Cytogenetic study of the effect of gossypol on mouse germ cells

The clastogenic and mutagenic activities of a new antifertility and antitumor agent gossypol were studied in the mouse male germ cells. Results of the present work indicate that at the doses 125 and 250 mg/kg the drug does not significantly increase frequencies of the micronuclei in the early spermatids and sperm head abnormalities. Hence, genotoxic influence can not be proposed as responsible for the antifertility effect of gossypol.     

Effect of gossypol on intracellular Ca2+ regulation in human hepatoma cells

Gossypol is a natural toxicant present in cottonseeds, and is hepatotoxic to animals and human. The effect of gossypol on cytosolic free Ca2+ levels ([Ca2+]i) in HA22/VGH human hepatocytes was explored using fura-2 as a fluorescent Ca2+ indicator. Gossypol increased [Ca2+]i in a concentration-dependent manner with an EC50 value of 2 microM. The Ca2+ signal was reduced by removing extracellular Ca2+ or by 10 microM La3+, but was not affected by nifedipine, verapamil or diltiazem. Pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) to deplete the endoplasmic reticulum Ca2+ partly reduced 10 microM gossypol-induced Ca2+ release; and conversely pretreatment with gossypol abolished thapsigargin-induced Ca2+ release. The Ca2+ release induced by 10 microM gossypol was not changed by inhibiting phospholipase C with 2 microM U73122 or by depleting ryanodine-sensitive Ca2+ stores with 50 microM ryanodine. Together, the results suggest that in human hepatocytes, gossypol induced a [Ca2+]i increase by causing store Ca2+ release from the endoplasmic reticulum in a phospholipase C-independent manner, and by inducing Ca2+ influx.     

Peroxisomes in human and mouse testis: differential expression of peroxisomal proteins in germ cells and distinct somatic cell types of the testis

The vital importance of peroxisomal metabolism for regular function of the testis is stressed by the severe spermatogenesis defects induced by peroxisomal dysfunction. However, only sparse information is available on the role and enzyme composition of this organelle in distinct cell types of the testis. In the present study, we characterized the peroxisomal compartment in human and mouse testis in primary cultures of murine somatic cells (Sertoli, peritubular myoid, and Leydig cells) and in GFP-PTS1 transgenic mice with a variety of morphological and biochemical techniques. Formerly, peroxisomes were thought to be absent in late stages of spermatogenesis. However, our results obtained by detection of different peroxisomal marker proteins show the presence of these organelles in most cell types in the testis, except for mature spermatozoa. Furthermore, we demonstrate a strong heterogeneity of peroxisomal protein content in various cell types of the human and mouse testis and show marked differences in structure, abundance, and localization of these organelles in spermatids, depending on their maturation. Highest and selective enrichment of the peroxisomal lipid transporters (ABCD1 and ABCD3) as well as ACOX2, the key regulatory enzyme of the beta-oxidation pathway 2 for side chain oxidation of cholesterol, were found in Sertoli cells, whereas Leydig cells were enriched in catalase and ABCD2. Our results suggest a cell type-specific metabolic function of peroxisomes in the testis and point to an important role for peroxisomes in spermiogenesis and in the lipid metabolism of Sertoli cells.     

Effect of elicitors on the production of gossypol and methylated gossypol in cotton hairy roots

The effect of two chemical elicitors, salicylic acid and methyl jasmonate, on the production of gossypol, 6-methoxygossypol, and 6,6′-dimethoxygossypol in Gossypium barbadense hairy roots was examined. Methyl jasmonate, but not salicylic acid, was found to increase the production of gossypol and its methylated forms, but with a concomitant reduction in culture growth. The optimal methyl jasmonate dose was between 100 and 300 μM for hairy roots harvested 7 days after elicitation. After 20 d of induction with 100 μM methyl jasmonate, an eightfold increase in the level of gossypol was observed in elicited cultures compared with control cultures, double the highest gossypol levels previously reported for any cotton tissue. A two to threefold increase in the level of 6-methoxygossypol and a slight increase in the levels of 6,6′-dimethoxygossypol were also observed. Although methyl jasmonate stimulated the production of both optical forms of gossypol, the distribution of the enantiomers was different between elicited and control cultures.     

