Differentiation, Multiplication and Control of Bloodstream Form Trypanosoma (Duttonella) vivax in Mice

.The growth and differentiation of Trypanosoma vivax was studied in intact and irradiated C3H/He and C57B1/6 mice. In irradiated (800 R) or intact C3H/He and irradiated (800 R) C57B1/6 mice, T. vivax parasitaemia increased rapidly then entered a plateau phase and thereafter declined in an antibody-independent remission phase. Throughout the infection, variations were observed in parasite morphology, density, DNA content, number of organisms with 2 nuclei and 2 kinetoplasts and infectivity of parasites for mice. Parasites in exponential phase had the highest number of members in the S, G₂ and M phases of the cell cycle as determined by staining with the interchalating dye Chromomycin A, and analysis on a flow cytometer. During this phase there were numerous parasites with 2 nuclei and 2 kinetoplasts and infectivity was high for mice. As the parasitaemia approached and entered the plateau phase, the proportion of organisms in the S, G₂ and M phases of the cell cycle as well as the number of those with 2 kinetoplasts decreased slightly; the number of organisms with 2 nuclei decreased rapidly; and parasites had a considerably reduced capacity to infect mice. Organisms from the remission phase contained only 1 nucleus and 1 kinetoplast and were not infective for mice. The study suggests that T. vivax organisms transit from dividing to committed non-dividing forms and that some non-dividing, non-infective T. vivax organisms remain trapped in the S, G₂ and M stages of the cell cycle and die without completing binary fission. In contrast to the above, parasite wave remission occurred in T.vivax-infected intact C57B1/6 mice during exponential growth when there were large numbers of dividing form organisms present in the bloodstream as determined by both DNA content and the proportion of parasites with 2 kinetoplasts and 2 nuclei. Clearance of T. vivax from the bloodstream of infected intact C57B1/6 mice coincided with the production of a parasite-specific antibody response. The studies are discussed with reference to the mode of induction of host protective antibody responses to exponentially growing T. vivax.     

Redistribution of bone marrow cells according to mitotic cycle phases after the action of agents altering the intracellular concentration of cyclic nucleotides

The method of flow cytofluorometry was used to determine the distribution of murine bone marrow cells along the phases of the cell cycle in normal mice (CBA X C57Bl) F1 after the whole body equal X-ray irradiation, after irradiation in combination with burn, after administration of imidazole, insulin and caffeine, and after the treatment of cells by acetylcholine. In non-irradiated mice insulin and caffeine induced an increased ratio of bone marrow cells in G0 and G1 and a reduced one in S phase. Imidazole increased the number of G2- and M-cells by 1.5 times only in regenerating bone marrow of irradiated mice. After X-ray irradiation of mice at a dose of 2.3 Gy, reducing the percentage of cells in S and increasing it in G2 and M phases, insulin, acetylcholine and caffeine were found to decrease the number of cells in G2 and M phases: besides, caffeine and acetylcholine increased the percentage of S-phase cells. The data obtained are discussed in terms of possibility of normalization of bone marrow cell proliferation broken after exposure to physical stress-factors.     

The involvement of two cdc2-related kinases (CRKs) in Trypanosoma brucei cell cycle regulation and the distinctive stage-specific phenotypes caused by CRK3 depletion

Cyclin-dependent protein kinases are among the key regulators of eukaryotic cell cycle progression. Potential functions of the five cdc2-related kinases (CRK) in Trypanosoma brucei were analyzed using the RNA interference (RNA(i)) technique. In both the procyclic and bloodstream forms of T. brucei, CRK1 is apparently involved in controlling the G(1)/S transition, whereas CRK3 plays an important role in catalyzing cells across the G(2)/M junction. A knockdown of CRK1 caused accumulation of cells in the G(1) phase without apparent phenotypic change, whereas depletion of CRK3 enriched cells of both forms in the G(2)/M phase. However, two distinctive phenotypes were observed between the CRK3-deficient procyclic and bloodstream forms. The procyclic form has a majority of the cells containing a single enlarged nucleus plus one kinetoplast. There is also an enhanced population of anucleated cells, each containing a single kinetoplast known as the zoids (0N1K). The CRK3-depleted bloodstream form has an increased number of one nucleus-two kinetoplast cells (1N2K) and a small population containing aggregated multiple nuclei and multiple kinetoplasts. Apparently, these two forms have different mechanisms in cell cycle regulation. Although the procyclic form can be driven into cytokinesis and cell division by kinetoplast segregation without a completed mitosis, the bloodstream form cannot enter cytokinesis under the same condition. Instead, it keeps going through another G(1) phase and enters a new S phase resulting in an aggregate of multiple nuclei and multiple kinetoplasts in an undivided cell. The different leakiness in cell cycle regulation between two stage-specific forms of an organism provides an interesting and useful model for further understanding the evolution of cell cycle control among the eukaryotes.     

