Early massive follicle loss and apoptosis in heterotopically grafted newborn mouse ovaries

Jun Liu1,3, Josiane Van der Elst1, Rudy Van den Broecke2 and Marc Dhont1,2

1 Infertility Centre, Department of Obstetrics and Gynaecology and 2 Department of Gynaecologic Oncology, Ghent University Hospital, De Pintelaan 185, 9000 Ghent, Belgium


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: Ovarian tissue cryopreservation and transplantation can be used to restore fertility to sterile females. A question that warrants further investigation is whether the follicular content is affected by the freeze–thawing and grafting procedure, and if so, to what extent and by what mechanism. METHODS AND RESULTS: Intact newborn mouse ovaries were allografted under the kidney capsule or were cryopreserved by slow freezing with dimethylsulphoxide as the cryoprotectant prior to grafting. Estrogenic activity of ovariectomized recipient mice, as revealed by vaginal cytology, resumed after 11 days of transplantation. At 14 days after transplantation, ovarian grafts were recovered and processed histologically for follicle number counting. The follicular content of grafts of fresh ovaries was 58% of that from ovaries of age-matched 14 day old mice. In frozen–thawed ovarian grafts, the follicular content was only 9% lower than that of fresh grafted ovaries. Apoptosis of follicular cells was investigated by DNA nick end labelling. We observed a marked increase in the staining of fragmentation of DNA shortly after transplantation (2–12 h) of fresh newborn mouse ovaries. CONCLUSIONS: The results of the present study indicate that transplantation rather than cryopreservation accounts for the major and early loss of primordial follicles in grafted newborn mouse ovaries.

Key words: apoptosis/cryopreservation/follicle/mouse ovary/transplantation


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
In the first attempts of ovarian cryopreservation (Deanesly, 1954Go; Parkes and Smith, 1954Go; Parkes, 1958Go; Parrott, 1960Go), mouse ovaries were cooled in a glycerol/saline solution to -79°C and live offspring were obtained after autografting, albeit at a low rate (Parrott, 1960Go). Moreover, when the follicular content of fresh and frozen–thawed ovarian tissue was compared, only ~5% of the follicle population survived the freeze–thawing process, with primordial follicles being the sole survivors (Deanesly, 1954Go; Green et al., 1956Go). Over recent years significant advances have been made in the field of cryobiology with the introduction of automated freezers and more efficient cryoprotectants (Newton et al., 1996Go). The structure of the ovary is well suited for tissue storage because primordial follicles are abundant, developmentally dormant and are located peripherally. Isolated mouse primordial follicles and murine ovarian tissue were effectively frozen in media with dimethylsulphoxide (DMSO) as cryoprotective agent, leading to restoration of fertility after grafting (Carroll and Gosden, 1993Go; Harp et al., 1994Go). Human ovarian tissue was successfully cryopreserved in DMSO and 1,2-propanediol in terms of morphological normal follicles although survival rates were not quantified (Hovatta et al., 1996Go). A murine study on the effect of cryoprotectants on follicle survival after freezing demonstrated that 81–94% of primordial follicles survived when DMSO or propanediol were used as cryoprotectants (Candy et al., 1997Go).

The ultimate goal of cryopreservation of the ovaries is to preserve fertile oocytes. There is no report so far for any species in which primordial follicles have been isolated, frozen, thawed and grown to maturity in vitro, though this has been demonstrated in vivo (Carroll and Gosden, 1993Go). Only one research team has achieved fertilization, embryonic development of mouse oocytes and live birth after complete growth from the primordial follicle stage in vitro by organ culture and subsequent oocyte–granulosa–complexes culture (Eppig and O'Brien, 1996Go). At present, this can only be achieved by ovarian tissue grafting. In recipients of orthotopic grafts of frozen ovarian tissue follicle growth and estrous cycles have been obtained [mouse (Harp et al., 1994Go; Cox et al., 1996Go), sheep (Baird et al., 1999Go), human (Oktay and Karlikaya, 2000Go)]. Live offspring after orthotopic grafting of frozen–thawed ovarian tissue has been reported in mice (Gunasena et al., 1997aGo,1997bGo; Sztein et al., 1998Go; Candy et al., 2000Go) and sheep (Gosden et al., 1994aGo). Apparently normal antral follicles have been found in either fresh or frozen ovarian tissue xenografted to immunodeficient mice in cat (Gosden et al., 1994bGo), marmoset monkey (Candy et al., 1995Go), and human (Oktay et al., 1998Go; Weissman et al., 1999Go; Gook et al., 2001Go; Van den Broeckeet al., 2001Go). Moreover, live mouse offspring were obtained by IVF of oocytes from cryopreserved primordial stages after sequential ovarian transplantation and in-vitro maturation (Liu et al., 2001Go).

