[Show abstract][Hide abstract] ABSTRACT: Adenomyosis, defined as ectopic endometrial tissue within the myometrium, can often be misdiagnosed as multiple uterine leiomyomata or endometrial thickening. We therefore performed a combined mRNA and long noncoding (lnc)RNA microarray and bioinformatic analysis of eutopic and ectopic endometria in women with adenomyosis to better understand its pathogenesis and help in the development of a semi-invasive diagnostic test. A total of 586 mRNAs were increased and 305 mRNAs decreased in the ectopic endometrium of adenomyosis compared with the eutopic endometrium, while 388 lncRNA transcripts were up-regulated and 188 down-regulated in ectopic compared with paired eutopic endometrial tissue. Bioinformatic analysis suggested a series of metabolic and molecular abnormalities in adenomyosis, which have many similarities with endometriosis. Furthermore, our study constitutes the first known report of lncRNA expression patterns in human adenomyosis ectopic and eutopic endometrial tissue.
No preview · Article · Jan 2016 · Molecular BioSystems
[Show abstract][Hide abstract] ABSTRACT: Primordial germ cells (PGCs) are embryonic founders of germ cells that ultimately differentiate into oocytes and spermatogonia. Embryonic proliferation of PGCs starting from E11.5 ensures the presence of germ cells in adulthood, especially in female mammals whose total number of oocytes declines after this initial proliferation period. To better understand mechanisms underlying PGC proliferation in female mice, we constructed a proteome profile of female mouse gonads at E11.5. Subsequent KEGG pathway analysis of the 3,662 proteins profiled showed significant enrichment of pathways involved in fatty acid degradation. Further, the number of PGCs found in in vitro cultured fetal gonads significantly decreased with application of etomoxir, an inhibitor of the key rate-limiting enzyme of fatty acid degradation carnitine acyltransferase I (CPT1). Decrease in PGCs was further determined to be the result of reduced proliferation rather than apoptosis. The inhibition of fatty acid degradation by etomoxir has the potential to activate the Ca(2+)/CamKII/5'-adenosine monophosphate-activated protein kinase (AMPK) pathway; while as an upstream activator, activated AMPK can function as activator of p53 to induce cell cycle arrest. Thus, we detected the expressional level of AMPK, phosphorylated AMPK (P-AMPK), phosphorylated p53 (P-p53) and cyclin-dependent kinase inhibitor 1 (p21) by Western blots, the results showed increased expression of them after treatment with etomoxir, suggested the activation of p53 pathway was the reason for reduced proliferation of PGCs. Finally, the involvement of p53-dependent G1 cell cycle arrest in defective proliferation of PGCs was verified by rescue experiments. Our results demonstrate that fatty acid degradation plays an important role in proliferation of female PGCs via the p53-dependent cell cycle regulation.
No preview · Article · Dec 2015 · Cell cycle (Georgetown, Tex.)
[Show abstract][Hide abstract] ABSTRACT: Uterine stromal cells undergo extensive proliferation and differentiation during postimplantation development, a process known as decidualization. While a range of signaling molecules have been demonstrated to play essential roles in this event, its potential epigenetic regulatory mechanisms remained largely unknown. Retinoblastoma binding protein 7 (Rbbp7) is a protein reported as a core component of many histone modification and chromatin remodeling complexes. In the present study, our in situ hybridization and immunochemistry analysis first revealed a spatiotemporal expression of Rbbp7 in the uterus during the periimplantation period. Observations of remarkable induction of Rbbp7 expression in uterine stromal cells in response to progesterone-nuclear receptor PR signaling pointed toward to its potential physiological significance during postimplantation uterine development. Employing stealth RNA knockdown approach combined with primary murine uterine stromal cell culture and in vitro induced decidualization model, we further demonstrated that Rbbp7 silencing compromises stromal cell decidualization via attenuating histone H4 acetylation and cyclin D3 expression. The results collectively suggested that Rbbp7 is a potentially functional player regulating normal histone acetylation modification and cyclin D3 expression in stromal cells during postimplantation decidual development.
Copyright 2015 by The Society for the Study of Reproduction.
