Primordial Oocytes Research Articles

Female-specific mutagenic response of mice to hycanthone

Male and female gametogeneses differ markedly in all mammals. While male germ cells are continuously being produced from stem cells throughout the reproductive life span, the number of female germ cells is fixed during prenatal development and, soon after birth, all of the oocytes are arrested in a modified diplotene, or dictyate, stage. Following puberty, dictyate oocytes are hormonally triggered to mature either singly or in groups, resulting in ovulation and the completion of the first meiotic division. It has been hypothesized that female mice are more susceptible to dominant lethal effects of intercalating agents that male mice because oocyte chromosomes, which are arrested in a diffuse state, are generally more accessable to intercalation than are the more condensed chromosomes present within most male germ cell stages. This hypothesis was further tested using the intercalating agent hycanthone methanesulfonate. Effects of hycanthone were studied in maturing and primordial oocytes and in male germ cells throughout spermatogenesis. No induction of dominent lethality was observed for treated males while a significant increase in embryonic death, expressed around the time of implantation, was observed in females that mated within 4.5 days after treatment. These effects were the result of dominant lethal mutations induced in maturing oocytes and not of maternal toxicity as indicated by the presence of chromosomal aberrations observed at first-cleavage metaphase of zygotes obtained from treated females. These results add support to the hypothesis that certain intercalating chemicals, which are not mutagenic to male mice, may be mutagenic to females and point to a need for more in-depth studies of female-specific mutagenesis.

Gonadal expression of c-kit encoded at the W locus of the mouse

Recently, it has been shown that the c-kit proto-oncogene is encoded at the white spotting (W) locus in mice. Mutations of this gene cause depletion of germ cells, some hematopoietic cells and melanocytes. In order to define further the role of c-kit in gametogenesis, we have examined its expression in late fetal and postnatal ovaries and in postnatal testis. By RNA blot analysis, c-kit transcripts were not detected in late fetal ovaries but appeared at birth. The relative amount reached a maximum in ovaries of juvenile mice, and decreased in adult ovaries. c-kit transcripts were present in increasing amounts in isolated primordial, growing and full-grown oocytes, as well as in ovulated eggs. Little was detected in early 2-cell embryos and none in blastocysts. In situ hybridization revealed c-kit transcripts in a few oocytes of late fetal ovaries and in all oocytes (from primordial to full-grown) in ovaries from juvenile and adult mice. Expression was also observed in ovarian interstitial tissue from 14 days of age onward. Using indirect immunofluorescence, the c-kit protein was detected on the surface of primordial, growing and full-grown oocytes, as well as on embryos at the 1- and 2-cell stages; little remained in blastocysts. In situ hybridization analysis of testes from mice of different ages demonstrated expression in spermatogonia from 6 days of age onward. Using information provided by determining the stage of the cycle of the seminiferous epithelium for a given tubule and by following the age dependence of labeling, it was concluded that the period of expression of c-kit extends from at least as early as type A2 spermatogonia through type B spermatogonia and into preleptotene spermatocytes. Leydig cells were labelled at all ages examined. The expression pattern in oocytes correlates most strongly with oocyte growth and in male germ cells with gonial proliferation.

Binding proteins for insulin-like growth factors in the human ovary: identification, follicular fluid levels and immunohistological localization of the 29–32 kd type 1 binding protein, IGF-bp1

