Mol. Hum. Reprod. Advance Access originally published online on March 25, 2004
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Molecular Human Reproduction, Vol. 10, No. 5, pp. 313-319, 2004
© European Society of Human Reproduction and Embryology 2004
Immunocytochemical detection and RT–PCR expression of leukaemia inhibitory factor and its receptor in human fetal and adult ovaries
1Infertility and IVF Unit, Department of Obstetrics and Gynecology, Rabin Medical Center, Beilinson Campus, Petah Tikva 49100 and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, 2Department of Obstetrics and Gynecology, Rabin Medical Center, Beilinson Campus, Petah Tikva and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel. 3Department of Obstetrics and Gynecology, Royal Victoria Hospital, McGill University, Montreal, Quebec H3A 1A1, Canada, 4The Felsenstein Medical Research Center, Beilinson Campus, Petah Tikva 49100 and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel, 5Department of Farm Animal Health, Veterinary Faculty, Utrecht University, Utrecht, The Netherlands and 6Department of Human Genetics, McGill University, Montreal, Quebec H3A 1A1, Canada
7 To whom correspondence should be addressed. e-mail: ronita{at}clalit.org.il
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ABSTRACT |
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The ability to mature human primordial follicles in vitro would assist fertility restoration. However, the signals initiating growth of primordial follicles are unknown. Growth factors such as leukaemia inhibitory factor (LIF) may play a role in this process. To investigate the expression of LIF and its receptor in early developing follicles, nine ovarian samples from adolescents/adults aged 13–43 years and 23 ovaries from human fetuses aged 19–33 gestational weeks were immediately fixed or frozen. The fixed samples were prepared for a study of immunocytochemical staining of LIF and its two receptor units (LIF-R and gp 130). mRNA was extracted from the frozen ovarian samples, and the expression of LIF, LIF-R and gp 130 was investigated by RT–PCR. Products were resolved by 10% polyacrylamide gel electrophoresis and image analysis. There was strong to moderate immunocytochemical staining for LIF and LIF-R in oocytes from the primordial follicular stages onwards, and very weak to moderate staining for gp 130. LIF-R was also detected in granulosa cells of primary and secondary follicles from adolescents/adults. Transcripts of LIF, LIF-R and gp 130 RNA were identified by RT–PCR in all samples. The immunocytochemical staining and mRNA expression of LIF and its receptor are consistent with the concept that LIF might be involved in growth initiation of human primordial follicles through its receptor.
Key words: Key words: gp 130/immunocytochemistry/LIF-R/primordial follicles/RT–PCR
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Introduction |
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Primordial germ cells (PGC) reach the human ovary from day 26 of pregnancy (for review see Gosden, 1995). At this point they are called oogonia. Their subsequent development can be divided into three phases: mitotic divisions, meiotic division, and follicular assembly. Follicular assembly commences during the 4th or 5th month of pregnancy, when a single flat layer of ovarian somatic cells surrounds the oocyte. In rats and mice, follicular formation takes place during the first 3 postnatal days (Ojeda et al., 2000). Most follicles in ovaries of women, as well as human fetuses, are unilaminar: 30–50 µm in size (for review see Gougeon, 1996): mostly primordial follicles, but also primary follicles with cuboidal granulosa cells (GC). The signals that trigger their growth are unknown. The ability to mature unilaminar follicles in vitro has major clinical implications, especially for former cancer patients (for review see Abir et al., 1998).
Various growth factors such as leukaemia inhibitory factor (LIF) might play a role in maturation of primordial follicles (for reviews see Gosden, 1995; Gougeon, 1996; Van den Hurk et al., 2000; Nilsson et al., 2002). LIF, a cytokine of the interleukin-6 family, is named for its ability to inhibit the proliferation of a myeloid leukaemia cell line (for reviews see Senturk and Arici, 1998; Lass et al., 2001). Its receptor consists of two glycoprotein (gp) units: a low-affinity LIF-
type I membrane receptor (LIF-R) and a ß-receptor unit (gp 130) that cannot bind LIF on its own (Gearing et al., 1994; Schiemann et al., 1995). When the two units merge, a high affinity receptor is formed (for review see Lass et al., 2001).
