ON THE PERSISTENCE OF OOCYTE NUCLEI FROM FETUS TO MATURITY IN THE LABORATORY MOUSE

Tritiated thymidine injected intraperitoneally into female mice midway through the gestation period was demonstrated by autoradiographic methods to be incorporated into the nuclei of oocytes of female embryos, observed at the pachytene stage of meiosis 2 to 4 days after the injection. The tritium label was also demonstrated in the oocyte nuclei of the daughters of similarly treated females at maturity (6 weeks post partum). It was also found that some follicle cells, likewise labeled with H³-thymidine in mid-fetal life, persisted to maturity with few or no intervening mitoses. The observations are presented in support of the prevailing view that individual oocytes which arise from germ cell primordia in fetal stages become the egg cells of the adult female mammal.     

Time of origin of cells in the histiogenesis of the spinal cord]

In order to establish the moment of appearance of neuroblasts and ectoglia of the spinal cord the autoradiographic study with the use of H3-thymidine and C14-thmidine injected to pregnant mice with the intervals between injections 121/2 or 24 hours was undertaken. It was establised that spinal neurons were removed from the nervous tube beginning from the 10th up to 13th days of embroyogenesis. The motoneurons of the anterior horn were the first to appear (10th-12th days), the neurons of the intermideate zone were the next to appear (11th - 12th days) and the last were the neurons of posterior horn (13th day). Beginning from the 13th day of embryogenesis there appeared the ectoglia which migrated following meurblasts two days later. The saturation of the grey matter with glial cells and the saturation of the white matter with Schwann cells was brought about by means of additional multiplication at the site of the glioblasts removed from the nervous tube. The main function of the matrix layer neuroepithelium of the nervous tube as a provider of cells to the spinal cord terminated on the 15th day of embryogenesis.     

DNA SYNTHESIS IN THE OOPLASM OF DROSOPHILA MELANOGASTER

Tritiated thymidine was injected into 2-day-old Drosophila melanogaster females, and tissue sections were prepared from the ovary for radioautography with both the light and electron microscopes. Besides the expected incorporation of H³-thymidine into nuclei of nurse cells and follicle cells, there was a relatively high level of incorporation of label into ooplasmic DNA. The highest level of incorporation occurred at stage 12. At the same time, the 15 nurse cell nuclei also incorporate thymidine in spite of the fact that they are breaking down and degenerating. The label in the ooplasm is not removed by extraction with DNase (although this removes nuclear label) unless extraction is preceded by a treatment with protease. Electron microscopic radioautography revealed that 36% of the silver grains resulting from decay of H³-thymidine are found over mitochondria, with a further 28% being located within 0.25 µ of these organelles. The remaining 36% of the silver grains was not found to be associated with any organelles, and it probably represents synthesis in the cytoplasm by the "storage DNA" characteristic of many eggs. It is suggested that one mechanism acting throughout the egg chamber is responsible for the synchronous synthesis of DNA in the degenerating nurse cells, in the mitochondria of the egg, and in the "storage DNA" of the ooplasm.     

TAURINE IN DEVELOPING RAT BRAIN: MATERNAL-FETAL TRANSFER OF [35S]TAURINE AND ITS FATE IN THE NEONATE

The transfer of [³⁵] taunne, injected intrapentoneally into pregnant rats (near term), to fetal tissues has been measured. Taurine can enter fetal brain as easily as it can fetal liver. In contrast, it cannot enter mature brain as easily as it can enter mature liver. After birth, [³⁵S] taurine, which had been injected into the dam before birth of the pups, continues to accumulate in the brain of the pups for some days. During the neonatal period, the concentration of taurine is decreasing, but the total pool of taurine in the brain is increasing rapidly. In order to help supply this increasing pool, the taurine present in the brain at birth appears to be conserved and an increasing amount of taurine is synthesized in situ. The net result during the neonatal period of development is that brain taurine specific radioactivity decreases and brain taurine has a very slow rate of turnover.     

