Bovine ovarian cortical pieces grafted to chick embryonic membranes: A model for studies on the activation of primordial follicles

R.A. Cushman, C.M. Wahl,1 and J.E. Fortune,2

Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca NY 14853, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: Little is known about the factors that control the initiation of growth of primordial follicles. Primordial follicles in pieces of fetal bovine ovarian cortex spontaneously activate in vitro and develop to the primary stage, but few follicles develop further. For decades, embryologists have grafted tissue to the chorioallantoic membrane (CAM) of chick embryos to study the development of various organs and structures. METHODS AND RESULTS: To test the hypothesis that grafting cortical pieces beneath the CAM membrane of 6-day-old chick embryos (`in ovo') would support the activation of primordial follicles and the growth of activated follicles to the secondary stage, ovarian cortical pieces from six bovine fetuses (6–8 months gestation) were placed either in ovo or in organ culture in serum-free medium (in vitro). Cortical pieces were retrieved after 0, 2, 4, 7, or 10 days in ovo or in vitro. Histological examination revealed a dramatic infiltration of the CAM-grafted cortical pieces with blood vessels. By day 2 in vitro, the number of primordial follicles had declined by 87% concomitant with a 3.5-fold increase in primary follicles (P < 0.01), providing evidence of the expected activation of primordial follicles. Unexpectedly, primordial follicles were not activated in CAM-grafted tissue, as shown by maintenance of their numbers and lack of increase in primary follicles during 10 days in ovo. In experiment 2, a subset of pieces was switched from culture to CAM grafts and from CAM grafts to culture on day 2. The CAM did not support the growth of primary follicles activated in vitro, apparently because the activated follicles did not survive the transfer (P < 0.05). However, primordial follicles maintained in ovo retained their ability to activate; after their removal from the CAM into culture, primordial follicles decreased in number and primary follicles increased in number within 2 days (P < 0.05). CONCLUSIONS: The CAM graft will provide a useful model for studying the factors involved in activation of primordial follicles.

Key words: bovine/folliculogenesis/ovary/primary follicle/primordial follicle


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The majority of the follicles in the mammalian ovary are in the dormant primordial pool in the outer, cortical region of the ovary, but <1% of these follicles grow to ovulatory size (Hirshfield, 1988Go). In recent years, a great deal of research has been focused on the final stages of follicular growth, immediately prior to ovulation. In contrast, relatively little is known about the initiation of growth of primordial follicles and the early growth of preantral follicles. The resting pool of primordial follicles is a resource that could be utilized for alleviating infertility, developing new forms of contraception, or delaying menopause if the signals that initiate primordial follicle growth were understood.

Most primordial follicles in bovine and baboon cortical pieces spontaneously activate and develop to the primary stage in serum-free culture (Wandji et al., 1996Go, 1997Go; Braw-Tal and Yossefi, 1997Go) but very few of these follicles continue to develop to the secondary stage. These results suggest that an inhibitor of medullary origin regulates activation in vivo and that separation of the cortex from the medulla causes primordial follicles to activate in vitro. This idea is supported by the results of Eppig and O'Brien who observed activation of only a small proportion of follicles when whole neonatal mouse ovaries were placed in organ culture (Eppig and O'Brien, 1996Go). However, when bovine cortical pieces were cultured in the presence of medullary tissue, activation was not inhibited (Derrar et al., 2000Go; J.Fortune and S.Kito, unpublished data).

