TY - JOUR
AU1 - Qu, Jianping
AU2 - Godin, Pierre Arnaud
AU3 - Nisolle, Michelle
AU4 - Donnez, Jacques
AB - Abstract The freezing of ovarian tissue and the growth of immature oocytes from primordial follicles is an interesting concept in ovarian tissue transplantation and in-vitro fertilization. In this study, the morphology and distribution of primordial follicles were studied in ovarian tissue from 24 women before and after cryopreservation. Cryopreservation did not significantly change either the morphology or number per unit volume of morphologically normal follicles in frozen ovarian tissue. Primordial follicles were predominant, accounting for 78.6% and 82.6% of total follicles in fresh and frozen ovarian tissues respectively. The distribution of follicles was extremely uneven in ovarian tissue. A large variation in follicle numbers was observed in ovarian tissue samples from patient to patient, and even in the same patient, indicating that the number of follicles counted in one sample of ovarian tissue may not represent the number of follicles in other tissue samples. Ovarian tissue could be frozen in the form of strips instead of fragments for fast processing and better viability of ovarian tissue in cryopreservation. The number of follicles in ovarian tissue declined with the increasing age of the patients. An immunohistochemical study showed that immunoreactivity for the epidermal growth factor (EGF) receptor was detected in primordial follicles of adult ovarian tissue. EGF receptor staining was most intense in the oocytes of primordial follicles. Weak staining for EGF receptor was observed in some surrounding pregranulosa cells. Immunohistochemical staining for EGF receptor was also present in the stromal cells of ovarian tissue, but to a much lesser degree. There was no significant difference in the immunohistochemical staining for EGF receptor in ovarian tissue before and after cryopreservation. cryopreservation, epidermal growth factor, ovarian tissue, primordial follicle, receptor Introduction Human ovaries contain numerous primordial follicles, which serve as a reservoir of gametes during the entire reproductive life of the female. The primordial follicle is the earliest form of ovarian follicle, consisting of an oocyte surrounded by a single layer of pregranulosa cells (Adashi, 1996). These follicles are located in the cortex of the ovary, and progressively enter the next phase of development under an unknown initiation signal. Only a few follicles develop to the Graafian follicle stage, when they are able to ovulate. The mechanism involved in the recruitment of primordial follicles to preantral follicles is unclear. It may require the presence of a factor secreted by the ovary or from an extragonadal source, which selectively causes some primordial follicles to enter the growth phase while keeping the others quiescent. Epidermal growth factor (EGF) is a 53 amino acid growth factor that shows mitogenic effects in a variety of mesodermal and ectodermal tissues, and is involved in regulating cell proliferation in mammals (Fisher and Lakshmanan, 1990). In females, EGF has been found to stimulate the secretion of several hormones in trophoblast cells of the placenta during pregnancy (Miyazawa, 1992; Qu and Thomas, 1995). EGF plays a role in oocyte maturation in vitro (Gomez et al., 1993; Singh et al., 1997) and also stimulates the proliferation of granulosa cells in vivo and in vitro (Feng et al., 1987; Roy and Greenwald, 1990). Transforming growth factor-alpha (TGF-α), with 50 amino acid residues, has a 35–40% homology with EGF (Fisher and Lakshmanan, 1990). EGF and TGF-α both bind to a single EGF receptor, a transmembrane glycoprotein, on the surface of cells. The EGF receptor is present in the ovary and corpus luteum (Ayyagari and Khan-Dawood, 1987; Feng et al., 1987) and in endometrium (Imai et al., 1995). There is evidence that EGF binds to thecal cells, atretic follicles and luteal cells. EGF and its receptor are expressed in the oocytes of primary and preantral follicles of human ovarian tissue (Maruo et al., 1993; Tamura et al., 1995). A recent report revealed the expression of TGF-α and EGF in human primordial and primary follicles (Reeka et al., 1998), though studies of the presence of EGF receptor in human primordial follicles are limited in number. Although the EGF receptor has been found in the primordial follicles of human fetuses (Bennett et al., 1996), its presence in primordial follicles of human adult ovarian tissue remains to be determined. The storage of ovarian tissue at low temperature is an attractive concept for in-vitro fertilization (IVF) and ovarian tissue transplantation. It has been demonstrated that cryopreservation of ovarian tissue does not substantially damage follicles in sheep (Gosden et al., 1994), primates (Candy et al., 1995) and humans (Hovatta et al., 1996; Oktay et al., 1998a). After freezing, primate and human ovarian tissues have been successfully grafted into immunodeficient animals (Candy et al., 1995; Newton et al., 1996; Oktay et al., 1998b). Normal follicles developed in cryopreserved marmoset and human ovarian tissues grafted into mice (Candy et al., 1995; Newton et al., 1996). In addition, small pieces of ovarian tissue rapidly became revascularized under the kidney capsules of host mice, while human primordial follicles grew to early antral stages in xenografts in severe combined immunodeficient (SCID) mice (Oktay et al., 1998b). The freezing of matured oocytes for clinical use is still in debate because of the adverse