The occurrence of pregnancy-associated endometrial alpha 1-globulin (alpha 1-PEG), a 29-32 kd insulin-like growth factor binding protein, now termed type 1 or IGF-bp1, has been examined in the human ovary by monoclonal and polyclonal antibody based radioimmunoassay and immunohistological techniques. Follicular fluids aspirated from 51 follicles of 32 women undergoing hyperstimulation involving buserelin or clomiphene-based protocols contained 35.5-276.0 ng/ml (mean 101.0 mg/ml) of immunoreactive IGF-bp1. Mean fluid concentrations were three times the level of IGF-bp1 detected in paired serum samples, available for 21 women. Immunoreactive IGF-bp1 in follicular fluid exhibited similar dose-response curves to purified protein and amniotic fluid and immunoreactive IGF-bp1 coeluted in gel filtration with a peak of [125I]-IGF-1 binding corresponding to the elution profile of purified IGF-bp1. Gel filtration also revealed the presence in follicular fluid of a greater than 100 kd binding protein with a binding capacity equal to IGF-bp1 under the conditions employed. A highly significant correlation (P less than 0.001) was found between follicular fluid progesterone and IGF-bp1 and a correlation of lower significance was found between oestradiol and IGF-bp1 (P less than 0.05). However, only low levels of immunoreactive IGF-bp1 were detected in supernatant media of granulosa cells in culture (range undetectable to 2.3 ng/ml). Employing monoclonal antibody-based immunohistology, immunoreactive IGF-bp1 was consistently associated with luteinized granulosa cells of corpora lutea rather than paraluteal cells and its intensity of reactivity appeared to reflect luteal phase steroid hormone profiles. No consistent reactivity was detected in preovulatory follicles and granulosa cells in culture, although reactivity was associated with primordial oocytes. Immunoreactive IGF-bp1 was detected in six of nine supernatant media of explants of luteal tissue obtained from five corpora lutea, with levels ranging from undetectable to greater than 200 ng/ml. These observations suggest that IGF-bp1 is primarily related to luteinization of the granulosa and the resultant luteal cells, and if produced by the luteal cells, additional exogenous factors are required to induce production by granulosa cells in vitro.

A retroviral gp70-related protein is expressed at specific stages during mouse oocyte maturation and in preimplantation embryos

Mouse monoclonal antibodies directed against different epitopes of the murine leukemia virus envelope protein gp70 were used to study the expression of retroviral related env proteins during mouse oocyte maturation, fertilization and blastocyst formation. A gp70-related antigen was detected with immunohistochemistry in growing oocytes but not in primordial and primary non-growing oocytes. Atretic oocytes were also negative. Both parthenogenetically activated and fertilized oocytes were positive. Two-cell stages showed a patchy distribution of the antigen. Later preimplantation stages were negative except for a markedly positive period during compaction of the morula and adhesion of the blastocyst.

Cholinoceptive properties of human primordial, preantral, and antral oocytes: In situ hybridization and biochemical evidence for expression of cholinesterase genes

Cholinoceptive properties of human primordial, preantral, and antral oocytes: In situ hybridization and biochemical evidence for expression of cholinesterase genes

Cholinoceptive properties of human primordial, preantral, and antral oocytes: in situ hybridization and biochemical evidence for expression of cholinesterase genes.

In addition to their well-known involvement in neuromuscular junctions and in brain cholinergic synapses, cholinergic mechanisms have been implicated in the growth and maturation of oocytes in various species. Functional acetylcholine receptors were electrophysiologically demonstrated in amphibian and mammalian oocyte membranes, and activity of the acetylcholine-hydrolyzing enzyme, acetylcholinesterase (AChE), was biochemically measured in the exceptionally big oocytes of the frog Xenopus laevis. However, biochemical methods could not reveal whether AChE was produced within the oocytes themselves or in the surrounding follicle cells. Furthermore, this issue is particularly important for understanding growth and fertilization processes in the much smaller human oocytes, in which the sensitivity of AChE biochemical measurements is far too low to be employed. To resolve this question, a molecular biology approach was combined with biochemical measurements on ovarian extracts and sections. To directly determine whether the human cholinesterase (ChE) genes are transcriptionally active in oocytes, and, if so, at what stages in their development, the presence of ChE mRNA was pursued. For this purpose frozen ovarian sections were subjected to in situ hybridization using 35S-labeled human ChE cDNA. Highly pronounced hybridization signals were localized within oocytes in primordial, preantral, and antral follicles, but not in other ovarian cell types, demonstrating that within the human ovary ChE mRNA is selectively synthesized in viable oocytes at different developmental stages.(ABSTRACT TRUNCATED AT 250 WORDS)

Ultrastructural Observations on the Germ Plasm in the Lizard Podarcis sicula: (germ plasm/germ cells/ultrastructure/reptiles).

Ultrastructural studies on embryos and adult females of Podarcis sicula revealed fibrogranular electron-dense aggregates in the cytoplasm of primordial germ cells, oogonia, and oocytes. The ultrastructural similarities of these aggregates to fibrogranular aggregates in germ cells of some animal species and their relationship with mitochondria, free ribosomes, as well as cisternae of the rough endoplasmic reticulum strongly suggest that they correspond to the germ plasm.

