Localization of Ubiquitin C-Terminal Hydrolase L1 in Mouse Ova and Its Function in the Plasma Membrane to Block Polyspermy
2006; Elsevier BV; Volume: 169; Issue: 5 Linguagem: Inglês
10.2353/ajpath.2006.060301
ISSN1525-2191
AutoresSatoshi Sekiguchi, Jungkee Kwon, Etsuko Yoshida, Hiroko Hamasaki, Shizuko Ichinose, Makoto Hideshima, Mutsuki Kuraoka, Akihisa Takahashi, Yoshiyuki Ishii, Shigeru Kyuwa, Keiji Wada, Yasuhiro Yoshikawa,
Tópico(s)Reproductive Biology and Fertility
ResumoProtein degradation is essential for oogenesis and embryogenesis. The ubiquitin-proteasome system regulates many cellular processes via the rapid degradation of specific proteins. Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) is exclusively expressed in neurons, testis, ovary, and placenta, each of which has unique biological activities. However, the functional role of UCH-L1 in mouse oocytes remains unknown. Here, we report the expression pattern of UCH-L1 and its isozyme UCH-L3 in mouse ovaries and embryos. Using immunocytochemistry, UCH-L1 was selectively detected on the plasma membrane, whereas UCH-L3 was mainly detected in the cytoplasm, suggesting that these isozymes have distinct functions in mouse eggs. To further investigate the functional role of UCH-L1 in mouse eggs, we analyzed the fertilization rate of UCH-L1-deficient ova of gad female mice. Female gad mice had a significantly increased rate of polyspermy in in vitro fertilization assays, although the rate of fertilization did not differ significantly from wild-type mice. In addition, the litter size of gad female mice was significantly reduced compared with wild-type mice. These results may identify UCH-L1 as a candidate for a sperm-oocyte interactive binding or fusion protein on the plasma membrane that functions during the block to polyspermy in mouse oocytes. Protein degradation is essential for oogenesis and embryogenesis. The ubiquitin-proteasome system regulates many cellular processes via the rapid degradation of specific proteins. Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) is exclusively expressed in neurons, testis, ovary, and placenta, each of which has unique biological activities. However, the functional role of UCH-L1 in mouse oocytes remains unknown. Here, we report the expression pattern of UCH-L1 and its isozyme UCH-L3 in mouse ovaries and embryos. Using immunocytochemistry, UCH-L1 was selectively detected on the plasma membrane, whereas UCH-L3 was mainly detected in the cytoplasm, suggesting that these isozymes have distinct functions in mouse eggs. To further investigate the functional role of UCH-L1 in mouse eggs, we analyzed the fertilization rate of UCH-L1-deficient ova of gad female mice. Female gad mice had a significantly increased rate of polyspermy in in vitro fertilization assays, although the rate of fertilization did not differ significantly from wild-type mice. In addition, the litter size of gad female mice was significantly reduced compared with wild-type mice. These results may identify UCH-L1 as a candidate for a sperm-oocyte interactive binding or fusion protein on the plasma membrane that functions during the block to polyspermy in mouse oocytes. Ubiquitin C-terminal hydrolase L1 (UCH-L1) is one of many deubiquitinating enzymes and is selectively and abundantly expressed in the ovary, placenta, testis, and neuronal cells.1Kwon J Kikuchi T Setsuie R Ishii Y Kyuwa S Yoshikawa Y Characterization of the testis in congenitally ubiquitin carboxy-terminal hydrolase-1 (Uch-L1) defective (gad) mice.Exp Anim. 2003; 52: 1-9Crossref PubMed Scopus (43) Google Scholar, 2Kwon J Wang YL Setsuie R Sekiguchi S Sakurai M Sato Y Lee WW Ishii Y Kyuwa S Noda M Wada K Yoshikawa Y Developmental regulation of ubiquitin C-terminal hydrolase isozyme expression during spermatogenesis in mice.Biol Reprod. 