Disruption of the Transmembrane Dense Core Vesicle Proteins IA-2 and IA-2 Causes Female Infertility
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《内分泌学杂志》
Experimental Medicine Section (A.K., S.N., A.L.N), Oral Infection and Immunity Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland 20892
Diabetes Research Laboratories, Oxford Centre for Diabetes, Endocrinology and Metabolism, Churchill Hospital (A.C.), Oxford OX3 7LJ, United Kingdom
Department of Human Anatomy and Genetics (J.F.M.), Oxford OX1 3QX, United Kingdom
Abstract
Female infertility is a worldwide problem affecting 10–15% of the population. The cause of the infertility in many cases is not known. In the present report, we demonstrate that alterations in two transmembrane structural proteins, IA-2 and IA-2, located in dense core secretory vesicles (DCV) of many endocrine and neuroendocrine cells, can result in female infertility. IA-2 and IA-2 are best known as major autoantigens in type 1 diabetes, but their normal function has remained an enigma. Recently we showed in mice that deletion of IA-2 and/or IA-2 results in impaired insulin secretion and glucose intolerance. We now report that double knockout (DKO), but not single knockout, female mice are essentially infertile. Vaginal smears showed a totally abnormal estrous cycle, and examination of the ovaries revealed normal-appearing oocytes but the absence of corpora lutea. The LH surge that is required for ovulation occurred in wild-type mice but not in DKO mice. Additional studies showed that the LH level in the pituitary of DKO female mice was decreased compared with wild-type mice. Treatment of DKO females with gonadotropins restored corpora lutea formation. In contrast to DKO female mice, DKO male mice were fertile and LH levels in the serum and pituitary were within the normal range. From these studies we conclude that the DCV proteins, IA-2 and IA-2, play an important role in LH secretion and that alterations in structural proteins of DCV can result in female infertility.
Introduction
DENSE CORE SECRETORY vesicles (DCVs) are present in neuroendocrine cells and are required for the regulated secretion of hormones and neurotransmitters (1). IA-2 and IA-2 are enzymatically inactive members of the protein tyrosine phosphatase family and are expressed as transmembrane proteins in the DCVs of many neuroendocrine cells including the pancreatic islets and pituitary (2, 3, 4, 5, 6, 7). These proteins are of considerable clinical interest because they are major autoantigens in type 1 diabetes and are widely used as diabetes-specific diagnostic and predictive markers (8). The function of IA-2 and IA-2 in secretory vesicles, however, has remained unclear.
Recently we succeeded in knocking out the individual IA-2 and IA-2 genes [single knockout (SKO)] and found that this resulted in abnormal glucose tolerance tests and impaired insulin secretion in response to glucose (9, 10). Because of the possible complementary role of these two proteins, we generated double knockout (DKO) mice. Fasting and nonfasting blood glucose and insulin levels remained within the normal range in DKO mice and did not differ from that of SKO mice (11). Insulin secretion, however, was more severely impaired in the DKO than in the SKO mice. In addition, as described here, deletion of both the IA-2 and IA-2 genes, in contrast to deletion of the individual genes, results in female infertility.
Materials and Methods
Mice
Targeted disruption of the individual IA-2 and IA-2 genes was described previously (9, 10, 11). C57BL/6Nci, eighth generation, IA-2+/– mice were mated with C57BL/6Nci, fourth generation, IA-2+/– mice, and the double-heterozygous outcomes were interbred to generate IA-2–/–/IA-2–/– mice. Mice were housed under a 12-h light, 12-h dark schedule, with lights on at 0600 h and maintained under specific-pathogen-free conditions. All procedures were approved by our Institutional Animal Care and Use Committee.
Western blot
Antibodies to mouse IA-2 and IA-2 were prepared as described previously (9, 10). Proteins from brains, separated by SDS-PAGE, were transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA). Blots were detected by SuperSignal (Pierce, Rockford, IL).