Cadmium-induced ovarian toxicity in hamsters, mice, and rats

The effects of cadmium on the female genital tract of Syrian hamsters (Cr:RGH), of four mouse strains ( BALB cAnNCr , DBA 2NCr , C57BL 6NCr , NFS NCr ), and two rat strains ( F344 NCr and WF NCr ) were studied by light microscopy after a single sc injection of cadmium chloride (CdCl 2). Experiments involved animals prior to and after sexual maturity, and employed CdCl 2 doses ranging from 20 to 47.5 μmol/kg. Animals were examined at intervals from 24 hr to 8 weeks following treatment. Syrian hamsters were most susceptible to CdCl 2-induced ovarian hemorrhagic necrosis at all ages tested. Most severe ovarian lesions occurred in immature hamsters, in mature hamsters at high doses, and shortly before ovulation at all doses. The small arteries of the developing follicles and interstitial stroma seemed selectively susceptible to CdCl 2 toxicity while the corpora lutea, mesothelium, primordial oocytes, and the rete ovarii appeared resistant. Pretreatment with zinc acetate markedly reduced the extent of ovarian lesions in hamsters. Although reduced in weight by 45%, the hamster ovaries recovered morphologically within 2 months after severe acute hemorrhagic necrosis. Uterine and cervical stromal hemorrhages were seen only in immature hamsters at doses of ≥ 30 μmol CdCl 2/kg. Of the mice, only the DBA 2NCr strain showed significant CdCl 2-induced ovarian hemorrhages, and these hemorrhages occurred at doses also producing lethal liver toxicity. Lesions of the uterus were rare. Rats showed dose- and age-dependent toxicity in the ovaries, uterus, cervix, and liver. CdCl 2 exposure in mature rats induced uterine lesions only in F344 rats, while acute ovarian and hepatic toxicity was less severe in mature animals of both strains. No lesions were noted after 7 days in mature WF rats. In both rats and mice, no cycle dependency of the ovarian lesions was evident.

Oocyte-specific expression and developmental regulation of ZP3, the sperm receptor of the mouse zona pellucida

The mouse zona pellucida is composed of three sulfated glycoproteins, encoded by the oocyte genome, that have important biological functions in preimplantation development. One of the zona gene products, ZP3, functions as the sperm receptor at fertilization. Our present data demonstrate that the ZP3 gene is transcribed in oocytes where its expression is developmentally regulated. Resting primordial oocytes do not express ZP3 mRNA, but these transcripts rapidly accumulate in growing oocytes so that they represent 0.1–0.2% of the total poly(A +) RNA. As oocytes complete their growth and undergo meiotic maturation, the abundance of ZP3 transcripts falls off dramatically; ovulated eggs contain less than 15% of peak levels. The oocyte-specific accumulation of ZP3 transcripts serves as an attractive system for further studies of factors that modulate developmentally regulated genes during mammalian oogenesis.

Nucleolus, nucleolar chromosomes, and nucleolus-associated chromatin from early diplotene to dictyotene in the human oocyte.

The shape, relationships, relative DNA content, and nucleolar activity of the short arm of acrocentric bivalents were studied in human oocytes from early diplotene to dictyotene. At the beginning of diplotene, the short arms of the previously paired chromosomes were again separated and displayed the same morphological features as in mitotic prophase chromosomes. They were connected only with the nucleolus. In situ hybridization and silver staining showed that the nucleolar organizer regions (NORs) were located in the peripheral region of the nucleolus. Tritiated-uridine incorporation was active. At birth, the relationships of the acrocentric short arms showed increasing complexity. The chromosomes ended in nucleolus-associated chromatin blocks of irregular shape, containing large quantities of DNA as demonstrated by intense binding of 3H-actinomycin D. The number of chromosomes converging on these chromatin blocks exceeded the number of acrocentrics, suggesting that heterochromatic regions of other chromosomes were associated with the short arm of acrocentrics. In the electron microscope, the NORs were represented by fibrillar centers located on the periphery of the nucleolus and consistently connected with the blocks of dense chromatin. These relationships remained unchanged in the primordial oocyte in the adult ovary. Persistence of 3H-uridine uptake showed that the oocyte was not at a "resting" stage. The possible cytogenetic consequences of these observations are discussed.

