Mixed germ cell tumor with embryonal carcinoma, choriocarcinoma, and epithelioid trophoblastic tumor in the ovary of a cynomolgus monkey.

A seven-year-old female cynomolgus monkey had a mass in the left ovary with metastasis to the lung and the right ovary. The mass of these organs showed three different characteristics, and its immunohistochemical profiles were consistent with embryonal carcinoma (EC), choriocarcinoma (CC), and epithelioid trophoblastic tumor (ETT). The EC was characterized with sheets and glandlike structures with large pleomorphic, single-nucleated epithelial cells that were immunohistochemically positive for α-fetoprotein, octamer-4, and CD30, and with multinucleated giant cells resembling syncytiotrophoblasts. The CC also represented biphasic proliferation of the cytotrophoblast positive for cytokeratin 7 (CK7), which showed negative immunoreactivity for all three of the above antibodies, and it was syncytiotrophoblast positive for human chorionic gonadotropin. The ETT showed numerous floating cells in an abundant eosinophilic extracellular matrix with vacuolated or eosinophilic cytoplasm and was immunohistochemically positive for CK7, p63, and α-inhibin, which features nodule or cordlike structures. Collectively, this neoplasm was identified as a mixed germ cell tumor with EC, CC, and ETT. To our knowledge, this is the first report of EC in nonhuman primates as a component of mixed germ cell tumor.

Inhibition of in vitro fertilization and early embryonic development in hamsters by gossypol.

We have previously reported an inhibitory effect of gossypol and its metabolite on bovine and mouse early embryonic development. In the present study, eggs were collected from oviducts of superovulated hamsters. Epididymal sperm were used for in vitro fertilization (IVF). Gossypol at 5, 10, and 30 micrograms/ml significantly inhibited the formation of 2 pronuclei by 45, 65 and 95%, respectively. On the first day of pregnancy, hamsters were given an intrauterine treatment of 200 micrograms of gossypol in 100 microliters of corn oil per uterine horn. On day 3, embryos from controls were in morula (65%) and early morula (17%) stages, while less than 2% of embryos from the gossypol-treated hamsters were in the morula stage. The numbers of embryo implantation sites on day 8 and pups in controls (14 +/- 2.0 and 12 +/- 1.5, respectively) were significantly higher than those in the gossypol-treated hamsters (8.5 +/- 2.0 and 4.0 +/- 1.5, respectively). Our results suggest that gossypol is able to affect fertilization, embryonic development, embryo implantation, and the number of pups in hamsters through a not-yet-defined mechanism.

Acceleration of embryo transport in superovulated adult rats.

The etiology of reduced fertility in rodents and humans after exogenous gonadotropin-induced superovulation is unknown. This study examines implantation failure in adult rats induced to superovulate by gonadotropin treatment. After multiple injections of pregnant mare serum gonadotropin (PMSG) followed by human chorionic gonadotropin (hCG), superovulation in adult rats was confirmed on day-1 of pregnancy, however, on day-13, no implantation sites were observed. Daily flushing of oviducts and uteri revealed that the number of embryos recovered from superovulated rats on day-2 was only one-third of that retrieved on day-1. The numbers of embryos retrieved on these days from the oviducts of control rats, however, were almost equivalent. On day-3, no embryos were retrieved from either the oviducts or uteri of superovulated rats, while all embryos from control rats were found in the oviducts. Most embryos recovered from superovulated rats showed normal development. Compared to levels in control rats, serum estradiol (E2) in superovulated rats increased significantly on days-2 and -3 of pregnancy, whereas, serum progesterone (P) levels remained unchanged. Thus, E2/P ratio increased on days-2 and -3 of pregnancy in superovulated adult rats coinciding with the timing of embryo transport acceleration. Our results suggest that superovulated oocytes can be fertilized and reach an early stage of development within the oviduct. Implantation failure in superovulated rats may be due to the accelerated embryo transport resulting from elevated E2/P ratio.

The effects of estrous cow serum on the in vitro maturation and fertilization of the bovine follicular oocyte.

The effect of serum obtained from a cow at the time of standing estrus (serum A), at ovulation (serum B), and at 24 h after ovulation (serum C) on the in vitro maturation and fertilization of bovine oocytes was examined. Of 144 (Group A), 159 (Group B), and 158 (Group C) oocytes, 77 (53.4%), 82 (51.6%) and 82 (51.9%) oocytes were characterized by expansion of cumulus cells, respectively. There was no significant difference in the effect of the three types of cow serum on the cumulus expansion (P < 0.05). Of 461 oocytes, 316 oocytes were cultured with sperm for fertilization, and 145 oocytes were cultured without sperm for evidence of parthenogenetic development. Of 56 (Group A), 56 (Group B), and 62 (Group C) oocytes with expanded cumulus cells, 19 (33.9%), 7 (12.5%), and 11 (17.7%) oocytes were cleaved, respectively, after exposed to the sperm for 24 h. There was a significant difference in the effect of the three types of cow serum on the fertilization rate (P < 0.05). A total of 145 oocytes was cultured in the absence of sperm and no evidence of parthenogenetic division was observed. The effect of the three types of serum obtained from the cow on the maturation of oocytes was not significant, but a significant difference did exist in the fertilization rate of oocytes. Cow serum obtained at the time of standing estrus had a beneficial effect on the fertilization rate of oocytes in vitro.

