Environmentally-relevant exposure to diethylhexyl phthalate (DEHP) alters regulation of double-strand break formation and crossover designation leading to germline dysfunction in Caenorhabditis elegans.

Exposure to diethylhexyl phthalate (DEHP), the most abundant plasticizer used in the production of polyvinyl-containing plastics, has been associated to adverse reproductive health outcomes in both males and females. While the effects of DEHP on reproductive health have been widely investigated, the molecular mechanisms by which exposure to environmentally-relevant levels of DEHP and its metabolites impact the female germline in the context of a multicellular organism have remained elusive. Using the Caenorhabditis elegans germline as a model for studying reprotoxicity, we show that exposure to environmentally-relevant levels of DEHP and its metabolites results in increased meiotic double-strand breaks (DSBs), altered DSB repair progression, activation of p53/CEP-1-dependent germ cell apoptosis, defects in chromosome remodeling at late prophase I, aberrant chromosome morphology in diakinesis oocytes, increased chromosome non-disjunction and defects during early embryogenesis. Exposure to DEHP results in a subset of nuclei held in a DSB permissive state in mid to late pachytene that exhibit defects in crossover (CO) designation/formation. In addition, these nuclei show reduced Polo-like kinase-1/2 (PLK-1/2)-dependent phosphorylation of SYP-4, a synaptonemal complex (SC) protein. Moreover, DEHP exposure leads to germline-specific change in the expression of prmt-5, which encodes for an arginine methyltransferase, and both increased SC length and altered CO designation levels on the X chromosome. Taken together, our data suggest a model by which impairment of a PLK-1/2-dependent negative feedback loop set in place to shut down meiotic DSBs, together with alterations in chromosome structure, contribute to the formation of an excess number of DSBs and altered CO designation levels, leading to genomic instability.

Separation and Loss of Centrioles From Primordidal Germ Cells To Mature Oocytes In The Mouse.

Oocytes, including from mammals, lack centrioles, but neither the mechanism by which mature eggs lose their centrioles nor the exact stage at which centrioles are destroyed during oogenesis is known. To answer questions raised by centriole disappearance during oogenesis, using a transgenic mouse expressing GFP-centrin-2 (GFP CETN2), we traced their presence from e11.5 primordial germ cells (PGCs) through oogenesis and their ultimate dissolution in mature oocytes. We show tightly coupled CETN2 doublets in PGCs, oogonia, and pre-pubertal oocytes. Beginning with follicular recruitment of incompetent germinal vesicle (GV) oocytes, through full oocyte maturation, the CETN2 doublets separate within the pericentriolar material (PCM) and a rise in single CETN2 pairs is identified, mostly at meiotic metaphase-I and -II spindle poles. Partial CETN2 foci dissolution occurs even as other centriole markers, like Cep135, a protein necessary for centriole duplication, are maintained at the PCM. Furthermore, live imaging demonstrates that the link between the two centrioles breaks as meiosis resumes and that centriole association with the PCM is progressively lost. Microtubule inhibition shows that centriole dissolution is uncoupled from microtubule dynamics. Thus, centriole doublets, present in early G2-arrested meiotic prophase oocytes, begin partial reduction during follicular recruitment and meiotic resumption, later than previously thought.

Effect of Sublethal Nickel Chloride Exposure on Crayfish, Astacus leptodactylus Ovary: An Ultrastructural, Autometallographic, and Electrophoretic Analyses.

Cytological responses in different organs of sentinel organisms have proven to be useful tools for characterizing the health status of those organisms and assessing the impact of environmental contaminants. Our study shows that nickel (II) accumulated in both germ cells (oogonia and developing oocytes) and somatic cells (muscle cells, follicle cells) in the Astacus leptodactylus ovary. Muscle cells from ovarian wall show disorganization and the disruption of cytoplasmic microtubules and pyknosis of the cell nucleus. Follicle cells, both those that surround the developing oocytes and also those that are not associated with the oocytes contained within the cytoplasm vacuoles of different sizes, degenerated mitochondria, myelin bodies, disorganized microtubules, and pyknotic nuclei. The most evident pathological phenomenon was the alteration and disorganization of the basal matrix, which separates the ovarian interstitium from ovarian follicles compartment. Exposure to nickel induces cytoplasmic vacuolation in oogonia and developing oocytes, structural alteration of the developing yolk granules and condensation of the nucleoli. Ultrastructural autometallography has shown grains of silver-enhanced nickel inside the cytoplasm of the muscle cells with altered morphology, including the cytoplasm, nucleus, and basal matrix of the follicle cells, and in intracisternal granules and developing yolk granules of the oocytes.