2004; 71: 515-521Crossref PubMed Scopus (86) Google Scholar, 3Sekiguchi S Takatori A Negishi T Kwon J Kokubo T Ishii Y Kyuwa S Yoshikawa Y Localization of ubiquitin carboxyl-terminal hydrolase-L1 in cynomolgus monkey placentas.Placenta. 2005; 26: 99-103Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar, 4Sekiguchi S Yoshikawa Y Tanaka S Kwon J Ishii Y Kyuwa S Wada K Nakamura S Takahashi K Immunohistochemical analysis of protein gene product 9.5, a ubiquitin carboxyl-terminal hydrolase, during placental and embryonic development in the mouse.Exp Anim. 2003; 52: 365-369Crossref PubMed Scopus (19) Google Scholar, 5Wilson PO Barber PC Hamid QA Power BF Dhillon AP Rode J Day IN Thompson RJ Polak JM The immunolocalization of protein gene product 9.5 using rabbit polyclonal and mouse monoclonal antibodies.Br J Exp Pathol. 1988; 69: 91-104PubMed Google Scholar, 6Kwon J Mochida K Wang YL Sekiguchi S Sankai T Aoki S Ogura A Yoshikawa Y Wada K Ubiquitin C-terminal hydrolase L-1 is essential for the early apoptotic wave of germinal cells and for sperm quality control during spermatogenesis.Biol Reprod. 2005; 73: 29-35Crossref PubMed Scopus (83) Google Scholar Recent studies suggest that UCH-L1 associates with monoubiquitin and prolongs ubiquitin half-life in neurons.7Osaka H Wang YL Takada K Takizawa S Setsuie R Li H Sato Y Nishikawa K Sun YJ Sakurai M Harada T Hara Y Kimura I Chiba S Namikawa K Kiyama H Noda M Aoki S Wada K Ubiquitin carboxy-terminal hydrolase L1 binds to and stabilizes monoubiquitin in neuron.Hum Mol Genet. 2003; 12: 1945-1958Crossref PubMed Scopus (326) Google Scholar Our previous work on UCH-L1 function in gad mice suggests that these mice are resistant to apoptotic stress in retinal cells and testicular germ cells.8Harada T Harada C Wang YL Osaka H Amanai K Tanaka K Takizawa S Setsuie R Sakurai M Sato Y Noda M Wada K Role of ubiquitin carboxy terminal hydrolase-L1 in neural cell apoptosis induced by ischemic retinal injury in vivo.Am J Pathol. 2004; 164: 59-64Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 9Kwon J Wang YL Setsuie R Sekiguchi S Sato Y Sakurai M Noda M Aoki S Yoshikawa Y Wada K Two closely related ubiquitin C-terminal hydrolase isozymes function as reciprocal modulators of germ cell apoptosis in cryptorchid testis.Am J Pathol. 2004; 165: 1367-1374Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar This observation is consistent with a recent report that the overexpression of UCH-L1 induces testicular germ cell apoptosis in Uchl1 transgenic mice.10Wang YL Liu W Sun YJ Kwon J Setsuie R Osaka H Noda M Aoki S Yoshikawa Y Wada K Overexpression of ubiquitin carboxyl-terminal hydrolase L1 arrests spermatogenesis in transgenic mice.Mol Reprod Dev. 2006; 73: 40-49Crossref PubMed Scopus (65) Google Scholar Furthermore, both UCH-L1 and UCH-L3, the predominant functional UCHs, are differentially expressed in testis during spermatogenesis. These results demonstrate that these enzymes have distinct functions in the testis and epididymis after apoptotic stress,9Kwon J Wang YL Setsuie R Sekiguchi S Sato Y Sakurai M Noda M Aoki S Yoshikawa Y Wada K Two closely related ubiquitin C-terminal hydrolase isozymes function as reciprocal modulators of germ cell apoptosis in cryptorchid testis.Am J Pathol. 