Injection Of Thymidine Research Articles

Passage of spermatozoa through the epididymis of the boar (Sus scrofa domesticus)

Previous studies of epididymal transit in the pig have had insufficient animal numbers to provide a comprehensive picture of a continuous process. The present study attempts to addresses this issue. Radioactively labeled thymidine was infused into the testicular arteries of 48 sexually rested young adult boars of the Goettingen Miniature Pig breed. Hemicastrations were performed in random order in 2 animals daily on Days 21–24 following thymidine injection and in 4 animals daily on Days 25–41 and 43, 45, 47 and 49. Sperm obtained from 12 epididymal sites between the proximal caput and distal cauda were autoradiographically examined to record the percentage of labeled sperm and labeling intensity at different times after thymidine infusion. An initial surge of labeled spermatozoa emerged in the proximal caput 28 days after thymidine injection. After 2 d labeled sperm had arrived at the distal caput and, after another 2, the bulk of labeled sperm was found in the corpus. From there the sperm advanced to the transition of corpus and cauda, where progress was arrested until Day11. On Day 12, transit was resumed and by Day 13 sperm had passed through the cauda and vacated the epididymis via the ductus deferens.

Differential cell proliferation and differentiation in developing and growing claws of turtles and alligator determine their shape

AbstractDifferential cell proliferation and differentiation in developing and growing claws of turtles and alligator determine their shape. Acta Zoologica (Stockolm). Morphogenesis and cell proliferation in claws of turtle and alligator have been analysed by immunolabelling and thymidine autoradiography. Proliferating cells are randomly distributed in the epidermis and mesenchyme at the tip of forming digits. Developing claws elongate with a dorsal curvature forming the unguis. Numerous proliferating keratinocytes are present in the unguis at 4 hr–1 day after injection of 5BrdU or by PCNA detection. Their size increases before being incorporated into the ungueal corneous layer. Immunolabelling at 3–6 days post‐injection of 5BrdU indicates that proliferating keratinocytes move towards the tip of the claw giving rise to the claw pad. The ventral side of the claw termed sub‐unguis shows a lower proliferation that expands the surface and produces thin corneocytes that are desquamated. Autoradiography and immunolabelling indicates that proliferating cells of growing turtle claws are present along most of the germinal epidermis 7 hr–1 day after injection of tritiated thymidine or 5BrdU. Low labelled corneocytes incorporated into the corneous layer are detected at 6–12 days post‐injection, generally localized towards the tip of the claw, indicating they have migrated distally. The study confirms that claws of reptiles have an extended proliferative zone instead of a localized proximal germinal matrix as in mammalian claws.

Higher environmental temperatures promote acceleration of spermatogenesis in vivo in mice (Mus musculus)

Temperature is considered a crucial modulator of reproductive activity and testis homeostasis. It is well known that elevated temperatures cause several effects on testicular components, particularly on germ cells, which might lead to the impairment of spermatogenesis and loss of male fertility. The present study aimed to evaluate the effects of different environmental temperatures on several morphofunctional testis parameters, with emphasis on duration of spermatogenesis and spermatogenic efficiency. Thirty sexually mature Swiss mice (Mus musculus) were allocated in three different experimental groups, being kept in vivarium for three weeks at 16 °C, 23 °C (control group) and 32 °C. In order to estimate the duration of spermatogenesis, three animals per each group received intraperitoneal injections of tritiated thymidine and the testes were perfused-fixed and routinely processed for histological, morphometrical and immunoperoxidase analyses. Although the lower temperature (16 °C) did not change most of the evaluated testicular parameters, our findings showed that higher environmental temperature (32 °C) is able to alter important testis parameters, resulting for instance in acceleration of spermatogenesis, alterations in the stages frequencies, increased number of germ and Leydig cells apoptosis and reduced Sertoli cell and spermatogenic efficiencies. As in many conditions infertile men exhibit higher mean scrotal temperature, we believe that experimental studies with mice involving temperature might represent an interesting approach to better understand the mechanisms related to human testis function and sperm production.

