A novel ubiquitin carboxyl terminal hydrolase is involved in toad oocyte maturation

p28, a 28kD protein from toad (Bufo bufo gargarizans) oocytes, was identified by using p13sucl-agarose affinity chromatography. Sequence homology analysis of the full-length cDNA of p28 (Gene Bank accession number: AF 314091) indicated that it encodes a protein containing 224 amino-acids with about 55% identities and more than 70% positives to human, rat or mouse UCH-L1, and contains homological functional domains of UCH family. Anti-p28 monoclonal antibody, on injecting into the oocytes, could inhibit the progesterone-induced resumption of meiotic division in a dose-dependent manner. The recombinant protein p28 showed similar SDS/PAGE behaviors to the native one, and promoted ubiquitin ethyl ester hydrolysis, a classical catalytic reaction for ubiquitin carboxyl terminal hydrolases (UCHs). The results in this paper reveal that a novel protein, p28, exists in the toad oocytes, is a UCH L1 homolog, was engaged in the process of progesterone-induced oocyte maturation possibly through an involvement in prot     

A novel ubiquitin carboxyl terminal hydrolase is involved in toad oocyte maturation

p28, a 28kD protein from toad (Bufo bufo gargarizans) oocytes, was identified by using p13suc1-agaroseaffinity chromatography. Sequence homology analysis of the full-length cDNA of p28 (Gene Bank accessionnumber: AF 314091) indicated that it encodes a protein containing 224 amino-acids with about 55% iden-tities and more than 70% positives to human, rat or mouse UCH-L1, and contains homological functionaldomains of UCH family. Anti-p28 monoclonal antibody, on injecting into the oocytes, could inhibit theprogesterone-induced resumption of meiotic division in a dose-dependent manner. The recombinant proteinp28 showed similar SDS/PAGE behaviors to the native one, and promoted ubiquitin ethyl ester hydrolysis,a classical catalytic reaction for ubiquitin carboxyl terminai hydrolases (UCHs). The results in this paperreveal that a novel protein, p28, exists in the toad oocytes, is a UCH L1 homolog, was engaged in theprocess of progesterone-induced oocyte maturation possibly through an involvement in protein turnover anddegradation.     

蟾蜍泛素羧基末端水解酶（tUCH）以不依赖其UCH活性的方式参与卵母细胞成熟调控

本实验室巳报道中华大蟾蜍卵母细胞的p28蛋白具泛素羧基末端水解酶活性,称作tUCH,它和哺乳类中发现的UCH Ll的氨基酸序列具高度同源性,二级结构同源性比较发现,二者可能具类似的功能。本文实验表明：未成熟卵母细胞和成熟卵母细胞的可溶性蛋白中均含有tUCH,约占提取物中蛋白质总量的2％。根据测定所得到的GST—tUCH和GST—UCH Ll对底物Ub—AMC的酶动力学参数,说明卵母细胞中tUCH可能与小鼠UCH L1有类似的生物学功能;anti—tUCH单抗可以与原核细胞表达的tUCH和显性失活突变类型tUCH C(90)S特异结合,但不识别小鼠的UCH Ll。Anti-tUCH单抗能够和tUCH结合但不能封闭它的UCH活性;当anti-tUCH单抗注入卵母细胞内,则孕酮诱导的生发泡破裂(germinal vesicle breakdown,GVBD)过程受到抑制,足见tUCH参与GVBD调节并不依赖其UCH活性。     

蟾蜍泛素羧基末端水解酶(tUCH)以不依赖其UCH活性的方式参与卵母细胞成熟调控

本实验室已报道中华大蟾蜍卵母细胞的p28蛋白具泛素羧基末端水解酶活性,称作tUCH,它和哺乳类中发现的UCH L1的氨基酸序列具高度同源性,二级结构同源性比较发现,二者可能具类似的功能。本文实验表明:未成熟卵母细胞和成熟卵母细胞的可溶性蛋白中均含有tUCH,约占提取物中蛋白质总量的2%。根据测定所得到的GST-tUCH和GST-UCH L1对底物Ub-AMC的酶动力学参数,说明卵母细胞中tUCH可能与小鼠UCH L1有类似的生物学功能;anti-tUCH单抗可以与原核细胞表达的tUCH和显性失活突变类型tUCH C(90)S特异结合,但不识别小鼠的UCH L1。Anti-tUCH单抗能够和tUCH结合但不能封闭它的UCH活性。当anti-tUCH单抗注入卵母细胞内,则孕酮诱导的生发泡破裂(germinal vesicle breakdown,GVBD)过程受到抑制,足见tUCH参与GVHD调节并不依赖其UCH活性。     