2004; 165: 1367-1374Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 11Kwon J Sekiguchi S Wang YL Setsuie R Yoshikawa Y Wada K The region-specific functions of two ubiquitin C-terminal hydrolase isozymes along the epididymis.Exp Anim. 2006; 55: 35-43Crossref PubMed Scopus (13) Google Scholar even though they have high (52%) amino acid sequence identity.12Kurihara LJ Semenova E Levorse JM Tilghman SM Expression and functional analysis of Uch-L3 during mouse development.Mol Cell Biol. 2000; 20: 2498-2504Crossref PubMed Scopus (67) Google Scholar The above data are in accordance with a number of studies that have linked inhibition of the ubiquitin-proteasome system (UPS) with suppression of apoptosis.8Harada T Harada C Wang YL Osaka H Amanai K Tanaka K Takizawa S Setsuie R Sakurai M Sato Y Noda M Wada K Role of ubiquitin carboxy terminal hydrolase-L1 in neural cell apoptosis induced by ischemic retinal injury in vivo.Am J Pathol. 2004; 164: 59-64Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 13Rasoulpour RJ Schoenfeld HA Gray DA Boekelheide K Expression of a K48R mutant ubiquitin protects mouse testis from cryptorchid injury and aging.Am J Pathol. 2003; 163: 2595-2603Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 14Yang Y Yu X Regulation of apoptosis: the ubiquitous way.FASEB J. 2003; 17: 790-799Crossref PubMed Scopus (176) Google Scholar, 15Baarends WM Wassenaar E Hoogerbrugge JW van Cappellen G Roest HP Vreeburg J Ooms M Hoeijmakers JH Grootegoed JA Loss of HR6B ubiquitin-conjugating activity results in damaged synaptonemal complex structure and increased crossing-over frequency during the male meiotic prophase.Mol Cell Biol. 2003; 23: 1151-1162Crossref PubMed Scopus (90) Google Scholar UCH-L1 is an important enzyme for UPS-dependent proteolysis and plays a regulatory role in the cell cycle and cellular proliferation. Thus, its expression in placenta is of considerable interest.3Sekiguchi S Takatori A Negishi T Kwon J Kokubo T Ishii Y Kyuwa S Yoshikawa Y Localization of ubiquitin carboxyl-terminal hydrolase-L1 in cynomolgus monkey placentas.Placenta. 2005; 26: 99-103Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar, 4Sekiguchi S Yoshikawa Y Tanaka S Kwon J Ishii Y Kyuwa S Wada K Nakamura S Takahashi K Immunohistochemical analysis of protein gene product 9.5, a ubiquitin carboxyl-terminal hydrolase, during placental and embryonic development in the mouse.Exp Anim. 2003; 52: 365-369Crossref PubMed Scopus (19) Google Scholar Recent studies reported that UPS controls the degradation of various substrates during gametogenesis and fertilization,16Baarends WM Roest HP Grootegoed JA The ubiquitin system in gametogenesis.Mol Cell Endocrinol. 1999; 151: 5-16Crossref PubMed Scopus (83) Google Scholar, 17Sutovsky P Ubiquitin-dependent proteolysis in mammalian spermatogenesis, fertilization, and sperm quality control: killing three birds with one stone.Microsc Res Tech. 2003; 61: 88-102Crossref PubMed Scopus (236) Google Scholar, 18Sutovsky P Manandhar G McCauley TC Caamano JN Sutovsky M Thompson WE Day BN Proteasomal interference prevents zona pellucida penetration and fertilization in mammals.Biol Reprod. 2004; 71: 1625-1637Crossref PubMed Scopus (115) Google Scholar, 19Sutovsky P McCauley TC Sutovsky M Day BN Early degradation of paternal mitochondria in domestic pig (Sus scrofa) is prevented by selective proteasomal inhibitors lactacystin and MG132.Biol Reprod. 2003; 68: 1793-1800Crossref PubMed Scopus (81) Google Scholar but relatively little is known about the functional role of the UPS in fertilization. UCH-L1 is expressed in oocytes in ovaries.5Wilson PO Barber PC Hamid QA Power BF Dhillon AP Rode J Day IN Thompson RJ Polak JM The immunolocalization of protein gene product 9.5 using rabbit polyclonal and mouse monoclonal antibodies.Br J Exp Pathol. 1988; 69: 91-104PubMed Google Scholar, 20Ellederova Z Halada P Man P Kubelka M Motlik J Kovarova H Protein patterns of pig oocytes during in vitro maturation.Biol Reprod. 