Follicular development in transplanted fetal and neonatal mouse ovaries is influenced by the gonadal status of the adult recipient

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1 FERTILITY AND STERILITY VOL. 74, NO. 2, AUGUST 2000 Copyright 2000 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A. Follicular development in transplanted fetal and neonatal mouse ovaries is influenced by the gonadal status of the adult recipient Shae-Lee Cox, Ph.D., a Jillian Shaw, Ph.D., b and Graham Jenkin, Ph.D. a,b Department of Physiology and Monash Institute of Reproduction and Development, Monash University, Clayton, Victoria, Australia Received September 13, 1999; revised and accepted February 16, Supported by the National Health and Medical Research Council, Canberra, Australia; Monash IVF, Melbourne, Australia; Serono, Melbourne, Australia; and Monash University Postgraduate Writing-Up Award, Clayton, Australia. Reprint requests: Shae-Lee Cox, Ph.D., Department of Physiology, Monash University, Wellington Rd, Clayton, 3168, Victoria, Australia (FAX: ; a Department of Physiology, Monash University b Monash Institute of Reproduction and Development, Monash University /00/$20.00 PII S (00)00635-X Objective(s): To investigate the pattern of follicular development in transplanted fetal and neonatal mouse ovaries in the presence or absence of the recipient s own ovaries. Design: Controlled experiment. Setting: Academic research laboratory, Department of Physiology, Monash University, Clayton, Australia. Intervention(s): Mouse ovaries from 16-day-old fetuses, 3-day-old neonates, and 10-day-old neonates were transplanted under the kidney capsule of adult female mice, which either retained their own ovaries in situ or were bilaterally ovariectomized. Main Outcome Measure(s): Histologic analysis. Result(s): By 4 weeks after transplantation, fetal and neonatal ovaries transplanted to ovariectomized recipients displayed a pattern of follicular development similar to that observed in in situ adult mouse ovaries. In contrast, follicular development did not progress beyond the early stage in fetal and 3-day-old ovaries transplanted to recipients that retained their in situ ovaries. However, 10-day-old ovaries transplanted to recipients that retained their in situ ovaries displayed full follicular development and corpora lutea formation by 8 weeks after transplantation. Conclusion(s): Follicular development in transplanted immature ovarian tissue is influenced by the age of the donor ovary and gonadal status of the recipient. (Fertil Steril 2000;74: by American Society for Reproductive Medicine.) (Fertil Steril 2000;74: by American Society for Reproductive Medicine.) Key Words: Ovary, transplantation, follicular development Ovarian transplantation can be used to restore fertility to sterile females in both animals and humans. Despite recent advances in this area, optimal transplantation conditions have not yet been established. The success of ovarian tissue transplantation in the long term will depend on the number of follicles that survive and the ability of these follicles to develop and ovulate. Reanastomosis of whole ovaries to a blood supply reduces the number of follicles that are lost because of ischemia. In contrast, follicle loss in whole ovaries or ovarian tissue pieces grafted without anastomosis to a blood supply can vary from 26 to 50 (1 4). As a consequence, the functional life span of the graft may be considerably reduced in comparison with that of in situ ovaries. Most follicles in adult ovarian tissue that survive transplantation are the primordial and small growing follicles (5, 6). Although Nugent et al. (7) showed that treatment with an antioxidant can increase the number of growing follicles that survive in grafted adult mouse ovarian tissue, the number of primordial follicles that survive remains unaffected. Thus, the use of fetal ovaries collected at a time in gestation when the ovary contains the maximum number of primordial oocytes, may be advantageous as it could allow a greater number of oocytes to survive and develop after transplantation. Previous studies in which fetal mouse ovaries were transplanted into adult recipients have shown that ovulation only occurs in grafts 366