Winter Hibernation and UCHL1-p34cdc2 Association in Toad Oocyte Maturation Competence

Currently, it is believed that toad oocyte maturation is dependent on the physiological conditions of winter hibernation. Previous antibody-blocking experiments have demonstrated that toad ubiquitin carboxyl-terminal hydrolase L1 (tUCHL1) is necessary for germinal vesicle breakdown during toad oocyte maturation. In this paper, we first supply evidence that tUCHL1 is highly evolutionarily conserved. Then, we exclude protein availability and ubiquitin carboxyl-terminal hydrolase enzyme activity as factors in the response of oocytes to winter hibernation. In the context of MPF (maturation promoting factor) controlling oocyte maturation and to further understand the role of UCHL1 in oocyte maturation, we performed adsorption and co-immunoprecipitation experiments using toad oocyte protein extracts and determined that tUCHL1 is associated with MPF in toad oocytes. Recombinant tUCHL1 absorbed p34^cdc2, a component of MPF, in obviously larger quantities from mature oocytes than from immature oocytes, and p13^suc1 was isolated from tUCHL1 with a dependence on the ATP regeneration system, suggesting that still other functions may be involved in their association that require phosphorylation. In oocytes from hibernation-interrupted toads, the p34^cdc2 protein level was significantly lower than in oocytes from toads in artificial hibernation, providing an explanation for the different quantities isolated by recombinant tUCHL1 pull-down and, more importantly, identifying a mechanism involved in the toad oocyte’s dependence on a low environmental temperature during winter hibernation. Therefore, in toads, tUCHL1 binds p34^cdc2 and plays a role in oocyte maturation. However, neither tUCHL1 nor cyclin B1 respond to low temperatures to facilitate oocyte maturation competence during winter hibernation.     

Molecular cloning and immunohistochemical localization of ubiquitin C-Terminal hydrolase expressed in testis of a teleost,the Nile Tilapia,Oreochromis niloticus

We previously produced four monoclonal antibodies to testicular proteins of a teleost, the Nile tilapia. One of the monoclonal antibodies, TAT(Testicular Antigen of Tilapia)-10, recognizes a Mr=27,000 protein (27 kD protein), which is present in A and early B type spermatogonia, spermatids, and spermatozoa in testis. In order to clarify the function of this protein, molecular cloning was conducted. The cDNA for the 27 kD protein contains a complete open reading frame encoding 220 amino acid residues. The predicted amino acid sequence of the 27 kD protein was homologous to those of the ubiquitin carboxy-terminal hydrolases (UCH) reported in mammals. The measurement of the ubiquitin-releasing activity of the recombinant 27 kD protein revealed that the protein is the active form of UCH. Northern blot analysis showed that the UCH mRNA was expressed in ovary and brain in addition to the testis. Immunohistochemical study showed that, in brain, UCH was localized especially on the olfactory organ including the olfactory bulb and olfactory epithelium in olfactory rosetta, suggesting the involvement of the protein in chemoreceptive function. In the Tilapia ovary, UCH localized especially in pre-vitellogenic oocytes, suggesting that the enzyme activity could be important in oocyte growth. This is the first report for the cDNA cloning and cellular localization of UCH in fish. J. Exp. Zool. 293:368-383, 2002.     

In-cell NMR spectroscopy of proteins inside Xenopus laevis oocytes

In-cell NMR is an application of solution NMR that enables the investigation of protein conformations inside living cells. We have measured in-cell NMR spectra in oocytes from the African clawed frog Xenopus laevis. ¹⁵N-labeled ubiquitin, its derivatives and calmodulin were injected into Xenopus oocytes and two-dimensional ¹H–¹⁵N correlation spectra of the proteins were obtained. While the spectrum of wild-type ubiquitin in oocytes had rather fewer cross-peaks compared to its in vitro spectrum, ubiquitin derivatives that are presumably unable to bind to ubiquitin-interacting proteins gave a markedly larger number of cross-peaks. This observation suggests that protein–protein interactions between ubiquitin and ubiquitin-interacting proteins may cause NMR signal broadening, and hence spoil the quality of the in-cell HSQC spectra. In addition, we observed the maturation of ubiquitin precursor derivative in living oocytes using the in-cell NMR technique. This process was partly inhibited by pre-addition of ubiquitin aldehyde, a specific inhibitor for ubiquitin C-terminal hydrolase (UCH). Our work demonstrates the potential usefulness of in-cell NMR with Xenopus oocytes for the investigation of protein conformations and functions under intracellular environmental conditions.Electronic Supplementary Material Supplementary material is available to authorized users in the online version of this article at .     