Developmental effects of imatinib mesylate on follicle assembly and early activation of primordial follicle pool in postnatal rat ovary.

Imatinib mesylate is an anti-cancer agent that competitively inhibits several receptor tyrosine kinases (RTKs). RTKs play important roles in the regulation of primordial follicle formation, the recruitment of primordial follicles into the pool of growing follicles and maturation of the follicles. In the present study, we investigated the effects of the tyrosine kinase inhibitor imatinib on primordial follicle assembly and early folliculogenesis in postnatal rats. Female Sprague-Dawley rats were treated with either imatinib (150mg/kg) or placebo (water) on postnatal days 2-4. Bilateral ovariectomy was performed on postnatal day 2 and 5. Histology, immunohistochemistry, and mRNA analysis were performed. Imatinib treatment was associated with increased density of the multi-oocyte follicles (P<0.01), oogonia (p<0.01) and germline clusters (P<0.05), decreased activation of primordial follicles, increased expression of c-Kit and AMH, and decreased protein expression of Kit-ligand and GDF9 when compared to age-matched controls. In conclusion, imatinib affects folliculogenesis in postnatal rat ovaries by delaying the cluster breakdown, follicular assembly and early activation of the primordial follicle pool.

Changes in ultrastructure and expression of steroidogenic factor-1 in ovaries of zebrafish Danio rerio exposed to glyphosate.

Glyphosate is a broad-spectrum organophosphate (OP) herbicide, highly soluble in water, and when applied in terrestrial systems it penetrates into soil, eventually reaching the aquatic community and affecting nontarget organisms. The aim of this study was to evaluate the toxicity of glyphosate on ovaries of zebrafish (Danio rerio). Ovaries (n = 18 per triplicate) were exposed to 65 µg/L of glyphosate [N-(phosphonomethyl) glycine] for 15 d. This concentration was determined according to Resolution 357/2005/CONAMA/Brazil, which establishes the permissible concentration of glyphosate in Brazilian inland waters. Nonexposed ovaries (n = 18 per triplicate) were used as control. Subsequently, morphology and expression of steroidogenic factor-1 (SF-1) of exposed and nonexposed ovaries was determined. No apparent changes were noted in general morphology of exposed and nonexposed ovaries. However, a significant increase in diameter of oocytes was observed after exposure to glyphosate. When ovarian ultrastructure was examined the presence of concentric membranes, appearing as myelin-like structures, associated with the external membranes of mitochondria and with yolk granules was found. After glyphosate exposure, immunohistochemistry and immunoblotting revealed greater expression of SF-1 in the oocytes, which suggests a relationship between oocyte growth and SF-1 expression. These subtle adverse effects of glyphosate on oocytes raised a potential concern for fish reproduction. These results contribute to understanding glyphosate-induced toxicity to nontarget organisms, showing subcellular and molecular impairments that may affect reproduction in +female fish.

Dexamethasone induces germ cell apoptosis in the human fetal ovary.

CONTEXT: The 21-hydroxylase deficiency is the most common cause of congenital adrenal hyperplasia. Pregnant women presenting a risk of genetic transmission may be treated with synthetic glucocorticoids such as dexamethasone (DEX) to prevent female fetus virilization. OBJECTIVE: The aim of this study was to assess the potential deleterious effects of DEX exposure on fetal ovarian development. SETTINGS: Human fetal ovaries, ranging from 8-11 weeks after fertilization, were harvested from material available after legally induced abortions. They were cultured in the absence or presence of DEX (2, 10, or 50 µm) over 14 d, and histological analyses were performed. RESULTS: The glucocorticoid receptor NR3C1 was present and the signaling pathway active in the fetal ovary as demonstrated by the expression of NR3C1 target genes, such as PLZF and FKBP5, in response to DEX exposure. DEX decreased germ cell density at the 10 and 50 µm doses. Exposure to DEX, even at the highest dose, did not change oogonial proliferation as monitored by 5-bromo-2'-deoxyuridine incorporation and significantly increased the apoptotic rate, detected with cleaved caspase 3 staining. Interestingly, the expression of the prosurvival gene KIT was significantly decreased in the presence of DEX during the course of the culture. CONCLUSION: We have demonstrated for the first time that in vitro exposure to high doses of DEX impairs human fetal oogenesis through an increase in apoptosis. These data are of high importance, and additional epidemiological studies are required to investigate the female fertility of those women who have been exposed to DEX during fetal life.