Effect of various agents on the cytoplasmic calcium concentration in cultured human muscle cells

1. We determined the cytoplasmic Ca2+ concentration ([Ca2+]i) in cultured human muscle cells using the fluorescent indicator Quin-2. 2. The [Ca2+]i was dependent on the external Ca2+ concentration. Acetylcholine in the presence of external Ca2+ caused a transient increase in [Ca2+]i. Inhibition by nifedipine indicated that this response was mediated through activated voltage-operated channels. In nominally Ca2(+)-free buffer acetylcholine did not markedly increase [Ca2+]i. Therefore, the increase in [Ca2+]i as a response to depolarization is mainly due to influx of external Ca2+. 3. Various concentrations of caffeine did not influence the [Ca2+]i. Dantrolene decreased [Ca2+]i, both in the presence and absence of external Ca2+. The reduction probably resulted from an action of dantrolene on the intracellular Ca2+ stores, since dantrolene did not influence 45Ca2+ influx or efflux and caffeine partially counteracted the reduction.     

棉酚对大鼠前列腺细胞增殖的影响

The effect of gossypol on prostate cells of rat was studied both in vitro and in vivo. For in vivo study, mature male Sprague Dawley rats were given gossypol orally, 5 mg/day, 5 times a week for one month. Then the prostates were examined histologically. By adding gossypol to the culture system directly, NbE-1 cells were used in vitro study. Several parameters, including histological structure, cell growth, DNA synthesis and mitotic cycle of cells were measured by different methods. The prostate size and weight in experimental rats decreased and significant differences between acini in controls and gossypol treated animals were noted. Acini of control prostate were full of protruding folds constituted by columnar epithelial cells, whereas, the most of the acini in treated animals were composing of cubic or square epithelial cells and epithelial folds were rarely seen. Thus, acini with normal appearance were about 14% less than that in controls. In the in vitro experiments, both cell proliferation and DNA synthesis decreased from 0 to 80% and from 0 to 90% respectively, when the concentrations of gossypol were increased from 0 to 20 micrograms/ml. It seems that 10 micrograms/ml of gossypol could cause the most significant inhibition in these two cellular functions. The cell mitotic assay showed that in treated groups, the cell number of S phase was decreased from 43 to 31%, this fact indicated that the inhibition on cell proliferation caused by gossypol might be due to preventing cells to enter S phase. Besides, inhibition effect on the proliferation of prostatic epithelial cells was dosage dependent and related to the duration of treatment.     

Occurrence of nuage in fetal human germ cells

Summary Germ cells of human fetuses aged 7–13 weeks were studied. Dense fibrous material called nuage was found in the cytoplasm of human fetal germ cells. The nuage increased in frequency in the germ cells during the interval studied. In oogonia the nuage occurred much more frequently than in spermatogonia and the frequency of nuage in oogonia increased earlier than in spermatogonia. The number of nuage was continuously at a higher level in oogonia than in spermatogonia. It is suggested that these findings support the theory that the nuage may act as a germ cell determinant.     

Ultrastructure of developing germ cells in the fetal human testis

Electron microscopic studies of the testis were performed on 12 human embryos and fetuses between 9 and 30 weeks post conceptionem. According to their ultrastructural features, the fetal germ cells could be divided into the following three stages of differentiation: 1) gonocytes, 2) intermediate cells, and 3) fetal spermatogonia. Sertoli cells were present among the germ cells in all the testes studied. The gonocytes showed the highest nucleo-cytoplasmic ratio. Their round nucleus contained a centrally located, prominent nucleolus. The cytoplasm displayed a well developed Golgi apparatus, lipid droplets and parallel arrays of short cisternae of the rough surfaced endoplasmic reticulum (rER). Microfilaments were numerous, particularly just beneath the cell membrane. The intermediate cells were found to extend several cytoplasmic processes and to contain a moderate number of long, branched and/or widened rER cisterna which were frequently connected to the perinuclear cisterna. Intermediate cells were often connected to one another by intercellular cytoplasmic bridges. The fetal spermatogonia also displayed cytoplasmic bridges. These cells showed the lowest nucleo-cytoplasmic ratio and more condensed nuclear chromatin. The mitochondria were situated close to the nucleus. Many of them were connected by a cementing substance. Lipid droplets and rER cisternae were rare in these cells. Infoldings of the inner nuclear membrane were often present in the gonocytes and in the intermediate cells, but were rarely observed in the fetal spermatogonia. Glycogen particles, polyribosomes, and chromatoid bodies ("nuage") were present in all the three germ cell types. With the maturation of the fetus, the number of gonocytes was found to decrease, whereas the number of fetal spermatogonia increased. The Sertoli cells also changed their ultrastructure, showing an increase in the number of rER cisternae, as well as of microfilaments, lipid droplets, and secondary lysosomes.     