Depletion of anaphase-promoting complex or cyclosome (APC/C) subunit homolog APC1 or CDC27 of Trypanosoma brucei arrests the procyclic form in metaphase but the bloodstream form in anaphase

The anaphase-promoting complex or cyclosome (APC/C) is a multiprotein subunit E3 ubiquitin ligase complex that controls segregation of chromosomes and exit from mitosis in eukaryotes. It triggers elimination of key cell cycle regulators such as securin and mitotic cyclins during mitosis by polyubiquitinating them for proteasome degradation. Seven core subunit homologs of APC/C (APC1, APC2, APC11, CDC16, CDC23, CDC27, and DOC1) were identified in the Trypanosoma brucei genome data base. Expression of six of them was individually ablated by RNA interference in both the procyclic and bloodstream forms of T. brucei. Only the CDC27- and APC1-depleted cells were enriched in the G2/M phase with inhibited growth. Further studies indicated that T. brucei APC1 and CDC27 failed to complement the corresponding deletion mutants of budding yeast. However, their depletion from procyclic-form T. brucei enriched cells with two kinetoplasts and an enlarged nucleus possessing short metaphase-like mitotic spindles, suggesting that APC1 and CDC27 may play essential roles in promoting anaphase in the procyclic form. Their depletion from the bloodstream form, however, enriched cells with two kinetoplasts and two nuclei connected through a microtubule bundle, suggesting a late anaphase arrest. This is the first time functional APC/C subunit homologs were identified in T. brucei. The apparent differential activities of this putative APC/C in two distinct developmental stages suggest an unusual function. The apparent lack of functional involvement of some of the other individual structural subunit homologs of APC/C may indicate the structural uniqueness of T. brucei APC/C.     

Quantification of Trypanosoma cruzi in the heart, lymph nodes and liver of experimentally infected mice, using limiting dilution analysis.

Limiting dilution analysis was used to quantify Trypanosoma cruzi in the lymph nodes, liver and heart of Swiss and C57Bl/10 mice. The results showed that, in Swiss and Bl/10 mice infected with T. cruzi Y strain, the number of parasites/mg of tissue increased during the course of the infection in both types of mice, although a greater number of parasites were observed in heart tissue from Swiss mice than from Bl/10. With regard to liver tissue, it was observed that the parasite load in the initial phase of infection was higher than in heart. In experiments using T. cruzi Colombian strain, the parasite load in the heart of Swiss and Bl/10 mice increased relatively slowly, although high levels of parasitization were nonetheless observable by the end of the infection. As for the liver and lymph nodes, the concentration of parasites was lower over the entire course of infection than in heart. Both strains thus maintained their characteristic tissue tropisms. The limiting dilution assay (LDA) proved to be an appropriate method for more precise quantification of T. cruzi, comparing favorably with other direct microscopic methods that only give approximate scores.     

Pairwise knockdowns of cdc2-related kinases (CRKs) in Trypanosoma brucei identified the CRKs for G1/S and G2/M transitions and demonstrated distinctive cytokinetic regulations between two developmental stages of the organism

Expression of the cdc2-related kinase 3 (CRK3) together with expression of CRK1, -2, -4, or -6, were knocked down in pairs in the procyclic and bloodstream forms of Trypanosoma brucei, using the RNA interference technique. Double knockdowns of CRK3 and CRK2, CRK4, or CRK6 exerted significant growth inhibition and enriched the cells in G2/M phase, whereas a CRK3 plus CRK1 (CRK3 + CRK1) knockdown arrested cells in both G1/S and G2/M transitions. Thus, CRK1 and CRK3 are apparently the kinases regulating the G1/S and G2/M checkpoint passages, respectively, whereas the other CRKs are probably playing only minor roles in cell cycle regulation. A CRK1 + CRK2 knockdown in the procyclic form was found to cause aberrant posterior cytoskeletal morphogenesis (X. M. Tu and C. C. Wang, Mol. Biol. Cell 16:97-105, 2005). A CRK3 + CRK2 knockdown, however, did not lead to such a change, suggesting that CRK2 depletion can lead to the abnormal morphogenesis only when procyclic-form cells are arrested in the G1 phase. The G2/M-arrested procyclic form produces up to 20% stumpy anucleated cells (zoids) in the population, suggesting that cytokinesis and cell division are not blocked by mitotic arrest but are apparently driven to completion by the kinetoplast cycle. In the bloodstream form, however, G2/M arrest resulted in little zoid formation but, instead, enriched a population of cells each containing multiple kinetoplasts, basal bodies, and flagella and an aggregate of multiple nuclei, indicating failure in entering cytokinesis. The two different cytokinetic regulations between two distinct stage-specific forms of the same organism may provide an interesting and useful model for further understanding the evolution of cytokinetic control among eukaryotes.     

