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Gamete Biology |
a Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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gametogenesis, ovary, spermatogenesis, steroid hormone receptors, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Using Northern blot and in situ analyses, several laboratories have shown that GCNF mRNA is expressed in mouse and Xenopus embryos [10–14]. Four different GCNF messages (2.4, 7.5, 8.5, and 10 kilobases ;obkb;cb) and only one GCNF transcript (7.5 kb) are found in Xenopus embryos and E8.5–E9.5 mouse embryos, respectively [10, 12, 13]. The expression of GCNF in mouse and Xenopus embryos indicates that GCNF may play a role in normal embryonic development during gastrulation and organogenesis. Indeed, recent targeted mutation studies have shown that GCNF is required for normal anteroposterior axis formation and somitogenesis in mouse and Xenopus embryos during early gestation [11, 13, 14].
In adult animals, expression of GCNF is quite unique for a nuclear receptor. It is predominantly expressed in the gonads of all species tested, including zebrafish, Xenopus, mice, rats, and humans [2, 6, 7, 15–17]. Multiple forms of GCNF transcripts (2.4, 3, 7.5, 8.5, and 10 kb) are found in the testes and ovaries of Xenopus and zebrafish [7, 10]. Two GCNF transcripts (2.2–2.4 and 7.4–8 kb) are expressed in the testes of various mammalian species, including mice, rats, and humans [3, 4, 15–17], and only one GCNF transcript (7.4–7.5 kb) is expressed in human ovaries [18, 19]. In the testes of mice and humans, two GCNF transcripts differ in their 3' untranslated regions [15, 19]. The expression of GCNF mRNA in gonads of zebrafish, mice, rats, and humans has been shown to be germ cell-specific [3, 4, 7, 15, 16, 20]. In mouse and rat testis, GCNF mRNA is expressed in postmeiotic round spermatids with the highest expression levels at stages VI–VIII of spermatogenesis [4, 15, 16, 20], whereas in zebrafish and humans, GCNF mRNA is expressed in pachytene spermatocytes, which are still in the first meiotic division during spermatogenesis [7, 17]. Similar to the germ cell-specific expression pattern of GCNF in males, GCNF mRNA is also exclusively expressed in developing germ cells in the ovaries of mice, Xenopus, and zebrafish [3, 7, 10, 15]. In Xenopus and zebrafish, GCNF is highly expressed in previtellogenic oocytes in the early stages of oogenesis [7, 10]. In the mouse ovary, GCNF mRNA was detected in primary, secondary, and preovulatory oocytes, but not in primordial follicles [3, 16]. The germ cell-specific expression pattern indicates that GCNF may be a transcription factor that plays a role in regulating some aspects of spermatogenesis and oogenesis. GCNF could also be a maternally supplied factor that is involved in early mouse embryonic development.
Although it is well documented that GCNF mRNA is expressed in the germ cells of murine gonads [3, 4, 15, 16, 20], little is known about the localization of GCNF protein in gonads and preimplantation embryos. To date, expression of GCNF protein in ovaries and fertilized oocytes during early embryonic developmental stages before implantation has not been reported. GCNF protein was reported to be present in mouse round spermatids by Western blot analysis [20, 21]. However, an immunofluorescence study showed that GCNF protein was present only in primary spermatocytes, but not in round spermatids [20]. The immunofluorescence results reported by Bauer et al. [20] appear to contradict the observation that GCNF mRNA is predominantly expressed in postmeiotic spermatogenic cells [4, 15, 16, 20]. Therefore, expression of GCNF protein in the mouse testis remains to be clarified. In this communication, we generated and characterized polyclonal antibodies against a GCNF-specific polypeptide. Using these antibodies we determined the presence of GCNF protein in mouse gonads during postnatal development. We also determined the presence of GCNF protein in unfertilized oocytes and preimplantation embryos at different stages. Our results show that GCNF protein is expressed in both the cytoplasm and nuclei of postmeiotic round and elongating spermatids in the testis, and in the cytoplasm of mouse oocytes in primary, secondary, and preovulatory follicles. Our data also show that GCNF is a maternal protein and is expressed in preimplantation mouse embryos. This study adds further weight to the theory that GCNF plays a role in reproduction and preimplantation embryonic development.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The fusion protein GST-GCNF expression vector was generated by insertion of a full-length mouse GCNF cDNA [3] into the EcoRI–XhoI site of pGEX-4T-1 vector (Promega, Madison, WI). The GCNF plasmid for in vitro translation was constructed by insertion of the full-length GCNF cDNA into the pIVEx2.4a vector (Roche Diagnostics, Indianapolis, IN). Cytomegalovirus (CMV)-HA-GCNF and CMV-Myc-GCNF vectors were constructed by inserting a full-length GCNF cDNA into the EcoRI–XhoI site of the pCMV-HA and pCMV-Myc vectors (BD Biosciences Clontech, Palo Alto, CA), respectively.
