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Orthotopic transplantation of LH receptor knockout and wild-type ovaries
Life Sciences 77 (2005) 2656 – 2662 www.elsevier.com/locate/lifescie
Orthotopic transplantation of LH receptor knockout and wild-type ovaries Daksha Chudgar 1, Zhenmin Lei, Ch.V. Rao* Division of Research, Department of Ob, Gyn and Women’s Health, 438 MDR Building, University of Louisville, Health Sciences Center, Louisville, KY 40292, United States Received 15 November 2004; accepted 16 March 2005
Abstract Luteinizing hormone (LH) receptor knockout animals have an ovarian failure due to an arrest in folliculogenesis at the antral stage. As a result, the animals have an infertility phenotype. The present study was undertaken to determine whether this phenotype could be reversed by orthotopic transplantation of wild-type ovaries. The results revealed that transplanting wild-type ovaries into null animals did not result in resumption of estrus cycles. Although the number of different types of follicles increased, none progressed to ovulation. The serum hormone profiles improved, reflecting the ovarian changes. The wild-type animals with null ovaries also failed to cycle and their ovaries and serum hormone levels were more like null animals with their own ovaries. Although the lack of rescue of null ovaries placed into wild-type animals was predicted, the failure of wild-type ovaries placed in null animals was not, which could be due to chronic exposure of transplanted tissue to high circulating LH levels and also possibly due to altered internal milieu in null animals. These findings may have implications for potential future considerations of grafting normal donor ovaries into women who have an ovarian failure resulting from inactivating LH receptor mutations. D 2005 Elsevier Inc. All rights reserved. Keywords: Ovaries; LH receptor knockout; Graffian follicles; Orthotopic ovarian transplantation; Serum hormone levels
* Corresponding author. Tel.: +1 502 852 5688; fax: +1 502 852 0881. E-mail address: [email protected] (C.V. Rao). URL: http://www.louisville.edu/medschool/obgyn/molrepro. 1 Present address: Division of Reproductive Endocrinology, Infertility and Genetics, Medical College of Georgia, Augusta, GA 30912-3305, United States. 0024-3205/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2005.03.024
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Introduction Ovary is a classical target of LH actions (Rao, 1982; Richards et al., 1995). The actions include the stimulation of folliculogenesis, thecal cells to produce increasing amounts of aromatizable androgens, rupture of preovulatory follicles to release oocytes, formation of corpus luteum and stimulation of its secretion of steroid and protein hormones (Rao, 1982; Richards et al., 1995). In the absence of LH actions, ovary fails, which has consequences for other organs because of their dependence on ovarian secretion of hormones (Rao, 2001a; Rao and Lei, 2002). Thus, animals without LH actions will have a phenotype (Rao, 2001a; Rao and Lei, 2002). It is possible to produce these animals by immunoneutralization of the LH, downregulation of its release by treatment with GnRH analogs or hypophysectomy. All these approaches are successful to a variable extent due to completeness of removal of residual LH activity. The contemporary approach to produce these animals will be to knockout either LH receptor gene or LHh subunit gene (Lei et al., 2001; Zhang et al., 2001; Ma et al., 2004). The LH receptor knockout animals have recently been developed by gene targeting in embryonic stem cells (Lei et al., 2001; Zhang et al., 2001). In the model developed in our laboratory, LH receptor gene has been completely silenced (Lei et al., 2001). In the other model, the extracellular receptor domain, which is presumed to be nonfunctional, continues to be transcribed (Zhang et al., 2001). The LH receptor knockout animals have ovarian failure (Lei et al., 2001; Zhang et al., 2001). Thus, their weight dramatically decreased and folliculogenesis was arrested at the antral stage (Lei et al., 2001; Zhang et al., 2001). The quantification revealed that the number of primordial, secondary and antral follicles was lower and atretic follicles was higher as compared with age matched wild-type siblings (Ponnuru et al., 2004). Neither the ovarian changes nor the folliculogenesis could be stimulated by normalizing their serum estradiol and progesterone levels or by treatment with pregnant mare serum gonadotropin (Rao, 2001a; Rao and Lei, 2002). We tested the hypothesis in the present study that the null phenotype could be reversed by orthotopic (placed in the position occupied by host ovary) transplantation of wild-type ovaries and the wild-phenotype could be changed to null by transplanting null ovaries.
