein is now known to play several distinct roles in fly development, including gonadal development and function (13, 14, 15, 16). Mammals have three hedgehog proteins: sonic hedgehog (Shh), Indian hedgehog (Ihh), and desert hedgehog (Dhh), which all show conservation of structure and function with the single hedgehog protein in Drosophila (14, 17). In mammalian embryos, the different hedgehog proteins play various roles in control of developmental processes, and the proteins are implicated in the etiology of several human congenital malformations and cancers (15, 18). Shh is most intensely studied and was found to be involved in establishment of the early left-right axis, regulation of ventral cell fates in the central nervous system, specification of the anterioposterior axis in developing limb, and morphogenesis of a variety of organs (15, 19). Ihh and Dhh have more restricted functions (20). An important role for Dhh in testis development was detected by analysis of the phenotype of Dhh knockout mice, which points to Dhh signaling in Sertoli-Leydig cell communication. However, that study did not reveal a role for Dhh in the ovary (21).
The amino acid sequences of Shh, Ihh, and Dhh are highly similar, and all three hedgehog proteins can evoke similar mechanistic responses in a number of in vitro and in vivo settings (17, 22). Shh, Ihh, and Dhh have comparable affinities for the same plasma membrane proteins involved in hedgehog signaling: patched homolog (Ptch) 1 and Ptch2 and hedgehog interacting protein 1 (Hip1) (22, 23). At the cell surface, the 12-transmembrane protein Ptch1 and its close homolog Ptch2 act as common hedgehog receptors, whereas the membrane-bound glycoprotein Hip1 binds hedgehog proteins, thereby preventing receptor binding (24). In the absence of ligand, Ptch1 represses signaling activity of the seven-transmembrane protein Smo (smoothened). Virtually all cells will express a low basal level of Ptch1, which provides inhibitory control of signaling by Smo. However, hedgehog binding functionally inactivates Ptch1, making Smo available to initiate intracellular signaling. Cyclopamine, an exogenous teratogenic compound, binds Smo and acts as a highly specific antagonist of hedgehog signaling (25, 26) (Fig. 1A). Signaling by Smo transduces all hedgehog signals, using the transcription factors Gli1, Gli2, and Gli3 as downstream effectors (27, 28, 29). Gli proteins are included in a complex that associates with the cytoskeleton and keeps full-length Gli out of the nucleus. This complex targets Gli2 and Gli3 for cleavage, producing shorter C-terminal forms containing the Zn-finger, which act as transcriptional repressors (30, 31, 32). Gli1 is not a target for cleavage and acts as a constitutive activator of transcription (Fig. 1A).
FIG. 1. Hedgehog signaling pathway. A, General representation of the hedgehog signaling pathway and its components: ligands (Shh, Ihh, and Dhh); membrane bound receptors (Ptch1, Ptch2); membrane-associated signal transducer (Smo); and intracellular transcriptional effectors (Gli1, Gli2, and Gli3). The exogenous teratogenic compound cyclopamine acts as a highly specific antagonist by blocking signaling, by direct binding to Smo. B, Northern blot analysis of expression of mRNAs encoding components of the hedgehog signaling pathway in 10.5dpc mouse embryos, adult mouse testis, and adult mouse ovary.
A functionally highly relevant aspect of hedgehog signaling is that several genes encoding components of the hedgehog signaling pathway are targets of hedgehog signaling. Exposure of cells, which will express a low basal level of Ptch1 to control Smo, to hedgehog proteins leads to strong induction of expression of the genes encoding Ptch1, Ptch2, Hip1, and Gli1 (24, 33, 34, 35). Induction of the transcription factor Gli1 provides the system with a transcriptional stimulus, which is embedded within a negative signaling feedback loop generated by up-regulation of the signaling antagonists Ptch1, Ptch2, and Hip1. This endows the hedgehog signaling network with a robust switch-like mechanism, which is essential to control induction events in embryogenesis (36, 37, 38).
Here we present evidence that Ihh and Dhh produced by granulosa cells act as ovarian paracrine factors, inducing target gene expression in the developing theca cell compartment. This suggests a role of hedgehog signaling in theca cell development in growing follicles, similar to the role of Dhh in Leydig cell development in testis (21, 39, 40). Oocytes take a special position because they are probably exposed to Ihh and Dhh from the surrounding granulosa cells, and they express the receptor Ptch1 but lack the signal transducer Smo.
The present results associate, for the first time, the hedgehog signaling pathway with somatic cell communication and cell differentiation events in mouse ovary.
Materials and Methods
Experimental animals
All experimental mice were of FVB/n strain and maintained in accordance with accepted standards of humane animal care as outlined in Ethical Guidelines for Care and Use of Laboratory Animals on a regular light-dark cycle (lights on between 0500 and 1900 h). For induction of superovulation, 22 d post partum (dpp) female mice were injected ip with 150 μl Folligonan (pregnant mare serum gonadotropin; 50 U/ml) between 1300 and 1400 h to stimulate follicular growth, followed after 47 h by ip injection of 150 μl Chorulon (chorionic gonadotropin; 50 U/ml) to induce ovulation and luteinization. Ovulation is expected to occur between 2200 and 2400 h; none of the superovulated females were set up for mating.
RNA isolation, Northern blotting, and RT-PCR analysis
Total RNA was extracted from 10.5 d post coitum (dpc) mouse embryos and adult (2.5 month old) mouse testis and ovaries using Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Enrichment for polyA+ RNA was performed using Dynabeads mRNA direct kit (Dynal, Oslo, Norway); an amount of 3 μg of polyA+ RNA was loaded in each lane, and the RNA was electrophoresed through a 1% agarose 3[N-morpholino]propanesulfonic acid-formaldehyde gel. Subsequently the RNA was transferred to nylon membrane and probed with 32P-labeled DNA probes generated by random priming of (partial) cDNA fragments for mouse Shh, Ihh, Dhh (17, 41), Ptch1 (34), Ptch2 (42), Smo (29), Gli1, Gli2, Gli3 (42), and a 430-bp mouse genomic DNA PstI fragment of the mouse Amh gene, containing exon 1 (43). Blots were hybridized with 2 x 106 cpm/ml hybridization buffer [50% vol/vol formamide, 7% (wt/vol) dextran sulfate, 5x saline sodium citrate (SSC), 5x Denhardt’s, 20 mM sodium phosphate buffer (pH 6.8), 1% (wt/vol) sodium dodecyl sulfate (SDS), 100 mg/ml denatured salmon sperm DNA, and 100 mg/ml yeast tRNA] at 65 C overnight (16 h). Blots were washed sequentially in 2x SSC/0.1% SDS and 0.2x SSC/0.1% SDS at 65 C and exposed to phosphoimager screens.
Oocytes from 14dpp and 21dpp mice were isolated by mechanical disruption of ovaries and follicles, followed by incubation in trypsin (1 mg/ml), collagenase (1 mg/ml), hyaluronidase (1 mg/ml), and DNaseI (10 μg/ml) in M2 medium (Sigma, St. Louis, MO) at 37 C. Superovulated oocytes were isolated by incubation of cumulus-oocyte complexes in hyaluronidase (1 mg/ml) and DNaseI (10 μg/ml) in M2 medium at 37 C. For each sample, 20 oocytes were collected in 50 μl TRIZOL, snap frozen and thawed twice, and total RNA precipitated in the presence of 0.2 μg/μl glycogen (Invitrogen). Samples were incubated with 1 U/μl RNase-free DNaseI (Promega, Fitsburg, WI) for 30 min at 37 C before reverse transcription. First-strand cDNA synthesis was performed using the Superscript first-strand synthesis system for RT-PCR (Invitrogen) using random hexamers as primers. An unfertilized oocyte was assumed to contain 0.43 ng total RNA, which was used to match an equivalent amount of whole-ovary RNA. PCR was performed using platinum Taq DNA polymerase (Invitrogen) in 20-μl aliquots at 94 C for 3 min, followed by 94 C for 0 sec, 55 C for 30 sec, 72 C for 30 sec, for 40 cycles; and finally 72 C for 10 min; and storage at 4 C until samples were analyzed on a 2% agarose gel in 0.04 M Tris-acetate, 1 mM EDTA buffer. Primer sets were designed to cross intron sequences to distinguish between genomic DNA and cDNA products. Sequences used to target mouse cDNAs can be found in the supplement to Materials and Methods (published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org).
In situ hybridization and immunohistochemistry
Mouse ovaries were prepared and hybridized with 35S-uridine 5-triphosphate-labeled RNA probes as previously described (44, 45, 46). To compare expression of different mRNAs, we used serial ovarian sections of 8 μm thickness. These sections were hybridized with 35S-uridine 5-triphosphate-labeled probe sequences generated by transcription of (partial) cDNA fragments for mouse Shh, Ihh, Dhh (17, 41), Ptch1 (34), Ptch2 (42), Smo (29), Gli1, Gli2, Gli3 (42), Hip1 (24), and a 430-bp mouse genomic DNA PstI fragment of the mouse Amh gene, containing exon 1 (43); all cDNA fragments were sequenced to verify the identity. As negative controls, antisense riboprobes were applied; this never resulted in any specific signal above background (not shown). Sections were counterstained with hematoxylin, and images were taken using an Axioplan 2 (Zeiss, Gottingen, Germany) equipped with a CoolSNAP-Pro color charge-coupled device camera (Media Cybernetics, Wokingham, UK).
