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School of Human Development (B.K.C.), University of Nottingham, Nottingham NG7 2UH, United Kingdom; Division of Biological Sciences (Institute of Cell and Molecular Biology) (E.E.T), University of Edinburgh, Edinburgh EH9 3JR, United Kingdom; Department of Obstetrics and Gynaecology (D.T.B.), University of Edinburgh, Edinburgh EH3 9EW, United Kingdom; and School of Biosciences (R.W.), Sutton Bonington Campus, University of Nottingham, Leicestershire LE12 5RD, United Kingdom
Address all correspondence and requests for reprints to: Dr. Bruce K. Campbell, School of Human Development, Floor D East Block, Queens Medical Centre, University of Nottingham, Nottingham NG7 2UH, United Kingdom. E-mail: bruce.campbell{at}nottingham.ac.uk.
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Abstract |
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Compared with the Hypo group, Hyper ewes had higher (P < 0.001) gonadotropins, had greatly enlarged grafts, had reestablished a normal follicular hierarchy 2 months earlier (P < 0.05), had higher (P < 0.05) levels of proliferating cell nuclear antigen expression in tertiary, preantral, and antral follicles, and had higher (P < 0.01) concentrations of inhibin A and estradiol. Compared with time zero controls, increases in the number of primary follicles and the rate of proliferation in primary and secondary follicles in both groups of autografts (P < 0.05) were also observed.
In conclusion, the results of this experiment provide the first evidence that gonadotropins can affect the rate of development of preantral follicles in vivo in a large monovulatory species. Furthermore data are presented to support the existence of a gonadotropin-independent intraovarian feedback loop regulating both the rate of primordial follicle initiation and primary and secondary follicle development.
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Introduction |
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We have previously reported that primordial follicles in thin strips of ovarian cortex remain viable after freezing to -196 C, that these grafts can restore apparently normal ovarian cycles and fertility when autografted onto the ovarian pedicle of young sheep (16), and that normal reproductive cycles can be restored for up to 22 months after autografting (17). This technique therefore represents a valuable intervention for restoration of fertility after treatment for malignant disease. In addition, this latter study also showed that autografting ovarian tissue results in a 2- to 5-fold increase in peripheral FSH and LH concentrations and that there is a marked delay of between 3 and 6 months from the time of autografting until first estrus (17). Studies on autografts in sheep (17) and human xenografts in SCID mice (18) have shown that the ischemia that occurs in the graft before revascularization results in the loss of virtually the entire growing follicle population and 35–50% of the primordial follicles, whereas cryopreservation damage results in the loss of only 7% of the oocytes if optimum freezing protocols are used. Previous estimates, based on mitotic index of granulosa cells at different stages of development, have suggested that the duration of folliculogenesis in sheep is between 4 and 6 months (4, 19), and the present results are therefore consistent with the hypothesis that the delay from autografting to resumption of cyclicity represents the period of time required for surviving primordial follicles to develop to a preovulatory follicle. Thus autografting appears to result in the synchronization of early follicle development, a characteristic, we reasoned, that could be used to develop a large animal model to study the control of early follicle development in an experimental, rather than observational context.
In the present experiment, we used this autograft model to examine one of the key unanswered questions concerning the physiological mechanisms controlling early folliculogenesis, namely the role of the gonadotropins. Despite the observation that mRNA for FSH receptors is expressed by the granulosa cells of secondary follicles in many species (3, 15, 20), numerous investigators have reported that follicle development proceeds to the small antral stage in hypophysectomized (20), hypogonadotrophic (21, 22, 23, 24), and FSH receptor or gonadotropin knockout (25, 26) individuals, strongly suggesting that FSH has no role in early folliculogenesis. Conversely, effects of FSH on early folliculogenesis in vivo have been reported in rodents (27, 28, 29), whereas effects in vitro have been reported by numerous investigators in many species (7, 30, 31). As detailed above, the ovarian autograft results in a marked increase in circulating FSH concentrations that can be attributed, at least in part, to depletion of the ovarian follicle population resulting in lower secretion of inhibin A (32). The experimental paradigm employed in this experiment therefore involves prevention of this increase in FSH after autografting, using a combined treatment with GnRH-agonist and low-dose estradiol, with examination of the quantitative and qualitative effects of this intervention on the follicle population during the subsequent restoration of the normal pattern of folliculogenesis.
