Conserved and divergent patterns of expression of DAZL, VASA and OCT4 in the germ cells of the human fetal ovary and testis
© Anderson et al; licensee BioMed Central Ltd. 2007
Received: 15 May 2007
Accepted: 18 December 2007
Published: 18 December 2007
Germ cells arise from a small group of cells that express markers of pluripotency including OCT4. In humans formation of gonadal compartments (cords in testis, nests in ovary) takes place during the 1st trimester (6–8 weeks gestation). In the 2nd trimester germ cells can enter meiotic prophase in females whereas in males this does not occur until puberty. We have used qRTPCR, Westerns and immunohistochemical profiling to determine which of the germ cell subtypes in the human fetal gonads express OCT4, DAZL and VASA, as these have been shown to play an essential role in germ cell maturation in mice.
OCT4 mRNA and protein were detected in extracts from both 1st and 2nd trimester ovaries and testes. In ovarian extracts a marked increase in expression of VASA and DAZL mRNA and protein occurred in the 2nd trimester. In testicular extracts VASA mRNA and protein were low/undetectable in 1st trimester and increased in the 2nd trimester whereas the total amount of DAZL did not seem to change. During the 1st trimester, germ cells were OCT4 positive but did not express VASA. These results are in contrast to the situation in mice where expression of Vasa is initiated in Oct4 positive primordial germ cells as they enter the gonadal ridge. In the 2nd trimester germ cells with intense cytoplasmic staining for VASA were present in both sexes; these cells were OCT4 negative. DAZL expression overlapped with both OCT4 and VASA and changed from the nuclear to the cytoplasmic compartment as cells became OCT4-negative. In males, OCT4-positive and VASA-positive subpopulations of germ cells coexisted within the same seminiferous cords but in the ovary there was a distinct spatial distribution of cells with OCT4 expressed by smaller, peripherally located, germ cells whereas DAZL and VASA were immunolocalised to larger (more mature) centrally located cells.
OCT4, DAZL and VASA are expressed by human fetal germ cells but their patterns of expression are temporally and spatially distinct. In the 1st trimester OCT4 was detected in most germ cells. In the 2nd trimester the onset of expression of VASA was associated with the formation of oocytes and spermatogonia both of which were OCT-4 negative. Relocation of DAZL from nucleus to cytoplasm paralleled the down regulation of OCT4 and the onset of expression of VASA. These data reveal similarities between the expression of key regulatory proteins within germ cells as they mature in male and female fetal human gonads suggesting that in the female these maturational changes are not determined by entry into meiosis.
The ovaries and testes both develop from an identical structure, the embryonic genital ridge . Germ cells do not originate from within the genital ridges but differentiate from clusters of cells located in the extra-embryonic proximal epiblast . Primordial germ cells (PGC) proliferate and migrate via the embryonal endoderm reaching the gonadal ridges at about 5th week of pregnancy in the human (reviewed in ). Within the genital ridge germ cells have been shown to express the pluripotency markers OCT4 (POU5F1, ) and NANOG  and human embryonic germ cell lines, capable of differentiating into a number of cell lineages, have been established from early gonadal tissue [6, 7]. We have previously reported that during the 1st trimester of pregnancy the germ cells (gonocytes) in the testicular cords all express OCT4 but that only a subpopulation of germ cells remains OCT4 positive during the 2nd trimester . In previous studies OCT4 positive germ cells were detected in the cortical region of human fetal ovaries recovered during the 2nd trimester ; however expression of OCT4 has not been investigated in the 1st trimester ovary.
Studies in knockout mice have identified a number of genes the expression of which is critical for germ cell survival and functional maturation in both ovary and testis. One such gene is Dazl, an RNA binding protein that is a member of a conserved gene family members of which include BOULE and DAZ [10, 11]. A second example is Mvh (mouse Vasa homologue) a gene that encodes an RNA helicase which is specific to the germ cell lineage . In mice Dazl mRNA is first detectable in post-migratory germ cells on e11.5 [11, 13] and the protein has been immunolocalised to the cytoplasm of fetal and adult germ cells . Targeted deletion of the Dazl gene results in germ cell loss in both males and females . In Dazl -/- females germ cell loss occurred in fetal ovaries at the time of meiotic entry and adult ovaries did not contain oocytes [14, 15]. In Dazl -/- males the pattern of germ cell loss is variable and in some studies has been reported to occur during fetal life  whilst in others it was associated with spermatogonial differentiation [14, 16] or entry to meiosis .