Another question of interest is the identity of factors needed to sustain growth in vitro once follicles have been activated. Attempts to stimulate their growth with FSH, activin, or serum-supplementation met with no or limited success (Fortune et al., 1998Go, 1999Go, 2000Go). Grafting tissue to the chorioallantoic membrane (CAM) of chick embryos, to study the development of various organs and structures, is a technique that has been used by embryologists for decades (Rudnick, 1944Go; Rawles, 1952Go). Tissue placed on the CAM is rapidly vascularized and the lack of an immune system at this stage of chick development prevents graft rejection. Therefore, pieces of bovine ovarian cortex were grafted beneath the chorioallantoic membrane of 6-day-old chick embryos to test the hypothesis that culture in ovo would support the activation of primordial follicles and the growth of activated follicles to the secondary stage.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Bovine fetal ovaries (n = 6; 6–8 months post-conception) were collected at an abattoir and transported to the laboratory as previously described (Wandji et al., 1996Go). Briefly, the ovaries were quartered at the abattoir and transported at ambient temperature (20–22°C) in Leibovitz's L-15 medium (Life Technologies, Grand Island, NY, USA) supplemented with 1% fetal bovine serum, 50 IU penicillin/ml and 50 µg streptomycin/ml (Life Technologies). At the laboratory, the cortex was dissected from the medullary tissue and cut into 0.5 mm3 pieces. Six pieces from each fetus were fixed immediately as day 0 controls.

Experiment 1
In this experiment, the capacity of the CAM graft environment to support the activation of primordial follicles and their continued growth was compared to serum-free organ culture. Prior to the experiment, a window was made in the shells of 3-day-old fertilized chicken eggs, the window was sealed with a piece of tape, and the eggs were maintained in an incubator at 37–38°C and 60% humidity. In preliminary experiments, ovarian cortical pieces were placed on the outer surface of the CAM of 6-day-old chick embryos, which is the `classical' technique. However, the grafts did not become vascularized. Therefore, we developed a modification of the classical technique, placing the cortical pieces beneath the developing CAM, between the CAM and the yolk sac (one piece/egg). The window was resealed with tape and the eggs were returned to the incubator (culture in ovo). Cortical pieces (n = four pieces/time point/fetus) were retrieved after 0, 2, 4, 7, or 10 days in ovo. The experiment was repeated with cortical pieces from six fetal ovaries.

Another set of cortical pieces from the same six fetal ovaries (n = four pieces/time point/fetus) was placed in 24-well Costar (Corning Inc., Corning, NY, USA) plates (two pieces/well) on uncoated culture plate inserts (Millicell-CM, 0.4 µm pore size; Millipore Corporation, Bedford, MA, USA) with 300 µl of Waymouth's medium MB 752/1 (Sigma Chemical Co., St. Louis, MO, USA) supplemented with antibiotics (50 µg/ml streptomycin sulphate and 75 µg/ml penicillin, Sigma), ITS+ (6.25 µg/ml insulin, 6.25 µg/ml transferrin, 6.25 ng/ml selenious acid, 1.25 mg/ml BSA, 5.35 µg/ml linoleic acid; Collaborative Biomedical Products, Becton Dickinson Labware, Bedford, MA, USA) and 25 mg/l pyruvic acid sodium salt (Sigma) and incubated at 38.5°C (culture in vitro). Cortical pieces were retrieved after 0, 2, 4, 7, or 10 days in vitro.

Experiment 2
Interestingly, in experiment 1, primordial follicles in CAM-grafted ovarian cortex remained healthy, but did not leave the resting pool. Therefore, experiment 2 was designed to determine if (i) primordial follicles in CAM grafts retain the ability to activate and (ii) if the CAM will support the growth to the secondary stage of the primary follicles resulting from activation in vitro of primordial follicles in cortical cultures. Experiment 2 was performed with further cortical pieces from four of the six ovaries used in experiment 1. The initial steps of experiment 2 were identical to those for experiment 1. Then, on day 2 of culture, a subset of cortical pieces (n = four pieces/time point/fetus) was transferred from in vitro to in ovo and a subset was transferred from in ovo to in vitro. Cortical pieces were retrieved after 0, 2, 4, 7, or 10 days in vitro or in ovo (n = four pieces/time point/fetus). The controls for this `crossover' experiment were cortical pieces from the same four fetuses cultured in vitro and in ovo.