The influence of serum and gonadotropins on in vitro maturation and fertilization of bovine oocytes.

The influence of estrous cow serum (ECS) or fetal calf serum (FCS) and their interaction with gonadotropins on in vitro bovine maturation and fertilization was evaluated. The addition of ECS or FCS to the medium significantly increased the percentage of oocyte maturation over that of Ham's F-10 medium alone (P<0.05). The addition of follicle stimulating hormone (FSH), human chorionic gonadotropin (hCG) or of FSH plus hCG to the medium provided no advantage in maturation over serum alone. However, FSH plus HCG added to the maturation medium significantly improved the frequency of pronucleus formation in both groups compared with a medium to which no gonadotropins were added (P<0.05). Both ECS and FCS (as medium supplements) promoted oocyte maturation. Although the addition of a combination of FSH plus hCG to the medium did not increase the rate of oocyte maturation, it did contribute to the high incidence of subsequent pronucleus formation.

Role of cyclic adenosine monophosphate (cAMP) in vitro on bovine oocyte maturation.

This experiment attempted to determine the effect of cAMP on maturation of bovine oocytes in chemically-defined, serum-free medium. Cumulus-oocyte complexes were incubated in modified DME/Ham F-12 medium containing dbcAMP at 0 (control), 10(-6), 10(-4) and 10(-2) M. After 18 and 24 hours of culture, the percentage of oocyte maturation between 0 (control) and 10(-2) M dbcAMP-treated groups were significant. Some oocytes were cultured with dbcAMP (10(-2) M) for 6, 12 and 24 hours followed by incubation in control medium to test the reversibility of inhibition or of any harmful effect of dbcAMP. The inhibitory effect of 10(-2) M dbcAMP on bovine oocyte maturation was reversed by transferring cumulus-oocyte complexes to the control medium. In addition, forskolin (0.12 and 0.24 mM) was effective (P < 0.01) in preventing the resumption of meiosis. The cAMP content of oocytes cultured with forskolin was not increased, although cumulus cells responded to forskolin with an increase in cAMP content. These results indicate that elevated levels of cAMP in the culture medium are important in regulating resumption of meiosis of bovine oocytes in vitro.

Fertilizability and subsequent developmental ability of bovine oocytes matured in medium containing epidermal growth factor (EGF).

We have previously shown that epidermal growth factor (EGF) is capable of promoting maturation of bovine cumulus-oocyte complexes in chemically defined serum-free medium. In this study, fertilizability and subsequent developmental capacity of bovine oocytes matured in EGF-containing medium were evaluated. Fetal bovine serum (FBS, 10%) and EGF at 10 ng/ml in Dulbecco's modified Eagles medium with Ham's nutrient mixture F-12 (DME/F12) significantly increased the rate of formation of two pronuclei compared with the rate obtained from DME-F12 alone (P<0.05). Early embryonic development was assessed during 48 h in culture. Data were evaluated in terms of cleavage and four- to eight-cell formation. Oocytes matured in 10 ng/ml EGF showed significantly higher rates of cleavage (P<0.01) and four- to eight-cell formation than did oocytes matured in control medium (P<0.05). Bovine oocytes matured in the presence of EGF can be normally fertilized and can cleave and develop in vitro up to the eight-cell stage.

Male reproductive toxicity study of nitrazepam in rats.

The main focus of this study is the optimal administration period concerning toxic effects on male fertility in rats. To assess functional and morphological changes induced in the testis by nitrazepam, male rats were administered the drug at doses of 0, 20, 40 or 80 mg/kg during pre-mating periods of 2, 4 or 9 weeks and then the 2 weeks of mating. At the end of the administration period the animals were sacrificed and sperm number, motility, abnormalities and histopathological changes in the testis were examined. Decreases in testis weight, epididymis weight, number of sperm in the testis and sperm motility were observed in the 40 and 80 mg/kg sections of the 2, 4 and 9 week pre-mating treated groups. Mating with untreated females revealed no adverse effects on copulation rate in any group; however, a remarkable decrease in pregnancy rate was noted in the 80 mg/kg section of the 2, 4 and 9 week treated groups. On histological examination, various degrees of localized necrosis in the seminiferous epithelium and Leydig cell hyperplasia were observed in the testis. No clear changes were observed in the 20 mg/kg section of the 2 week pre-mating administration group, but at the 4 week time point, necrosis of spermatogenic cells began to appear. The primary morphological event was evident in spermatocytes with necrosis of the cytoplasm observed from 4 weeks after administration of nitrazepam, although sperm motility and sperm head counts were unaffected. From these findings, examination of sperm characteristics and histopathological changes in the testis are important parameters for evaluation of drugs inducing testicular damage. We conclude that a 4 week administration period is sufficient to detect effects of nitrazepam on male fertility.