The interaction between ubiquitin C-terminal hydrolase 37 and glucose-regulated protein 78 in hepatocellular carcinoma

The ubiquitin C-terminal hydrolase (UCH) is a subfamily of deubiquitinating enzymes, which consists of four members: UCH-L1, UCH-L3, UCH37, and BRCA1-associated protein-1. Although there is growing evidence that UCH enzymes and human malignancies are closely correlated, there have been few studies on UCH37, especially on its interactions with other proteins. In the current study, a functional proteomic analysis was performed to screen UCH37-interacting proteins in hepatocellular carcinoma (HCC), and glucose-regulated protein 78 was identified as one interacting with UCH37, which was confirmed by co-immunoprecipitation and confocal laser scanning microscopy analysis, suggesting that their interaction could provide a new insight into the mechanism of HCC.     

UCH‐L1 promotes invasion of breast cancer cells through activating Akt signaling pathway

As a de‐ubiquitin enzyme, ubiquitin C‐terminal hydrolase (UCH)‐L1 has been shown to be overexpressed in several human cancers. However, the function of UCH‐L1 in invasion of breast cancers is still unclear. Here we report that the expression of UCH‐L1 is significantly higher in cancer cells with higher invasive ability. While ectopic UCH‐L1 expression failed to alter cell proliferation in MCF‐7 cells, it caused a significant upregulation of cellular invasion. Furthermore, siRNA mediated knockdown of UCH‐L1 led to suppression of invasion in UCH‐L1 overexpressing MCF‐7 cells. In order to identify molecular mechanisms underlying these observations, a novel in vitro proximity‐dependent biotin identification method was developed by fusing UCH‐L1 protein with a bacterial biotin ligase (Escherichia coli BirA R118G, BioID). Streptavidin magnetic beads pulldown assay revealed that UCH‐L1 can interact with Akt in MCF‐7 cells. Pulldown assay with His tagged recombinant UCH‐L1 protein and cell lysate from MCF‐7 cells further demonstrated that UCH‐L1 preferentially binds to Akt2 for Akt activation. Finally, we demonstrated that overexpression of UCH‐L1 led to activation of Akt as evidenced by upregulation of phosphorylated Akt. Thus, these findings demonstrated that UCH‐L1 promotes invasion of breast cancer cells and might serve as a potential therapeutic target for treatment of human patients with breast cancers.     

Raf-1 protein kinase is important for progesterone-induced Xenopus oocyte maturation and acts downstream of mos.

In somatic cells, the Raf-1 serine/threonine protein kinase is activated by several polypeptide growth factors. We investigated the role of Raf-1 in progesterone-induced meiotic maturation of Xenopus laevis oocytes. Raf-1 enzymatic activity and phosphorylation (reflected by a mobility shift on sodium dodecyl sulfate gels) were increased in oocytes following progesterone stimulation. The increase in Raf-1 activity was concurrent with an elevation in the activity of mitogen-activated protein (MAP) kinase. When RNA encoding an oncogenic form of Raf-1 (v-Raf) was injected into immature oocytes, MAP kinase mobility shift, germinal vesicle breakdown, and histone H1 phosphorylation increased markedly. When RNA encoding a dominant-negative version of Raf-1 was injected, progesterone-induced oocyte maturation was blocked. When RNA encoding Xenopus mos (mosxe) was injected into oocytes, Raf-1 and MAP kinase mobility shifts were observed after several hours. Also, when antisense mosxe oligonucleotides were injected into oocytes, progesterone-induced Raf-1 and MAP kinase mobility shifts were blocked. Finally, when antisense mosxe oligonucleotides were coinjected with v-Raf RNA into oocytes, histone H1 kinase activation, germinal vesicle breakdown, and MAP kinase mobility shift occurred. These findings suggest that Raf-1 activity is required for progesterone-induced oocyte maturation and that Raf-1 is downstream of mosxe activity.     