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Materials and Methods |
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Hormone preparations
The GnRH-agonist (GnRHa; buserelin 1 mg/ml; Aventis Pharmac, Kent, UK) was administered by osmotic minipumps (2ML4: release rate 6 µl/h; Charles River UK, Kent, UK) that were replaced every 4 wk (22). The minipumps were placed sc in the flank either under general anesthesia at the time of surgery or under local anesthesia subsequently. Estradiol implants (2 cm) were prepared from crystalline estradiol-17ß (Sigma, Dorset, UK) and SILASTIC brand tubing (Dow Corning, Midland, MI) as previously described (33) and placed sc in the front axilla under general anesthesia at the time of initial surgery and left in place for the duration of the experiment. This size of implant would be expected to give estradiol concentrations of 4–5 pg/ml, the level commonly observed during the follicular phase in normal sheep (33, 34).
Experimental treatment
All animals were bilaterally ovariectomized and two fresh cortical patches (1 mm thick x 4–5 mm square) were autografted onto the ovarian pedicle on each side (16). Two additional patches from each animal were fixed immediately in 4% paraformaldehyde to act as time zero controls. At the time of autografting, half (n = 12) of the animals received both GnRHa and estradiol implants to prevent the postcastration rise in gonadotropins (hypogonadotrophic; Hypo), and this treatment was continued for the duration of the experiment. The remaining 12 autografts received no further treatment and were therefore expected to have elevated gonadotropins (hypergonadotrophic; Hyper). In half of the animals from each treatment group, ovarian tissue was recovered from the left ovarian pedicle after 1 month and from the right ovarian pedicle after 3 months of grafting (designated 1/3; n = 6/group). In the remaining animals, designated 2/4, ovarian tissue was recovered from the left ovarian pedicle after 2 months and from the right ovarian pedicle after 4 months of grafting (n = 6/group). Ovarian tissue was immediately fixed in 4% paraformaldehyde and processed for histological analysis. Thus, we had available ovarian tissue from six ewes in both the Hyper and Hypo groups at 1, 2, 3, and 4 months after grafting.
Blood sampling
Jugular venous blood samples were collected by venipuncture every 2–3 d for the entire experimental period. In addition, before graft retrieval, timed samples of ovarian venous blood were collected for estimation of ovarian estradiol and inhibin A secretion as previously described (35). After collection the blood was centrifuged at 4 C and the plasma stored at -20 C before RIA.
Hormone assays
Plasma concentrations of LH, FSH, estradiol (36, 37) and inhibin A (38) were determined using previously described RIAs. The sensitivity of the assays for LH, FSH, estradiol, and inhibin A were 0.2 µg/liter (National Institute of Diabetes and Digestive and Kidney Diseases, oLH, S23), 0.3 µg/liter (U.S. Department of Agriculture, oFSH, SIAFP-RP2), 14 ng/liter, and 30 ng/liter (rhInhibin A), respectively. The intra- and intercoefficients of variation for all the immunoassays were less than 12% in the 20–80% effective dose range.
Histological analysis
Ovarian tissue was fixed overnight in 4% paraformaldehyde in PBS 0.01 M (pH 7.6) (PBS, Sigma) and transferred to 70% ethanol until processing. The fixed tissue was embedded in paraffin after the usual dehydration steps. Serial sections of 5 µm from each block were cut and every fifth section dewaxed and rehydrated in decreasing concentrations of alcohol (90, 70, and 30% and distilled water), followed by two washes of 5 min in PBS before staining with hematoxylin and eosin (BDH, Poole, UK) for estimation of follicle number and stage of development. The intervening sections were stored and a subsample processed for immunohistochemistry if histological analysis revealed the presence of ovarian follicles.
