Arrest at primordial follicle (PMF) stage and growth initiation into primary follicles (PRIF) are the crucial process for the female fertility. However, factors and mechanisms regulate these processes are poorly understood. Previously, using suppression subtractive hybridization (SSH) between day1 and day5 mouse ovaries, we found abundantly expressed 15 ESTs (expressed sequence tags) in day5-subtracted cDNA library, by comparison with day1-subtracted cDNA library. Among 15 ESTs, 5-25 clone (RIKEN cDNA E330009P21 gene) was a novel EST contig and selected for further study. The present study was conducted by EST clustering to obtain the full-length sequence containing that novel EST contig and further evaluated the expression of the novel gene in the mouse ovary. Experimental study to find novel gene by the in silico method and characterize its expression in ovary. We performed EST clustering and cloned long sequence of 5-25 clone by 5' Rapid Amplification of cDNA Ends (RACE). This novel sequence was similar to the registered BC085247 gene (F-box and WD40-repeat-containing protein; FBWD40). Differential expression of FBWD40 mRNA was evaluated at various postnatal stage by real-time PCR. To determine the tissue distribution of FBWD40 expression, we performed Northern blot and in situ hybridization. Expression of FBWD40 transcript was ovary-specific and highly expressed in 5-day mouse ovary and decreased thereafter. Moreover, localization revealed that the expression of FBWD40 transcript is oocyte-specific from primordial to the preovulatory follicles, and detected more in nucleus. Localization of protein expression by immunohistochemistry and Western blot is in processing. We succeeded in silico finding of the novel gene that is ovary- and oocyte-specific. This is the first report of oocyte-specific expression of the FBWD40-like sequence. We suggest that oocyte-specific expression of the FBWD40 mRNA imply its significant role(s) in growth and maturation of oocytes and/or follicles during early folliculogenesis.

Siah is murine homologue of the Drosophila seven in absentia gene (sina) and has three homologues (siah1a, siah1b, and siah2). To elucidate the unique function of Siah family members, we investigated the differential expression of the three members during ovarian folliculogenesis. Experimental study using mouse ovarian organ culture system in a Millicell CM inserts. Measurement of mRNA transcripts by quantitative real time PCR analysis. Total RNA was extracted from various mouse tissues and developmentally sampled ovaries and testes (1-day, 5-day, 2-week, 3-week, 4-week, 6-week). To investigate the expression pattern of these transcripts during the early folliculogenesis in vitro, ovaries from ICR neonates (the day of birth) were cultured for 4 days (for primordial to primary transition) or 8 days (for secondary follicle formation). The highly homologous Siah1a and 1b was measured using the UTR-specific primers (3’UTR for 1a and 5’UTR for 1b). Siah1a was ubiquitously expressed in many tissues including ovary and testis, whereas Siah1b was ovary-specific. Siah2 was expressed tissue-specific in brain, kidney, and ovary, but not in testis. In the ovary, all of the homologues were oocyte-specific and highly expressed in neonatal 1-day ovaries. Siah1a and Siah2 expression was monotonous independent of ovarian development. Interestingly, only Siah1b was gradually decreased according to the ovarian developmental stages. However, developmentally decreased endogenous Shia1b was induced during in vitro culture on day 8. This is the first report regarding the potential unique function of Siah1b as an oocyte-specific regulator for folliculogenesis. We conclude that it will be very valuable to evaluate mRNA expression of Siah1b as a novel developmental marker, related in vitro as well as in vivo ovarian folliculogenesis.

