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Section on Women’s Health Research, Developmental Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health (Z.-B.T., L.M.N.), Bethesda, Maryland 20892; and the Department of Pediatrics, Georgetown University Medical Center (Z.-B.T.), Washington, D.C. 20007
Address all correspondence and requests for reprints to: Lawrence M. Nelson, M.D., Section on Women’s Health Research, Developmental Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health, Building 10, Room 10N262, Bethesda, Maryland 20892-1862. E-mail: nelsonl{at}cc1.nichd.nih.gov
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Introduction |
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Ovarian folliculogenesis is a dynamic process that begins with the formation of primordial follicles and extends through the completion of the ovarian life cycle at menopause. During gestation, fetal oocytes complete the prophase of the first meiotic division and enter into the dictyate, a prolongation of the diplotene stage. Perinatally, mouse oocytes recruit a single layer of flattened granulosa cells to form primordial follicles that represent the entire reservoir of female germ cells (13). Mechanisms responsible for the maintenance of primordial follicles and their subsequent initiation into growth and development remain largely unknown. The most salient characteristics of follicular growth are the striking increase in the diameter of the oocyte (from 12 to 65 µm) and the dramatic proliferation of the surrounding granulosa cells (14). Interactions between the two cell types are integral to this process with the ovarian autocrine/paracrine growth factors such as GDF-9 and the c-Kit system (15, 16, 17). Such interactions are particularly important in early follicular growth before gonadotropins become the major determinants of folliculogenesis.
In recent years, it has been proposed that the immune and
endocrine systems interact in regulating ovarian function (18). Unlike
the testes, the ovary is not an immunologically privileged site, and
macrophages, lymphocytes, and polymorphonuclear granulocytes reside
within the organ. The immune cytokines, such as interleukin-1 and tumor
necrosis factor-
, can affect ovarian function in vitro
(19, 20), although the importance of the intraovarian resident immune
cells in ovarian physiology is uncertain (21). Clinically, the ovarian
immune cells might contribute to ovarian diseases (21). Nevertheless,
the pathogenic mechanism that might provoke the lymphocytes to affect
the ovary is still unknown.
Here, we report that mice with autoimmune oophoritis produce antibodies that bind to a 125-kDa protein localized within the cytoplasm of growing oocytes. Using immune serum from these mice, we have isolated and characterized a novel complementary DNA (cDNA) encoding an oocyte-specific protein that may play a role in autoimmune premature ovarian failure.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Immunohistochemistry and immunoblotting
Normal mouse (B6A, NIH Swiss) ovarian frozen sections were
incubated with mouse serum (20 C, 2 h, diluted 1:20). After
washing with 0.05% Tween-20 in PBS, the sections were incubated (20 C,
2 h, diluted 1:360) with a fluorescein isothiocyanate-conjugated
goat antimouse IgG antibody(Sigma Chemical Co., St. Louis,
MO) and examined by fluorescent microscopy.
Growing oocytes were dissected from 14-day-old mouse ovaries (B6A and NIH Swiss) and solubilized in 0.125 M Tris-HCl, pH 6.8, and 1% SDS. The proteins were separated by SDS-PAGE and transferred onto a nitrocellulose filter at 100 V for 1 h for immunoblotting (23). Mouse sera (1:50 dilution) were incubated with the filter (20 C, 2 h). The filter was washed with Tris-buffered saline buffer containing 0.2% Tween-20 (three times, 20 min each time). Alkaline phosphatase-conjugated goat antimouse IgG antibody (Stratagene, La Jolla, CA) and 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT) as the substrates were used to detect the filter-bound primary antibodies.
Screening ovarian cDNA library
Cells from a mouse ovarian cDNA library (1 x
106 plaque-forming units) (24), a gift from Dr. Jurrien
Dean at NIH, were screened with pooled TX-3 female mouse serum (1:50).