Methods Animals LH receptor knockout mice were generated by gene targeting in embryonic stem cells and maintained according to the NIH guidelines for the Care and Use of Laboratory Animals. All the studies have been approved by the Animal Care and Use Committee at our institution. The animals were maintained on 12 h light–dark cycles with food and water provided ad libitum. Adult male and female heterozygous mice (129/SVS) were mated to obtain wild-type (+/+), heterozygous (+/ ) and homozygous ( / ) animals. The zygosity was determined by PCR of tail genomic DNA. Orthotopic ovarian transplantation Ten to 15-day old donor mice were sacrificed to remove both their ovaries. The removal was coordinated with bilateral ovariectomy of 45–65 day old recipients and immediately placing the donor
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ovaries into bursa and covering them with bursa membrane so that they would stay within the cavity. Then the abdominal wall was closed with sutures or staples and the animals were transferred back to the cages for recovery. The recipient animals were observed for cyclicity by vaginal smears, which were obtained by flushing the vaginas twice with saline and smearing the cells on slides. After air drying and heating, the slides were stained with Dip Quick Stain (Jorgensen Labs., Inc., Loveland, CO) and examined under a microscope. At the end of 35–40 days, the recipient animals were sacrificed and the transplanted ovaries were located under a magnifying lens ( 150–300) and removed. The recovery of transplanted ovaries varied from 33% to 50%. The animal groups consisted of transplantation of wild-type ovaries into null and wild-type animals and null ovaries into wild-type animals. At the time of removal of transplanted ovaries, trunk blood was collected and serum was separated for measurement of estradiol, progesterone and LH levels by their corresponding radioimmunoassays. Measurement of serum hormone levels The serum volume obtained from each animal was not adequate for the measurement of all hormones. Therefore, three serum pools each for WT–null (donor–recipient), null–WT and WT–WT animals, were made by combining the samples. The hormone levels were quantified in pools in duplicate by the 3rd generation estradiol RIA kit (Diagnostic Systems Laboratories Inc., Webster, TX), progesterone RIA kit (Diagnostic Products Corp., Los Angeles, CA) and LH RIA kit (Amersham Pharmacia Biotech, Piscataway, NJ). The kit instructions were followed in the measurements. The inter- and intra-assay coefficient of variations were less than 10% in the range of levels found in the samples. The crossreactivity was less than 1% for the other hormones. Processing of ovaries and quantification of follicles Eight pairs each of transplanted ovaries from WT–null, null–WT and WT–WT animals were immediately processed upon removal. They were fixed overnight in 10% formalin and embedded in paraffin. Seven-micrometer thick sections were cut and every 7th section was stained with hematoxylin–eosin. Different types of follicles were counted by the previously described fractionator and nucleator principle (Mayhew, 1991; Flaws et al., 1997). Thus, primordial follicles were identified by an intact oocyte with a visible nucleolus surrounded by a single layer of flattened pregranulosa cells. Primary follicles were identified by an enlarged oocyte with a visible nucleolus surrounded by single layer of cuboidal granulosa cells. Secondary follicles were identified the same as the primary except two or more layers of cuboidal granulosa cells surrounded the oocyte. Follicles with any size antrum were counted as antral follicles. To obtain an estimate of the total number of follicles present in each ovary, numbers counted were multiplied by a factor of 49. All the data were stored and analyzed using a computerized video based interactive Bioquant Image Analysis System. Statistical analysis The data were presented as means and their standard errors. Significant differences were obtained by ANOVA and Duncan’s multiple range tests (Steele, 1960).
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Results Neither null animals with wild-type ovaries nor wild-type animals with null ovaries exhibited estrus cycles. In contrast, 3 of 5 wild-type animals with transplanted wild-type ovaries showed estrous cycles and 2 out of 6 became pregnant. As expected from the lack of cyclicity, none of the null and wild-type animals with wild-type and null ovaries, respectively, became pregnant. Null animals with wild-type ovaries had lower estradiol and progesterone levels and higher LH levels, compared with wild-type animals with transplanted wild-type ovaries (Table 1). However, only the progesterone level difference reached statistical significance. Wild-type animals with null ovaries had significantly lower estradiol and progesterone and higher LH levels (Table 1). Wild-type ovaries transplanted into null animals had a higher number of primordial, primary, secondary and antral follicles and a lower number of naked oocytes as compared with wild-type ovaries transplanted into wild-type animals (Table 2). However, like the serum estradiol and LH levels, these differences did not reach statistical significance. Null ovaries transplanted into wild-type animals had a lower number of primordial and secondary follicles and a higher number of naked oocytes as compared with wild-type ovaries transplanted into wild-type or null animals (Table 2). However, only the differences in primordial follicle and naked oocyte numbers reached statistical significance when compared with wild-type ovaries transplanted into null animals. Only wild-type ovaries transplanted into wild-type animals contained corpora lutea, suggesting that ovulations must have occurred, which is consistent with the cyclicity of animals (Table 2).