Granulosa cells surround the oocyte within the ovarian follicle and play an essential role in creating conditions required for oocyte as well as follicular development. Previously, we sought to compile a list of genes expressed during mouse early folliculogenesis using cDNA microarray, and found differential expression of eight genes related to ion transport, especially potassium ion. To elucidate their involvement in the regulation of ovarian follicular development, we characterized the expression of 8 genes. Those were Atp1a1, Atp1b3, Gria1, Kcna5, Kcnab1, Kcnj8, Kcnk1, and RIKEN 2410004N11 (human K+ channel tetramerization protein (GMRP-1) like sequence). Confirmation by quantitative real-time PCR analysis after cDNA microarray To characterize a group of ion transport-related genes, total RNA was extracted from various mouse tissues including ovaries according to developmental stages (1-day, 5-day, 2-week, 3-week, 4-week), and the specific mRNA expressions were measured by quantitative real-time PCR analysis. Expression of Gria1 (ovary, uterus, brain), Kcna5 (ovary, brain, heart), and Kcnab1 (ovary, testis, brain) was tissue-specific and differentially regulated during folliculogenesis whereas the others were ubiquitously expressed in various tissues. During the ovarian development, expression of Gria1 and Kcna5 transcripts was gradually decreased, whereas that of Kcnab1 transcript was gradually increased. Expression of the other 5 genes was not changed according to the ovarian development. This differential expression pattern of different ion transport-related genes suggested intricate, spatial, temporal regulatory mechanisms on the ovarian folliculogenesis through the regulation of ion transport system. Importance of regulation of potassium ion is advocated.

ObjectiveIn the previous study, we found the 28 novel genes including hypothetical proteins using suppression subtractive hybridization (SSH) between day1 and day5 mouse ovaries. The present study was conducted to further characterize the expression of novel genes during the mouse ovarian folliculogenesis.DesignExperimental study using SSH, Real-time PCR, RT-PCR,in situ hybridizationMaterials and methodsThe 28 novel genes were analyzed based on the description of mouse reference genomic contigs to determine mouse chromosomal localization. We performed the mouse chromosomal alignment in the NCBI database (http: //ww.ncbi.nlm.nih.gov) and Ensembl database (http: //www.ensemble.org), and found 15 genes as single copy genes in the mouse chromosome. These 15 selected novel genes were characterized further by molecular biological methods.ResultsAll 15 genes were expressed in various tissues and none was ovary-specific. Of those 15 genes, however, 10 genes showed high expression in day1 ovaries and decreased thereafter developmental stages. The in situ hybridization was conducted for those 10 genes and revealed that 3 genes were oocyte-specific. Those are hypothetical proteins LOC228358 and LOC232560(c1q), and mKIAA0978 protein. The mRNA expression was cytoplasmic from primordial to the preovulatory follicles.ConclusionWe succeeded in finding 3 novel oocyte-specific genes by using subtractive hybridization, Real-time PCR and in situ hybridization. This study suggests that these novel genes may play role(s) in early follicular development in the mouse oocyte. ObjectiveIn the previous study, we found the 28 novel genes including hypothetical proteins using suppression subtractive hybridization (SSH) between day1 and day5 mouse ovaries. The present study was conducted to further characterize the expression of novel genes during the mouse ovarian folliculogenesis. In the previous study, we found the 28 novel genes including hypothetical proteins using suppression subtractive hybridization (SSH) between day1 and day5 mouse ovaries. The present study was conducted to further characterize the expression of novel genes during the mouse ovarian folliculogenesis. DesignExperimental study using SSH, Real-time PCR, RT-PCR,in situ hybridization Experimental study using SSH, Real-time PCR, RT-PCR,in situ hybridization Materials and methodsThe 28 novel genes were analyzed based on the description of mouse reference genomic contigs to determine mouse chromosomal localization. We performed the mouse chromosomal alignment in the NCBI database (http: //ww.ncbi.nlm.nih.gov) and Ensembl database (http: //www.ensemble.org), and found 15 genes as single copy genes in the mouse chromosome. These 15 selected novel genes were characterized further by molecular biological methods. The 28 novel genes were analyzed based on the description of mouse reference genomic contigs to determine mouse chromosomal localization. We performed the mouse chromosomal alignment in the NCBI database (http: //ww.ncbi.nlm.nih.gov) and Ensembl database (http: //www.ensemble.org), and found 15 genes as single copy genes in the mouse chromosome. These 15 selected novel genes were characterized further by molecular biological methods. ResultsAll 15 genes were expressed in various tissues and none was ovary-specific. Of those 15 genes, however, 10 genes showed high expression in day1 ovaries and decreased thereafter developmental stages. The in situ hybridization was conducted for those 10 genes and revealed that 3 genes were oocyte-specific. Those are hypothetical proteins LOC228358 and LOC232560(c1q), and mKIAA0978 protein. The mRNA expression was cytoplasmic from primordial to the preovulatory follicles. All 15 genes were expressed in various tissues and none was ovary-specific. Of those 15 genes, however, 10 genes showed high expression in day1 ovaries and decreased thereafter developmental stages. The in situ hybridization was conducted for those 10 genes and revealed that 3 genes were oocyte-specific. Those are hypothetical proteins LOC228358 and LOC232560(c1q), and mKIAA0978 protein. The mRNA expression was cytoplasmic from primordial to the preovulatory follicles. ConclusionWe succeeded in finding 3 novel oocyte-specific genes by using subtractive hybridization, Real-time PCR and in situ hybridization. This study suggests that these novel genes may play role(s) in early follicular development in the mouse oocyte. We succeeded in finding 3 novel oocyte-specific genes by using subtractive hybridization, Real-time PCR and in situ hybridization. This study suggests that these novel genes may play role(s) in early follicular development in the mouse oocyte.