Ovarian transplantation can be used to restore fertility to sterile females (Gosden et al., 1994aGo; Gunasena et al., 1997aGo; Sztein et al., 1998Go; Baird et al., 1999Go; Candy et al., 2000Go; Cox et al., 2000Go; Liu et al., 2000Go). A question that warrants further investigation is whether the follicular content is affected by the freeze–thawing and grafting procedure, and if so, to what extent. A shortened fertile lifespan (Gunasena et al., 1997aGo; Sztein et al., 1998Go) and reduced litter sizes (Sztein et al., 1998Go) of recipient mice carrying ovarian grafts have been reported. Only one study (Candy et al., 2000Go) reported that the mean number of mouse pups per litter and the mean number of litters per female were similar in recipients of grafts of fresh and frozen ovaries and in untreated control females of the same strain.

We therefore wanted to investigate follicle survival and developmental potential after allotransplantation of fresh and frozen–thawed newborn mouse ovaries by comparing the number of follicles and by analysis of follicular cell apoptosis after ovarian tissue grafting.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Animals
Female B6CBF1 (C57bl/6jxCBA/Ca) mice bred in the Central Animal House of the Ghent University Hospital were used throughout the study. The mice were housed with free access to food and water and with 14:10 h light:dark cycles. Every cage containing one CBA/Ca male and two C57bl/6j females was checked twice per day (in the morning and evening) for the delivery of newborn mice. The date of the birth of newborn mice was day 0. Approval for this study was obtained from the Animal Research Ethical Committee of the Ghent University Hospital. One day old female F1 mice were used for collection of newborn mouse ovaries. Female F1 mice 10–12 weeks old were used as recipients for ovarian transplantation.

Cryopreservation of newborn mouse ovaries
Mouse ovaries from 1 day old mice were slowly cryopreserved using a modification of a published method (Gosden et al., 1994aGo). Briefly, the intact ovaries were recovered and suspended in a cryoprotectant medium of Leibovitz L-15 supplemented with 10% (v/v) fetal bovine serum (FBS; Life Technologies, Merelbeke, Belgium) and 1.5 mol/l DMSO (Sigma, Bornem, Belgium) on crushed ice. Each ovary was then drawn up into the middle of a 0.25 ml plastic freezing straw (Type-2A 175, Industrie de la Médicine Vétérinaire, L'Aigle, France) with a small volume of cryoprotectant medium. The straws were sealed with polyvinyl chloride powder and held on ice for 20 min before they were placed in a programmable biological freezer (Planer Biomed, Sunbury, Middlesex, UK) pre-cooled to 0°C. The straws were cooled at -2°C/min to -7°C and held for 5 min, seeded manually, held at -7°C for a further 10 min, cooled to -40°C at -0.3°C/min, further cooled to -140°C at -10°C/min and finally transferred to liquid nitrogen (LN2) for storage. For thawing of the ovaries, the straws were removed from LN2, held in air for 20 s and transferred to a water bath at room temperature for 10-20 s. The contents of straws were emptied into L-15 medium with 10% FBS. The cryoprotective agent was removed by repeated rinsing. The mouse ovaries were kept in this medium at room temperature until transplantation.