For immunohistochemistry, adult mouse (2.5 months old) ovaries were dissected and fixed overnight in 4% paraformaldehyde in PBS at 4 C, dehydrated, paraffin embedded, sectioned (8 μm), and mounted on SuperFrost Plus microscope slides. Immunohistochemistry was performed as previously described (9), using anti-Amh (C-20, Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1:1000 and antibody AB80 against vertebrate hedgehog proteins (47).
Organ culture of ovaries from neonatal mice
Intact ovaries from 2dpp mice were organ cultured for 6 d as previously described (48, 49). In brief, each dissected ovary was placed on a quarter piece of a Millicell-CM membrane (0.4 μm pore size; Millipore Corp., Medford, MA) floating on top of 750 μl culture medium, all contained within a single well of a 4-well plate (Nunc, Roskilde, Denmark). Ovarian culture medium was Waymouth MB752/1 + L-glutamine (Life Technologies, Inc.-Invitrogen, Paisley, Scotland, UK) supplemented with 0.23 mM pyruvic acid (Sigma), 0.5 x antibiotic/antimycotic solution (Life Technologies-Invitrogen), 3 mg/ml BSA (True Cohn crystalline powder; MP Biomedicals, Irvine, CA), and 10% (vol/vol) fetal calf serum. Cyclopamine and its derivative 3-keto-N-aminoethyl-aminocaproyl-dehydrocinnamoyl (KAAD)-cyclopamine inhibit hedgehog pathway activation by binding directly to Smo (26). The KAAD derivative has a 10- to 20-fold higher potency than cyclopamine (25) and was obtained from Toronto Research Chemicals (Toronto, Canada). A stock solution of KAAD-cyclopamine was prepared by dissolving 1 mg in 250 μl methanol (5.7 mM), which was stored at –20 C. Ovaries were cultured in the absence (control; methanol) or presence of 57 nM and 0.57 μM KAAD-cyclopamine. The cultures were incubated at 37 C and gassed with 5% CO2 in air. Culture medium was refreshed every other day, and ovaries were fixed in 4% paraformaldehyde/PBS after 6 d of culture and subsequently subjected to in situ hybridization analysis as described above.
Results
Mouse ovary expresses a relatively high level of Ihh mRNA
To obtain a precise quantitative and qualitative indication of mRNA expression, we performed Northern blot analysis of adult mouse whole-ovary mRNA (poly-A+ RNA). For reasons of comparison, we also included mRNA from 10.5dpc mouse embryos and adult mouse testis, in which we detected mRNAs for Shh and Dhh, respectively (Fig. 1B). This confirms published data (17, 21). Like the adult testis, the adult ovary also contains Dhh mRNA. However, in the ovary we find a relatively high level of Ihh mRNA, which is not expressed at a detectable level in the testis (Fig. 1B).
In embryo, testis, and ovary, mRNAs encoding Ptch1, Ptch2, and Smo are present (Fig. 1B). The Northern blot signal for Smo mRNA in adult testis tissue is quite low, but this is explained by developmental dilution of Smo mRNA when the developing testis becomes populated with an increasing number of spermatogenic cells with no or a very low level of Smo mRNA; in prenatal (18.5dpc) and postnatal (8.5dpp) testis tissue, a much higher expression of Smo mRNA was detected (not shown).
The presence of Ihh, Dhh, and Smo mRNAs in mouse ovary, together with a significant level of mRNA transcribed from the hedgehog target gene Ptch1, provides a first indication for an active hedgehog signaling pathway.
Ihh and Dhh mRNAs are located in granulosa cells of preantral and antral follicles, and Ptch1 expression is induced in the adjacent theca cell compartment
For cellular localization of the expression of Ihh and Dhh mRNAs, and expression of the target gene Ptch1, we carried out in situ hybridization on adult mouse ovary serial sections. Expression of Amh mRNA was included for comparison.
The results show that Ihh and Dhh mRNAs are present in granulosa cells in preantral and antral follicles (Fig. 2, C–F), whereas Shh mRNA is not detected using RT-PCR and in situ hybridization (data not shown). For expression of the Ptch1 gene, a low background mRNA level is observed over the whole ovary, but expression of this target gene is clearly up-regulated in cells surrounding the follicles (Fig. 2, G and H), which at a higher magnification (not shown) were found to represent cells located in the theca cell compartment. A basal level of Ptch1 expression, independent of hedgehog signaling, is observed in many tissues and likely serves to control the activity of a low level of Smo (15, 29, 50). Hedgehog signaling from granulosa cells to the mesenchymal theca cell compartment, resulting in induced Ptch1 gene expression, is present throughout the preantral and antral stages of follicle development. Corpora lutea do not express Ihh and Dhh and only a basal level of Ptch1 mRNA (Fig. 2, asterisks).
FIG. 2. Cellular localization of Amh, Ihh, Dhh, and Ptch1 mRNAs in the postnatal mouse ovary. In situ hybridization localizes the expression of Amh (A and B), Ihh (C and D), and Dhh (E and F) mRNAs to granulosa cells of preantral and antral follicles, whereas hedgehog receptor and target gene Ptch1 (G and H) mRNA is localized to cells in the surrounding theca cell compartment. Shown are hematoxylin-stained bright-field images (A, C, E, and G) with corresponding dark-field images (B, D, F, and H) of serial whole adult mouse ovary sections. Closed arrowheads point to large antral follicles in which granulosa cells no longer express Amh mRNA (A and B) but still express Ihh and Dhh (C, D, E, and F), whereas expression of Ptch1 remains up-regulated in the theca cell compartment (G and H). Corpora lutea (asterisks) do not express Amh (A and B), Ihh (C and D), Dhh (E and F), or Ptch1 (G and H), whereas atretic follicles rapidly lose expression of Ihh, Dhh, and Ptch1 (arrows). Note that expression of the hedgehog target gene Ptch1 is induced in the theca cell layer, whereas oocytes (open arrowheads, H) lack this response. Scale bar, 200 μm.
Down-regulation of Amh mRNA expression in granulosa cells in antral follicles (Fig. 2, A and B) is in agreement with published data (9, 51) and clearly precedes down-regulation of Ihh and Dhh in antral follicles (Fig. 2, closed arrowheads). Expression of Ihh, Dhh, and Ptch1 mRNAs is rapidly lost in atretic follicles (Fig. 2, arrows; and supplemental Fig. 1, published on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org). Immunohistochemical staining shows that Ihh/Dhh and Amh proteins are limited to granulosa cells of preantral and antral follicles (Fig. 3A, left follicle) and are down-regulated in atretic follicles, which are characterized by degenerating cells with pyknotic nuclei (Fig. 3A, arrowheads in right follicle).
FIG. 3. Oocytes do not express Ihh and Dhh and lack expression of Smo. A, Immunostaining of adult mouse ovary, showing Amh and hedgehog (Ihh/Dhh) staining of granulosa cells. Like Amh protein, coimmunostaining for both Ihh and Dhh proteins shows that hedgehog is detected in granulosa cells of healthy growing follicles (left follicle), whereas expression is diminished in an atretic follicle (right follicle). Arrowheads point to regions with many pyknotic nuclei representing apoptotic granulosa cells, characteristic for atretic follicles. Scale bar, 100 μm. B, Expression of genes encoding components of the hedgehog signaling pathway in oocytes. Total RNA was isolated from oocytes of 14 and 21dpp superovulated ovaries and adult mouse whole ovaries. The RNA was incubated either in the presence (+) or absence (–) of reverse transcriptase (RT), and each PCR was performed on an amount of cDNA equivalent to that of half an oocyte. Oocytes lack expression of Ihh, Dhh, Ptch2, and Amh mRNAs and also mRNA encoding the essential hedgehog signal transducer Smo.
Oocytes lack Smo and are nonresponsive to granulosa cell-derived Ihh and Dhh
It is to be expected that oocytes are exposed to hedgehog proteins (Ihh and Dhh) from granulosa cells. However, no induction of Ptch1 expression is detected in oocytes (Fig. 2, G and H, open arrowheads). This implies that Ihh and Dhh from granulosa cells are not involved in communication toward the developing oocyte. However, the in situ hybridization technique does not preclude that transcripts for hedgehog signaling components might be present in developing oocytes at a relatively low level. Therefore, we also used the more sensitive RT-PCR with primer sets for Ihh, Dhh, Ptch1, Ptch2, Smo, Amh, Gdf9, and zona pellucida glycoprotein 3 (Zp3). Oocytes were isolated free of granulosa cell contamination, as was confirmed by the absence of mRNA for Amh (Fig. 3B), a factor that is known to be produced by granulosa cells in growing follicles (51). Gdf9 and Zp3 are oocyte-specific genes (52, 53), and the present RT-PCR results show an enrichment of the respective mRNAs in oocytes, compared with whole ovary, using the same amount of total RNA from oocytes and whole ovary in the RT-PCR (Fig. 3B). As concluded from the mRNA expression data, oocytes do not produce Ihh or Dhh themselves, and the oocytes also lack expression of Smo, indicating that they are not capable of responding to hedgehog from granulosa cells (Fig. 3B). There is some expression of Ptch1 mRNA in oocytes, not of Ptch2, which probably represents the above-described hedgehog independent basal expression.
Hedgehog signaling in primary and ovulatory stages of follicle development
Developing follicles in adult mice were classified as previously described (6, 54). We characterized expression of Ihh and Dhh mRNAs and target gene responses in the mesenchymal pre-theca and theca cell compartment, during primary and ovulatory stages of follicle development, in more detail.