2 placed in bilaterally ovariectomized (BLO) recipients (8, 9). However, it has not yet been determined if the proportion of follicles at each stage of follicular development in these fully developed fetal grafts is normal. These earlier studies from our laboratory (8, 9) also showed that follicular development in fetal ovaries was inhibited in the presence of the recipient s own in situ ovaries. The use of fetal tissue for human clinical transplantation raises a myriad of ethical issues (10). Our previous studies using fetal mouse ovarian tissue have raised an important question: Does the inhibition of follicular development, observed in fetal ovaries transplanted into adult recipients that retain their own ovaries, also occur in ovaries that contain follicles at more advanced stages of follicular development at the time of transfer? The existing literature is ambiguous in this respect (11, 12). Investigation into the types and proportion of follicles present in fetal and neonatal ovarian grafts in the presence or absence of the recipient s own remaining ovaries would be of benefit in situations where the full restoration of ovarian function and continued fertility of the recipient after transplantation is important. Therefore, this study specifically examined the number and type of follicles present in ovarian grafts from immature animals in the presence or absence of the in situ ovaries of the adult recipients. MATERIAL AND METHODS Animals Inbred Balb/c females were used throughout this study. Ovaries were collected from fetuses at day 16 of gestation (n 32) and from pups 3 (n 14) and 10 (n 14) days after birth. Graft recipients were 6 8-week-old virgin females (n 121). All mice were obtained from the Central Animal House at Monash University, Australia, and were housed under a 12-hour light/dark regime at 22 C. Ethical approval for these studies was obtained from the Physiology Animal Ethics Committee of Monash University and conforms with the conditions laid down by the NH&MRC/ CSIRO/AAC Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (1997). Collection of Ovaries for Transplantation Fetuses and neonates were decapitated, and their abdomens were opened to identify the gonads. The ovaries were dissected free and transferred to phosphate-buffered saline (Sigma, St. Louis, MO); they were kept at room temperature until required for transplantation. Control fetal (n 4), 3-day-old (n 6), and 10-day-old (n 5) ovaries were placed into Bouin s solution in preparation for histologic examination. Transplantation and Ovariectomy Procedures Fetal and neonatal ovaries were transplanted underneath the kidney capsule of BLO or intact adult female recipients. TABLE 1 Classification of mouse ovarian follicles. Follicle type Primordial Pre Early Late Description Up to one layer of flattened follicular cells One or more layers of cuboidal follicular cells without an antrum Growing follicle with scattered areas of fluid Mature follicle containing a single continuous antrum Briefly, the recipient was anesthetized with avertin [2.5 solution of tribromoethanol; Aldrich, Milwaukee, WI and tertiary amyl alcohol (Aldrich) diluted in distilled water]; a dorsolateral incision was made into the skin and peritoneum to expose the kidney. A small incision was made into the kidney capsule and one fetal or neonatal ovary was inserted under the capsule. The kidney was returned to its normal anatomical position. If the recipient was to be BLO, both of the in situ ovaries were removed by cautery at the junction of the oviduct and uterus. Six ovaries were randomly chosen for histologic examination to serve as controls (control adult ovaries) for comparison with follicular development in fetal and neonatal grafts. The remaining ovaries were discarded. The incision was closed with 9-mm michel clips (Clay Adams, Sparks, MD). Experimental Design BLO (n 48) and intact (n 47) recipients each had one fetal or neonatal ovary transplanted under the left kidney capsule. BLO and intact recipients of fetal ovaries were killed by cervical dislocation at 2, 4, 6, or 8 weeks after transplantation (6 mice per group). BLO and intact recipients of 3-day-old and 10-day-old ovaries were killed by cervical dislocation at 4 or 8 weeks after transplantation. A subgroup of intact recipients that received fetal (n 6), 3-day-old (n 5), or 10-day-old (n 5) ovaries were BLO at 4 weeks after transplantation to see if full follicular development could be subsequently induced in these grafts. These recipients were killed at 4 weeks after being BLO as this was the time at which full follicular development (defined by the presence of follicles and corpora lutea) was observed in grafts transplanted to BLO recipients. Histologic Examination of Ovaries Ovaries were fixed in Bouin s fixative for 24 hours, embedded in paraffin wax, serial sectioned at 7 m thickness, and stained with hematoxylin and eosin. Follicles were counted and classified in every third section to obtain an estimate of the total number of follicles in each ovary. Follicles were classified according to the number of layers of granulosa cells surrounding each oocyte (Table 1). The nucleolus of the oocyte within the follicle was used as a marker for follicle counting. Follicles were considered FERTILITY & STERILITY 367

