This study was designed to assess the release of nitric oxide (NO) from porcine granulosa cells. The cells obtained from medium-sized ovarian follicles (3-6 mm diameter) were cultured for 48 h with ovine FSH to be developed. A significant accumulation of nitrite was observed during development, showing an increase in NO synthesis. The mRNA of the endothelial NO synthase was detected in the granulosa cells, as revealed by RT-PCR. Furthermore, nitrite was accumulated in the culture medium after 1-h stimulation with 12-O-tetradecanoylphorbol 13-acetate (TPA), but not with 4α-phorbol, 4β-phorbol 13-monoacetate (4β-PMA) and forskolin. TPA led to an increase in the nitrite content in a dose-dependent manner. NO was measured directly in the cell suspension with an amperometric NO sensor. The NO sensor signal gradually increased to reach a plateau (5-15 pA) after exposure to TPA in the presence of L-arginine, but not D-arginine. The concentration of NO was approximately 5.5-11 nM, but no detectable change was observed after the addition of 4α-phorbol or 4β-PMA. These results suggest that the synthesis of NO is promoted at least in part through the activation of protein kinase C in granulosa cells.
Cyclic Merino ewes were immunized three times against 5α−androstane-3α,17β-diol (3α-diol) at three week intervals (weeks 0,3 and 6). After the third immunization, estrous cycles were synchronized by a series of PG injections and intensive blood samples were obtained for gonadotropin analyses during the mid-luteal phase and during the early follicular phase. Ovulation rates were determined after each immunization and subsequent cloprostenol (PG)-induced luteolysis (weeks 3, 6, 8 and 11). After the second immunization, daily blood samples were obtained and concentrations of plasma progesterone (P) were measured. Immunization against 3α-diol significantly increased the ovulation rate after the second and third immunizations and at week 10 (P<0.01, P<0.01 and P<0.05 respectively). It also induced anovulation in several ewes. Plasma concentrations of P were significantly increased by 3α-diol immunization (P<0.01). Pulse frequency of LH and concentrations of FSH were also increased during the luteal phase (P<0.01 and P<0.05 respectively). There was a significant correlation between the mean concentrations of FSH during the luteal phase and the subsequent ovulation rates (P<0.01). These results indicate that immunization against 3α-diol increases ovulation rate in ewes and its effect was, at least partially, exerted by increased plasma concentrations of FSH.
The ultrasonic features of the uterine horn in dairy cows were compared with the macro- and microscopic features to find reliable landmarks for ultrasonography. The uterine horns removed from 16 slaughtered Holstein cows were immersed in water and examined with ultrasonography in cross-section. Then these uterine horns were examined macroscopically and microscopically in cross-section. In another part of our experiment, layers in the uterine were removed from the uterine wall under ultrasonic observation and the remaining tissues were examined macroscopically and microscopically. Also a 21-G needle was placed vertically into successive layers of cross-sections of the uterine horn under macroscopic observation and then the tissues were examined ultrasonically to determine where the tip of the needle was. Then a small amount of Indian ink was injected through the needle into each layer and the locations of the ink deposits were determined histologically. The uterine wall was visualized in 5 layers on the ultrasonic images: inner echogenic layer, slightly echogenic elliptical layer, central echogenic layer, slightly echogenic arched layer and outer echogenic layer, from inside to outside. Through macro- and microscopic examinations, it was found that the slightly echogenic elliptical layer corresponded to the circular muscle layer, and the slightly echogenic arched layer corresponded to the longitudinal muscle layer. In the present study, it was demonstrated that the 5 layers imaged by ultrasonography corresponded histologically to the endometrium, myometrium (circular muscle layer, stratum vasculare, longitudinal muscle layer) and perimetrium, from inside to outside, respectively.
Determination of the expression level and localization of extracellular matrix (ECM) components is crucial for understanding the mechanism of maintenance and remodeling of the ovarian follicle structure. Previously, we demonstrated species-specific differences in the process of apoptosis in granulosa and theca cells during follicular atresia. In the present study, we histochemically compared the localization of type I, III and IV collagens and the expression of their mRNA. In healthy porcine or bovine follicles, type III and IV collagens were mainly distributed in theca interna layers, and strong to moderate expression of their mRNAs were observed in granulosa and theca interna layers, respectively. During follicular atresia, marked decreases in type I and IV collagens were observed in porcine follicles, while no changes were seen in bovine follicles. These findings indicate that these ECMs have important roles in follicular development and/or degeneration, and that species-specific differences in their production reflect differences in the regulatory mechanism of granulosa cell apoptosis between atretic porcine and bovine follicles.