LIF has been found to regulate the growth and differentiation of the pre-migratory and post-migratory PGC in the developing murine (Cheng et al., 1994; De Felici, 2000) and porcine gonad (Durcova-Hills et al., 1998; Shim and Anderson, 1998) and possibly also in humans (Shamblott et al., 1998). In laboratory studies, LIF, when combined with other growth factors such as stem cell factor (SCF), increased the number of pachytene-stage murine oogonia (Lyrakou et al., 2002) and promoted growth of rat primordial follicles in culture (Nilsson et al., 2002).
The immunocytochemical detection and molecular expression of LIF and its receptors in early developing ovarian follicles in women and human fetuses is unknown. The aim of the present study was to investigate this issue at the mRNA level (RT–PCR) and the protein level (immunocytochemistry) in order to identify the presence of the transcripts and localize the antigens within the ovary.
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Materials and methods |
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Human adult and fetal ovaries
Ovarian samples were obtained from 23 aborted human fetuses aged 19–33 gestational weeks (Table I). All but six pregnancy terminations (by prostaglandin induction) were performed because of fetal anatomical malformations or chromosomal abnormalities (Table I). Normal fetuses originated from legal terminations conducted because of psychiatric problems of the mothers. Our departmental pregnancy termination policy mandates feticide for all fetuses >21 gestational weeks. Therefore, only fetal specimens shown to be non-apoptotic in a previous study (Abir et al., 2002) were used for the present analyses (see also Discussion).
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In addition, small ovarian biopsies were donated from nine pre-menopausal adolescents/women aged 13–43 years undergoing various gynaecological laparoscopic procedures (Table II). In half of the patients, the procedures were performed to obtain ovarian tissue for cryopreservation prior to chemotherapy for different types of cancer (for review see Abir et al., 1998).
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All ovarian samples were cut to be as uniform in size as possible (
2x2 mm) and fixed immediately in neutral buffered formalin. A portion of every sample was frozen immediately for subsequent RNA extraction. Approval for this experiment was obtained from the ethics committee of our institute and an informed consent was obtained from all the women, guardians and mothers.
Cryopreservation of ovarian tissue
Five to seven tissue slices measuring 1–2 mm were placed in cryogenic vials (Nalge Nunc International, Denmark) filled with a 1.5 dimethylsulphoxide (DMSO; Sigma, USA) and 0.1 mol/l sucrose solution (Sigma) (Newton et al., 1998) and kept on ice for half an hour. The samples were then frozen slowly in a programmable freezer (Kryo 10; series 10/20; Planer Biomed, UK) and immediately placed in liquid nitrogen. The slices were cryopreserved–stored for 3–24 months until RNA extraction.
Immunocytochemistry for LIF, LIF-R and gp 130
The fixed specimens were rehydrated in a graded series of ethanol, embedded in paraffin, and sectioned (5 µm). Unstained sections were placed on OptiPlus positive-charged microscope slides (BioGenex Laboratories, USA) for immunocytochemistry and were deparaffinized and rehydrated, quenched in 3% H2O2 (Vitamed, Israel) in the dark to block endogenous peroxidase activity, and rinsed with ethanol. The sections were then microwaved first under full power (800 W) and then under 75% power, with citrate buffer at pH 6.0 (diluted in distilled water from a x10 citrate buffer solution at pH 6.0) (Dako Corporation, USA) to enhance antigen retrieval. The antigen retrieval procedure was terminated by cooling and rinsing the sections in distilled water and phosphate-buffered saline (PBS) at pH 7.4 (Biological Industries, Israel).
