Impaired Regulation of Gonadotropins Leads to the Atrophy of the Female Reproductive System in klotho-Deficient Mice
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《内分泌学杂志》
Department of Pathology and Tumor Biology (R.T., T.F., Y.N., Y.T., Y.-I.N.), Graduate School of Medicine, Kyoto University, Yoshida Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan
Core Research for Evolutional Science and Technology (T.F., Y.N., Y.-I.N.), Japan Science and Technology Corporation, Tokyo 170-0013, Japan
Teaching and Research Support Center (Y.I.), Department of Pathology (R.Y.O.), School of Medicine, Tokai University, Boseidai, Ishihara-city, Kanagawa 259-1193, Japan
Abstract
klotho-Deficient mice exhibit a syndrome resembling human premature ageing, with multiple pathological phenotypes in tissues including reproductive organs. It was proposed that Klotho might possess the hormonal effects on many organs. In this study, the female reproductive system of klotho mice was examined to reveal the mechanism that brought the female sterility by histological and molecular approaches. We observed cessation of ovarian follicular maturation at the preantral stage and the presence of numerous atretic ovarian follicles and atrophic uteri. In situ hybridization analysis revealed that LH receptor and aromatase P450 were not expressed in the ovaries. These results suggest the impairment of gonadal development during the antral transition process. We next addressed the responsible organs for the failure of antral transition. Transplantation of klotho ovaries to wild-type mice resulted in the ability to bear offspring. Administration of FSH or GnRH induced advanced maturation of ovaries and uteri in klotho mice. These results indicate that the female reproductive organs in klotho mice are potentially functional and that klotho gene deficiency leads to the atrophy of reproductive organs via impairment of the hypothalamic-pituitary axis. Absence of the estrus cycle and constant low trends of both FSH and LH levels were found in female klotho mice. Immunohistochemical analysis revealed that the production of both FSH and LH were decreased in pituitary gland. Taken together, our findings suggest the involvement of klotho in the regulatory control of pituitary hormones.
Introduction
WE ESTABLISHED A mouse mutant exhibiting a syndrome resembling human ageing that includes short lifespan, atrophy of outer genital organs, impaired maturation of gonadal cells, sterility of both sexes, osteopenia, arteriosclerosis, skin atrophy, growth retardation, ectopic calcification, impaired glucose metabolism, atrophy of thymus, and pulmonary emphysema (1). We also identified the responsible gene, klotho, which is expressed mainly in the kidney and choroid plexus and weakly in some other organs including the pituitary gland and ovary (1, 2). The klotho gene encodes a membrane protein containing the KL domains that show homology to -glucosidase, which is secreted after the cleavage in the extracellular domain (1, 3, 4). The secreted Klotho protein was found in sera and cerebrospinal fluid (5). Atrophic tissues in klotho mouse are different from those that express klotho, suggesting that Klotho or other soluble factor(s) might exert hormonal effects. So far, a function for secreted Klotho protein has been implicated by the partial rescue of some organs by ectopic expression of the membrane form of Klotho (6, 7). Alternatively, the possibility exists that pituitary hormones might mediate Klotho activity (1). Ovarian follicular development is mainly regulated by pituitary gonadotropins, FSH and LH (8, 9). Both FSH and LH are synthesized in gonadotrophs of the anterior pituitary, stored in secreting granules (10), and secreted in response to GnRH signals from the hypothalamus. Ovarian follicular growth proceeds to the preantral stage independently of gonadotropin regulation (11, 12). Further development requires the FSH signal through the FSH receptor, exclusively expressed in the granulosa cells (13). FSH signal induces granulosa cell proliferation and expression of several genes including LH receptor (LHR) and aromatase P450 (P450arom) (14). P450arom is encoded by the cyp19 gene and catalyzes the final step in the biosynthesis of estrogens from androgens (15). Estrogens, produced by the collaborative actions of granulosa and theca cells (8), possess many functions affecting the reproductive system (16), bone and mineral metabolism (17, 18), or others.
In this study, we examined the female genital organs of klotho mice and attempted to determine how Klotho exerts its function. klotho mice exhibited hypogonadotropic hypogonadism (19), probably due to the lack of GnRH stimulation. These results indicate that klotho deficiency affects the regulation of the endocrine system by the hypothalamic-pituitary axis.
Materials and Methods
Animals
Wild-type (+/+) and homozygous klotho mice (kl/kl) were obtained by mating heterozygous mice (kl/+) carrying the insertional mutation in the klotho gene locus (TA20; C3H/J and C57BL/6J mixed background) (1). All animals were maintained and used according to accepted standards of human animal care and use under specific pathogen-free conditions at Kyoto University (Kyoto, Japan).
Histology
Ovaries and uteri were dissected out, fixed with 4% paraformaldehyde in PBS for 16 h, dehydrated through an ethanol series (70, 95, 100%), penetrated with chloroform, embedded in paraffin, and microtome-sectioned to a thickness of 6 μm. Specimens were dewaxed through xylene, 100, 90, 70, and 50% ethanol, and processed for staining with hematoxylin and eosin or for in situ hybridization analysis.
Fragments of pituitary were fixed by immersion with 1% glutaraldehyde in phosphate buffer (pH 7.4) for 2 h at room temperature, dehydrated in graded methanol at –30 C, and embedded in Lowicryl K4M at –20 C. It took 24 h at –20 C and a subsequent 48 h at room temperature for the polymerization of Lowicryl K4M using UV rays. Immunoelectron microscopic staining was done on ultrathin sections using the protein A gold method after applying primary antibodies. Immunoreacted ultrathin sections were examined with a JEOL 1200EX electron microscope (20).