effects of cooling and cryoprotectants which may damage the microtubular system and meiotic spindle and induce polyploidy, raising questions about the safety of this methodology (Pickering and Johnson, 1987; Tucker et al., 1996; Oktay et al., 1998a). Nevertheless, immature oocytes are less likely to be injured by cooling and cryoprotectants because they are small, not well developed, with few organelles, no zona pellucida, and are relatively metabolically quiescent and undifferentiated. Therefore, ovarian tissue containing immature oocytes in primordial follicles may be better suited to cryopreservation and grafting. Advances in developing new technologies for the growth of immature mammalian oocytes open up the possibility of using immature human oocytes from very early follicular stages to full maturity in vivo and in vitro (Eppig and O'Brien, 1996; Wandji et al., 1997; Oktay et al., 1998a). The maturation of human oocytes in primordial and primary follicles in vitro or in vivo, in combination with the cryopreservation of ovarian tissues, may benefit many infertile patients, provide follicles for young cancer patients undergoing chemo- or radiotherapy and for prematurely menopausal women, and revolutionize IVF (Nayudu, 1994; Gosden and Rutherford, 1997; Donnez and Bassil, 1998). We attempted to set up a bank of human ovarian tissue for the IVF programme in our research unit. To improve the technology for the cryopreservation of ovarian tissue and the growth of oocytes from early follicles in vitro and in vivo, we studied: (i) the distribution of follicles in ovarian tissue samples from patients; (ii) the influence of cryopreservation on the morphology and number of surviving follicles in ovarian tissue; (iii) the expression of EGF receptors in primordial follicles in ovarian tissue; and (iv) the possible effect of freezing on the immunoreactivity of the EGF receptor in the follicles of frozen ovarian tissue. Materials and methods Subjects Twenty-four female patients aged between 21 and 41 years, who underwent infertility evaluation, were selected for this study. All patients had regular ovulatory cycles. They underwent diagnostic laparoscopy and biopsy of ovarian tissue. Approval of this study was obtained from the Ethics Committee of the Catholic University of Louvain. Ovarian tissue After biopsy, ovarian tissue was immediately transported to the laboratory in Leibovitz L-15 medium supplemented with glutamax (Gibco, Paisley, UK) at 4°C. Ovarian tissue from 14 patients was dissected into small pieces (1 mm3). Some pieces of tissue were fixed in Bouin's solution, and immediately underwent histological study; these were assigned to the fresh fragment group. The other pieces of tissue were processed using a programmable freezer and stored in liquid nitrogen as the frozen fragment group. Ovarian tissue from another 10 patients was cut in two different ways. First, ovarian tissue was dissected in the same way as described above. Second, ovarian tissue was cut into strips (1×1×4–6 mm). A small piece was cut from the same strip, fixed in Bouin's solution, studied histologically, and used as the fresh control fragment. The remaining strip was frozen and stored in liquid nitrogen, and assigned to the frozen strip group. After various periods of time, frozen fragments and strips were thawed and fixed in Bouin's solution for further histological studies. Freezing Ovarian tissue samples were frozen using a modification of a published method (Gosden et al., 1994). Briefly, Leibovitz medium containing 2% human albumin (Red Cross, Brussels, Belgium) and 10% dimethylsulphoxide (DMSO) (Sigma, St Louis, MO, USA) was dispensed into (1 ml/vial) cryogenic vials (Simport, Quebec, Canada) precooled on ice. Ovarian tissue fragments (five to six pieces) or strips (one to two pieces) were suspended in the cryoprotective medium, and were equilibrated in the medium for 30 min on ice before cooling was initiated. The cryotubes were cooled in a programmable freezer (Kryo 10, Series III, Planer, Sunbury on Thames, UK) using the following programmes: (i) cooled from 0°C to –8°C at –2°C/min; (ii) seeded manually by touching the cryotubes with forceps prechilled in liquid nitrogen; (iii) cooled to –40°C at –0.3°C/min; (iv) cooled to –150°C at –30°C/min; and (v) transferred to liquid nitrogen (–196°C) immediately for storage. Thawing The cryogenic vials were thawed at room temperature for 2 min, and immersed in a water bath at 37°C for another 2 min. The ovarian tissues were immediately transferred from the vials to tissue culture dishes (Becton Dickinson, Meylan Cedex, France) in Leibovitz medium, and subsequently washed three times with fresh medium to remove cryoprotectant before further processing. Histological study Fixed fresh and frozen samples of ovarian tissue, fragments or strips were dehydrated with ethanol, cleared with toluene and embedded in paraffin. A continuous series of ovarian tissue sections of 6 μm thickness was prepared from each ovarian fragment and mounted on glass slides. The odd slides were stained with haematoxylin and eosin for morphological study and follicle counting, and the even slides were immunohistochemically stained later and not used for counting follicles. Areas of tissue sections of different shapes were viewed under the microscope incorporating a micrometer and calculated individually according to geometrical formulae. Morphological changes associated with the death of follicles were observed microscopically in the sections from each paraffin tissue block, including increased