H3-THYMIDINE DERIVATIVE POOLS IN RELATION TO MACRONUCLEAR DNA SYNTHESIS IN TETRAHYMENA PYRIFORMIS

The formation of a soluble H³-thymidine derivative pool has been examined in Tetrahymena pyriformis as a function of macronuclear DNA synthesis during the cell life cycle. An autoradiographic technique which allows the detection of water-soluble materials within a cell has shown that these cells do not take up and retain exogenous H³-thymidine during G1 or G2. Uptake of H³-thymidine is restricted to the S period of the cell cycle. Additional autoradiographic experiments show, however, that a soluble pool of H³-thymidine derivatives persists from the end of one DNA synthesis period to the beginning of the next synthesis period in the subsequent cell cycle. Since this persisting pool cannot be labeled with H³-thymidine, the pool does not turn over during non-S periods.     

Reutilization by maternal and embryonal cells of nuclear materials from H3-thymidine labeled lymphocytes introduced into the lumen of intestine of rats

Summary H³-thymidine labeled lymphocytes from thymus and lymph nodes of donor rats were washed and injected in to the intestine of recipient rats on the 11th and 19th day of gestation; subsequent labeling of maternal and embryonal cells was studied autoradiographically 24 hours after injection. In 12-day embryos, numerous stem cells or hemocytoblasts were labeled frequently intensely. In 20-day embryos, stem cells or hemocytoblasts scattered throughout the liver were often labeled. In other fetal tissues at this stage, cells in thymus, spleen, mesenteric lymph node and intestine were labeled but scarcely and weakly. In mothers, labeling in lymphoid tissues was scarce but definite, in thymus, mesenteric lymph node and spleen. These results suggest that nuclear materials from lymphocytes emigrated into the intestinal canal of the mother could be reutilized by maternal and embryonal cells.     

Metabolic DNA in the hepatocyte nuclei in newborn rats.

The behaviour in time of labelled nuclear DNA in the hepatocytes of newborn rats was studied using autoradiographic and biochemical techniques in two groups of experiments. In the first group H-3-thymidine was injected to the mothers at the 16th day of pregnancy and the amount of labelled DNA was evaluated in the newborns after delivery. In the second group H-3-thymidine was injected to the newborns two hours after birth and the labelled DNA was studied at the same time intervals as the first group. The amount of labelled thymidine incorporated into the first group of animals remains constant for the first three days of life, thereafter a reduction in specific activity of DNA is observed concomitant with an increase of the percentage of labelled nuclei and a decrease of the number of grains per nucleus. These results show that mitotic divisions, which are absent during the first three days of life, take place between the third and sixth days of life. The decrease of the specific activity is therefore due to dilution and not to loss of labelled DNA. In the second group of experiments the DNA labelled with H-3-thymidine shows a decrease by about 30--40% per day during the first three days of life accompanied by a decrease in the number of grains per nucleus without changes in the percentage of labelled nuclei. These data show that DNA synthesized during the first day after birth is metabolically unstable, unlike that synthesized during foetal life.     

Persistent vaginal cornification in mice treated with estrogen prenatally.

Twelve of 14 female mice of the ICR strain which had received a single injection of 50 mug estradiol-17beta on day 17 of fetal life exhibited irreversible cornification or stratification of the vaginal epithelium which persisted after ovariectomy until sacrifice performed 42-48 days later. Eight of the 12 mice had corpora lutea in their ovaries removed at 3-5 months of age. A similar injection of estradiol on day 15 of fetal life induced irreversible cornification or stratification of the vaginal epithelium in 6 of 12 females and only one of the 6 had corpora lutea in its ovaries when removed at 3-5 months. Mice given the same dose of estradiol on the day of birth or at 3 days of postnatal age invariably had ovaries bearing follicles of varying sizes and hypertrophied interstitial tissue but no corpora lutea. Changes in the vaginal epithelium in these animals were less remarkable as compared to that in prenatally treated mice.     