Discussion Ovarian transplantation is an old technique that has mainly been used as a research tool as well as for preserving genetic material of endangered animals and safeguarding women’s fertility in cases of premature failure, radiation and chemotherapy of cancer and bone marrow recipient patients (Goding, 1966; Nugent et al., 1997; Guanasena et al., 1998; Meirow et al., 1999; Kim et al., 2001; Wolvekamp et al., 2001). The technique offers a choice of transplantation sites of which subcutaneous is the most convenient and under the kidney capsule offers the best opportunity for rapid revascularization and survival of transplanted tissue (Rumery and Blandau, 1976; Gosden et al., 1994a; Cox et al., 1996). Orthotopic transplantation is necessary, if the fertility testing is required (Jones and Krohn, 1960; Guanasena et al., 1997). Fresh or frozen ovaries can be transplanted with the best results obtained with autotransplants and when the donor ovaries came from fetal or neonatal animals, which are rich in primordial follicles, and when recipients were bilaterally ovariectomized adult animals (Deanesly, 1956;
Table 1 Serum hormone levels Donor–Recipient
Estradiol (pg/ml)
WT–Null Null–WT WT–WT
15.6 F 0.4 11.5 F 0.5T 21.1 F 0.6
T p b 0.05 compared with WT–WT.
Progesterone (ng/ml) 2.8 F 0.8T 2.6 F 0.6T 6.8 F 1.4
LH (ng/ml) 17.9 F 3.0 29.6 F 4.0T 12.6 F 1.3
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Table 2 Follicle count in the transplanted ovaries Donor–recipient WT–Null Null–WT WT–WT
Total number per ovary Primordial
Primary
Secondary
Antral
Naked oocytes
4961 F1005 2527 F 913T 3397 F 259
2067 F 341 1519 F 231 1674 F 403
1681 F 526 849 F 276T 1253 F 362
609 F 288 549 F 151 421 F130
245 F 76 859 F 107T 459 F 141
Corpora lutea Absent Absent Present
T p b 0.05 compared with WT–Null.
Gosden et al., 1994b; Candy et al., 1995; Newton et al., 1996; Baird et al., 1999; Cox et al., 2000; Imthurn et al., 2000; Laschke et al., 2002). We used orthotopic transplantation to test the hypothesis that the null phenotype could be reversed if its ovaries were replaced with wild-type ovaries and wild-phenotype could be reversed to null if its ovaries were replaced with null ovaries. The ovaries were transplanted into bilaterally ovariectomized recipients followed by allowing adequate time for recovery and function of the animals. The results revealed that transplantation of wild-type ovaries into null animals did not result in resumption of estrus cycles. However, the number of different types of follicles increased without their progression to ovulation and the number of naked oocytes decreased as compared with wild-type ovaries placed in wild-type animals. The increased follicular numbers without progression to ovulation could be due to initial stimulation followed by desensitization of transplanted ovaries by the elevated LH and FSH levels in the null animals. The lower serum progesterone levels and the absence of corpora lutea also further substantiate the lack of progression of follicular growth and ovulation. As LH is capable of acting on multiple tissues in the body (Rao, 2001b), the failure of wild-type ovaries in null animals could also be due to alterations in the immune and other systems (Rao and Lei, 2002; Lei and Rao, 2003; Yu et al., 2003, 2004). Wild-type animals with null ovaries also failed, which was expected due to the absence of LH receptors in ovaries. Thus, the animals did not cycle and the number of primordial and secondary follicles was lower and the number of naked oocytes was higher than wild-type ovaries placed into wildtype or null animals. The follicle numbers in nontransplanted null or wild-type ovaries were higher than the corresponding transplanted ovaries (Ponnuru et al., 2004), suggesting that the transplantation procedure itself may have led to follicular loss due to inadequate blood supply during a critical period following grafting. The hormone levels in wild-type animals with null ovaries were similar to null animals with their own ovaries (Rao and Lei, 2002), suggesting the lack of follicular growth and an absence of negative feedback regulation of LH. The hormone profiles in null animals with wild-type ovaries was somewhat improved. However, this improvement was only partial, as their serum estradiol and progesterone levels were still lower and LH levels were higher as compared with wild-type animals with transplanted wildtype ovaries. Not all the wild-type animals with transplanted wild-type ovaries cycled or became pregnant, which probably has to do with an incomplete restoration of disrupted vascular connections within the time frame of the experiments. The previous success rates of fertility among animals which received orthotopic ovarian transplantation varied greatly, which was probably due to the differences in surgical techniques, strain, age of the animals, etc. (Jones and Krohn, 1960; Guanasena et al., 1997; Candy et al., 2000). The lack of pregnancy in WT–null and null–WT animals was due to lack of
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ovulation. In addition, uterus was atrophied and estradiol and progesterone therapy could not completely reverse morphological and gene expression uterine phenotype and the ability of uterus to implant donor blastocysts (Rao and Lei, 2002; Lin et al., 2005). Some of the differences in serum hormone levels and follicle numbers were not dramatic between the transplantation groups, which is expected considering that ovarian and feedback changes will be slow. Perhaps these differences would have magnified had we allowed the animals to live longer. In summary, null phenotype was not reversed by replacement with wild-type ovaries, but as expected, wild-phenotype was reversed to null by replacing with null ovaries. These findings may have implications for potential future considerations of grafting normal donor ovaries into women who have ovarian failure resulting from inactivating LH receptor mutations. Acknowledgement This work was supported by NIH grant, R01 HD040204.