Ovary transplantation
Ovary transplantation was performed as described previously (21, 22, 23), using 4-wk-old klotho mice as donors and 6-wk-old severe combined immunodeficiency disease (SCID) mice as recipients to avoid immunological rejection of tissues. The ovaries of anesthetized klotho mice were harvested and kept in prewarmed culture medium. Recipient SCID female mice were anesthetized with Avertin. A dorsolateral incision about 1.0 cm long was made in the lumber region on the either side of the midline. Through the incision, the gonadal tissues were gently pulled outside the body. The fat pad of the tissue was fastened to a cotton gauze by forceps to operate easily. The ovarian bursa was cut to make a small incision at the opposite side of the oviduct. Then, the ovary was removed by cutting the ovarian stalk with scissors. After bleeding stopped, donor ovaries from each klotho mouse were transplanted into the empty bursas of each recipient SCID mouse. Then, the bursas were placed back and incisions closed using wound clips. Two weeks after operation, treated female SCID mice were mated with 8-wk-old BALB/c male mice. Offspring born to recipient mice were examined for coat color and confirmed by genotyping PCR.
Gonadotropin treatment
To induce ovulation, 5-wk-old mice were injected intraperitoneally with 5 IU pregnant mare’s serum gonadotropin (PMSG) (Serotropin, Teikoku Hormone Mfg. Co., Ltd., Tokyo, Japan). Forty-eight hours after treatment, 5 IU human chorionic gonadotropin (hCG) (Sankyo Co., Ltd., Tokyo, Japan) was administered. Ovarian tissues were collected 48 h after PMSG treatment or 24 h after hCG treatment.
FSH and GnRH treatments
Female klotho mice at 4–5 wk of age were injected sc with 4 μg ovine FSH (Biogenesis, Ltd., Poole, UK), 20% polyvinyl pyrrolidone (Nacalai Tesque, Inc., Kyoto, Japan), 0.9% NaCl (24, 25), or 300 ng GnRH (Bachem, Bubendorf, Switzerland), 20% polyvinyl pyrrolidone, or 0.9% NaCl twice a day for 3 d. For long-term experiments, 50 ng GnRH in 0.9% NaCl was administered sc to female klotho mice at 4–5 wk of age every 2 h for 5 d (26), and then ovaries and uteri were dissected out and used for histological analysis and in situ hybridization analysis.
In situ hybridization
In situ hybridization was performed as described (27). Processed slides were counterstained with nuclear fast red. RNA probes used were designed basically as previously described (28). Sense and antisense probes for FSHR were designed from nucleotides 367-1266 of GenBank clone AF095642, LHR probes were designed for 592-1331 of M81310, and P450arom probes were designed for 42–659 of D00659. These regions were subcloned into pBluescript (Stratagene, La Jolla, CA) vectors and used to synthesize RNA probes with DIG RNA Labeling kit (Roche Molecular Biochemicals Corp., Mannheim, Germany) according to the manufacturer’s protocol.
Measurement of serum levels of gonadotropins and steroids by RIA
Aliquots of blood were collected from 7- to 8-wk-old female wild-type and klotho mice. Sera were prepared at 4 C and kept at –80 C until the assay. Measurements of FSH and LH levels in sera were performed by SRL, Inc. (Tokyo, Japan) using the rat FSH [125I] and LH [125I] assay systems (BIOTRAK, Amersham Pharmacia biotech UK, Ltd., Buckinghamshire, UK), according to the manufacturer’s instruction. Measurements of estradiol and progesterone levels in sera were performed by SRL, Inc. using the DPC estradiol kit and DPC progesterone (Diagnostic Products Corp., Los Angeles, CA), respectively, according to the manufacturer’s instruction. The inter- and intraassay coefficients of variation were within 5–12%. Limits of detection for the hormone assay were 0.09 ng/tube (FSH), 0.08 ng/tube (LH), 12.2 pg/ml (estradiol), and 0.06 ng/ml (progesterone). Sera (200–500 μl) were used for measurements.
Statistical analysis
FSH, LH, estradiol, and progesterone concentrations in sera of wild-type and klotho mice were compared by F test for variance and Wilcoxon-Mann-Whitney test for median. Statview software (SAS Institute, Inc., Cary, NC) was used for the analysis. Differences between the two groups were considered statistically significant at P < 0.05.
Results
To investigate the female reproductive system of klotho mice, ovaries and uteri were examined histologically. Marked differences were not found between 12-d-old klotho mice and wild-type littermates (Fig. 1), except that granulosa cells appeared slightly more layered in ovarian follicles of klotho mice than those of wild-type mice. At 8 wk old, apparent differences were observed. Although ovaries of wild-type mice contained corpora lutea and follicles at every stage of development including preovulatory follicles (Fig. 2, A and B), ovaries of klotho homozygous littermates did not contain any follicles beyond the preantral stage or corpora lutea (Fig. 2, D and E). Instead, many atretic follicles were observed (Fig. 2E, arrowheads). More than 50 ovaries of randomly selected klotho mice were examined, and this was the case in all of them. The uteri recovered from 8-wk-old klotho mice were lean and atrophic, whereas those of wild-type mice were healthy (Fig. 2, C and F). These differences emerged around 3 wk of age and became apparent at 7 wk of age (data not shown).
Next, we examined the expression of genes at the specific stage of follicular maturation by in situ hybridization. At the age of 8 wk, FSHR was sufficiently expressed in some follicles of both wild-type and klotho mice (Fig. 3, A and D). P450arom (cyp19) was expressed in antral follicles of wild-type mice (Fig. 3B, arrows) but could not be detected in klotho mouse ovaries (Fig. 3E). LHR was abundantly expressed in mural granulosa cells of preovulatory follicles, luteal cells, and theca cells in ovaries of 8-wk-old wild-type mice (Fig. 3C), whereas it was not detectable in ovaries of 8-wk-old klotho mice, even in the theca cells (Fig. 3F).