The number of oogonia and somatic cells in the human female embryo and fetus in relation to whether or not exposed to maternal cigarette smoking.

BACKGROUND: Prenatal exposure to maternal cigarette smoking or compounds of cigarette smoke is associated with serious reproductive hazards such as apoptotic death of oogonia in murine offspring and decreased fecundability in human offspring. The present study addresses potential effects of in utero exposure to cigarette smoking. METHODS: Twenty-nine human first-trimester ovaries from legal abortions [aged 38-64 days post-conception (p.c.)] were collected. Mothers filled out a questionnaire about their smoking habits and delivered a urine sample for cotinine analysis. The ovarian cell numbers were estimated using stereological methods. RESULTS: A non-linear correlation between the numbers of oogonia and somatic cells in relation to age of the embryo/fetus was shown in 28 ovaries, including the first estimates performed in ovaries younger than 47 days p.c. Prenatal exposure to smoke showed a significant decrease in the number of somatic cells (P < or = 0.01). The number of oogonia was not significantly associated with prenatal exposure to maternal smoking (P < or = 0.09). The ratio between the two cell types decreased considerably from 1:45 to 1:23 from 38 to 46 days p.c. and was not affected by smoking. CONCLUSIONS: Oogonia proliferate and/or invade the developing ovary at a much faster relative rate than somatic cells. In utero exposure to maternal smoking significantly reduces the number of somatic cells from Days 38 to 64 p.c. Since oocytes cannot survive without being enclosed by somatic cells in a follicle, reduction in the somatic cells number may have long-range consequences on the number of oocytes available in adult life and on the future fertility of female offspring exposed to smoking in utero.

Lanosterol metabolic product(s) is involved in primordial folliculogenesis and establishment of primordial follicle pool in mouse fetal ovary.

The primordial follicles present in neonatal ovary represent the fecundity of a female throughout her reproductive life. Germ cell meiosis and apoptosis are two important events during primordial folliculogenesis. In this study, through focusing on the cytochrome P450 lanosterol 14 alphademethylase (CYP51) and its lanosterol metabolic product(s), we explored the possible regulatory mechanism of the initiation of germ cell meiosis and primordial follicle formation. The expression of CYP51 could be detected in both oocytes and granulosa cells during primordial folliculogenesis by immunochemistry. RS21745, which leads to the reduction of lanosterol metabolic product(s) level, inhibited the primordial follicle formation in a dose-dependent manner, and thus postpone the establishment of the primordial follicle pool when the mouse fetal ovaries were cultured in serum-free medium. In contrast, the number of primordial follicle increased significantly with the accumulation of the lanosterol metabolic products caused by 0.025, 0.0625, and 0.125 microM AY9944-A-7 supplements. AY9944-A-7 also up-regulated the expression of meiotic diplotene stage marker gene msy2 and primordial follicle formation regulatory gene fig-alpha. Furthermore, AY9944-A-7 decreased the expression of apoptosis gene bax and significantly prevented oocyte apoptosis from 15.37 +/- 1.97% to 3.68 +/- 0.27% (P < 0.01) in neonatal ovary in vitro. In conclusion, our results indicate that lanosterol metabolic product(s) is involved in the primordial folliculogenesis by regulating the oocyte meiosis and apoptosis.

A progestin and an estrogen regulate early stages of oogenesis in fish.

Using two species of teleost fish, Japanese huchen (Hucho perryi) and common carp (Cyprinus carpio), we investigated whether sex steroids are involved in early oogenesis in vitro. Ovarian fragments were cultured to examine the effects of a progestin, 17alpha, 20beta-dihydroxy-4-pregnen-3-one (DHP), and an estrogen, estradiol-17 beta (E2). DHP and E2 significantly promoted DNA synthesis in ovarian germ cells, as judged by 5-bromo-2-deoxyuridine (BrdU) incorporation into these cells. Furthermore, to detect the initiation of the first meiotic division of early oogenesis, we assessed ultrastructurally the occurrence of synaptonemal complexes (SCs) and analyzed by immunohistochemistry the expression of a meiosis-specific marker, Spo11. In huchen, a higher percentage of oocytes with SC was seen in DHP-treated ovarian fragments than in control or E2-treated ovarian fragments. Spo11 was expressed in germ cells after DHP treatment of carp ovarian explants. These data suggest that the progression of germ cells through early oogenesis involves two sex steroids: E2, which acts directly on oogonial proliferation, and DHP, which acts directly on the initiation of the first meiotic division of oogenesis.