Full-text · Article · Jun 2015 · Biology of Reproduction
[Show abstract][Hide abstract] ABSTRACT: In mammals, pronucleus formation, a landmark event for egg activation and fertilization, is critical for embryonic development. However, the mechanisms underlying pronucleus formation remain unclear. Increasing evidence has shown that the transition from a mature egg to a developing embryo and the early steps of development are driven by the control of maternal cytoplasmic factors. Herein, a two-dimensional-electrophoresis-based proteomic approach was used in metaphase II and parthenogenetically activated mouse eggs to search for maternal proteins involved in egg activation, one of which was poly(rC)-binding protein 1 (PCBP1). Phosphoprotein staining indicated that PCBP1 displayed dephosphorylation in parthenogenetically activated egg, which possibly boosts its ability to bind to mRNAs. We identified 75 mRNAs expressed in mouse eggs that contained the characteristic PCBP1-binding CU-rich sequence in the 3'-UTR. Among them, we focused on H2a.x mRNA, as it was closely related to pronucleus formation in Xenopus oocytes. Further studies suggested that PCBP1 could bind to H2a.x mRNA and enhance its stability, thus promoting mouse pronucleus formation during parthenogenetic activation of murine eggs, while the inhibition of PCBP1 evidently retarded pronucleus formation. In summary, these data propose that PCBP1 may serve as a novel maternal factor that is required for determining the normal timing of pronucleus formation.
No preview · Article · Apr 2015 · Cellular and Molecular Life Sciences CMLS
[Show abstract][Hide abstract] ABSTRACT: DDX3X is a highly conserved DEAD-box RNA helicase that participates in RNA transcription, RNA splicing, and mRNA transport, translation, and nucleo-cytoplasmic transport. It is highly expressed in metaphase II (MII) oocytes and is the predominant DDX3 variant in the ovary and embryo. However, whether it is important in mouse early embryo development remains unknown. In this study, we investigated the function of DDX3X in early embryogenesis by cytoplasmic microinjection with its siRNA in zygotes or single blastomeres of 2-cell embryos. Our results showed that knockdown of Ddx3x in zygote cytoplasm led to dramatically diminished blastocyst formation, reduced cell numbers, and an increase in the number of apoptotic cells in blastocysts. Meanwhile, there was an accumulation of p53 in RNAi blastocysts. In addition, the ratio of cell cycle arrest during 2-cell to 4-cell transition increased following microinjection of Ddx3x siRNA into single blastomeres of 2-cell embryos compared with control. These results suggest that Ddx3x is an essential gene associated with cell survival and cell cycle control in mouse early embryos, and thus plays key roles in normal embryo development.
[Show abstract][Hide abstract] ABSTRACT: Dairy products are considered as nutrient-dense foods and consumed by many people in western countries, as well as an increasing number of Asian people. Excessive and frequent application of pesticides on vegetables and fruits leads to a potential health hazard to consumers. The organophosphate insecticide chlorpyrifos has been reported to bind with human and bovine serum albumin. Thus, it is necessary to explore the interaction between food protein and chlorpyrifos. In this study, equilibrium dialysis and fluorescence spectra were used to demonstrate binding of milk proteins to chlorpyrifos. The amount of milk protein bound was 0.03 ± 0.01 mg/g. Moreover, the milk protein-chlorpyrifos complexes were stable at pH 3.5 to 9.5 and ion concentrations from 0.1 to 1.0 M. The amount of chlorpyrifos bound to milk proteins decreased to 50% after being in vitro digested by pepsin and trypsin. The results showed that the interaction between food proteins and the pesticide might partially remove the insecticide and reduce the concentration of pesticide absorbed into the blood and, thus, alleviate the corresponding toxicity.
No preview · Article · Apr 2014 · Journal of Dairy Science
[Show abstract][Hide abstract] ABSTRACT: Many pregnant women suffer miscarriages during early gestation, but the description of these early pregnancy losses (EPL) can be somewhat confusing because of the complexities of early development. Thus, the identification of proteins with different expression profiles related to early pregnancy loss is essential for understanding the comprehensive pathophysiological mechanism. In this study, we report a gel-free tandem mass tags- (TMT-) labeling based proteomic analysis of five placental villous tissues from patients with early pregnancy loss and five from normal pregnant women. The application of this method resulted in the identification of 3423 proteins and 19647 peptides among the patient group and the matched normal control group. Qualitative and quantitative proteomic analysis revealed 51 proteins to be differentially abundant between the two groups (≥1.2-fold, Student's
). To obtain an overview of the biological functions of the proteins whose expression levels altered significantly in EPL group, gene ontology analysis was performed. We also investigated the twelve proteins with a difference over 1.5-fold using pathways analysis. Our results demonstrate that the gel-free TMT-based proteomic approach allows the quantification of differences in protein expression levels, which is useful for obtaining molecular insights into early pregnancy loss.