The filter-bound primary antibodies were detected by alkaline
phosphatase-conjugated goat antimouse IgG antibody and BCIP/NBT
substrates using a picoBlue Immunoscreening Kit
(Stratagene). The positive clones were plaque purified,
and plasmids were excision rescued according to the manufacturer’s
directions (Stratagene). Subsequent screening and plaque
purifications were carried out using a 32P-labeled cDNA
probe from the original clone.
Northern hybridization
Total RNA was isolated from 21-day-old mouse ovaries (NIH Swiss)
using RNAzol B (Tel-Test, Inc., Friendswood, TX), and
polyadenylated [poly(A)+] RNA was purified by Oligotex-dT
columns (QIAGEN, Chatsworth, CA). Both mouse ovarian total
RNA and poly(A)+ RNA were separated by 1%
agarose/formaldehyde gel electrophoresis and transferred onto a
nitrocellulose filter. The RNA blot was hybridized (68 C, 2 h)
with 32P-labeled cDNA probe (
1 kb) in QuikHyb solution
(Stratagene). After a final wash (65 C, 0.1 x
SSC-0.1% SDS), hybridization signals were detected by
autoradiography.
Ribonuclease (RNase) protection assay
Antisense RNA probe was prepared by in vitro
transcription (23) using OP1 cDNA [nucleotides (nt) 1848–1987] as a
template, T7 RNA polymerase, and [-32P]UTP (3000
Ci/mmol; ICN Biomedicals, Inc., Irvine, CA). After
examining RNA integrity by gel electrophoresis, 5 µg mouse tissue
total RNA (Ambion, Inc., Austin, TX) was incubated (42 C,
24 h) with the probe (2 x 104 cpm) in 20 µl
hybridization buffer as suggested by the manufacturer of the RNase
protection assay kit (Ambion, Inc.). After digestion (37
C, 1 h) with RNase A/T1 mixture and precipitation, the
hybridization samples were separated on 5% denaturing polyacrylamide
gel for autoradiography. 32P-Labeled antisense RNA for
mouse ß-actin served as a control probe.
In situ hybridization
Ovaries from 12-day-old NIH Swiss mice were fixed in 4%
paraformaldehyde and sectioned (5 µm). Both
[35S]UTP-labeled sense and antisense probes were
synthesized by in vitro transcription using OP1 cDNA as
templates (nt 2149–2831). Short probes (200–400 nt) were prepared by
alkaline hydrolysis and hybridized (60 C, 24 h) to the ovarian
sections. After dipping in Kodak NTB-2 emulsion, the slides were
exposed for 5 days and developed in Kodak develop D-19 and Kodak Fixer
(Molecular Histology, Inc., Gaithersburg, MD).
Rapid amplification of cDNA ends (RACE)-PCR
Using 5'-RACE-PCR (Life Technologies, Grand Island,
NY), RT to synthesize cDNA was carried out by an OP1-specific
oligonucleotide (5'-GCCTCTGTCACTTCATC-3') using mouse ovarian total RNA
(1 µg) as template. After terminal deoxynucleotidyltransferase
tailing, the cDNAs were amplified using 5'-RACE abridged
anchor primer (5'-GG-CCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG-3') and
OP1-specific 3'-primer (5'-CTGGTCCTTGGTCTTTCTGGATTG-3'). Conditions for
PCR reactions were 95 C for 5 min; 35 cycles of 95 C for 1 min, 63 C
for 0.5 min, and 72 C for 2 min; followed by 72 C for 10 min. The PCR
products were subcloned into TA cloning vector
(Invitrogen, San Diego, CA) for DNA sequencing.
DNA sequencing
A dimethylsulfoxide-modified dideoxy chain termination method
(25) was employed to determine cDNA sequence using
[-35S]deoxy-ATP (Amersham, Arlington
Heights, IL) and the Sequenase sequencing kit (U.S. Biochemical Corp., Cleveland, OH). Both strands of the cDNA were sequenced
by T3 and T7 as well as specific internal oligonucleotide primers. The
computer software of the Genetic Computer Group (1991) and PCGene
(IntelliGenetics, Inc., Mountain View, CA) were used for
sequence analysis.