Because the vaginal opening was too narrow to recover the vaginal smear and amount of serum volume was too small, it was difficult to determine the stage of estrus cycle in individual klotho mice. Accordingly, we selected mice randomly to collect serum samples and measured the concentration of hormones. The serum FSH levels of 7- to 8-wk-old female wild-type (+/+) and klotho (kl/kl) mice were 8.3 ± 6.0 ng/ml (mean ± SD, n = 30) and 5.2 ± 1.4 ng/ml (n = 8), respectively (Fig. 4A). The serum LH levels of 7- to 8-wk-old female wild-type and klotho mice were 2.3 ± 1.8 ng/ml (n = 26) and 1.8 ± 0.2 ng/ml (n = 5), respectively (Fig. 4B). The serum estradiol levels of 7- to 8-wk-old female wild-type and klotho mice were 25.4 ± 26.8 pg/ml (n = 6) and 20.7 ± 12.1 pg/ml (n = 8), respectively (Fig. 4C). The serum progesterone levels of 7- to 8-wk-old female wild-type and klotho mice were 3.5 ± 2.8 pg/ml (n = 6) and 16.9 ± 5.9 pg/ml (n = 8), respectively (Fig. 4D). Serum concentrations of both gonadotropins, FSH and LH, showed significantly smaller variance in klotho mice than in wild-type mice (P < 0.001). This might reflect the nonexistence of the estrus cycle in female klotho mice. Significant differences in the median were obtained only for progesterone (P < 0.01) but not for FSH (P = 0.053), LH (P = 0.957), and estradiol (P = 0.082). It was reported that low levels of klotho gene expression were detected in ovary (1). To examine whether the marked gonadal abnormalities in klotho mice are primarily due to the defects in ovaries, we carried out two types of experiments. First, we examined the effect of ovary transplantation. Ovaries of 4-wk-old klotho homozygotes (kl/kl) (coat color is agouti) were transplanted into ovarian bursas of 6-wk-old wild-type SCID (albino) females, and recipient mice were subsequently bred to wild-type BALB/c (albino) males. All six recipient females became pregnant. A total of 34 pups (5.7 pups/litter) were born, 26 (4.3 pups/litter) of which were agouti and heterozygous for the klotho locus (kl/+) (Table 1). These results indicate that klotho homozygous ovaries could give rise to offspring when transplanted to wild-type mice. Second, we examined the effects of treatment with gonadotropins. Administration of PMSG to 5-wk-old female klotho mice induced antral formation of ovarian follicles (data not shown). Subsequent administration of hCG after PMSG treatment induced continuation of follicular maturation and small number of ovulation in klotho ovaries (Fig. 5B), whereas maturation of ovarian follicles was never observed in klotho mice injected with saline (Fig. 5A). Functionally, PMSG/hCG treatment appeared to stimulate growth and maturation of the uterus, with a considerable increase in uterine size and development of endometrium (data not shown). These results indicate that female gonads in klotho mice are potentially functional and suggest that the severe atrophy of reproductive organs may be due to a deficiency of serum factors.
To determine the extra-ovarian factor(s) lacking in klotho mice, which are necessary to gonadal maturation, we examined the response of klotho mouse gonads to these hormones. Administration of purified FSH and GnRH had effects on the maturation of gonads in klotho mice. The ovaries of FSH-treated klotho mice developed follicles that expressed P450arom and LHR (Fig. 6, E and F), whereas vehicle-treated klotho mouse ovaries scarcely expressed those (Fig. 6, B and C). To examine whether the Klotho protein is necessary for gonadotropin production or secretion, we further examined the effects of GnRH on klotho mice. This treatment also induced the expression of these two genes (Fig. 6, H and I). P450 aromatase was induced almost as much as seen in wild-type mice (data not shown). Induction of LHR was detected only in theca cells after treatment with GnRH (Fig. 6I). FSHR expression was observed in vehicle-, FSH-, and GnRH-treated klotho mouse ovaries (Fig. 6, A, D, and G). To confirm this effect, we carried out the thorough treatment of GnRH for klotho mice. This long-term GnRH treatment resulted in further maturation of gonads (Fig. 7). The ovarian follicles matured to the preovulatory stage (Fig. 7C). Growth of uteri with marked increase in endometrial glands was observed (Fig. 7D). Effects of this long-term GnRH treatment were confirmed by in situ hybridization analysis. LHR were induced not only in theca cells but also mural granulosa cells of klotho mice (data not shown).
To reveal what is affected by the klotho deficiency in the pituitary gland, microscopic analysis was performed. Sections of female pituitary gland were immunologically stained for FSH or LH (Fig. 8). The number of FSH positive cells varied between animals in case of wild-type animals. FSH signals were also detected in the mutant animals; however, the number of positive cells was generally less in the mutants. Signals for LH were hardly observed in the klotho mice, whereas those were clearly detected in the wild-type mice. Because it is known that both LH and FSH are produced and secreted by identical cells, these suggest that the production of pituitary hormones is decreased in the mutant animals, although the hormone producing cells exist in the mutant pituitary. Further analysis for LH was performed by the immunoelectromicroscopy. The number of immunogold particles in secretory granules was clearly less in klotho mice compared with wild-type animals (Fig. 8, C and F). These also suggest that the LH production is decreased in the mutant mice, whereas the LH-producing cells exist in the mutant pituitary.