Spermatogenesis in a neotropical marsupial species, Philander frenatus (Olfers, 1818)

Despite the singular morphology of the male genital system and the different reproductive strategies of marsupials, little emphasis has been given to the testis morphology and spermatogenic kinetics in this mammalian order. The present study aimed to investigate the testis function and the duration of spermatogenesis in the southeastern four-eyed opossum, Philander frenatus. Testes of six adult males were routinely processed for histological and stereological analyses. In order to determine the duration of spermatogenesis, intratesticular injections of tritiated thymidine were performed 1h, 13days and 21days before the sacrifice. Based on the development of the acrosomic system, ten stages of the seminiferous epithelium cycle were characterized. The mean body and testis weights for the P. frenatus were respectively 326±20g and 0.4±0.05g, providing a gonadosomatic index of 0.3±0.02%. The most advanced germ cell types labeled at 1h, 13days and 21days after thymidine injections were, respectively, preleptotene spermatocytes at stage IV, pachytene spermatocytes at stage IV and diplotene spermatocytes at stage IX. Based on the stages frequencies and the most advanced labeled germ cells, each spermatogenic cycle and the entire spermatogenic process lasted respectively 13.5±0.5 and 60.9±2.4days. When compared to the vast majority of eutherian mammals already investigated, these data indicate that the Philander frenatus presents a relatively long duration of spermatogenesis.

Spermatogenic efficiency in the spiny rat, Trinomys moojeni (Rodentia: Echimyidae)

The spiny rat ( Trinomys moojeni) is a rodent found in the Atlantic Forest, which is considered one of the most diverse and threatened biomes in the world. Knowledge on reproductive biology and physiology is critical to conservation and species management, allowing the prevention of extinction and the use of males in natural and artificial reproduction programs. The main objectives of the present study were to investigate the testis structure as well as spermatogenic and Sertoli cell efficiency in the spiny rat captured in the Caraça Natural Reserve, a fragment of the Atlantic Forest located in the State of Minas Gerais, Brazil. Ten sexually mature spiny rats were analyzed. Intraperitoneal injections of tritiated thymidine were administered to estimate duration of spermatogenesis. The testes were perfused-fixed in buffered glutaraldehyde and routinely processed for histological and morphometric analyses as well as the characterization of the stages of seminiferous epithelium cycle. Volume density (%) of seminiferous tubules and Leydig cells were 97 ± 0.3 and 0.3 ± 0.02, respectively. The duration of one spermatogenic cycle and total duration of spermatogenesis were 8.6 ± 0.1 and 38.5 ± 0.5 days, respectively. Due to the very great volume density of the seminiferous tubules, short duration of spermatogenesis, tubule length per gram of testis (∼40 m), great Sertoli cell efficiency (∼15 spermatids per Sertoli cell) and large number of Sertoli cells per testis gram (53 million), spermatogenic efficiency in the spiny rat (82 million) is by far the greatest of the mammalian species investigated thus far.

The seminiferous epithelium cycle and its duration in different breeds of dog (Canis familiaris)

Testis structure and function in dogs are relatively poorly investigated. The aim of the present study was to carry out a comparative investigation of the stages of the seminiferous epithelium cycle and its duration in different breeds of dog. Fifty-six sexually mature dogs (mongrel, n = 12; pinscher, n = 12; beagle, n = 5; American pit bull, n = 9; poodle, n = 12; and Labrador retriever, n = 6) were analysed. Intratesticular injections of tritiated thymidine were given to determine the duration of spermatogenesis. Orchiectomy was performed at different time periods following injection (1 h, 2 and 4 weeks). Testis fragments were embedded in plastic and routinely prepared for histological and autoradiographic evaluations. Eight stages were characterized based on the acrosome system. Significant (P < 0.05) differences were found for the frequencies of the different stages characterized (except Stages V, VI and VIII), particularly for the mongrel. Stage IV (when spermiation occurs) was the most frequent in all six breeds (~25%), whereas Stages II and VIII were the least frequent (< 8%). Each spermatogenic cycle and the total duration of spermatogenesis lasted 13.73 +/- 0.03 and 61.9 +/- 0.14 days, respectively, for the mongrel, poodle, pinscher, beagle, and Labrador retriever. These values were approximately 10% lower (P < 0.03) for the American pit bull (12.55 +/- 0.26 and 56.5 +/- 1.17 days, respectively). To our knowledge, this is the first comprehensive study to perform a careful investigation of stage frequencies and seminiferous epithelium cycle duration in this very important domestic species.