Assessment of follicular and oocyte survival and growth
Upon retrieval from culture or from beneath the CAM, cortical pieces were fixed for 1 h in 2.5% glutaraldehyde, 2.5% formaldehyde in 0.075mol/l cacodylate buffer, pH 7.3. The pieces were embedded in LR White plastic (EMS, Fort Washington, PA, USA) and 2 µm sections were cut with a glass knife. For each piece of ovarian cortex, every other set of 10 consecutive sections was mounted on gelatin-coated slides and stained with Toluidine blue. Sections were collected as sets of 10 so that each follicle could be assessed in multiple adjacent sections to accurately stage early primary follicles. Every other set of 10 was collected to insure that follicles would not be counted twice. In order to avoid counting or measuring a follicle twice, only the largest cross section in each set of 10 consecutive sections was used and only follicles with the germinal vesicle present in that section were counted and measured. Between 18 and 31 sections were examined for each treatment and time point.

Follicles were classified as: (i) primordial, one layer of flattened and/or small cuboidal somatic cells around the oocyte (van Wezel and Rodgers, 1996Go); (ii) primary, a single layer of large cuboidal granulosa cells around the oocyte and (iii) secondary, two complete layers of granulosa cells. Health of follicles was classified as described previously (Wandji et al., 1996Go). Briefly, follicles were classified as: (i) healthy, intact basal lamina, oocyte with no more than three cytoplasmic vacuoles, intact germinal vesicle and nucleolus; (ii) early atretic, oocyte with more than three cytoplasmic vacuoles and beginning of chromatin condensation, (iii) moderately atretic, fragmentation of the oocyte cytoplasm and nucleolus, heavy condensation of the oocyte chromatin, or (iv) late atretic, oocyte completely fragmented or absent.

Sections were examined under an inverted microscope equipped with Hoffman modulation contrast optics, and the image was projected onto a video monitor. The diameters of individual healthy (i.e. Class 1) follicles and enclosed oocytes were measured by a computer-driven image analysis program (NIH Image; NIH, Bethesda, MD, USA). Each follicle and its oocyte were measured in two dimensions, and the two measurements were averaged.

Statistical analysis
Mean numbers of total primordial and primary follicles per section, mean numbers of healthy primordial and primary follicles per section, and mean diameters of healthy primordial and primary follicles and their oocytes were calculated for cortical pieces from each fetus (n = 6 in experiment 1 and n = 4 in experiment 2) for each day and treatment. Overall means were then calculated. Data were log transformed if Hartley's test indicated heterogeneity of variance among the means. Differences among means were tested by ANOVA with treatment, day and the interaction of treatment and day as the independent variables. Differences among individual means were tested after ANOVA using Duncan's multiple range test.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Experiment 1
On day 0, freshly isolated bovine ovarian cortex contained mostly primordial follicles (Figures 1A and 2GoGo). Vascularization of the cortical pieces transplanted beneath the CAM was evident at the earliest time of graft retrieval (day 2) and was extensive after 10 days in ovo (Figure 1BGo). After only 2 days in vitro, follicular populations in cultured cortical pieces had shifted dramatically compared with day 0, the number of primordial follicles had decreased and the number of primary follicles had increased (Figure 1CGo). In cortical pieces retrieved on day 2 and later, the number of primordial follicles had declined by 87%, concomitant with a 3.5-fold increase in primary follicles (P < 0.01), providing evidence of the expected activation of primordial follicles (Figure 2Go). Diameters of the few primordial follicles that were not activated and their oocytes increased over the 10 day period in culture (follicles: from 27.2 ± 0.5 µm to 32.5 ± 0.5 µm; oocytes: from 20.4 ± 0.2 µm to 23.9 ± 0.6 µm; P < 0.05). Primary follicles increased in diameter progressively during the 10 days of culture (from 34.9 ± 0.6 µm to 44.3 ± 1.7 µm; P < 0.05) and their oocytes were larger by day 10 (from 23.5 ± 0.4 µm to 25.6 ± 0.7 µm; P < 0.05).