Transplantation procedure
The recipient mice were anaesthetised by i.p. injection of 100 µl of sodium pentobarbital (Nembutal; Sanofi, Brussels, Belgium) diluted 1:4 in phosphate-buffered saline (PBS; Sigma). The kidney was exteriorized through a dorsal-horizontal incision. A small hole was torn in the kidney capsule using fine watchmakers' forceps under aseptic conditions. The intact fresh or frozen-thawed newborn mouse ovaries were inserted underneath the capsule through the small hole. Both recipient in-situ ovaries were removed by cautery at the top of the uterine horns. Finally, the body wall incisions and skin were closed. The transplantation process was performed at room temperature. The duration of the grafting process was ~30 min in each experimental trial.

Validation of grafting newborn mouse ovaries by vaginal cytology
Female F1 mice 10–12 weeks old were randomly assigned to one of four experimental groups: sham-operated controls (group I, n = 5); ovariectomized controls (group II, n = 5); ovariectomized mice receiving fresh ovarian allotransplants (group III, n = 6); and ovariectomized animals receiving cryopreserved ovarian allotransplants (group IV, n = 6). Ovarian transplants were collected from 1 day old newborn female F1 mice for immediate transplantation to group III or for cryopreservation prior to transplantation to group IV. Sham-operated (group I) animals received the surgical procedure of kidney externalization and replacement back to the abdominal cavity, and suturing of the body wall incisions. Both ovaries of each recipient mouse in groups II, III and IV were removed. Group II animals received no further treatment, and the incisions were closed. In group III and IV animals, newborn fresh or frozen-thawed mouse ovaries were inserted under the renal capsule, respectively.

All animals were allowed to recover for a week before vaginal cytology was examined. These animals were monitored 5-7 times per week for ~1 month. The vaginal wall of each recipient was scraped gently and the cells were mixed with a drop of PBS on a clean glass slide. The stage of the estrous cycle was determined from the cell types observed with an inverted microscope with Hoffman contrast modulation. To evaluate the developmental potential of ovarian grafts, the recipients of group III and IV were autopsied 6 weeks after transplantation. The ovarian grafts were dissected out and placed in Bouin's solution for histological processing as described below.

Histology and follicle counting
In a second set of experiments, fresh and frozed-thawed newborn mouse ovaries were transplanted bilaterally under the kidney capsule of the same recipients. The recipient mice (n = 3) were anaesthetized as described above. In each recipient, one fresh newborn mouse ovary was grafted underneath the left kidney capsule and one frozen-thawed newborn mouse ovary underneath the right kidney capsule. Recipients' ovaries were removed. Two weeks after transplantation, recipient mice were killed by cervical dislocation. Ovarian grafts were removed from the kidney and fixed in Bouin's fluid. Fixed ovaries were embedded in paraffin, serially sectioned at 5 µm and stained with haematoxylin and eosin. The sections were examined serially for the presence of follicles. Follicles were classified as follows: (i) primordial follicles with one layer of flattened pregranulosa cells surrounding the oocytes; (ii) primary follicles with one layer of cuboid granulosa cells; (iii) preantral follicles with two or more layers of granulosa cells and no antrum; (iv) antral follicles with an antral cavity. To avoid counting follicles more than once, follicles were counted in the section where the dark-staining nucleolus was seen within the nucleus of the oocyte. The diameter of the nucleolus of oocytes in preantral and antral follicles is ~7-10 µm, and thus there is a risk of overcounting. Much attention was paid to avoid double counting in adjacent sections when dealing with preantral or antral follicles. The diameter of the nucleolus as a marker for primordial follicles is estimated to be ~2 µm (Jones and Krohn, 1961Go), so the risk of overcounting is reduced in 5 µm thick sections. Age-matched prepubertal mouse ovaries recovered from 2 week old mice were histologically processed as described above as in-vivo development control.