Functional and ultrastructural characteristics of two types of rat granulosa cell cultured in the presence of FSH or transforming growth factor alpha (TGF-alpha).

Granulosa cells were isolated from 15-day-old, 25-day-old or PMSG-primed rats and were separated by Percoll gradient (20 to 60%) into five fractions. The cells in fraction 2 were mostly small cells (6.96-9.57 microns) and fractions 3 and 4 had a relatively high population of large cells (10.96-13.05 microns) which were sorted to collect a pure population of large cells. Aliquots of small or large cells were cultured separately in serum-free defined DMEM/F-12 medium containing 50 ng FSH ml-1, or 10 ng transforming growth factor alpha (TGF-alpha) ml-1 for 3 days. In PMSG-primed rats, the large cells produced 3.2-fold more progesterone than did small cells (with FSH/TGF alpha: 255 +/- 35.0 versus 77.32 +/- 14.5 x 10(-6) ng, day 1). Large and small cells from 25-day-old rats produced similar amounts of progesterone (FSH/TGF alpha: 65.68 +/- 9.6 versus 78.25 +/- 12.3 x 10(-6) ng, day 1). In 15-day-old rats, large and small cells produced very low concentrations of progesterone (FSH/TGF alpha: 4.69 +/- 1.2 versus 2.66 +/- 1.0 x 10(-6) ng, day 1). Large cells from PMSG-primed rats had characteristics of steroidogenic cells, i.e. smooth endoplasmic reticulum and well-developed mitochondria with tubular cristae compared with small cells, whereas small and large cells from 25- and 15-day-old rats contained the regularly occurring organelles without the endoplasmic reticulum of the smooth variety and mitochondria with lamellar cristae. This study shows that the heterogeneity of granulosa cells is related to size, metabolic response to FSH, TGF-alpha or to both factors and morphological features, all of which may be associated with the transition from preantral to preovulatory stages of follicle differentiation.

Steroidogenic acute regulatory (StAR) protein (p25) and prohibitin (p28) from cultured rat ovarian granulosa cells.

This study has identified and characterized two intracellular proteins (25 and 28 kDa) during ongoing differentiation of rat granulosa cells isolated from preantral and early antral follicles. The identity of p25 was confirmed as the mitochondria associated StAR protein by western blotting analysis. In the culture conditions used, this protein was expressed only when granulosa cells were stimulated with FSH to produce progesterone. It is apparent that the steroidogenic differentiation of granulosa cells affects StAR expression. Amino acid sequence analysis of p28 identified it as prohibitin and was corroborated by western blot analysis with antibodies specific for rat prohibitin. During the ongoing differentiation of granulosa cells there were changes in the expression of p28/prohibitin. Although prohibitin is constitutively expressed in granulosa cells, there is an increase in the more acidic isoform of prohibitin when oestrogen concentrations are raised by increased production or exogenous addition. This increase in this acidic isoform of prohibitin is due to phosphorylation. It is possible that oestrogen induces phosphorylation of prohibitin and, thus, may be involved in the regulation of granulosa cell proliferation and the ontogeny of the ovarian follicle.

DNase I interaction on muscle Z-line.

The effect of deoxyribonuclease I on muscle Z-line structures was re-examined. Under conditions of deoxyribonuclease I activation (presence of the divalent cation Ca2+ and Mg2+), a deoxyribonuclease I preparation did not affect Z-line structure if phenylmethylsulfonylfluoride, an inhibitor of serine proteases, was also present. In the absence of protease inhibitor, both Z-lines and M-lines were digested, even in the presence of EDTA and EGTA as inhibitors of deoxyribonuclease I. These electron microscopic observations were consistent with the following results from sodium dodecyl sulphate gel electrophoresis: when the protease was inhibited but deoxyribonuclease I was activated, myofibrillar proteins remained essentially intact. However, degradation of proteins in both rabbit psoas and chicken pectoralis myofibrils was observed in the presence of deoxyribonuclease I inhibitors when the protease inhibitor was absent. Our data strongly suggest that the interaction of deoxyribonuclease I with Z-line proteins previously reported is most likely due to contamination of the deoxyribonuclease I fraction by the serine-type proteases.

The protein synthesis of human full term placenta cell in monolayer culture system.

Protein synthesis of human placenta from cesarean section was analyzed by SDS gel electrophoresis using full term cell culture system. The qualitative pattern of cytoskeletal proteins before and after culture was also examined. After trypsinization, cytotrophoblasts were cultured for 20 days in the humidified incubator of 5% CO2 in 95% air. The confluency was obtained in 10 days after inoculation. The pattern of SDS-PAGE showed several protein bands including actin (43,000 Da) and desmin (55,000 Da) as major constituents of 12 and 20 day cultures. The significant differences between band appearances in samples before and after culturing were noted. The present results indicated that myosin may not be synthesized in high content, differing from previous observations. Cytoskeletal protein production seemed to be markedly enhanced in the cultured system.