Neuronal recruitment in adult zebra finch brain during a reproductive cycle

Previous studies suggest that adult neurogenesis and neuronal replacement are related to the acquisition of new information. The present study supports this hypothesis by showing that there is an increase in new neuron recruitment in brains of adult male and female zebra finches that coincides with the need to memorize vocalizations of nestlings before they fledge. We counted [(3)H]-Thymidine labeled neurons 40 days after [(3)H]-Thymidine injections. These counts were made in the parents' brains at the time eggs hatched, at the time juveniles fledged and still needed parental care, and at the time juveniles were already independent. We focused on nidopallium caudale (NC), a brain region which plays a role in sound processing. Recruitment of new NC neurons increased at the time the young fledged, followed by a significant decrease when the young reached independence. We suggest that this increase enables parents to recognize their own young when they are still dependent on parental feeding, yet easily lost among other fledglings in the colony. We saw no such increase in neuronal recruitment in the olfactory bulb, suggesting anatomical specificity for the effect seen in NC. We also found a preliminary, positive correlation between number of fledglings and number of new NC neurons in the parents' brain at fledging, suggesting that the number of neurons recruited is sensitive to the number of young fledged.

Keratinocyte stem cells, label-retaining cells and possible genome protection mechanisms.

Stem cells are the crucial cells upon which the entire tissue is dependent. Here we define and discuss what is meant by and known about keratinocyte stem cells. One way in which these cells have been studied is by their ability to retain radioactivity labelled thymidine for long periods of time (label retaining cells, LRCs). The underlying mechanism has been assumed in the past to be slow cycling but a more likely explanation is the selective segregation of old and new DNA strands (Cairns's hypothesis). Experiments in the small intestine indicate that the stem cells here are selectively sorting their DNA and becoming LRCs. A possible role for p53 in stem cell biology is presented.

Effet synchroniseur du rythme circadien de la corticostérone sur la vitesse de synthèse du DNA dans le foie du jeune rat

Post-natal liver proliferation is synchronized by corticosterone circadian variation from the 21st day of life in rats. After tritiated thymidine injection, we compare the labelling index, the specific radioactivity of DNA, the DNA synthesis rate, the plasma corticosterone in un-rhythmed rats at day 15 and in rhythmed rats at day 25. No variation of these parameters is found during the 15th nycthemeron. During the 25th nycthemeron, the corticosterone circadian wave peaking at 18h is followed by the circadian variations of the 3 proliferation parameters peaking at 24 h.

Undifferentiated cells in the snail myocardium are capable of DNA synthesis and myodifferentiation.

Cellular mechanisms of heart-muscle growth in the snail Achatina fulica have been studied using cytophotometry and electron microscopic autoradiography. Cytophotometric DNA measurements showed that the snail cardiomyocytes are mononucleated cells with diploid nuclei. Ultrastructural analysis of the snail myocardium revealed that, in addition to mature myocytes, it contains small roundish undifferentiated cells (UCs) and poorly differentiated muscle cells. EM autoradiography detected silver grains over the nuclei of UCs 2 h after injection of tritiated thymidine ([(3)H]Tdr), while the nuclei of both mature and poorly differentiated myocytes remained unlabeled. In EM autographs of the myocardial tissue fixed 14 days after [(3)H]Tdr administration, labeled myonuclei were evident, which may suggest some myodifferentiation of prelabeled UCs. Many labeled UCs persist for 14 days after a single [(3)H]Tdr injection, suggesting that not all UCs undergo myodifferentiation after passing through the cell cycle, and that those that do not can enter the next cycle. UCs in the snail myocardium presumably provide not only reserve but also stem cells for myocytes. Thus, the heart muscle of the adult snail consists of mononucleated diploid myocytes with blocked proliferative activity and a renewable population of precursor myogenic cells. The results obtained suggest that the growth of this muscle involves a myoblastic mechanism of myogenesis; this mechanism differs from that of vertebrate cardiac muscle growth, which is non-myoblastic-that is, based on proliferation or polyploidization of cardiomyocytes. Evolutionary aspects of cellular mechanisms of the heart-muscle growth are discussed.

Cell kinetic studies in murine ventral tongue epithelium: cell cycle progression studies using double labelling techniques.