Mouse Oocyte Killing by Neutrons: Target Considerations

Highly radiosensitive primordial mouse oocytes, the principal cells at genetic risk in the female, have been studied using 0.43 MeV neutrons. Analysis of the survival curve (D37 = 0.055 Gy) indicates that the diameter of the radiosensitive target (assumed spherical and of unit density) is larger than that of the nucleus but not of the oocyte, inplicating a non-nuclear but sub-cellular target. This is consistent with results from 3H-thymidine incorporated in DNA. Our efforts to identify the extraordinarily radiosensitive lethality target in these primordial oocytes suggest it is the plasma membrane. Monte Carlo calculations for 0.43 MeV neutrons show that at the D37 only a single proton recoil will traverse the plasma membrane, consistent with the observed exponential survival curve. A highly sensitive non-DNA target for mouse oocyte killing may importantly influence interpretations of genetic mutation data from mice and their use in estimating genetic risk in humans.

Mouse Oocyte Killing by Neutrons: Target Considerations

Highly radiosensitive primordial mouse oocytes, the principal cells at genetic risk in the female, have been studied using 0.43 MeV neutrons. Analysis of the survival curve (D37 = 0.055 Gy) indicates that the diameter of the radiosensitive target (assumed spherical and of unit density) is larger than that of the nucleus but not of the oocyte, inplicating a non-nuclear but sub-cellular target. This is consistent with results from 3H-thymidine incorporated in DNA. Our efforts to identify the extraordinarily radiosensitive lethality target in these primordial oocytes suggest it is the plasma membrane. Monte Carlo calculations for 0.43 MeV neutrons show that at the D37 only a single proton recoil will traverse the plasma membrane, consistent with the observed exponential survival curve. A highly sensitive non-DNA target for mouse oocyte killing may importantly influence interpretations of genetic mutation data from mice and their use in estimating genetic risk in humans.

The effect of intraovarian injection of benzo( a)pyrene on primordial oocyte number and ovarian aryl hydrocarbon [benzo( a)pyrene] hydroxylase activity

The effect of intraovarian (io) injection of benzo(a)pyrene (BP) on primordial oocyte number, ovarian aryl hydrocarbon hydroxylase (AHH, EC 1.14.14.1) activity, and hepatic AHH activity and P-450 content was investigated in C57BL/6N (B6) and DBA/2N (D2) mice. Ovaries were exteriorized through dorsolumbar incisions under ether anesthesia, and 1.0 μl of corn oil or corn oil containing BP was injected directly into the ovary. Bilateral io injection of BP was ovotoxic in a time and dose dependent manner. Primordial oocyte destruction was maximal 8 days after injection in both B6 and D2 mice. Unilateral io injection of BP destroyed oocytes only in the treated ovary. The threshold dose for primordial oocyte destruction was similar in both strains, approximately 50 ng/ovary. However, ED50's were different; 1.1 μg/ovary in B6 and 8.9 μg/ovary in D2 mice. Intraperitoneal treatment with α-naphthoflavone (80 mg/kg) inhibited the oocyte destruction by io treatment with BP (10 μg/ovary) in both B6 and D2 mice. In B6 mice, ovarian AHH activity was induced by BP in the treated ovary after either bilateral, or unilateral io treatment. However, io BP treatment did not change ovarian AHH activity in D2 mice. Hepatic AHH activity and P-450 content was not altered by io BP treatment in either mouse strain. These results strengthen the hypothesis that the ovary has the capability of metabolizing BP to ovotoxic products.