Transition of primordial follicles to primary follicles (follicle recruitment) starts to take place in mice between 2.5 and 4.5dpp, and the first growing follicles appear in the medulla part of the ovary. Ovaries at 2.5dpp after birth contain only primordial follicles, but these follicles do not contain detectable levels of hedgehog mRNAs (data not shown). Hence, it is concluded that granulosa cells of primordial follicles do not express Ihh or Dhh and do not up-regulate Ptch1 gene expression in the immediate surrounding mesenchymal stroma cells. However, at 4.5dpp many primary follicles are present throughout the medullary region, and granulosa cells in these follicles express both Ihh and Dhh mRNAs (Fig. 4, A–D). In adult ovary, Ihh and Dhh mRNAs are detected in granulosa cells of primary follicles, with Dhh mRNA readily detectable at the type 3a follicle stage (<20 follicle cells in largest cross-section), followed by Ihh mRNA at the type 3b follicle stage (21–60 follicle cells in largest cross-section) (Fig. 4, G–J).
FIG. 4. Initiation of expression of Ihh, Dhh, Ptch1, and Gli1 mRNAs in primary follicles of early neonatal and adult mouse ovaries. In 4.5dpp mouse ovaries, the first primary follicles clearly express Ihh (A and B), Dhh (C and D), and Ptch1 (E and F). Similarly, primary follicles of adult ovaries express Ihh (G and H), Dhh (I and J), Ptch1 (K and L), and Gli1 (M and N) mRNAs. Note that Dhh is expressed as soon as granulosa cells take up a cuboidal appearance (I and J, arrowhead). Scale bar, 100 μm (A–F) and 50 μm (G–N).
Theca cells originate from mesenchymal precursor cells near primary follicles (11). Induced expression of the hedgehog target genes Ptch1 and Gli1 was found in mesenchymal stroma cells surrounding primary follicles, in 4.5dpp neonatal ovary and also in adult mouse ovary (Fig. 4, E, F, and K–N). This implies that Ptch1 and Gli1 act as early marker genes in differentiation of the theca cell lineage and points to a possible role for Ihh or Dhh or both in the initial phase of theca cell recruitment and development.
To determine expression of mRNAs encoding hedgehog signaling components around ovulation, ovaries from adult mice were analyzed by in situ hybridization after induction of superovulation with Folligonan (pregnant mare serum gonadotropin) and Chorulon (chorionic gonadotropin) (see Materials and Methods). Three hours after injection of Chorulon, loss of expression of Ihh and Dhh mRNAs was seen in some granulosa cells closest to the basal lamina (arrowheads in Fig. 5, A and B; and supplemental Fig. 2). This loss continues at later stages of ovulation, resulting in absence of Ihh and Dhh mRNAs from granulosa cells in the preovulatory follicles that are ready to undergo ovulation. During preovulatory stages, expression of Ptch1 (Fig. 5, C, F, I, and L) and Gli1 mRNAs (supplemental Fig. 2) in the theca cell compartment is gradually lost, reflecting the loss of expression of Ihh and Dhh in granulosa cells. In the same ovarian cross-sections, throughout the ovulation induction procedure, preantral follicles maintain Ihh and Dhh expression and also expression of the target genes Ptch1 and Gli1 (Fig. 5; and supplemental Fig. 2), which indicates that hedgehog signaling in the preantral follicles is not changed by the gonadotropic treatment. Taken together, this ovulation experiment clearly demonstrates dynamic synchrony between expression of hedgehog mRNAs and target gene induction.
FIG. 5. Ihh and Ptch1 mRNAs in mouse ovary during and after induced superovulation. Ovaries were analyzed before ovulation at 1600 h (A–C), during ovulation at 2200 h (D-F), and midnight (G–I) and the morning after ovulation at 0900 h (J–L). Expression of Ihh is lost, going from preovulatory (B) to ovulatory (E and H) and postovulatory (K) follicles but is maintained in preantral follicles (H and K). Expression of Ptch1 mRNA is also lost, but there is a low basal level in periovulatory follicles (F, I, and L) and maintenance of a high level in the theca cell compartment of preantral follicles (I and L). Scale bar, 100 μm.
Genes encoding additional components of the hedgehog signaling pathway
We further examined the postnatal mouse ovary for in situ expression of gene transcripts encoding additional protein components of the hedgehog signaling pathway. The seven-transmembrane signal transducer Smo is expressed throughout the mouse ovarian tissue, including all stages of growing follicles, the corpus luteum, and the interfollicular mesenchyme (Fig. 6, A and B), with exception of the oocyte (Figs. 6, A and B, and 3B, arrowheads). Expression of Smo is found in many tissues and is balanced by an ubiquitous low level of Ptch1, which is required to restrict signaling activity to cells that are exposed to hedgehog proteins. Smo is not a target gene for hedgehog signaling. In contrast to Smo but similar to Ptch1, the genes encoding the second hedgehog receptor Ptch2 and the binding protein Hip1 are targets of hedgehog signaling. Ptch2 and Hip1 mRNAs both are found to be up-regulated in the theca cell layer of growing follicles (Fig. 6, C–F) but not in the oocyte (arrowheads in Figure 6, C–F).
FIG. 6. Expression of genes encoding additional components of the hedgehog signaling pathway in postnatal mouse ovary. In situ expression in adult mouse ovary of mRNAs encodes the membrane-associated signal transducer Smo (A and B); the second hedgehog receptor Ptch2 (C and D); Hip1 (E and F); and the intracellular transducers Gli1 (G and H), Gli2 (I and J), and Gli3 (K and L). Note that Ptch2, Hip1, and Gli1 are target genes of hedgehog signaling and are expressed in theca cell layers, similar to induced expression of Ptch1 mRNA (not shown in this figure), but not oocytes (arrowheads). Scale bar, 100 μm.
In different cell types, basal expression of the transcriptional regulators Gli2 or Gli3 is required for initiation of hedgehog signaling to allow for activation of the pathway and induction of Gli1 expression (31, 55, 56, 57). Gli3 mRNA is found at near-to-background level, but there is clear expression of Gli2 mRNA, which is seen at a basal level throughout the tissue, perhaps with a slight accumulation of mRNA in theca cells surrounding some growing follicles (Fig. 6, I–L). Expression of the hedgehog target gene Gli1 starts in mesenchymal cells surrounding the primary follicle (Fig. 4) and is maintained in the theca cell compartment of preantral and antral follicles (Fig. 6, G and H).
Cyclopamine down-regulates target gene activation in ovarian mesenchymal cells
The steroidal alkaloid cyclopamine and its derivative KAAD-cyclopamine specifically block cellular responses to vertebrate hedgehog signaling by directly binding to the essential signal transducer Smo (26). KAAD-cyclopamine blocks hedgehog signaling both in vitro and in vivo, with a 10- to 20-fold higher potency but with a similar or lower toxicity, compared with cyclopamine (25, 58). We set up organ cultures of whole ovaries isolated from 2dpp mice, in which follicles are at the primordial stage and do not express Ihh or Dhh (data not shown). Individual ovaries were cultured for 6 d, either in the absence (control) or presence of 57 nM or 0.57 μM KAAD-cyclopamine (Fig. 7). The addition of KAAD-cyclopamine did not affect the histological morphology of the ovaries during the 6-d culture period. Both in the absence and presence of KAAD-cyclopamine, the cultured ovaries were found to contain developing follicles up to the type 4 preantral stage with two to three layers of granulosa cells (Fig. 7, A–C). Growing follicles in ovaries that were cultured for 6 d in the absence of KAAD-cyclopamine express mRNAs for Amh and Ihh in granulosa cells and Ptch1 and Gli1 in surrounding stromal cells (Fig. 7, D, G, J, and M). No changes in the expression of Amh, Ihh, Ptch1, and Gli1 were observed at 5.7 nM KAAD-cyclopamine (data not shown). When the ovaries were cultured in the presence of 57 nM or 0.57 μM KAAD-cyclopamine, expression of Amh and Ihh mRNAs in granulosa cells of growing follicles is maintained (Fig. 7, E, F, H, and I), but expression of both Ptch1 and Gli1 in the stromal cells is strongly down-regulated in the presence of 57 nM KAAD-cyclopamine (Fig. 7, K and N) and even further down-regulated at 0.57 μM KAAD-cyclopamine (Fig. 7, L and O). A low level of Ptch1 mRNA remains present in ovaries cultured in the presence of 57 nM and 0.57 μM KAAD-cyclopamine and represents the low basal level that is independent of hedgehog signaling, encoding a basal level of Ptch1 that controls Smo in the absence of hedgehog ligands (Fig. 7L; and data not shown).
FIG. 7. KAAD-cyclopamine inhibits the expression of Ptch1 and Gli1 mRNAs in cultured neonatal mouse ovaries. Ovaries were isolated from 2dpp mice and cultured as described in Materials and Methods for 6 d in the absence (control) or presence of 57 nM or 0.57 μM KAAD-cyclopamine, indicated above each panel. The top panel (A–C) shows representative hematoxylin-eosin-stained sections of the cultured ovaries. Scale bar, 50 μm. The cultured ovaries were sectioned for in situ hybridization to detect Amh, Ihh, Ptch1, and Gli1 mRNAs. For Amh (D–F) and Ihh (G–I), only the bright-field images are shown, whereas for Ptch1 (J–L) and Gli1 (M–O), the figure also includes the dark-field images. Scale bar, 100 μm.