Potentiation of ATP-induced currents due to the activation of P2X receptors by ubiquitin carboxy-terminal hydrolase L1

Mammalian neuronal cells abundantly express a de-ubiquitinating isozyme, ubiquitin carboxy-terminal hydrolase L1 (UCH L1). Loss of UCH L1 function causes dying-back type of axonal degeneration. However, the function of UCH L1 in neuronal cells remains elusive. Here we show that overexpression of UCH L1 potentiated ATP-induced currents due to the activation of P2X receptors that are widely distributed in the brain and involved in various biological activities including neurosecretion. ATP-induced inward currents were measured in mock-, wild-type or mutant (C90S)-UCH L1-transfected PC12 cells under the conventional whole-cell patch clamp configuration. The amplitude of ATP-induced currents was significantly greater in both wild-type and C90S UCH L1-transfected cells, suggesting that hydrolase activity was not involved but increased level of mono-ubiquitin might play an important role. The increased currents were dependent on cAMP-dependent protein kinase (PKA) and Ca2+ and calmodulin-dependent protein kinase (CaMKII) but not protein kinase C. In addition, ATP-induced currents were likely to be modified via dopamine and cyclic AMP-regulated phosphoprotein (DARPP-32) that is regulated by PKA and phosphatases. Our finding shows the first evidence that there is a relationship between UCH L1 and neurotransmitter receptor, suggesting that UCH L1 may play an important role in synaptic activity.     

The potential role of ubiquitin c-terminal hydrolases in oncogenesis

Deubiquitinating enzymes (DUBs), capable of removing ubiquitin (Ub) from protein substrates, are involved in numerous biological processes. The ubiquitin C-terminal hydrolases (UCHs) subfamily of DUBs consists of four members: UCH-L1, UCH-L3, UCH37 and BRCA1-associated protein-1 (BAP1). UCH-L1 possesses deubiquitinating activity and dimerization-dependent ubiquitin ligase activity, and functions as a mono-ubiquitin stabilizer; UCH-L3 does both deubiquitinating and deneddylating activity, except dimerization or ligase activity, and unlike UCH-L1, can interact with Lys48-linked Ub dimers to protect it from degradation and in the meanwhile to inhibit its hydrolase activity; UCH37 is responsible for the deubiquitinating activity in the 19S proteasome regulatory complex, and as indicated by the recent study, UCH37 is also associated with the human Ino80 chromatin-remodeling complex (hINO80) in the nucleus and can be activated via transient association of 19S regulatory particle- or proteasome-bound hRpn13 with hINO80; BAP1, binding to the wild-type BRCA1 RING finger domain, is regarded as a tumor suppressor, but for such suppressing activity, as demonstrated otherwise, both deubiquitinating activity and nucleus localization are required. There is growing evidence that UCH enzymes and human malignancies are closely correlated. Previous studies have shown that UCH enzymes play a crucial role in some signalings and cell-cycle regulation. In this review, we provided an insight into the relation between UCH enzymes and oncogenesis.     

Casein kinase G may be the target of spermine during progesterone-induced oocyte maturation

Casein kinase G (CKG) with more than 2500-fold enrichraent was purified from Bufo bufo gargarizans ovaries. The catalytic activity of the enzyme was found to be associated with its 42 kD subunit, and its 26 kD subunit was found to be the major tsrget for the enzyme autophos phorylation. Each fuU-grown oocyte contained 1.9 units of CKG corresponding to an intracellular concentration of 93 nM. After injecting an amount of 0,38 units of the enzyme into the oocyte, approximately 50% of the progesterone-induoed maturation was inhibited. The inhibitory effect was enhanced in oocytes pretreated with spermine, which was consistent with the results that the enzyme was activated in vitro in the presence of spermine, The MPF-induced oocyte maturation was delayed and even prohibited in the kinase-microinjected oocytes. A 55 kD oocyte protein was identified as an suhstrate of CKG both in vivo and in vitro, and the enhancement of the 55 kD protein phosphory[ation was associated with kinase inhibition on maturation and on protein synthesis in kinase-microinjected oocytes. As the endogenous spermine level decreased in the course of progesteroneinduced oocyte maturation. 55 kD protein was dephosphorylated, Heparin, a specific inhibitor of CKG, potentiated the progesterone-induced oocyte maturation. Altogether the experimental reSults indicated Strongly that CKG may be the physiological target of spermine.     