VASA is a member of the DEAD box family of genes first identified in Drosophila where it was shown to be essential for female germ cell development . In mice with targeted deletions of Mvh ovarian function appeared normal but males were infertile with demise of germ cells at the zygotene stage of meiosis . Mvh has been detected in germ cells on e12.5 immediately after colonisation of the gonad but not in migratory PGCs [12, 19, 20]. Information on expression of VASA in human fetal gonads is limited but protein was detected in OCT4 negative cells in a testis at 21 weeks gestation . First trimester ovaries have not been examined but in the 2nd trimester VASA has been localised to oocytes within primordial follicles .
In the present paper we have extended our studies on the germ cell subpopulations within the human fetal ovaries and testes to determine when and where DAZL and VASA proteins are expressed and have performed co-staining for OCT4 as this is commonly used to delineate the germ 'stem cell' population [8, 22, 23]. These studies have revealed a dynamic and overlapping pattern in the expression of these germ cell markers, revealed that nuclear localisation of DAZL parallels the onset of VASA expression, and highlighted similarities in the development of male and female germ cell lineages.
Expression of mRNAs for OCT4, DAZL and VASAin first and second trimester gonads
Western analysis of total protein levels
DAZL protein was detected in both 1st and 2nd trimester testes (Figure 2D) but only in the 2nd trimester ovaries (Figure 2C). OCT4 was detected in extracts from ovaries and testes obtained during both 1st and 2nd trimesters (Figure 2E, F).
Immunolocalisation of OCT4, DAZL and VASA revealed differential patterns of expression in both ovary and testis
Co-localisation of OCT4 and DAZL
Differential expression of OCT4 and VASA
Differential and overlapping expression of DAZL and VASA
Germ cells within the male and female gonads develop from an apparently homogeneous bi-potential population of PGC. In both male and female human fetal gonads germ cells initially proliferate but thereafter their fates diverge with female germ cells entering meiotic prophase as early as 11 weeks gestation (9 weeks post conception; ) whereas meiotic entry is does not occur until puberty in males. In rodents male germ cells removed from the testicular environment and placed in vitro can spontaneously enter meiosis emphasising the common developmental potential of the germ cells in the two sexes and the critical role played by the testicular environment in modifying germ cell fate [25, 26].
In the current study we have used OCT4 immunohistochemistry to identify the subpopulation of putative 'germ stem' cells in human fetal testes and ovaries. Studies from our own laboratory  have already established that expression of OCT4 is restricted to the gonocyte population of human testicular germ cells whilst others  have reported that this protein is localised to oogonia in the ovary. Expression of DAZL and VASA was compared to OCT4 as studies in knockout mice have reported that both genes are essential for germ cell maturation in that species [14, 19]. Analysis of ovarian extracts revealed a striking increase in the total amount of both DAZL and VASA mRNA and protein in the ovary as development progressed into the 2nd trimester. A similar trend in expression was noted in the testes but was less striking. Expression of OCT4 was maintained in both ovary and testis at all gestational ages examined consistent with persistence of a putative germ 'stem cell' population for many months in the human gonads although the extent to which these cells can acquire/maintain pluripotency in vitro remains unresolved (reviewed in ). In the 2nd trimester testis all seminiferous cords contained a mixture of OCT4 positive and OCT4 negative germ cells a situation which is different to that in rodents where the expression of OCT4 is down regulated synchronously in all germ cells within the cords before birth [29, 30]. In the human fetal ovary expression of OCT4 protein was restricted to oogonia in the peripheral zone of the organ, in agreement with the observations of Stoop et al.. In mice expression of Oct4 mRNA is extinguished in a rostro-caudal wave coincident with meiotic entry of germ cells .
Co-incident and overlapping patterns of expression of these germ cell specific proteins were revealed by fluorescent co-immunolocalisation on fixed specimens from a range of gestational ages. Previous studies have immunolocalised DAZL in a small number of samples recovered in the 2nd trimester [32–34] but we believe this is the first study to document expression in 1st trimester samples and to compare expression with that of OCT4 and VASA. In the 1st trimester, DAZL was detected in the nuclear compartment in OCT4 positive germ cells in both sexes. In a previous study we immunolocalised DAZL to the nuclei of male germ cells in a single sample at 17 weeks gestation and in the cytoplasm of female germ cells at 15 weeks . Other studies have claimed that DAZL is present in both the cytoplasm and nucleus of male germ cells at 20–21 weeks . We have extended these observations and demonstrated that DAZL is localised to the nuclear compartment in OCT4 positive cells (gonocytes and oogonia). In the ovaries localisation of DAZL to the cytoplasmic compartment appeared to be transient occurring as OCT4 expression but thereafter the amount of protein declined and was low in the largest ovarian germ cells, i.e. those that had formed into primordial follicles and those approaching that stage. This was evident when a morphometric analysis was performed showing that the germ cells immunopositive for DAZL alone were smaller than those containing both DAZL and VASA, which were in turn smaller than those only immunopositive for VASA.