Thereafter, all sections were incubated with the primary antibodies: for LIF, an anti-human monoclonal mouse IgG2B antibody (MAB250, clone 9824, 500 µg/ml; R&D Systems Europe Ltd, UK) (1:10, 1:15); for LIF-R a specific anti-human polyclonal goat IgG antibody (Extracellular Domain, AF-249-NA, 250 µg/ml; R&D Systems Europe Ltd) (1:20, 1:30), for gp 130, an anti-human monoclonal mouse IgG1 antibody (Extracellular Domain, MAB628, clone 28105, 500 µg/ml; R&D Systems Europe Ltd) (1:10). Two negative control solutions were used. The first consisted of a normal mouse IgGa2 antibody for LIF and gp 130 (Santa Cruz Biotechnology, USA) (1:10), and a goat IgG antibody (ChromPure goat IgG, whole molecule; Jackson ImmunoReasearch Laboratories, USA) for LIF-R (1:20). The second negative control solutions were prepared by mixing 1:50 the diluted primary antibodies (R&D Systems Europe Ltd) with recombinant human LIF (10 µg/ml, Sigma); recombinant human LIF sR (50 µg/ml; R&D Systems Europe Ltd) or recombinant human soluble gp 130 (10 µg/ml; R&D Systems Europe Ltd) respectively, followed by 
1 h of incubation before application. All the sections were then rinsed and incubated with biotinylated anti-rabbit, anti-mouse and anti-goat immunoglobulins in PBS containing carrier protein and 15 mmol/l sodium azide (Link from Dako LSAB+ System, HRP; Dako). After further rinsing, the sections were incubated with streptavidin conjugates to horseradish peroxidase-containing carrier protein and antimicrobial agents (streptavidin HRP from Dako LSAB+ system, HRP; Dako) and then exposed to a diaminobenzidine urea H2O2 solution in distilled water (Sigma Fast tablets; Sigma) and counterstained with Mayer’s haematoxylin (Pioneer Research Chemicals Ltd, UK) (purple–blue staining). Brown staining indicated detection of either LIF, LIF-R or gp 130. Unless otherwise stated, all dilutions were performed with PBS at pH 7.6 (Biological Industries), the main rinsing solution was PBS (Biological Industries), and the incubations were carried out at room temperature. Two sections per slice were stained for LIF, LIF-R and gp 130.
The number of follicles per section was counted with an image analyser (analySIS, Soft Imaging System; Digital Solutions for Imaging and Microscopy System, GmbH, Germany), and the follicles were classified as follows (Gougeon, 1996): primordial follicles containing an oocyte surrounded by a single layer of flat GC; primary follicles with cuboidal GC, secondary follicles surrounded by more than one layer of cuboidal GC; and atretic follicles with pyknotic follicular cells, with eosinophilia of the ooplasm and clumping of the chromatin material. Follicular counts and their division into classes represent the number of follicles per class included in the immunocytochemistry study.
RNA extraction
RNA samples were extracted from the same ovarian samples used for immunocytochemistry. They were partially thawed at 37°C and then rapidly removed from the semi-frozen DMSO solution and placed in TRizol Reagent (Pioneer Research Chemicals) at room temperature and homogenized. To obtain RNA fractions (supernatants) from these homogenates, chloroform (Biolab, Israel) was added first and then a solution of phenol–chloroform–isoamyl alcohol (Sigma), followed by centrifugation at 4°C. To obtain RNA pellets, the RNA fractions were mixed with isopropanol (Biolab) and kept overnight at –20°C, followed by centrifugation at 4°C. Ethanol (75%) was added to stabilize the pellet. The samples were kept at –80°C until RT–PCR was performed.