DNA nick end labelling by in-situ TUNEL analysis
Grafted fresh ovaries were recovered from recipients at 2 h (T = 2 h), 12 h (T = 12 h), 24 h (T = 24 h), day 3 (T = 3 days) and day 7 (T = 7 days) after transplantation in a third set of experiments. Ovaries fixed immediately after removal from in-situ served as in-vivo control. Ovaries fixed after the duration of processing between ovary removal from in situ and closure of each experiment (~30 min) served as time zero control (T = 0 h). Ovaries from 2, 4 and 8 day old mice were fixed as corresponding in-vivo controls for grafts recovered at 24 h, 3 days and 7 days after transplantation. Three ovaries grafted unilaterally were included in each condition. The tissue was fixed in 4% phosphate-buffered formaldehyde at 4°C overnight, embedded in paraffin by the standard method, cut into 5 µm thickness sections, and mounted on SuperFrost® Plus slides. Tissue sections were subsequently deparaffinized by heating at 60°C for 5 min and washing twice in xylene for a total of 10 min. The sections were then rehydrated through a graded series of alcohols and double distilled water.

In-situ TUNEL [terminal deoxynucleotidyl transferase (TdT)- mediated dUTP nick end labelling] analyses were carried out according to the instructions of a commercial assay kit (In Situ Cell Death Detection Kit—Fluorescein; Boehringer Mannheim, Mannheim, Germany) with some modifications. Briefly, tissue sections were treated with 20 µg/ml proteinase K in 10 mmol/l Tris-HCl (pH 7.4-8.0) for 25 min at room temperature and washed twice in PBS. Sections were incubated for 1 h at 37°C with 50 µl TUNEL reaction mixture in a humidified dark chamber. A second set of tissue sections was incubated with 50 µl reaction buffer without TdT as a negative control. As a positive control, a third set of sections was treated with 1 µg/ml RQ1 RNase-free DNase (Promega, Leiden, The Netherlands) for 15 min at room temperature to induce non-specific breaks in DNA. The reaction was stopped by washing the sections in PBS three times. A small amount of Vectashield mounting medium (Vector Laboratories, Burlingame, CA, USA) was placed on each section to preserve fluorescence and prevent rapid photobleaching. The tissue sections were covered with coverslips. Tissue sections were examined using a fluorescence microscope (Axioplan II; Zeiss, Zaventem, Belgium) equipped with an epi-illumination single band emitter filter cassette for the illumination of green (FITC). Fluorescein-12-dUTP, once conjugated to the 3'-OH ends of fragmented DNA, stains the nuclei of apoptotic cells green.

Statistics
All data are expressed as means ± SEM. One-way analysis of variance followed by Newman-Keuls post t-test was used to compare the mean numbers of follicles present in fresh or frozen grafted ovaries and 2 week old mouse ovaries. The proportion of follicles in different developmental classes was compared by means of {chi}2 test. P <= 0.05 was considered significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Vaginal cytology
Daily sequential vaginal cytology showed cornified epithelial cells in all animals receiving fresh ovarian grafts (group III) and cryopreserved grafts (group IV) starting at 11 days after transplantation. Observation was continued until 32 days after transplantation and the intervals between the appearance of epithelial cells were 2.7 ± 1.0 and 3.5 ± 1.7 days for a duration of 1.0 ± 0.5 and 1.2 ± 0.4 consecutive days respectively. Estrous cyclicity was seen 7-8 days after surgery in sham-operated animals (group I). About 1 week after surgery, smears from ovariectomized animals (group II) revealed a total absence of all cells with the exception of a few cornified epithelial cells.

Qualitative evaluation of follicle development in grafted ovaries
Six week old established grafts from fresh (group III) and frozen-thawed newborn mouse ovarian grafts (group IV) showed a morphologically similar appearance. Histological examination showed that transplanted grafts in both the fresh and frozen groups contained follicles at all stages of folliculogenesis and corpora lutea (Figure 1Go).



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Figure 1. Histological sections illustrating the appearance of a grafted fresh ovary (A) and a grafted frozen–thawed ovary (B), isolated from newborn mice and allotransplanted under thekidney capsule for 6 weeks. Note the presence of primordial follicles (arrows) and different classes of growing follicles (F) and corpora lutea (CL) in the grafted ovarian tissue. K = kidney tissue. Bars = 100 µm.