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Figure 1. Morphology of ovarian cortical pieces before or during culture in vitro or beneath the chick chorioallantoic membrane (CAM). (A) Freshly isolated ovarian cortical piece containing predominantly primordial follicles (black arrow) and a primary follicle (white arrow). (B) A piece of bovine ovarian cortex 10 days after transplantation beneath the CAM. Note the dramatic vascularization that has occurred. (C) Ovarian cortex cultured for 2 days in medium supplemented with ITS+ (for composition see text). Almost all follicles appear to have been activated to become primary follicles (arrow). (D) Ovarian cortex after 10 days beneath the CAM. Most of the follicles have remained at the primordial stage (arrow). (E) Ovarian cortex that was grafted beneath the CAM from day 0–2 and then cultured from day 2–4. Note that activation has occurred (arrow). (F) Ovarian cortex that was cultured from day 0–2 and then maintained as a CAM graft from day2–10. Note the atretic primary follicles (arrow). In panels A and CF, bar = 33 µm; in panel B, bar = 375 µm.

 


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Figure 2. Numbers of (A) primordial and (B) primary follicles (mean per histological section ± SEM, n = 6 fetuses) in fetal bovine ovarian cortex after 0, 2, 4, 7, or 10 days in vitro or beneath the CAM (in ovo). Within each follicular stage and each experimental condition, bars with no common superscripts are different (P < 0.01).

 
In contrast to the results in vitro, primordial follicles were not activated in CAM-grafted tissue (Figures 1D and 2GoGo), as shown by maintenance of their numbers and the lack of increase in primary follicles during 10 days in ovo (Figure 2Go). There was no change in the diameters of primordial follicles or their oocytes in cortical pieces grafted beneath the CAM (P = NS). Likewise, when cortical pieces were placed beneath the CAM the diameters of primary follicles and their oocytes did not change during the 10 day period.

In freshly isolated cortical pieces, about 13 and 55% of primordial and primary follicles, respectively, were in some stage of atresia. Neither the incidence nor the degree of atresia changed with time in culture or culture condition.

Experiment 2
Experiment 2 was a `crossover' experiment conducted to determine if (i) primordial follicles in cortical tissue placed beneath the CAM still retained the ability to activate, and (ii) primary follicles activated in vitro could grow to the secondary stage in cortical pieces grafted beneath the CAM. Morphometric analysis of serial sections showed the expected lack of activation of primordial follicles when ovarian cortical pieces were grafted beneath the CAM (Figure 3AGo, right). In contrast, when cortical pieces were retrieved from the CAM on day 2 and placed in organ culture, a decrease (P < 0.01) in the number of primordial follicles (Figure 3AGo, middle right) and an increase in the number of primary follicles were evident by day 4 (Figure 1EGo and Figure 3BGo, middle left). Numbers of primordial and primary follicles in cortical pieces fixed on days 7 and 10, (i.e. after 5 and 8 days, respectively, in culture after removal from beneath the CAM) were similar to day 4 (P > 0.05, Figure 3Go, middle right). These changes are similar to those that occurred in cortical pieces placed in organ culture on day 0 in this (Figure 3Go, left) and the previous (Figure 2Go) experiments. The percentages of primordial and primary follicles in some stage of atresia in the cortical pieces transferred from in ovo to in vitro were not different from those observed in day 0 controls or in cortical pieces placed in culture on day 0.



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Figure 3. Numbers of (A) primordial and (B) primary follicles (mean per histological section ± SEM, n = 4 fetuses) in fetal bovine ovarian cortex after 0, 2, 4, 7, or 10 days in vitro (left), in ovo (right), or after transfer between in vitro/in ovo (middle). Within each follicular stage and each experimental condition, bars with no common superscripts are different (P < 0.05). Arrows indicate when `crossovers' occurred.