Discussion
To date, some cases of single-gene mutations resulting in sterility have been reported (29). In this study, we examined the cause of female sterility in klotho mice and inferred the mechanism by which Klotho protein exerts systemic effects. Although faint expression of klotho in the ovary and pituitary has been detected by RT-PCR, and atrophic phenotypes of these organs in klotho mice were reported (1), whether the absence of klotho in these regions is the primary cause has not been confirmed. In this study, we demonstrate that female gonads of klotho mice are out of estrus cycles, but they are potentially functional because transplanted ovaries could give rise to offspring (Table 1), and gonadotropin administration resulted in maturation of ovarian follicles (Figs. 5 and 6).
We have not observed either matured ovarian follicles or the vaginal opening of klotho female mice under normal conditions. At 12 d old, there was no apparent difference in gonadal organs between the klotho female and wild-type littermate (Fig. 1). At 8 wk old, there were apparent differences between genotypes (Figs. 2 and 3). Phenotypes in female reproductive system in klotho mice become apparent around the weaning stage the same as phenotypes observed in other organs.
Previous studies proposed the possibility that the soluble Klotho protein may exert hormonal effects on some organs including ovary to maintain their function (6, 7). In contrast to these studies, it was suggested that disrupted regulation of pituitary hormones induced the appearance of klotho-deficient phenotypes (1). We demonstrate here that sterility in klotho females is mainly caused by the absence of proper stimulus by gonadotropins. All of the following support this idea. First, few follicles were observed to develop beyond the preantral stage to antral stage in klotho mice (Fig. 2), confirmed by the nonexpression of both LHR and P450arom, the maker genes of antral stage (Fig. 3). These ovarian phenotypes of klotho mice are very similar to those of hypophysectomized mice (25). The primary stimulus for antral transition is believed to be FSH (11, 30). Second, constant low levels of FSH and LH in sera were observed in klotho mice (Fig. 4). The production of FSH and LH was decreased in the pituitary of klotho mouse revealed by the immunohistochemistry (Fig. 8). Third, exogenous administration of purified FSH alone was sufficient to induce antral transition in klotho ovaries (Fig. 6). Nevertheless, the possibility that the Klotho protein might exert synergistic effects with gonadotropins on follicular maturation remains. Roles of ovarian intrinsic Klotho also remain to be elucidated. Several local factors are known to modulate or partially substitute the effects of gonadotropins on ovarian follicular maturation (31).
Higher serum level of progesterone in klotho mice was observed in this study (Fig. 4). The mechanism that leads to this elevation is not clear yet. Because LH levels were constantly low in the klotho mice, other factor(s) may be involved in this induction. Overactivation of adrenal gland might be a reason for this elevation (29). Slight elevation of serum ACTH was observed in the klotho mice (Toyama, R., unpublished data). This could be due to either the direct abnormality of hypothalamic-adrenal axis or feedback regulation, as observed in congenital adrenal hyperplasia (32).
Although the atrophy of gonadotrophs might be attributed to the deficiency of Klotho protein in the pituitary, the predominant cause of their dysfunction seems due to the deficiency of hypothalamic GnRH. Gonadotrophs of klotho mice sustained ability to develop ovarian follicles in response to exogenous GnRH injection. This result suggests potential reactivity of pituitary gonadotrophs and subsequent production of functional gonadotropins.
The mechanisms leading to the deficiency of GnRH stimulation in klotho mice remain to be elucidated. Loss of klotho gene expression in GnRH-producing cells might be the cause of dysregulation of GnRH. However, if any, deficiency of klotho in GnRH-producing cells should lead to dysregulation of only hypothalamic-pituitary-gonadal axis and its derived effects. We have not yet identified the klotho-expressing cells in hypothalamus because of its faint expression. Deficiency of klotho expression in choroid plexus might be the primary cause because klotho gene expression is abundant in choroid plexus in the brain (1). Absence of Klotho protein in this region might produce an imbalance in the cerebrospinal fluid such as dysregulation of mineral, sugar, protein, and Klotho concentrations. This imbalance would lead to failure of proper response by the endocrine system of the hypothalamus and pituitary gland. Decreased number of secretory granules in GH-producing cells of klotho mice was reported (1). Various stresses, such as abnormal mineral balance, itself could be the other candidate for the cause of sterility. Impaired up-regulation of serum phosphorus and calcium levels caused by impaired vitamin D metabolism in klotho mice was also reported (33).
It is possible that klotho mice cannot trigger the onset of puberty and stay in the prepubertal stage. Vaginal opening was not observed in klotho female mice (1). Abnormal circulation or absence of higher level of gonadotropins observed in this study might reflect the prepubertal absence of GnRH pulse. A train of GnRH discharges is necessary to initiate the onset of puberty (34). Different immunoreactivity between two gonadotropins in the gonadotrophs might reflect the different requirement for the antral follicular development at the onset of puberty (35).
In this study, we revealed the potential reactivities, despite the atrophic phenotypes, of reproductive organs including the uterus, ovary, and gonadotrophs in female klotho mice. This is the first report demonstrating that some of the pathological phenotypes in klotho mice result from abnormal regulation of pituitary hormones. In other words, this suggests that Klotho might be involved in the regulatory control of hypothalamic-pituitary-axis and that the function of Klotho should be located upstream of gonadotropins.
Acknowledgments
We thank Drs. A. Imura, T. Yoshida, M. Hoshino, S. Yoshida, and all the members of our lab at Kyoto University for discussion and help. We also thank T. Obata, M. Iizuka, and Y. Kurotaki for technical assistance.
Footnotes
First Published Online September 22, 2005
Abbreviations: hCG, Human chorionic gonadotropin; LHR, LH receptor; P450arom, aromatase P450; PMSG, pregnant mare’s serum gonadotropin; SCID, severe combined immunodeficiency disease.