RT–PCR
Frozen total RNA samples were centrifuged at 4°C, 13 000 g for 30 min. After the supernatant was completely removed, the pellets containing RNA were resuspended in 50 µl RNAse-free diethyl procarbonate (DEPC)-treated water. The concentration of each sample was measured by spectrophotometer (Cary UV 100, USA), and the sample was stored at –70°C. A total of 0.5 µg RNA was used for cDNA synthesis in the presence (RT+) or absence (RT–) of reverse transcriptase. The final reaction mixture containing RNA 10 µl, Oligo DT (dT) 2 µl, 10 mmol/l dNTP, 2 µl 5xRT buffer 4 µl, 0.1 mol/l dithiothreitol 2 µl, RNAsin 1 µl, and 100 IU of M-MLV reverse transcriptase (BRL) was incubated at 37°C for 1 h. The reaction was terminated by heating at 95°C for 5 min. cDNA amplification was performed with two rounds of hemi-nested PCR primers to increase the specificity and yield of the PCR product. The total 50 µl PCR reaction contained 5 µl reverse-transcribed cDNA, 5 µl of 10xPCR buffer (Qiagen), 50 mmol/l of each dNTP, 2.5 IU of Taq polymerase, and 0.4 µmol/l of each primer. Each reaction was overlaid with 50 µl of mineral oil and heated to 95°C for 4 min. PCR was carried out essentially as previously described (Ao et al., 1994), for 25–29 cycles with outer primers and 30–35 cycles with inner, hemi-nested primers at the appropriate annealing temperatures for each set (Table II). For the hemi-nested PCR, 2 µl of primary product was added to 28 µl of freshly prepared mix, as above. The amplified product was subjected to 10% polyacrylamide gel electrophoresis using pBR322 Hae III digested DNA marker as a reference for fragment size, and stained with ethidium bromide. LIF, LIF-R and gp 130 PCR products from human fetal and adult ovaries were purified with QIAquick Purification Kit (Qiagen) and sequenced commercially.
Statistical analysis
Data from the follicular counts were statistically analysed by a 
2-test, Fisher’s exact test and analysis of variance.
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Results |
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Immunocytochemical detection of LIF, LIF-R and gp 130
LIF, LIF-R (strong to moderate staining) and gp 130 (very weak to moderate staining) were detected in oocytes from primordial, primary and secondary follicles in all samples studied. LIF-R was detected in GC of primary and secondary follicles from the adolescents/women but not from the fetuses, and in some of the stromal cells of all the specimens studied. Figures 1, 2 and 3 show the immunocytochemical staining of LIF, LIF-R and gp 130 respectively. The negative controls did not stain for LIF, LIF-R or gp 130 (blue-purple) (Figures 1C, 2C, 3B).
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Follicular counts
A total of 158 follicles was counted in the stained samples from the adolescents/women (20 ± 36 follicles per section). Most of the follicles were primordial (93.7%); two secondary follicles were identified. A total of 5399 follicles was counted in the stained ovarian sections from fetuses (P < 0.0001 compared with follicles from adolescents/women; 154 ± 165 follicles per section); 94.7% were primordial and only five were secondary. The total number of primordial follicles was significantly greater than the number of primary and secondary follicles (P < 0.0001). No atretic follicles were detected in any of the sections.
Detection of LIF, LIF-R and gp 130 transcripts
The cDNA amplification primers for LIF, LIF-R and gp 130 were designed to span introns so that genomic DNA contamination could be eliminated. Hemi-nested RT–PCR analysis was performed on seven adult and fetal samples (Figure 4). All yielded the expected fragment sizes (Table III and Figure 4). There was no difference in the expression pattern of the genes between adolescents/women and fetal samples. However, because the RT–PCR analysis was not quantitative, the presence of differential transcript levels among these genes and between the adolescents/women and fetal samples could not be distinguished. Constitutively expressed HPRT gene was used as the positive control for the RT reaction. There was no contamination of genomic DNA in any of the samples tested, and all negative controls (RT–) processed without reverse transcriptase yielded no amplification product. Sequence analysis of the product confirmed the identity as LIF, LIF-R and gp 130 (data not shown). Sequences corresponding to positions 498 to 703, positions 610 to 881, as well as positions 1185 to 1365 were identical to the human LIF, LIF-R and gp 130 GeneBank-published mRNA sequences respectively (www.ncbi.nlm. nih.gov/).