The dorsal and ventral epithelia on the murine tongue exhibit very pronounced circadian rhythms in terms of the cell cycle. These rhythms are such that three injections of tritiated thymidine 3 h apart spanning the circadian peak in S phase cells labelled between 40 and 50% of the basal cells. Injection of bromodeoxyuridine generally gave slightly lower labelling indices. Approximately the same proportion (54% of the basal cells) could be accumulated in metaphase over a 24-h period using vincristine as a stathmokinetic agent. The experiments reported here using mouse ventral tongue epithelium use double-labelling approaches to address the question: what proportion of the approximately 50% of the basal cells that are proliferating have a 24-h cell cycle and can therefore be labelled by a similar labelling protocol the following day? The results suggest a heterogeneity amongst the proliferating basal cells, similar to the heterogeneity proposed for the dorsal tongue epithelium. Although not all the basal component has been accounted for, the data presented here suggest that about 20% of the basal cells may have a cell cycle time of 24 h, about 30% appear to have a longer cell cycle time (48 or 72 h), while about 20% of the basal cells appear to be postmitotic maturing G1 cells, awaiting the appropriate signals for migration into the suprabasal layer.

Effect of myenteric denervation on intestinal epithelium proliferation and migration of suckling and weanling rats.

The effects of myenteric denervation on the cell kinetics of the intestinal epithelium of suckling and weanling rats were investigated. The myenteric plexus of an ileal segment was partially ablated by serosal application of benzalkonium chloride (BAC) in three groups of rats: those that underwent surgery at 13 days and were killed 15 (13/28-day-old) or 23 (13/36-day-old) days after treatment, and those that were operated at 21 days (21/36-day-old) and were killed 15 days after treatment. The extent of denervation was assessed in whole-mount preparations. The cell bodies of myenteric neurones were stained by NADH-diaphorase histochemical technique. Cell proliferation was estimated by the mitotic index (MI) and morphometric analysis of villus and crypt lengths using an image analysis system. Thickness of the muscle layers was also assessed by morphometry. Cell migration on the villi was estimated by the position of the leading labelled cell 24 h after tritiated thymidine injection. The number of neurones was reduced by around 80% in rats operated at 13 days, and reduced by 98% in those operated at 21 days. The thickness of the muscle layers was increased in all groups of treated animals. MI was significantly higher 15 days after BAC-treatment in the 13/28 group. Morphological changes in the intestinal mucosa were observed 15 days after BAC-treatment, when there was an increase in villus height (13/28 group) and crypt depth (13/28 and 21/36 groups). Cell migration rate was accelerated in the 21/36 group. No differences where found in the 13/36 group. These results show the strong effect of myenteric ablation on cell proliferation and migration in the ileal epithelium in the first 15 days of treatment in suckling and in weanling rats, and the subsequent recovery of intestinal mucosa homeostasis later on.

Review: Hepatic Oval Cells: "Knocking on Forbidden Doors"

Hepatic oval “stem” cells were first described in 1956 by E. Farber (1), and are considered to be a cell capable of developing into hepatocytes or bile duct epithelial cells. Other investigators suggest that they are the progeny of a hepatic stem cell. Numerous in vivo and in vitro studies have documented a central role of oval cells in liver biology and carcinogenesis (2–4). In addition to the effects seen in the liver, oval cells (or cells very similar to oval cells) have been implicated in the architecture of the regenerating pancreas after injury (5–6) and they may be involved in the regeneration of other organs as well. While there is a precise purpose for this cell population in the liver, the mechanism(s) by which oval cells activate, proliferate and differentiate are poorly understood. Despite forty years of research and 300 publications on hepatic oval “stem” cells their ontogeny remains a mystery. “Oval cell proliferation” describes the occurrence of nonparenchymal cells detected in large numbers at early stages of carcinogenesis. The population is heterogeneous and contains cells that differ in their differentiation state and potential in their lineage commitment. “Oval cell compartment” is also used to describe the cells invading the parenchyma after the administration of certain carcinogens (7–8). Farber (1991) argued that although oval cells might be the progenitors of hepatocelluar carcinomas (HCC) induced by 3 9 methyl-4 9 -dimethyl-amino-azo-benzene (DMAB), this conclusion can not be generalized to other models of hepatocarcinogenesis (3). It is his view that cell lineage analyses cannot be achieved in a culture system(s) and that the markers used in studies of hepatocarcinogenesis can be considered reliable lineage markers for work done in vivo (3). Farber also reported that in each oval cell compartment model the oval cells will express different isoenzyme profiles, however, certain hepatic alpha-fetoprotien (AFP) and biliary cytokeratin-19 (CK-19) markers will always be expressed (3). In the DMAB model, the single most important conclusion provided by this data is that hepatocytes could be generated from nonparenchymal progenitor cells (3,9). This model establishes the key principle that some type of progenitor cell exists even if it turns out that precursor cell differentiation occurs only infrequently. Evarts et al. (1987 and 1989) show that the oval cell compartment is activated extensively in a 2-Acetylaminofluorene/two thirds partial-hepatecomy (2-AAF/PHx) model. This model is a variation of the SoltFarber protocol (2,10) and has become the industry standard. Figure 1 represents a timeline for this type of protocol. Under these conditions hepatocyte proliferation is heavily suppressed, and it was possible to label oval cells with [3H]-thymidine. This procedure allowed them the ability to follow the fate of the labeled cells. Four to five days after [3H]-thymidine injection, the label was found in basophilic hepatocytes expressing AFP and albumin (2). The authors concluded that there was a precursor-product relationship between the labeled cells of the oval cell compartment and the basophilic hepatocytes. Further studies, presented by the same investigators , show that proliferation of the oval cells and hepatocyte differentiation at early stages of carcinogenesis are associated with the activation of the stellate or Ito cells, which may reg-