 
As in experiment 1, there was an increase in the diameters of healthy primordial and primary follicles and their oocytes in vitro, and there was no change in diameters of these follicles or their oocytes in ovo (data not shown). In cortical pieces removed from the CAM and placed in vitro on day 2, diameters of the few unactivated primordial follicles and their oocytes were larger by day 4, and they continued to grow in culture (day 2 versus day 10: 27.8 ± 0.8 µm versus 30.8 ± 0.1 µm for follicles and 20.4 ± 0.4 µm versus 22.6 ± 0.6 for oocytes; P < 0.05). There was also an increase in the average diameter of primary follicles (day 2 versus day 10: 35.7 ± 0.9 µm versus 41.2 ± 1.6 µm; P < 0.05) and their oocytes (day 2 versus day 10: 23.1 ± 0.9 µm versus 25.0 ± 0.7; P < 0.05) following transfer to culture, in a pattern that mimicked growth of primary follicles and oocytes in cortical pieces placed in organ culture on day 0.

In the reciprocal crossover experiment, cortical pieces were placed in organ culture for 2 days and then grafted beneath the CAM. In vitro, most primordial follicles were activated during the first 2 days in culture, as expected (Figure 3Go, left). When cortical pieces, containing activated follicles, were transferred to the CAM on day 2, numbers of primary and primordial follicles did not change significantly through to day 10, the last time of tissue retrieval, similar to results for pieces that were organ-cultured for the entire 10-day experimental period (Figure 3Go, middle left versus left). However, in contrast with the results for all other experimental situations, there was an increase in the percentage of primary follicles showing signs of atresia after transfer of cortical pieces from culture to the CAM. As shown in Figure 4BGo (middle left) and Figure 1FGo, the number of healthy primary follicles declined precipitously between day 2 and day 10, indicating a loss of healthy primary follicles present in the cortical pieces when they were transferred to the CAM on day 2, but no decline when pieces remained in culture (Figure 4BGo, left). In addition, the healthy follicles present in the transferred pieces and their oocytes, did not increase in diameter, as did follicles in vitro. In fact, the few healthy primary follicles remaining on day 10 were smaller than the average diameter of the much larger number of follicles present on day 2 (day 2 versus day 10: 40.0 ± 1.0 versus 34.5 ± 0.2 µm; P < 0.05). In contrast, transfer from culture to the CAM did not affect the numbers (Figure 3AGo), health (Figure 4AGo), or diameters (data not shown) of the primordial follicles.



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Figure 4. Number of healthy (A) primordial and (B) primary follicles in pieces of fetal bovine ovarian cortex after 0, 2, 4, 7, or 10 days in vitro, in ovo, or after transfer between in vitro/in ovo (middle). Within each follicular stage and each experimental condition, bars with no common superscripts are different (P < 0.05). Arrows indicate when `crossovers' occurred.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
These results show that primordial follicles in bovine cortical pieces placed beneath the CAM of 6-day-old chick embryos do not spontaneously activate, in dramatic contrast to the wholesale, spontaneous activation of primordial follicles in cultured cortical pieces in this and previous studies (Wandji et al., 1996Go, 1997Go; Braw-Tal and Yossefi, 1997Go). It is unlikely that CAM-grafted pieces are exposed to substances or conditions that are detrimental to the primordial follicles, because the primordial follicles were as healthy, after 10 days in ovo, as follicles in freshly isolated cortical pieces. In addition, in experiment 2, primordial follicles in CAM-grafted cortical pieces activated and began to grow when cortical pieces were placed in organ culture after 2 days in ovo. This shows that their capacity to activate was equivalent to that of primordial follicles in freshly isolated tissue. The very different results, observed when cortical pieces were cultured in ovo versus in vitro, although unexpected, provide new and unique opportunities to test hypotheses about the mechanisms of activation of primordial follicles.