[Show abstract][Hide abstract] ABSTRACT: Ovarian physiology and pathology are important areas of scientific research. Efforts have been made to identify the ovary-related transcriptomes in different species. However, the proteomic studies are limited. The rhesus monkey is very similar to humans, and it is widely used in the study of reproductive biology and medicine. In this study, using an optimized proteomics platform, we successfully identified 5723 rhesus ovarian proteins, of which 4325 proteins were consistently identified in all three replicates and with at least 2 unique peptides. The 4325 proteins were chosen for further analysis. Through gene ontology and pathway analyses, we obtained a preliminary understanding of the function of these proteins. A random immunohistochemistry analysis was used to determine the expression of proteins in various cell types. By comparing the genes identified in this study with genes that were reported to have relatively high levels of expression in human oocytes, we obtained genes that were predicted to play roles in maintenance of normal ovarian physiology. Searching the identified genes from this study against the MGI database gave us a list of proteins those exist in the rhesus monkey ovary and are important for female mouse reproduction as well. The overlap of genes in this study and the genes whose abnormal expression or dysfunction were reported to be associated with human polycystic ovary syndrome (PCOS) and premature ovarian failure (POF) prompted us to use the rhesus monkey to study these two common causes of female infertility. This study may provide a basis for future studies of human reproductive disorders using the rhesus monkey as a model.
Full-text · Article · Jan 2014 · Molecular BioSystems
[Show abstract][Hide abstract] ABSTRACT: RING box protein-1 (RBX1) is an essential component of Skp1-cullin-F-box protein (SCF) E3 ubiquitin ligase and participates in diverse cellular processes by targeting various substrates for degradation. However, the physiological function of RBX1 in mouse oocyte maturation remains unknown. Here, we examined the expression, localization and function of RBX1 during mouse oocyte meiotic maturation. Immunofluorescence analysis showed that RBX1 displayed dynamic distribution during the maturation process: it localized around and migrated along with the spindle and condensed chromosomes. Rbx1 knockdown with the appropriate siRNAs led to a decreased rate of first polar body extrusion and most oocytes were arrested at metaphase I. Moreover, downregulation of Rbx1 caused accumulation of Emi1, an inhibitor of the anaphase-promoting complex/cyclosome (APC/C), which is required for mouse meiotic maturation. In addition, we found apparently increased expression of the homologue disjunction-associated protein securin and cyclin B1, which are substrates of APC/C E3 ligase and need to be degraded for meiotic progression. These results indicate the essential role of the SCF(βTrCP)-EMI1-APC/C axis in mouse oocyte meiotic maturation. In conclusion, we provide evidence for the indispensable role of RBX1 in mouse oocyte meiotic maturation.
[Show abstract][Hide abstract] ABSTRACT: In the mammalian ovary, primordial follicles are generated early in life and remain dormant for prolonged periods. Their growth resumes via primordial follicle activation and they continue to grow until the preovulatory stage under the regulation of hormones and growth factors, such as estrogen, follicle stimulating hormone (FSH), and insulin-like growth factor-I (IGF-I). Both FSH and IGF-I activate the PI3K/Akt signaling pathway in granulosa cells (GCs), yet it remains inconclusive whether the PI3K pathway is crucial for follicle growth. In this study, we investigated the p110δ-isoform (encoded by the Pik3cd gene) of PI3K catalytic subunit expression in the mouse ovary and its function in fertility. Pik3cd null females were subfertile, exhibited fewer growing follicles and more atretic antral follicles in the ovary, and responded poorly to exogenous gonadotropins compared to controls. Ovary transplantation showed that Pik3cd null ovaries responded poorly to FSH stimulation in vitro; this confirmed that the follicle growth defect was intrinsically ovarian. In addition, estradiol (E2)-stimulated follicle growth and GC proliferation in preantral follicles was impaired in Pik3cd null ovaries. FSH and E2 substantially activated the PI3K/Akt pathway in GCs of control mice, but not in those of Pik3cd null mice. However, primordial follicle activation and oocyte meiotic maturation were not affected by Pik3cd knockout. Taken together, our findings indicate that the p110δ-isoform of the PI3K catalytic subunit is a key component of the PI3K pathway for both FSH and E2-stimulated follicle growth in ovarian granulosa cells; however, it is not required for primordial follicle activation and oocyte development.