2004; 71: 1533-1539Crossref PubMed Scopus (87) Google Scholar Oocytes, as well as spermatogonia in testis, have multiple potentials and activities for development. However, the function of UCH-L1 during oogenesis is unclear. RFPL4 (ret finger protein-like 4) and FAM (fat facets in mouse) are involved in regulating oogenesis.21Suzumori N Burns KH Yan W Matzuk MM RFPL4 interacts with oocyte proteins of the ubiquitin-proteasome degradation pathway.Proc Natl Acad Sci USA. 2003; 100: 550-555Crossref PubMed Scopus (63) Google Scholar, 22Pantaleon M Kanai-Azuma M Mattick JS Kaibuchi K Kaye PL Wood SA FAM deubiquitylating enzyme is essential for preimplantation mouse embryo development.Mech Dev. 2001; 109: 151-160Crossref PubMed Scopus (58) Google Scholar RFPL4 is highly expressed during oogenesis and functions as an E3 ubiquitin ligase to target proteins for proteasomal degradation.21Suzumori N Burns KH Yan W Matzuk MM RFPL4 interacts with oocyte proteins of the ubiquitin-proteasome degradation pathway.Proc Natl Acad Sci USA. 2003; 100: 550-555Crossref PubMed Scopus (63) Google Scholar FAM is a developmentally regulated substrate-specific deubiquitinating enzyme that is required for preimplantation of the mouse embryo.22Pantaleon M Kanai-Azuma M Mattick JS Kaibuchi K Kaye PL Wood SA FAM deubiquitylating enzyme is essential for preimplantation mouse embryo development.Mech Dev. 2001; 109: 151-160Crossref PubMed Scopus (58) Google Scholar Thus, the UPS might be important during oocyte development and differentiation of the embryo after fertilization. Here, we analyzed the functional role of UCH-L1 using mouse oocytes and embryos. Our results indicate that UCH-L1 is selectively expressed on the plasma membrane of mouse ova, where it may regulate membrane penetration by spermatozoa. In addition, the unique expression patterns of UCH-L1 and UCH-L3 suggest that these proteins have distinct functions during oogenesis and embryogenesis. Our results therefore provide strong evidence that UCH-L1 functions in the polyspermy block during mammalian fertilization. We used 8-week-old BDF1, gad (CBA/RFM),23Kikuchi T Mukoyama M Yamazaki K Moriya H Axonal degeneration of ascending sensory neurons in gracile axonal dystrophy mutant mouse.Acta Neuropathol (Berl). 1990; 80: 145-151Crossref PubMed Scopus (46) Google Scholar, 24Saigoh K Wang YL Suh JG Yamanishi T Sakai Y Kiyosawa H Harada T Ichihara N Wakana S Kikuchi T Wada K Intragenic deletion in the gene encoding ubiquitin carboxy-terminal hydrolase in gad mice.Nat Genet. 1999; 23: 47-51PubMed Scopus (0) Google Scholar and Uchl3 knockout (C57BL/6J)12Kurihara LJ Semenova E Levorse JM Tilghman SM Expression and functional analysis of Uch-L3 during mouse development.Mol Cell Biol. 2000; 20: 2498-2504Crossref PubMed Scopus (67) Google Scholar, 25Kurihara LJ Kikuchi T Wada K Tilghman SM Semenova E Levorse JM Loss of Uch-L1 and Uch-L3 leads to neurodegeneration, posterior paralysis and dysphagia.Hum Mol Genet. 2001; 10: 1963-1970Crossref PubMed Scopus (83) Google Scholar female and male mice. BDF1 mice were purchased from Nihon SLC, Inc. (Hamamatsu, Japan). The gad mouse is an autosomal recessive mutant that was obtained by crossing CBA and RFM mice. The gad line was maintained by intercrossing for more than 20 generations.23Kikuchi T Mukoyama M Yamazaki K Moriya H Axonal degeneration of ascending sensory neurons in gracile axonal dystrophy mutant mouse.Acta Neuropathol (Berl). 1990; 80: 145-151Crossref PubMed Scopus (46) Google Scholar, 24Saigoh K Wang YL Suh JG Yamanishi T Sakai Y Kiyosawa H Harada T Ichihara N Wakana S Kikuchi T Wada K Intragenic deletion in the gene encoding ubiquitin carboxy-terminal hydrolase in gad mice.Nat Genet. 1999; 23: 47-51PubMed Scopus (0) Google Scholar The Uchl3 knockout mouse was generated by standard methods using homologously recombinant ES cells from 129SV mice.12Kurihara LJ Semenova E Levorse JM Tilghman SM Expression and functional analysis of Uch-L3 during mouse development.Mol Cell Biol. 2000; 20: 2498-2504Crossref PubMed Scopus (67) Google Scholar, 25Kurihara LJ Kikuchi T Wada K Tilghman SM Semenova E Levorse JM Loss of Uch-L1 and Uch-L3 leads to neurodegeneration, posterior paralysis and dysphagia.Hum Mol Genet. 