Reduction of primordial follicles caused by maternal treatment with busulfan promotes endometrial adenocarcinoma development in donryu rats.

Ovarian dysfunction leading to hormonal imbalance plays a crucial role in uterine carcinogenesis in rats as well as women. However, the effects of a reduction in primordial follicles at birth on uterine adenocarcinoma development have hitherto not been determined. The present study was therefore conducted using female Donryu rats, a high incidence rat strain of uterine adenocarcinoma. The animals were maternally exposed to 2.5 or 5.0 mg/kg of busulfan on gestation day 14 to reduce primordial follicles, and were then initiated by intrauterine treatment with N-ethyl-N'-nitro-N-nitrosoguanidine at 11 weeks of age. Both busulfan treatment doses caused earlier occurrence of persistent estrus, with dose-dependence as compared to controls. At 15 months of age, the rats were euthanized. The incidence of uterine adenocarcinomas and multiplicity of uterine neoplastic lesions were significantly increased by the 5.0 mg/kg, but not the 2.5 mg/kg busulfan treatment. Morphologically, the ovaries exposed to busulfan treatment exhibited severe atrophy, with few or no follicles and corpus lutea. Serum 17beta-estradiol (E2), progesterone, and inhibin levels were significantly decreased in the busulfan treatment groups, with a clear dose-relation. Interestingly, only the 5.0 mg/kg busulfan treatment elevated the E2/progesterone ratio. These results provide evidence that the reduction of primordial follicles promotes uterine adenocarcinoma development in rats in association with an earlier occurrence of the persistent estrus status.

Continuing primordial germ cell differentiation in the mouse embryo is a cell-intrinsic program sensitive to DNA methylation.

The initial cohort of mammalian gametes is established by the proliferation of primordial germ cells in the early embryo. Primordial germ cells first appear in extraembyronic tissues and subsequently migrate to the developing gonad. Soon after they arrive in the gonad, the germ cells cease dividing and undertake sexually dimorphic patterns of development. Male germ cells arrest mitotically, while female germ cells directly enter meiotic prophase I. These sex-specific differentiation events are imposed upon a group of sex-common differentiation events that are shared by XX and XY germ cells. We have studied the appearance of GCNA1, a postmigratory sex-common germ cell marker, in cultures of premigratory germ cells to investigate how this differentiation program is regulated. Cultures in which proliferation was either inhibited or stimulated displayed a similar extent of differentiation as controls, suggesting that some differentiation events are the result of a cell-intrinsic program and are independent of cell proliferation. We also found that GCNA1 expression was accelerated by agents which promote DNA demethylation or histone acetylation. These results suggest that genomic demethylation of proliferative phase primordial germ cells is a mechanism by which germ cell maturation is coordinated.

O-ethylthymidine adducts are the most relevant damages for mutation induced by N-ethyl-N-nitrosourea in female germ cells of Drosophila melanogaster.

Responses to genotoxic agents vary not only among organisms, test systems, and cellular stages, but also between sexes; little, however, is known about the mutagenic consequences of chemical exposures to female germ cells. In this study, the mutagenicity of N-ethyl-N-nitrosourea (ENU) was analyzed in female germ cells of Drosophila melanogaster using the recessive-lethal test and the vermilion system, which simultaneously generates information on induced mutation frequency and mutation spectrum. ENU was mutagenic in all stages of oogenesis, although there were differences among the stages. In mature and immature oocytes, ENU-induced mutations in the vermilion locus were 43.5% A:T-->G:C transitions, 39.1% A:T-->T:A transversions, 8.7% G:C-->A:T transitions, and 8.7% A:T-->C:G transversions, indicating that the most important premutagenic lesions induced by this chemical are O(4)-ethylthymine and O(2)-ethylthymine. The low frequency of mutation involving O(6)-ethylguanine (i.e., G:C-->A:T transitions) could be a consequence of the repair of these lesions by O(6)-methylguanine DNA methyltransferase. Comparison of these results with those previously obtained in male germ cells stresses the importance of the repair activity of the analyzed cells, because the mutation spectrum in female germ cells was similar to the spectrum obtained with repair-proficient spermatogonial cells and different from repair-deficient postmeiotic cells. The results also indicate that studies with female germ cells could be an alternative to the use of premeiotic male germ cells, especially when the analysis of these cells is difficult or almost impossible and when studies of in vivo DNA repair in premeiotic germ cells are performed.