References Baird, D.T., Webb, R., Campbell, B.K., Harkness, L.M., Gosen, R.G., 1999. Long-term ovarian function in sheep after ovariectomy and transplantation of autografts stored at 196 8C. Endocrinology 140, 462 – 471. Candy, C.J., Wood, M.J., Whittingham, D.G., 1995. Follicular development in cryopreserved marmoset ovarian tissue after transplantation. Human Reproduction 10, 2334 – 2338. Candy, C.J., Wood, M.J., Whittingham, D.G., 2000. Restoration of a normal reproductive lifespan after grafting of cryopreserved mouse ovaries. Human Reproduction 15, 1300 – 1304. Cox, S.L., Shaw, J., Jenkin, G., 1996. Transplantation of cryopreserved fetal ovarian tissue to adult recipients in mice. Journal of Reproduction and Fertility 107, 315 – 322. Cox, S.L., Shaw, J., Jenkin, G., 2000. Follicular development in transplanted fetal and neonatal mouse ovaries is influenced by the gonadal status of the adult recipient. Fertility and Sterility 74, 366 – 371. Deanesly, R., 1956. Cyclic function in ovarian grafts. Journal of Endocrinology 13, 211 – 220. Flaws, J.A., Abbud, R., Mann, R.J., Nilson, J.H., Hirshfield, A.N., 1997. Chronically elevated luteinizing hormone depletes primordial follicles in the mouse ovary. Biology of Reproduction 57, 1233 – 1237. Goding, J.R., 1966. Ovarian autotransplantation with vascular anastomoses and its application to the study of reproductive physiology in the ewe. Journal of Physiology 186, 86 – 87. Gosden, R.G., Boultron, M.I., Grant, K., Webb, R., 1994a. Follicular development from ovarian xenografts in SCID mice. Journal of Reproduction and Fertility 101, 619 – 623. Gosden, R.G., Baird, D.T., Wade, J.C., Webb, R., 1994b. Restoration of fertility to oophorectomized sheep by ovarian autografts stored at 196 8C. Human Reproduction 9, 597 – 603. Guanasena, K.T., Villines, P.M., Critser, E.S., Critser, J.K., 1997. Live births after autologous transplant of cryopreserved mouse ovaries. Human Reproduction 12, 101 – 106. Guanasena, K.T., Lakey, J.R., Villines, P.M., Bush, M., Raath, C., Critser, E.S., McGann, L.E., Critser, J.K., 1998. Antral follicles develop in xenografted cryopreserved African elephant (Loxodonta africana) ovarian tissue. Animal Reproduction Science 53, 265 – 275. Imthurn, B., Cox, S.-L., Jenkin, G., Trounson, A.O., Shaw, J.M., 2000. Gonadotropin administration can benefit ovarian tissue grafted to the body wall: implications for human ovarian grafting. Molecular and Cellular Endocrinology 163, 141 – 146. Jones, E.C., Krohn, P.L., 1960. Orthotopic ovarian transplantation in mice. Journal of Endocrinology 20, 135 – 146. Kim, S.S., Battaglia, D.E., Soules, M.R., 2001. The future of human ovarian cryo-preservation and transplantation: fertility and beyond. Fertility and Sterility 75, 1049 – 1056.