Study of the effects of aging on macromolecular synthesis in mouse steroid secreting cells using microscopic radioautography.

The effects of aging on DNA, RNA and protein synthesis in adrenal gland cortical cells and testicular Leydig cells of ddY mice at various ages (from prenatal day 19 to postnatal days 1, 3, 7, 14, months 1 and 6 and 1 and 2 years after birth) were examined using light and electron microscopic (EM) radioautography after labeling with [3H]-thymidine, [3H]-uridine and [3H]-leucine. The percentage of the labeled cells in the adrenal glands after [3H]-thymidine injection was greatest in the zona glomerulosa of the cortex and the medulla on embryonic day 19, in the zona fasciculata and zona reticularis of the cortex on postnatal day 1 and gradually decreased with aging. EM radioautography revealed that well developed cell organelles such as smooth surfaced endoplasmic reticulum, Golgi apparatus, mitochondria with tubular cristae and lipid droplets were more frequently observed in the cytoplasm of unlabeled cells as compared to cells labeled with [3H]-thymidine in the three zones of the cortex. The effects of aging on RNA synthesis in adrenal glands after [3H]-uridine injection revealed that all types of cells of the adrenal gland were labeled. In each cell, silver grains were localized over the nuclei and cytoplasm and were more dense in the nuclei than the cytoplasm. Grain counts were highest in the cortex and medulla on fetal day 19 and then gradually decreased with aging from postnatal day 14 to 1 year after birth. In the cortex, the number of silver grains was higher in the zona glomerulosa than in the other zones from fetal day 19 to 1 year and grain counts were higher in the medulla in the embryonic stage as compared to the postnatal stages. However, the number of labeled mitochondria and the mitochondrial labeling index increased with aging. The [3H]-thymidine labeling index in the Leydig cells of the testis was low at embryonic and early postnatal stages, increased slightly at 6 months and reached a peak at 9 months which was maintained at a relatively high level in senescence. The number of the silver grains over the nuclei and cytoplasm of Leydig cells due to [3H]-uridine labeling was observed from embryonic day 19 and increased from month 3 onwards. From adult to senescence, [3H]-uridine incorporation was maintained at high levels in the nuclei and was relatively low in the cytoplasm. The effects of aging on [3H]-leucine incorporation in Leydig cells were also examined. The labeling indices between embryonic and early postnatal stages showed no obvious differences although the number of the silver grains in both cytoplasm and nucleus increased from 6 months onwards and was maintained at high levels until senescence. From these results, it was concluded that the effects of aging on DNA, RNA and protein synthesis in steroid secreting cells such as adrenal gland cortical cells and testicular Leydig cells of mice correlated with the hormonal changes observed with aging.