Recombinant protein
A near full-length OP1 cDNA (nt 74–3438) was cloned into
pTrcHisC (Invitrogen), a bacterial expression vector that
contains an Xpress epitope at the N-terminus of recombinant protein.
Protein expression was induced by isopropylthio-ß-galactoside
(1 mM) for 3 h in Escherichia coli (TOP
10). Bacterial lysate was used for SDS-PAGE and immunoblotting. The
recombinant proteins were detected by anti-Xpress antibody (1:1000;
Invitrogen) and different mouse sera (1:50). Alkaline
phosphatase-conjugated goat antimouse IgG antibody and BCIP/NBT
substrates were used to visualize the recombinant protein as described
above.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, a), whereas
serum from control mice did not (Fig. 1A, b and c). To characterize the
protein(s) detected by the immune serum, normal oocytes were isolated
for immunoblotting with immune and control sera. As shown in Fig. 1B, the immune serum (diluted 1:50) detected a 125-kDa oocyte protein that
was not observed with control serum. The specificity of the immune
serum was substantiated by its inability to react with purified mouse
zonae pellucidae (data not shown), a major constituent of the growing
mouse oocyte (26, 27). Thus, the major antigen detected by the immune
sera from mice with ovarian autoimmunity was a 125-kDa protein located
within the oocyte cytoplasm.
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).
The first methionine in the open reading frame was in a context
(ACAATGGGT), recognized as the sequence for a
vertebrate initiator codon (28), and a polyadenylation signal (AATAAA)
was located just 12 bp upstream of the poly(A)+ tail. The
sequence of the cloned OP1 cDNA and deduced polypeptide chain (Fig. 2) had no significant homology with DNA
and protein sequences in GenBank. Computer analysis revealed three
potential antigenic sites with high hydrophilicity present in the OP1
protein: amino acids 133–138 (SKEEDE), 761–766 (KDDDMK), and
1101–1106 (SDEDDR) (29). The protein had no structural motifs,
indicating that it was secreted or compartmentalized in the nucleus or
plasma membrane. These data were consistent with the
immunohistochemical data that localized OP1 within the oocyte
cytoplasm. Two identical highly charged regions (11 residues of 28)
present near the amino-terminus were not detected in other proteins
currently in GenBank, and a leucine zipper motif raises the possibility
that OP1 may interact with other proteins in the cytoplasm. There was a
typical ATP/GTP binding motif (P-loop) in OP1 from amino acids 197–204
(GRPGVGKS) (30), and the protein contained several kinase
phosphorylation sites for cAMP- and cGMP-dependent protein kinases,
protein kinase C, and casein kinase II (31, 32, 33).
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). The size of the OP1 transcript
corresponded to the isolated cDNA (3447 bp), assuming the presence of a
poly(A)+ tail (150–200 nt) and the 5'-noncoding regions
(68 and 119 nt). Using a RNase protection assay (Fig. 3B) and an
OP1-specific probe (180 nt), OP1 transcripts were detected in total RNA
isolated from the ovary (lane 10), but not from nine other mouse
tissues. As a control, a ß-actin-specific probe (300 nt) was used to
detect ß-actin transcripts in RNA isolated from all tissues (data not
shown).
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). Synthetic antisense OP1 transcripts
hybridized with the endogenous OP1 RNA within the cytoplasm of growing
oocytes as well as the resting oocytes that were localized at the
periphery of the ovary. As a control, synthetic sense OP1 transcripts
did not hybridize to the ovarian tissue. This restricted expression of
the Op1 gene was in accord with the immunolocalization of
the OP1 protein in the cytoplasm of growing oocytes.