Effect of follicle-stimulating hormone on different cell sub-populations in the ovary of newly hatched chicks treated during embryonic development.

1. Cell sub-populations of the ovary of newly-hatched chicks were assessed following follicle stimulating hormone (FSH) treatment during embryonic development. Changes in cell number and the amount of oestradiol in serum were determined. 2. White Leghorn chick embryos received 1 microgram FSH applied to the chorioallantoic membrane at 13, 15, and 17 d of incubation. Within 24 h after hatching, animals were killed and blood was collected. The left ovary was immediately removed then weighed and processed by an enzymatic-mechanical dissociation method for total cell count. An air-drying method was also used for meiotic preparations to study the germinal cells. 3. The pre-follicular ovary is able to respond to FSH by inducing an increase both in the serum oestradiol concentration and in the number of steroidogenic cells and of poorly differentiated cells of the ovarian medulla. 4. FSH increases the number of oogonia, which are responsible for a sharp increase in the total population of germ cells in the FSH-treated ovary. 5. It is possible that FSH acts to increase the proliferation of oogonia and a delay in the meiotic prophase through a change in the microenvironment rather than by a direct effect on germ cells.

Characterization of a method for quantitating food consumption for mutation assays in Drosophila.

Quantitation of food consumption is necessary when determining mutation responses to multiple chemical exposures in the sex-linked recessive lethal assay in Drosophila. One method proposed for quantitating food consumption by Drosophila is to measure the incorporation of 14C-leucine into the flies during the feeding period (Thompson and Reeder: Environmental Mutagenesis 10:357-365, 1987). Three sources of variation in the technique of Thompson and Reeder have been identified and characterized. First, the amount of food consumed by individual flies differed by almost 30% in a 24 hr feeding period. Second, the variability from vial to vial (each containing multiple flies) was around 15%. Finally, the amount of food consumed in identical feeding experiments performed over the course of 1 year varied nearly 2-fold. The use of chemical consumption values in place of exposure levels provided a better means of expressing the combined mutagenic response. In addition, the kinetics of food consumption over a 3 day feeding period for exposures to cyclophosphamide which produce lethality were compared to non-lethal exposures. Extensive characterization of lethality induced by exposures to cyclophosphamide demonstrate that the lethality is most likely due to starvation, not chemical toxicity.

A simple method for the identification of compounds which inhibit tubulin polymerization in filarial worms.

The incubation in vitro of excised ovaries of Dirofilaria immits in medium containing mebendazole between 10(-5) and 10(-8) M for four or six hours results in the accumulation of up to 20% of oogonial cells in arrested mitotic metaphase. In aceto-orcein-stained squashes of the tissue, cells possess condensed chromosomes but no detectable spindle microtubules. Similar results were obtained with colchicine, but the lowest effective concentration of this drug was 10(-7) M. This procedure affords a simple and rapid method for detecting compounds capable of inhibiting tubulin polymerization in filarial worms.

[Differential cell mutability during oogenesis and exposure to ethyleneimine and ethylmethane sulfonate in different Drosophila melanogaster lines]. / Differentsial'naia mutabil'nost' kletok v khode oogeneza pri deistvii étilenimina i étilmetansul'fonata u raznykh linii Drosophila melanogaster.

The rate of recessive sex-linked lethal mutations (RLM) was estimated by brood pattern method at different stages of oogenesis, initially, in the wild-type R-86 strain of Drosophila melanogaster after treatment with EI and EMS. The former which is known to induce dominant lethals in mature oocytes of the 14th stage with a high frequency was equally effective in inducing RLM in oocytes of different age and in oogonia. EMS which does not induce dominant lethals when used as vapour was shown to increase RLM frequency in mature fraction of oocytes (the 14A stage only). Similar type of different mutability was found in mutagen-sensitive strain mus-201G1 and in the control 3-4 strain having the same genetical background as mus mutation. Female germ cells of mus-201G1 strain appeared to have a higher mutability in the case of EI, though no differences in mutability between these strains after EMS treatment were registered. The data are discussed in view of the specificity of primer damages occurring as a result of comparable mutagens action and participation of different repair systems in elimination of these damages.