Preparation of GCNF Fusion Proteins and P19 Nuclear Extract
The GST-GCNF fusion protein was expressed in bacterial BL21 (DE3) cells and purified by glutathione affinity columns according to the manufacturer's protocol (BD Biosciences Clontech). Fusion proteins HA-GCNF and Myc-GCNF were obtained from Cos-1 cells that were transiently transfected with pCMV-HA-GCNF or pCMV-Myc-GCNF plasmid for 48 h as previously described [21]. Protein extract from Cos-1 cells (CMV) transfected with the empty pCMV-HA vector were also prepared as negative controls for Western blot analyses. In vitro-translated GCNF was obtained using the TNT T7-coupled reticulocyte lysate system (Promega).
P19 cell nuclear extract was prepared as previously described [22]. Briefly, P19 cells were harvested and resuspended in a low-salt buffer (10 mM Hepes pH 7.9, 1.5 mM MgCl2, 10 mM KCl, and 2 mM dithiothreitol ;obDTT;cb). The cell suspension was then subject to homogenization using a type B pestle glass Dounce homogenizer for 10 up-and-down strokes. Cell nuclei were spun down, resuspended in a high-salt extraction buffer (20 mM Hepes pH 7.9, 20% glycerol, 0.55 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM PMSF, and 2 mM DTT), and then homogenized to lyse the nuclear membranes. After centrifugation the supernatant was collected and then dialyzed against a dialysis buffer (20 mM Hepes pH 7.9, 20% glycerol, 0.1 M KCl, 0.2 mM EDTA, 5 mM MgCl2, 0.5 mM PMSF, and 2 mM DTT). The dialyzed solution was then centrifuged to obtain the supernatant (P19 cell nuclear extract).
Anti-GCNF Polyclonal Antibodies
A GCNF-specific polypeptide (amino acids 477–495, LPLLFKVV-LHSCKTSTVKE) was synthesized, purified by high-performance liquid chromatography, conjugated to keyhole limpet hemocyanin (KLH), and then injected into rabbits to generate rabbit anti-GCNF antibodies by Bethyl Laboratories, Inc. (Montgomery, TX). Preimmune and hyperimmune sera were collected and passed through HiTrap Protein A HP affinity columns (Amersham Pharmacia Biotech, Piscataway, NJ) to generate rabbit preimmune immunoglobulin G (IgG) and rabbit anti-GCNF IgG, respectively.
Electrophoresis Mobility Shift Assay
Electrophoresis mobility shift assays (EMSAs) were performed as previously described [23]. The sequences of the DR0 oligonucleotides were 5'-CTGACTGGGTAAGGTCAAGGCTATTCTAAAGTCGA-3' (forward) and 5'-TCAGTCGACTTTAGAATAGCCTTGACCTTACCCAG-3' (reverse). The probe was radiolabeled by fill-in reaction with Klenow fragment in the presence of [
-32P]dATP and [-32P]dCTP. Two microliters of in vitro-translated GCNF or 15 µg of P19 cell nuclear extract were used in binding assays, and 1 µl (0.81 µg protein) of purified GCNF antibodies or rabbit preimmune serum was used for EMSA.
Animal Tissue Preparations
Healthy adult C57BL/6 mice were purchased from Jackson Laboratory (Bar Harbor, ME) and maintained on a 14L:10D cycle, with free access to food and water in the vivarium of the Baylor College of Medicine. Five-, 12-, 21-, and 56-day-old animals were obtained by intercrossing healthy adult C57BL/6 males and females in the vivarium. Animals were killed with carbon dioxide, and ovaries, testes, and epididymides were trimmed free of fat and then subject either to homogenization or fixation. Whole-cell homogenates from adult mouse testes, caput epididymides, cauda epididymides, and 12-day-old testes were prepared as described previously [24]. Sperm were collected from the cauda regions of adult epididymides, and then extracted with a Laemmli sample buffer [25]. Testes and ovaries from 5-, 12-, 21-, and 56-day-old mice were fixed in 4% paraformaldehyde, dehydrated, and then embedded in paraffin. Paraffin-embedded tissues were sectioned at 7 µm of thickness by a Leica Histoslide 2000 Slide microtome (Leica Microsystems AG, Wetzlar, Germany). All experiments in this study were approved by the Animal Welfare Committee of Baylor College of Medicine.