INCORPORATION OF H3-THYMIDINE INTO SHOOT AND ROOT APICES OF CERATOPTERIS THALICTROIDES

Gifford , Ernest M., Jr . (U. California, Davis.) Incorporation of H³-thymidine into shoot and root apices of Ceratopteris thalictroides. Amer. Jour. Bot 47(10): 834–837. Illus. 1960.—The localization of tritiated thymidine in apical meristems of Ceratopteris thalictroides by the autoradiographic method is described. Intact, floating plants of the fern were placed in 1/2 strength Hoagland's inorganic nutrient solution containing H³-thymidine (10 μc/ml.) for 3 days. The material was killed, dehydrated and embedded in paraffin. Autoradiographic stripping film (AR 10 Kodak) was applied to serial sections. After an appropriate exposure period, the film was developed and the sections with the superimposed film were stained lightly with Harris' hematoxylin. The autoradiographs revealed the presence of the H³-thymidine in nuclei of the large, individualized apical cells of shoots and roots which is proof of DNA synthesis. In no instances were these nuclei unlabeled. If endomitotic reduplication is excluded the results of these studies lend support to the concept that apical cells actually do divide and perhaps at a higher rate than envisioned by other workers. Considerable cytoplasmic labeling occurred and its significance to general problems of DNA synthesis is discussed.     

Early androgen treatment and male and female sexual behavior in mice

To investigate the role of neonatal androgen stimulation in the development of the potential for masculine and feminine sexual behavior in the mouse, different groups of mice were hormonally manipulated early in life. One group of female mice was administered testosterone propionate (TP) within 24 hr of birth; a second group of females was given a control injection of oil on the day of birth; a third group of females received an injection of TP on the 10th day after birth. A group of males received a control injection of oil on the day of birth. All mice were gonadectomized at about 30 days of age. At 60 days of age, mice were injected with estrogen and progesterone and tested for sexual receptivity; several weeks later all mice were injected with TP and tested for male sexual behavior. Female behavior: Females given oil at birth and females given TP on the 10th day after birth showed high levels of sexual receptivity as adults following estrogen-progesterone treatment. Females given TP on the day of birth, and male mice, rarely exhibited lordosis following estrogen-progesterone treatment. Male behavior: Most mice, regardless of genetic sex or neonatal treatment, mounted in adulthood following administration of exogenous androgen. There was little difference in mounting frequency between groups, suggesting that exogenous or endogenous androgen stimulation of the neonatal mouse does not facilitate adult mounting behavior. These data for the mouse are in essential agreement with existing data for the rat, and indicate that sexual behavioral differentiation induced by androgen stimulation in infancy is best characterized as an inhibition of the potential to display feminine sexual behavior in adulthood.     

Incorporation of a phospholipid precursor into the hemochorionic and yolk sac placentas associated rat embryos.

Pregnant Long-Evans rats were subjected to a teratogenic regimen, i.e., were fed a synthetic diet lacking folic acid and containing 9-methylpteroylglutamic acid on the 11th to 14th days of gestation. Experimental and control pregnant rats injected with 10 muCi of [2-14C] ethanolamine on the 14th day were killed 1 or 2 days later. The total radioactivity and radioactivities of phosphatidylethanolamine (PE), phosphatidylcholine (PC), and lysophosphatidylethanolamine (LPE) were determined in chloroform extracts of homogenates and subcellular fractions prepared from hemochorionic and yolk sac placentas and maternal liver. The distribution of radioisotope into PC and PE of control and experimental yolk sac placentas was similar, and paralleled the distribution in maternal liver. However, the distribution of radioisotope into PC and PE of the hemochorionic placentas did not parallel that of the maternal liver, and radiolabeled PC accumulated faster in experimental placentas than in controls. We suggest that the ability of the hemochorionic placenta to synthesize PC from PE was impaired by the teratogenic regimen, and that the organ took up relatively more PC from the maternal plasma. We propose that this teratogen-induced shift from placental lecithin synthesis to selective lecithin uptake underlies the previous finding of an increased accumulation of radio-labeled PC in embryos from pregnant females subjected to this teratogenic regimen (Chepenik and Waite, '73).     

