Ubiquitin carboxyl-terminal hydrolase L1 contributes to the oocyte selective elimination in prepubertal mouse ovaries.

Apoptosis of abnormal oocytes is essential for defective oocyte elimination during prepubertal ovary development, and the ubiquitin system regulates the cell apoptosis via the degradation of specific proteins. Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) is a component of the ubiquitin system, and the UCH-L1-dependent apoptosis is important for spermatogenesis. In the present study, the change in the number of follicles and the expression of UCH-L1 in oocytes were determined in prepubertal mouse ovaries by immunohistochemical techniques. A significant decrease in the follicular pool was found in prepubertal mouse ovaries during the period of day 21 to day 28 after birth, and accordingly, the UCH-L1 protein expression was increased, to some degree in association with Jun activation domain-binding protein 1 (Jab1) and cyclin-dependent kinase inhibitor p27(Kipl). The increased UCH-L1 protein, together with the corresponding changes of Jab1 was detected in morphologically abnormal oocytes of prepubertal ovaries. Through the immunofluorescent colocalization, UCH-L1 was shown concentrating in abnormal oocytes, and a parallel change in Jab1 was also seen. The affinity analysis confirmed the interaction between UCH-L1 and Jab1 in ovaries. These results suggest that UCH-L1 plays an important role, possibly in association with Jab1 and p27(Kipl), in selective elimination of abnormal oocytes during mouse prepubertal development.

[Uterine expression of adenovirus E4 promoter-binding protein 4 (E4BP4) during the embryo implantation period in mice].

Adenovirus E4 promoter-binding protein 4 (E4BP4), a mammalian basic leucine zipper (bZIP) nuclear transcription factor, was identified to be involved in cell survival and proliferation. Previous research data showed that the expression of E4BP4 gene was up-regulated at the implantation sites on day 5 pregnancy of mouse. The aim of the present study was to examine the expression pattern of E4BP4 gene in uterus during pre-implantation period, and at the implantation sites and inter-implantation sites during the implantation process, through the Northern blot, in situ hybridization, Western blot and immunohistochemistry analyses. It was found that, (1) during the pre-implantation period, the expression of E4BP4 gene was gradually increased; (2) its expression was sharply up-regulated at the implantation sites compared with that at the inter-implantation sites during the embryo implantation process; (3) the uterine expression of E4BP4 gene was not embryo-dependent, but observed in artificially induced decidulization of pseudopregnant mouse; (4) both E4BP4 mRNA and E4BP4 protein were localized in stromal cells and decidual cells around the uterine lumen. These results indicated that E4BP4 may play a critical role in embryo implantation process by promoting stromal cell proliferation and inhibiting decidual cell apoptosis.

Rat oocytes in early tertiary follicles can attain appropriate maturation in combination culture system.

We have developed a "combination culture system", composed of primary differentiated granulosa cells, proestrus anterior pituitary lobe and delta-4-androstenedione. This cultured system effectively induce the maturation of oocytes from early tertiary follicles of diethylstilbestrol (DES)-treated rats. After DES stimulation, an average of 189 oocytes were obtained from early tertiary follicles of unilateral ovaries; 78% of these oocytes, when cultured in this culture system, extruded the first polar body, a criterion of oocyte maturation. Of the mature oocytes, the rate of normal fertilization and egg cleavage were 88% and 93%, respectively. After 96 h of culturing in vitro, 59% of the zygotes developed into morulae or blastocysts. Embryos implanted at the two-cell stage were able to develop into healthy individuals. Thus the combination culture system provides an experimental model in which the molecular mechanisms of egg maturation, fertilization, and embryonic development can be studied.

[Localization of EGFP-DPF-1 expressed and secreted by HeLa cells in oocytes].