Intra- and interindividual epigenetic variation in human germ cells

Epigenetics represents a secondary inheritance system that has been poorly investigated in human biology. The objective of this study was to perform a comprehensive analysis of DNA methylation variation between and within the germlines of normal males. First, methylated cytosines were mapped using bisulphite modification-based sequencing in the promoter regions of the following disease genes: presenilins (PSEN1 and PSEN2), breast cancer (BRCA1 and BRCA2), myotonic dystrophy (DM1), and Huntington disease (HD). Major epigenetic variation was detected within samples, since the majority of sperm cells of the same individual exhibited unique DNA methylation profiles. In the interindividual analysis, 41 of 61 pairwise comparisons revealed distinct DNA methylation profiles (P=.036 to 6.8 x 10(-14)). Second, a microarray-based epigenetic profiling of the same sperm samples was performed using a 12,198-feature CpG island microarray. The microarray analysis has identified numerous DNA methylation-variable positions in the germ cell genome. The largest degree of variation was detected within the promoter CpG islands and pericentromeric satellites among the single-copy DNA fragments and repetitive elements, respectively. A number of genes, such as EED, CTNNA2, CALM1, CDH13, and STMN2, exhibited age-related DNA methylation changes. Finally, allele-specific methylation patterns in CDH13 were detected. This study provides evidence for significant epigenetic variability in human germ cells, which warrants further research to determine whether such epigenetic patterns can be efficiently transmitted across generations and what impact inherited epigenetic individuality may have on phenotypic outcomes in health and disease.     

Identification of sperm subpopulations in canine ejaculates: effects of cold storage and egg yolk concentration

The aim of this study was to evaluate the effects of cold storage and egg yolk concentration on the distribution of spermatozoa within the different subpopulations. Twenty ejaculates from 4 dogs were collected, diluted in either TRIS buffer containing 20% (TEY20) or 10% centrifuged egg yolk (TEY10) and cooled following a conventional protocol. The kinematic parameters of individual spermatozoa were evaluated in fresh ejaculates and after 24 and 72 h of preservation at 5°C. A multivariate clustering procedure separated 54,261 motile spermatozoa into four subpopulations: Subpopulation 1 consisting of poorly active and non-progressive spermatozoa (19.80%), Subpopulation 2 consisting of slow and low-linear spermatozoa (25.21%), Subpopulation 3 consisting of high speed and progressive spermatozoa (23.88%), and Subpopulation 4 consisting of highly active but non-progressive spermatozoa (31.11%). Although, cold storage had a significant (P<0.05) effect on both the frequency distribution of spermatozoa within subpopulations and the motion characteristics of each subpopulation, the sperm subpopulation structure was perfectly maintained after cold storage. Subpopulations 1 and 2 significantly (P<0.001) decreased during cold storage (Subpopulation 1: 26.6, 16.9 and 18.4%; and Subpopulation 2: 33.6, 21.3 and 24.0%, respectively, for fresh, 24 and 72 h post-cooled), whereas Subpopulations 3 and 4 significantly (P<0.05) increased (Subpopulation 3: 16.7, 27.6 and 24.3%, and Subpopulation 4: 23.1, 34.1 and 33.4%, respectively, for fresh, 24 and 72 h post-cooled). Regarding the relative percentage of spermatozoa within each extender, Subpopulation 3 was more frequently observed in TEY20 after both 24 and 72 h of cold storage. Significant correlations (P<0.05) were found between the proportions of spermatozoa assigned to Subpopulation 3 in the fresh ejaculates and those in stored samples after 24 h (r=0.48498). In conclusion, cold storage significantly modified both the specific parameters and the distribution of spermatozoa within subpopulations. These changes did not affect the general motile sperm structure present in dog, which is conserved during cold storage. The analysis of the changes observed in structures of subpopulations also suggests that the TEY20 provide more effective preservation of dog semen during cold storage.     