Histology
Antibodies against LH, FSH, ACTH, prolactin, and TSH were purchased from Dako (Carpinteria, CA). Pituitaries, ovaries, and testis were collected in 10% neutral-buffered formalin and processed for paraffin embedding. Sections were stained with hematoxylin and eosin. Sections of pituitaries were incubated with primary antibodies followed by biotin-conjugated second antibody and streptavidin-horseradish peroxidase.
Measurement of hormones in serum and pituitary
Vaginal smears from IA-2+/+/IA-2+/+, and IA-2–/–/IA-2–/– female mice were collected daily to determine the stage of the estrous cycle over 3 wk. Smears were streaked on slides and stained with Giemsa solution. At 1830 h, plasma from IA-2+/+/IA-2+/+ and IA-2–/–/IA-2–/– mice were collected and stored at –20 C until assayed. Plasma LH concentrations were measured using Biotrak LH immunoassay system (Amersham Biosciences, Piscataway, NJ). At the times indicated, male and female mice were killed and pituitaries were harvested and frozen on dry ice. Blots loaded with equivalent amounts of protein were incubated with goat antibodies to LH (R-16), FSH (C-19), or actin (C-11) (Santa Cruz Biotechnology, Santa Cruz, CA) and then horseradish peroxidase-linked antigoat Ig (Dako).
Induced ovulation
Three-month-old IA-2–/–/IA-2–/– female mice were injected ip with 5 IU of pregnant mare serum gonadotropin (PMSG) or saline between 1300 and 1500 h. After 48 h, the mice were injected with 5 IU human chorionic gonadotropin (hCG) or saline. Forty-eight hours later, the mice were killed and the ovaries harvested for histology. PMSG and hCG were purchased from Sigma Chemical Co. (St. Louis, MO).
Statistical analysis
Statistical analysis was performed using the Student’s t test for unpaired comparisons. Values are presented as mean ± SEM. P < 0.05 was considered significant.
Results
Wild-type (WT) (IA-2+/+/IA-2+/+), SKO (IA-2–/–/IA-2+/+; IA-2 +/+/IA-2–/–) and DKO (IA-2–/–/IA-2–/–) mice were generated as described (11). Western blot analysis (Fig. 1A) showed strong IA-2 bands in the IA-2+/+/IA-2+/+ and IA-2+/+/IA-2–/– mice but total absence in the IA-2–/–/IA-2+/+ and the IA-2–/–/IA-2–/– mice. Conversely, strong IA-2 bands were seen in the IA-2+/+/IA-2+/+ and IA-2–/–/IA-2+/+ mice but were totally absent in the IA-2+/+/IA-2–/– and IA-2–/–/IA-2–/– mice. The knockout of both IA-2 and IA-2 was further confirmed by immunostaining of pituitary tissue with antibodies specific for IA-2 or IA-2. IA-2- and IA-2-stained cells are seen in IA-2+/+/IA-2+/+ but not in IA-2–/–/IA-2–/– mice (Fig. 1B).
Earlier experiments showed that the deletion of neither IA-2 nor IA-2 had any affect on fertility (9, 10). To determine whether deletion of both IA-2 and IA-2 would affect fertility, WT, double-heterozygous, and DKO male mice were mated with WT female mice. As seen in Fig. 1C, there was no difference in the litter size of the different groups. Similarly, when WT or heterozygous females were mated with normal WT males, all of the females gave birth and there was no difference in the size of their litters. However, when DKO females were mated with WT males, only two of the 12 DKO females gave birth and the litter sizes were very small. Thus, the majority of the females in which both IA-2 and IA-2 were knocked out were infertile.