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Discussion |
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The present study evaluated the concurrent immunocytochemical detection and RT–PCR expression of LIF and its two receptor units (LIF-R, gp 130) in human adolescents/adult ovaries and fetal ovaries. The immunocytochemical studies detected all three antigens—proteins to the oocytes of primordial, primary and secondary follicles. LIF-R was detected in GC from primary and secondary ovarian follicles from adolescents/women. Transcripts of LIF, LIF-R and gp 130 were expressed in all adult and fetal ovaries tested.
The findings for human fetal ovaries should be considered with caution, as most of the ovaries were derived from abnormal fetuses after feticide. The availability of human fetal ovaries for research is extremely limited, especially at late gestational ages, when most abortions are performed because of fetal abnormalities. However, because the spectrum of abnormalities was very wide, it is very unlikely that they were all connected with ovarian developmental defects. As all the fetuses underwent feticide, we sought to avoid the inclusion of ovarian tissue containing dead follicles by use of fetal specimens that were previously found (Abir et al., 2002) to contain cells with very low apoptotic rates (0–5% in germ cells and 
1% in GC). By contrast, specimens that originated from fetuses that had been dead for >24 h showed very strong apoptosis staining: the tissue was so distorted that differences between apoptosis and necrosis could not be distinguished (Abir et al., 2002). Moreover, seven of the ovarian specimens used in the present study survived in culture for 4 weeks, with an increase in 17ß-estradiol secretion (Biron-Shental et al., 2004), further supporting the viability of the ovarian follicles.
Data on the detection of LIF or its receptor in mammalian ovaries remain sparse. Transcripts for LIF-R were expressed in the developing murine gonad, and a fluorescence-activated cell sorter localized it to the surface of the PGC (Cheng et al., 1994). These results agree with our findings of LIF-R staining and the presence of transcripts in all the ovaries examined. Gp 130 was detected in germ cells of mice throughout development (Molyneaux et al., 2003). In ovaries of adult mice, a weak stain was detected in primordial follicles, with higher intensity in growing oocytes. Our present study showed very weak–moderate staining for gp 130 in oocytes from all the human samples studied, and it is therefore likely that most of the signalling of LIF in the human ovary is done via its low affinity LIF-R receptor. Another group reported LIF protein detection in rat GC of primordial and primary follicles, but no or very low staining in oocytes, and high LIF protein staining in both oocytes and GC of secondary and antral follicles (Nilsson et al., 2002). By contrast, we failed to identify by immunocytochemistry LIF in GC of human follicles, but only in their oocytes, although LIF transcripts were identified in the ovarian mRNA samples. The possibility of the differential expression of these genes in fetal and adult ovaries could not be due to the qualitative nature of our RT–PCR assay. The RT–PCR assay used in this study was a hemi-nested approach. Therefore, we could only show the presence or absence of the transcripts in the samples analysed. The possibility of higher expression of some genes due to higher follicular numbers in the fetal ovaries compared with adult ovaries could not be ruled out.
SCF and LIF regulate survival and differentiation of both migratory and postmigratory PGC already settled in the murine gonad, primarily through their anti-apoptotic function (Cheng et al., 1994; De Felici, 2000). One study in a mouse model showed that each factor alone was able to reduce PGC apoptosis (Pesce et al., 1993), whereas another study showed that only their combination led to a substantial reduction in apoptosis: LIF alone only slightly reduced apoptosis and SCF had no effect (Morita et al., 1999). The addition of an anti-gp 130 antibody blocked the survival of the postmigratory colonizing murine PGC (Koshimizu et al., 1996), and the addition of anti-LIF-R antiserum abolished PGC survival in culture (Cheng et al., 1994). In the pig too, either LIF or SCF (Shim and Anderson, 1998) or their combination (Durcova-Hills et al., 1998) was essential for the survival and proliferation of PGC. The effect of LIF on human PGC is unknown. When LIF was added to the culture medium of human post-migratory PGC from fetuses aged 5–9 gestational weeks, there was no indication of its possible benefit (Shamblott et al., 1998).