Collection of Ovulated Oocytes and Preimplantation Mouse Embryos
Ovulated oocytes and mouse embryos at 1-, 2-, 4-, 8-, 16-cell, and blastocyst stages were collected as described previously [26, 27]. The cumulus cells surrounding the oocytes were removed by hyaluronidase (Sigma Chemical Co., St. Louis, MO) [27]. The zona pellucida of oocytes and early embryos was removed by brief exposure to warm, acidified Tyrodes solution (pH 2.1–2.5; Irvine Scientific, Santa Ana, CA) [27]. After rinsing twice in PBS, the oocytes and early embryos were fixed with 4% paraformaldehyde (in PBS) at room temperature for 1 h. The fixed cells were permeabilized by incubation with 0.2% Triton in PBS at room temperature for 30 min. After two washes in PBS, the permeabilized cells were immediately used for immunofluorescent staining.
Western Blot Analysis
Testes and caput and cauda epididymide homogenates (50 µg), purified GSTGCNF (5 ng), Cos-1 cell protein extracts, CMV (40 µg) and CMVGCNF (20–40 µg), P19 cell nuclear extract (15 µg), cauda sperm extract (50 µg), were separated in 10%–12% SDS-PAGE gels, and then transferred onto polyvinylidene fluoride Immobilon-P membranes (Millipore Corporation, Bedford, MA). The membranes were blocked in 5% nonfat milk in Tris-buffered saline (TBS; 137 mM NaCl and 20 mM Tris pH 7.6) at room temperature for 3 h, and then incubated with polyclonal rabbit anti-GCNF antibodies diluted 1:300 in TBS at room temperature for 3 h. The membranes were washed in TBST (0.1% Tween-20 in TBS) three times at room temperature for 15 min, and then incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology) for 1 h at room temperature. After extensive washes with TBST the membranes were processed using an enhanced chemiluminescence system (Boehringer-Mannheim, Indianapolis, IN) according to the manufacturer's protocol. These experiments were repeated on samples from more than three animals.
Immunohistochemistry
Immunohistochemical studies of GCNF in testicular and ovarian sections were performed using a Rabbit ImmunoCruz Staining System (Santa Cruz Biotechnology) according to the manufacturer's protocol. Briefly, tissue samples were dewaxed, rehydrated, and then heated at 95°C in 10 mM sodium citrate buffer, pH 6.0 for 5 min to unmask antigens. Endogenous peroxidase activity of samples was quenched by incubation with 3% H2O2. Tissue samples were blocked and then incubated either with rabbit IgG isolated from the preimmune sera or with purified anti-GCNF antibodies (1:300 dilution) at room temperature for 2 h. After extensive washes these samples were incubated with biotinylated goat anti-rabbit secondary antibodies followed by a HRP-streptavidin complex. Positive signals were visualized by incubation in peroxidase substrate using diaminobenzidine (DAB) as the chromogen. Slides were then counterstained in 0.05% (w/v) methyl green (Sigma). These tissue slides were examined with a Zeiss Axioskop 2 microscope (Carl Zeiss, Inc., Thornwood, NY).
Permeabilized oocytes and preimplantation embryos were incubated in blocking solution (1% IgG free BSA, 5% donkey serum, and 0.1% Triton in PBS) for 1 h and then incubated with either rabbit preimmune serum IgG or anti-GCNF polyclonal antibodies (1:300 dilution in blocking solution) at room temperature for 3 h. After two washes in PBST (0.1% Tween-20 in PBS) at room temperature for 30 min, these cells were incubated with Cy3-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) at a 1:400 dilution in blocking buffer for 1 h. After two washes in PBST, cells were stained with 10 µg/ml Hoechst 33258 (Sigma) for 1 h. After extensive washes in PBS, the cells were mounted on glass slides using a Vectashield mounting medium for fluorescence (Vector Laboratories, Burlingame, CA). Oocytes and preimplantation embryos were then examined with a Zeiss Axioskop 2 immunofluorescence microscope (Carl Zeiss).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To determine the specificity of anti-GCNF polyclonal antibodies, Western blot analysis was performed to characterize their ability to recognize recombinant GCNF (HA-GCNF and Myc-GCNF), GST-GCNF fusion protein, and endogenous GCNF protein in the P19 cell nuclear extract (Fig. 1). As shown in Figure 1, A and B, a 61-kDa protein band was detected by GCNF antibodies only in the protein extract from Cos-1 cells transfected with a pCMV-HA-GCNF or pCMV-Myc-GCNF plasmid, but not in control Cos-1 cells transfected with a CMV-HA vector. These antibodies also recognized a 58-kDa endogenous GCNF protein in the P19 cell nuclear extract (Fig. 1A) and the GST-GCNF fusion protein expressed in bacterial cells by Western blot analysis (Fig. 1B). The specificity of the anti-GCNF antibodies was further confirmed in EMSAs (Fig. 1C). In vitro translated GCNF and endogenous GCNF in P19 cell nuclear extract bound to DR0 DNA oligonucleotides forming a GCNF-DNA complex (Fig. 1C) as previously reported [8, 21]. Addition of anti-GCNF antibodies reduced the mobility of these complexes (Fig. 1C).