2001; 10: 1963-1970Crossref PubMed Scopus (83) Google Scholar The knockout line was back-crossed several times with C57BL/6J mice. gad mice were maintained at our institute, and Uchl3 knockout mice were maintained at the National Institute of Neuroscience, National Center of Neurology and Psychiatry (Tokyo, Japan). Animal care and handling were in accordance with institutional regulations and were approved by the Animal Care and Use Committee of the University of Tokyo. Female mice were superovulated by intraperitoneal injections with 5 IU of pregnant mare serum gonadotropin (Sankyo, Tokyo, Japan) for 48 hours, followed by 5 IU of human chorionic gonadotropin (Sankyo). Ovulated eggs were collected from the ampullae of oviducts by the scratching method 16 hours after human chorionic gonadotropin injection and placed in 400-μl droplets of Toyoda, Yokoyama, and Hoshi (TYH)26Kito S Hayao T Noguchi-Kawasaki Y Ohta Y Hideki U Tateno S Improved in vitro fertilization and development by use of modified human tubal fluid and applicability of pronucleate embryos for cryopreservation by rapid freezing in inbred mice.Comp Med. 2004; 54: 564-570PubMed Google Scholar containing 0.4 mg/ml bovine serum albumin (Sigma-Aldrich, St. Louis, MO). Spermatozoa were collected from the cauda epididymis of male mice and preincubated for 1 hour in 400 μl of TYH to allow capacitation before insemination. After capacitation, the sperms were introduced into the fertilization medium at a final concentration of 150 spermatozoa/μl. At 4 hours after insemination, 0.05% hyaluronidase (Sigma-Aldrich) was added to the medium for 5 minutes. The eggs were washed thoroughly three times and then cultured in potassium simplex optimized medium (KSOM).26Kito S Hayao T Noguchi-Kawasaki Y Ohta Y Hideki U Tateno S Improved in vitro fertilization and development by use of modified human tubal fluid and applicability of pronucleate embryos for cryopreservation by rapid freezing in inbred mice.Comp Med. 2004; 54: 564-570PubMed Google Scholar After fertilization, all embryos were incubated in a humidified atmosphere of 5% CO2 in air at 37°C in 100-μl drops of KSOM overlaid with mineral oil. To analyze fertilization in gad mice, gad (n = 5) and wild-type (CBA/RFM) (n = 5) female mice were superovulated. Ovulated oocytes of gad and wild-type mice were fertilized with wild-type spermatozoa. Total protein of ovary extracts (10 μg/lane), oocytes, or embryos (20 oocytes or embryos per lane) was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis using 12.5% gels. Proteins were electrophoretically transferred to polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA) and blocked with 1% bovine serum albumin in TBS-T [50 mmol/L Tris base, pH 7.5, 150 mmol/L NaCl, and 0.1% (w/v) Tween 20]. The membranes were incubated individually with primary antibodies against UCH-L1, UCH-L3,2Kwon J Wang YL Setsuie R Sekiguchi S Sakurai M Sato Y Lee WW Ishii Y Kyuwa S Noda M Wada K Yoshikawa Y Developmental regulation of ubiquitin C-terminal hydrolase isozyme expression during spermatogenesis in mice.Biol Reprod. 