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Keywords:

  • germ cells;
  • Hbp1;
  • HMG box;
  • testis;
  • gonad development

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES

HMG box containing protein 1 (HBP1) is a high mobility group domain transcriptional repressor that regulates proliferation in differentiated tissues. We have found mouse Hbp1 to be expressed strongly in the embryonic mouse testis from approximately 12.5 days post coitum, compared with low levels of expression in the embryonic ovary. Expression of Hbp1 is maintained in the developing testis beyond the onset of spermatogenesis after birth. Whole-mount in situ hybridisation analysis showed that expression of Hbp1 in the XY gonad is localized within the developing testis cords, the precursors of the seminiferous tubules. Expression of Hbp1 is not apparent in testis cords of gonads from homozygous We mutant embryos, which lack germ cells. In situ hybridisation analysis on cryosectioned embryonic testis indicated that Hbp1 expression resembles that of the germ cell marker Oct4. We conclude that Hbp1 is up-regulated specifically in germ cells of the developing XY gonad. The expression of Hbp1 in XY germ cells appears to correlate with the onset of mitotic arrest in these cells. Developmental Dynamics 230:366–370, 2004. © 2004 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES

The establishment of the germline is one of the most critical stages in embryogenesis and one that, at first glance, appears not to be conserved among metazoans. In lower organisms, the germline is generally set aside very early in development, such as in Drosophila where germ cells are the first to be formed in the embryo (Rongo and Lehmann, 1996). By contrast, germ cells of mammalian species are not established until embryogenesis is well under way. In the mouse embryo, the progenitors of the primordial germ cells (PGCs) derive from the proximal epiblast and migrate into the adjoining extraembryonic mesoderm (Lawson and Hage, 1994), within which primordial germ cells are first detectable around 7.2 days post coitum (dpc) as a cluster of cells expressing high levels of alkaline phosphatase activity (Ginsberg et al., 1990). Between approximately 8.5 and 11 dpc, these cells migrate along the hindgut into the genital ridges, continuing to proliferate until 2–3 days after their establishment in the genital ridge (reviewed by McLaren, 2000).

Shortly after the germ cells arrive at their destination, the molecular decision as to whether the bipotential gonad will proceed down the male or female developmental pathways is made, after the expression of the Y-linked male sex determining gene Sry in the XY genital ridge from around 10.5–11.5 dpc. Expression of Sry results in the differentiation of Sertoli cells, a supporting cell lineage that surrounds the germ cells to form the testis cords, the precursors of the adult seminiferous tubules. In the XX gonad, PGCs enter meiosis around 13.5 dpc, signifying their commitment to the oogenic pathway. This finding marks one of the earliest detectable female-specific changes in gonad morphology. The continued presence of meiotic germ cells is essential for further development of the ovary (McLaren, 1991). Male PGCs must be prevented from entering meiosis during foetal development and are held in mitotic arrest until shortly after birth when spermatogenesis is initiated (McLaren and Southee, 1997). How these processes are conducted at a molecular level remains a mystery.

We previously have undertaken an expression screening approach to identify genes expressed differentially between the male and female mouse gonad during the critical period of sex determination, based on the presumption that such genes are good candidates for involvement in sex determination and gonad development (Bowles et al., 2000; Bullejos et al., 2001; Koopman et al., 2002). One of the many candidate genes we have identified as being up-regulated in the embryonic testis by this method is that encoding HMG box containing protein 1 (HBP1), which we now find to be strongly up-regulated in germ cells of the developing embryonic testis from approximately 12.5 dpc, compared with expression in the developing ovary.

HBP1, like members of the TCF/LEF (Novak and Dedhar, 1999) and SOX (Wilson and Koopman, 2002) families, is a sequence-specific HMG box transcription factor with significant roles in the regulation of proliferation and differentiation. Expression of Hbp1 in vitro leads to cell cycle arrest even in the presence of optimal proliferation signals (Tevosian et al., 1997). In vivo, HBP1 has been shown to regulate proliferation within differentiated adult tissue; after partial hepatectomy in transgenic mice carrying Hbp1 under a hepatocyte-specific promoter, as little as a twofold increase in the level of HBP1 protein is sufficient to delay progression of hepatocytes through G(1) to the peak of S phase by 10–12 hr (Shih et al., 2001). HBP1 can function as a direct transcriptional repressor of cell cycle targets such as the promoters for n-Myc (Tevosian et al., 1997) and the CDK inhibitor p21 (Gartel et al., 1998). Sequence-specific activation of promoters such as that for histone H1(0) has also been shown for HBP1, indicating that HBP1 plays a vital role in chromatin remodeling events during arrest of cell proliferation in differentiating cells (Zhuma et al., 1999; Lemercier et al., 2000).