No preview · Article · Jul 2013 · Molecular Endocrinology
[Show abstract][Hide abstract] ABSTRACT: Global transcriptional silencing in fully grown oocytes is a critical event during mammalian oogenesis. However, how this event is regulated remains elusive. Here, we provide evidence that poly(rC)-binding protein 1 (PCBP1), a protein found by us previously to be present in metaphase II (MII) mouse oocytes, participates in maintenance of the transcriptionally silent state in fully grown mouse oocytes. Knocking down Pcbp1 by microinjection of its specific siRNAs into fully grown germinal vesicle (GV) oocytes resulted in remarkable changes in their transcriptional state, including the disequilibrium between the number of oocytes with an NSN (non-surrounded nucleolus) and those with a SN (surrounded nucleolus), and obvious transcriptional reactiviation in oocytes with a SN configuration as evidenced by BrUTP incorporation assay and immunofluorescent labeling of phosphorylated RNA polymerase II CTD and trimethylated H3 lysine 4, markers for active transcription. Furthermore, in a comprehensive microarray analysis of the preovulatory oocyte transcriptome, an incredible number of nearly 4,000 transcripts were upregulated in the Pcbp1 knockdown groups. These data indicate that lack of the function of PCBP1 disrupts the quiescent status of transcription in the fully grown oocytes, and hence supporting a role of this protein in the regulation of global transcriptional silcencing in fully grown mouse oocytes.
[Show abstract][Hide abstract] ABSTRACT: Gestational diabetes mellitus (GDM) is one type of diabetes that presents during pregnancy and significantly increases the risk of a number of adverse consequences for the fetus and mother. The microRNAs (miRNA) have recently been demonstrated to abundantly and stably exist in serum and to be potentially disease-specific. However, no reported study investigates the associations between serum miRNA and GDM.
We systematically used the TaqMan Low Density Array followed by individual quantitative reverse transcription polymerase chain reaction assays to screen miRNAs in serum collected at 16-19 gestational weeks. The expression levels of three miRNAs (miR-132, miR-29a and miR-222) were significantly decreased in GDM women with respect to the controls in similar gestational weeks in our discovery evaluation and internal validation, and two miRNAs (miR-29a and miR-222) were also consistently validated in two-centric external validation sample sets. In addition, the knockdown of miR-29a could increase Insulin-induced gene 1 (Insig1) expression level and subsequently the level of Phosphoenolpyruvate Carboxy Kinase2 (PCK2) in HepG2 cell lines.
Serum miRNAs are differentially expressed between GDM women and controls and could be candidate biomarkers for predicting GDM. The utility of miR-29a, miR-222 and miR-132 as serum-based non-invasive biomarkers warrants further evaluation and optimization.
[Show abstract][Hide abstract] ABSTRACT: Epigenetic regulation is considered one of the most important mechanisms by which changes in gene expression occur without changes in the underlying DNA sequence. More and more studies have shown that this kind of regulation plays a very important role during the process of early embryonic development. Methylation of histones is a special process in epigenetic regulations that plays a dual role: some activate gene expression, while some inhibit it; trimethylation of histone 3 lysine 4 has been shown to be a marker of gene expression activation. Previous research has led us to focus on the role of WDR82, which has been shown to recognize a subunit in the methyltransferases complex that catalyzes H3K4me3 in early embryonic development. Although it has been shown that a defect in WDR82 causes dysfunction of SETD1A/SETD1B and results in loss of H3K4me3 in human cell lines, the exact role of WDR82 in the methyltransferases complex during early embryonic development is not clear. Our study has shown that a defect in WDR82 causes dysfunction of SETD1A/SETD1B and affects the normal H3K4me3 status in the transcription start region of POU5F1, which then causes the down-regulation of POU5F1 as well as its downstream factors STAT3/BIRC5, which are responsible for the extremely high apoptotic rates of blastocysts. Finally, the result of a blocked WDR82 consists of stunted embryonic development and death. Thus, WDR82 can be considered a key epigenetic regulation-related factor crucial in the normal growth and development of embryos.