The length of the cycle of seminiferous epithelium in goats (Capra hircus)

This is the first report in literature showing the length of the seminiferous epithelium cycle in goats. In the present study, the duration of spermatogenesis was estimated using intratesticular injections of tritiated thymidine. Animals were castrated at 4 h, 7 days, and 11 days after injections. The duration of each spermatogenic cycle in goats is 10.6 ± 0.5 days (SEM). Considering that the total duration of spermatogenesis takes about 4.5 cycles of seminiferous epithelium, spermatogenesis was estimated to last 47.7 days. The approximate primary spermatocytes life span is 14.1 days, while spermiogenesis in goats lasts 14.9 days. Staging in goats was based on the tubular morphology, where 8 stages of the cycle are yielded for all species. The relative stage frequencies in goats, based on 400 seminiferous tubule cross sections for each animal were as follows: stage 1: 15.8 ± 1.0%; stage 2: 12.8 ± 0.5%; stage 3: 20.5 ± 0.9%; stage 4: 10.7 ± 0.7%; stage 5: 11.6 ± 0.6%; stage 6: 9.3 ± 1.1%; stage 7: 7.6 ± 0.4%; stage 8: 11.7 ± 0.6%. The pre-meiotic, meiotic and post-meiotic phases’ relative frequencies were 49.1%, 10.7% and 40.2%, respectively. The duration of spermatogenesis in goats is very similar to that found in rams.

Neuron loss in the mouse hippocampus following prenatal injection of tritiated thymidine or saline

To investigate possible effects of injections of tritiated thymidine ([3H]dThd) into pregnant mice or the injection procedure itself on the proliferation of neuronal precursor cells in the fetuses, pregnant mice received intraperitoneal injections of either [3H]dThd or saline on embryonic days 12, 14, and 19, while their offspring remained untreated. A second group of dams was not injected but their male offspring received a subcutaneous injection of again either [3H]dThd or saline on postnatal day 10. Then total numbers of hippocampal pyramidal cells (areas CA1 to CA3) and granular cells (dentate gyrus) were determined stereologically for 20-day-old as well as for 80-day-old male pups. No significant differences were found for the mean total number of pyramidal cells between the investigated groups of pups. However, the mean total number of granular cells was significantly reduced in those groups in which the dams had received an intraperitoneal injection, irrespective of whether [3H]dThd or saline was injected. This revives the repeated warning in the literature to consider the effect of the injection procedure on the developing brain when interpreting possible effects of agents administered during pregnancy.

Seminiferous epithelium cycle and its duration in capybaras (Hydrochoerus hydrochaeris)

Although capybara is the largest rodent in the world and largely distributed in Central and South America, there is no report in the literature concerning the cycle of seminiferous epithelium in this species. In the present study, the length of spermatogenic cycle was estimated using intratesticular injections of tritiated thymidine. Animals were sacrificed at 1 h, 8 days, and 17 days after injections. The duration of one spermatogenic cycle in capybaras is 11.9 ± 0.1 days (SEM). Spermatogenesis was estimated to last 53.6 days, when considering that the total duration of spermatogenesis takes about 4.5 cycles of seminiferous epithelium. The approximate life span of primary spermatocytes is 19.1 days, while spermiogenesis lasts 16.7 days. Staging in capybaras was based on the spermatid nuclei shape and location of spermatids, named tubular morphology method, which consists of 8 stages in all species. The relative stage frequencies in capybaras, based on the analysis of approximately 200 cross sections of seminiferous tubule for each of the ten animals were as follow: stage 1: 14.0 ± 1.5%; stage 2: 15.1 ± 1.0%; stage 3: 15.7 ± 1.1%; stage 4: 14.6 ± 1.1%; stage 5: 8.7 ± 0.7%; stage 6: 7.0 ± 0.7%; stage 7: 9.4 ± 0.9%; stage 8: 15.5 ± 1.0%. The pre-meiotic, meiotic and post-meiotic phases relative frequencies were 44.8%, 14.6% and 40.6%, respectively. Compared to most rodents investigated so far, the duration of spermatogenesis in capybaras is relatively long.

Progenitor cell cycling during hair cell regeneration in the vestibular and auditory epithelia of the chick.