Aside from the HMG domain, which is implicated in sequence-specific binding and sharp bending of DNA (Deckert et al., 1999), the HBP1 protein contains conserved activation and repression domains, including two motifs required for interaction with retinoblastoma (Rb) family members (Lavender et al., 1997; Tevosian et al., 1997). HBP1 interacts specifically with the retinoblastoma family members Rb and p130, which are up-regulated as differentiation proceeds, but not with p107, which is down-regulated with differentiation (reviewed by Yee et al., 1998). As such, HBP1 appears to have dual repressor/activator functions, which are determined by promoter context and cell type and can repress transcription by both sequence-dependent and -independent means. HBP1 uses both of these methods to repress the Wnt–β-catenin pathway (Sampson et al., 2001). HBP1 can repress Wnt pathway target genes such as cyclin D1 and other cell cycle genes by directly binding and repressing regulatory sequences within their promoters. However, HBP1-mediated inhibition of Wnt signalling does not require DNA binding, as HBP1 can also bind and inhibit active complexes of TCF4 and β-catenin, blocking DNA binding of TCF/LEF family members to their target sites by direct protein–protein interaction (Sampson et al., 2001).

Here, we investigate the expression of Hbp1 in mouse embryonic male germ cells, and discuss what role this factor may have in the arrest of proliferation and promotion of differentiation in this developmental system.

RESULTS AND DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES

In a previous study (Bowles et al., 2000), male- and female-enriched gonadal cDNA libraries were constructed by using poly(A)+ RNA isolated from 12.5 to 13.5 dpc embryonic testis and ovary. By the same approach, earlier-stage male- and female-enriched libraries were constructed by using RNA from 11.5 ± 0.5 dpc tissue. Random clones were sequenced from the male-enriched (male vs. female subtracted) library 11M. Two identical clones, 11M47 and 11M52, were found to correspond to the C-terminus of the open reading frame and some 3′-UTR sequence from the mouse high mobility group (HMG) box containing protein 1 gene (Hbp1; nucleotides 1495-1635 of GenBank NM153198). The domain structure of this factor has been determined (Lavender et al., 1997; Tevosian et al., 1997).

Whole-mount in situ hybridization (WISH) analysis revealed that Hbp1 exhibits higher levels of expression in 13.5 dpc embryonic testis than in the ovary at the same stage (Fig. 1). Prominent expression of Hbp1 was evident in embryonic testis cords, with background levels of expression observed in the adjoining mesonephric tissue.

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Figure 1. Whole-mount in situ hybridization analysis of the time course of Hbp1 expression in embryonic mouse gonads between 11.5 and 18.0 days post coitum (dpc). Male (M) gonads, on the left in each panel, show stronger expression than female (F) gonads from approximately 12.5 dpc. Expression of Hbp1 is prominent in the testis cords of the developing male gonad.

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In view of available evidence that HBP1 is a cell cycle inhibitor that negatively regulates cell proliferation in differentiated tissues through a variety of methods (Tevosian et al., 1997; Shih et al., 2001), we considered that Hbp1 might have a role in control of the cell cycle in the developing gonad and set out to investigate its expression in more detail. To this end, additional WISH analyses were performed to establish the onset and time course of expression. We found that expression of Hbp1 was strongly up-regulated around 12.5 dpc in the male gonad, and continued throughout development of the testis in utero. Expression levels of Hbp1 in the ovary remained low throughout this period (Fig. 1).