To determine the cause of infertility, the estrous cycle of the DKO females was studied by examining the cellular composition (i.e. ratio of leukocytes to epithelial cells) of vaginal smears. These studies revealed the total absence of a normal estrous cycle (data not shown). To see whether the absence of the normal estrous cycle was in any way caused by abnormalities in the ovaries, histological studies were carried out. The number and appearance of oocytes and antral follicles in the DKO females was similar to WT females, but corpora lutea were absent and lipid droplets were found in stromal cells (Fig. 2, A and B). The possibility that the infertility, abnormal estrous cycle, and absence of corpora lutea was a result of impaired secretion of gonadotropins was investigated by measuring plasma LH levels. As seen in Fig. 3A, during proestrus, there was the expected LH surge in the WT mice, but no surge could be detected in plasma of the DKO mice at any of the times (i.e. daily) tested, and a consistent low level persisted throughout. This suggested that ovulation was being inhibited as a result of the failure of the ovaries to receive sufficient gonadotropin stimulation from the pituitary. To investigate this possibility, the DKO females were treated with PMSG and hCG. Within 96 h, large numbers of corpora lutea appeared (Fig. 2C). hCG alone was not as effective as the combination of PMSG and hCG (not shown).
These findings raised the possibility that the primary cause of infertility in the DKO female mice resided at the level of the pituitary. Immunohistochemical studies showed that IA-2 colocalized with LH in the same cells in the pituitary (Fig. 2D), but no gross difference was observed in the appearance or number of cells expressing LH, FSH, ACTH, prolactin, GH and TSH in the DKO vs. the WT mice (Fig. 2E). However, Western blots (Fig. 3, B–D) showed that whereas the concentration of LH in pituitary lysates of young prepubertal (24 d old) DKO mice was nearly the same as in WT (Fig. 3B), at 4 months of age, the DKO mice showed nearly a 75% decrease in LH (Fig. 3C) and a 60% decrease in FSH (Fig. 3D) compared with WT mice at proestrus.
In contrast to the DKO females, the DKO male mice showed no evidence of infertility, and the histology and weight of the testes did not appear to be different from that of the WT mice (Fig. 4, A and B). Plasma LH and FSH levels also were within the normal range in both the DKO and WT mice (Fig. 4, C and D). However, with increasing age, the LH in pituitary lysates showed a slight (34%), but not statistically significant, decrease in the DKO compared with the WT mice (Fig. 4, E and F).
Discussion
Several lines of evidence now support the idea that IA-2 and IA-2 are positive regulators of secretion. First, gene-targeting experiments showed that the deletion of IA-2 or IA-2 alone or in combination inhibited insulin secretion (9, 10, 11). Second, knockdown of IA-2 with IA-2 small interfering RNA in MIN-6 cells resulted in inhibition of both regulated and basal insulin secretion (12). Third, knockdown of IA-2 in MIN-6 cells resulted in a decrease in the number of insulin-containing vesicles (12). Because IA-2 and IA-2 are integral components of DCV in many different neuroendocrine cells throughout the body, this suggested that IA-2 and IA-2 might affect the secretion of other hormones. The current experiments show that this is the case with LH. The effect of the knockout of IA-2 and IA-2 on secretion, however, is subtle. In the DKO mice, insulin and glucose levels remain within the normal range (11). Only when the mice are stressed by administration of a high-glucose load does the defect in insulin secretion become apparent. The same is the case with LH secretion. Only when the LH surge needed for ovulation and the production of corpora lutea is examined (13) does the impairment of secretion and its biological effect, infertility, become apparent. The secretion of other pituitary hormones also might be affected as a result of the deletion of IA-2 and IA-2. A prime candidate is FSH, which colocalizes with LH in the same cells. Our experiments showed a substantial decrease in FSH in the pituitary of the DKO female mice compared with the WT female mice (Fig. 3D). Basal FSH levels in the plasma of the DKO and WT females, however, were essentially the same, and because of the absence of a regular estrous cycle, an FSH surge was not detected in the DKO females at any of the times examined (data not shown).
The secretion of LH from the pituitary is a complex multistep process involving stimulation by GnRH found in DCV in the hypothalamus (14). Although not yet examined, it is quite possible that the deletion of IA-2 and IA-2 also may inhibit the secretion of GnRH. If this turns out to be the case, then the infertility reported here might be the result of a cascade beginning with the failure of impaired secretion of GnRH to provide sufficient stimulation for the release of LH and that, in turn, this is superimposed upon the already impaired ability of DCV in the pituitary to secrete LH.