2004; 71: 515-521Crossref PubMed Scopus (86) Google Scholar monoubiquitin (U5379, Sigma-Aldrich), zona pellucida 2 (ZP2; Santa Cruz Biotechnology, Santa Cruz, CA), and zona pellucida 3 (ZP3) (Santa Cruz Biotechnology). After thorough rinsing, blots were further incubated with peroxidase-conjugated goat anti-rabbit IgG (DakoCytomation, Glostrup, Denmark) for 1 hour at room temperature. Immunoreactions were visualized by enhanced chemiluminescence (ECL Plus; GE Healthcare UK Ltd. Amersham Place, Little Chalfont, Buckinghamshire, UK). Each immunoreactive band was quantified using commercially available software (Quantity One; PDI, Upper Saddle River, NJ). Negative control extracts of ovary or oocytes were obtained from gad and Uchl3 knockout mice. Ovaries of BDF1 female and gad mice were fixed in 4% paraformaldehyde, embedded in paraffin wax, and then sectioned at 4-μm thickness. Ovary sections of gad mice were stained with hematoxylin and eosin (H&E). Light microscopy was used for routine observations. For immunohistochemical staining, the sections were incubated with Block Ace (Dainippon Sumitomo Pharma, Osaka, Japan) for 1 hour at room temperature followed by incubation overnight at 4°C with a rabbit polyclonal antibody against UCH-L1 and UCH-L3.2Kwon J Wang YL Setsuie R Sekiguchi S Sakurai M Sato Y Lee WW Ishii Y Kyuwa S Noda M Wada K Yoshikawa Y Developmental regulation of ubiquitin C-terminal hydrolase isozyme expression during spermatogenesis in mice.Biol Reprod. 2004; 71: 515-521Crossref PubMed Scopus (86) Google Scholar The sections were then incubated with biotinylated goat anti-rabbit IgG (DAKO), which was followed by incubation with streptavidin-biotin-horseradish peroxidase complex (sABC kit; DAKO). Immunoreactivity was visualized by treating the sections with 3,3′-diaminobenzidine tetroxide (Dojin Kagaku, Kumamoto, Japan). Finally, the sections were counterstained with hematoxylin. Negative control ovaries were obtained from gad and Uchl3 knockout mice. For immunocytochemical staining, whole oocytes or embryos were fixed for 30 minutes with 4% paraformaldehyde in phosphate-buffered saline (PBS) and 0.2% (w/v) Triton X-100 (ICN Biomedicals, Aurora, OH) in PBS for 30 minutes. Nonspecific binding of immunoglobulins was blocked by incubation with Block Ace (Dainippon Pharmaceutical, Ltd.) for 1 hour at room temperature. The sections were then incubated with primary antibodies against UCH-L1, UCH-L3, and lectin with Rhodamine-Lens Culinaris Agglutinin (Vector Laboratories, Burlingame, CA). The sections were then incubated with Alexa 488-conjugated goat anti-rabbit IgG (Molecular Probes, Eugene, OR) and propidium iodide (Molecular Probes). Stained sections were observed under a confocal laser microscope (Laser Scanning Microscope 510; Carl Zeiss, Jena, Germany). Negative control oocytes were obtained from gad and Uchl3 knockout mice by superovulation. Normal oocytes were identified by the presence of the first polar body.27Wang WH Abeydeera LR Han YM Prather RS Day BN Morphologic evaluation and actin filament distribution in porcine embryos produced in vitro and in vivo.Biol Reprod. 1999; 60: 1020-1028Crossref PubMed Scopus (117) Google Scholar, 28Ju JC Tseng JK Nuclear and cytoskeletal alterations of in vitro matured porcine oocytes under hyperthermia.Mol Reprod Dev. 2004; 68: 125-133Crossref PubMed Scopus (75) Google Scholar The frequency of normal oocytes was calculated by counting the normal oocytes in the total superovulated oocytes of gad (n = 173) and wild-type (n = 148) mice. To determine the fertilization rate, putative fertilized eggs (by in vitro fertilization) were fixed in acetic alcohol (1:3, glacial acetic acid/ethanol) and then stained with 1% aceto-orcein to visualize pronuclei and assess sperm penetration and incidence of monospermic and polyspermic fertilization. Polyspermic fertilization was defined as the presence of three or more pronuclei. The rate of polyspermic fertilization was calculated by counting the polyspermic eggs among the total fertilized eggs of gad (n = 71) and wild-type (n = 56) mice. Ovulated mature oocytes and zygotes of BDF1 mice were fixed with 4% paraformaldehyde, and frozen sections were prepared for electron microscopy. The sections were incubated with an antibody against UCH-L1.2Kwon J Wang YL Setsuie R Sekiguchi S Sakurai M Sato Y Lee WW Ishii Y Kyuwa S Noda M Wada K Yoshikawa Y Developmental regulation of ubiquitin C-terminal hydrolase isozyme expression during spermatogenesis in mice.Biol Reprod. 