Accepted for publication September 15, 2005.
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Core Research for Evolutional Science and Technology (T.F., Y.N., Y.-I.N.), Japan Science and Technology Corporation, Tokyo 170-0013, Japan
Teaching and Research Support Center (Y.I.), Department of Pathology (R.Y.O.), School of Medicine, Tokai University, Boseidai, Ishihara-city, Kanagawa 259-1193, Japan
Abstract
klotho-Deficient mice exhibit a syndrome resembling human premature ageing, with multiple pathological phenotypes in tissues including reproductive organs. It was proposed that Klotho might possess the hormonal effects on many organs. In this study, the female reproductive system of klotho mice was examined to reveal the mechanism that brought the female sterility by histological and molecular approaches. We observed cessation of ovarian follicular maturation at the preantral stage and the presence of numerous atretic ovarian follicles and atrophic uteri. In situ hybridization analysis revealed that LH receptor and aromatase P450 were not expressed in the ovaries. These results suggest the impairment of gonadal development during the antral transition process. We next addressed the responsible organs for the failure of antral transition. Transplantation of klotho ovaries to wild-type mice resulted in the ability to bear offspring. Administration of FSH or GnRH induced advanced maturation of ovaries and uteri in klotho mice. These results indicate that the female reproductive organs in klotho mice are potentially functional and that klotho gene deficiency leads to the atrophy of reproductive organs via impairment of the hypothalamic-pituitary axis. Absence of the estrus cycle and constant low trends of both FSH and LH levels were found in female klotho mice. Immunohistochemical analysis revealed that the production of both FSH and LH were decreased in pituitary gland. Taken together, our findings suggest the involvement of klotho in the regulatory control of pituitary hormones.
Introduction
WE ESTABLISHED A mouse mutant exhibiting a syndrome resembling human ageing that includes short lifespan, atrophy of outer genital organs, impaired maturation of gonadal cells, sterility of both sexes, osteopenia, arteriosclerosis, skin atrophy, growth retardation, ectopic calcification, impaired glucose metabolism, atrophy of thymus, and pulmonary emphysema (1). We also identified the responsible gene, klotho, which is expressed mainly in the kidney and choroid plexus and weakly in some other organs including the pituitary gland and ovary (1, 2). The klotho gene encodes a membrane protein containing the KL domains that show homology to -glucosidase, which is secreted after the cleavage in the extracellular domain (1, 3, 4). The secreted Klotho protein was found in sera and cerebrospinal fluid (5). Atrophic tissues in klotho mouse are different from those that express klotho, suggesting that Klotho or other soluble factor(s) might exert hormonal effects. So far, a function for secreted Klotho protein has been implicated by the partial rescue of some organs by ectopic expression of the membrane form of Klotho (6, 7). Alternatively, the possibility exists that pituitary hormones might mediate Klotho activity (1). Ovarian follicular development is mainly regulated by pituitary gonadotropins, FSH and LH (8, 9). Both FSH and LH are synthesized in gonadotrophs of the anterior pituitary, stored in secreting granules (10), and secreted in response to GnRH signals from the hypothalamus. Ovarian follicular growth proceeds to the preantral stage independently of gonadotropin regulation (11, 12). Further development requires the FSH signal through the FSH receptor, exclusively expressed in the granulosa cells (13). FSH signal induces granulosa cell proliferation and expression of several genes including LH receptor (LHR) and aromatase P450 (P450arom) (14). P450arom is encoded by the cyp19 gene and catalyzes the final step in the biosynthesis of estrogens from androgens (15). Estrogens, produced by the collaborative actions of granulosa and theca cells (8), possess many functions affecting the reproductive system (16), bone and mineral metabolism (17, 18), or others.
In this study, we examined the female genital organs of klotho mice and attempted to determine how Klotho exerts its function. klotho mice exhibited hypogonadotropic hypogonadism (19), probably due to the lack of GnRH stimulation. These results indicate that klotho deficiency affects the regulation of the endocrine system by the hypothalamic-pituitary axis.
Materials and Methods
Animals
Wild-type (+/+) and homozygous klotho mice (kl/kl) were obtained by mating heterozygous mice (kl/+) carrying the insertional mutation in the klotho gene locus (TA20; C3H/J and C57BL/6J mixed background) (1). All animals were maintained and used according to accepted standards of human animal care and use under specific pathogen-free conditions at Kyoto University (Kyoto, Japan).
Histology
Ovaries and uteri were dissected out, fixed with 4% paraformaldehyde in PBS for 16 h, dehydrated through an ethanol series (70, 95, 100%), penetrated with chloroform, embedded in paraffin, and microtome-sectioned to a thickness of 6 μm. Specimens were dewaxed through xylene, 100, 90, 70, and 50% ethanol, and processed for staining with hematoxylin and eosin or for in situ hybridization analysis.
Fragments of pituitary were fixed by immersion with 1% glutaraldehyde in phosphate buffer (pH 7.4) for 2 h at room temperature, dehydrated in graded methanol at –30 C, and embedded in Lowicryl K4M at –20 C. It took 24 h at –20 C and a subsequent 48 h at room temperature for the polymerization of Lowicryl K4M using UV rays. Immunoelectron microscopic staining was done on ultrathin sections using the protein A gold method after applying primary antibodies. Immunoreacted ultrathin sections were examined with a JEOL 1200EX electron microscope (20).