eosinophilia, shrinkage of the cytoplasm, and karyolysis, pyknosis and karyorrhexis of the nucleus. The number of follicles was counted in individual tissue sections. If one follicle was present in several continuous sections, it was only counted in the first section in order to avoid repetition. Unfortunately, no computerized image analysis system was available in our laboratory. Follicles were classified according to the stages of development as follows (Gougeon, 1991, 1996): (i) primordial follicle: one layer of flattened cells around the oocyte; (ii) primary follicle: one layer of cuboidal cells around the oocyte; (iii) secondary follicle: more than two layers of cuboidal cells around the oocyte. Immunohistochemical study Immunohistochemical staining was performed using an avidin–biotin–immunoperoxidase system. The sections were deparaffinized with toluene, rehydrated in ethanol and quenched in 3% hydrogen peroxide for 30 min at 37°C to block the activity of endogenous peroxidase. After washing in water and Tris-buffered solution (TBS; Tris–HCl 50 mmol/l, NaCl 150 mmol/l, pH 7.6), normal goat serum (Dako, Glostrup, Denmark) was applied for 1 h at 37°C to minimize non-specific binding. The antibody against human EGF receptor (Oncogene Research Products, Cambridge, MA, USA) was used as the primary antibody at a dilution of 1:50 and added to the sections for overnight incubation in a sealed humidified chamber at 4°C. After washing with TBS, biotinylated secondary antibody (1:250) (Boehringer Mannheim, Brussels, Belgium) was applied, and the specimens were incubated for 30 min at room temperature. Streptomycin–avidin–peroxidase conjugate (Boehringer Mannheim) was added for 30 min at room temperature. The chromogenic reaction was developed by incubation with a freshly prepared solution of 3,3′-diaminobenzidine (Dako). The sections were counterstained with Mayer's haematoxylin (Merck, Darmstadt, Germany), mounted with a coverslip, and evaluated using microscopy. Optimal working dilutions were determined by serial titration. Control procedures were undertaken to assure the specificity of the immunological reaction. Two negative controls were included simultaneously by replacing the primary antibodies with: (i) phosphate-buffered saline (PBS); and (ii) rabbit IgG (Dako) at the same dilution for the specific primary antibody. In some experiments, the antibody was preabsorbed with EGF receptor peptide (Oncogene Research Products), resulting in the absence of staining for EGF receptor in ovarian tissue sections. Sections of normal human endometrium of the uterus, known to contain EGF receptor (Imai et al., 1995), served as the positive controls. Statistical analysis Means between individual groups were analysed by the Wilcoxon signed rank test. The correlation between follicle number and patients' age was analysed by linear regression. A P-value < 0.05 was considered statistically significant. Results Morphology and distribution of primordial follicles before and after cryopreservation The morphology of ovarian tissue fragments was studied in 24 patients. Cryopreservation did not result in any significant change in the morphology of ovarian tissue as stained by haematoxylin and eosin (Figure 1). Only a few cells showed hypereosinophilia and shrinkage of the cytoplasm and/or karyolysis, pyknosis and karyorrhexis of the cell nuclei in both fresh and frozen–thawed tissue fragments. No histological differences were identified microscopically in either follicles or stromal cells in ovarian tissue samples before and after freezing. The total number of follicles varied within the range of 0 to 64.6 follicles/mm3 for fresh tissue fragments, and 0 to 105.7 follicles/mm3 for frozen tissue fragments (Table I). There was no significant difference in the mean number of morphologically normal follicles between the two groups. In ovarian tissue, primordial follicles were predominant, accounting for 78.6% of the total follicles in fresh tissue fragments and 82.6% in frozen tissue fragments (Table II). These primordial follicles were often identified by their round shape and were embedded within the dense connective tissue of the ovarian cortex. Primary follicles were found less frequently, and secondary follicles only occasionally in either fresh or frozen ovarian tissue fragments. The majority of primary follicles were distributed in the stroma of the cortex or at the corticomedullary junction. The rarely observed secondary follicles were localized mainly at the corticomedullary junction or some region of the medulla beneath the cortex. The distribution of follicles was extremely uneven in ovarian tissue. A large variation in follicle numbers was observed in ovarian tissue samples from patient to patient (Figure 2). Furthermore, even though some tissue samples were originally obtained from the same patient, the number of follicles counted in one sample of ovarian tissue did not match the number found in another tissue sample. In one patient, follicle numbers varied from a high density of 64.6 follicles/mm3 in fresh tissue fragments to no follicle at all in frozen fragments. In another patient, follicles were observed at a density of 45.5 follicles/mm3 in frozen ovarian tissue fragments, but no follicle was found in fresh ovarian tissue fragments. Freezing of ovarian tissue strips To investigate whether ovarian tissue could be frozen in the form of a tissue strip instead of a fragment, the morphology and number of follicles were observed in frozen tissue strips and compared with those in frozen fragments