 
Quantification of survival and resumption of follicular growth after grafting
All fresh (n = 3) and frozen (n = 3) newborn mouse ovarian grafts were recovered 2 weeks after transplantation underneath the kidney capsule of ovariectomized recipients. The number of follicles in each grafted ovary and in 2-week-old mouse ovaries were counted (Table IGo). Grafts of fresh ovaries and frozen ovaries contained similar numbers of primordial follicles. The number of primordial follicles in fresh grafted ovaries and frozen grafted ovaries were significantly lower than that in the ungrafted 2 week old mouse ovaries. Two weeks after transplantation, surviving primordial follicles were recruited for growth to primary, preantral and antral stages. The number of follicles in primary and preantral stages was not significantly different among fresh grafted ovaries, frozen grafted ovaries and 2 week old mouse ovaries. The grafts of fresh ovaries and frozen ovaries contained more antral follicles than 2 week old mouse ovaries, but the difference was not significant. Approximately 70% of the total follicle population comprised primordial follicles in both 2 week old grafted ovary groups. By comparison, in ungrafted in-vivo 2 week old mouse ovaries, primordial follicles comprised 84% of the follicle population (P < 0.0001).


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Table I. Number of follicles in 2 week old grafts after transplantation of fresh or frozen-thawed newborn mouse ovaries, and in 2 week old mouse ovaries
 
The total number of follicles present in the grafted ovaries was expressed as a proportion of the total number of follicles in the 2 week old mouse ovaries (Table IIGo). The average number of follicles per intact 2 week old mouse ovary was slightly less than 4000. Grafts of fresh ovaries contained 58% of the follicles in 2 week old ovaries while frozen-thawed ovarian grafts contained 49%.


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Table II. Survival of total follicle content after grafting fresh or frozen-thawed newborn mouse ovaries
 
DNA fragmentation in grafted ovaries
A few scattered TUNEL-positive primordial oocytes or surrounding granulosa cells were present throughout the cortical region of in-vivo newborn mouse ovaries (Figure 2A,BGo). About 30 min manipulation of ovaries in medium before grafting (T = 0) slightly induced positive staining for DNA fragmentation in the medulla region (Figure 2CGo). In T = 2 h and T = 12 h ovarian grafts, a marked increase of staining of DNA fragmentation was detected in granulosa cells or stroma cells especially in the medulla region (Figure 2D,EGo). In T = 24 h ovarian grafts, a low level of staining for DNA fragmentation in cortical and medulla regions was observed except in the region of hilum (Figure 2FGo). With increasing time from T = 3 days to T = 7 days after grafting, ovarian grafts showed a low amount of TUNEL-positive cells (Figure 2GGo) similar to that observed in in-situ ovarian tissue of the age-matched mice (Figure 2HGo); a few oocytes displayed staining for apoptotic DNA fragmentation in the cortical region (photographs of T = 7 day grafts and in-vivo control ovaries are not shown).



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Figure 2. Representative slide sections of mouse ovarian grafts and in-vivo ovaries processed for apoptosis using TUNEL [terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labelling] staining. (A) Day 1 newborn mouse ovary showing a few scattered TUNEL-positive primordial oocytes or granulosa cells in the cortical region. (B) A x4 enlargement of apoptotic cells in A (marked by the white arrows). (C) The ovary after about 30 min manipulation during the process of grafting showed slightly positive staining for DNA fragmentation in the medulla region. Ovarian grafts after 2 h (D) and 12 h (E) of operation showed a marked increase in the number of granulosa cells or stroma cells stained in the medulla region. (F) After 24 h of grafting, a low level of TUNEL-positive staining was observed in medulla regions. However, a high level staining was maintained in the region of hilum (h). (G) Ovarian graft after 3 days showed a low number of positive cells. (H) Ovary from 4 day old mouse displayed a few oocytes stained positively in the cortical region. (I) Negative control: minimal background in the tissue incubated without TdT. (J) Positive control: all of the cells in the section are fluorescently tagged. Bar = 40 µm in B and 150 µm in all other panels.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
In this study, viability of intact newborn mouse ovaries, containing predominantly primordial follicles, allografted under the kidney capsule was evaluated by vaginal cytology, follicle count and apoptosis analysis. Estrous cyclicity, follicular recruitment and growth were restored after allografting but a substantial loss of follicle content was observed, mainly due to the grafting procedure. The most significant and original feature of this study is that it is shown for the first time that acute follicular cell death occurs shortly after grafting, probably through an apoptotic mechanism.