To determine which cell type(s) within the testis cords express Hbp1, gonads were dissected from 13.5 dpc male embryos homozygous for the extreme allele of dominant white spotting (We/We; Buehr et al., 1993). These gonads lack germ cells. WISH analysis revealed that Hbp1 was not expressed in the testis cords of gonads from 13.5 dpc male We/We embryos (Fig. 2A). Similarly, reverse transcriptase-polymerase chain reaction (RT-PCR) amplification of total RNA extracted from 13.5 dpc male We/We and wild-type embryonic gonads showed that transcripts for Hbp1 were significantly reduced in We/We male gonads (Fig. 2B), like those of the germ cell marker Scp3 (synaptonemal complex protein gene 3, also known as Sycp3), which is expressed in premeiotic male germ cells at this stage in development (Di Carlo et al., 2000). This finding indicates expression of Hbp1 in the embryonic testis is dependent on the presence of germ cells and suggests two possibilities: that Hbp1 is expressed specifically in the germline, and/or that Hbp1 is expressed in surrounding somatic lineages in the testis cords such as Sertoli cells, as a result of germ cell signalling to those cells.

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Figure 2. Reduced Hbp1 expression in gonads from 13.5 days post coitum male We/We embryos, which lack germ cells. A: Whole-mount in situ hybridization analysis comparing Hbp1 expression in We/We and wild-type (+/+) littermate gonads. B: Reverse transcriptase-polymerase chain reaction amplification from total RNA extracted from We/We and wild-type (+/+) littermate gonads, using primers specific for Hbp1, Hprt (a ubiquitously expressed gene included as a positive control), Sox9 (a Sertoli cell marker), and Scp3 (a marker of early meiotic and premeiotic germ cells).

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To establish which of these possibilities is the case, in situ hybridization (ISH) analysis was performed on cryosections of embryonic testis (Fig. 3). The expression pattern of Hbp1 closely resembled that of the germ cell marker Oct4, exhibiting the characteristic ring-like staining pattern of germ cells, distributed throughout the central region of the testis cords. The expression patterns of Hbp1 and Oct4 contrasted with that of the Sertoli cell marker Amh, which was characteristic of the more peripheral, stellate pattern of staining exhibited by Sertoli cells. This finding, together with the We/We expression data, indicates that Hbp1 is expressed in the germ cells of the developing testis.

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Figure 3. In situ hybridization analysis on cryosectioned embryonic testis (14.0 days post coitum). A–C: The expression pattern of Hbp1 (A) is most similar to that of Oct4 (B), a marker of germ cells, and differs from Amh (C), a marker of Sertoli cells. Expression in germ cells is characterized by ring-like patterns of staining toward the core of each testis cord. Scale bars = 50 μm in A–C.

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Germ cells of the developing XY gonad cease proliferation around 13.5 dpc, and remain in mitotic arrest until the onset of spermatogenesis, approximately 6 days post partum (McLaren, 2000). The specific up-regulation of Hbp1 in male germ cells around the same time as they exit the cell cycle, along with the established role of this factor in the control of proliferation in differentiated tissues (Shih et al., 2001), suggests a role for HBP1 in male-specific arrest in proliferation of male germ cells. At the same stage in the ovary, XX primordial germ cells enter meiotic prophase I; the presence of meiotic germ cells is essential for the continuation of ovarian development (McLaren, 1991) and has been shown to inhibit testis cord formation in gonad culture experiments (Yao et al., 2003). In utero, male germ cells are actively prevented from entering meiosis (McLaren and Southee, 1997), presumably under the influence of some meiosis-inhibiting (or spermatogenesis-promoting) signal originating from the local somatic cell environment of the testis (McLaren, 1984). It is not known whether male-specific mitotic arrest is part of the mechanism by which XY germ cells are prevented from entry to meiosis.

The present observations raise the possibility that HBP1 may respond to diffusible Sertoli cell factors produced in response to SRY or SOX9 to promote this male-specific arrest in proliferation. However, HBP1 is not capable of preventing XY germ cells from entering meiosis postnatally, as spermatogenesis proceeds in the presence of Hbp1 expression in the testis; Hbp1 has been shown previously to be expressed in the adult testis by Northern analysis (Lemercier et al., 2000) and was detected by us in newborn and week-old testis by RT-PCR analysis (data not shown).