3 TABLE 2 Types of oocytes/follicles present in control fetal, 3-dayold, and 10-day-old mouse ovaries at the time of collection. Age of ovary oocytes/follicles per ovary (no. of ovaries) primordial oocyte/follicles pre follicles 16 days, fetal 10,089 1,195 a (4) 100 a 0 a 3 days 2, b (6) 98 b 2 b 10 days 1, b (5) 91 b 9 c Note: Significant difference (P.05) between each donor age group are indicated by superscript a,b,c. atretic whenever two or more pyknotic nuclei were found in a section or when the oocyte showed signs of degeneration such as fragmentation, loss of nuclear membrane, or thinning of the cumulus oophorous. Atretic follicles in which the nucleolus was undetected were counted at the section in which their cross-sectional area was largest, to avoid overestimation of the number of atretic follicles in grafts. Corpora lutea were counted at the section in which their crosssectional area was largest. Statistical Analysis Results are presented as the mean SE. A one-way analysis of variance was used to compare the total number of follicles, the types of follicles and corpora lutea between treatment and control groups within each donor ovary age group. Differences between groups were compared using Tukey-Kramer multiple comparisons test. Treatments were considered significantly different when P.05. RESULTS Control day-16 fetal mouse ovaries contained only primordial oocytes with no attached follicular cells, whereas control 3-day-old and 10-day-old neonatal ovaries contained primordial and pre follicles (Table 2). The number of primordial oocytes in control fetal ovaries was significantly greater than the number of primordial follicles that were found in 3-day-old and 10-day-old ovaries (Table 2). Threeday-old and 10-day-old ovaries contained pre follicles with only one layer of follicular cells. However, 10-day-old ovaries also contained pre follicles at more advanced stages of follicular development (i.e., with more than one layer of follicular cells). Fetal, 3-day-old, and 10-day-old donor ovaries reached full follicular development within 4 weeks of transplantation to BLO recipients, with the proportion of follicles at all stages of development being similar to that observed in the control adult ovaries (Table 3). In contrast, follicular development was suspended at the early stage of development in fetal, 3-day-old and 10-day-old donor ovaries at 4 weeks after transplantation into intact recipients (Table 4). Arrested follicular development was also observed in fetal and 3-day-old donor ovaries at 8 weeks after transplantation into intact recipients. However, 10-day-old donor ovaries exhibited full follicular development, including corpora lutea, at 8 weeks after transplantation to intact recipients (Table 4). Grafts at all donor ages that were transplanted to intact recipients that were subsequently BLO, 4 weeks after transplantation, exhibited full follicular development similar to that observed in the control adult ovaries (Table 4). DISCUSSION For ovarian transplantation to be successful, not only do follicles and oocytes within ovarian grafts need to survive, TABLE 3 Follicle types present in fetal and neonatal ovaries after transplantation to bilaterally ovariectomized recipient mice. Age donor ovary Weeks after grafting follicles per ovary (no. of ovaries) primordial pre early late atretic follicles corpora lutea Fetal 2 1, a (4) 83.0 a 17.0 a,c 0.0 a 0.0 a 0.0 a a 4 2, a (5) 91.0 a 7.0 b 0.8 a 0.2 a 1.0 a a 6 1, a (6) 94.0 a 4.0 b 0.6 a 0.4 a 1.0 a a 8 1, a (4) 98.0 a 1.5 b 0.2 a 0.1 a 0.2 b a 3 days 4 1, a,c (6) 86.0 a 11.0 a 1.0 a 0.0 a 2.0 a a a (4) 73.0 a,c 22.0 a 1.0 a 1.0 a 3.0 a a 10 days 4 1, (2) a (5) 81.0 a,c 16.0 a 0.5 a 0.5 a 2.0 a a Adult control NA 1, (6) Note: Significant difference (P.05) are indicated by different superscripts between treatment groups within each donor age group ( a,b ) and between all groups and the adult control group ( c ). NA not applicable. 368 Cox et al. Transplantation of immature mouse ovaries Vol. 74, No. 2, August 2000

4 TABLE 4 Follicle types present in fetal and neonatal ovaries after transplantation to intact recipient mice. Age of donor ovary Weeks after grafting follicles per ovary (no. of ovaries) primordial pre early late atretic follicles corpora lutea Fetal 2 1, a (4) 83.0 a 17.0 b 0.0 a 0.0 a 0.0 a 0.0 a 4 1, a (5) 88.0 a 9.0 a 1.0 a 0.0 a 2.0 a 0.0 a 6 1, a (3) 97.0 a 0.9 a 0.1 a 0.0 a 2.0 a,b 0.0 a 8 3, a (4) 90.0 a 3.7 a,b 0.3 a 0.0 a 6.0 b 0.0 a BLO at 4 weeks 8 1, a (4) 86.0 a 12.0 a,b 0.7 a 0.5 b 0.8 a b 3 days 4 1, a (4) 83.0 a,b 13.0 a 1.0 a 0.0 a 3.0 a 0.0 a 8 1, a (5) 85.0 a 12.0 a 1.0 a 0.0 a 2.0 a 0.0 a BLO at 4 weeks a (3) 85.0 b 12.0 a 0.8 a 0.2 a 2.0 b b 10 days a (5) 91.0 a 6.8 a 0.2 a 0.0 a 2.0 a 0.0 a 8 1, a (3) 89.0 a 8.0 a,b 1.0 a 0.0 a 2.0 a b BLO at 4 weeks 8 1, a (4) 76.0 a 20.0 b 0.5 a 0.5 b 3.0 a b Note: Significant differences (P.05) between treatment groups within each donor age group are indicated by different superscripts ( a,b ). BLO at 4 weeks intact recipients were bilaterally ovariectomized 4 weeks after grafting. but the follicles must also be able to grow to maturity and the oocytes must be able to be released during ovulation. Reports