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, anti-Xpress antibody identified an epitope in the N-terminal region of
the recombinant OP1 (125 kDa) and control chloramphenicol
acetyltransferase (32 kDa). The recombinant OP1 reacted specifically
with the immune serum from mice with autoimmune premature ovarian
failure (Fig. 5E). We obtained the same results from three separate
pools of the immune sera containing antibodies against the oocyte
cytoplasm. Taken together, these data suggested that protein encoded by
the OP1 cDNA was the ooplasm protein bound by the immune serum from
mice with ovarian autoimmunity.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study we show that the mice with autoimmune oophoritis produce antibodies that bind to a 125-kDa protein located within the oocyte cytoplasm. Using immune serum from affected mice to screen an expression library, we cloned ovarian cDNA that contains an open reading frame encoding a novel protein, OP1. The primary structure of OP1 was deduced from the nucleic acid sequence and predicts a cytoplasmic protein with a molecular mass of 125,502 Da. The gene encoding OP1 is expressed uniquely in oocytes, and recombinant OP1 protein reacts specifically with immune serum from mice with autoimmune premature ovarian failure. Although these data are consistent with OP1 being the oocyte-specific protein recognized by immune sera from mice with autoimmune premature ovarian failure, they do not demonstrate disease causality. To establish a sequential relationship between the development of autoimmune ovarian disease and the production of these autoantibodies, we plan to measure anti-OP1 antibodies in serum samples from mice in the process of developing premature ovarian failure. In addition, efforts are currently underway to immunize mice susceptible to autoimmune oophoritis to induce tolerance to OP1 and determine the effect on the course of disease.
There are striking similarities between human autoimmune premature ovarian failure and the autoimmune oophoritis that develops in the mice after neonatal thymectomy. Both have similar histological distribution of the ovarian lymphocytic infiltration (36, 37), the production of antiovary autoantibodies (4, 5, 6, 7, 9) and a reduced natural killer cell activity (38, 39). The susceptibility to the ovarian disorder in both appears to be associated with the genes outside the major histocompatibility complex (40, 41, 42). T cells have been strongly implicated in the pathogenesis of autoimmune premature ovarian failure in the mouse model, and the oophoritis can be adoptively transferred by cells with a T helper phenotype (11, 12). To define the pathogenesis of T cell-mediated autoimmune diseases, it is of primary importance to identify the antigens targeted by the T cells.
In some cases the inciting antigen in autoimmune disease is a common target of both autoreactive B and T cells. For example, in nonobese diabetic mice the appearance of autoantibodies against glutamic acid decarboxylase (GAD) is coincident with the onset of the T cell-mediated destruction of the insulin-producing ß-islet cells of the pancreas. GAD has been confirmed as the inciting antigen based on experiments showing that induction of specific tolerance to this molecule ameliorates the entire spectrum of this disease (34, 35). In murine autoimmune gastritis, another organ-specific autoimmune disorder induced by neonatal thymectomy, the ß-subunit of H/K adenosine triphosphatase is a common target of autoreactive B and T cells. Similar to GAD, the ß-subunit of H/K adenosine triphosphatase has been shown to be the inciting antigen by induction of specific tolerance to this protein (43). Interestingly, the protein that serves as the inciting antigen for the organ-specific autoimmune disease often performs a function critical to the specific organ. The function of the OP1 protein is presently unknown. However, the OP1 molecule has an ATP-/GTP-binding domain and multiple phosphorylation sites of protein kinases. This suggests a potential role for OP1 in the signal transduction and intracellular regulation in the oocyte.
Our data suggest that OP1 is one of important ovarian-specific antigens in the mouse model of autoimmune premature ovarian failure. However, it remains to be determined whether the antigen(s) responsible for disease in the thymectomized mouse model pertain to human disease. Antibodies against oocyte proteins are detected infrequently in women with premature ovarian failure, but that may primarily reflect the low sensitivity of immunological methods. Women with premature ovarian failure as a group are known to have circulating antibodies that are capable of precipitating more radiolabeled ovarian protein than control serum (14). The specific proteins precipitated by these antibodies have not been characterized, and the identification of etiological antigens remains an important goal in understanding the human disease. By characterizing the human OP1 protein, it may be possible to establish an assay to determine whether a subset of patients has circulating anti-OP1 antibodies and, thus, gain further insight into the pathogenesis of human autoimmune premature ovarian failure.
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Acknowledgments |
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Received December 7, 1998.
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References |
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inhibits gonadotropin hormonal action in
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