Ovary transplantation
Ovary transplantation was performed as described previously (21, 22, 23), using 4-wk-old klotho mice as donors and 6-wk-old severe combined immunodeficiency disease (SCID) mice as recipients to avoid immunological rejection of tissues. The ovaries of anesthetized klotho mice were harvested and kept in prewarmed culture medium. Recipient SCID female mice were anesthetized with Avertin. A dorsolateral incision about 1.0 cm long was made in the lumber region on the either side of the midline. Through the incision, the gonadal tissues were gently pulled outside the body. The fat pad of the tissue was fastened to a cotton gauze by forceps to operate easily. The ovarian bursa was cut to make a small incision at the opposite side of the oviduct. Then, the ovary was removed by cutting the ovarian stalk with scissors. After bleeding stopped, donor ovaries from each klotho mouse were transplanted into the empty bursas of each recipient SCID mouse. Then, the bursas were placed back and incisions closed using wound clips. Two weeks after operation, treated female SCID mice were mated with 8-wk-old BALB/c male mice. Offspring born to recipient mice were examined for coat color and confirmed by genotyping PCR.
Gonadotropin treatment
To induce ovulation, 5-wk-old mice were injected intraperitoneally with 5 IU pregnant mare’s serum gonadotropin (PMSG) (Serotropin, Teikoku Hormone Mfg. Co., Ltd., Tokyo, Japan). Forty-eight hours after treatment, 5 IU human chorionic gonadotropin (hCG) (Sankyo Co., Ltd., Tokyo, Japan) was administered. Ovarian tissues were collected 48 h after PMSG treatment or 24 h after hCG treatment.
FSH and GnRH treatments
Female klotho mice at 4–5 wk of age were injected sc with 4 μg ovine FSH (Biogenesis, Ltd., Poole, UK), 20% polyvinyl pyrrolidone (Nacalai Tesque, Inc., Kyoto, Japan), 0.9% NaCl (24, 25), or 300 ng GnRH (Bachem, Bubendorf, Switzerland), 20% polyvinyl pyrrolidone, or 0.9% NaCl twice a day for 3 d. For long-term experiments, 50 ng GnRH in 0.9% NaCl was administered sc to female klotho mice at 4–5 wk of age every 2 h for 5 d (26), and then ovaries and uteri were dissected out and used for histological analysis and in situ hybridization analysis.
In situ hybridization
In situ hybridization was performed as described (27). Processed slides were counterstained with nuclear fast red. RNA probes used were designed basically as previously described (28). Sense and antisense probes for FSHR were designed from nucleotides 367-1266 of GenBank clone AF095642, LHR probes were designed for 592-1331 of M81310, and P450arom probes were designed for 42–659 of D00659. These regions were subcloned into pBluescript (Stratagene, La Jolla, CA) vectors and used to synthesize RNA probes with DIG RNA Labeling kit (Roche Molecular Biochemicals Corp., Mannheim, Germany) according to the manufacturer’s protocol.
Measurement of serum levels of gonadotropins and steroids by RIA
Aliquots of blood were collected from 7- to 8-wk-old female wild-type and klotho mice. Sera were prepared at 4 C and kept at –80 C until the assay. Measurements of FSH and LH levels in sera were performed by SRL, Inc. (Tokyo, Japan) using the rat FSH [125I] and LH [125I] assay systems (BIOTRAK, Amersham Pharmacia biotech UK, Ltd., Buckinghamshire, UK), according to the manufacturer’s instruction. Measurements of estradiol and progesterone levels in sera were performed by SRL, Inc. using the DPC estradiol kit and DPC progesterone (Diagnostic Products Corp., Los Angeles, CA), respectively, according to the manufacturer’s instruction. The inter- and intraassay coefficients of variation were within 5–12%. Limits of detection for the hormone assay were 0.09 ng/tube (FSH), 0.08 ng/tube (LH), 12.2 pg/ml (estradiol), and 0.06 ng/ml (progesterone). Sera (200–500 μl) were used for measurements.
Statistical analysis
FSH, LH, estradiol, and progesterone concentrations in sera of wild-type and klotho mice were compared by F test for variance and Wilcoxon-Mann-Whitney test for median. Statview software (SAS Institute, Inc., Cary, NC) was used for the analysis. Differences between the two groups were considered statistically significant at P < 0.05.
Results
To investigate the female reproductive system of klotho mice, ovaries and uteri were examined histologically. Marked differences were not found between 12-d-old klotho mice and wild-type littermates (Fig. 1), except that granulosa cells appeared slightly more layered in ovarian follicles of klotho mice than those of wild-type mice. At 8 wk old, apparent differences were observed. Although ovaries of wild-type mice contained corpora lutea and follicles at every stage of development including preovulatory follicles (Fig. 2, A and B), ovaries of klotho homozygous littermates did not contain any follicles beyond the preantral stage or corpora lutea (Fig. 2, D and E). Instead, many atretic follicles were observed (Fig. 2E, arrowheads). More than 50 ovaries of randomly selected klotho mice were examined, and this was the case in all of them. The uteri recovered from 8-wk-old klotho mice were lean and atrophic, whereas those of wild-type mice were healthy (Fig. 2, C and F). These differences emerged around 3 wk of age and became apparent at 7 wk of age (data not shown).
Next, we examined the expression of genes at the specific stage of follicular maturation by in situ hybridization. At the age of 8 wk, FSHR was sufficiently expressed in some follicles of both wild-type and klotho mice (Fig. 3, A and D). P450arom (cyp19) was expressed in antral follicles of wild-type mice (Fig. 3B, arrows) but could not be detected in klotho mouse ovaries (Fig. 3E). LHR was abundantly expressed in mural granulosa cells of preovulatory follicles, luteal cells, and theca cells in ovaries of 8-wk-old wild-type mice (Fig. 3C), whereas it was not detectable in ovaries of 8-wk-old klotho mice, even in the theca cells (Fig. 3F).