The physiological events during oocyte maturation are still poorly understood, and the molecular approach such as discovery of cellular signaling pathway is very important. To address this issue, we analyzed the change in phosphorylation of seven target proteins in isolated GV, GVBD, MI, and MII (in vitro and vivo) mouse oocytes by using Bio-Plex phosphoprotein assay system (Bio-Rad). Bead-based multiplex phosphorylation assays that detect the phosphorylation of 7 proteins to maximize the information obtained from a single sample at a time in lysates of isolated stage-specific mouse oocytes. Fully grown, GV-intact oocytes were collected in the presence of 0.2 mM IBMX in M2 medium and cultured in M16 medium for 2, 8, and 16 hours to obtain GVBD, MI, and MII (in vitro) oocytes, respectively. Superovulated MII (in vivo) oocytes were obtained after 16 hours of hCG injection, and cumulus cells were removed by treatment with hyaluronidase. Seven target proteins are Akt, GSK-3α/β, IκBα, STAT3 including three MAP kinases, namely ERK1/2, JNK, p38 MAPK. Lysates were obtained from 100 oocytes at each stage, and the experiment was repeated three times. The fold change in phosphorylation state was calculated in comparison to that of GV oocyte as a control. There were two groups, major and minor, of molecules in changing phosphorylation amounts. The phosphorylation of three MAP kinases and that of STAT3 was changed over 3 folds up to 20 folds, while phosphorylation of the other three signal molecules, Akt, GSK-3α/β, and IκBαwas less than 3 folds. Phosphorylation started from GVBD stages and maximized at MI-MII. All of changes except Akt were statistically significant (p<0.05). We succeeded in measuring phosphorylation of seven different important proteins at once in a single lysate sample. We firstly found that the three MAP kinases, ERK1/2, p38, and JNK, are involved in regulating the mouse oocyte maturation. Results of the present study initiate the study of several signaling pathways at once during the oocyte maturation.

Objective: Using suppression subtractive hybridization (SSH), we previously found that the wee1 kinase was one of the highly expressed genes in the primordial follicles (PMF) than in primary follicles (PRI). We hypothesized that the wee1 kinase may inhibit meiotic cell cycle of the primary oocytes by inhibitory phosphorylation of the cdc2. The present study was conducted to find out roles of the wee1 kinase in the primary oocytes of the primordial and primary follicles. Design: Experimental study using SSH, Laser capture microdissection (LCM), RT-PCR, immunohistochemisty (IHC), and immunoblot. Materials and Methods: Since PMF and PRI are too small to isolate in pure, we used day1 ovaries consist mainly of PMF, and day5 ovaries consist of PMF and PRI for searching genes differentially expressed in PMF vs. PRI by SSH. In the day1-subtractive cDNA library, wee1 kinase was one of the most abundantly expressed genes. To confirm the differential wee1 mRNA expression, the same number of each stage follicles was collected using LCM and analyzed by RT-PCR. Wee1 mRNA and protein expression was localized by in situ hybridization and IHC, respectively. Day1 ovaries were enzymatically and mechanically dissociated to isolate oocytes from the somatic cells including pre-granulosa cells. We conducted RT-PCR for cell cycle machinery components related to wee1 function, such as cdc2, cyclin B1, cdc25C, and immunoblot for cdc2 and phosphorylated cdc2 at 15 tyrosine (cdc2-p). Results: We found that the every cells in the ovary expressed wee1 mRNA, but oocytes highly expressed wee1 protein. Wee1 protein expression was solely cytoplasmic. Oocytes at the primordial follicles expressed mRNA for all components for cell cycle machinery, except cdc25C. Protein expression for every component except cdc25C in the oocytes was confirmed by IHC, and those were all cytoplasmic, too. We also confirmed the expression of the cdc2 and cdc2-p proteins in the oocytes of the PMF by immunoblot. Conclusion: We concluded that the high level of wee1 expression in the oocytes might sustain the meiotic cell cycle arrest of oocytes as a tonic inhibitor from the resting primordial follicles to the growing follicles. Wee1 would arrest meiosis by exerting inhibitory phosphorylation of cdc2 and/or by sequestering cdc2 and cyclin B1 in the oocyte cytoplasm. Supported by: a grant of the Korea Health 21 R&D Project, Ministry of Health & Welfare, Republic of Korea (01-PJ10-PG6-01GN13-0002).