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Detection of GCNF Protein in Mouse Testis
To determine whether GCNF protein is present in the testis, we performed Western blot analysis using anti-GCNF polyclonal antibodies. As shown in Figure 1B, a 58-kDa GCNF protein band was detected in the homogenates of adult mouse testis, but not in 12-day-old mouse testis (juvenile). No GCNF protein was detected in the sperm extract from adult cauda epididymis, nor in homogenates from adult caput and cauda epididymides (Fig. 1B). To determine the localization of GCNF protein in the testis, immunohistochemical studies were performed on testis sections prepared from 5-, 12-, 21-, and 56-day-old animals using anti-GCNF antibodies. Preimmune serum IgG was used as a negative control. As shown in Figure 2, a–d, no positive signals were detected in 5- and 12-day-old testis sections incubated either with anti-GCNF polyclonal antibodies or with preimmune serum IgG. However, strong positive staining was observed in testicular sections from 21- and 56-day-old animals incubated with anti-GCNF polyclonal antibodies (Fig. 2, f, h, j, and l). These positive signals were not observed in sections incubated either with preimmune serum IgG (Fig. 2, e, g, i, and k) or with anti-GCNF polyclonal antibodies that had been preincubated with GCNF immunogen polypeptide (data not shown). Positive staining was localized in both the cytoplasm and nuclei of round spermatids in 21-day-old animals (Fig. 2h), and in postmeiotic spermatogenic cells (round and elongating spermatids) in 56-day-old animals (Fig. 2l). No staining was observed in Sertoli cells, spermatogonia, or pachytene spermatocytes in the seminiferous tubules of either 21-day-old or 56-day-old animals. The highest level of GCNF expression was observed in elongating spermatids.
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Localization of GCNF Protein in the Ovary
To determine the localization of GCNF protein in the ovary, we performed immunohistochemical studies on ovarian sections prepared from 5-, 12-, 21-, and 56-day-old animals using anti-GCNF antibodies. Preimmune serum IgG was used as a negative control. As expected, no positive signals were observed in the sections incubated with preimmune serum IgG (Fig. 3, a, c, e, g, and i). Weak brown staining was observed in oocytes of primary follicles in 5-day-old animals, but not in somatic cells or primordial follicles (Fig. 3b). At Day 12 after birth, positive staining was more evident in oocytes of primary follicles (P, Fig. 3d). At day 21, strong positive staining was observed in oocytes of both primary (P, Fig. 3, f and h) and secondary follicles (S, Fig. 3f). At Day 56, positive signals were observed in oocytes of primary, secondary, and preovulatory (G) follicles (Fig. 3j). These positive signals were present in the cytoplasm of oocytes at all stages (Fig. 3, d, f, and h). No GCNF protein was detected in somatic cells or oocytes of primordial follicles in all tested ovaries.