Because the vaginal opening was too narrow to recover the vaginal smear and amount of serum volume was too small, it was difficult to determine the stage of estrus cycle in individual klotho mice. Accordingly, we selected mice randomly to collect serum samples and measured the concentration of hormones. The serum FSH levels of 7- to 8-wk-old female wild-type (+/+) and klotho (kl/kl) mice were 8.3 ± 6.0 ng/ml (mean ± SD, n = 30) and 5.2 ± 1.4 ng/ml (n = 8), respectively (Fig. 4A). The serum LH levels of 7- to 8-wk-old female wild-type and klotho mice were 2.3 ± 1.8 ng/ml (n = 26) and 1.8 ± 0.2 ng/ml (n = 5), respectively (Fig. 4B). The serum estradiol levels of 7- to 8-wk-old female wild-type and klotho mice were 25.4 ± 26.8 pg/ml (n = 6) and 20.7 ± 12.1 pg/ml (n = 8), respectively (Fig. 4C). The serum progesterone levels of 7- to 8-wk-old female wild-type and klotho mice were 3.5 ± 2.8 pg/ml (n = 6) and 16.9 ± 5.9 pg/ml (n = 8), respectively (Fig. 4D). Serum concentrations of both gonadotropins, FSH and LH, showed significantly smaller variance in klotho mice than in wild-type mice (P < 0.001). This might reflect the nonexistence of the estrus cycle in female klotho mice. Significant differences in the median were obtained only for progesterone (P < 0.01) but not for FSH (P = 0.053), LH (P = 0.957), and estradiol (P = 0.082). It was reported that low levels of klotho gene expression were detected in ovary (1). To examine whether the marked gonadal abnormalities in klotho mice are primarily due to the defects in ovaries, we carried out two types of experiments. First, we examined the effect of ovary transplantation. Ovaries of 4-wk-old klotho homozygotes (kl/kl) (coat color is agouti) were transplanted into ovarian bursas of 6-wk-old wild-type SCID (albino) females, and recipient mice were subsequently bred to wild-type BALB/c (albino) males. All six recipient females became pregnant. A total of 34 pups (5.7 pups/litter) were born, 26 (4.3 pups/litter) of which were agouti and heterozygous for the klotho locus (kl/+) (Table 1). These results indicate that klotho homozygous ovaries could give rise to offspring when transplanted to wild-type mice. Second, we examined the effects of treatment with gonadotropins. Administration of PMSG to 5-wk-old female klotho mice induced antral formation of ovarian follicles (data not shown). Subsequent administration of hCG after PMSG treatment induced continuation of follicular maturation and small number of ovulation in klotho ovaries (Fig. 5B), whereas maturation of ovarian follicles was never observed in klotho mice injected with saline (Fig. 5A). Functionally, PMSG/hCG treatment appeared to stimulate growth and maturation of the uterus, with a considerable increase in uterine size and development of endometrium (data not shown). These results indicate that female gonads in klotho mice are potentially functional and suggest that the severe atrophy of reproductive organs may be due to a deficiency of serum factors.
To determine the extra-ovarian factor(s) lacking in klotho mice, which are necessary to gonadal maturation, we examined the response of klotho mouse gonads to these hormones. Administration of purified FSH and GnRH had effects on the maturation of gonads in klotho mice. The ovaries of FSH-treated klotho mice developed follicles that expressed P450arom and LHR (Fig. 6, E and F), whereas vehicle-treated klotho mouse ovaries scarcely expressed those (Fig. 6, B and C). To examine whether the Klotho protein is necessary for gonadotropin production or secretion, we further examined the effects of GnRH on klotho mice. This treatment also induced the expression of these two genes (Fig. 6, H and I). P450 aromatase was induced almost as much as seen in wild-type mice (data not shown). Induction of LHR was detected only in theca cells after treatment with GnRH (Fig. 6I). FSHR expression was observed in vehicle-, FSH-, and GnRH-treated klotho mouse ovaries (Fig. 6, A, D, and G). To confirm this effect, we carried out the thorough treatment of GnRH for klotho mice. This long-term GnRH treatment resulted in further maturation of gonads (Fig. 7). The ovarian follicles matured to the preovulatory stage (Fig. 7C). Growth of uteri with marked increase in endometrial glands was observed (Fig. 7D). Effects of this long-term GnRH treatment were confirmed by in situ hybridization analysis. LHR were induced not only in theca cells but also mural granulosa cells of klotho mice (data not shown).
To reveal what is affected by the klotho deficiency in the pituitary gland, microscopic analysis was performed. Sections of female pituitary gland were immunologically stained for FSH or LH (Fig. 8). The number of FSH positive cells varied between animals in case of wild-type animals. FSH signals were also detected in the mutant animals; however, the number of positive cells was generally less in the mutants. Signals for LH were hardly observed in the klotho mice, whereas those were clearly detected in the wild-type mice. Because it is known that both LH and FSH are produced and secreted by identical cells, these suggest that the production of pituitary hormones is decreased in the mutant animals, although the hormone producing cells exist in the mutant pituitary. Further analysis for LH was performed by the immunoelectromicroscopy. The number of immunogold particles in secretory granules was clearly less in klotho mice compared with wild-type animals (Fig. 8, C and F). These also suggest that the LH production is decreased in the mutant mice, whereas the LH-producing cells exist in the mutant pituitary.
