Sperm replacement in sperm-storage tubules causes last-male sperm precedence in chickens.

1. This study elucidated the last-male sperm precedence (LMSP) mechanism in chickens by examining replacement in storage tubules (SSTs) after multiple artificial inseminations (AI) and the effects of seminal plasma (SP) and male breed on sperm replacement in SSTs.2. Hens were artificially inseminated with fluorescent dye-labelled spermatozoa from White Leghorn (WL) chickens. Secondary AI was conducted 3 d later with sperm labelled with different nuclear fluorescent dye. Percentage of first and second inseminated sperm in SSTs and their logarithmic odds were calculated. The effect of SP on LMSP was examined using (1) Lake's solution-washed sperm before second insemination, and (2) SP injected continuously after first insemination. Effect of breed difference on sperm replacement was investigated using Barred Plymouth Rock (BP) sperm.3. Successive WL-sperm inseminations at three-day intervals caused > 70% stored sperm replacement in SSTs. Although SP removal from sperm from second insemination significantly decreased replacement, its intra-vaginal injection did not affect release. Secondary insemination using BP sperm significantly increased replacement.4. Sperm replacement is a major factor favouring LMSP in domestic chickens. Two fluorescent staining of sperm, and intra-vaginal multiple AI technique have enabled visualisation, differentiation, and quantification of multiple inseminated sperm stored in the SSTs.

Effects of testosterone on production of perivitelline membrane glycoprotein ZPC by granulosa cells of Japanese quail (Coturnix japonica).

Avian perivitelline membrane, an investment homologous to the zona pellucida of mammalian oocytes, is composed of at least two glycoproteins. Previous studies have indicated that one of the components, a glycoprotein homologous to mammalian ZPC, is produced and secreted by the granulosa cells of developing follicles of the chicken ovary. In the present study, we evaluated the expression and regulation of ZPC in Japanese quail (Coturnix japonica) granulosa cells both in vivo and in vitro. Western blot analysis of the SDS-solubilized granulosa layer using anti-quail ZPC antiserum showed that the amount of ZPC increased in parallel with follicular development. Northern blot analysis of total RNA using cDNA of quail ZPC showed that the increase in mRNA expression was also correlated with follicular development. To investigate the regulation of ZPC production, the granulosa cells were cultured in a medium containing steroid hormones such as progesterone, estradiol-17ss, or testosterone. By measuring ZPC protein and mRNA with Western and Northern blot analyses, respectively, we found that addition of testosterone maintained ZPC contents in the culture of the granulosa cells, and that ZPC mRNA expression was high in the culture with testosterone compared to the control. These results suggest that testosterone stimulates ZPC protein production at the gene transcription level.

Characterization of progressive changes in ZPC of the vitelline membrane of quail oocyte following oviductal transport.

The inner layer of the vitelline membrane of avian oocyte is equivalent to the zona pellucida of mammalian oocytes or to the vitelline envelope of amphibian oocytes. One of the two major glycoproteins in the inner layer of quail vitelline membrane, formerly called 33-kDa glycoprotein, is homologous to mammalian ZPC, one of the components of zona pellucida. Quail ZPC is found to have different mobilities on SDS-polyacrylamide gel electrophoresis depending on whether it is obtained from the preovulatory follicle or from the laid eggs. In order to characterize the progressive changes in the molecular size of quail ZPC during the oviductal transport, the inner layer isolated from the follicle was incubated in vivo in various regions of the oviduct and subjected to Western blot analysis with anti-quail ZPC antiserum. The quail ZPC of the inner layer incubated in infundibulum reduced its apparent molecular weight, exhibiting the same electrophoretic mobility as that of laid eggs. The similar reduction in molecular weight was observed after the in vitro incubation of the inner layer with the extracts of infundibulum. From the comparison of the N-terminal amino acid sequences, it was found that the first 26 residues of the quail ZPC in follicular oocytes are missing from the ZPC of laid eggs. In addition, lectin blot analysis suggested the modification of oligosaccharide chains during the oviductal transport. These results represent the first description in the avian oviduct of the presence of protease, which is similar to oviductin, a trypsin-like protease involved in the hydrolysis of a major component of the vitelline envelope of amphibian oocytes. Mol. Reprod. Dev. 55:175-181, 2000.

Effects of oestradiol-17beta and testosterone on progesterone production in the cultured granulosa cells of Japanese quail.

In order to study the effects of steroid hormones on steroidogenesis in the avian ovary, quail granulosa cells were cultured with follicle stimulating hormone (FSH), oestradiol-17beta or testosterone. The progesterone content of the medium during the culture period of 66 h and the following 3 h of incubation with luteinising hormone (LH), was measured by radioimmunoassay. When FSH, oestradiol-17beta or testosterone were added during the 66 h culture, subsequent progesterone production by the cells during 3 h of incubation with LH was significantly increased. However, testosterone also stimulated progesterone production in the medium during the 66 h culture period, whereas FSH oroestradiol-17beta did not. Addition of staurosporine during culture inhibited both LH-stimulated progesterone production and testosterone-stimulated progesterone production. These results indicate that the processes during which granulosa cells acquired responsiveness to LH, and testosterone stimulates progesterone production might both be mediated by a staurosporine-sensitive protein kinase C-dependent pathway in quail granulosa cells.

Involvement of mitogen-activated protein kinase in transforming growth factor alpha-stimulated cell proliferation in the cultured granulosa cells of the Japanese quail.

The avian granulosa cells proliferate during follicular growth phase and differentiate to produce progesterone in response to luteinizing hormone (LH) when the follicle becomes the largest. In order to study the involvement of mitogen-activated protein (MAP) kinase in proliferation of the granulosa cells in avian species, quail granulosa cells were cultured for 66 h with various hormones (follicle stimulating hormone (FSH), LH, progesterone, estradiol-17beta, testosterone), or growth factors (transforming growth factor alpha (TGF alpha), epidermal growth factor (EGF), insulin-like growth factor I (IGF-I), IGF-II), and the presence of immunodetectable MAP kinase was examined in the cell lysates. When the granulosa cells were cultured with TGF alpha, the cell number as well as the incorporation of [3H]thymidine was increased. Other hormones or growth factors caused no significant increase in cell numbers. Stimulation of the cells with TGF alpha for 10 min caused a retarded mobility of MAP kinase in the gel of SDS-PAGE. Both the increases in [3H]thymidine incorporation and the retarded mobility were inhibited by the presence of a tyrosine kinase inhibitor, genistein, indicating the importance of phosphorylation of protein during the TGF alpha-stimulation.

Circulating blood group-related antigen(s) in cancer patients detected by the monoclonal antibodies produced against hepatocellular carcinoma cell line.

Two murine monoclonal antibodies (MAbs) S1 and S3 produced against a human hepatoma cell line were found to recognize both H (type II) antigen and difucosyl type II structure (Y-antigen). Both MAbs reacted with hepatoma tissues obtained from surgical resection. A double determinant enzyme immunoassay (DDEI) employing these MAbs successfully detected the corresponding antigen(s) in the supernatant which is either released or shed from the immunizing hepatoma cell line. DDEI also revealed that these antibodies reacted with the corresponding antigen(s) in the sera from cancer patients. Among the sera from O blood group donors, individuals with primary hepatoma showed markedly higher levels of antigen concentration than the normal control group. Several cases with gall bladder, lung and pancreas carcinomas, which are of O blood type, also had significantly higher levels of antigen. One case with ovarian cancer of B blood type showed higher antigen concentration than normal O blood group donors suggesting a change in the carbohydrate structure of the corresponding antigen(s) in the serum. These data suggest that the release or the shedding of the antigen(s) from the cells may increase due to the malignant transformation, resulting in higher amounts of the antigen(s) in the serum of certain cancer patients.

[NK activities in cancer patients--their modification by an immunopotentiator].

NK activity of patients with cancer against gastric carcinoma cell line KATO-III, lung carcinoma cell line A-549 as well as K-562 was significantly lower than that in the control group. We investigated in vitro effects of immunopotentiator, OK-432, on the effector cells of both healthy donors and cancer patients. NK activity of healthy donors was clearly augmented after treatment of effector cells with OK-432. NK activity of some patients with cancer, however, showed no augmentation after OK-432 treatment. These results might be helpful for monitoring cancer patients from immunological view point.