in 10 patients. There was no significant morphological difference in cellular components between frozen tissue fragments and strips. Only very few cells were found with hypereosinophilia and shrinkage of the cytoplasm and nuclear changes including karyolysis, pyknosis and karyorrhexis in both frozen fragments and strips, even in the centre of a strip as long as 5 mm (1×1×5 mm). Follicle number was in the range of 0 to 62.4 follicles/mm3 in frozen fragments, similar to the number observed in frozen strips (0 to 65.8 follicles/mm3; not significant). Age distribution of follicles in ovarian tissue A negative correlation was observed between the number of follicles in ovarian tissue and the age of patients. Figure 3 shows the age distribution of follicle numbers in fresh and frozen ovarian tissue fragments in patients aged 21–41 years. The number of follicles in ovarian tissue declined with the increasing age of the patients in a linear regression (r = –0.485, P = 0.016 in fresh ovarian tissue fragments, and r = –0.515, P = 0.01 in frozen ovarian tissue fragments). Expression of epidermal growth factor receptor in primordial follicles In an immunohistochemical study, immunoreactivity for the EGF receptor was observed in primordial follicles in fresh ovarian tissue (Figure 4 Middle). In a primordial follicle, the most intense staining for EGF receptor was localized in the oocyte. Weak immunohistochemical staining for EGF receptor was also detected in some surrounding pregranulosa cells in primordial follicles. Although immunoreactivity for the EGF receptor was also observed in stromal cells in ovarian tissue, the intensity of staining was much weaker than that observed in oocytes in primordial follicles. In the control sections, replacing the primary antibody with non-immune rabbit serum resulted in the complete inhibition of staining in primordial follicles and stromal cells in ovarian tissue (Figure 4 Upper), indicating the specificity of immunohistochemical staining for the EGF receptor in primordial follicles. To study the influence of freezing on EGF receptor immunoreactivity, immunohistochemical staining was also observed in frozen ovarian tissue in comparison with fresh tissue. Cryopreservation did not result in a significant change in the staining pattern for EGF receptor in frozen ovarian tissue (Figure 4 Lower). The intensity of staining for EGF receptor in the oocytes of primordial follicles from frozen ovarian tissue was similar to that found in fresh ovarian tissue. The most intense staining for EGF receptor was localized in the oocytes of primordial follicles. The signal for EGF receptor was much weaker in the stromal cells surrounding the follicles, compared with that in the oocytes. Weak immunoreactivity for EGF receptor was found in pregranulosa cells surrounding the oocytes in primordial follicles in frozen ovarian tissue. Discussion Advances in the technology of cryopreservation have confirmed that frozen–thawed ovarian tissue can be grafted to restore cyclical function and fertility, resulting in successful pregnancies in mice (Harp et al., 1994; Cox et al., 1996). Studies of the sheep ovary, which resembles the human ovary in size and composition, have shown that cryopreservation of ovarian tissue is also feasible in large animal species (Gosden et al., 1994). These animals became pregnant following transplantation of frozen–thawed ovarian tissue slices. Recent studies in the mouse have further demonstrated that cryopreserved primordial follicles extracted from mouse ovaries were able to reverse infertility in oophorectomized animals (Carroll and Gosden, 1993). A live newborn was reported after continuous culture of mouse follicles from the primordial stage (Eppig and O'Brien, 1996), indicating that it will be possible to produce mature oocytes for IVF by growing primordial follicles in vitro. This technique has also proved successful in large animal species, where primordial follicle growth has been initiated in cultures of fetal bovine, primate and human ovarian tissues (Zhang et al., 1995; Wandji et al., 1996, 1997). Grafts of frozen marmoset ovarian tissue developed follicles at all stages of folliculogenesis, including large antral follicles, 21–32 weeks after transplantation in the renal capsule of ovariectomized nude mice (Candy et al., 1995). Cryopreservation of human ovarian tissue has been extensively studied previously. In addition to an early study (Grischenko et al., 1987) in which cryopreserved human ovarian tissue, when transplanted subcutaneously in women, restored their menstrual cycles, it was also reported that cryopreserved human ovarian tissue could be successfully transplanted beneath the kidney capsule of immunodeficient mice (Newton et al., 1996). A histological study (Hovatta et al., 1996) showed that, after cryopreservation, the surviving follicles appeared morphologically normal in frozen–thawed human ovarian tissue in comparison with those from fresh ovarian tissue. Approximately two-thirds of the follicles were able to survive freeze–thawing, as measured by histological examination after frozen human ovarian tissue has been cultured in vitro for 15 days (Hovatta et al., 1997). A cryoprotectant is essential for minimizing ice formation and the survival of ovarian tissue after freezing and thawing. Cryopreservation using ethylene glycol or DMSO resulted in the survival of 84% or 74% of the follicles respectively, compared with only 44% when propylene glycol was used, and 10% with glycerol, in frozen–thawed human ovarian tissue grafted