It is well known that an injection of prostaglandin F2α (PGF2 α) or its analogue during the mid luteal phase of the estrous cycle induces a rapid decrease in plasma progesterone (P4) concentration, followed by luteolysis in the cow. There is evidence that a potent vasoactive peptide, endothelin-1 (ET-1), is produced in the bovine corpus luteum (CL), and that it is directly involved in luteolysis. We previously found that ET-1 concentrations in the peripheral plasma increase during the period of luteolysis and ovulation in cows. However, it is not clear whether the elevation of peripheral plasma ET-1 concentration observed during luteolysis and ovulation originates exclusively from the ovary and/or CL. Such a profile of plasma ET-1 concentration may be affected by the age of female calves as well as the activity of the ovary. Thus, we aimed to 1) determine in detail the changes in plasma ET-1 and P4 concentrations during the estrous cycle in the cow, 2) investigate plasma changes in ET- 1 and P4 concentrations in new-born, 120-day-old and 240-day-old female calves, and 3) examine the effect of luteolytic injection of PGF2α analogue on the plasma ET-1 concentrations in the animals in this study. The peripheral plasma ET-1 concentrations in the cycling cows showed a pulsatile increase. They reached the their highest level (13.66 pg/ml) around the time of luteolysis and estrus, dropped significantly during Days 2-12 (early to mid luteal phase) and Days 13-19 (late luteal phase) (p<0.05), and then increased again on Days 20-22 (p<0.05) when the next estrus appeared. In the peripheral plasma of newborn, 120-day- old and 240-day-old female calves, P4 concentrations remained at low levels (0.1-0.2 ng/ml). ET-1 concentrations in these animals were lower than those in the cycling cows, and remained at low levels throughout the experimental period. Moreover, plasma ET-1 concentrations, unlike those in the cycling cow, did not change after a luteolytic PGF2α injection. In conclusion, the results of the present study gave the first detailed information that plasma ET-1 concentrations increase in a pulsatile manner after the onsets of spontaneous luteolysis and PGF2α-induced luteolysis in cycling cows, but not in female calves. The results suggest that the changes in the plasma ET-1 concentrations during the estrous cycle directly correlate with the cyclic changes in the ovarian function and the uterus.
The mouse myogenin gene regulatory region carrying the β-galactosidase (LacZ) gene was fused to a neomycin resistant gene, and introduced into ESD3 embryonic stem (ES) cells by electroporation. Transfected cells were expanded, and Southern hybridization was performed using the LacZ DNA sequence as a probe. The sublines of transformed cells were selected by karyotype analysis and by their ability to form embryoid bodies (EBs); some sublines were used for analysis of the transgene expression. The transgene was not expressed by the transformed cells in an undifferentiated state. In suspended EBs produced in hanging drops, the transgene was expressed within certain limited parts of the EBs. In some EBs, the transgene was expressed as patches in the edge of simple embryoid bodies (SEBs) and in the inner wall of cystic embryoid bodies (CEBs). In the differentiation culture system, the transgene was expressed at a cellular level, and polarization of the cells which expressed LacZ was observed. The results suggest that our sublines could be useful as skeletal muscle-specific cell markers for analysis of mouse myogenesis.
We have shown that 48-h fasting suppresses luteinizing hormone (LH) secretion in female rats which is enhanced by an estrogenic milieu and mediated by noradrenergic neurons. A similar fasting protocol yielded a suppression of LH secretion in male rats in a testosterone-dependent manner. In this experiment, we intended to identify specific nuclei in the hypothalamus and caudal brainstem that are activated during fasting for 6, 24, 30 or 48 h in castrated male rats with or without chronic testosterone treatment. Fasting started at 1300 h (lights-off at 1900 h). In testosterone-treated castrates, Fos-like-immunoreactive (FL-ir) cells were significantly increased in the paraventricular nucleus, A2 region of the nucleus of the solitary tract, supraoptic nucleus and amygdala 6 h after the onset of fasting but not 24, 30 or 48 h after the onset. On the other hand, FL-ir cells were not significantly different in those areas among fasted castrates compared with unfasted controls. In the A2 region of testosterone-treated castrates, tyrosine hydroxylase- and dopamine-β-hydroxylase-immunoreactive (TH-ir, DBH-ir) cells did not exhibit Fos-like immunoreactivity (FLI). Our results suggest that the absence of food may activate neurons in discrete hypothalamic and brainstem nuclei in male rats at the beginning of fasting and this activation requires an androgenic milieu. The absence of FLI in TH- or DBH-ir cells in the A2 region would indicate that transmitter systems other than catecholaminergic neurons may be involved during the very early stage of 48-h fasting-induced suppression of LH secretion.
This study characterized immunological and biological activities of the bovine placental lactogen (bPL) secreted by the placental explant obtained on Day 55, 130, 140, 146 and 270 of pregnancy. Tissues were minced and cultured for 24 hours. The bPL concentration and lactogenic activity of culture media were determined by radioimmunoassay using an anti-bPL antiserum and lymphoma cell (Nb2) bioassay, respectively. The bPL concentration in the media conditioned with cotyledons and caruncles gradually increased as gestation progressed. At term of gestation, fetal cotyledons produced a three-fold greater concentration of bPL than maternal caruncles. Lactogenic activity in conditioned media was higher in fetal cotyledons than in caruncles. Lactogenic activity in the conditioned media decreased in both the cotyledon and caruncle at term of gestation. Anti-bPL monoclonal antibody revealed a positive band of bPL in Western blot analysis, using the medium conditioned with cotyledons and caruncles collected in mid-pregnancy, however, no immunopositive band was detected in the medium conditioned with maternal caruncles at term of gestation. These results show that biological and immunochemical characteristics of bPL secreted by maternal caruncle and fetal cotyledon are different and undergo change with the advancement of gestation.