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Laschke, M.W., Menger, M.D., Vollmar, B., 2002. Ovariectomy improves neovascularization and microcirculation of freely transplanted ovarian follicles. Journal of Endocrinology 172, 535 – 544. Lei, Z.M., Rao, Ch.V., 2003. Cytokine storm in LH receptor knockout female mice. Journal of the Society for Gynecologic Investigation 10 (Supplement), 205A. (Abstract 346). Lei, Z.M., Mishra, S., Zou, W., Xu, B., Foltz, M., Li, X., Rao, Ch.V., 2001. Targeted disruption of luteinizing hormone/human chorionic gonadotropin receptor gene. Molecular Endocrinology 15, 184 – 200. Lin, D.X., Lei, Z.M., Li, X., Rao, Ch.V., 2005. Targeted disruption of LH receptor gene revealed the importance of uterine LH signaling. Molecular Cellular Endocrinology 234, 105 – 116. Ma, X., Dong, Y., Matzuk, M., Kumar, T.R., 2004. Targeted disruption of luteinizing hormone h-subunit leads to hypogonadism, defects in gonadal steroidogenesis and infertility. Proceedings of the National Academy of Sciences of the United States of America 101, 17294 – 17299. Mayhew, T.M., 1991. The new stereological methods for interpreting functional morphology from slices of cells and organs. Experimental Physiology 76, 639 – 665. Meirow, D., Fasouliotis, S.J., Nugent, D., Schenker, J.G., Gosden, R.G., Rutherford, A.J., 1999. A laparoscopic technique for obtaining ovarian cortical biopsy specimens for fertility conservation in patients with cancer. Fertility and Sterility 71, 948 – 951. Newton, H., Aubard, Y., Rutherford, A., Sharma, V., Gosden, R., 1996. Low temperature storage and grafting of human ovarian tissue. Human Reproduction 11, 1487 – 1491. Nugent, D., Meirow, D., Brook, P.F., Aubard, Y., Gosden, R.G., 1997. Transplantation in reproductive medicine — previous experience, present knowledge and future prospects. Human Reproduction Update 3, 267 – 280. Ponnuru, P., Lei, Z.M., Rao, Ch.V., 2004. Ovarian aging as a function of LH receptor gene dose. Journal of the Society for Gynecologic Investigation 11 (Supplement), 131A. (Abstract #180). Rao, Ch.V., 1982. Receptors for gonadotropins in human ovaries. In: Muldoon, T.G., Mahesh, V.B., Perez-Ballester, B. (Eds.), Recent Advances in Fertility Research. Developments in Reproductive Endocrinology Part A. Liss, New York, pp. 123 – 135. Rao, Ch.V., 2001a. Multiple novel roles of luteinizing hormone. Fertility and Sterility 76, 1097 – 1100. Rao, Ch.V., 2001b. An overview of the past, present and future of nongonadal LH/hCG actions in reproductive biology and medicine. Seminars in Reproductive Medicine 19, 7 – 17. Rao, Ch.V., Lei, Z.M., 2002. Consequences of targeted inactivation of LH receptors. Molecular and Cellular Endocrinology 187, 57 – 67. Richards, J., Fitzpatrick, S., Clemens, J., Morris, J., Alliston, T., Sirois, J., 1995. Ovarian cell differentiation: a cascade of multiple hormones, cellular signals and regulated genes. Recent Progress in Hormone Research 50, 223 – 254. Rumery, W.L., Blandau, R.J., 1976. The response of transplants of cultured fetal mouse ovaries in kidneys of ovariectomized adult mice. Journal of Morphology 149, 421 – 436. Steele, R.G.D., 1960. Principles and Procedures of Statistics, with Special Reference to the Biological Sciences. McGraw-Hill, New York. Wolvekamp, M.C., Cleary, M.L., Cox, S.L., Shaw, J.M., Jenkin, G., Trounson, A.O., 2001. Follicular development in cryopreserved common wombat ovarian tissue xenografted to nude rats. Animal Reproduction Science 65, 135 – 147. Yu, Y., Lei, Z.M., Rao, Ch.V., 2003. Adrenals phenotype in LH receptor knockout animals. Biology of Reproduction 68 (Supplement 1), 223. (Abstract #270). Yu, Y., Lei, Z.M., Rao, Ch.V., 2004. The development of obesity in LH receptor knockout females. Journal of the Society for Gynecologic Investigation 11 (Supplement), 132A. (Abstract #182). Zhang, F.P., Poutanen, M., Wilbertz, J., Huhtaniemi, I., 2001. Normal prenatal but arrested postnatal sexual development of luteinizing hormone receptor knockout (LuRKO) mice. Molecular Endocrinology 15, 172 – 183.