2004; 71: 515-521Crossref PubMed Scopus (86) Google Scholar Subsequently, the ABC method was performed as indicated by the supplier, and the peroxidase reaction was developed in diaminobenzidine. Immunostained sections were fixed in 2.5% glutaraldehyde, postfixed in 1% OsO4, dehydrated in a graded series of ethanol, and embedded in Epon 812.29Ishibashi S Sakaguchi M Kuroiwa T Yamasaki M Kanemura Y Shizuko I Shimazaki T Onodera M Okano H Mizusawa H Human neural stem/progenitor cells, expanded in long-term neurosphere culture, promote functional recovery after focal ischemia in Mongolian gerbils.J Neurosci Res. 2004; 78: 215-223Crossref PubMed Scopus (151) Google Scholar Ultrathin sections were cut with an ultramicrotome, stained with uranyl acetate,29Ishibashi S Sakaguchi M Kuroiwa T Yamasaki M Kanemura Y Shizuko I Shimazaki T Onodera M Okano H Mizusawa H Human neural stem/progenitor cells, expanded in long-term neurosphere culture, promote functional recovery after focal ischemia in Mongolian gerbils.J Neurosci Res. 2004; 78: 215-223Crossref PubMed Scopus (151) Google Scholar and examined with an electron microscope H-7100 (Hitachi, Hitachinaka, Japan). gad (n = 12) and wild-type (CBA/RFM) (n = 15) female mice and wild-type (CBA/RFM) (n = 9) male mice were subjected to a breeding study. Three female mice of the same genotype were housed with one male mouse per cage. Cages were monitored daily at midday, and the appearance of a vaginal plug was recognized as day 0.5 of gestation. The number of pups, litters, and litter size of gad and wild-type female mice were recorded. The mean and SD were calculated for all data (presented as mean ± SD). The Student's t-test was used for all statistical analyses. We first used Western blotting to address whether both UCH-L1 and UCH-L3 levels are expressed in ovaries during proestrus, estrus, metestrus, and diestrus (Figure 1A). Both proteins were detected at all estrous cycle stages in wild-type mice. As expected, UCH-L1 was not detected in gad ovaries and UCH-L3 was not detected in Uchl3 knockout ovaries. The levels of UCH-L1 and UCH-L3 in ovaries did not change throughout the estrous cycle (Figure 1A). We next used immunohistochemistry to address whether both UCH-L1 and UCH-L3 are present in developing follicles (primordial, primary, secondary, tertiary, and mature follicles; Figure 1B). In the ovary, UCH-L1 staining was most intense at the plasma membrane of oocytes in developing follicles (Figure 1B, a–e), whereas UCH-L3 staining was concentrated in the cytoplasm of oocytes (Figure 1B, g–k). The oocytes of gad and Uchl3 knockout mice ovaries were negative for UCH-L1 and UCH-L3, respectively (Figure 1B, f and l). Using Western blotting, we examined the levels of both UCH-L1 and UCH-L3 in mature oocytes and during embryogenesis (zygote, two cells, four cells, eight cells, morulas, and blastocysts). UCH-L1 and UCH-L3 were detected in mature oocytes and during all of the embryonic stages we tested; the level of UCH-L1 was essentially constant in all cases, but the level of UCH-L3 was lower in the blastocyst stage (Figure 2A). We further analyzed the distribution of UCH-L1 and UCH-L3 in ovulated mature oocytes and during embryogenesis using immunocytochemistry (Figure 2B). UCH-L1 immunoreactivity was intense on the plasma membrane of oocytes and developing embryos but not in the cytoplasm of eggs (Figure 2B, a–g). Furthermore, UCH-L1 immunoreactivity was observed continuously during embryogenesis (Figure 2B, c–g). In the blastocyst stage, UCH-L1 was observed