The observed down-regulation of Ptch1 and Gli1 mRNA expression by the antagonist KAAD-cyclopamine, provides direct evidence that hedgehog target gene expression in the pre-theca cell compartment of developing follicles depends on local hedgehog signaling.
Discussion
Hedgehog signaling in theca cell development
The present study exposes the mammalian ovary as a novel site of active hedgehog signaling. For the three mammalian hedgehog ligands, we show that Ihh and Dhh, but not Shh, are expressed in the mouse ovary during reproductive life. Expression of Ihh and Dhh localizes to granulosa cells of growing follicles, whereas expression of the hedgehog signaling target genes Ptch1, Ptch2, Gli1, and Hip1 is induced in the theca cell compartment. In contrast, expression of these hedgehog target genes is not induced in oocytes. Expression of Ptch1 and Gli1 mRNAs is strongly down-regulated in cultured ovaries treated with the specific hedgehog signaling antagonist KAAD-cyclopamine, providing direct evidence that induced target gene expression in the ovarian mesenchymal cells requires hedgehog signaling.
Initiation of follicle growth, often referred to as follicle recruitment, is defined as the transition of a nongrowing primordial follicle (type 2) to an early primary follicle (type 3a). During this transition, granulosa cells increase in number and transform from a squamous to a cuboidal morphology, and growth of the oocyte is initiated (6). Granulosa cells of primordial follicles do not express Ihh or Dhh, and there is no detectable induction of target gene expression in any ovarian cell type in neonatal mice at 2dpp, when the ovary contains only primordial follicles. Observations on adult mouse ovary show that Dhh mRNA is first detected when granulosa cells take up a cuboidal morphology during the transition from type 2 to type 3a primary follicle stages, whereas expression of Ihh initiates at the type 3b primary follicle stage. Induced expression of the hedgehog target genes Ptch1 and Gli1 is first detected in mesenchymal cells adjacent to granulosa cells between the type 3a and type 3b primary follicle stages. The results suggest that hedgehog signaling does not play part in the initiation of follicle growth but rather starts to act early after the transition from the primordial to the primary follicle stage.
Formation of theca cells with steroidogenic capacity is a crucial functional aspect of follicle development, but little is known regarding regulation of recruitment of theca precursor cells and their development into steroidogenic theca cells. Mesenchymal pre-theca cells most likely are located within the ovarian stroma and may have stem cell properties, but theca stem cells have not been identified yet (3, 59, 60). Primordial and primary follicles lack morphologically differentiated theca cells, and the first steroidogenic theca cells appear when preantral follicles contain two to three layers of granulosa cells (11). These theca cells respond to LH and gain expression of steroidogenic enzymes and the capacity to produce androgens. Pre-theca cells, however, do not express the LH receptor, and theca cell recruitment and early differentiation are LH independent, so that locally produced paracrine factors are possible candidates to induce theca cell differentiation (11). Mesenchymal pre-theca cells surrounding the primary follicle show an increase in DNA replication (59) and onset of the expression of several genes characteristic of theca cells, including the genes encoding bone morphogenetic protein (Bmp) 4 (61), the LH receptor (11), and also Ptch1 and Gli1, as described herein. Initiation of pre-theca cell recruitment and differentiation starts as early as the primary follicle stage, and it appears that theca differentiation factors must be secreted by primary follicles. Induction of hedgehog target gene expression initiates in pre-theca cells at the primary follicle stage, and both Ihh and Dhh are excellent candidates to act as early theca differentiation factors. In Dhh knockout mice, no ovarian phenotype is observed (21), which indicates that Ihh might act as the predominant signal in the ovary. This does not exclude, however, that the actions of the two hedgehog ligands in the ovary are partially redundant. Testis lacks local Ihh production so that only Dhh can act as Leydig cell differentiation factor, which explains the occurrence of a testicular phenotype in Dhh knockout mice (21, 39, 62, 63).
The testicular phenotype of Dhh knockout mice revealed that hedgehog signaling plays an essential role in testis tissue organization. In these knockout mice, the testis shows dysregulation of formation of the tubular wall and defects in differentiation of interstitial Leydig cells (21, 39, 62, 63). In formation of the tubular wall, peritubular myoid cells interact with Sertoli cells, and both cell types contribute protein components to the basal lamina (64). Dhh signaling seems essential for communication between Sertoli cells, peritubular myoid cells, and the interstitial Leydig cell compartment. In the ovary, the paracrine action of Ihh and Dhh might be important, somewhat similar to the role of hedgehog signaling in testis, for development of a proper tissue organization, with an intact follicular basal lamina and a well-structured steroidogenic theca cell compartment.
As a next step, we aim to evaluate the ovarian phenotype of a granulosa cell-specific Ihh conditional knockout on the Dhh knockout background. This type of analysis may yield information to explain the precise role of hedgehog signaling in ovarian function, which also will be relevant in relation to our recent observations that active hedgehog signaling is present in human ovarian follicles (results not shown).
Hedgehog signaling in germ line cells
The Caenorhabditis elegans genome contains a gene ptc-1, which encodes a membrane protein similar to mammalian Ptch1/Ptch2 (65). However, true homologs of hedgehog and smoothened genes have not been detected in C. elegans (66), which indicates that ptc-1 is involved in signaling or other activities not related to a functional hedgehog signaling pathway. C. elegans ptc-1 has been shown to be important for cytokinesis of germ cells; ptc-1 null mutant C. elegans is sterile, and the gonads contain multinucleate germ cells (67).
In Drosophila ovary, hedgehog is produced by somatic cells of the terminal filament and the adjacent cap cells situated at the distal tip of the germarium (68, 69). Hedgehog signaling in Drosophila ovary has been studied using induced ptc-LacZ expression, which is detected in somatic cells extending from the terminal filament up to and including the somatic stem cells (68). This ovarian hedgehog signaling has been shown to control proliferation and maintenance of ovarian somatic stem cells (70). Germ line cells are located near the distal tip of the germarium, very close to the cellular source of hedgehog, but ptc-LacZ expression is not detected in these germ line cells (68). The Drosophila ovarian germ line cells express a basal level of ptc protein, but the cubitus interruptus protein, which is the Drosophila homolog of the mammalian Gli transcription factors, is not present (68). There are a few indications that ptc and smo in Drosophila germ line cells might play some role as cell-autonomous components of the hedgehog signaling pathway, in embryonic germ cell migration (71). However, most available data suggest that there is lack of hedgehog signaling in Drosophila ovarian germ line cells.
In mouse oocytes in growing follicles, we did not observe induced expression of the hedgehog target genes Ptch1, Ptch2, Hip1, and Gli1. We conclude that these oocytes, in diplotene stage of meiotic prophase, do not respond to granulosa-derived Ihh and Dhh. This lack of response can be explained by lack of expression of Smo in oocytes because we have not detected Smo mRNA in oocytes using RT-PCR or in situ hybridization. Also in spermatogenic germ line cells, expression of Smo mRNA is probably absent or very low, as described herein. It can be suggested that male and female germ line cells are not responsive to hedgehog signaling during gametogenesis. This would be in agreement with observations on expression of a Ptch1-LacZ knock-in gene construct in a mouse model that has been used to demonstrate active hedgehog signaling in target cells (34). In target cells, hedgehog signaling activates the endogenous Ptch1 promoter above basal level, leading to LacZ activity from expression of the Ptch1-LacZ knock-in construct. In this mouse model, LacZ activity in the testis is mainly found in peritubular and interstitial cells but not in Sertoli cells and germ line cells (21, 39, 62, 63).
In summary, the available data indicate that gonadal hedgehog signaling, in animal species ranging from worms and flies to mammals, is not directly involved in control of germ line cells. In Drosophila ovary, the main role of hedgehog signaling is related to control of proliferation and maintenance of somatic stem cells. In mammalian gonads, Sertoli cells in testis and granulosa cells in ovary provide for hedgehog signaling as an important factor in the communication between the cellular compartments that perform gametogenesis and steroidogenesis.
Acknowledgments
We thank Prof. A. P. McMahon (Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA) for cDNA probes representing hedgehog signaling components and for the rabbit polyclonal antibody Ab80.
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Address all correspondence and requests for reprints to: Dr. Mark Wijgerde, Department of Reproduction and Development, Erasmus University Medical Center, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands. E-mail: m.wijgerde@erasmusmc.nl.
Abstract
Follicle development in the mammalian ovary requires interactions among the oocyte, granulosa cells, and theca cells, coordinating gametogenesis and steroidogenesis. Here we show that granulosa cells of growing follicles in mouse ovary act as a source of hedgehog signaling. Expression of Indian hedgehog and desert hedgehog mRNAs initiates in granulosa cells at the primary follicle stage, and we find induced expression of the hedgehog target genes Ptch1 and Gli1, in the surrounding pre-theca cell compartment. Cyclopamine, a highly specific hedgehog signaling antagonist, inhibits this induced expression of target genes in cultured neonatal mouse ovaries. The theca cell compartment remains a target of hedgehog signaling throughout follicle development, showing induced expression of the hedgehog target genes Ptch1, Ptch2, Hip1, and Gli1. In periovulatory follicles, a dynamic synchrony between loss of hedgehog expression and loss of induced target gene expression is observed. Oocytes are unable to respond to hedgehog because they lack expression of the essential signal transducer Smo (smoothened). The present results point to a prominent role of hedgehog signaling in the communication between granulosa cells and developing theca cells.