Objective: Specific mRNA degradation by sequence specific double-stranded RNA (dsRNA), namely RNAi is a useful method with which to study gene function. Previously, we found that the MTi7 was one of the highly expressed genes in the primary follicles, and the MTi7 mRNA expression was oocyte-specific. To determine the role of MTi7 in the folliculogenesis, dsRNA targeting MTi7 was produced. To evaluate whether the MTi7 dsRNA is working, it was microinjected into the mouse oocytes and embryos. The present study was conducted to determine the role(s) of MTi7 in the mouse oocytes and embryos by RNAi.Design: Experimental study using RNAi, semi-quantitative RT-PCR, and immunofluoresence staining.Materials and Methods: The dsRNAs were prepared by in vitro transcription and annealing of the equimolar amounts of the sense and antisense single stranded RNAs followed by purification of the dsRNA. A gene with known function, c-mos dsRNA was used as a control for injection into the oocytes with germinal vesicle (GV). Purified MTi7 dsRNA was microinjected into the GV, zygotes with pronucleus (PN), and 2-cell embryos. Changes in the mRNA expression after dsRNA injection was measured by semi-quantitative RT-PCR 18–20 hours after microinjection. Changes in morphology were observed under the microscope, and changes in the intracellular organelles were analyzed by immunofluoresence staining.Results: MTi7 expressed in GV, MII, and 2-cell embryos and reduced thereafter. The relative amount of c-mos or MTi7 compared to that of control was determined after microinjection. We found 60%-90% decrease in c-mos and MTi7 mRNA, in control and experiment group, respectively. The MTi7 dsRNA-microinjected GV oocytes were alive and showed several shapes such as GVBD, PN, and uneven cytoplasmic distribution. Some oocytes had polar body-like structure in the perivitelline space. By propidium iodide and tubulin staining, arrested chromosomes before division, several nuclei, and uneven distribution of tubulin was observed. However, almost all MTi7 dsRNA-microinjected PN and 2-cell embryos were arrested at that stage, while medium-injected control embryos developed to the next stages.Conclusion: We report here that RNAi provides an outstanding approach to study the function of a gene with unknown functions. We concluded that the MTi7 is a gene related to the cell division in the early stage embryos, but it seems likely that the MTi7 has different roles in the oocytes compared to the embryos. Objective: Specific mRNA degradation by sequence specific double-stranded RNA (dsRNA), namely RNAi is a useful method with which to study gene function. Previously, we found that the MTi7 was one of the highly expressed genes in the primary follicles, and the MTi7 mRNA expression was oocyte-specific. To determine the role of MTi7 in the folliculogenesis, dsRNA targeting MTi7 was produced. To evaluate whether the MTi7 dsRNA is working, it was microinjected into the mouse oocytes and embryos. The present study was conducted to determine the role(s) of MTi7 in the mouse oocytes and embryos by RNAi. Design: Experimental study using RNAi, semi-quantitative RT-PCR, and immunofluoresence staining. Materials and Methods: The dsRNAs were prepared by in vitro transcription and annealing of the equimolar amounts of the sense and antisense single stranded RNAs followed by purification of the dsRNA. A gene with known function, c-mos dsRNA was used as a control for injection into the oocytes with germinal vesicle (GV). Purified MTi7 dsRNA was microinjected into the GV, zygotes with pronucleus (PN), and 2-cell embryos. Changes in the mRNA expression after dsRNA injection was measured by semi-quantitative RT-PCR 18–20 hours after microinjection. Changes in morphology were observed under the microscope, and changes in the intracellular organelles were analyzed by immunofluoresence staining. Results: MTi7 expressed in GV, MII, and 2-cell embryos and reduced thereafter. The relative amount of c-mos or MTi7 compared to that of control was determined after microinjection. We found 60%-90% decrease in c-mos and MTi7 mRNA, in control and experiment group, respectively. The MTi7 dsRNA-microinjected GV oocytes were alive and showed several shapes such as GVBD, PN, and uneven cytoplasmic distribution. Some oocytes had polar body-like structure in the perivitelline space. By propidium iodide and tubulin staining, arrested chromosomes before division, several nuclei, and uneven distribution of tubulin was observed. However, almost all MTi7 dsRNA-microinjected PN and 2-cell embryos were arrested at that stage, while medium-injected control embryos developed to the next stages. Conclusion: We report here that RNAi provides an outstanding approach to study the function of a gene with unknown functions. We concluded that the MTi7 is a gene related to the cell division in the early stage embryos, but it seems likely that the MTi7 has different roles in the oocytes compared to the embryos.