into the renal capsule of SCID mice (Newton et al., 1996). The extent of follicular survival was presumed to be in part related to the speed of cryoprotectant permeation into ovarian tissue specimens (Candy et al., 1997). A quantitative study using NMR spectroscopy (Newton et al., 1998) revealed that at 4°C, ethylene glycol and DMSO permeated human ovarian tissue more efficiently than either propylene glycol or glycerol. Recently, it was reported (Oktay et al., 1998b) that human primordial follicles could develop to antral-secretory stages in ovarian tissue xenografts in SCID/hpg mice as measured by morphological examination and proliferating cell nuclear antigen (PCNA) immunostaining. Administration of follicle stimulating hormone (FSH) was required to promote the growth of follicles beyond the two-layer stage in the mice. These encouraging results raise hopes for the clinical use of ovarian tissue banking, although at present we have not reached the goal of applying cryopreserved human ovarian tissue to the treatment of patients (Moomjy and Rosenwaks, 1998; Newton, 1998; Weissman et al., 1999). However, the usefulness of ovarian tissue autografting may be limited in patients with ovarian failure caused by endocrine- or immunology-related problems, or in those in whom the ovarian tissue is contaminated with viable cancer cells. In studies of tissue cryopreservation and transplantation, human ovarian tissue has mainly been collected by biopsy from patients undergoing laparoscopy in clinics. Usually, only one sample is obtained at biopsy. Whether this ovarian tissue sample contains a sufficient number of primordial follicles is vital for the success of autografting or allografting of ovarian tissue in vivo and the maturation of oocytes in vitro. In the present study, we investigated the distribution of follicles in fragments of human ovarian tissue, obtained by biopsy from 24 women, before and after cryopreservation. Our results showed that the number of follicles in ovarian tissue samples differed from patient to patient, ranging from 0 to 64 follicles/mm3. This implies great variation in follicular distribution in ovarian tissue between individual patients. We also observed that even in the same patient, the number of follicles varied to a great extent between tissue fragments. These results suggest that follicles are unevenly scattered in the cortex of the ovary and that the number of follicles counted in one ovarian tissue fragment may not represent the number found in another. Although it is not yet known how many follicles will be needed for grafting or growth of follicles in vitro, it is proposed that gynaecologists collect multiple samples from different parts of the ovary at biopsy in order to reduce the risk of the lack of follicles in obtained ovarian tissue fragments. The inverse relationship between the age of women and their rate of fertility is well established (Menken et al., 1986). It has been reported that the number of primordial follicles in the ovaries declines from more than 250 000 at menarche to hundreds or thousands at the end of reproductive life (Baker, 1963). At the age of 51 years (the median age of menopause in Western society), only 1000 follicles remain in the ovaries (Faddy et al., 1992). In our study, we also observed a negative correlation between follicular number and the age of the study subjects. Follicular density in ovarian tissue declined with patient age between 21 and 41 years. Our observation is consistent with the findings of others (Lass et al., 1997), who reported a negative correlation between follicular density and ovarian volume, and the age of women. In the present study, we found that primordial follicles were distributed predominantly in the outer cortical region of the ovary, accounting for 78.6–82.6% of total follicles. By contrast, primary follicles were sparsely scattered, and secondary follicles could only be found occasionally. This observation indicates that the number of primordial follicles is much greater than primary and secondary follicles in the ovaries. Primordial follicles should therefore be considered as a major potential source of oocytes for future use in IVF programmes in clinics after maturation in vitro or in vivo. The viability of ovarian tissue after cryopreservation is crucial in the transplantation of ovarian tissue or in-vitro maturation of oocytes. Viability is influenced by many factors, including the method of freezing, the choice of cryoprotectant, and the time lapse between the sample being taken in the operating theatre and the tissue being processed and frozen in the laboratory. In the present study, we used DMSO as a cryoprotectant for freezing ovarian tissue, as previously described. We did not observe any significant change in the morphology of the cortex and medulla of ovarian tissue before and after cryopreservation. Neither was there any decrease in the number of morphologically normal follicles. In cryopreservation, tissue samples are generally cut into small fragments (about 1 mm3) to allow the cryoprotectant to penetrate to the centre of the tissue mass. However, such a method for ovarian tissue processing is time-consuming and may influence the viability of follicles after freezing. It is also difficult in routine clinical practice to freeze ovarian tissue, especially with large ovarian samples taken by ovariectomy. To avoid cell death, fresh ovarian tissue should be processed not more than 1 h after the samples have been obtained in the operating theatre. In some experiments of this