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Presence of GCNF Protein in Unfertilized Oocytes and Preimplantation Mouse Embryos
To determine whether GCNF protein is present in ovulated oocytes and preimplantation embryos, we performed immunofluorescence studies on these samples using anti-GCNF antibodies (Fig. 4). Preimmune serum IgG was used as a negative control. Positive GCNF signals in the samples stained with Cy3 were shown with red fluorescence (a''–n''), whereas the nuclei of early mouse embryos stained with Hoechst 33258 were shown with blue fluorescence (a'–n'). No positive signals were observed in ovulated oocytes (Fig. 4a'') or mouse embryos at 1-cell (Fig. 4c''), 2-cell (Fig. 4e''), 4-cell (Fig. 4g''), 8-cell (Fig. 4i''), 16-cell (Fig. 4k''), and blastocyst (Fig. 4m'') stages after incubation with preimmune serum IgG. However, strong positive GCNF signals were observed in unfertilized oocytes (Fig. 4b'') and in 1-cell (Fig. 4d''), 2-cell (Fig. 4f''), 4-cell (Fig. 4h''), 8-cell (Fig. 4j''), and 16-cell (Fig. 4l'') mouse embryos and blastocysts (Fig. 4n'') after incubation with polyclonal anti-GCNF antibodies. These positive signals were localized in both the cytoplasm and nuclei of early mouse embryos (Fig. 4d'', f'', h'', j'', l'', and n''). The same results were obtained using another anti-GCNF antibody raised against a GCNF polypeptide from amino acid residues 437–454 (CQDFTEYKYTHQPHRFPD, data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Because only the 7.5-kb GCNF mRNA is detectable in 20-day-old testis by Northern blot analysis [15, 16], our results also indicate that GCNF protein can be translated from the 7.5-kb GCNF mRNA at Day 21 after birth. Adult mouse testis contains two GCNF transcripts (2.3 kb and 7.5 kb) [15, 16] that differ only in the 3' untranslated region [15]. If the 2.3-kb GCNF transcript is translationally competent, it appears that both GCNF transcripts produce the same protein because there is one band on the Western blot (Fig. 1B).
The presence of GCNF protein in the nuclei of postmeiotic spermatogenic cells indicates that GCNF may function as a transcription factor that binds to DR0 elements of potential GCNF target genes, and then regulates transcription of these genes during spermiogenesis. Protamine 1 and protamine 2 are two potential GCNF target genes in the testis, because multiple DR0 binding motifs are present in the promoters of these two genes [21]. GCNF has been shown to bind to these DR0 elements in protamine gene promoters [21, 29]. The expression of the GCNF gene, determined at both the mRNA and protein levels (Fig. 2) [15, 16, 20], is turned on earlier than that of the protamine genes [30]. A decrease in the amount of protamines disrupts nuclear formation and normal sperm function, and haploinsufficiency of protamine 1 or 2 causes male infertility [31]. Therefore, GCNF may be involved in the regulation of male fertility by regulating the expression of protamine genes during spermatogenesis.
For the first time, we report here that GCNF protein is present not only in growing and mature oocytes of the mouse ovary (Fig. 3), which is consistent with our previous in situ RNA hybridization studies [3, 15], but also in ovulated oocytes and early mouse embryos from 1- to 16-cell stages and blastocysts (Fig. 4). The presence of GCNF protein in preovulatory and ovulated oocytes (Figs. 3 and 4) indicates that GCNF is a maternal protein. Because no GCNF protein was detected in the epididymis (Fig. 1B), which stores millions of mature sperm for ejaculation [32], and no protein synthesis occurs in 1-cell mouse embryos [26, 33], GCNF protein in mouse embryos at the 1-cell stage is most likely derived from ovulated oocytes. Whether GCNF protein in early mouse embryos at 2-cell to 16-cell and blastocyst stages is derived from ovulated oocytes, capacitated sperm, or zygotes (or all these) remains to be determined. The lack of nuclear GCNF staining in oocytes may be due to the inability of GCNF antibodies to access the GCNF protein in the nuclei. It is known that endogenous GCNF exists in a large complex called the TRIF complex [8]. In this large complex, GCNF epitopes are likely to be masked by their association with other factors. In addition, GCNF bound to DNA in the nuclei could also mask epitopes.
In combination with previous results [3, 15, 16], this study indicates that GCNF may play a role during gametogenesis and early embryonic development before implantation. It is our goal to determine the function of GCNF during these processes, we have deleted the GCNF gene in the germline using a conventional knockout strategy [14]. GCNF-null mutant mice die at 9.5–10.5 days postcoitum, indicating that GCNF is essential for normal embryonic development after gastrulation [14]. However, this embryonic lethality of GCNF-null mutants prevents us from analyzing the role of GCNF during reproduction. The role of GCNF during preimplantation embryonic development could be also masked by the maternal GCNF protein from a wild-type GCNF allele in a GCNF heterozygous mother. Therefore, generation of testis- and ovary-specific GCNF knockout mice using the Cre/LoxP system [34] is imperative for characterizing the function of this gene during male and female reproduction and preimplantation embryonic development.
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FOOTNOTES |
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2 Correspondence: Austin J. Cooney, Department of Molecular and Cellular Biology, Baylor College of Medicine, BCMM-M733, One Baylor Plaza, Houston, TX 77030. FAX: 713 790 1275; acooney{at}bcm.tmc.edu
Received: 5 June 2002.
First decision: 4 July 2002.
Accepted: 5 August 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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