Sections were examined on a DMRB microscope (Leica Microsystem UK, Milton Keynes, Hertfordshire, UK) and follicles classified as primordial, primary, secondary, tertiary, preantral, early antral, and antral according to established criteria (15). Briefly, primordial follicles had a single layer of flattened granulosa cells; primary follicles had a single layer of granulosa cells with at least one of these cells being cuboidal; secondary and tertiary follicles had two and three layers of cuboidal granulosa cells, respectively; preantral follicles had multiple layers of granulosa cells; and antral follicles had a discrete antral cavity. Clearly degenerative follicles with few granulosa cells, pyknotic nuclei, and a shrunken oocyte were few and not counted. Because proliferating cell nuclear antigen (PCNA) expression was used as an objective means to assess the functional status of follicles, more subjective morphological estimates of follicular atresia were not employed. To avoid double counting of follicles, preantral follicles were counted only if the section went through the oocyte nucleolus. The low density of antral follicles in these sections meant that identification of such follicles between consecutive sections was straightforward and hence double counting unlikely. Counting of follicles in consecutive sections was continued until at least 50 follicles had been counted from each patch or until the entire patch had been sectioned. Because ovarian tissue was often surrounded by connective tissue, the number of sections cut per block varied greatly according to the orientation of ovarian tissue to the sectional plane.
Immunohistochemistry of proliferating cell nuclear antigen
Sections of 5 µm were dewaxed in xylene for 10 min and rehydrated in decreasing concentrations of alcohol (90, 70, and 30% and distilled water), followed by two washes of 5 min in PBS. Antigen retrieval was carried out by microwaving the sections in citrate buffer (0.01 M, pH 6.0) at full power (800 W) for 10 min; they were then left to rest in the buffer for 20 min until cool. After two washes in PBS, the sections were incubated 3% hydrogen peroxide in methanol for 30 min. After a further two washes in PBS, the slides were incubated in blocking buffer, consisting of 20% vol/vol normal horse serum (Vectastain kit, Vector, Peterborough, UK) in PBS for 30 min and then washed again in PBS before addition of a 1:50 dilution of monoclonal PCNA antibody (Novacastra, Newcastle-upon-Tyne, UK) in blocking buffer for 60 min at 37 C. After a further two washes in PBS, the slides were incubated with a 1:500 dilution of biotinylated horse antimouse immunoglobulin (Vectastain kit, Vector) in blocking buffer for 30 min at room temperature. Negative controls were carried out by replacing the primary antibody with a 1:100 dilution of mouse immunoglobulin (Sigma). The section were again washed twice in PBS for 5 min before incubation in avidin-biotin peroxidase complex (ABC-HRP, Dako Cytomation, Cambridgeshire, UK) for 30 min at room temperature. After a final wash in PBS and Tween 20, bound antibodies were visualized in brown by incubation in 3–3'diaminobenzidine (Dako Cytomation), the sections were counterstained with Harris hematoxylin (BDH), dehydrated through increasing concentration of alcohols, and mounted with coverslips. Each run of PCNA staining contained tissue from time zero control, Hyper and Hypo groups, and the same positive control. Conditions between PCNA runs were kept as similar as possible.
The slides were observed using a DMRB microscope (Leica) and the images captured onto computer. The intensity of PCNA staining in at least three randomly chosen replicate areas at equidistant points around the membrana granulosa in each follicle was determined using NIH Image software. Comparison of the relationship between PCNA staining intensity and manual counts of the proportion of cells staining for PCNA within the same area across a range of follicle sizes showed a highly significant correlation (n = 30; r = 0.95; P < 0.001).
Statistical analysis
Statistical analysis of LH and FSH profile data were performed by repeated-sample ANOVA with log-transformed data being partitioned on the basis of treatment and time (ANOVA). Ovarian estradiol and inhibin secretion data were analyzed by ANOVA with data being partitioned on the basis of treatment and time of collection. Because full quantification of follicle numbers was not carried out, the number of follicles in each class was converted to a percentage of the total number of follicles counted for each animal at each time point and compared by 
2 or Fisher’s exact test as appropriate. For both secretion data and follicle numbers, independence between samples taken from the same individual at 2-month intervals was assumed. The mean PCNA staining intensity of granulosa cells in each follicle were expressed as a percentage relative to the mean value in that follicle class at time zero collection and analyzed by ANOVA, with data being partitioned on the basis of treatment and time of collection. Because these data were found to vary according to treatment but not time of collection, results were pooled across time of collection for presentation. In all cases, two-way ANOVA was followed by Bonferroni post hoc analysis.