Differentiation of human germ cell tumor cells

Human germ cell tumors are an excellent model for investigating the mechanism of human early embryogenesis as well as cellular differentiation. Three human EC cell lines, NCR-G 2, 3 and 4 were newly established from testicular mixed embryonal carcinomas in vitro, G3 and G4 cells were capable of somatic cell differentiation. The G3 cells demonstrated the most noticeable antigenetical changes with the administration of retinoic acid. SSEA-1 appeared on some cells whereas expression of HLA-A, B, C as well as 2H2, 2D7 and 5D4 antigens tended to be reduced in G3 cell line. 2H2, 2D7 and 5D4 antigens which we recently produced were immature human EC specific cell surface antigens, defined by mouse monoclonal antibodies, obtained immunization with G2 cells. The production of hCG, high molecular weight cytokeratin and intercellular matrices such as type IV collagen and laminin were inducible in G3 cells. Thus, G3 cells are thought to be one of the most pluripotent human EC cells. These findings clearly indicate that the EC cell lines we established provide an opportunity to study differentiation mechanism of human germ cell tumors and also human somatic cells.     

Study on the concentration of circulating primordial germ cells (cPGCs) in early chick embryos

Experiments were conducted to elucidate the factor that influences the concentration of circulating primordial germ cells (cPGCs) in two-day old chick embryos. The concentration of cPGCs was observed to be highest at stage 14 (66.9 +/- 23.2 microliters) and decreased thereafter. However, considerable egg to egg variations in cPGC concentration, especially at stages 13, 14, 15, and 16 were observed. After conducting experiments to elucidate the source of egg to egg variation in cPGC concentration among embryos, it was revealed that there are hens that lay eggs which contain either constantly high (more than 80 PGCs/microliter) or constantly low (less than 30 PGCs/microliter) concentration of cPGCs. The results obtained from the present experiments showed that one of the major source of egg to egg variation in the concentration of cPGCs was due to the individual differences among females that produced the eggs.     

Effect of low-level radiation on the death of male germ cells

Hormetic and adaptive responses induced by low-level radiation in hematopoietic and immune systems have been observed, as shown by stimulatory effects on cell growth and resistance to subsequent radiation-induced cytogenetic damage. However, in terms of cell death by apoptosis, the effects of low-level radiation are controversial: Some studies showed decreased apoptosis in response to low-level radiation while others showed increased apoptosis. This controversy may be related to the radiation doses or dose rates and also, more importantly, to the cell types. Testes are one of the most radiosensitive organs. The loss of male germ cells after exposure to ionizing radiation has been attributed to apoptosis. In the present study, the effects of low-level radiation at doses up to 200 mGy on mouse male germ cells in terms of apoptosis and the expression of apoptosis-related proteins were examined at different times after whole-body exposure of mice to low-level radiation. In addition, the effect of pre-exposure to low-level radiation on subsequent cell death induced by high doses of radiation was examined to explore the possibility of low-level radiation-induced adaptive response. The results showed that low-level radiation in the dose range of 25-200 mGy induced significant increases in apoptosis in both spermatogonia and spermatocytes, with the maximal effect at 75 mGy. The increased apoptosis is most likely associated with Trp53 protein expression. Furthermore, 75 mGy low-level radiation given pre-irradiation led to an adaptive response of seminiferous germ cells to subsequent high-level radiation-induced apoptosis. These results suggest that low-level radiation induces increased apoptosis in male germ cells but also induces a significant adaptive response that decreases cell death after a subsequent high-dose irradiation.     

Effect of inorganic lead on the primordial germ cells in the mouse embryo

Embryos from mice exposed to lead chloride (20 micrograms/gm body weight) by a single intravenous injection on day 8 of gestation were examined regarding the number and distribution of their primordial germ cells on 4 consecutive days of development. The cells, visualized by histochemical staining for alkaline phosphatase, showed a normal body distribution but were significantly fewer at all four stages compared with those of control embryos of corresponding age. Furthermore, the staining of the primordial germ cells was much weaker in the embryos from the lead-treated dams than in those from control dams, suggesting that the lead had interfered with the production or activity of alkaline phosphatase. It is assumed that these effects could have contributed to the fertility reduction previously observed in female offspring of mice exposed to lead at an early stage of pregnancy.     

Intercellular connections between germ cells in the developing human ovary

Summary An electron microscopic examination of human fetal ovaries reveals the presence of intercellular bridges between developing germ cells. The bridges are characterized by a band of electron-dense material beneath the lateral limiting membrane, and cell organelles are seen within the confines of these connections. Their general morphology is similar to that described in ovaries of other species. The possible functional significance of these connections is discussed.This work was supported by grants from the Edward G. Schlieder Educational Foundation, New Orleans, Louisiana State University and HD-03288 from the National Institute of Child Health and Human Development.The authors would like to thank Miss Cathy Chase for her technical assistance.