The eukaryotic expression plasmid pEGFP-N1/DPF-1 was constructed through fusing rabbit oviduct-specific glycoprotein (DPF-1) gene to the 5' terminus of eGFP gene. After transfecting the recombinant plasmid into HeLa cells, we got some cell strains expressing and secreting eGFP-DPF-1 stably. The apparent molecular weight of the fusion protein was up to 120 KD indicating the fusion protein was modified. Rabbit oocyte-cumulus cells complexes (COC), the cumulus cells-deprived oocytes or oviductal oocytes were co-cultured with the cell strain or cultured in the conditioned medium derived from the cell strain to observe the distribution of DPF-1 in oocytes after incubation. The results revealed that DPF-1 but not GFP alone associated with zona pellucidae (ZP) of oocytes, especially in the inner layer of ZP, and was evenly distributed in dot-like shape on the outer surface of the cytoplasmic membrane. In contrast, the cumulus cells around the oocyte did not interfere the association of DPF-1 with oocyte, and no trace of DPF-1 was found in the cumulus cells. The experiment proved that the oviductin expressed and secreted by eukaryotic cells in vitro could bind to oocytes, and was directly observed by using green fluorescent protein as a tracer. These results provided some interesting informations for the further studying on the functions of the oviductin.

[Cloning, expression, and antibody production of mouse ISP2].

A cDNA encoding mouse implantation serine proteinase 2 ISP2 was amplified from total cDNAs of mouse uterus implantation sites on D4.5 of pregnancy by PCR, and sequenced GenBank accession No. A442918 . DNA sequencing indicated that the ISP2 cDNA had an unreported 204 bp sequence at 3' untranslated region besides the open reading frame encoding 279 amino acid residues, which was identical with literature. In order to obtain recombinant ISP2 rISP2 an expression plasmid pGEX-4T-2/ISP2 was constructed and transformed into E.coli BL21(DE3) strain. Expressed fusion protein GST-ISP2 was purified by SDS-PAGE and digested with thrombin, and the digestion mixture was subjected to SDS-PAGE again to recover rISP2. Rabbits were immunized using rISP2 as immunogen, and the polyclonal anti-ISP2 antisera were obtained. Immunohistochemical analysis using this antisera showed specific and high expression of ISP2 in mouse endometrial gland epithelium in early pregnancy.

[Developmental stage-dependence of cyclin B1 protein localization and gene expression in rabbit spermatogenesis].

By means of in situ hybridization and immunohistochemistry, the protein localization and gene expression of cyclin B1 in spermatogenic cells were characterized during the spermatogenesis of rabbits. The results showed that the cyclin B1 mRNA in rabbit spermatogenic epithelium was expressed dominantly in primary spermatocytes. The expression was observed in round spermatids with a gradual decline in the process of metamorphosis of the spermatids, but not in elongated spermatids and sperms. Cyclin B1 protein was expressed in mitotic spermatogonia and meiotic spermatocytes and was observed predominantly in round and elongated spermatids. These results indicate that the expression of cyclin B1 mRNA and localization of cyclin B1 protein are dependent on the developmental stages of spermatogenic cells.

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 p13(suc1)-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 protein turnover and degradation.

Antibodies against the C-terminal peptide of rabbit oviductin inhibit mouse early embryo development to pass 2-cell stage.

A full-length rabbit oviductin cDNA(1909bp) was cloned. It consists of a 5'-UTR of 52bp, an open reading frame (ORF) of 1374bp and a 3'-UTR of 483bp and has more than 80% homology with that of other mammal oviductins. N-terminal peptide (NTP) (384 residues) and C-terminal peptide (CTP) (73 residues) of deduced protein precursor has about 80% and 50% identity with that of other mammals respectively. Fusion proteins GST-NTP 368(1R-368N)and GST-CTP73 (369F-441A) were expressed and purified. NH2-terminal of CTP sequencing reveals that the purified protein is consistent with the deduced one. In order to study the function of NTP and CTP the mouse anti-NTP and rabbit anti-CTP antisera were prepared. Tissue-specific (skeleton muscle, oviduct, uterus, ovary, liver, heart and brain) analysis indicated that rabbit oviductin was only found in oviduct. The conditioned medium derived from the rabbit oviduct mucosa epithelial cells has a function of overcoming the early embryonic development block of Kunming mouse cultured in vitro. Anti-CTP antiserum could totally inhibit the early embryo development at 2-cell stage cultured in the conditioned culture medium, but anti-NTP antiserum couldn't. There was a positive relationship between the ratio of early embryos at development block and the dosage of anti-CTP antiserum added in the conditioned culture medium. These results suggest that oviductin has a function not only on fertilization, but also on the release of early embryonic development block, and the later function domain of rabbit oviductin may be situate in its C-terminal.