Estrous cyclicity of ovariectomized recipient mice was restored after transplantation. Six week grafts from both fresh and frozen groups contained all stages of growing follicles and corpora lutea, indicating the occurrence of ovulation. Cryopreservation did not have a detrimental effect on grafted ovarian function. However, optimal transplantation conditions still need to be investigated. Even though sufficient follicles can survive and estrous cycles be restored, many follicles in the grafts died during the procedure. The present study shows that ~58% of the follicle population remained in fresh ovarian grafts. Only a further 9% of follicles were lost when the tissue was cryopreserved prior to transplantation. The major loss of follicles occurred acutely after grafting rather than after the freeze-thaw procedure. This confirms results obtained previously in different species which show that a substantial loss of follicles occurs after transplantation (Felicio et al., 1983Go; Candy et al., 1997Go; Baird et al., 1999Go; Nisolle et al., 2000Go). These results are also in agreement with the reports on the shortened fertile lifespan (Gunasena et al., 1997aGo; Sztein et al., 1998Go) and reduced litter sizes (Sztein et al., 1998Go) of recipient mice. The follicular losses can be attributed, at least partly, to ischaemia-reperfusion injury resulting from lipid peroxidation and other free radical-mediated effects in the grafts (Nugent et al., 1998Go). There was an appreciable improvement in survival rates from 45 to 72% when host animals were administered the antioxidant {alpha}-tocopherol, and this indicates that follicle loss is partly due to reactive oxygen species generated during ischaemia and reperfusion (Nugent et al., 1998Go). Therefore, steps should be taken to minimize lipid peroxidation during ischaemia in ovarian grafts, such as chilling the tissue to reduce the production of reactive metabolites before reperfusion, and adding free radical scavengers (e.g. superoxide dismutase) to the medium (Parks et al., 1982Go).

Studies on the influence of ovariectomy of the recipient on the survival and growth of ovarian grafts are contradictory. Retarded growth of ovarian grafts has been reported after transplantation of ovarian tissue to intact recipients (Cox et al., 1996Go,2000Go). The removal of the recipient's ovaries gives rise to two physiological changes: circulating gonadotrophins are elevated and factors secreted by the intact recipient ovaries are eliminated. FSH stimulates granulosa cell mitosis, follicle maturation and inhibits granulosa cell apoptosis. Gonadotrophins also play a positive role in the vascular response after transplantation (Dissen et al., 1994Go). Therefore, high levels of FSH in ovariectomized recipients may facilitate follicular survival and development in transplanted ovaries. We also noticed that the proportion of growing follicles in grafted ovaries was higher than that in-vivo 2 week old mouse ovaries. This accelerated growth of follicles might be a response to the rise in concentration of circulating gonadotrophins in ovariectomized recipients. The inter- and intra-ovary development of follicles is regulated by some unknown mechanisms. Dominant follicles can inhibit the growth of other follicles in the contralateral ovary as well as in the same ovary. The phenomenon was also confirmed with follicles in culture (Spears et al., 1996Go). It is therefore likely that the dominant follicles within the ovaries of intact recipients play an inhibitory effect on the follicle growth in the ovarian grafts. The accelerated rate of depletion of the ovarian grafts reserve in ovariectomized recipients may also relate to the observations of reduced fertile lifespan and reduced litter sizes (Gunasena et al., 1997aGo; Sztein et al., 1998Go).