Germ cell studies in mice after prolonged exposure to nitrous oxide

Male and female Swiss Webster (SW) mice, age 13 to 14 weeks, were exposed by inhalation for 4 hr per day, 5 days per week, for 14 weeks, to either room air, 0.5% nitrous oxide, 5.0% nitrous oxide, or 50% nitrous oxide. Murine germ cells were examined for evidence of injury after this exposure. A group of male mice were treated with methyl methanesulfonate (MMS) as a positive control for sperm abnormalities while a group of female mice were treated with 3-methylcholanthrene (3-MC) as a positive control for oocyte destruction. There were no significant differences among the four inhalation exposure groups in testes weight, percentage of abnormally shaped sperm, sperm count, or histologic appearance of the testes; the mean percentage (±SE) of abnormal sperm ranged from 8.9 ± 2.4 (5.0% nitrous oxide) to 13.5 ± 0.5 (50% nitrous oxide) with a concurrent control value of 10.4 ± 2.3%. In the positive control experiment, 25.2 ± 4.1% of sperm from mice treated with MMS were abnormal compared with 2.5 ± 0.3% of sperm from mice treated with saline ( p < 0.001), indicating that sperm of SW mice are sensitive to chemical damage. There was no significant difference between the mean number of oocytes in mice treated with 50% nitrous oxide (33.3 ± 14.4) and in control mice (29.8 ± 8.0). In the positive control experiment, mice treated with 3-MC had significantly fewer ( p < 0.001) primordial oocytes, 67.2 ± 19.5 compared with control mice, 222.4 ± 21.9, indicating that this strain is sensitive to chemical damage of the ovary. Thus, murine germ cells showed no evidence of toxic effects due to prolonged exposure to nitrous oxide.

Female germ cell loss from radiation and chemical exposures

Female germ cells in some mammals are extremely sensitive to killing by ionizing radiation, especially during development. Primordial oocytes in juvenile mice have an LD50 of only 6-7 rad, and the germ cell pool in squirrel monkeys is destroyed by prenatal exposure of 0.7 rad/day. Sensitivity varies greatly with species and germ cell stage. Unusually high sensitivity has not been found in macaques and may not occur in man, but this has not been established for all developmental stages. The exquisite oocyte radiosensitivity in mice apparently reflects vulnerability of the plasma membrane, not DNA, which may have implications for estimating human genetic risks. Germ cells can be killed also by chemicals. Such oocyte loss, with similarities to radiation effects, is under increasing study, including chemotherapy observations in women. More than 75 compounds have been tested in mice, with in vivo toxicity quantified by oocyte loss; certain chemicals apparently act on the membrane.

Female Germ Cell Loss From Radiation and Chemical Exposures

Female germ cell in some mammals are extremely sensitive to killing by ionizing radiation, especially during development. Primordial oocytes in juvenile mice have an LD30 of only 6‐7 rad, and the germ cell pool in squirrel monkeys is destroyed by prenatal exposure of 0.7 rad/day. Sensitivity varies greatly with species and germ cell stage. Unusually high sensitivity has not been found in macaques and may not occur in man, but this has not been established for all developmental stages. The exquisite oocyte radiosensitivity in mice apparently reflects vulnerability of the plasma membrane, not DNA, which may have implications fur estimating human genetic risks. Germ cells can be killed also by chemicals. Such oocyte loss, with similarities to radiation effects, is under increasing study, including chemotherapy observations in women. More than 75 compounds have been tested in mice, with in vivo toxicity quantified by oocyte loss; certain chemicals apparently act on the membrane.

RRNA accumulation and protein synthetic patterns in growing mouse oocytes.

The rRNA contents of mouse primordial oocytes, three stages of growing oocytes, full-grown oocytes, and ovulated ova have been measured by hybridization of RNA samples to excess 3H-DNA complementary to rRNA. Since it was known from previous work that rRNA is stable, the results when plotted against days of oocyte growth indicated that rRNA was synthesized at a constant rate over the first 9 days of growth and about 1.5 times faster in the last 5 days. The maximum value of 0.3 ng per oocyte was attained by about 14 days of growth in oocytes 59 micrometers in diameter, well below the maximum diameter of 77 micrometers for full-grown oocytes. The stability of proteins synthesized in mid-growth phase oocytes was measured by labeling for 5 h with 35S-methionine and then following the decline of incorporated label during a 48h chase; 40% of the label decayed with a half-life of 11 h. and 60% was apparently stable. The two-dimensional electrophoretic patterns of labeled proteins synthesized by growing and full-grown oocytes were compared. The principal change was the appearance or great increase in intensity of several spots in full-grown oocytes as compared to growing oocytes. Egg proteins separated on a two-dimensional gel were visualized by silver staining. The cytoskeletal proteins actin, tubulin, and putative intermediate filament protein, as well as putative lactate dehydrogenase, were synthesized in growing and full-grown oocytes, and accumulated to form a significant portion of bulk egg protein.