in the outer layer cells of the trophectoderm (Figure 2B, g). UCH-L3 immunoreactivity was seen in the cytoplasm of oocytes and developing embryos (Figure 2B, i–o), as was detected in ovaries (Figure 2A). In the blastocyst stage, UCH-L3 was observed in the inner cells (Figure 2B, o). The oocytes and developing embryos of gad and Uchl3 knockout mice were negative for UCH-L1 and UCH-L3, respectively (Figure 2B, h and p). The cortical granule is located right under the plasma membrane in oocytes. This organelle is unique to oocytes and plays an important role in fertilization. At the light microscope level, it is difficult to determine whether UCH-L1 immunoreactivity localizes to the plasma membrane or cortical granule. To address this issue, we investigated the subcellular localization of UCH-L1 in ovulated mature oocytes and zygotes at the ultrastructural level (Figure 3). Intense UCH-L1 immunoreactivity was observed on the plasma membrane of ovulated mature oocytes (Figure 3, A and B) and zygotes (Figure 3, C and D). However, the intensity of the plasma membrane immunoreactivity was greater in mature oocytes than in zygotes. Therefore, we concluded that the distribution of UCH-L1 may change after fertilization. To assess whether gad mice have morphologically normal ovaries, we used histology to compare ovaries from gad and wild-type (CBA/RFM) mice. gad mouse ovaries had morphologically normal oocytes, follicles, and corpora lutea (Figure 4, A and B). In addition, gad mice had a normal estrous cycle (data not shown). We further assessed gad mouse ovarian function by comparison with wild-type mice using the ovulation test. The total number of ovulated oocytes and the normal oocyte ovulation rate of superovulated oocytes did not differ significantly between gad mice and wild-type mice (Figure 4, C and D). To analyze the fertilization rate in UCH-L1-deficient embryos of gad female mice, we assessed fertilization by in vitro fertilization using wild-type spermatozoa. For mice, the presence of more than three or more pronuclei defines polyspermic fertilization. Fertilized eggs from gad mice had characteristics consistent with this definition of polyspermy (Figure 5, B and C). By contrast, wild-type eggs showed normal zygotic nuclei (Figure 5A). The fertilized eggs of gad mice had a significantly higher rate of polyspermy (27 ± 0.04%) compared with wild-type mice (2 ± 0.02%) (Figure 5E). However, the fertility of gad mouse eggs (46 ± 0.06%) did not differ significantly from that of wild-type mice (43 ± 0.09%) (Figure 5D). To further evaluate the high polyspermic fertilization rate of gad female mice, we characterized the litter size of these mice after mating with wild-type male mice. gad female mice exhibited normal puberty and estrous cycle, as assessed by vaginal opening and vaginal smear, but they had reduced fertility, as evidenced by a significant decrease in litter size (3 ± 2.0) compared with wild-type mice (6.6 ± 1.3) (Table 1).Table 1Breeding Study of gad and Wild-Type Female Mice with Wild-Type Male MiceGenotype (n)LittersPupsLitter sizeWild type (15)15996.6 ± 1.3gad/gad (12)11333 ± 2.0*Significantly different from wild-type mice, P < 0.001.Results represent the mean ± SD.* Significantly different from wild-type mice, P < 0.001. Open table in a new tab Results represent the mean ± SD. Our previous study demonstrated that UCH-L1 stabilizes monoubiquitin in testes and that the level of monoubiquitin is decreased in male gad mice.8Harada T Harada C Wang YL Osaka H Amanai K Tanaka K Takizawa S Setsuie R Sakurai M Sato Y Noda M Wada K Role of ubiquitin carboxy terminal hydrolase-L1 in neural cell apoptosis induced by ischemic retinal injury in vivo.Am J Pathol. 2004; 164:
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