Tritium labelling and cytochemistry of extra DNA in Acheta

Females of Acheta domesticus were injected with H³-thymidine and H³-uridine at various stages of development in order to study DNA and RNA synthesis in the DNA body present in the oocytes. Staining with alkaline fast green, azure B and the Feulgen reaction were employed as cytochemical tests. The following main results were obtained.

1. The DNA body appears in the oogonia at interphase as a Feulgen positive spherical structure 2 microns in diameter and is seen in subsequent mitotic divisions as a slightly smaller structure of variable shape. H³-thymidine autoradiography discloses that the DNA present in this body is synthesised at a different time from the chromosomal DNA.
2. At interphase and during the early prophase of meiosis the DNA body increases in size becoming a large Feulgen positive sphere 6 microns in diameter. Small nucleoli are present within this body. The DNA of the body is complexed with histone as revealed by alkaline fast green staining. H³-thymidine labelling discloses that it is at these stages that the bulk of the DNA synthesis takes place in the body.
3. Every oocyte contains a DNA body, and no body of comparable size or shape seems to be present in the male meiotic prophase.
4. At pachytene and diplotene the DNA body acquires the appearance of a “puff”. Two zones can be distinguished inside the DNA body: (1) an inner core of DNA and an outer shell of RNA. The inner core is Feulgen positive and stains light green with azure B, the outer shell is Feulgen negative and stains purple-violet with azure B, as does the cytoplasm. From the inner DNA core many Feulgen positive fibrils radiate into the outer RNA shell. These fibrils appear unstained or slightly greenish with Azure B, forming a transparent network in a purple-violet background. This gives the body the typical appearance of a “puff”. H³-uridine incorporation reveals that the RNA synthesis occurs in the outer RNA shell of the body and in the chromosomes. RNase treatment removes the H³-uridine incorporated into these regions.
5. At the end of diplotene the DNA body starts to disintegrate. The DNA core breaks up into minor components and the outer RNA zone also begins to disintegrate. By late diplotene the whole body has vanished, releasing DNA, histone and RNA into the nucleus. Subsequently the nuclear envelope disintegrates as it regularly does at the end of prophase of meiosis.
6. The simplest interpretation of the above results is that the DNA body represents hundreds of copies of the genes of the nucleolar organizing region.

     

KINETICS OF ROD OUTER SEGMENT RENEWAL IN THE DEVELOPING MOUSE RETINA

The kinetics of rod outer segment renewal in the developing retina have been investigated in C57BL/6J mice. Litters of mice were injected with [³H]amino acids at various ages and killed at progressively later time intervals. Plastic 1.5 µm sections of retina were studied by light microscope autoradiography. The rate of outer segment disk synthesis, as judged by labeled disk displacement away from the site of synthesis, is slightly greater than the adult level at 11–13 days of age; it rises to more than 1.6 times the adult rate between days 13 and 17, after which it falls to the adult level at 21–25 days. The rate of disk disposal, as measured by labeled disk movement toward the site of disposal, is less than 15% of the adult level at 11–13 days of age; it rises sharply to almost 70% of the adult level by days 13–15 and then more gradually approaches the adult rate. The net difference in rates of synthesis and disposal accounts for the rapid elongation of rod outer segments in the mouse between days 11 and 17 and the subsequent, more gradual elongation to the adult equilibrium length reached between days 19 and 25. The changing rate of outer segment disk synthesis characterizes the late stages of cytodifferentiation of the rod photoreceptor cells.     