HBP1 has been shown to regulate proliferation by repression of the Wnt–β-catenin pathway (Sampson et al., 2001). The involvement of Wnt family genes in gonad development currently appears limited to the actions of Wnt4 and Wnt7a, which are involved in the establishment (Wnt4; Vainio et al., 1999) and subsequent male-specific degradation (Wnt7a; Parr and McMahon, 1998) of the Müllerian duct, the progenitor of the female internal genitalia. We have not detected expression of Hbp1 within the developing Müllerian duct (Figs. 1, 2, and data not shown). Wnt4 has also been implicated in the postmeiotic maintenance of XX germ cells, with Wnt4-/- female mice showing a marked reduction in oocyte development (Vainio et al., 1999). Wnt4 is expressed in the genital ridges of both sexes up until 11.5 dpc, whereafter expression persists in the XX gonad but is down-regulated in the XY gonad (Vainio et al., 1999). In the developing ovary, Wnt4 is thought to repress male-specific migration of mesonephric cells into the gonad, preventing steroid production and the formation of testis-specific vasculature (Jeays-Ward et al., 2003). Further studies will be required to determine whether the actions of WNT4 in germ or somatic cell lineages of the male gonad are opposed by expression of Hbp1, and to establish the precise role of Hbp1 in male germ cell development.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES

Isolation of Mouse Hbp1 From Male-Enriched Subtracted Library

Total RNA was isolated from 11.5 to 12.5 dpc male and female embryonic gonads by using Trizol reagent (Invitrogen). Poly(A)+ RNA was isolated by using a Oligotex mRNA kit (QIAGEN). Suppression-subtraction hybridization (Diatchenko et al., 1996) was carried out by using a PCR-Select cDNA subtraction kit (Clontech) as outlined previously (Bowles et al., 2000). Male-enriched and female-enriched libraries were produced, which were cloned into pGEM-T Easy Vector (Promega). Random clones from the male-enriched library were sequenced using vector primers. Identical clones 11M47 and 11M52 from the male-enriched library showed high homology to mouse Hbp1.

In Situ Hybridization

RNA in situ hybridization on whole-mount and cryosectioned embryonic mouse tissue was carried out by using standard methods (Wilkinson and Nieto, 1993; Hargrave and Koopman, 1999). Riboprobes for Oct4 (Scholer et al., 1990) and Amh (Münsterberg and Lovell-Badge, 1991) were synthesized as described previously. Clone 11M52 was used as a template for synthesis of a riboprobe for Hbp1; riboprobes generated against other regions of the Hbp1 coding sequence and 3′-UTR region gave identical results by means of ISH analysis (data not shown).

RT-PCR

Total RNA was isolated from embryonic, newborn, and adult mouse gonads with Trizol reagent (Invitrogen). A total of 0.5–2 μg of total RNA was used as a template for first-strand cDNA synthesis using oligo(dT) as primer, in the presence of MMLV reverse transcriptase (Invitrogen). One microliter of cDNA was subsequently used as a template for PCR amplification using Taq polymerase (New England Biolabs). Primers used were as follows: HPRT-1A, 5′-CCTGCTGGATTACATTAAAGCACT-3′; HPRT-1B, 5′-GTCAAGGGCATATCCAACAACAAA-3′; SCP3D, 5′-ACAACAAGAGGAAATACAGAA-3′; SCP3E, 5′-GAGAGAACAACTATTAAAACA-3′; SOX9.5B, 5′-GTGGCAAGTATTGGTCAA-3′; SOX9.5C, 5′-GAACAGACTCACATCTCT-3′; 11M52fwd, 5′-GTACACATTAGAAGCAAAGGCT-3′; 11M52rev, 5′-GTACTTATGCAGATGCAGACTT-3′.

PCR reactions were denatured at 95°C for 2 min, followed by 30 cycles of 95°C for 30 sec, 55°C for 30 sec, and 72°C for 60 sec. Amplified products were separated on a 1.5% agarose gel by using a 1-kb DNA ladder (Invitrogen) for size comparison.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES

The authors thank Dr. Cate Browne for critical reading of the manuscript. J.M.S. is the recipient of an Australian Postgraduate Award and a supplementary scholarship from the Institute for Molecular Bioscience. P.K. is a Professorial Research Fellow of the Australian Research Council.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES