An exogenous gene (GFP&lacZ/pkkv4-lacZ) introduced into the germinal crescent region (GCR) of avian embryos was confirmed to be successfully transferred to the gonads via the primordial germ cells (PGCs). The hatched chicks were raised until the stage of sexual maturation. The incorporation of pkkv4-lacZ was detected in both male and female DNA-treated chickens (DNA-chicken). Female DNA-chicken were subjected to artificial insemination using sperm from normal male chickens according to routine methods. Fertilized eggs obtained from female DNA-chicken were incubated for 72 h and the expression of green fluorescent protein (GFP) gene in the embryos of offspring was detected under a fluorescent microscope. Following the examination for GFP the embryos removed from the yolk were also examined by X-gal staining to detect the expression of the lacZ gene in offspring, and the expression of pkkv4-lacZ was clearly detected. Finally, the presence of pkkv4-lacZ in the extracts from embryos was determined by polymerase chain reaction (PCR) analysis. In male DNA-chicken, the presence of injected DNA was also confirmed in the extracts from sperm. These results suggest that the exogenous gene introduced into the GCR of chicken embryos migrated successfully to the gonad, resulting in incorporation into the offspring and spermatozoa of DNA-chicken.

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Bovine cumulus cells after oocyte maturation were cultured under serum-starvation condition or grown to confluence. Their cell size, cell cycle phases, and developmental potential as donors for nuclear transfer (NT) were investigated. The majority of trypsinized cells of both cultures were found within the medium size range (15-20 μm). Flow cytometric analysis revealed that small- and medium-sized cells had significantly higher percentages of nuclei existing in the G0/G1 phase (95-98%) than large-sized cells (82%) regardless of the culture conditions. Serum-starved cells of different sizes (small, 9-14 μm; medium, 15-20 μm; large, 21-26 μm) had the same fusion rate, cleavage, development to blastocysts, and blastocyst cell number of NT embryos. NT embryos reconstituted from serum-starved and confluent medium-sized cells showed the same developmental rate. These results indicate that the most available medium-sized cells derived from both serum-starvation and confluent cultures can be used as a G0/G1-phase nuclei source.

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The effect of basic fibroblast growth factor (bFGF) on the production of prostaglandin (PG) F₂α and PGE₂, and the intracellular mechanisms of its action, were investigated in cultured bovine mid-luteal cells (days 8-12 of the estrous cycle). The cells were cultured for 24 h and then exposed to varying concentrations of bovine recombinant bFGF (rbFGF, 1-100 ng/ml) for a further 24 h. A 24-h stimulation with the highest concentration of rbFGF resulted in increases in both PGF₂α and PGE₂ production by mid-luteal cells (P<0.05). Both U-73122 (an inhibitor of phospholipase (PL) C, 10^-6 M) and anthranilic acid (ACA; an inhibitor of PLA₂, 10^-5 M) inhibited the rbFGF-induced PGF₂α and PGE₂ production (P<0.05). Moreover, following down-regulation of protein kinase C with a tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate (PMA, 10^-6 M), the stimulatory effect of rbFGF was no longer evident. These results suggest that locally produced bFGF may play a role as one of the autocrine and/or paracrine stimulators of the production of PGs in bovine corpus luteum, and the stimulatory effect of bFGF on the production of PGs is mediated via the PLC-PKC-PLA₂ pathway

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The expression of cytoplasmic dynein and kinesin in rat Sertoli cells was examined using immunohistochemistry with light and electron microscopies, and the existence of such bi-directional motors in the cells was confirmed. Cytoplasmic dynein was localized in the ectoplasmic specializations (ESs), mitochondria, and endoplasmic reticula. Among microtubules, an intense reaction of dynein was also detected, while, the reaction of kinesin changed in intensity in a stage-dependent manner. The most distinct reaction of kinesin in Sertoli cells was detected at stages VII-VIII, while a weak reaction was observed at stages IX-XIV. No positive reaction was detected at stages I-VI. The intracellular localization of kinesin was detected in the ES (stage X) and endoplasmic reticulum, but a defined reaction was not observed among microtubules. These results suggested that an active bi-directional organelle transport is performed in rat Sertoli cells, and the system may relate to the elongation and release of spermatids, and to the migration of membranous organelles such as mitochondria and endoplasmic reticula. In addition, the sliding movement of microtubules may be carried out mainly by cytoplasmic dynein.

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The presence of gonadotropin-releasing hormone (GnRH)-like protein has been previously demonstrated in the bovine follicle. This GnRH-like protein was purified and concluded to be histone H2A. However, neither GnRH peptide nor specific GnRH-immunoreactivity (GnRH-IR) has been demonstrated in the bovine follicle so far. Thus, this study focused on the detection of specific GnRH-IR using the second-antibody enzyme immunoassay in isolated bovine mature follicles, and on an examination of the direct effect of luteinizing hormone (LH), endothelin-1 (ET-1), and cytokines on the GnRH-IR using an in vitro microdialysis system (MDS). We further examined a cross-reactivity of the GnRH antibody with bovine histone H2A. GnRH-IR was detected in microdialyzed perfusate from isolated bovine mature follicles at 4.40 ± 0.35 pg/ml (mean ± SEM). Bovine histone H2A showed no GnRH-IR at all. Heat treatment of the extract (100 C for 10 min) did not affect the GnRH-IR. Single infusion of LH, ET-1, or cytokines into the MDS did not affect the GnRH-IR. However, infusion of ET-1 after LH exposure increased the GnRH-IR. These results demonstrate the presence of specific GnRH-IR, that is different from histone H2A, in microdialyzed perfusate of isolated bovine mature follicles in vitro, suggesting that the GnRH-IR may reflect a role of GnRH-like peptide or some peptide structurally similar to GnRH in the local regulation of mature follicles.

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To identify sperm glycosidases specific to sugar residues found in the zona pellucida of pig oocytes, frozen-thawed ejaculated spermatozoa were treated experimentally and assayed for activities of α-L-fucosidase, α-D-mannosidase, β-D-galactosidase and N-acetyl-β-D-glucosaminidase (GlcNAc’ase). When spermatozoa were incubated with calcium ionophore A23187 for 1 h, about 70% were acrosome-reacted and about 72, 46, 56 and 35% of the activities of α-L-fucosidase, α-D-mannosidase, β-D-galactosidase and GlcNAc’ase, respectively, were lost from spermatozoa. However, lower levels of α-L-fucosidase, α-D-mannosidase and β-D-galactosidase activity, and a higher level of GlcNAc’ase activity were released from spermatozoa treated with protease. Furthermore, treating spermatozoa with the detergent n-octylglucoside caused the release of similar amounts of α-L-fucosidase and β-D-galactosidase activity and higher amounts of α-D-mannosidase and GlcNAc’ase activity, compared with those treated with calcium ionophore. Protease and n-octylglucoside treatments released similar amounts GlcNAc’ase activity. These results suggest that GlcNAc’ase is present mainly in the plasma membrane of pig spermatozoa, but that α-L-fucosidase, α-D-mannosidase and β-D-galactosidase are mainly found in the acrosome matrix including the outer acrosomal membrane, and that α-D-mannosidase may also be located in the inner acrosomal membrane. Maximal activity of α-D-mannosidase was at around physiological pH (7-8), but the other three glycosidases showed maximal activity at pH 4-5. When cumulus-free pig oocytes matured in culture were inseminated in the presence of various glycosidase inhibitors, the number of spermatozoa bound to the zona pellucida and the proportion of oocytes penetrated were greatly reduced by the inhibitors of α-L-fucosidase, α-D-mannosidase and β-D-galactosidase, but not by an inhibitor of GlcNAc’ase. These results indicate that α-L-fucosidase, α-D-mannosidase and β-D-galactosidase are all important glycosidases involved in pig sperm-zona interaction.

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In pelagic egg-spawning marine fish, hepatically derived vitellogenin undergoes limited proteolysis within the follicles during early oocyte maturation followed by additional degradation during late oocyte maturation. This study demonstrates the role of cathepsin D expressed in late maturating oocytes of round scad Decapterus maruadsi, in relation to ovarian cathepsins B and L that are possible agents causing yolk degradation. Ovarian cathepsins B, D and L in late oocyte lysate were isolated by gel filtration, and their respective molecular masses were estimated as 26.7, 34.9 and 29.9 kDa. The effect of pepstatin, a potent inhibitor of cathepsin D, was examined on their activities in the oocyte lysate and isolates of cathepsins. In both sources, the activity of cathepsin D was completely inhibited whereas that of cathepsin L was not inhibited. However, the activity of cathepsin B in the lysate was significantly reduced while no inhibition of its activity was observed in the isolate. Results indicate that ovarian cathepsin D is involved in activation of coexistent procathepsin B during late oocyte maturation in scad.

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The present study aimed to examine the possibility that bovine somatotropin (BST) treatment after artificial insemination (AI) may improve the fertility by the stimulation of corpus luteum (CL) function in seasonally anestrous ewes. The experiment was conducted at the Tawa Field Station, Shibecha-cho in Hokkaido, Japan, during the non-breeding season (May, 1995). Eighty-nine mature (2 to 3 years old) Suffolk and Suffolk-crossed ewes were treated with controlled internal drug release devices containing 0.3 g progesterone (CIDR-G) for 12 days. Ewes were injected intramuscularly with 500 IU PMSG the day before CIDR removal. Eighty-seven ewes were assigned to an insemination of frozen semen on a fixed-time basis (42 to 50 h) after removal of CIDR. On the 11th day after AI (13 days after CIDR removal), 43 inseminated ewes were received 100 mg of recombinant BST, while the remaining 44 ewes were given vehicle as controls. The BST administration appeared to reduce pregnancy (65.1% vs. 72.7%) and lambing (51.2 vs. 65.9%) rates, prolificacy (145.5% vs. 165.5%) and proportion of ewes rearing more than two lambs (47.6% vs. 60.7%), as compared with controls, although there was no signifiant difference. BST-treated ewes had higher plasma GH concentrations (endogenous GH plus exogenous BST) at Day 19 (8 days after BST treatment) as compared with controls in both pregnant and non-pregnant ewes (P<0.05). Also, in the control group, plasma GH concentration of pregnant ewes was higher than that of non-pregnant ewes (P<0.05). Plasma progesterone (P) and estradiol-17β (E) cencentrations at Day 19 were lower in BST-treated ewes than that of control ewes (P<0.05). In conclusion, an administration with 100 mg of BST on Day 11 after AI during the non-breeding season resulted in the suppression of the ovarian function 1 week thereafter. Consequently, the conception rate and lambing rate did not improve by the present BST treatment in the ewe.

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