The sexual function of mature rat males after prenatal modulation of cholinergic system

The data obtained have shown that prenatal exposure of pregnant rat females of 9-19-day pregnancy to N-cholinolytics as compared to M-cholinolytics produce long-term behavioural changes in pubescent rat progeny. Pubescent rat progeny had low dynamics of gaining sexual experience and decreased sexual activity with equal disturbance of motivation and coitus. The number of males with absence of sexual activity was above that of the control group. We suggest that sexual dysfunction of offspring adulthood was provoked by introduction of ganglerone (N-cholinolytic) which had been injected on 9-11 and 12-14 days of gestation, and metamyzil (M-cholinolytic) injected on 9-11 days of gestation. Apparently, regulation of neuronal mechanisms for sexual function is disturbed as a consequence of lasting change in neurotransmitter activity. It is suggested that dopaminergic activity in brain limbic structures was affected the most. The significant decrease in blood testosterone values has also been elucidated.     

ACTIVITY OF MARGINAL AND PLATE MERISTEMS DURING LEAF DEVELOPMENT OF XANTHIUM PENNSYLVANICUM

The mitotic and biosynthetic activities of the marginal and plate meristems were studied during the entire course of leaf development of Xanthium pennsylvanicum. In contrast to statements in the literature, marginal meristem activity is long in duration, as assayed by the mitotic counts and H³-thymidine incorporation. This me istem is active 23 days. The plate meristem is active for an additional 3 days after cessation of cell division in the marginal meristem, but the total duration of its mitotic activity is also approximately 23 days. Numerous periclinal cell divisions of the plate meristem form additional cell layers and contribute to the growth of the lamina in thickness. Incorporation of H³-thymidine increased during the course of leaf development. Cells between plastochronic ages 0 and 2.0 incorporated more of the radioisotopic precursor than those of younger leaf primordia. The uptake and incorporation of H³-thymidine into nuclear DNA was more sluggish during the early stages of development than in the more expanded leaves. No DNA synthesis was demonstrated after cessation of cell division in the leaf lamina. Metabolic or endomitotic DNA synthesis after leaf plastochron index (LPI) 3.0 seems improbable. No significant differences in the incorporation of H³-thymidine could be demonstrated between the marginal and plate meristems. This would indicate no distinct biosynthetic differences between the two meristems. The definitions of the marginal and plate meristems of Xanthium leaves were formulated in view of the above findings.     

Prenatal and Neonatal Diazepam Administration Synchronizes Testicular Malate Dehydrogenase Circadian Rhythms in Young Rats

Rat or hamster pups exposed to constant light or darkness since birth exhibit many circadian rhythms synchronized with those of the mother. During early development, a number of cues derived from the maternal circadian system synchronize the fetal and neonatal circadian clock. Maternal pineal sympathetic denervation during early pregnancy disrupts maternal synchronization of parotid α-amylase and testicular malate dehydrogenase circadian rhythms in rat pups. Maternal pineal sympathetic denervation was used to study potential agents able to synchronize the fetal or neonatal circadian clock. Melatonin injection to denervated pregnant mothers prevents the pineal sympathetic denervation effect on those circadian rhythms. We now studied the synchronizing effect of a benzodiazepine compound, diazepam. This GABA_A agonist synchronized testicular malate dehydrogenase (MDH) activity of pups when it was injected to sympathetic denervated pregnant dams (a daily dose at 07:00 or 19:00 h from the 14 th to the 20 th day of gestation) or orally administered to the pups (a daily dose at 19:00 h from the 10 th to 24 th day of life). Co-injection of diazepam and GABA_A antagonist, flumazenil, blocked the synchronizing effect of diazepam. The results demonstrate that diazepam has a synchronizing effect on the development of the circadian clock in rats and suggest that modulation of maternal GABA_A could participate in mammalian maternal synchronization.     