Introduction
IN THE MAMMALIAN OVARY, developing follicles represent the functional units that carry out and coordinate the two most important ovarian functions, gametogenesis and steroidogenesis (1, 2, 3). During adult reproductive life, recruitment of follicles from the primordial pool is a continuous process that results in formation of primary follicles and sets the basis for subsequent follicle development (4). In a developing follicle, proliferation and differentiation of the somatic granulosa and theca cell compartments, and development of the oocyte, are highly coordinated. This requires intercellular communication between these cell types and compartments (5). When a primordial follicle enters the pool of growing primary follicles, the flattened granulosa cells become cuboidal and change from a quiescent to a proliferative state (6). From this stage, granulosa cells secrete proteins like stem cell factor (or Kit ligand) and anti-Müllerian hormone (Amh), which are implicated in control of follicle development (7, 8, 9). In a primary follicle, the oocyte starts to produce growth differentiation factor (Gdf) 9. As shown by the ovarian phenotype of Gdf9 knockout mice, this factor is important for development of follicles beyond the primary follicle stage and formation of the first theca cells (10). Pre-theca cells originate from mesenchymal precursor cells within the ovarian cortical stroma around the primary follicles but do not emerge as fully differentiated steroidogenic theca cells before the onset of the preantral follicle stage (11). The role of Gdf9 involves action on granulosa cells (12). Little is known about factors from granulosa cells involved in formation and differentiation of pre-theca cells.
Hedgehog proteins form a small and highly conserved family of intercellular signaling molecules, whose first known member was initially described as a protein encoded by a segment polarity gene in Drosophila, and that protein is now known to play several distinct roles in fly development, including gonadal development and function (13, 14, 15, 16). Mammals have three hedgehog proteins: sonic hedgehog (Shh), Indian hedgehog (Ihh), and desert hedgehog (Dhh), which all show conservation of structure and function with the single hedgehog protein in Drosophila (14, 17). In mammalian embryos, the different hedgehog proteins play various roles in control of developmental processes, and the proteins are implicated in the etiology of several human congenital malformations and cancers (15, 18). Shh is most intensely studied and was found to be involved in establishment of the early left-right axis, regulation of ventral cell fates in the central nervous system, specification of the anterioposterior axis in developing limb, and morphogenesis of a variety of organs (15, 19). Ihh and Dhh have more restricted functions (20). An important role for Dhh in testis development was detected by analysis of the phenotype of Dhh knockout mice, which points to Dhh signaling in Sertoli-Leydig cell communication. However, that study did not reveal a role for Dhh in the ovary (21).
The amino acid sequences of Shh, Ihh, and Dhh are highly similar, and all three hedgehog proteins can evoke similar mechanistic responses in a number of in vitro and in vivo settings (17, 22). Shh, Ihh, and Dhh have comparable affinities for the same plasma membrane proteins involved in hedgehog signaling: patched homolog (Ptch) 1 and Ptch2 and hedgehog interacting protein 1 (Hip1) (22, 23). At the cell surface, the 12-transmembrane protein Ptch1 and its close homolog Ptch2 act as common hedgehog receptors, whereas the membrane-bound glycoprotein Hip1 binds hedgehog proteins, thereby preventing receptor binding (24). In the absence of ligand, Ptch1 represses signaling activity of the seven-transmembrane protein Smo (smoothened). Virtually all cells will express a low basal level of Ptch1, which provides inhibitory control of signaling by Smo. However, hedgehog binding functionally inactivates Ptch1, making Smo available to initiate intracellular signaling. Cyclopamine, an exogenous teratogenic compound, binds Smo and acts as a highly specific antagonist of hedgehog signaling (25, 26) (Fig. 1A). Signaling by Smo transduces all hedgehog signals, using the transcription factors Gli1, Gli2, and Gli3 as downstream effectors (27, 28, 29). Gli proteins are included in a complex that associates with the cytoskeleton and keeps full-length Gli out of the nucleus. This complex targets Gli2 and Gli3 for cleavage, producing shorter C-terminal forms containing the Zn-finger, which act as transcriptional repressors (30, 31, 32). Gli1 is not a target for cleavage and acts as a constitutive activator of transcription (Fig. 1A).
FIG. 1. Hedgehog signaling pathway. A, General representation of the hedgehog signaling pathway and its components: ligands (Shh, Ihh, and Dhh); membrane bound receptors (Ptch1, Ptch2); membrane-associated signal transducer (Smo); and intracellular transcriptional effectors (Gli1, Gli2, and Gli3). The exogenous teratogenic compound cyclopamine acts as a highly specific antagonist by blocking signaling, by direct binding to Smo. B, Northern blot analysis of expression of mRNAs encoding components of the hedgehog signaling pathway in 10.5dpc mouse embryos, adult mouse testis, and adult mouse ovary.
A functionally highly relevant aspect of hedgehog signaling is that several genes encoding components of the hedgehog signaling pathway are targets of hedgehog signaling. Exposure of cells, which will express a low basal level of Ptch1 to control Smo, to hedgehog proteins leads to strong induction of expression of the genes encoding Ptch1, Ptch2, Hip1, and Gli1 (24, 33, 34, 35). Induction of the transcription factor Gli1 provides the system with a transcriptional stimulus, which is embedded within a negative signaling feedback loop generated by up-regulation of the signaling antagonists Ptch1, Ptch2, and Hip1. This endows the hedgehog signaling network with a robust switch-like mechanism, which is essential to control induction events in embryogenesis (36, 37, 38).
Here we present evidence that Ihh and Dhh produced by granulosa cells act as ovarian paracrine factors, inducing target gene expression in the developing theca cell compartment. This suggests a role of hedgehog signaling in theca cell development in growing follicles, similar to the role of Dhh in Leydig cell development in testis (21, 39, 40). Oocytes take a special position because they are probably exposed to Ihh and Dhh from the surrounding granulosa cells, and they express the receptor Ptch1 but lack the signal transducer Smo.
The present results associate, for the first time, the hedgehog signaling pathway with somatic cell communication and cell differentiation events in mouse ovary.
Materials and Methods
Experimental animals
All experimental mice were of FVB/n strain and maintained in accordance with accepted standards of humane animal care as outlined in Ethical Guidelines for Care and Use of Laboratory Animals on a regular light-dark cycle (lights on between 0500 and 1900 h). For induction of superovulation, 22 d post partum (dpp) female mice were injected ip with 150 μl Folligonan (pregnant mare serum gonadotropin; 50 U/ml) between 1300 and 1400 h to stimulate follicular growth, followed after 47 h by ip injection of 150 μl Chorulon (chorionic gonadotropin; 50 U/ml) to induce ovulation and luteinization. Ovulation is expected to occur between 2200 and 2400 h; none of the superovulated females were set up for mating.
RNA isolation, Northern blotting, and RT-PCR analysis
Total RNA was extracted from 10.5 d post coitum (dpc) mouse embryos and adult (2.5 month old) mouse testis and ovaries using Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Enrichment for polyA+ RNA was performed using Dynabeads mRNA direct kit (Dynal, Oslo, Norway); an amount of 3 μg of polyA+ RNA was loaded in each lane, and the RNA was electrophoresed through a 1% agarose 3[N-morpholino]propanesulfonic acid-formaldehyde gel. Subsequently the RNA was transferred to nylon membrane and probed with 32P-labeled DNA probes generated by random priming of (partial) cDNA fragments for mouse Shh, Ihh, Dhh (17, 41), Ptch1 (34), Ptch2 (42), Smo (29), Gli1, Gli2, Gli3 (42), and a 430-bp mouse genomic DNA PstI fragment of the mouse Amh gene, containing exon 1 (43). Blots were hybridized with 2 x 106 cpm/ml hybridization buffer [50% vol/vol formamide, 7% (wt/vol) dextran sulfate, 5x saline sodium citrate (SSC), 5x Denhardt’s, 20 mM sodium phosphate buffer (pH 6.8), 1% (wt/vol) sodium dodecyl sulfate (SDS), 100 mg/ml denatured salmon sperm DNA, and 100 mg/ml yeast tRNA] at 65 C overnight (16 h). Blots were washed sequentially in 2x SSC/0.1% SDS and 0.2x SSC/0.1% SDS at 65 C and exposed to phosphoimager screens.
Oocytes from 14dpp and 21dpp mice were isolated by mechanical disruption of ovaries and follicles, followed by incubation in trypsin (1 mg/ml), collagenase (1 mg/ml), hyaluronidase (1 mg/ml), and DNaseI (10 μg/ml) in M2 medium (Sigma, St. Louis, MO) at 37 C. Superovulated oocytes were isolated by incubation of cumulus-oocyte complexes in hyaluronidase (1 mg/ml) and DNaseI (10 μg/ml) in M2 medium at 37 C. For each sample, 20 oocytes were collected in 50 μl TRIZOL, snap frozen and thawed twice, and total RNA precipitated in the presence of 0.2 μg/μl glycogen (Invitrogen). Samples were incubated with 1 U/μl RNase-free DNaseI (Promega, Fitsburg, WI) for 30 min at 37 C before reverse transcription. First-strand cDNA synthesis was performed using the Superscript first-strand synthesis system for RT-PCR (Invitrogen) using random hexamers as primers. An unfertilized oocyte was assumed to contain 0.43 ng total RNA, which was used to match an equivalent amount of whole-ovary RNA. PCR was performed using platinum Taq DNA polymerase (Invitrogen) in 20-μl aliquots at 94 C for 3 min, followed by 94 C for 0 sec, 55 C for 30 sec, 72 C for 30 sec, for 40 cycles; and finally 72 C for 10 min; and storage at 4 C until samples were analyzed on a 2% agarose gel in 0.04 M Tris-acetate, 1 mM EDTA buffer. Primer sets were designed to cross intron sequences to distinguish between genomic DNA and cDNA products. Sequences used to target mouse cDNAs can be found in the supplement to Materials and Methods (published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org).