Two studies on human populations have provided preliminary evidence that expression of DAZL is important for normal functioning of the human germ line. The first study reported a strong association between several common single nucleotide polymorphisms (SNPs) and age at menopause in a sample population of 324 women . In a second study a patient with premature ovarian failure at age 34 was found to contain a homozygous mutation of DAZL (Arg to Gly at 115) in a region of the protein critical for RNA binding . The situation in men is complicated by the presence of multiple copies of the closely related DAZ gene on the Y chromosome, deletions of which have been frequently documented as a cause of male infertility . However a patient with a homozygous mutation in DAZL (Asn 10 Cys) was reported to be azoospermic .
The marked increase in expression of VASA between 9 and 14 weeks gestation suggested to us that this was coincident with the entry of female germ cells into meiosis. However increases in both protein and mRNA were also demonstrated in male germ cells suggesting that the onset of expression of VASA was associated the maturation of the gonocytes into prespermatogonia rather than meiotic entry per se. In the ovary VASA was detected in the cytoplasm of in slightly larger, more mature germ cells than DAZL, and was also present in oocytes within primordial follicles. Previous studies that have detected expression of VASA in cytoplasm of germ cells within the fetal ovary and testes at 17 weeks gestation [9, 21, 37]. Co-staining of sections with OCT4 and VASA has extended these findings by demonstrating that VASA protein is not expressed in gonocytes or oogonia in the 1st trimester and provides a useful method for identifying the different populations of germ cells present within the 2nd trimester gonads.
Examination of the 3' UTR of the Mvh mRNA has revealed a number of putative Dazl-binding sites that are conserved between human, rat and mouse . Our data provide some additional support for a role for DAZL in initiating expression of VASA although further studies are required preferably using isolated human germ cells. DAZL has also been implicated in the regulation of other conserved germ-cell RNA-binding proteins including PUM2 , the human homolog of Pumilio that is required for maintenance of germ line stem cells in Drosophila and Caenorhabditis elegans.
By documenting the differential and partially overlapping patterns of expression of OCT4, DAZL and VASA proteins in the human fetal ovary and testis we have gained new insight into parallels in the maturation of germ cells in these two organs. Our findings clearly demonstrate changes in overall germ cell maturation with increasing gestation, and indicate a switch from OCT4 expression in the less mature germ cells (gonocytes, peripheral oogonia) to VASA expression in the more mature germ cells (prespermatogonia, oogonia in nests, oocytes). In testicular samples germ cells at different stages of maturation co-exist within the same seminiferous cords. Both male and female germ cells showed a similar pattern of change with development, despite the onset of meiosis in the female but not in the male, suggesting that although increases in expression of DAZL and VASA in the ovary may be related to meiotic entry there are underlying developmental patterns common to both sexes.
Tissue recovery and processing
Human fetal gonads were obtained following termination of pregnancy during the first (61–64 days gestation, n = 11) and 2nd trimesters (14 to 19 weeks gestation, n = 29). Women gave written consent according to national guidelines  and the study was approved by the Lothian Ethics Committee. Termination of pregnancy was induced by treatment with mifepristone (200 mg, orally), followed by misoprostol (Pharmacia, Surrey UK; 200 mg every 3 h, per vaginam). None of the terminations was for reasons of fetal abnormality, and all fetuses used in this study appeared morphologically normal. Gestational age was determined by ultrasound examination before termination and was confirmed by subsequent direct measurement of foot length in 2nd trimester samples. The sex of the 1st trimester fetal gonads was determined by PCR for the SRY gene as detailed in . Testes and ovaries were dissected carefully from the fetal abdominal cavity and either snap frozen and stored at -70°C prior to extraction of total protein or RNA, or fixed in Bouins fluid for 2–3 h, stored in 70% ethanol and processed into paraffin wax using standard methods.
