Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca NY 14853, USA
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Abstract |
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Key words: bovine/folliculogenesis/ovary/primary follicle/primordial follicle
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Introduction |
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Most primordial follicles in bovine and baboon cortical pieces spontaneously activate and develop to the primary stage in serum-free culture (Wandji et al., 1996, 1997
; Braw-Tal and Yossefi, 1997
) 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, 1996
). However, when bovine cortical pieces were cultured in the presence of medullary tissue, activation was not inhibited (Derrar et al., 2000
; J.Fortune and S.Kito, unpublished data).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., 1998, 1999
, 2000
). 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, 1944
; Rawles, 1952
). 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.
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Materials and methods |
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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 3738°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, 1996); (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., 1996
). 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.
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Results |
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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 3A, 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 3A
, middle right) and an increase in the number of primary follicles were evident by day 4 (Figure 1E
and Figure 3B
, 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 3
, middle right). These changes are similar to those that occurred in cortical pieces placed in organ culture on day 0 in this (Figure 3
, left) and the previous (Figure 2
) 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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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 3, 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 3
, 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 4B
(middle left) and Figure 1F
, 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 4B
, 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 3A
), health (Figure 4A
), or diameters (data not shown) of the primordial follicles.
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Discussion |
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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, 1978), 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., 1994a
; Nugent et al., 1998
). 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., 1994a
). 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., 1994a
; Nugent et al., 1998
), or when neonatal mouse ovaries were placed in organ culture (Eppig and O'Brien, 1996
). 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, 1996; Herrmann and Spanel-Borowski, 1998
). 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., 2001
). After 8 days in vivo or in vitro, activation had occurred, as expected, (Eppig and O'Brien, 1996
) 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., 1999), 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., 1992
) 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., 1996, 1997
), 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., 1997; Parrot and Skinner, 1999
), growth differentiation factor-9 (Vitt et al., 2000
), 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 13 weeks later revealed primordial follicles, but few or no growing follicles (Gosden et al., 1994b; Newton et al., 1996
; Baird et al., 1999
). 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.
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Acknowledgements |
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Notes |
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2 To whom correspondence should be addressed at: T6-012B VRT, Cornell University, Ithaca, NY 14853, USA.E-mail: jf11{at}cornell.edu
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References |
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Submitted on June 19, 2001; accepted on September 12, 2001.