Precisely how IA-2 and IA-2 regulate secretion is under intense investigation, and at least three different hypotheses have been proposed. The first is that IA-2/IA-2 interact with proteins associated with the cytoskeleton system thereby affecting trafficking of secretory vesicles (15). The second is that the cytoplasmic domain of IA-2 forms heterodimers with other protein tyrosine phosphatases and thereby influences signaling associated with secretion (16). The third is that IA-2 stabilizes DCVs (12). When IA-2 is overexpressed, there is an increase in the number of DCVs and the content of hormones in neuroendocrine cells, whereas when IA-2 is knocked down in cell lines by small interfering RNA, there is a decrease in the content of hormones in neuroendocrine cells (12).
A variety of factors are known to cause female infertility including alterations in gonadotropins and their receptors (17). The current experiments show for the first time that alterations in integral structural proteins of DCVs also can cause female infertility. These findings raise the possibility that at the human level, mutations in IA-2/IA-2 or the degree of expression of these and perhaps other structural proteins of DCVs may be one of the explanations for the 25–30% of fully investigated, but still unexplained causes of infertility (18).
Footnotes
First Published Online November 3, 2005
Abbreviations: DCV, Dense core secretory vesicle; DKO, double knockout; PMSG, pregnant mare serum gonadotropin; SKO, single knockout; WT, wild type.
Accepted for publication October 27, 2005.
References
Burgoyne RD, Morgan A 2003 Secretory granule exocytosis. Physiol Rev 83:581–632
Lan MS, Lu J, Goto Y, Notkins AL 1994 Molecular cloning and identification of a receptor-type protein tyrosine phosphatase, IA-2, from human insulinoma. DNA Cell Biol 13:505–514
Lan MS, Wasserfall C, Maclaren NK, Notkins AL 1996 IA-2, a transmembrane protein of the protein tyrosine phosphatase family, is a major autoantigen in insulin-dependent diabetes mellitus. Proc Natl Acad Sci USA 93:6367–6370
Lu J, Li Q, Xie H, Chen ZJ, Borovitskaya AE, Maclaren NK, Notkins AL, Lan MS 1996 Identification of a second transmembrane protein tyrosine phosphatase, IA-2, as an autoantigen in insulin-dependent diabetes mellitus: precursor of the 37-kDa tryptic fragment. Proc Natl Acad Sci USA 93:2307–2311
Solimena M, Dirkx Jr R, Hermel JM, Pleasic-Williams S, Shapiro JA, Caron L, Rabin DU 1996 ICA512, an autoantigen of type I diabetes, is an intrinsic membrane protein of neurosecretory granules. EMBO J 15:2102–2114
Wasmeier C, Hutton JC 1999 Secretagogue-dependent phosphorylation of phogrin, an insulin granule membrane protein tyrosine phosphatase homologue. Biochem J 341:563–569
Roberts C, Roberts GA, Lobner K, Bearzatto M, Clark A, Bonifacio E, Christie MR 2001 Expression of the protein tyrosine phosphatase-like protein IA-2 during pancreatic islet development. J Histochem Cytochem 49:767–776
Notkins AL 2002 Immunologic and genetic factors in type 1 diabetes. J Biol Chem 277:43545–43548
Saeki K, Zhu M, Kubosaki A, Xie J, Lan MS, Notkins AL 2002 Targeted disruption of the protein tyrosine phosphatase-like molecule IA-2 results in alterations in glucose tolerance tests and insulin secretion. Diabetes 51:1842–1850
Kubosaki A, Gross S, Miura J, Saeki K, Zhu M, Nakamura S, Hendriks W, Notkins AL 2004 Targeted disruption of the IA-2 gene causes glucose intolerance and impairs insulin secretion but does not prevent the development of diabetes in NOD mice. Diabetes 53:1684–1691
Kubosaki A, Nakamura S, Notkins AL The dense core vesicle proteins IA-2 and IA-2: metabolic alterations in double knockout mice. Diabetes, in press
Harashima S, Clark A, Christie MR, Notkins AL 2005 The dense core transmembrane vesicle protein IA-2 is a regulator of vesicle number and insulin secretion. Proc Natl Acad Sci USA 102:8704–8709
Parkening TA, Collins TJ, Smith ER 1982 Plasma and pituitary concentrations of LH, FSH, and prolactin in aging C57BL/6 mice at various times of the estrous cycle. Neurobiol Aging 3:31–35