study, ovarian tissue was cut into strips (1×1×4–6 mm) for cryopreservation. After freezing both ovarian tissue strips and fragments, no significant differences were found between the two sample types in terms of morphological changes and follicular numbers. This suggests that ovarian tissues may be cryopreserved in the form of strips instead of fragments. Compared with ovarian tissue fragments, tissue strips have the advantage of easier handling for storage in the hospital's ovarian bank, with a better chance of having sufficient follicles either for autografting in vivo or for maturation in vitro. In humans, primordial follicles are an early form of ovarian follicle. As yet, the mechanism involved in the development of primordial follicles to preantral follicles has not been clarified. It has been found that primary imprinting during oocyte growth has a crucial effect on both the expression and repression of maternal alleles during embyrogenesis (Obata et al., 1998). EGF is a growth factor known for its numerous functions in the regulation of cell transformation and differentiation (Fisher and Lakshmanan, 1990). In women, EGF plays an important role in regulating the secretion of hormones in trophoblast cells in the placenta during pregnancy (Miyazawa, 1992; Qu and Thomas, 1995). In the ovary, EGF may also regulate the function of follicular development (Das et al., 1991; Lonergan et al., 1996; Goud et al., 1998). It has been shown that EGF altered the growth of granulosa cells in rats (Bendell and Dorrington, 1990), and inhibited the action of FSH to augment aromatase activity in human granulosa cells in vitro (Steikampf et al., 1988). In addition, EGF or TGF-α stimulated the proliferation of porcine granulosa cells (Leal et al., 1990), while EGF, either alone or in combination with insulin-like growth factor I, stimulated cumulus expansion in bovine oocytes matured in vitro (Lorenzo et al., 1994). A recent report (Goud et al., 1998) showed that addition of EGF to maturation medium, and maintenance of the cumulus during culture, improved the nuclear and cytoplasmic maturation of human oocytes in vitro. Furthermore, EGF exposure stimulated the growth of human preantral follicles and induced the expression of TGF-β type II receptor in almost all follicular cells in the culture, implying a role for EGF in the regulation of preantral folliculogenesis (Roy and Kole, 1998) There is increasing evidence that the EGF receptor is present in human ovaries (Maruo et al., 1993; Tamura et al., 1995; Bennett et al., 1996; Reeka et al., 1998). In two of these studies (Maruo et al., 1993; Tamura et al., 1995), the immunolocalization of EGF and its receptor in human ovaries during follicular growth and regression was investigated. These authors found immunoreactivity for EGF and EGF receptor in the oocytes of primary and preantral follicles. More recently, it was reported (Reeka et al., 1998) that both TGF-α and EGF were expressed in the oocytes of primordial and primary follicles. The staining intensity for TGF-α and EGF decreased in the preantral follicle and finally disappeared. By contrast, EGF receptor expression was observed in the granulosa cells of antral follicles. EGF and its receptors were identified by immunohistochemical staining in the ovarian tissue of human fetuses aged 12–24 weeks (Bennett et al., 1996). Expression of EGF receptor was localized in the oocytes, suggesting the presence of EGF receptor in the primordial follicles of fetal ovaries. In the present study, we observed that immunoreactivity for the EGF receptor was present in primordial follicles in human adult ovarian tissue. The strong immunohistochemical staining for EGF receptor was localized in the oocyte, while weak staining was also present in some surrounding pregranulosa cells. In a later study, we also observed the expression of EGF receptor in primary and secondary follicles. Our results corroborate the previous findings of others (Bennett et al., 1996), and suggest that the EGF receptor can be expressed in the follicles from very early stages of folliculogenesis. Following a later report on the expression of TGF-α and EGF in primordial follicles (Reeka et al., 1998), these results further suggest that the EGF receptor may be a mediator for the action of EGF/TGF-α in the regulation of the growth of oocytes in primordial follicles. The observations of this study are not consistent with an earlier report (Maruo et al., 1993) which noted the lack of EGF receptors in primordial follicles. The reason for such a discrepancy is unknown, but one possible explanation is that different techniques were used, for example the fixatives for ovarian tissue staining. This study was one part of our research project on human ovarian tissue banking and the in-vitro maturation of oocytes in early follicles. The presence of EGF receptor in follicles suggests that EGF or TGF-α may be involved in regulating the growth of follicles in human ovarian tissue from very early developmental stages. We assume that EGF or TGF-α may be used in the culture of early follicles from frozen ovarian tissue to promote the growth of immature oocytes in vitro. To exclude the possibility that freeze–thawing would alter or damage the EGF receptors in ovarian tissue after cryopreservation, we compared the immunoreactivity of the EGF receptor between frozen–thawed and fresh ovarian tissues and, indeed, identified no such effect. In conclusion, the EGF receptor may be expressed in the primordial follicles of human adult ovarian tissue. It would, therefore, be worthwhile in the future to study the effect of EGF/TGF-α and their receptors on the in-vitro growth of oocytes in primordial follicles from frozen ovarian tissue. Table I. Number of follicles/mm3 in fresh and frozen fragments of ovarian tissue* Patient no.  