No preview · Article · Dec 2010 · Biology of Reproduction
[Show abstract][Hide abstract] ABSTRACT: Membrane modifications in sperm cells represent a key step in sperm capacitation; however, the molecular basis of these modifications is not fully understood. Ezrin is the best-studied member of the ezrin/radixin/merlin family. As a cross-linker between the cortical cytoskeleton and plasma membrane proteins, ezrin contributes to remodeling of the membrane surface structure. Furthermore, activated ezrin and the Rho dissociation inhibitor, RhoGDI, promote the formation of cortical cytoskeleton-polymerized actin through Rho activation. Thus, ezrin, actin, RhoGDI, Rho and plasma membrane proteins form a complicated network in vivo, which contributes to the assembly of the structure of the membrane surface. Previously, we showed that ezrin and RhoGDI1 are expressed in human testes. Thus, we sought to determine whether the ezrin-RhoGDI1-actin-membrane protein network has a role in human sperm capacitation. Our results by Western blot indicate that ezrin is activated by phosphorylation of the threonine567 residue during capacitation. Co-immunoprecipitation studies revealed that, during sperm capacitation, the interaction between ezrin and RhoGDI1 increases, and phosphostaining of two dimensional electrophoresis gels showed that RhoGDI1 is phosphorylated, suggesting that RhoGDI1 dissociates from RhoA and leads to actin polymerization on the sperm head. We speculate that activated ezrin interacts with polymerized actin and the glycosylated membrane protein cd44 after capacitation. Blocking sperm capacitation using ezrin- or actin-specific monoclonal antibodies decreases their acrosome reaction (AR) rate, but has no effect on the AR alone. Taken together, our results show that a network consisting of ezrin, RhoGDI1, RhoA, F-actin and membrane proteins functions to influence the modifications that occur on the membrane of the sperm head during human sperm capacitation.
Preview · Article · Sep 2010 · Asian Journal of Andrology
[Show abstract][Hide abstract] ABSTRACT: The testis is the male gonad responsible for spermatogenesis and male hormone secretion. The complicated processes of spermatogenesis and steroidogenesis determine the complexity of protein expression control in the testis. In this study, the heterogeneity of human testis proteins was investigated using 2-dimensional gel electrophoresis. A total of 847 protein spots corresponding to 462 unique proteins were identified successfully by mass spectrometry. Notable heterogeneity was evidenced by the presence of more than 1 spot with different molecular weight and/or Isoelectric point values for each of 180 different proteins. Analysis of the detected peptides of these proteins indicated that this heterogeneity was partly the result of alternative splicing and/or proteolysis. SP_PIR_Keywords analysis suggested that alternative initiation sites and various forms of posttranslational modification may also contribute toward this heterogeneity. Using Pro-Q Diamond phosphostain, 68 spots representing 52 proteins were stained, confirming the presence of phosphorylated forms of these proteins in the human testis. These data were used to establish a proteome reference database, which can be accessed over the Internet (http://reprod.njmu.edu.cn/2d). This database provides an initial reference map of the human testis and serves as a useful resource for comparative proteomics studies of the human testis under normal and pathological states. The abundant protein heterogeneity observed in this study and further investigation of its biological significance will contribute toward understanding protein expression regulation in the human testis and will generate insight into the molecular mechanism of spermatogenesis.
Full-text · Article · Jul 2010 · Journal of Andrology
[Show abstract][Hide abstract] ABSTRACT: Spermiogenesis is a unique process in mammals during which haploid round spermatids mature into spermatozoa in the testis. Its successful completion is necessary for fertilization and its malfunction is an important cause of male infertility. Here, we report the high-confidence identification of 2116 proteins in mouse haploid germ cells undergoing spermiogenesis: 299 of these were testis-specific and 155 were novel. Analysis of these proteins showed many proteins possibly functioning in unique processes of spermiogenesis. Of the 84 proteins annotated to be involved in vesicle-related events, VAMP4 was shown to be important for acrosome biogenesis by in vivo knockdown experiments. Knockdown of VAMP4 caused defects of acrosomal vesicle fusion and significantly increased head abnormalities in spermatids from testis and sperm from the cauda epididymis. Analysis of chromosomal distribution of the haploid genes showed underrepresentation on the X chromosome and overrepresentation on chromosome 11, which were due to meiotic sex chromosome inactivation and expansion of testis-expressed gene families, respectively. Comparison with transcriptional data showed translational regulation during spermiogenesis. This characterization of proteins involved in spermiogenesis provides an inventory of proteins useful for understanding the mechanisms of male infertility and may provide candidates for drug targets for male contraception and male infertility.