Discussion
To date, some cases of single-gene mutations resulting in sterility have been reported (29). In this study, we examined the cause of female sterility in klotho mice and inferred the mechanism by which Klotho protein exerts systemic effects. Although faint expression of klotho in the ovary and pituitary has been detected by RT-PCR, and atrophic phenotypes of these organs in klotho mice were reported (1), whether the absence of klotho in these regions is the primary cause has not been confirmed. In this study, we demonstrate that female gonads of klotho mice are out of estrus cycles, but they are potentially functional because transplanted ovaries could give rise to offspring (Table 1), and gonadotropin administration resulted in maturation of ovarian follicles (Figs. 5 and 6).
We have not observed either matured ovarian follicles or the vaginal opening of klotho female mice under normal conditions. At 12 d old, there was no apparent difference in gonadal organs between the klotho female and wild-type littermate (Fig. 1). At 8 wk old, there were apparent differences between genotypes (Figs. 2 and 3). Phenotypes in female reproductive system in klotho mice become apparent around the weaning stage the same as phenotypes observed in other organs.
Previous studies proposed the possibility that the soluble Klotho protein may exert hormonal effects on some organs including ovary to maintain their function (6, 7). In contrast to these studies, it was suggested that disrupted regulation of pituitary hormones induced the appearance of klotho-deficient phenotypes (1). We demonstrate here that sterility in klotho females is mainly caused by the absence of proper stimulus by gonadotropins. All of the following support this idea. First, few follicles were observed to develop beyond the preantral stage to antral stage in klotho mice (Fig. 2), confirmed by the nonexpression of both LHR and P450arom, the maker genes of antral stage (Fig. 3). These ovarian phenotypes of klotho mice are very similar to those of hypophysectomized mice (25). The primary stimulus for antral transition is believed to be FSH (11, 30). Second, constant low levels of FSH and LH in sera were observed in klotho mice (Fig. 4). The production of FSH and LH was decreased in the pituitary of klotho mouse revealed by the immunohistochemistry (Fig. 8). Third, exogenous administration of purified FSH alone was sufficient to induce antral transition in klotho ovaries (Fig. 6). Nevertheless, the possibility that the Klotho protein might exert synergistic effects with gonadotropins on follicular maturation remains. Roles of ovarian intrinsic Klotho also remain to be elucidated. Several local factors are known to modulate or partially substitute the effects of gonadotropins on ovarian follicular maturation (31).
Higher serum level of progesterone in klotho mice was observed in this study (Fig. 4). The mechanism that leads to this elevation is not clear yet. Because LH levels were constantly low in the klotho mice, other factor(s) may be involved in this induction. Overactivation of adrenal gland might be a reason for this elevation (29). Slight elevation of serum ACTH was observed in the klotho mice (Toyama, R., unpublished data). This could be due to either the direct abnormality of hypothalamic-adrenal axis or feedback regulation, as observed in congenital adrenal hyperplasia (32).
Although the atrophy of gonadotrophs might be attributed to the deficiency of Klotho protein in the pituitary, the predominant cause of their dysfunction seems due to the deficiency of hypothalamic GnRH. Gonadotrophs of klotho mice sustained ability to develop ovarian follicles in response to exogenous GnRH injection. This result suggests potential reactivity of pituitary gonadotrophs and subsequent production of functional gonadotropins.
The mechanisms leading to the deficiency of GnRH stimulation in klotho mice remain to be elucidated. Loss of klotho gene expression in GnRH-producing cells might be the cause of dysregulation of GnRH. However, if any, deficiency of klotho in GnRH-producing cells should lead to dysregulation of only hypothalamic-pituitary-gonadal axis and its derived effects. We have not yet identified the klotho-expressing cells in hypothalamus because of its faint expression. Deficiency of klotho expression in choroid plexus might be the primary cause because klotho gene expression is abundant in choroid plexus in the brain (1). Absence of Klotho protein in this region might produce an imbalance in the cerebrospinal fluid such as dysregulation of mineral, sugar, protein, and Klotho concentrations. This imbalance would lead to failure of proper response by the endocrine system of the hypothalamus and pituitary gland. Decreased number of secretory granules in GH-producing cells of klotho mice was reported (1). Various stresses, such as abnormal mineral balance, itself could be the other candidate for the cause of sterility. Impaired up-regulation of serum phosphorus and calcium levels caused by impaired vitamin D metabolism in klotho mice was also reported (33).
It is possible that klotho mice cannot trigger the onset of puberty and stay in the prepubertal stage. Vaginal opening was not observed in klotho female mice (1). Abnormal circulation or absence of higher level of gonadotropins observed in this study might reflect the prepubertal absence of GnRH pulse. A train of GnRH discharges is necessary to initiate the onset of puberty (34). Different immunoreactivity between two gonadotropins in the gonadotrophs might reflect the different requirement for the antral follicular development at the onset of puberty (35).
In this study, we revealed the potential reactivities, despite the atrophic phenotypes, of reproductive organs including the uterus, ovary, and gonadotrophs in female klotho mice. This is the first report demonstrating that some of the pathological phenotypes in klotho mice result from abnormal regulation of pituitary hormones. In other words, this suggests that Klotho might be involved in the regulatory control of hypothalamic-pituitary-axis and that the function of Klotho should be located upstream of gonadotropins.
Acknowledgments
We thank Drs. A. Imura, T. Yoshida, M. Hoshino, S. Yoshida, and all the members of our lab at Kyoto University for discussion and help. We also thank T. Obata, M. Iizuka, and Y. Kurotaki for technical assistance.
Footnotes
First Published Online September 22, 2005
Abbreviations: hCG, Human chorionic gonadotropin; LHR, LH receptor; P450arom, aromatase P450; PMSG, pregnant mare’s serum gonadotropin; SCID, severe combined immunodeficiency disease.
Accepted for publication September 15, 2005.
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