Orthotopic transplantation of LH receptor knockout and wild-type ovaries Daksha Chudgar 1, Zhenmin Lei, Ch.V. Rao* Division of Research, Department of Ob, Gyn and Women’s Health, 438 MDR Building, University of Louisville, Health Sciences Center, Louisville, KY 40292, United States Received 15 November 2004; accepted 16 March 2005
Abstract Luteinizing hormone (LH) receptor knockout animals have an ovarian failure due to an arrest in folliculogenesis at the antral stage. As a result, the animals have an infertility phenotype. The present study was undertaken to determine whether this phenotype could be reversed by orthotopic transplantation of wild-type ovaries. The results revealed that transplanting wild-type ovaries into null animals did not result in resumption of estrus cycles. Although the number of different types of follicles increased, none progressed to ovulation. The serum hormone profiles improved, reflecting the ovarian changes. The wild-type animals with null ovaries also failed to cycle and their ovaries and serum hormone levels were more like null animals with their own ovaries. Although the lack of rescue of null ovaries placed into wild-type animals was predicted, the failure of wild-type ovaries placed in null animals was not, which could be due to chronic exposure of transplanted tissue to high circulating LH levels and also possibly due to altered internal milieu in null animals. These findings may have implications for potential future considerations of grafting normal donor ovaries into women who have an ovarian failure resulting from inactivating LH receptor mutations. D 2005 Elsevier Inc. All rights reserved. Keywords: Ovaries; LH receptor knockout; Graffian follicles; Orthotopic ovarian transplantation; Serum hormone levels
* Corresponding author. Tel.: +1 502 852 5688; fax: +1 502 852 0881. E-mail address: [email protected] (C.V. Rao). URL: http://www.louisville.edu/medschool/obgyn/molrepro. 1 Present address: Division of Reproductive Endocrinology, Infertility and Genetics, Medical College of Georgia, Augusta, GA 30912-3305, United States. 0024-3205/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2005.03.024
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Introduction Ovary is a classical target of LH actions (Rao, 1982; Richards et al., 1995). The actions include the stimulation of folliculogenesis, thecal cells to produce increasing amounts of aromatizable androgens, rupture of preovulatory follicles to release oocytes, formation of corpus luteum and stimulation of its secretion of steroid and protein hormones (Rao, 1982; Richards et al., 1995). In the absence of LH actions, ovary fails, which has consequences for other organs because of their dependence on ovarian secretion of hormones (Rao, 2001a; Rao and Lei, 2002). Thus, animals without LH actions will have a phenotype (Rao, 2001a; Rao and Lei, 2002). It is possible to produce these animals by immunoneutralization of the LH, downregulation of its release by treatment with GnRH analogs or hypophysectomy. All these approaches are successful to a variable extent due to completeness of removal of residual LH activity. The contemporary approach to produce these animals will be to knockout either LH receptor gene or LHh subunit gene (Lei et al., 2001; Zhang et al., 2001; Ma et al., 2004). The LH receptor knockout animals have recently been developed by gene targeting in embryonic stem cells (Lei et al., 2001; Zhang et al., 2001). In the model developed in our laboratory, LH receptor gene has been completely silenced (Lei et al., 2001). In the other model, the extracellular receptor domain, which is presumed to be nonfunctional, continues to be transcribed (Zhang et al., 2001). The LH receptor knockout animals have ovarian failure (Lei et al., 2001; Zhang et al., 2001). Thus, their weight dramatically decreased and folliculogenesis was arrested at the antral stage (Lei et al., 2001; Zhang et al., 2001). The quantification revealed that the number of primordial, secondary and antral follicles was lower and atretic follicles was higher as compared with age matched wild-type siblings (Ponnuru et al., 2004). Neither the ovarian changes nor the folliculogenesis could be stimulated by normalizing their serum estradiol and progesterone levels or by treatment with pregnant mare serum gonadotropin (Rao, 2001a; Rao and Lei, 2002). We tested the hypothesis in the present study that the null phenotype could be reversed by orthotopic (placed in the position occupied by host ovary) transplantation of wild-type ovaries and the wild-phenotype could be changed to null by transplanting null ovaries.