In situ hybridization and immunohistochemistry
Mouse ovaries were prepared and hybridized with 35S-uridine 5-triphosphate-labeled RNA probes as previously described (44, 45, 46). To compare expression of different mRNAs, we used serial ovarian sections of 8 μm thickness. These sections were hybridized with 35S-uridine 5-triphosphate-labeled probe sequences generated by transcription of (partial) cDNA fragments for mouse Shh, Ihh, Dhh (17, 41), Ptch1 (34), Ptch2 (42), Smo (29), Gli1, Gli2, Gli3 (42), Hip1 (24), and a 430-bp mouse genomic DNA PstI fragment of the mouse Amh gene, containing exon 1 (43); all cDNA fragments were sequenced to verify the identity. As negative controls, antisense riboprobes were applied; this never resulted in any specific signal above background (not shown). Sections were counterstained with hematoxylin, and images were taken using an Axioplan 2 (Zeiss, Gottingen, Germany) equipped with a CoolSNAP-Pro color charge-coupled device camera (Media Cybernetics, Wokingham, UK).
For immunohistochemistry, adult mouse (2.5 months old) ovaries were dissected and fixed overnight in 4% paraformaldehyde in PBS at 4 C, dehydrated, paraffin embedded, sectioned (8 μm), and mounted on SuperFrost Plus microscope slides. Immunohistochemistry was performed as previously described (9), using anti-Amh (C-20, Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1:1000 and antibody AB80 against vertebrate hedgehog proteins (47).
Organ culture of ovaries from neonatal mice
Intact ovaries from 2dpp mice were organ cultured for 6 d as previously described (48, 49). In brief, each dissected ovary was placed on a quarter piece of a Millicell-CM membrane (0.4 μm pore size; Millipore Corp., Medford, MA) floating on top of 750 μl culture medium, all contained within a single well of a 4-well plate (Nunc, Roskilde, Denmark). Ovarian culture medium was Waymouth MB752/1 + L-glutamine (Life Technologies, Inc.-Invitrogen, Paisley, Scotland, UK) supplemented with 0.23 mM pyruvic acid (Sigma), 0.5 x antibiotic/antimycotic solution (Life Technologies-Invitrogen), 3 mg/ml BSA (True Cohn crystalline powder; MP Biomedicals, Irvine, CA), and 10% (vol/vol) fetal calf serum. Cyclopamine and its derivative 3-keto-N-aminoethyl-aminocaproyl-dehydrocinnamoyl (KAAD)-cyclopamine inhibit hedgehog pathway activation by binding directly to Smo (26). The KAAD derivative has a 10- to 20-fold higher potency than cyclopamine (25) and was obtained from Toronto Research Chemicals (Toronto, Canada). A stock solution of KAAD-cyclopamine was prepared by dissolving 1 mg in 250 μl methanol (5.7 mM), which was stored at –20 C. Ovaries were cultured in the absence (control; methanol) or presence of 57 nM and 0.57 μM KAAD-cyclopamine. The cultures were incubated at 37 C and gassed with 5% CO2 in air. Culture medium was refreshed every other day, and ovaries were fixed in 4% paraformaldehyde/PBS after 6 d of culture and subsequently subjected to in situ hybridization analysis as described above.
Results
Mouse ovary expresses a relatively high level of Ihh mRNA
To obtain a precise quantitative and qualitative indication of mRNA expression, we performed Northern blot analysis of adult mouse whole-ovary mRNA (poly-A+ RNA). For reasons of comparison, we also included mRNA from 10.5dpc mouse embryos and adult mouse testis, in which we detected mRNAs for Shh and Dhh, respectively (Fig. 1B). This confirms published data (17, 21). Like the adult testis, the adult ovary also contains Dhh mRNA. However, in the ovary we find a relatively high level of Ihh mRNA, which is not expressed at a detectable level in the testis (Fig. 1B).
In embryo, testis, and ovary, mRNAs encoding Ptch1, Ptch2, and Smo are present (Fig. 1B). The Northern blot signal for Smo mRNA in adult testis tissue is quite low, but this is explained by developmental dilution of Smo mRNA when the developing testis becomes populated with an increasing number of spermatogenic cells with no or a very low level of Smo mRNA; in prenatal (18.5dpc) and postnatal (8.5dpp) testis tissue, a much higher expression of Smo mRNA was detected (not shown).
The presence of Ihh, Dhh, and Smo mRNAs in mouse ovary, together with a significant level of mRNA transcribed from the hedgehog target gene Ptch1, provides a first indication for an active hedgehog signaling pathway.
Ihh and Dhh mRNAs are located in granulosa cells of preantral and antral follicles, and Ptch1 expression is induced in the adjacent theca cell compartment
For cellular localization of the expression of Ihh and Dhh mRNAs, and expression of the target gene Ptch1, we carried out in situ hybridization on adult mouse ovary serial sections. Expression of Amh mRNA was included for comparison.
The results show that Ihh and Dhh mRNAs are present in granulosa cells in preantral and antral follicles (Fig. 2, C–F), whereas Shh mRNA is not detected using RT-PCR and in situ hybridization (data not shown). For expression of the Ptch1 gene, a low background mRNA level is observed over the whole ovary, but expression of this target gene is clearly up-regulated in cells surrounding the follicles (Fig. 2, G and H), which at a higher magnification (not shown) were found to represent cells located in the theca cell compartment. A basal level of Ptch1 expression, independent of hedgehog signaling, is observed in many tissues and likely serves to control the activity of a low level of Smo (15, 29, 50). Hedgehog signaling from granulosa cells to the mesenchymal theca cell compartment, resulting in induced Ptch1 gene expression, is present throughout the preantral and antral stages of follicle development. Corpora lutea do not express Ihh and Dhh and only a basal level of Ptch1 mRNA (Fig. 2, asterisks).
FIG. 2. Cellular localization of Amh, Ihh, Dhh, and Ptch1 mRNAs in the postnatal mouse ovary. In situ hybridization localizes the expression of Amh (A and B), Ihh (C and D), and Dhh (E and F) mRNAs to granulosa cells of preantral and antral follicles, whereas hedgehog receptor and target gene Ptch1 (G and H) mRNA is localized to cells in the surrounding theca cell compartment. Shown are hematoxylin-stained bright-field images (A, C, E, and G) with corresponding dark-field images (B, D, F, and H) of serial whole adult mouse ovary sections. Closed arrowheads point to large antral follicles in which granulosa cells no longer express Amh mRNA (A and B) but still express Ihh and Dhh (C, D, E, and F), whereas expression of Ptch1 remains up-regulated in the theca cell compartment (G and H). Corpora lutea (asterisks) do not express Amh (A and B), Ihh (C and D), Dhh (E and F), or Ptch1 (G and H), whereas atretic follicles rapidly lose expression of Ihh, Dhh, and Ptch1 (arrows). Note that expression of the hedgehog target gene Ptch1 is induced in the theca cell layer, whereas oocytes (open arrowheads, H) lack this response. Scale bar, 200 μm.
Down-regulation of Amh mRNA expression in granulosa cells in antral follicles (Fig. 2, A and B) is in agreement with published data (9, 51) and clearly precedes down-regulation of Ihh and Dhh in antral follicles (Fig. 2, closed arrowheads). Expression of Ihh, Dhh, and Ptch1 mRNAs is rapidly lost in atretic follicles (Fig. 2, arrows; and supplemental Fig. 1, published on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org). Immunohistochemical staining shows that Ihh/Dhh and Amh proteins are limited to granulosa cells of preantral and antral follicles (Fig. 3A, left follicle) and are down-regulated in atretic follicles, which are characterized by degenerating cells with pyknotic nuclei (Fig. 3A, arrowheads in right follicle).
FIG. 3. Oocytes do not express Ihh and Dhh and lack expression of Smo. A, Immunostaining of adult mouse ovary, showing Amh and hedgehog (Ihh/Dhh) staining of granulosa cells. Like Amh protein, coimmunostaining for both Ihh and Dhh proteins shows that hedgehog is detected in granulosa cells of healthy growing follicles (left follicle), whereas expression is diminished in an atretic follicle (right follicle). Arrowheads point to regions with many pyknotic nuclei representing apoptotic granulosa cells, characteristic for atretic follicles. Scale bar, 100 μm. B, Expression of genes encoding components of the hedgehog signaling pathway in oocytes. Total RNA was isolated from oocytes of 14 and 21dpp superovulated ovaries and adult mouse whole ovaries. The RNA was incubated either in the presence (+) or absence (–) of reverse transcriptase (RT), and each PCR was performed on an amount of cDNA equivalent to that of half an oocyte. Oocytes lack expression of Ihh, Dhh, Ptch2, and Amh mRNAs and also mRNA encoding the essential hedgehog signal transducer Smo.