Etude histologique et ultrastructurale de la gonade d'Helix aspersa Müller à l'éclosion

The gonad of young Helix aspersa, studied by light and electron microscopy during the first post-hatching week, showed a compact structure. Eventually, a lumen appeared as the cells moved apart. The gonad was composed of stem and germinal cells. The latter cells were represented by primordial germ cells and oocytes. With our method of rearing, the female germinal line differentiated first.

The biochemical and genetic characteristics of murine ovarian aryl hydrocarbon (Benzo[ a]pyrene) hydroxylase activity and its relationship to primordial oocyte destruction by polycyclic aromatic hydrocarbons

Primordial oocyte destruction by polycyclic aromatic hydrocarbons requires metabolic activation of the polycyclic hydrocarbon to an ovotoxic metabolite. The first and perhaps subsequent step(s) in the metabolism of polycyclic aromatic hydrocarbons to the proximate ovotoxin occurs via a microsomal cytochrome P-450-dependent monooxygenase. The role of aryl hydrocarbon (benzo[ a]pyrene) hydroxylase (AHH, EC 1.14.14.2) in activation of polycyclic hydrocarbons to ovotoxic products was studied in 11 inbred and two F 1 heterozygote murine strains. The polycyclic aromatic hydrocarbon responsive C57BL/6N and C57BL/6J mice had two- to threefold increases in ovarian AHH activity after polycyclic hydrocarbon treatment. The polycyclic aromatic hydrocarbon nonresponsive DBA/2N and DBA/2J mice had no change in ovarian AHH activity after similar polycyclic hydrocarbon treatment. The polycyclic aromatic hydrocarbon responsive C57BL/6N and C57BL/6J mice had more rapid primordial oocyte destruction after polycyclic hydrocarbon treatment than the nonresponsive DBA/2N and DBA/2J mice. The (DBA/2N)(C57BL/6N)F 1 and (DBA/2J)(C57BL/6J)F 1 mice were responsive to polycyclic hydrocarbons with two- to threefold increases in ovarian AHH activity after treatment, however, the rate of oocyte destruction was similar to that observed in the nonresponsive DBA/2N and DBA/2J mice. Survey of seven additional inbred strains of mice failed to demonstrate a strong correlation between either absolute ovarian AHH activity or fold induction of ovarian AHH activity and oocyte destruction. Similarly, treatment with 2,3,7,8-tetrachlorodibenzo- p-dioxin, a potent inducer of ovarian AHH activity in both polycyclic hydrocarbon responsive and nonresponsive strains, did not alter the rate of oocyte destruction after treatment with benzo[ a]pyrene or 3-methylcholanthrene. Although metabolic activation of polycyclic aromatic hydrocarbons to an ovotoxin is necessary for primordial oocyte destruction, strain differences in sensitivity to oocyte destruction reflect the biological sum of activation, detoxification, and repair. Therefore, differences in ovarian AHH activity or inducibility may not reflect differences in oocyte destruction after polycyclic hydrocarbon treatment.

Morphology of oocyte and follicle destruction by polycyclic aromatic hydrocarbons in mice

The carcinogenic polycyclic aromatic hydrocarbons, benzo( a)pyrene, 3-methylcholanthrene, and 7,12-dimethylbenz( a)anthracene, destroy primordial oocytes in the mouse ovary. The rate of oocyte destruction was proportional to the activity of the ovarian microsomal cytochrome P-450-dependent monooxygenase, aryl hydrocarbon (benzo( a)pyrene) hydroxylase (EC 1.14.14.2) as well as to the carcinogenicity of the polycyclic hydrocarbon. After treatment with benzo( a)pyrene or 3-methylcholanthrene only primordial oocyte destruction occurred, and no evidence of toxicity was observed in surrounding granulosa or ovarian stromal cells. 7,12-Dimethylbenz( a)anthracene was much more toxic and destroyed large follicles and oocytes in addition to primordial oocytes and primary follicles. Seven weeks after treatment with 3-methylcholanthrene the ovary had the bland afollicular appearance characteristic of ovarian failure. These three polycyclic aromatic hydrocarbons are capable of producing premature ovarian failure in rodents.