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Results |
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). In the implanted Hypo group, FSH and LH concentrations were profoundly suppressed to concentrations of 0.57 ± 0.02 µg/liter and 1.1 ± 0.04 µg/liter for the entire experimental period (P < 0.001; Fig. 1). There was no difference in FSH and LH profiles between the 1/3 and 2/4 groups and removal of one graft at 1 or 2 months in these groups, respectively, had no immediate effect on peripheral LH and FSH concentrations (Fig. 1).
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). In contrast, cortical grafts from the Hypo group remained small and contained no visible antral follicles at any time after grafting (Fig. 2B). This difference in graft development was reflected by endocrine investigations into the level of ovarian hormone production by autografts from the two experimental groups. These results showed a linear increase in inhibin A production, with increasing time after grafting in the Hyper group (P < 0.001), whereas in the Hypo group inhibin A production remained low at each time point (Fig. 3), with the Hypo group being significantly different (P < 0.01) from the Hyper group at 2, 3, and 4 months after grafting. Similarly, estradiol production was absent in the Hypo group at each time point but was observed in the Hyper group 3 and 4 months after autografting, leading to significant differences (P < 0.01) between the experimental groups at these time points (Fig. 3).
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. These data are based on total follicle counts of 4121, 2393, and 2203 for time zero controls, Hyper, and Hypo groups, respectively. The average number of sections per animal required to achieve these counts was 11 ± 1.1 (mean ± SEM) with a range of 4–57. The follicle population before treatment (time zero) exhibited the normal hierarchy with primordial follicles representing 93% of all follicles counted with a progressive decline in the proportion of follicles with increasing stages of development (Fig. 4). In ovarian tissue recovered within 1 month of grafting, no animal in either treatment group had any normal follicles past the tertiary stage of development, and as a result the follicle distributions differed significantly from time zero in both treatment groups (P < 0.01; Fig. 4A). Relative to time zero, there was an increased proportion of primary follicles in both Hyper and Hypo groups 1 month after grafting with this difference being statistically significant (P < 0.05) in the Hyper group. The Hyper group had similar proportions of secondary and tertiary follicles as time zero controls but no preantral or antral follicles. In contrast, whereas the Hypo group at the same time point had a similar proportion of secondary follicles as controls, these animals had no tertiary, preantral, or antral follicles (P < 0.05; Fig. 4A).
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). In contrast, after the same time period the follicle population in the Hypo group differed markedly from controls (P < 0.05) in that these animals had a higher proportion of primary follicles (P < 0.05), a similar proportion of secondary follicles, but no normal tertiary, preantral, or antral follicles. By 3 months after grafting, both groups had a higher proportion of growing follicles than that observed at time zero, although as mentioned above, there were no antral follicles in the Hypo group.
Within 4 months of grafting, these effects were no longer evident, and both experimental treatment groups exhibited a similar follicle hierarchy (Fig. 4D) that closely resembled that observed at time zero, apart from an increase (P < 0.01) in the proportion of antral follicles in the Hyper group (Fig. 4D).
Qualitative effects on the follicle population
The level of gonadotropin stimulation appeared to have a qualitative effect on the early follicle population. Follicles from the Hyper group appeared healthy and contained a large number of proliferating cells, as detected by PCNA expression (Fig. 5, A–C). In contrast, although not showing classical signs of atresia (pyknotic nuclei), multilaminar preantral follicles from the Hypo group often had a vacuolated appearance and low levels of PCNA staining (Fig. 5, D–F).
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). These data show that PCNA staining intensity was increased in the Hyper group in all follicle classes. In contrast, PCNA staining intensity in Hypo animals did not differ from controls in tertiary, preantral, and antral follicles but was elevated (P < 0.05) in primary and secondary follicles levels similar to those observed in the Hyper group in these follicle classes (Fig. 6).