on the influence of ovariectomy of the recipient on the survival and function of ovarian grafts are conflicting. Most studies involving transplantation of ovarian tissue to BLO recipients have reported subsequent functional graft development (8, 11 13). In contrast, retarded (8, 11) or normal growth (13) of ovarian tissue has been observed after transplantation of ovarian tissue to recipients that retained their in situ ovaries. The exact reasons why this difference exists is unclear, but it is plausible that factors secreted by the intact adult ovaries may directly, or indirectly, arrest development of the transplanted ovary. The results of this present study indicate that the pattern of follicular development in fetal, 3-day-old, and 10-day-old ovaries 4 weeks after transplantation to BLO adult recipients is the same as that observed in fully functional adult ovaries (Table 3). The ability of the fetal graft to display full follicular development after transplantation suggests that they have the potential to restore continued long-term fertility to the recipient. Studies in our laboratory have shown that fresh and frozen-thawed fetal mouse ovaries transplanted to the ovarian bursa of adult recipient mice restore continued fertility for up to 1 year (14). Although hormonal profiles of the mice in this study were not evaluated, Baird et al. (15) have shown that BLO juvenile ewes that received cryopreserved autotransplanted ovaries exhibit elevated FSH and LH levels up to 22 months after grafting. However, the pattern of hormonal changes throughout the estrous cycles of each of the ewes that received ovarian grafts was similar to that observed in normal ovulating ewes, indicating that long-term ovarian function is not adversely altered in the grafted tissue. In contrast to ovaries transplanted to BLO recipients, oocytes/follicles within fetal and 3-day-old ovaries that were transplanted to mice in which both of the recipient s in situ ovaries remained intact failed to progress beyond the early stage. These inhibited grafts were, however, able to reach full follicular development after subsequent removal of the recipient s own ovaries. Although the lack of late (preovulatory) follicles within grafts taken from recipients that retained their in situ ovaries for the duration of the experiment was not statistically significantly different from that observed in grafts taken from recipients that were BLO at 4 weeks after transplantation, it is of biological significance, as oocytes will only be released at ovulation naturally after reaching the late stage of follicular development. These studies indicate that the difference in levels of gonadotropins between BLO and intact animals may be responsible and/or factors secreted by the recipient s in situ ovaries may inhibit development of fetal and 3-day-old donor ovaries. In the mouse, the levels of circulating gonadotropins are known to rise after bilateral ovariectomy (FSH ng/ml, Cox et al. unpublished data) and are significantly higher than those observed in an intact animal of the same age ( ng/ml, Cox et al. unpublished data). We propose that the high FSH levels in BLO recipients may play a role in supporting follicular development in transplanted fetal and early neonatal ovaries. FSH has a number of roles in follicular development. For instance, FSH is thought to be responsible for the recruitment of follicles for maturation and ovulation in the next cycle, as well as antrum formation. Even though recruitment was similar in grafts taken from both BLO and intact animals, other FSH-dependent events such as antrum formation FERTILITY & STERILITY 369

5 were absent in grafts removed from intact recipients but not in BLO recipients, indicating that the levels of gonadotropins may be responsible for this difference. Further evidence that gonadotropins are involved in supporting follicular development in grafted immature ovaries is demonstrated by the ability of exogenous pregnant mare serum gonadotropin (PMSG) and hcg to cause follicular development and ovulation, respectively, in fetal ovaries transplanted to intact recipients (Cox et al., unpublished observations). Lack of gonadotropins may not be solely responsible for the inhibition of follicular development observed in immature ovaries transplanted into intact recipients. There is increasing evidence of interovarian and extraovarian feedback mechanisms existing within the ovary. Dominant follicles can inhibit the growth of other follicles in culture (16). Although the factor(s) responsible for this inhibition has not yet been elucidated, there is increasing evidence from a number of species that this factor(s) is present in the follicular fluid of follicles (17 19). It also appears that this factor(s) acts systemically, because dominant follicles can inhibit the growth of follicles in the contralateral ovary. The purported presence of a systemic factor