Age (years)  Fresh fragments      No. of frag.a  Volume (mm3)b  Primord folliclesc  Primary follicles (n)  Second folliclesd (n)  Total follicles (n)  No. of folliclese(/mm3)  1  21  8  3.560  218  10  2  230  64.6  2  21  4  0.845  7  1  1  9  10.7  3  23  9  3.614  44  6  1  51  14.1  4  24  4  2.272  93  5  5  103  45.3  5  25  5  1.212  13  4  1  18  14.9  6  26  3  1.776  44  14  3  61  34.3  7  26  3  0.997  7  4  0  11  11.0  8  26  5  1.888  28  4  1  33  17.5  9  28  4  1.984  26  4  0  30  15.1  10  28  2  0.621  35  3  0  38  61.2  11  30  11  5.896  13  3  0  16  2.7  12  32  6  3.354  0  0  0  0  0.0  13  32  6  3.750  9  2  0  11  2.9  14  33  6  4.886  16  3  0  19  3.9  15  34  4  1.617  1  0  1  2  1.2  16  35  2  0.750  0  0  0  0  0.0  17  35  4  1.461  1  1  0  2  1.4  18  36  5  3.978  60  5  3  68  17.1  19  37  5  1.973  10  2  0  12  6.1  20  37  4  1.274  9  3  1  13  10.2  21  37  8  6.795  6  2  0  8  1.2  22  37  6  2.077  51  12  1  64  30.8  23  38  7  2.887  25  7  0  32  11.1  24  41  2  0.695  6  1  0  7  10.1  Mean  30.9  5.1  2.506  30.1  3.9  1.0  34.9  16.1  SEM  1.2  0.5  0.341  9.4  1.1  0.3  10.0  3.7  Patient no.  Age (years)  Fresh fragments      No. of frag.a  Volume (mm3)b  Primord folliclesc  Primary follicles (n)  Second folliclesd (n)  Total follicles (n)  No. of folliclese(/mm3)  1  21  8  3.560  218  10  2  230  64.6  2  21  4  0.845  7  1  1  9  10.7  3  23  9  3.614  44  6  1  51  14.1  4  24  4  2.272  93  5  5  103  45.3  5  25  5  1.212  13  4  1  18  14.9  6  26  3  1.776  44  14  3  61  34.3  7  26  3  0.997  7  4  0  11  11.0  8  26  5  1.888  28  4  1  33  17.5  9  28  4  1.984  26  4  0  30  15.1  10  28  2  0.621  35  3  0  38  61.2  11  30  11  5.896  13  3  0  16  2.7  12  32  6  3.354  0  0  0  0  0.0  13  32  6  3.750  9  2  0  11  2.9  14  33  6  4.886  16  3  0  19  3.9  15  34  4  1.617  1  0  1  2  1.2  16  35  2  0.750  0  0  0  0  0.0  17  35  4  1.461  1  1  0  2  1.4  18  36  5  3.978  60  5  3  68  17.1  19  37  5  1.973  10  2  0  12  6.1  20  37  4  1.274  9  3  1  13  10.2  21  37  8  6.795  6  2  0  8  1.2  22  37  6  2.077  51  12  1  64  30.8  23  38  7  2.887  25  7  0  32  11.1  24  41  2  0.695  6  1  0  7  10.1  Mean  30.9  5.1  2.506  30.1  3.9  1.0  34.9  16.1  SEM  1.2  0.5  0.341  9.4  1.1  0.3  10.0  3.7  Patient no.  Age (years)  Frozen fragments      No. of frag.a  Volume (mm3)b  Primord folliclesc (n)  Primary follicles (n)  Second folliclesd (n)  Total follicles (n)  No. of folliclese(/mm3)  *Data derived from more than 8 months work; e.g. in patient no. 11, 24 frozen fragments of ovarian tissue were examined. Each paraffin block embedded one to three tissue fragments, giving a total of 155 slides. Each slide contained 2–3 bands, with each band consisting of 9–12 sections. In all, >4000 sections of frozen ovarian tissue were prepared for this patient.  aNumber of fragments.  bVolume of ovarian tissue observed microscopically (mm3); only odd-numbered slides contributed.  cPrimordial follicles.  dSecondary follicles.  eNumber of follicles observed per mm3.  1  21  4  1.249  0  0  0  0  0.0  2  21  6  2.857  288  11  3  302  105.7  3  23  5  2.646  137  26  2  165  62.4  4  24  5  3.443  155  6  2  163  47.3  5  25  4  1.086  9  4  0  13  12.0  6  26  3  1.090  0  0  0  0  0.0  7  26  5  2.273  27  1  0  28  12.3  8  26  4  2.087  73  14  1  88  42.2  9  28  3  1.179  56  6  2  64  54.3  10  28  5  1.917  66  7  1  74  38.6  11  30  24  16.371  56  5  1  62  3.8  12  32  4  0.988  39  6  0  45  45.5  13  32  5  1.747  8  0  0  8  4.6  14  33  3  0.798  1  1  1  3  3.8  15  34  15  4.357  1  1  1  3  0.7  16  35  15  8.096  13  5  1  19  2.3  17  35  3  0.946  0  0  0  0  0.0  18  36  4  1.924  20  3  0  23  12.0  19  37  3  1.273  28  1  1  30  23.6  20  37  4  1.293  0  0  0  0  0.0  21  37  7  3.682  2  0  0  2  0.5  22  37  5  1.877  55  17  1  73  38.9  23  38  8  3.104  17  3  0  20  6.4  24  41  3  0.864  1  0  0  1  1.2  Mean  30.9  6.1  2.735  43.8  4.9  0.7  49.4  21.6  SEM  1.2  1.0  0.676  13.6  1.3  0.2  14.6  5.6  Patient no.  Age (years)  Frozen fragments      No. of frag.a  Volume (mm3)b  Primord folliclesc (n)  Primary follicles (n)  Second folliclesd (n)  Total follicles (n)  No. of folliclese(/mm3)  *Data derived from more than 8 months work; e.g. in patient no. 11, 24 frozen fragments of ovarian tissue were examined. Each paraffin block embedded one to three tissue fragments, giving a total of 155 slides. Each slide contained 2–3 bands, with each band consisting of 9–12 sections. In all, >4000 sections of frozen ovarian tissue were prepared for this patient.  aNumber of fragments.  bVolume of ovarian tissue observed microscopically (mm3); only odd-numbered slides contributed.  cPrimordial follicles.  dSecondary follicles.  eNumber of follicles observed per mm3.  