In other animal studies, a combination of LIF, SCF and insulin-like growth factor-1 added to cultured mid-pregnancy (13.5 day) ovaries containing oogonia (Morita et al., 1999; Lyrakou et al., 2002) promoted their survival in culture (Morita et al., 1999) and led to a significant increase in the number of meiotic pachytene cells (Lyrakou et al., 2002). Cell proliferation, however, measured by bromodeoxyuridine incorporation, was not observed (Morita et al., 1999). The addition of LIF and insulin to the culture medium of day 4 rat ovaries, which contain an exclusive population of primordial follicles, led to a larger number of developing follicles compared with LIF alone (Nilsson et al., 2002). The addition of insulin alone had no effect. When an anti-LIF antibody was added to the culture medium, it slightly decreased the number of developing follicles. Together, these studies (Morita et al., 1999; Lyrakou et al., 2002; Nilsson et al., 2002) indicate a possible synergistic effect of various growth factors, including LIF, on the development of oogonia (Morita et al., 1999; Lyrakou et al., 2002) and primordial follicles (Nilsson et al., 2002) in rodents.
Female mice lacking the LIF gene were found to be fertile but their blastocysts failed to implant and develop (Stewart et al., 1992). This suggested a role of LIF in implantation, similar to findings in humans (for review see Lass et al., 2001; Aghajanova et al., 2003). Mice deficient in LIF-R had normal PGC at birth (Ware et al., 1995), but did not survive beyond 1 day, so germ cell development at later stages could not be followed. The findings of normal germ cell number and development in mice in the presence of LIF-related deficiencies (Stewart et al., 1992; Ware et al., 1995) can be explained by a synergy of LIF with other growth factors or complementary effects of other growth factors on LIF. Apparently, when the LIF system is disrupted, other complementary factors may control the deficiency process. Although Yoshida et al. (1998) reported a significant reduction in the number of PGC in gonads of gp 130-deficient mice, a more recent study (Molyneaux et al., 2003) demonstrated the decrease only in male fetuses. However, they also showed that female mice with a germ cell-specific loss of gp 130 function had a slight reduction in the number of primary follicles and a major defect in ovulation. These complex effects of gp 130 deficiencies are probably due to the involvement of gp 130 in signal transduction pathways also of other cytokines besides LIF, such as oncostatin M.
Our finding of the detection of LIF and its receptor in oocytes of human fetal and adult primordial follicles, as well as their transcripts in the respective ovaries, suggests a possible benefit of adding LIF to cultured primordial follicles from human fetuses as well as from women. Alternatively, a combination of LIF with other growth factors such as SCF (Lyrakou et al., 2002) or insulin (Nilsson et al., 2002) might promote folliculogenesis as in rodents (Lyrakou et al., 2002; Nilsson et al., 2002). Media supplementations with growth factors might assist researchers in developing a successful in vitro maturation system for human primordial follicles.
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Acknowledgements |
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The authors are indebted to Ms G.Ganzach from the Editorial Board of Rabin Medical Center for the English editing and to the staff of the Gynecology Ward for their help in locating suitable patients; and to the Ultrasound Unit for identifying fetal gender. This project was partially sponsored by the Brazilian Friends of the Israel Cancer Association and a research grant from Tel Aviv University (Leo Mintz Fund).
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Submitted on December 16, 2003; resubmitted on January 22, 2004; accepted on January 27, 2004.
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R. Abir, B. Fisch, S. Jin, M. Barnnet, A. Ben-Haroush, C. Felz, G. Kessler-Icekson, D. Feldberg, S. Nitke, and A. Ao Presence of NGF and its receptors in ovaries from human fetuses and adults Mol. Hum. Reprod., April 1, 2005; 11(4): 229 - 236. [Abstract] [Full Text] [PDF]
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