Wiesak T 2002 Role of LH in controlled ovarian stimulation. Reprod Biol 2:215–272
Ort T, Maksimova E, Dirkx R, Kachinsky AM, Berghs S, Froehner SC, Solimena M 2000 The receptor tyrosine phosphatase-like protein ICA512 binds the PDZ domains of 2-syntrophin and nNOS in pancreatic -cells. Eur J Cell Biol 79:621–630
Gross S, Blanchetot C, Schepens J, Albet S, Lammers R, den Hertog J, Hendriks W 2002 Multimerization of the protein-tyrosine phosphatase (PTP)-like insulin-dependent diabetes mellitus autoantigens IA-2 and IA-2 with receptor PTPs (RPTPs). Inhibition of RPTP enzymatic activity. J Biol Chem 277:48139–48145
Achermann JC, Jameson JL 1999 Fertility and infertility: genetic contributions from the hypothalamic-pituitary-gonadal axis. Mol Endocrinol 13:812–818
Evers JL 2002 Female subfertility. Lancet 360:151–159(Atsutaka Kubosaki, Shinichiro Nakamura, )
Diabetes Research Laboratories, Oxford Centre for Diabetes, Endocrinology and Metabolism, Churchill Hospital (A.C.), Oxford OX3 7LJ, United Kingdom
Department of Human Anatomy and Genetics (J.F.M.), Oxford OX1 3QX, United Kingdom
Abstract
Female infertility is a worldwide problem affecting 10–15% of the population. The cause of the infertility in many cases is not known. In the present report, we demonstrate that alterations in two transmembrane structural proteins, IA-2 and IA-2, located in dense core secretory vesicles (DCV) of many endocrine and neuroendocrine cells, can result in female infertility. IA-2 and IA-2 are best known as major autoantigens in type 1 diabetes, but their normal function has remained an enigma. Recently we showed in mice that deletion of IA-2 and/or IA-2 results in impaired insulin secretion and glucose intolerance. We now report that double knockout (DKO), but not single knockout, female mice are essentially infertile. Vaginal smears showed a totally abnormal estrous cycle, and examination of the ovaries revealed normal-appearing oocytes but the absence of corpora lutea. The LH surge that is required for ovulation occurred in wild-type mice but not in DKO mice. Additional studies showed that the LH level in the pituitary of DKO female mice was decreased compared with wild-type mice. Treatment of DKO females with gonadotropins restored corpora lutea formation. In contrast to DKO female mice, DKO male mice were fertile and LH levels in the serum and pituitary were within the normal range. From these studies we conclude that the DCV proteins, IA-2 and IA-2, play an important role in LH secretion and that alterations in structural proteins of DCV can result in female infertility.
Introduction
DENSE CORE SECRETORY vesicles (DCVs) are present in neuroendocrine cells and are required for the regulated secretion of hormones and neurotransmitters (1). IA-2 and IA-2 are enzymatically inactive members of the protein tyrosine phosphatase family and are expressed as transmembrane proteins in the DCVs of many neuroendocrine cells including the pancreatic islets and pituitary (2, 3, 4, 5, 6, 7). These proteins are of considerable clinical interest because they are major autoantigens in type 1 diabetes and are widely used as diabetes-specific diagnostic and predictive markers (8). The function of IA-2 and IA-2 in secretory vesicles, however, has remained unclear.
Recently we succeeded in knocking out the individual IA-2 and IA-2 genes [single knockout (SKO)] and found that this resulted in abnormal glucose tolerance tests and impaired insulin secretion in response to glucose (9, 10). Because of the possible complementary role of these two proteins, we generated double knockout (DKO) mice. Fasting and nonfasting blood glucose and insulin levels remained within the normal range in DKO mice and did not differ from that of SKO mice (11). Insulin secretion, however, was more severely impaired in the DKO than in the SKO mice. In addition, as described here, deletion of both the IA-2 and IA-2 genes, in contrast to deletion of the individual genes, results in female infertility.