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Discussion |
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The 2- to 3-fold increase in FSH and LH concentrations observed in untreated autografts was consistent with our earlier observations (17, 32), and the use of combined GnRH-agonist and steroid supplementation was highly effective in preventing this increase. Although the fact that both gonadotropins were affected in parallel means that we cannot unequivocally attribute effects of gonadotropin-modulation on preantral follicle development to either FSH or LH alone, a number of lines of evidence support the idea that these affects are more likely to be mediated by FSH. First, as its name implies, FSH has a well-established role in modulating granulosa cell proliferation and differentiation (42) and is known to induce antrum formation (43). Conversely, the main role of LH in nonovulatory follicles is the control of thecal steroidogenic enzyme activity (44). Second, FSH receptors develop during the very earliest stages of follicle development (15), whereas thecal LH receptors are not observed until the preantral stages (15). Third, whereas greatly elevated FSH concentrations are associated with an enhancement of follicle development (39), LH alone does not stimulate antral follicle development and superphysiological LH concentrations are associated with inhibition of antral follicle growth (45). Finally, in vitro studies have shown that FSH alone is capable of stimulating preantral follicle development (7, 30, 31). Thus, it appears likely that the effects of gonadotropin modulation on preantral follicle development observed in the present experiment are mediated by FSH rather than LH, although the possibility of a joint mode of action cannot be disregarded, especially as androgens have been shown to affect preantral follicle development in vitro in cows (Telfer, E. E., D. G. Armstrong, and B. K. Campbell, unpublished observations). Unfortunately, using the current paradigm, it would be difficult to modulate the gonadotropins separately due to the technical complexity and amount of material required to replace either gonadotropin after full suppression for such a protracted period.
Whereas it is clear from the present experiment that modulation of gonadotropins in autografts had no effect on the rate of primordial follicle initiation or the number of primary and secondary follicles that develop, in both groups there was an increase in the number of primary follicles in autografts relative to time zero controls between 1 and 3 months (Fig. 4), suggesting that the rate of recruitment of primordial follicle is increased in these animals. This observation is supported by an apparent increase in the rate of granulosa cell proliferation in primary and secondary follicles in both groups of autografts, relative to time zero controls (Fig. 6). Because the follicular hierarchy has been destroyed in the autografts, this observation supports the hypothesis that the rate of initiation is controlled by factors secreted by more mature growing follicles. A hypothesis first put forward to explain the common observation in many species that the rate of primordial follicle initiation is inversely correlated with the size of the growing pool of follicles (46, 47, 48). This hypothesis has been supported by more recent experiments using this model that focused on the period immediately after grafting to show a marked but transient increase in the rate of initiation of primordial follicle development in ovarian cortical strips as early as 1 wk after transplantation (Baird, D. T., E. E. Telfer, C. Souza, and B. K Campbell, unpublished observations). A similar increase in the rate of initiation is also observed in cortical strips cultured in vitro (11, 49). Collectively, these observations all support the existence of a gonadotropin-independent intraovarian feedback loop regulating both the rate of primordial follicle recruitment and early follicle development. The identity of this putative factor(s) is unknown, but members of the TGFß superfamily such as activin (50) and anti-Mullerian hormone (51) have been suggested as possible candidates.
The major finding from this work was that modulation of gonadotropins altered the pattern of early follicle development in autografts in such a way that a normal follicular hierarchy was reestablished within 2 months in hypergonadotrophic animals, compared with 4 months in hypogonadotrophic animals. This effect was observed primarily by morphometric analysis of the follicle population (Fig. 4) but was supported by qualitative differences in the growing follicle population (Figs. 5 and 6) and functional differences in the synthetic capacity of the growing follicle population of steroid (estradiol) and peptide (inhibin) markers of follicular somatic cell differentiation. As discussed above, the most likely explanation for these observations is that the large elevation in FSH observed in untreated autografts accelerates the rate of early folliculogenesis through stimulation of somatic cell proliferation and differentiation. However, the presence of FSH did not seem to be an absolute determinant of the ability of follicle development to proceed to the antral stage, under the influence of a tightly coordinated cascade of local factors (1).