THE TIMING OF MEIOSIS AND DNA SYNTHESIS DURING EARLY OOGENESIS IN THE TOAD, XENOPUS LAEVIS

Recently metamorphosed female Xenopus laevis toads were injected with tritiated thymidine. Animals were kept at 20°C and were sacrificed 1–23 days after isotope injection. Radio-autographs of squash preparations of the ovaries were made. The progress of labeled germ cell nuclei was followed to obtain information on the time course of early meiosis and extra-chromosomal DNA synthesis. Premeiotic S was estimated to take not more than 7 days. Leptotene takes 4 days, zygotene takes 5 days, and pachytene was estimated to be completed in about 18 days. The major period of amplification of the extrachromosomal DNA occurs in pachytene and takes about 13 days. A low level of synthesis was observed before and after this period, in zygotene and late pachytene-early diplotene, extending the total time for extrachromosomal DNA synthesis during meiosis to about 18 days. These data allowed the calculation to be made that one round of replication of the amplified DNA takes between 1.2 and 3.0 days. It was also found that in both oogonial and premeiotic interphases, the nucleolus-associated DNA shows asynchronous (probably late) labeling with respect to the chromosomes.     

The effect of carbohydrate metabolic changes during pregnancy on the capacity of liver mitochondria in rat progeny to oxidize the lipid intermediate]

Respiration rate, respiration control and ADP of liver mitochondria were studies in one-, ten-, and twenty-day-old rats born of females who had received subcutaneous insulin injections (0.25 U/100 g body weight) on days 5-7, 11-13 and 19-21 of pregnancy or glucose (1 g/100 g body weight, in the morning before feeding). Caprylate, an intermediate of the lipid metabolism, was used as the substrate for oxidation. In the control, caprylate oxidation in one-day old rats occurred at a low rate without providing for synthesis of ATP from ADP and phosphate. Insulin administration and alimentary hyperglycemia in females on days 5-7 of pregnancy had no significant effect on respiration rate of liver mitochondria in progeny of all ages tested. Administration of the above preparations on days 11-13 and 19-21 of pregnancy improved caprylate oxidation in mitochondria of the newborn rats. In other series the difference between experiments and the control was insignificant. Metabolic changes in the newborns are shown to be related to hyperinsulinemia in pregnant females.     

The pattern of DNA replication in the chromosomes of Puschkinia libanotica

The uptake of H³-thymidine into the chromosomes of Puschkinia libanotica has been studied in plants possessing or lacking a heterochromatic B chromosome. The pattern of H³-thymidine uptake by the A chromosomes at the end of the S phase is similar in plants of both genotypes. Regions around the centromere take up more H³-thymidine at the end of S than do more distal regions. The rate of uptake into the heterochromatin of the B chromosome increases towards the end of S, but there is no evidence that synthesis in the B chromosome carries on after the completion of DNA synthesis in the euchromatic A complement. It is proposed that at the end of the S phase more replicons in the heterochromatin of the B chromosome are engaged in DNA synthesis than in euchromatin.     

Photo-thermal regulation of diapause in Trichogramma piceum Djur. (Hymenoptera, Trichogrammatidae)

Interaction of the photoperiodic conditions of development of maternal females (day lengths of 2 to 22 h at 20°C) with the thermal regime of development of their progeny (temperature of 12 to 15°C at day length of 12 h) in determination of prepupal diapause in Trichogramma piceum was studied under laboratory conditions. At 15°C the diapause was practically absent. At lower temperatures, the proportion of diapausing prepupae was maximal (25% of larvae at 14°C, 70% of larvae at 13°, and 80% of larvae at 12°C) if the maternal females developed under short day conditions (10–12 h). When maternal females developed at day lengths of 18–20 h, diapause was rarely recorded at all temperatures, while ultra-short (less than 8–10 h) days also caused a decrease in the proportion of diapausing progeny. The right (ecologically important) threshold of this maternal long-day photoperiodic response was about 14–15 h independently of the temperature during the progeny development. These results make it possible to clarify the mechanism of the “maternal photoperiodic correction of the progeny thermal response.” Although the impact of the maternal photoperiodic response can be revealed only within a very narrow thermal range, the relative strength of the diapause-inducing effect of different day lengths is independent of the temperature regimen of the progeny development.