Materials and Methods
Mice
Targeted disruption of the individual IA-2 and IA-2 genes was described previously (9, 10, 11). C57BL/6Nci, eighth generation, IA-2+/– mice were mated with C57BL/6Nci, fourth generation, IA-2+/– mice, and the double-heterozygous outcomes were interbred to generate IA-2–/–/IA-2–/– mice. Mice were housed under a 12-h light, 12-h dark schedule, with lights on at 0600 h and maintained under specific-pathogen-free conditions. All procedures were approved by our Institutional Animal Care and Use Committee.
Western blot
Antibodies to mouse IA-2 and IA-2 were prepared as described previously (9, 10). Proteins from brains, separated by SDS-PAGE, were transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA). Blots were detected by SuperSignal (Pierce, Rockford, IL).
Histology
Antibodies against LH, FSH, ACTH, prolactin, and TSH were purchased from Dako (Carpinteria, CA). Pituitaries, ovaries, and testis were collected in 10% neutral-buffered formalin and processed for paraffin embedding. Sections were stained with hematoxylin and eosin. Sections of pituitaries were incubated with primary antibodies followed by biotin-conjugated second antibody and streptavidin-horseradish peroxidase.
Measurement of hormones in serum and pituitary
Vaginal smears from IA-2+/+/IA-2+/+, and IA-2–/–/IA-2–/– female mice were collected daily to determine the stage of the estrous cycle over 3 wk. Smears were streaked on slides and stained with Giemsa solution. At 1830 h, plasma from IA-2+/+/IA-2+/+ and IA-2–/–/IA-2–/– mice were collected and stored at –20 C until assayed. Plasma LH concentrations were measured using Biotrak LH immunoassay system (Amersham Biosciences, Piscataway, NJ). At the times indicated, male and female mice were killed and pituitaries were harvested and frozen on dry ice. Blots loaded with equivalent amounts of protein were incubated with goat antibodies to LH (R-16), FSH (C-19), or actin (C-11) (Santa Cruz Biotechnology, Santa Cruz, CA) and then horseradish peroxidase-linked antigoat Ig (Dako).
Induced ovulation
Three-month-old IA-2–/–/IA-2–/– female mice were injected ip with 5 IU of pregnant mare serum gonadotropin (PMSG) or saline between 1300 and 1500 h. After 48 h, the mice were injected with 5 IU human chorionic gonadotropin (hCG) or saline. Forty-eight hours later, the mice were killed and the ovaries harvested for histology. PMSG and hCG were purchased from Sigma Chemical Co. (St. Louis, MO).
Statistical analysis
Statistical analysis was performed using the Student’s t test for unpaired comparisons. Values are presented as mean ± SEM. P < 0.05 was considered significant.
Results
Wild-type (WT) (IA-2+/+/IA-2+/+), SKO (IA-2–/–/IA-2+/+; IA-2 +/+/IA-2–/–) and DKO (IA-2–/–/IA-2–/–) mice were generated as described (11). Western blot analysis (Fig. 1A) showed strong IA-2 bands in the IA-2+/+/IA-2+/+ and IA-2+/+/IA-2–/– mice but total absence in the IA-2–/–/IA-2+/+ and the IA-2–/–/IA-2–/– mice. Conversely, strong IA-2 bands were seen in the IA-2+/+/IA-2+/+ and IA-2–/–/IA-2+/+ mice but were totally absent in the IA-2+/+/IA-2–/– and IA-2–/–/IA-2–/– mice. The knockout of both IA-2 and IA-2 was further confirmed by immunostaining of pituitary tissue with antibodies specific for IA-2 or IA-2. IA-2- and IA-2-stained cells are seen in IA-2+/+/IA-2+/+ but not in IA-2–/–/IA-2–/– mice (Fig. 1B).