Methods Animals LH receptor knockout mice were generated by gene targeting in embryonic stem cells and maintained according to the NIH guidelines for the Care and Use of Laboratory Animals. All the studies have been approved by the Animal Care and Use Committee at our institution. The animals were maintained on 12 h light–dark cycles with food and water provided ad libitum. Adult male and female heterozygous mice (129/SVS) were mated to obtain wild-type (+/+), heterozygous (+/ ) and homozygous ( / ) animals. The zygosity was determined by PCR of tail genomic DNA. Orthotopic ovarian transplantation Ten to 15-day old donor mice were sacrificed to remove both their ovaries. The removal was coordinated with bilateral ovariectomy of 45–65 day old recipients and immediately placing the donor
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ovaries into bursa and covering them with bursa membrane so that they would stay within the cavity. Then the abdominal wall was closed with sutures or staples and the animals were transferred back to the cages for recovery. The recipient animals were observed for cyclicity by vaginal smears, which were obtained by flushing the vaginas twice with saline and smearing the cells on slides. After air drying and heating, the slides were stained with Dip Quick Stain (Jorgensen Labs., Inc., Loveland, CO) and examined under a microscope. At the end of 35–40 days, the recipient animals were sacrificed and the transplanted ovaries were located under a magnifying lens ( 150–300) and removed. The recovery of transplanted ovaries varied from 33% to 50%. The animal groups consisted of transplantation of wild-type ovaries into null and wild-type animals and null ovaries into wild-type animals. At the time of removal of transplanted ovaries, trunk blood was collected and serum was separated for measurement of estradiol, progesterone and LH levels by their corresponding radioimmunoassays. Measurement of serum hormone levels The serum volume obtained from each animal was not adequate for the measurement of all hormones. Therefore, three serum pools each for WT–null (donor–recipient), null–WT and WT–WT animals, were made by combining the samples. The hormone levels were quantified in pools in duplicate by the 3rd generation estradiol RIA kit (Diagnostic Systems Laboratories Inc., Webster, TX), progesterone RIA kit (Diagnostic Products Corp., Los Angeles, CA) and LH RIA kit (Amersham Pharmacia Biotech, Piscataway, NJ). The kit instructions were followed in the measurements. The inter- and intra-assay coefficient of variations were less than 10% in the range of levels found in the samples. The crossreactivity was less than 1% for the other hormones. Processing of ovaries and quantification of follicles Eight pairs each of transplanted ovaries from WT–null, null–WT and WT–WT animals were immediately processed upon removal. They were fixed overnight in 10% formalin and embedded in paraffin. Seven-micrometer thick sections were cut and every 7th section was stained with hematoxylin–eosin. Different types of follicles were counted by the previously described fractionator and nucleator principle (Mayhew, 1991; Flaws et al., 1997). Thus, primordial follicles were identified by an intact oocyte with a visible nucleolus surrounded by a single layer of flattened pregranulosa cells. Primary follicles were identified by an enlarged oocyte with a visible nucleolus surrounded by single layer of cuboidal granulosa cells. Secondary follicles were identified the same as the primary except two or more layers of cuboidal granulosa cells surrounded the oocyte. Follicles with any size antrum were counted as antral follicles. To obtain an estimate of the total number of follicles present in each ovary, numbers counted were multiplied by a factor of 49. All the data were stored and analyzed using a computerized video based interactive Bioquant Image Analysis System. Statistical analysis The data were presented as means and their standard errors. Significant differences were obtained by ANOVA and Duncan’s multiple range tests (Steele, 1960).
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Results Neither null animals with wild-type ovaries nor wild-type animals with null ovaries exhibited estrus cycles. In contrast, 3 of 5 wild-type animals with transplanted wild-type ovaries showed estrous cycles and 2 out of 6 became pregnant. As expected from the lack of cyclicity, none of the null and wild-type animals with wild-type and null ovaries, respectively, became pregnant. Null animals with wild-type ovaries had lower estradiol and progesterone levels and higher LH levels, compared with wild-type animals with transplanted wild-type ovaries (Table 1). However, only the progesterone level difference reached statistical significance. Wild-type animals with null ovaries had significantly lower estradiol and progesterone and higher LH levels (Table 1). Wild-type ovaries transplanted into null animals had a higher number of primordial, primary, secondary and antral follicles and a lower number of naked oocytes as compared with wild-type ovaries transplanted into wild-type animals (Table 2). However, like the serum estradiol and LH levels, these differences did not reach statistical significance. Null ovaries transplanted into wild-type animals had a lower number of primordial and secondary follicles and a higher number of naked oocytes as compared with wild-type ovaries transplanted into wild-type or null animals (Table 2). However, only the differences in primordial follicle and naked oocyte numbers reached statistical significance when compared with wild-type ovaries transplanted into null animals. Only wild-type ovaries transplanted into wild-type animals contained corpora lutea, suggesting that ovulations must have occurred, which is consistent with the cyclicity of animals (Table 2).
Discussion Ovarian transplantation is an old technique that has mainly been used as a research tool as well as for preserving genetic material of endangered animals and safeguarding women’s fertility in cases of premature failure, radiation and chemotherapy of cancer and bone marrow recipient patients (Goding, 1966; Nugent et al., 1997; Guanasena et al., 1998; Meirow et al., 1999; Kim et al., 2001; Wolvekamp et al., 2001). The technique offers a choice of transplantation sites of which subcutaneous is the most convenient and under the kidney capsule offers the best opportunity for rapid revascularization and survival of transplanted tissue (Rumery and Blandau, 1976; Gosden et al., 1994a; Cox et al., 1996). Orthotopic transplantation is necessary, if the fertility testing is required (Jones and Krohn, 1960; Guanasena et al., 1997). Fresh or frozen ovaries can be transplanted with the best results obtained with autotransplants and when the donor ovaries came from fetal or neonatal animals, which are rich in primordial follicles, and when recipients were bilaterally ovariectomized adult animals (Deanesly, 1956;
Table 1 Serum hormone levels Donor–Recipient
Estradiol (pg/ml)
WT–Null Null–WT WT–WT
15.6 F 0.4 11.5 F 0.5T 21.1 F 0.6
T p b 0.05 compared with WT–WT.