No preview · Article · Mar 2010 · Journal of Proteome Research
[Show abstract][Hide abstract] ABSTRACT: Compaction, occurring at the eight-cell stage of mouse development, is the process of cell flattening and polarization by which cellular asymmetry is first established. During this process many molecules and organelles undergo polarized distribution, but the cytoskeletal basis for these distribution specifications remains to be explored. The present study focused on cofilin1, an actin-binding protein that depolymerizes actin filaments. We showed that cofilin1 expression decreased at the compaction stage, and that down-regulation of cofilin1 expression by siRNA microinjection accelerated compaction. Continuous observation using time-lapse video miscroscopy confirmed these findings. That is, the embryonic cells microinjected with anti-cofilin1 antibody exhibit earlier adherence properties compared to uninjected cells. Pronuclear microinjection of a site-directed mutated cofilin1 plasmid, in which cofilin1 is sustained in its active form produced embryos with blastomeres that did not adhere, suggesting that inactivation of cofilin1 is critical for cell flattening and adherence. Fluorescein-phalloidin staining indicated that decreased cofilin1 expression promoted the formation of the apical pole, which is a marker for polarity. Scanning electron microscopy results demonstrated the appearance of microvilli on the outer face of blastomeres in cofilin1 knockdown embryos. Our results suggest that cofilin1 plays an important role in cortical cytoplasmic organization during embryo compaction.
[Show abstract][Hide abstract] ABSTRACT: The mature mouse oocyte contains the full complement of maternal proteins required for fertilization, reprogramming, zygotic gene activation (ZGA), and the early stages of embryogenesis. However, due to limitations of traditional proteomics strategies, only a few abundantly expressed proteins have yet been identified. Our laboratory applied a more effective strategy: one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D SDS-PAGE) and reverse-phase liquid chromatography tandem mass spectrometry (RP-LC-MS/MS) were employed to analyze the mature oocyte proteome in depth.
Using this high-performance proteomic approach, we successfully identified 625 different proteins from 2700 mature mouse oocytes lacking zona pellucidae. This is the largest catalog of mature mouse oocyte proteins compiled to date. According to their pattern of expression, we screened 76 maternal proteins with high levels of mRNA expression both in oocytes and fertilized eggs. Many well-known maternal effect proteins were included in this subset, including MATER and NPM2. In addition, our mouse oocyte proteome was compared with a recently published mouse embryonic stem cell (ESC) proteome and 371 overlapping proteins were identified.
This proteomics analysis will be a valuable resource to aid in the characterization of important maternal proteins involved in oogenesis, fertilization, early embryonic development and in revealing their mechanisms of action.
[Show abstract][Hide abstract] ABSTRACT: The assembly of primordial follicles early in ovarian development and subsequent transition to primary follicles are critical processes in ovarian biology. Inappropriate coordination of these processes contributes to ovarian pathologies such as premature ovarian failure and infertility. To better understand the molecular mechanisms involved in primordial follicle assembly and development, 2-D PAGE and MALDI-TOF/TOF technologies were used to construct a comparative proteome profile of the immature rat ovary at specific time-points (0, 24, 48, and 72 h postpartum). A total of 154 differential protein spots corresponding to 134 different proteins were definitively identified between any two time-points. Further cluster analysis showed four expression patterns, and each pattern correlated with specific cell processes that occur during early ovarian development. Seven proteins were randomly selected to verify expression patterns using Western blotting, and subsequently immunohistochemistry was performed to further investigate their cellular localization. Additionally, detailed functional analyses of these differentially expressed proteins were performed. Elucidation of how these changes in protein expression level coordinate primordial follicles assembly and development is intended to provide a better understanding of these critical biological processes early in ovarian development and will provide potential therapeutic molecular targets to regulate ovarian function and treat ovarian disease.