Earlier experiments showed that the deletion of neither IA-2 nor IA-2 had any affect on fertility (9, 10). To determine whether deletion of both IA-2 and IA-2 would affect fertility, WT, double-heterozygous, and DKO male mice were mated with WT female mice. As seen in Fig. 1C, there was no difference in the litter size of the different groups. Similarly, when WT or heterozygous females were mated with normal WT males, all of the females gave birth and there was no difference in the size of their litters. However, when DKO females were mated with WT males, only two of the 12 DKO females gave birth and the litter sizes were very small. Thus, the majority of the females in which both IA-2 and IA-2 were knocked out were infertile.
To determine the cause of infertility, the estrous cycle of the DKO females was studied by examining the cellular composition (i.e. ratio of leukocytes to epithelial cells) of vaginal smears. These studies revealed the total absence of a normal estrous cycle (data not shown). To see whether the absence of the normal estrous cycle was in any way caused by abnormalities in the ovaries, histological studies were carried out. The number and appearance of oocytes and antral follicles in the DKO females was similar to WT females, but corpora lutea were absent and lipid droplets were found in stromal cells (Fig. 2, A and B). The possibility that the infertility, abnormal estrous cycle, and absence of corpora lutea was a result of impaired secretion of gonadotropins was investigated by measuring plasma LH levels. As seen in Fig. 3A, during proestrus, there was the expected LH surge in the WT mice, but no surge could be detected in plasma of the DKO mice at any of the times (i.e. daily) tested, and a consistent low level persisted throughout. This suggested that ovulation was being inhibited as a result of the failure of the ovaries to receive sufficient gonadotropin stimulation from the pituitary. To investigate this possibility, the DKO females were treated with PMSG and hCG. Within 96 h, large numbers of corpora lutea appeared (Fig. 2C). hCG alone was not as effective as the combination of PMSG and hCG (not shown).
These findings raised the possibility that the primary cause of infertility in the DKO female mice resided at the level of the pituitary. Immunohistochemical studies showed that IA-2 colocalized with LH in the same cells in the pituitary (Fig. 2D), but no gross difference was observed in the appearance or number of cells expressing LH, FSH, ACTH, prolactin, GH and TSH in the DKO vs. the WT mice (Fig. 2E). However, Western blots (Fig. 3, B–D) showed that whereas the concentration of LH in pituitary lysates of young prepubertal (24 d old) DKO mice was nearly the same as in WT (Fig. 3B), at 4 months of age, the DKO mice showed nearly a 75% decrease in LH (Fig. 3C) and a 60% decrease in FSH (Fig. 3D) compared with WT mice at proestrus.
In contrast to the DKO females, the DKO male mice showed no evidence of infertility, and the histology and weight of the testes did not appear to be different from that of the WT mice (Fig. 4, A and B). Plasma LH and FSH levels also were within the normal range in both the DKO and WT mice (Fig. 4, C and D). However, with increasing age, the LH in pituitary lysates showed a slight (34%), but not statistically significant, decrease in the DKO compared with the WT mice (Fig. 4, E and F).
Discussion
Several lines of evidence now support the idea that IA-2 and IA-2 are positive regulators of secretion. First, gene-targeting experiments showed that the deletion of IA-2 or IA-2 alone or in combination inhibited insulin secretion (9, 10, 11). Second, knockdown of IA-2 with IA-2 small interfering RNA in MIN-6 cells resulted in inhibition of both regulated and basal insulin secretion (12). Third, knockdown of IA-2 in MIN-6 cells resulted in a decrease in the number of insulin-containing vesicles (12). Because IA-2 and IA-2 are integral components of DCV in many different neuroendocrine cells throughout the body, this suggested that IA-2 and IA-2 might affect the secretion of other hormones. The current experiments show that this is the case with LH. The effect of the knockout of IA-2 and IA-2 on secretion, however, is subtle. In the DKO mice, insulin and glucose levels remain within the normal range (11). Only when the mice are stressed by administration of a high-glucose load does the defect in insulin secretion become apparent. The same is the case with LH secretion. Only when the LH surge needed for ovulation and the production of corpora lutea is examined (13) does the impairment of secretion and its biological effect, infertility, become apparent. The secretion of other pituitary hormones also might be affected as a result of the deletion of IA-2 and IA-2. A prime candidate is FSH, which colocalizes with LH in the same cells. Our experiments showed a substantial decrease in FSH in the pituitary of the DKO female mice compared with the WT female mice (Fig. 3D). Basal FSH levels in the plasma of the DKO and WT females, however, were essentially the same, and because of the absence of a regular estrous cycle, an FSH surge was not detected in the DKO females at any of the times examined (data not shown).