Oocytes lack Smo and are nonresponsive to granulosa cell-derived Ihh and Dhh
It is to be expected that oocytes are exposed to hedgehog proteins (Ihh and Dhh) from granulosa cells. However, no induction of Ptch1 expression is detected in oocytes (Fig. 2, G and H, open arrowheads). This implies that Ihh and Dhh from granulosa cells are not involved in communication toward the developing oocyte. However, the in situ hybridization technique does not preclude that transcripts for hedgehog signaling components might be present in developing oocytes at a relatively low level. Therefore, we also used the more sensitive RT-PCR with primer sets for Ihh, Dhh, Ptch1, Ptch2, Smo, Amh, Gdf9, and zona pellucida glycoprotein 3 (Zp3). Oocytes were isolated free of granulosa cell contamination, as was confirmed by the absence of mRNA for Amh (Fig. 3B), a factor that is known to be produced by granulosa cells in growing follicles (51). Gdf9 and Zp3 are oocyte-specific genes (52, 53), and the present RT-PCR results show an enrichment of the respective mRNAs in oocytes, compared with whole ovary, using the same amount of total RNA from oocytes and whole ovary in the RT-PCR (Fig. 3B). As concluded from the mRNA expression data, oocytes do not produce Ihh or Dhh themselves, and the oocytes also lack expression of Smo, indicating that they are not capable of responding to hedgehog from granulosa cells (Fig. 3B). There is some expression of Ptch1 mRNA in oocytes, not of Ptch2, which probably represents the above-described hedgehog independent basal expression.
Hedgehog signaling in primary and ovulatory stages of follicle development
Developing follicles in adult mice were classified as previously described (6, 54). We characterized expression of Ihh and Dhh mRNAs and target gene responses in the mesenchymal pre-theca and theca cell compartment, during primary and ovulatory stages of follicle development, in more detail.
Transition of primordial follicles to primary follicles (follicle recruitment) starts to take place in mice between 2.5 and 4.5dpp, and the first growing follicles appear in the medulla part of the ovary. Ovaries at 2.5dpp after birth contain only primordial follicles, but these follicles do not contain detectable levels of hedgehog mRNAs (data not shown). Hence, it is concluded that granulosa cells of primordial follicles do not express Ihh or Dhh and do not up-regulate Ptch1 gene expression in the immediate surrounding mesenchymal stroma cells. However, at 4.5dpp many primary follicles are present throughout the medullary region, and granulosa cells in these follicles express both Ihh and Dhh mRNAs (Fig. 4, A–D). In adult ovary, Ihh and Dhh mRNAs are detected in granulosa cells of primary follicles, with Dhh mRNA readily detectable at the type 3a follicle stage (<20 follicle cells in largest cross-section), followed by Ihh mRNA at the type 3b follicle stage (21–60 follicle cells in largest cross-section) (Fig. 4, G–J).
FIG. 4. Initiation of expression of Ihh, Dhh, Ptch1, and Gli1 mRNAs in primary follicles of early neonatal and adult mouse ovaries. In 4.5dpp mouse ovaries, the first primary follicles clearly express Ihh (A and B), Dhh (C and D), and Ptch1 (E and F). Similarly, primary follicles of adult ovaries express Ihh (G and H), Dhh (I and J), Ptch1 (K and L), and Gli1 (M and N) mRNAs. Note that Dhh is expressed as soon as granulosa cells take up a cuboidal appearance (I and J, arrowhead). Scale bar, 100 μm (A–F) and 50 μm (G–N).
Theca cells originate from mesenchymal precursor cells near primary follicles (11). Induced expression of the hedgehog target genes Ptch1 and Gli1 was found in mesenchymal stroma cells surrounding primary follicles, in 4.5dpp neonatal ovary and also in adult mouse ovary (Fig. 4, E, F, and K–N). This implies that Ptch1 and Gli1 act as early marker genes in differentiation of the theca cell lineage and points to a possible role for Ihh or Dhh or both in the initial phase of theca cell recruitment and development.
To determine expression of mRNAs encoding hedgehog signaling components around ovulation, ovaries from adult mice were analyzed by in situ hybridization after induction of superovulation with Folligonan (pregnant mare serum gonadotropin) and Chorulon (chorionic gonadotropin) (see Materials and Methods). Three hours after injection of Chorulon, loss of expression of Ihh and Dhh mRNAs was seen in some granulosa cells closest to the basal lamina (arrowheads in Fig. 5, A and B; and supplemental Fig. 2). This loss continues at later stages of ovulation, resulting in absence of Ihh and Dhh mRNAs from granulosa cells in the preovulatory follicles that are ready to undergo ovulation. During preovulatory stages, expression of Ptch1 (Fig. 5, C, F, I, and L) and Gli1 mRNAs (supplemental Fig. 2) in the theca cell compartment is gradually lost, reflecting the loss of expression of Ihh and Dhh in granulosa cells. In the same ovarian cross-sections, throughout the ovulation induction procedure, preantral follicles maintain Ihh and Dhh expression and also expression of the target genes Ptch1 and Gli1 (Fig. 5; and supplemental Fig. 2), which indicates that hedgehog signaling in the preantral follicles is not changed by the gonadotropic treatment. Taken together, this ovulation experiment clearly demonstrates dynamic synchrony between expression of hedgehog mRNAs and target gene induction.
FIG. 5. Ihh and Ptch1 mRNAs in mouse ovary during and after induced superovulation. Ovaries were analyzed before ovulation at 1600 h (A–C), during ovulation at 2200 h (D-F), and midnight (G–I) and the morning after ovulation at 0900 h (J–L). Expression of Ihh is lost, going from preovulatory (B) to ovulatory (E and H) and postovulatory (K) follicles but is maintained in preantral follicles (H and K). Expression of Ptch1 mRNA is also lost, but there is a low basal level in periovulatory follicles (F, I, and L) and maintenance of a high level in the theca cell compartment of preantral follicles (I and L). Scale bar, 100 μm.
Genes encoding additional components of the hedgehog signaling pathway
We further examined the postnatal mouse ovary for in situ expression of gene transcripts encoding additional protein components of the hedgehog signaling pathway. The seven-transmembrane signal transducer Smo is expressed throughout the mouse ovarian tissue, including all stages of growing follicles, the corpus luteum, and the interfollicular mesenchyme (Fig. 6, A and B), with exception of the oocyte (Figs. 6, A and B, and 3B, arrowheads). Expression of Smo is found in many tissues and is balanced by an ubiquitous low level of Ptch1, which is required to restrict signaling activity to cells that are exposed to hedgehog proteins. Smo is not a target gene for hedgehog signaling. In contrast to Smo but similar to Ptch1, the genes encoding the second hedgehog receptor Ptch2 and the binding protein Hip1 are targets of hedgehog signaling. Ptch2 and Hip1 mRNAs both are found to be up-regulated in the theca cell layer of growing follicles (Fig. 6, C–F) but not in the oocyte (arrowheads in Figure 6, C–F).
FIG. 6. Expression of genes encoding additional components of the hedgehog signaling pathway in postnatal mouse ovary. In situ expression in adult mouse ovary of mRNAs encodes the membrane-associated signal transducer Smo (A and B); the second hedgehog receptor Ptch2 (C and D); Hip1 (E and F); and the intracellular transducers Gli1 (G and H), Gli2 (I and J), and Gli3 (K and L). Note that Ptch2, Hip1, and Gli1 are target genes of hedgehog signaling and are expressed in theca cell layers, similar to induced expression of Ptch1 mRNA (not shown in this figure), but not oocytes (arrowheads). Scale bar, 100 μm.
In different cell types, basal expression of the transcriptional regulators Gli2 or Gli3 is required for initiation of hedgehog signaling to allow for activation of the pathway and induction of Gli1 expression (31, 55, 56, 57). Gli3 mRNA is found at near-to-background level, but there is clear expression of Gli2 mRNA, which is seen at a basal level throughout the tissue, perhaps with a slight accumulation of mRNA in theca cells surrounding some growing follicles (Fig. 6, I–L). Expression of the hedgehog target gene Gli1 starts in mesenchymal cells surrounding the primary follicle (Fig. 4) and is maintained in the theca cell compartment of preantral and antral follicles (Fig. 6, G and H).
Cyclopamine down-regulates target gene activation in ovarian mesenchymal cells
The steroidal alkaloid cyclopamine and its derivative KAAD-cyclopamine specifically block cellular responses to vertebrate hedgehog signaling by directly binding to the essential signal transducer Smo (26). KAAD-cyclopamine blocks hedgehog signaling both in vitro and in vivo, with a 10- to 20-fold higher potency but with a similar or lower toxicity, compared with cyclopamine (25, 58). We set up organ cultures of whole ovaries isolated from 2dpp mice, in which follicles are at the primordial stage and do not express Ihh or Dhh (data not shown). Individual ovaries were cultured for 6 d, either in the absence (control) or presence of 57 nM or 0.57 μM KAAD-cyclopamine (Fig. 7). The addition of KAAD-cyclopamine did not affect the histological morphology of the ovaries during the 6-d culture period. Both in the absence and presence of KAAD-cyclopamine, the cultured ovaries were found to contain developing follicles up to the type 4 preantral stage with two to three layers of granulosa cells (Fig. 7, A–C). Growing follicles in ovaries that were cultured for 6 d in the absence of KAAD-cyclopamine express mRNAs for Amh and Ihh in granulosa cells and Ptch1 and Gli1 in surrounding stromal cells (Fig. 7, D, G, J, and M). No changes in the expression of Amh, Ihh, Ptch1, and Gli1 were observed at 5.7 nM KAAD-cyclopamine (data not shown). When the ovaries were cultured in the presence of 57 nM or 0.57 μM KAAD-cyclopamine, expression of Amh and Ihh mRNAs in granulosa cells of growing follicles is maintained (Fig. 7, E, F, H, and I), but expression of both Ptch1 and Gli1 in the stromal cells is strongly down-regulated in the presence of 57 nM KAAD-cyclopamine (Fig. 7, K and N) and even further down-regulated at 0.57 μM KAAD-cyclopamine (Fig. 7, L and O). A low level of Ptch1 mRNA remains present in ovaries cultured in the presence of 57 nM and 0.57 μM KAAD-cyclopamine and represents the low basal level that is independent of hedgehog signaling, encoding a basal level of Ptch1 that controls Smo in the absence of hedgehog ligands (Fig. 7L; and data not shown).