An interesting question is whether even a small proportion of primordial follicles become activated in CAM grafts. It is possible that under these conditions a few primordial follicles are leaving the dormant pool over time, similar to the slow pace of activation in vivo (Hirshfield and Midgley, 1978Go), but that the change is below the level of our ability to detect. If this were true, a longer culture period would be needed to observe a significant change in the primordial pool. Because the grafts can only be maintained for 10 days (after that time the increasing size of the chick hinders graft retrieval) this may not be enough time to determine if any activation occurs in the CAM grafts. However, activation does occur when cortical pieces are grafted to the kidney capsule of severely compromised immunodeficient (SCID) mice and in this situation, growth to the antral stage can occur after several months (Gosden et al., 1994aGo; Nugent et al., 1998Go). Activation in vivo is a much slower process than the wholesale spontaneous activation that occurs in vitro. This suggests that activation in ovo may be more similar to that which occurs in vivo. Ovarian cortical tissue, placed beneath the CAM of 6-day-old chick embryos, was rapidly vascularized. Similar effects have been observed when other tissues were grafted to the CAM, and when ovine cortical pieces were grafted beneath the kidney capsule of SCID mice (Gosden et al., 1994aGo). There are other situations in which activation of primordial follicles is restrained. Wholesale spontaneous activation did not appear to occur when cortical pieces were placed beneath the kidney capsule of SCID mice (Gosden et al., 1994aGo; Nugent et al., 1998Go), or when neonatal mouse ovaries were placed in organ culture (Eppig and O'Brien, 1996Go). Taken together, these data suggest that spontaneous activation is inhibited by contact with a vascular system and/or by close contact with non-cortical regions of the ovary.

Alternatively, differences in the nutrient and/or oxygen supply to the CAM graft may explain the inhibition of activation that occurs in ovo. The cortical region of the bovine ovary is poorly vascularized in vivo (van Wezel and Rodgers, 1996Go; Herrmann and Spanel-Borowski, 1998Go). It is possible that spontaneous activation occurs in vitro, because the cortical pieces are placed in an environment that is richer in nutrients and/or oxygen than in the intact ovary. In ovo, the chick membranes form a bursa-like structure around the vascularized cortical pieces that might result in a microenvironment that is actually closer to the situation in vivo than are the conditions in serum-free organ culture. On the other hand, the inhibition of spontaneous activation in the CAM-grafted cortical pieces, combined with the dramatic vascularization, suggests that there may be a specific factor in the blood of chick embryos that restrains activation of primordial follicles. More recent studies in our laboratory with newborn mouse ovaries (which contain only newly formed primordial follicles) have provided the first information about follicle activation in CAM grafts of intact ovaries (Cushman et al., 2001Go). After 8 days in vivo or in vitro, activation had occurred, as expected, (Eppig and O'Brien, 1996Go) but the number and percentage of primary follicles were higher in vitro, suggesting that some normal restraint of activation is diminished in vitro. Interestingly, activation was negligible in ovo, showing that, at least in newborn mouse ovaries, the inhibition imposed by the situation in ovo is almost complete, in contrast to what occurs in newborn mouse ovaries in vivo during the same time period. Taken together with the results presented here for bovine cortical pieces, these data strongly suggest that chick embryos produce an inhibitor of follicle activation.

Further research will be needed to determine the identity of this putative inhibitor of activation, but one candidate is anti-mullerian hormone (AMH). In AMH knockout mice, the pool of primordial follicles was depleted more rapidly than in wild-type controls (Durlinger et al., 1999Go), and in the heterozygotes the primordial pool was depleted at a rate intermediate between the knockouts or the wild-type controls. The developing chick gonad produces AMH in both males and females (DiClemente et al., 1992Go) and, therefore, we hypothesize that gonad-derived AMH in the chick embryo circulation is restraining activation of the primordial follicles in our CAM grafts. In support of this hypothesis, preliminary experiments showed that destruction of chick gonads is followed by activation of primordial follicles in ovo (R.A.Cushman, C.M.Wahl and J.E.Fortune, unpublished data).