We have previously reported that 3–6 months is required from autografting to restoration of normal estrous cycles (17) and noted that this figure agreed with histological estimates of the duration of folliculogenesis in sheep, derived from mitotic indices (4, 19). In both this and our previous experiments (17), the FSH concentrations in untreated autografts were 3-fold higher than normal, and in the current experiment significant antral follicle development and ovarian inhibin and estradiol production was observed within 2–3 months of autografting. Our previous estimate, however, was most likely confounded by the fact that the autografts were replaced toward the end of the breeding season so that an earlier reestablishment of the follicular hierarchy may not have been evident. In contrast, the suppression model resulted in FSH concentrations that were 5-fold lower than those observed in these prepubertal animals before treatment, and this profound suppression would be expected to reveal the maximal affect that gonadotropins can exert on preantral follicle development. Under these conditions, antral follicles were observed within 4 months of autografting, but no inhibin or estradiol secretion was evident at this time. It would therefore appear that the duration of folliculogenesis may be under some degree of endocrine control and this observation has important practical relevance (see below). Further experiments using normogonadotrophic autografts are currently being conducted to determine the duration of early folliculogenesis when gonadotropin concentrations are in the normal physiological range. Additional work is also required to examine the effect of these endocrine disturbances on the developmental competence of the oocyte within these follicles.
In the current paper, we used PCNA expression as a marker of functional status rather than morphological appearance, which we feel is more subjective and may not accurately reflect the developmental potential of individual follicles, especially among the preantral follicle population. The advent of techniques such as in situ hybridization and immunohistochemistry has enabled other more objective and quantifiable means to assess the functional status of a follicle. The proliferative marker, PCNA, was chosen for this work because preantral follicle growth occurs largely as a result of the proliferation of the somatic cell component of the ovarian follicle. Our experience with relating the appearance of proliferative, differentiative and apoptotic markers to morphological characteristics has been that only at the very terminal stages of morphological atresia does one find an exclusive relationship between the presence of these markers and morphological atresia and that even morphologically healthy follicles exhibit vastly different patterns of expression (52, 53 , and Campbell, B. K., unpublished observations) and contain apoptotic cells (54). From these types of observations has come the view that each follicle is sitting in a state of equilibrium between survival and death factors (54) and that morphological appearance of the earlier signs of atresia in a follicle may therefore not preclude subsequent normal development after local and/or endocrine environmental change. Using PCNA as a marker, we have been able to show novel and clear effects of peripheral FSH concentrations on early folliculogenesis, which correlate with our observations of the gross appearance of the grafts, morphometric analysis, and the endocrine activity of the follicle population.
The results of this experiment have strategic relevance in terms of the development of culture systems for preantral follicles, the clinical application of the autograft technique to restore fertility, and the design of stimulation regimes for assisted conception. The aim of any culture system for preantral follicles from species such as the human, cow, or sheep, in which it normally takes several months to pass through this stage in vivo, is to accelerate the rate of development without altering the sequence of developmental steps. The results of the present study indicate that the inclusion of FSH at doses up to 5 times the physiological dose in vitro might be beneficial in accelerating the rate of preantral follicle development. In the rodent, FSH has long been included at very high doses (7) to stimulate the development of ovulatory follicles from isolated preantral follicles. Recent advances in culture methodology in the ruminant (30, 55) and human (11) have allowed the culture of multilaminar preantral follicles to the antral stage, but in these culture systems, both physiological and superphysiological doses of FSH have not been shown to have a major effect on the rate of follicle growth in vitro. However, these cultures are normally performed in the presence of doses of insulin and/or IGF-I that could potentially mask the effects of FSH in vitro (40, 56). The observation that the rate of preantral follicle development may be under some degree of endocrine control in vivo has clinical significance. Thus, in situations in which the recruitable cohort of antral follicles for ovarian stimulation is depleted, as in older women, it may be possible to stimulate an increase in the number of these follicles by long-term, low-dose gonadotropin treatment.
In conclusion, the results of this experiment provide strong evidence that, contrary to accepted dogma, gonadotropins can affect the rate of development of preantral follicles in vivo in a large monovulatory species that has a prolonged period of preantral follicle development. Further data are presented to support the existence of a gonadotropin-independent intraovarian feedback loop regulating both the rate of primordial follicle initiation and primary and secondary follicle development. These results highlight the value of the cortical autograft as a large animal model system to study the control of early follicle development in an experimental rather than observational manner in monovulatory species.
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Acknowledgments |
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Footnotes |
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Abbreviation: PCNA, Proliferating cell nuclear antigen.
Received August 29, 2003.
Accepted for publication December 12, 2003.
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
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