FIG. 7. KAAD-cyclopamine inhibits the expression of Ptch1 and Gli1 mRNAs in cultured neonatal mouse ovaries. Ovaries were isolated from 2dpp mice and cultured as described in Materials and Methods for 6 d in the absence (control) or presence of 57 nM or 0.57 μM KAAD-cyclopamine, indicated above each panel. The top panel (A–C) shows representative hematoxylin-eosin-stained sections of the cultured ovaries. Scale bar, 50 μm. The cultured ovaries were sectioned for in situ hybridization to detect Amh, Ihh, Ptch1, and Gli1 mRNAs. For Amh (D–F) and Ihh (G–I), only the bright-field images are shown, whereas for Ptch1 (J–L) and Gli1 (M–O), the figure also includes the dark-field images. Scale bar, 100 μm.
The observed down-regulation of Ptch1 and Gli1 mRNA expression by the antagonist KAAD-cyclopamine, provides direct evidence that hedgehog target gene expression in the pre-theca cell compartment of developing follicles depends on local hedgehog signaling.
Discussion
Hedgehog signaling in theca cell development
The present study exposes the mammalian ovary as a novel site of active hedgehog signaling. For the three mammalian hedgehog ligands, we show that Ihh and Dhh, but not Shh, are expressed in the mouse ovary during reproductive life. Expression of Ihh and Dhh localizes to granulosa cells of growing follicles, whereas expression of the hedgehog signaling target genes Ptch1, Ptch2, Gli1, and Hip1 is induced in the theca cell compartment. In contrast, expression of these hedgehog target genes is not induced in oocytes. Expression of Ptch1 and Gli1 mRNAs is strongly down-regulated in cultured ovaries treated with the specific hedgehog signaling antagonist KAAD-cyclopamine, providing direct evidence that induced target gene expression in the ovarian mesenchymal cells requires hedgehog signaling.
Initiation of follicle growth, often referred to as follicle recruitment, is defined as the transition of a nongrowing primordial follicle (type 2) to an early primary follicle (type 3a). During this transition, granulosa cells increase in number and transform from a squamous to a cuboidal morphology, and growth of the oocyte is initiated (6). Granulosa cells of primordial follicles do not express Ihh or Dhh, and there is no detectable induction of target gene expression in any ovarian cell type in neonatal mice at 2dpp, when the ovary contains only primordial follicles. Observations on adult mouse ovary show that Dhh mRNA is first detected when granulosa cells take up a cuboidal morphology during the transition from type 2 to type 3a primary follicle stages, whereas expression of Ihh initiates at the type 3b primary follicle stage. Induced expression of the hedgehog target genes Ptch1 and Gli1 is first detected in mesenchymal cells adjacent to granulosa cells between the type 3a and type 3b primary follicle stages. The results suggest that hedgehog signaling does not play part in the initiation of follicle growth but rather starts to act early after the transition from the primordial to the primary follicle stage.
Formation of theca cells with steroidogenic capacity is a crucial functional aspect of follicle development, but little is known regarding regulation of recruitment of theca precursor cells and their development into steroidogenic theca cells. Mesenchymal pre-theca cells most likely are located within the ovarian stroma and may have stem cell properties, but theca stem cells have not been identified yet (3, 59, 60). Primordial and primary follicles lack morphologically differentiated theca cells, and the first steroidogenic theca cells appear when preantral follicles contain two to three layers of granulosa cells (11). These theca cells respond to LH and gain expression of steroidogenic enzymes and the capacity to produce androgens. Pre-theca cells, however, do not express the LH receptor, and theca cell recruitment and early differentiation are LH independent, so that locally produced paracrine factors are possible candidates to induce theca cell differentiation (11). Mesenchymal pre-theca cells surrounding the primary follicle show an increase in DNA replication (59) and onset of the expression of several genes characteristic of theca cells, including the genes encoding bone morphogenetic protein (Bmp) 4 (61), the LH receptor (11), and also Ptch1 and Gli1, as described herein. Initiation of pre-theca cell recruitment and differentiation starts as early as the primary follicle stage, and it appears that theca differentiation factors must be secreted by primary follicles. Induction of hedgehog target gene expression initiates in pre-theca cells at the primary follicle stage, and both Ihh and Dhh are excellent candidates to act as early theca differentiation factors. In Dhh knockout mice, no ovarian phenotype is observed (21), which indicates that Ihh might act as the predominant signal in the ovary. This does not exclude, however, that the actions of the two hedgehog ligands in the ovary are partially redundant. Testis lacks local Ihh production so that only Dhh can act as Leydig cell differentiation factor, which explains the occurrence of a testicular phenotype in Dhh knockout mice (21, 39, 62, 63).
The testicular phenotype of Dhh knockout mice revealed that hedgehog signaling plays an essential role in testis tissue organization. In these knockout mice, the testis shows dysregulation of formation of the tubular wall and defects in differentiation of interstitial Leydig cells (21, 39, 62, 63). In formation of the tubular wall, peritubular myoid cells interact with Sertoli cells, and both cell types contribute protein components to the basal lamina (64). Dhh signaling seems essential for communication between Sertoli cells, peritubular myoid cells, and the interstitial Leydig cell compartment. In the ovary, the paracrine action of Ihh and Dhh might be important, somewhat similar to the role of hedgehog signaling in testis, for development of a proper tissue organization, with an intact follicular basal lamina and a well-structured steroidogenic theca cell compartment.
As a next step, we aim to evaluate the ovarian phenotype of a granulosa cell-specific Ihh conditional knockout on the Dhh knockout background. This type of analysis may yield information to explain the precise role of hedgehog signaling in ovarian function, which also will be relevant in relation to our recent observations that active hedgehog signaling is present in human ovarian follicles (results not shown).
Hedgehog signaling in germ line cells
The Caenorhabditis elegans genome contains a gene ptc-1, which encodes a membrane protein similar to mammalian Ptch1/Ptch2 (65). However, true homologs of hedgehog and smoothened genes have not been detected in C. elegans (66), which indicates that ptc-1 is involved in signaling or other activities not related to a functional hedgehog signaling pathway. C. elegans ptc-1 has been shown to be important for cytokinesis of germ cells; ptc-1 null mutant C. elegans is sterile, and the gonads contain multinucleate germ cells (67).
In Drosophila ovary, hedgehog is produced by somatic cells of the terminal filament and the adjacent cap cells situated at the distal tip of the germarium (68, 69). Hedgehog signaling in Drosophila ovary has been studied using induced ptc-LacZ expression, which is detected in somatic cells extending from the terminal filament up to and including the somatic stem cells (68). This ovarian hedgehog signaling has been shown to control proliferation and maintenance of ovarian somatic stem cells (70). Germ line cells are located near the distal tip of the germarium, very close to the cellular source of hedgehog, but ptc-LacZ expression is not detected in these germ line cells (68). The Drosophila ovarian germ line cells express a basal level of ptc protein, but the cubitus interruptus protein, which is the Drosophila homolog of the mammalian Gli transcription factors, is not present (68). There are a few indications that ptc and smo in Drosophila germ line cells might play some role as cell-autonomous components of the hedgehog signaling pathway, in embryonic germ cell migration (71). However, most available data suggest that there is lack of hedgehog signaling in Drosophila ovarian germ line cells.
In mouse oocytes in growing follicles, we did not observe induced expression of the hedgehog target genes Ptch1, Ptch2, Hip1, and Gli1. We conclude that these oocytes, in diplotene stage of meiotic prophase, do not respond to granulosa-derived Ihh and Dhh. This lack of response can be explained by lack of expression of Smo in oocytes because we have not detected Smo mRNA in oocytes using RT-PCR or in situ hybridization. Also in spermatogenic germ line cells, expression of Smo mRNA is probably absent or very low, as described herein. It can be suggested that male and female germ line cells are not responsive to hedgehog signaling during gametogenesis. This would be in agreement with observations on expression of a Ptch1-LacZ knock-in gene construct in a mouse model that has been used to demonstrate active hedgehog signaling in target cells (34). In target cells, hedgehog signaling activates the endogenous Ptch1 promoter above basal level, leading to LacZ activity from expression of the Ptch1-LacZ knock-in construct. In this mouse model, LacZ activity in the testis is mainly found in peritubular and interstitial cells but not in Sertoli cells and germ line cells (21, 39, 62, 63).
In summary, the available data indicate that gonadal hedgehog signaling, in animal species ranging from worms and flies to mammals, is not directly involved in control of germ line cells. In Drosophila ovary, the main role of hedgehog signaling is related to control of proliferation and maintenance of somatic stem cells. In mammalian gonads, Sertoli cells in testis and granulosa cells in ovary provide for hedgehog signaling as an important factor in the communication between the cellular compartments that perform gametogenesis and steroidogenesis.
Acknowledgments
We thank Prof. A. P. McMahon (Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA) for cDNA probes representing hedgehog signaling components and for the rabbit polyclonal antibody Ab80.
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