We investigated nucleotide-labeling patterns during ongoing hair cell regeneration in the avian vestibular epithelium and during drug-induced regeneration in the avian auditory epithelium. For utricle experiments, post-hatch chicks received an injection of bromodeoxyuridine (BrdU) and were allowed to survive from 2 hours to 110 days after the injection. Utricles were fixed and immunoreacted to detect BrdU. The number of BrdU-labeled nuclei in the hair cell and support cell layers of the utricular sensory epithelium changes significantly between 2 hours and 110 days post-BrdU. At 2 hours, most labeled cells are isolated, while by 5-10 days, the majority of labeled cells are organized in pairs that are most frequently composed of a hair cell and a support cell. Pairs of labeled cells are seen as late as 110 days. Clusters of more than 3 labeled cells are uncommon at all time-points. The total number of labeled cells increases approximately 1.5-fold between 5 and 60 days post-BrdU. This increase is due primarily to a rise in the number of labeled support cells, and it is likely that it represents additional rounds of division by a subset of cells that were labeled at the time of the BrdU injection. There is a significant decrease in labeled nuclei in the hair cell layer between 60 and 110 days post-BrdU, suggesting that hair cells die during this period. To investigate support cell recycling in the drug-damaged auditory epithelium, we examined nucleotide double labeling after separate injections of BrdU and tritiated thymidine. A small number of support cells that incorporate BrdU administered at 3 days post-gentamicin treatment also label with tritiated thymidine administered between 17 and 38 hours later. We conclude that a small population of support cells recycles during regeneration in both the normal utricle and the drug-damaged basilar papilla.

Neurogenesis and Commitment of Corticospinal Neurons inreeler

In the homozygous (but not the heterozygous) reeler mutant, disruption of neuron migration leads to a major perturbation of the cortical environment that in turn could modify (1) the specification of neuronal fate and (2) the proliferation dynamics of cortical precursors. To investigate these issues, tritiated thymidine injections during cortical neurogenesis were coupled with postnatal injections of a retrograde tracer in the spinal cord to accurately measure the neurogenesis of corticospinal neurons in the heterozygous and homozygous mutant. The homozygous reeler shows (1) strict conservation of area-specific timetables of corticospinal neuron generation; (2) neurons with the appropriate birthdates show an enhanced probability of projecting to the spinal cord; (3) during early stages of corticogenesis, there is a reduced rate of neuron production followed at later stages by an increased rate of neuron production; and (4) these changes in the rate of neuron production were shown to be at least partially attributable to changes in the proportions of differentiative divisions. Taken together, our results show that in the developing cortex, the neurogenesis and specification of a given neuronal phenotype are partially controlled by the postmigratory compartment. On the other hand, neither areal identity nor the chronology of production of layer-specific neuronal phenotype seems to depend on the integrity of the cellular environment.

Influence of olfactory innervation on neurogenesis in the developing olfactory bulb of the frog, Xenopus laevis.

Previous research on development of the Xenopus olfactory bulb from our laboratory has shown that mitral cells begin to differentiate after olfactory axons make contact with the olfactory bulb, and the number of olfactory axons is correlated with the number of mitral cells throughout development. In embryos, removal of all afferent innervation before the mitral cells begin to differentiate results in a failure of the bulb to form; removal of half the olfactory axons, results in development of half the normal number of mitral cells. At larval stages, transection of the olfactory nerve results in a decrease in the number of neurons in the olfactory bulb. Thus, the olfactory axons play a major role in stimulating or maintaining development of the olfactory bulb neurons. Since we have found that neurogenesis in the bulb continues through metamorphosis, the goal of the current study was to determine whether olfactory axons influence cell genesis and/or neuronal maturation in the larval olfactory bulb. To study cell genesis, we cut the olfactory nerves, and 6 days later, injected the animals with [3H]thymidine. After 6 hr, the animals were killed and the tissue was processed for autoradiography. The number of labeled cells in the ventricular zone of the olfactory bulb was determined in every fifth section through the bulb in control and experimental animals. There was no statistical difference (Mann-Whitney rank sum test) in the number of labeled ventricular cells between controls and experimentals. Thus, olfactory axon innervation does not appear to play a role in precursor cell division during larval development. To study the influence of olfactory axon innervation on the ability of newly generated neurons to mature, we followed the same procedures. However, the animals were killed 21 days after the [3H]thymidine injection. The results from this experiment showed that there are many fewer labeled mitral cells in the experimental animals at 21 days. Together these results suggest that sensory deafferentation influences mitral cell differentiation or survival even during late stages of larval development.