As in our previous studies with bovine and baboon ovarian cortex (Wandji et al., 1996Go, 1997Go), there was significant growth of primary follicles and their oocytes during 10 days in vitro. In contrast, there was no appreciable change in the size of the primordial and primary follicles or their oocytes during the 10 days beneath the CAM. The lack of measurable growth of primary follicles in the CAM grafts may be an artifact of the low numbers of primary follicles present. However, it is also possible that there is a factor in the embryonic circulatory system that inhibits growth of primary follicles.

When cortical pieces were removed from beneath the CAM after 2 days and placed in vitro, spontaneous activation occurred. Therefore, it appears that primordial follicles placed beneath the CAM retain the ability to activate. Because treatments can be applied to CAM grafts, they provide an excellent model for investigating the role of kit ligand (Yoshida et al., 1997Go; Parrot and Skinner, 1999Go), growth differentiation factor-9 (Vitt et al., 2000Go), and other factors suggested to control activation of primordial follicles.

When follicles were activated in culture and grafted beneath the CAM on day 2, there was a dramatic decrease in both the number and diameter of healthy primary follicles during the 8 days beneath the CAM. Similarly, when ovine or human cortical pieces were grafted or transplanted to SCID mice, histological analysis of grafts recovered 1–3 weeks later revealed primordial follicles, but few or no growing follicles (Gosden et al., 1994bGo; Newton et al., 1996Go; Baird et al., 1999Go). The authors of these studies concluded that the preferential loss of growing follicles was due to their greater susceptibility to temporary ischaemia prior to revascularization of the tissue. The decrease in diameter of the healthy follicles in ovarian cortex placed beneath the CAM after 2 days in vitro suggests two possibilities. The first is that even those activated follicles classified as healthy have begun to undergo atresia and the decrease in diameter is due to shrinkage. The second, and perhaps more intriguing, is that the larger follicles actually undergo atresia first and that the smaller follicles are able to resist the damage caused by the ischaemic condition longer. Alternatively there might be some activation occurring in the CAM graft, and smaller newly activated primary follicles are replacing the larger activated follicles that are undergoing atresia. Again, this is difficult to determine because there are so few primordial follicles remaining to be activated after 2 days of organ culture.

In conclusion, grafting ovarian cortical pieces beneath the chick CAM appears to restrain the wholesale, spontaneous activation of primordial follicles that is observed in vitro. Because the primordial follicles in CAM grafts are capable of activation and further growth when they are removed from beneath the CAM and placed in culture, the CAM graft provides a unique model for investigating the roles of factors involved in primordial follicle activation and primary follicle growth in primates and domestic species. Such studies will help to elucidate the mechanisms involved in activation of primordial follicles into the growing pool, and could lead to the development of methods to grow follicles from the primordial stage to a point where the oocytes could be harvested for IVF. Alternatively, therapies could be developed to inhibit activation, resulting in new methods of contraception and methods to delay the onset of menopause.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The authors thank Taylor Packing (Wyalusing, PA, USA) for the donation of fetal bovine ovaries, Mr C.Murphy for technical assistance and Dr J.J.Eppig for critical reading of the manuscript. This work was funded by the NIH (HD35168), the USDA (00–35203–9151), and a National Research Service Award, F32 HD08624 (R.A.C.)


    Notes
 
1 Present address: Department of Biological and Chemical Sciences, Wells College, Aurora, NY 13020, USA Back

2 To whom correspondence should be addressed at: T6-012B VRT, Cornell University, Ithaca, NY 14853, USA.E-mail: jf11{at}cornell.edu Back


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 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
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Submitted on June 19, 2001; accepted on September 12, 2001.





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