The meiotic process is initiated in most mammalian species during prenatal life or shortly after birth, when the oogonial mitosis is accomplished and oogonia transform into oocytes. The oocyte reaches the diplotene stage of prophase just before or immediately after birth. At this stage, by a mechanism not fully understood, the meiotic process is arrested. The chromosomes decondense and resume their transcriptional activity. The oocyte, with a prominent nucleus is referred to as germinal vesicle, in which state it may persist throughout infancy and beyond the onset of puberty. Between 1 and 2 days after birth in mice, most oocytes are surrounded by follicular cells to form the primordial follicles (Hirshfield, 1994Go). Marked strain differences were noticed, however, in the stages of development attained on the first day after birth (Jones and Krohn, 1961Go). The primordial follicle consists of a small oocyte, a few flattened granulosa cells, and a basement membrane. They lie in the outer cortex of the ovary and represent the pool of resting follicles. In all species, the store of primordial follicles decreases with time. In women, menopause is the consequence of the exhaustion of the pool of such follicles (Gougeon et al., 1994). In sheep, comparison of the population of primordial follicles in ovaries of 2 and 8 year old ewes has demonstrated that 8 primordial follicles disappear from the reserve pool every day (Driancourt et al., 1985Go). Since granulosa cell death is never visualized in such follicles, it may be assumed that oocyte death is the cause of the death within the primordial follicles (Reynaud and Driancourt, 2000Go). The apoptotic nature of cell death during folliculogenesis in in-situ ovaries is supported by two lines of experimental evidence: internucleosomal DNA fragmentation was observed by agarose gel electrophoresis and the TUNEL method.

A systematic time-scheduled apoptosis analysis has been performed on ovarian grafts by the TUNEL technique for the first time in this study. The principal result of this study is that follicular cell death probably occurs through an apoptotic mechanism in newly grafted ovaries. About 30 min of manipulation of ovaries before grafting caused additional cell death in the medullar region, while the appearance of large numbers of TUNEL-positive cells was detected at 2 h until 12 h after transplantation. No further accumulation of apoptotic cells was observed at subsequent time intervals. This can be explained by the fact that apoptotic cells are quickly removed through phagocytosis by neighbouring cells in the tissue (Vaux, 1993Go). The distribution of apoptotic cells showed that granulosa cells and stroma cells were more prone to be affected by the procedure than primordial oocytes. It can be assumed that the somatic cells are more active metabolically than the primordial oocytes, and thus are more easily affected by the ischaemia caused by transplantation. From the relationship with the oocytes in histological sections, it is speculated that the positive-staining cells are granulosa cells and stroma cells; however, it is difficult to distinguish granulosa from stroma cells. In both ovarian grafts at day 3 or day 7 and fresh ovaries from age-matched controls, only a few TUNEL-positive oocytes were observed. It may be inferred therefore that the death of granulosa cells and stroma cells in the grafted ovaries eventually gives rise to the follicle loss.

In conclusion, this study has shown that fresh and frozen–thawed newborn mouse ovaries display normal ovarian function after transplantation to ovariectomized recipient mice. Primordial follicles in ovarian grafts can be recruited and may develop to primary, preantral, antral and ovulatory stages under the capsule of the kidney. Apoptosis of follicle cells occurred shortly after the transplantation procedure, which accounted for the loss of approximately half of the total content of primordial follicles. The results of this study thus show that transplantation rather than cryopreservation caused the majority of primordial follicle loss. To improve the results of ovarian transplantation, an appropriate protocol should be developed that minimizes follicular depletion and ensures a long fertile lifespan of the transplanted ovarian tissue.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The authors acknowledge Dr V.Schelfhout (Goormaghtigh Institute of Pathology, Ghent University Hospital) for assistance in tissue processing. Mrs V.David is thanked for her help in taking care of the mice. This study is supported by a research grant from the Bijzonder Onderzoeksfonds of the Ghent University, Belgium (grant No. BOF01112199).


    Notes
 
3 To whom correspondence should be addressed. E-mail: jun.liu{at}rug.ac.be Back

Submitted on March 9, 2001; resubmitted on September 11, 2001


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on March 9, 2001; resubmitted on September 11, 2001; accepted on November 8, 2001.