The secretion of LH from the pituitary is a complex multistep process involving stimulation by GnRH found in DCV in the hypothalamus (14). Although not yet examined, it is quite possible that the deletion of IA-2 and IA-2 also may inhibit the secretion of GnRH. If this turns out to be the case, then the infertility reported here might be the result of a cascade beginning with the failure of impaired secretion of GnRH to provide sufficient stimulation for the release of LH and that, in turn, this is superimposed upon the already impaired ability of DCV in the pituitary to secrete LH.
Precisely how IA-2 and IA-2 regulate secretion is under intense investigation, and at least three different hypotheses have been proposed. The first is that IA-2/IA-2 interact with proteins associated with the cytoskeleton system thereby affecting trafficking of secretory vesicles (15). The second is that the cytoplasmic domain of IA-2 forms heterodimers with other protein tyrosine phosphatases and thereby influences signaling associated with secretion (16). The third is that IA-2 stabilizes DCVs (12). When IA-2 is overexpressed, there is an increase in the number of DCVs and the content of hormones in neuroendocrine cells, whereas when IA-2 is knocked down in cell lines by small interfering RNA, there is a decrease in the content of hormones in neuroendocrine cells (12).
A variety of factors are known to cause female infertility including alterations in gonadotropins and their receptors (17). The current experiments show for the first time that alterations in integral structural proteins of DCVs also can cause female infertility. These findings raise the possibility that at the human level, mutations in IA-2/IA-2 or the degree of expression of these and perhaps other structural proteins of DCVs may be one of the explanations for the 25–30% of fully investigated, but still unexplained causes of infertility (18).
Footnotes
First Published Online November 3, 2005
Abbreviations: DCV, Dense core secretory vesicle; DKO, double knockout; PMSG, pregnant mare serum gonadotropin; SKO, single knockout; WT, wild type.
Accepted for publication October 27, 2005.
References
Burgoyne RD, Morgan A 2003 Secretory granule exocytosis. Physiol Rev 83:581–632
Lan MS, Lu J, Goto Y, Notkins AL 1994 Molecular cloning and identification of a receptor-type protein tyrosine phosphatase, IA-2, from human insulinoma. DNA Cell Biol 13:505–514
Lan MS, Wasserfall C, Maclaren NK, Notkins AL 1996 IA-2, a transmembrane protein of the protein tyrosine phosphatase family, is a major autoantigen in insulin-dependent diabetes mellitus. Proc Natl Acad Sci USA 93:6367–6370
Lu J, Li Q, Xie H, Chen ZJ, Borovitskaya AE, Maclaren NK, Notkins AL, Lan MS 1996 Identification of a second transmembrane protein tyrosine phosphatase, IA-2, as an autoantigen in insulin-dependent diabetes mellitus: precursor of the 37-kDa tryptic fragment. Proc Natl Acad Sci USA 93:2307–2311
Solimena M, Dirkx Jr R, Hermel JM, Pleasic-Williams S, Shapiro JA, Caron L, Rabin DU 1996 ICA512, an autoantigen of type I diabetes, is an intrinsic membrane protein of neurosecretory granules. EMBO J 15:2102–2114
Wasmeier C, Hutton JC 1999 Secretagogue-dependent phosphorylation of phogrin, an insulin granule membrane protein tyrosine phosphatase homologue. Biochem J 341:563–569
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