1  21  4  1.249  0  0  0  0  0.0  2  21  6  2.857  288  11  3  302  105.7  3  23  5  2.646  137  26  2  165  62.4  4  24  5  3.443  155  6  2  163  47.3  5  25  4  1.086  9  4  0  13  12.0  6  26  3  1.090  0  0  0  0  0.0  7  26  5  2.273  27  1  0  28  12.3  8  26  4  2.087  73  14  1  88  42.2  9  28  3  1.179  56  6  2  64  54.3  10  28  5  1.917  66  7  1  74  38.6  11  30  24  16.371  56  5  1  62  3.8  12  32  4  0.988  39  6  0  45  45.5  13  32  5  1.747  8  0  0  8  4.6  14  33  3  0.798  1  1  1  3  3.8  15  34  15  4.357  1  1  1  3  0.7  16  35  15  8.096  13  5  1  19  2.3  17  35  3  0.946  0  0  0  0  0.0  18  36  4  1.924  20  3  0  23  12.0  19  37  3  1.273  28  1  1  30  23.6  20  37  4  1.293  0  0  0  0  0.0  21  37  7  3.682  2  0  0  2  0.5  22  37  5  1.877  55  17  1  73  38.9  23  38  8  3.104  17  3  0  20  6.4  24  41  3  0.864  1  0  0  1  1.2  Mean  30.9  6.1  2.735  43.8  4.9  0.7  49.4  21.6  SEM  1.2  1.0  0.676  13.6  1.3  0.2  14.6  5.6  View Large Table II. Percentages of follicles at different stages in ovarian tissue Tissue  Primordial follicle (%)  Primary follicle (%)  Secondary follicle (%)  Values are mean ± SEM.  Fresh fragments  78.6 ± 2.6  16.3 ± 2.5  5.2 ± 2.4  Frozen fragments  82.6 ± 4.3  13.1 ± 2.5  4.4 ± 2.2  Tissue  Primordial follicle (%)  Primary follicle (%)  Secondary follicle (%)  Values are mean ± SEM.  Fresh fragments  78.6 ± 2.6  16.3 ± 2.5  5.2 ± 2.4  Frozen fragments  82.6 ± 4.3  13.1 ± 2.5  4.4 ± 2.2  View Large Figure 1. View large Download slide View large Download slide Morphology of human ovarian tissue before and after cryopreservation. (Upper) Primordial (open arrowhead) and primary follicles (closed arrowhead) in a fresh ovarian tissue fragment. A degenerated follicle was also observed in this fresh tissue section. (Lower) Normal morphology of five primordial follicles (open arrowhead) and one primary follicle (closed arrowhead) in a cryopreserved ovarian tissue fragment. Hypereosinophilia was present in one degenerated follicle in the section. (Haematoxylin and eosin staining; scale bars = 35 μm.) Figure 1. View large Download slide View large Download slide Morphology of human ovarian tissue before and after cryopreservation. (Upper) Primordial (open arrowhead) and primary follicles (closed arrowhead) in a fresh ovarian tissue fragment. A degenerated follicle was also observed in this fresh tissue section. (Lower) Normal morphology of five primordial follicles (open arrowhead) and one primary follicle (closed arrowhead) in a cryopreserved ovarian tissue fragment. Hypereosinophilia was present in one degenerated follicle in the section. (Haematoxylin and eosin staining; scale bars = 35 μm.) Figure 2. View largeDownload slide The relationship between the volume of ovarian tissue fragments and the number of follicles in (A) fresh and (B) frozen ovarian tissue. Figure 2. View largeDownload slide The relationship between the volume of ovarian tissue fragments and the number of follicles in (A) fresh and (B) frozen ovarian tissue. Figure 3. View largeDownload slide Correlation between follicle numbers in human ovarian tissue and the age of patients (range: 20–41 years). (A) The number of follicles in fresh fragments of ovarian tissue declined with increasing age of the patients (r = –0.485, P = 0.016). (B) The number of follicles in frozen fragments of ovarian tissue declined with increasing age of the patients (r = –0.515, P = 0.010). Figure 3. View largeDownload slide Correlation between follicle numbers in human ovarian tissue and the age of patients (range: 20–41 years). (A) The number of follicles in fresh fragments of ovarian tissue declined with increasing age of the patients (r = –0.485, P = 0.016). (B) The number of follicles in frozen fragments of ovarian tissue declined with increasing age of the patients (r = –0.515, P = 0.010). Figure 4. View large Download slide View large Download slide View large Download slide Expression of EGF receptors in primordial follicles in human ovarian tissue before and after cryopreservation. (Upper) Negative immunohistochemical staining with non-immune rabbit serum was observed in the oocytes of primordial follicles after cryopreservation (the control slide). (Middle) Intense brown immunohistochemical staining for EGF receptors was observed in the oocytes of primordial follicles before cryopreservation. (Lower) Intense brown immunohistochemical staining for EGF receptors was observed in the oocytes of primordial follicles after cryopreservation. (Scale bars = 35 μm.) Figure 4. View large Download slide View large Download slide View large Download slide Expression of EGF receptors in primordial follicles in human ovarian tissue before and after cryopreservation. (Upper) Negative immunohistochemical staining with non-immune rabbit serum was observed in the oocytes of primordial follicles after cryopreservation (the control slide). (Middle) Intense brown immunohistochemical staining for EGF receptors was observed in the oocytes of primordial follicles before cryopreservation. (Lower) Intense brown immunohistochemical staining for EGF receptors was observed in the oocytes of primordial follicles after cryopreservation. (Scale bars = 35 μm.) 1 To whom correspondence should be addressed at: Department of Gynaecology, Hospital of Saint Luc, Catholic University of Louvain (UCL), Avenue Hippocrate 10, UCL 10/9502, 1200 Brussels, Belgium The authors would like to thank D.Toussaint, and F.Casanas-Roux for excellent technical assistance in cryopreservation and morphological studies respectively. 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TI - Distribution and epidermal growth factor receptor expression of primordial follicles in human ovarian tissue before and after cryopreservation
JF - Human Reproduction
DO - 10.1093/humrep/15.2.302
DA - 2000-02-01
UR - https://www.deepdyve.com/lp/oxford-university-press/distribution-and-epidermal-growth-factor-receptor-expression-of-nw0p0iFBFi
SP - 302
EP - 310
VL - 15
IS - 2
DP - DeepDyve
ER -