RNA Extraction and RT PCR
Total RNA was extracted from fetal gonads using the RNeasy Mini Kit (Qiagen, UK) for mid trimester gonads and the RNeasy Micro Kit for 1st trimester gonads (with on-column DNase digestion Qiagen, UK) The RNA was primed for reverse transcription with oligo(dT) primer (Applied Biosystems, UK) at 65°C for 10 min. The entire reaction was added to a total volume of 38 μl containing dNTP to 1 mmol/l, dithiothreitol (DTT) to 10 mmol/l, 1× Expand Reverse Transcriptase (RT) buffer and 120 IU RNasin ribonuclease inhibitor (Promega Ltd, UK). One half (19 μl) of this reaction was added to 1 μl water (RT negative reaction) to act as a negative control to confirm the effiency of the DNase treatment. Fifty IU of Expand Reverse Transcriptase (Roche Diagnostics Ltd) was added to the remaining 19 μl (RT positive reaction) and both reactions were incubated for 1 h at 42°C. Reactions were stored at -20°C until required.
Real-time quantitative PCR
Primers used for quantitative PCR
Western blot analysis
Fetal gonads were homogenised in 1× RIPA buffer containing: 25 mM Tris, 1% Triton, 0.05% sodium deoxycholate, 0.1% SDS and 150 mM NaCl. Total protein was measured using the Protein Assay DC Kit from Bio-Rad (Hemel Hempstead, Herts, UK). Samples were denatured in 1× reduced sample buffer containing: 625 mM Tris (pH 6.8), 5% glycerol, 2% SDS, 0.0025% bromophenol Blue, 2.5% β-mercaptoethanol and 5 μg of total protein was loaded onto individual wells in a SDS-PAGE gel made with 10% (w/v) acrylamide; samples of pre-stained protein size markers were run on each gel (SeeBlue plus 2 prestained standard, Invitrogen, UK). Following separation of proteins they were transferred onto PVDF Immobilon-Fl membranes (Millipore UK Ltd, Watford, Herts); non-specific binding sites were blocked by incubating membranes in Odyssey blocking buffer (LI-COR, Lincoln, Nebraska, USA). All membranes were incubated with primary antibodies diluted in Odyssey blocking buffer overnight at 4°C. Proteins were detected using rabbit-anti-VASA (DDX4MVH, Abcam, Cambridge UK) diluted 1 in 500 together with mouse anti-β tubulin (cat no. T-4026, Sigma) diluted 1 in 300, mouse anti-DAZL (gift from Professor Howard Cook, MRC Human Genetics Unit, Edinburgh) diluted 1 in 500, with rabbit anti-β-tubulin (cat no. sc-9104, Santa Cruz) diluted 1 in 1000, or with goat anti-OCT4 (Santa Cruz) together with rabbit anti-β-tubulin (Santa Cruz) diluted 1 in 1000. Membranes were washed in PBS containing 0.1% Tween and bound antibodies were detected using fluorescently labelled secondary antibodies: goat anti-rabbit 680 (Invitrogen, Paisley, UK) and goat anti-mouse 800 (Invitrogen, Paisley, UK) for VASA and β-tubulin respectively; goat anti-mouse 680 (Invitrogen, Paisley, UK) and goat anti-rabbit 800 (Rockland, USA) for DAZL plus β-tubulin; while OCT4 and β-tubulin, were detected using donkey anti-goat 680 (Invitrogen Paisley, UK) and goat anti-rabbit 800 (Rockland, USA). Secondary antibodies were all diluted 1 in 10000 in Odyssey blocking buffer and incubated for 1 h at room temperature; bound fluorescent secondary antibodies were visualised using a LI-COR-Odyssey Infrared Imager (LI-COR Biosciences, Lincoln Nebraska).
Antibodies used for immunofluorescent immunohistochemistry
DAZL 1/500+VASA 1/300
VASA + OCT 4
VASA 1/300+OCT 41/200
CAGP + Tyr-Cy3(OCT4)
DAZL + OCT 4
DAZL 1/500 + OCT 4 1/200
RAMB+Streptavidin 546 (DAZL)
Three representative non-adjacent tissue sections from ovaries at gestational ages 14, 16 and 19 weeks immunostained for DAZL and VASA were analysed. The cell diameter of all immunopositive germ cells on each section was calculated as the average of two orthogonal measurements and classified as expressing DAZL, VASA or both. Data were analysed by analysis of variance with Tukey-Kramer post hoc testing.
We thank Joan Crieger and Sheila Macpherson for their hard work in support of this study.
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