Essential role of maternal UCHL1 and UCHL3 in fertilization and preimplantation embryo development

Post-translational protein modification by ubiquitination, a signal for lysosomal or proteasomal proteolysis, can be regulated and reversed by deubiquitinating enzymes (DUBs). This study examined the roles of UCHL1 and UCHL3, two members of ubiquitin C-terminal hydrolase (UCH) family of DUBs, in murine fertilization and preimplantation development. Before fertilization, these proteins were associated with the oocyte cortex (UCHL1) and meiotic spindle (UCHL3). Intracytoplasmic injection of the general UCH-family inhibitor ubiquitin-aldehyde (UBAL) or antibodies against UCHL3 into mature metaphase II oocytes blocked fertilization by reducing sperm penetration of the zona pellucida and incorporation into the ooplasm, suggesting a role for cortical UCHL1 in sperm incorporation. Both UBAL and antibodies against UCHL1 injected at the onset of oocyte maturation (germinal vesicle stage) reduced the fertilizing ability of oocytes. The subfertile Uchl1(gad-/-) mutant mice showed an intriguing pattern of switched UCH localization, with UCHL3 replacing UCHL1 in the oocyte cortex. While fertilization defects were not observed, the embryos from homozygous Uchl1(gad-/-) mutant females failed to undergo morula compaction and did not form blastocysts in vivo, indicating a maternal effect related to UCHL1 deficiency. We conclude that the activity of oocyte UCHs contributes to fertilization and embryogenesis by regulating the physiology of the oocyte and blastomere cortex.     

Structural basis for the specificity of ubiquitin C-terminal hydrolases.

The release of ubiquitin from attachment to other proteins and adducts is critical for ubiquitin biosynthesis, proteasomal degradation and other cellular processes. De-ubiquitination is accomplished in part by members of the UCH (ubiquitin C-terminal hydrolase) family of enzymes. We have determined the 2.25 A resolution crystal structure of the yeast UCH, Yuh1, in a complex with the inhibitor ubiquitin aldehyde (Ubal). The structure mimics the tetrahedral intermediate in the reaction pathway and explains the very high enzyme specificity. Comparison with a related, unliganded UCH structure indicates that ubiquitin binding is coupled to rearrangements which block the active-site cleft in the absence of authentic substrate. Remarkably, a 21-residue loop that becomes ordered upon binding Ubal lies directly over the active site. Efficiently processed substrates apparently pass through this loop, and constraints on the loop conformation probably function to control UCH specificity.     

Proteomic analysis of brain proteins in the gracile axonal dystrophy (gad) mouse, a syndrome that emanates from dysfunctional ubiquitin carboxyl-terminal hydrolase L-1, reveals oxidation of key proteins

Ubiquitin carboxyl-terminal hydrolase L-1 (UCH L-1) is a crucial enzyme for proteasomal protein degradation that generates free monomeric ubiquitin. Our previous proteomic study identified UCH L-1 as one specific target of protein oxidation in Alzheimer's disease (AD) brain, establishing a link between the effect of oxidative stress on protein and the proteasomal dysfunction in AD. However, it is unclear how protein oxidation affects function, owing to the different responses of proteins to oxidation. Analysis of systems in which the oxidized protein displays lowered or null activity might be an excellent model for investigating the effect of the protein of interest in cellular metabolism and evaluating how the cell responds to the stress caused by oxidation of a specific protein. The gracile axonal dystrophy (gad) mouse is an autosomal recessive spontaneous mutant with a deletion on chromosome 5 within the gene encoding UCH L-1. The mouse displays axonal degeneration of the gracile tract. The aim of this proteomic study on gad mouse brain, with dysfunctional UCH L-1, was to determine differences in brain protein oxidation levels between control and gad samples. The results showed increased protein oxidation in thioredoxin peroxidase (peroxiredoxin), phosphoglycerate mutase, Rab GDP dissociation inhibitor alpha/ATP synthase and neurofilament-L in the gad mouse brain. These findings are discussed with reference to the effect of specific protein oxidation on potential mechanisms of neurodegeneration that pertain to the gad mouse.     

Characterization of the testis in congenitally ubiquitin carboxy-terminal hydrolase-1 (Uch-L1) defective (gad) mice.