Nitric oxide (NO) has recently emerged as a potential regulator of follicular development because of its involvement in the regulation of several physiological functions of the ovary. NO influences apoptotic cell death of follicular cells as a follicle survival factor. The present study was conducted (1) to investigate the mechanism involved in the protective effect of NO on spontaneously induced follicular apoptosis in serum‐free condition and (2) to determine the role of NO on the expression of mRNAs and proteins for HSP70 and Bax. Preovulatory follicles obtained from PMSG‐primed rats were cultured for 24 hr in serum‐free medium with or without sodium nitroprusside (SNP), a NO generator. Granulosa cells within follicles incubated in medium alone for 24 hr exhibited extensive apoptosis. Treatment of SNP in the culture medium blocked this onset of apoptosis. Both mRNA and protein levels of HSP70 were highly increased with SNP than those of control group. On the contrary, those of Bax were suppressed with SNP treatment. Results of the present study suggest that NO prevents rat preovulatory follicular apoptosis in vitro by stimulating HSP70 and suppressing Bax expression. Mol. Reprod. Dev. 61:504–510, 2002. © 2002 Wiley‐Liss, Inc.

Objective: Production of a mature oocyte is a complex process during which oocyte and follicular cells maintain a close association. The present study was conducted to investigate the involvement of various connexins in oocyte-granulosa cell interactions during development of follicles and meiotic maturation of oocytes. Design: Follicle isolation, COC isolation, RT-PCR, IVM, IVF. Materials/Methods: Mouse: Follicles at different sizes (small: 200–400 μm; large: >450 μm) were mechanically isolated from PMSG-primed mouse ovaries, and punctured to get cumulus-oocyte complex (COC). Expression of various connexins (CX32, CX37, and CX43) was examined by RT-PCR. Maturation and fertilization capacity of the COCs were measured. Results: When the isolated mouse follicles were punctured, there were three types of COCs as follows. Oocytes were released themselves (denude) or with partially attached (partial) or tightly attached (intact) cumulus cells. The ratio between denuded/partial oocytes and intact COC was 74% (SD/P) and 26% (SI) in small follicles, while 41% (LD/P) and 59% (LI) in large follicles, respectively. Maturation and fertilization rates of the released oocytes were similar among SI, LD/P, LI groups, but those were always lower in SD/P oocytes. When connexin expression was evaluated in the mouse, 1) all groups of COCs (SI, LD/P, LI), except SD/P, showed the similar expression pattern for CX32, CX37 and CX43, 2) COCs in SD/P group had lower CX32, CX43 and higher CX37 expression compared to those three groups of COCs, and 3) among three connexins, CX32 was expressed at lower level compared to the other two connexins. Conclusions: Results of the present study suggest that 1) different combination of connexins may involve in different stages of follicular development, 2) the different connection between cumulus cells and oocytes may affect further developmental capacity of the oocytes, and 3) the absence of CX32 and low expression of CX43 in SD/P oocytes may result in low maturation and fertilization capacity of these oocytes. Supported by: This work was supported by a grant (HMP-98-M-5-0054) from the Good Health R & D Project, Ministry of Health & Welfare, Republic of Korea.