is supported by the demonstration that, in a variety of species, the removal of one ovary results in an increase in the ovulation rate in the remaining ovary (20 22). It is therefore likely that an inhibitory factor(s) produced by the dominant follicles within the ovaries of intact recipients is responsible for the inhibition of follicle growth beyond the stage in the fetal and 3-day-old donor ovarian grafts. Although follicular development in 3-day-old donor ovaries transplanted into intact recipients is inhibited at 8 weeks after transplantation, a different picture emerges for 10-dayold donor ovaries under the same transplantation conditions. Full follicular development was observed in 10-day-old donor ovaries at 8 weeks after transplantation into intact recipients. Follicular development in these grafts appears to be slower than grafts of the same donor age transplanted into BLO recipients. Thus, the 10-day-old ovary can overcome, or is not subjected to, the inhibitory effects of either the low levels of gonadotropins and/or factors produced by the recipient s in situ intact ovaries. This may result from the follicles within the 10-day-old donor ovary being at a more advanced stage of development than those within the 3-dayold donor ovaries (Table 2). Alternatively, ovarian development may be further advanced in 10-day-old neonates, since Ben-Or (23) has suggested that the interstitial cells in the ovary play a role in follicle development and responsiveness of follicles to gonadotropins. In the general discussion of Ben-Or s paper it is suggested that the interstitial cells of the very young ovaries (3-day-old neonates) are functionally different from those of older ovaries and that these cells produce a substance(s) that inhibits follicle growth. It is clear that further studies are needed to clarify why follicular development in 10-day-old mouse ovaries, but not younger ovaries, reaches the point of ovulation when placed in intact adult recipients. Ovarian banking is of particular value to some cancer patients. Pieces of ovary can be recovered by laparoscopy on short notice, before cancer therapies begin, with the possibility of them being autotransplanted back into the patient after remission, thus restoring fertility. The results of this study have important implications for pediatric cancer patients, who at present have no other options available to them for conserving their fertility. These patients could have their ovarian tissue cryopreserved and returned years later at child-bearing age. Based on the results of this study, it may be important to take into account the age of the patient at the time of ovarian tissue storage and whether any remaining ovarian tissue in the patient is functional at the time of transplantation. In conclusion, this study has shown that fetal and neonatal ovaries display normal ovarian function after transplantation to BLO adult recipients, but not after transplantation to recipients that retain their in situ ovaries. The recipient s in situ ovaries may therefore either directly or indirectly inhibit the development of the transplanted fetal and early neonatal ovaries. However, during neonatal development, the ovary acquires the ability to overcome the inhibitory environment of the intact adult recipient. Thus it appears that, in the mouse, the age of the donor ovary and the gonadal status of the recipient need to be taken into consideration for ensuring full follicular development of transplanted ovarian tissue. Acknowledgment: The authors thank Sue Meier, Ph.D., Dairing Research Corporation, New Zealand, for assistance in preparing the slides for histological examination. References 1. Green SH, Smith AU, Zuckerman S. The numbers of oocytes in ovarian autografts after freezing and thawing. J Endocrin 1956;13: Jones EC, Krohn PL. Orthotopic transplantation in mice. J Endocrin. 1960;20: Newton H, Aubard Y, Sharma V, Rutherford AJ, Gosden RG. The low temperature storage and grafting of human ovarian tissue into SCID mice. Hum Reprod 1996;11(7): Candy CJ, Wood MJ, Whittingham DG. Effects of cryoprotectants on the survival of follicles in frozen mouse ovaries. J Reprod Fert 1997; 110: Candy CJ, Wood MJ, Whittingham DG. Follicular development in cryopreserved marmoset ovarian tissue after transplantation. Hum Reprod 1995;10: Oktay K, Newton H, Mullan J, Gosden RG. Development of human primordial follicles to stages in hpg/scid mice stimulated with follicle stimulating hormone. Hum Reprod 1998;13(5): Nugent D, Newton H, Gallivan L, Gosden R. Protective effect of vitamin E on ischaemic-repurfusion injury in ovarian grafts. J Reprod Fert 1998;114(2): Cox S-L, Shaw JM, Jenkin G. Transplantation of cryopreserved fetal ovarian tissue to adult recipients in mice. J Reprod Fert 1996;107: Cox et al. Transplantation of immature mouse ovaries Vol. 74, No. 2, August 2000

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