The gracile axonal dystrophy (gad) mice are known to have a deletion within the gene encoding ubiquitin carboxy-terminal hydrolase-1 (Uch-L1) and show hereditary sensory deterioration and motor paresis. Expression of Uch-L1 is reported to be almost limited to the nervous system and testis. To understand whether Uch-L1, one of the major ubiquitin carboxy-terminal hydrolase (UCH) isozymes in the testis, affects spermatogenesis and other UCH isozymes (Uch-L3, L4 and L5) expression in the testis, we compared the testis between gad, hetero and wild type mice by histological, immunohistochemical analyses and RT-PCR. Histological analysis in 25-week-old gad mice showed shrinking of seminiferous tubules, decreasing total number of cells and enlargement of remaining cells in seminiferous tubules. By immunohistochemistry, a significant decrease (p < 0.05) in the number of proliferating cell nuclear antigen (PCNA) positive cells was observed. Expression of other UCH isozyme mRNAs was not apparently affected by Uch-L1 deficiency in 25-week-old gad mice. This study is the first report on the testis of gad mutant mouse.     

Xp38gamma/SAPK3 promotes meiotic G(2)/M transition in Xenopus oocytes and activates Cdc25C

We have studied the role of p38 mitogen-activated protein kinases (MAPKs) in the meiotic maturation of Xenopus oocytes. Overexpression of a constitutively active mutant of the p38 activator MKK6 accelerates progesterone-induced maturation. Immunoprecipit ation experiments indicate that p38gamma/SAPK3 is the major p38 activated by MKK6 in the oocytes. We have cloned Xenopus p38gamma (Xp38gamma) and show that co-expression of active MKK6 with Xp38gamma induces oocyte maturation in the absence of progesterone. The maturation induced by Xp38gamma requires neither protein synthesis nor activation of the p42 MAPK-p90Rsk pathway, but it is blocked by cAMP-dependent protein kinase. A role for the endogenous Xp38gamma in progesterone-induced maturation is supported by the inhibitory effect of kinase-dead mutants of MKK6 and Xp38gamma. Furthermore, MKK6 can rescue the inhibition of oocyte maturation by anthrax lethal factor, a protease that inactivates MAPK kinases. We also show that Xp38gamma can activate the phosphatase XCdc25C, and we identified Ser205 of XCdc25C as a major phosphorylation site for Xp38gamma. Our results indicate that phosphorylation of XCdc25C by Xp38gamma/SAPK3 is important for the meiotic G(2)/M progression of Xenopus oocytes.     

Identification of a 26S proteasome-associated UCH in fission yeast

We have identified a 26S proteasome-associated ubiquitin carboxyl-terminal hydrolase (UCH) in Schizosaccharomyces pombe. The gene (designated uch2+) encodes a protein containing a UCH catalytic domain at its N-terminus and a short extension at its C-terminus. uch2+ is nonessential as the uch2 null mutant strain showed no significant difference from the wild-type strain. The GFP-tagged Uch2p is localized predominantly to the nuclear periphery, which is similar to the 26S proteasome localization. Deletion of the C-terminal extension of Uch2p resulted in a drastic change of its subcellular localization: it showed a generally diffused distribution instead of a perinuclear pattern. Glycerol gradient centrifugation analysis and coimmunoprecipitation studies of fission yeast extracts using anti-Mts4p antiserum suggest that Uch2p is associated with the 26S proteasome and the association of Uch2p with the 26S proteasome is mediated by its C-terminal extension.     

Assay for characterization of human follicular oocyte maturation inhibitor using Xenopus oocytes

Human follicular fluid from healthy mature Graafian follicles and from pathologic ovarian cyst fluid was found to be inhibitory to progesterone-induced meiotic maturation of oocytes from the South African clawed toad, Xenopus laevis. Human follicular fluid but not human serum, collected from the same individuals, demonstrated a linear dose-response inhibition on the maturation of oocytes in the Xenopus assay system. These findings indicate that the human follicular and cyst fluids contain oocyte maturation inhibitor (OMI). This human OMI was inactivated when subjected to a boiling water bath for 2 min. The OMI action was shown to be reversible in its inhibitory action. The fact that OMI can act directly on the oocyte was demonstrated by its inhibitory action on maturation in defolliculated oocytes. The findings demonstrate that the inhibitory action of human OMI is not species-specific. Xenopus oocytes provide a simple, readily available, year-round bioassay material for testing follicular oocyte maturation inhibitor.