Progesterone (ng/ml) 2.8 F 0.8T 2.6 F 0.6T 6.8 F 1.4
LH (ng/ml) 17.9 F 3.0 29.6 F 4.0T 12.6 F 1.3
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Table 2 Follicle count in the transplanted ovaries Donor–recipient WT–Null Null–WT WT–WT
Total number per ovary Primordial
Primary
Secondary
Antral
Naked oocytes
4961 F1005 2527 F 913T 3397 F 259
2067 F 341 1519 F 231 1674 F 403
1681 F 526 849 F 276T 1253 F 362
609 F 288 549 F 151 421 F130
245 F 76 859 F 107T 459 F 141
Corpora lutea Absent Absent Present
T p b 0.05 compared with WT–Null.
Gosden et al., 1994b; Candy et al., 1995; Newton et al., 1996; Baird et al., 1999; Cox et al., 2000; Imthurn et al., 2000; Laschke et al., 2002). We used orthotopic transplantation to test the hypothesis that the null phenotype could be reversed if its ovaries were replaced with wild-type ovaries and wild-phenotype could be reversed to null if its ovaries were replaced with null ovaries. The ovaries were transplanted into bilaterally ovariectomized recipients followed by allowing adequate time for recovery and function of the animals. The results revealed that transplantation of wild-type ovaries into null animals did not result in resumption of estrus cycles. However, the number of different types of follicles increased without their progression to ovulation and the number of naked oocytes decreased as compared with wild-type ovaries placed in wild-type animals. The increased follicular numbers without progression to ovulation could be due to initial stimulation followed by desensitization of transplanted ovaries by the elevated LH and FSH levels in the null animals. The lower serum progesterone levels and the absence of corpora lutea also further substantiate the lack of progression of follicular growth and ovulation. As LH is capable of acting on multiple tissues in the body (Rao, 2001b), the failure of wild-type ovaries in null animals could also be due to alterations in the immune and other systems (Rao and Lei, 2002; Lei and Rao, 2003; Yu et al., 2003, 2004). Wild-type animals with null ovaries also failed, which was expected due to the absence of LH receptors in ovaries. Thus, the animals did not cycle and the number of primordial and secondary follicles was lower and the number of naked oocytes was higher than wild-type ovaries placed into wildtype or null animals. The follicle numbers in nontransplanted null or wild-type ovaries were higher than the corresponding transplanted ovaries (Ponnuru et al., 2004), suggesting that the transplantation procedure itself may have led to follicular loss due to inadequate blood supply during a critical period following grafting. The hormone levels in wild-type animals with null ovaries were similar to null animals with their own ovaries (Rao and Lei, 2002), suggesting the lack of follicular growth and an absence of negative feedback regulation of LH. The hormone profiles in null animals with wild-type ovaries was somewhat improved. However, this improvement was only partial, as their serum estradiol and progesterone levels were still lower and LH levels were higher as compared with wild-type animals with transplanted wildtype ovaries. Not all the wild-type animals with transplanted wild-type ovaries cycled or became pregnant, which probably has to do with an incomplete restoration of disrupted vascular connections within the time frame of the experiments. The previous success rates of fertility among animals which received orthotopic ovarian transplantation varied greatly, which was probably due to the differences in surgical techniques, strain, age of the animals, etc. (Jones and Krohn, 1960; Guanasena et al., 1997; Candy et al., 2000). The lack of pregnancy in WT–null and null–WT animals was due to lack of
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ovulation. In addition, uterus was atrophied and estradiol and progesterone therapy could not completely reverse morphological and gene expression uterine phenotype and the ability of uterus to implant donor blastocysts (Rao and Lei, 2002; Lin et al., 2005). Some of the differences in serum hormone levels and follicle numbers were not dramatic between the transplantation groups, which is expected considering that ovarian and feedback changes will be slow. Perhaps these differences would have magnified had we allowed the animals to live longer. In summary, null phenotype was not reversed by replacement with wild-type ovaries, but as expected, wild-phenotype was reversed to null by replacing with null ovaries. These findings may have implications for potential future considerations of grafting normal donor ovaries into women who have ovarian failure resulting from inactivating LH receptor mutations. Acknowledgement This work was supported by NIH grant, R01 HD040204.
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