脾虚证小鼠胸腺细胞周期和免疫细胞化学的实验研究

本文用ＦＣＭ和免疫细胞化学技术检测了小鼠胸腺细胞的活动周期,Ｂｒｄｕ＋细胞和表达Ｔｈｙｌ．２抗原的Ｔ细胞。小鼠包括对照组,利血平诱发的脾虚组,自然恢复组和健脾汤治疗后复健组。ＦＣＭ和免疫细胞化学结果显示,脾虚组小鼠Ｇ１期细胞堆积,Ｓ期和Ｇ２＋Ｍ期细胞减少。同时胸腺内Ｂｒｄｕ＋细胞和Ｔ细胞也相应减少。经过健脾汤治疗后,除Ｇ１期细胞外,Ｓ期和Ｇ２＋Ｍ期细胞,Ｂｒｄｕ＋细胞和Ｔ细胞均显著增加,并且几乎达正常水平。但是自然恢复组在恢复细胞活动周期正常化,以及恢复Ｂｒｄｕ＋细胞和Ｔ细胞方面比复健组缓慢。说明健脾汤对脾虚小鼠的胸腺细胞有促进ＤＮＡ合成和细胞增殖功能。     

Apical migration of nuclei during G2 is a prerequisite for all nuclear motion in zebrafish neuroepithelia

Nuclei in the proliferative pseudostratified epithelia of vastly different organisms exhibit a characteristic dynamics - the so-called interkinetic nuclear migration (IKNM). Although these movements are thought to be intimately tied to the cell cycle, little is known about the relationship between IKNM and distinct phases of the cell cycle and the role that this association plays in ensuring balanced proliferation and subsequent differentiation. Here, we perform a quantitative analysis of modes of nuclear migration during the cell cycle using a marker that enables the first unequivocal differentiation of all four phases in proliferating neuroepithelial cells in vivo. In zebrafish neuroepithelia, nuclei spend the majority of the cell cycle in S phase, less time in G1, with G2 and M being noticeably shorter still in comparison. Correlating cell cycle phases with nuclear movements shows that IKNM comprises rapid apical nuclear migration during G2 phase and stochastic nuclear motion during G1 and S phases. The rapid apical migration coincides with the onset of G2, during which we find basal actomyosin accumulation. Inhibiting the transition from G2 to M phase induces a complete stalling of nuclei, indicating that IKNM and cell cycle continuation cannot be uncoupled and that progression from G2 to M is a prerequisite for rapid apical migration. Taken together, these results suggest that IKNM involves an actomyosin-driven contraction of cytoplasm basal to the nucleus during G2, and that the stochastic nuclear movements observed in other phases arise passively due to apical migration in neighboring cells.     

Influence of growth hormone and thyroxine on cell kinetics in the proximal tibial growth plate of Snell dwarf mice

In Snell dwarf mice, the influence of short-term treatment with human growth hormone (hGH) or thyroxine on the proliferative and sulphation activity of the proximal tibial growth plate was studied. By autoradiographic methods, the [3H]methylthymidine incorporation after a single injection was measured, after 2 hr incorporation time. The labelling index was calculated and the number of labelled mitoses was counted. In addition, the distribution of the labelled nuclei over the proliferating and degenerating zones was determined by continuous labelling for 25 and 73 hr. In untreated dwarf mice after [3H]-methylthymidine administration, the number of labelled nuclei in the growth plate is low. Labelling occurs, as expected, mainly in the cells of the proliferative zones. The number of labelled nuclei in control dwarf mice was similar after 25 and 73 hr continuous labelling. This suggests that many cells are in a resting G0 or prolonged G1 phase. Both hGH and T4 treatment induce a significant increase of the number of labelled nuclei per growth plate and of the number of mitoses. Since hormonal treatment induces a small number of mitoses after 2 hr incorporation of the label, the minimal G2 phase of the cell cycle is less than 2 hr. In addition, treatment with hGH and T4 stimulates chondrocytes in the zone of proliferative and hypertrophic cells to actively incorporate [35S]-sulphate.     

Growth phase and medium ph modulate the expression of proteinase activities and the development of megasomes in axenically cultivated Leishmania (Leishmania) amazonensis amastigote-like organisms

Leishmania (Leishmania) amazonensis LV79 (MPRO/BR/72/M1841) has been adapted to grow at 33 C as amastigote-like (AL) organisms in modified UM-54 medium initially adjusted to a pH of 4.8-5.0. Axenic cultures could be routinely restarted from parasites recovered from footpad lesions obtained by inoculation of BALB/c mice with preadapted culture stages. Morphological features, proteinase activities, and infectivity of AL organisms were examined during the in vitro growth cycle, and differences were found between log- and stationary-phase parasites. Stationary-phase AL organisms were morphologically similar to lesion amastigotes, did not react with a paraflagellar rod-specific monoclonal antibody in western blots, and contained proteinase activities resolving identically to the enzymes of lesion amastigotes in gelatin gels. Whereas typical megasomes could be identified in about a third of the stationary-phase AL population, the organelles were rarely seen in log-phase organisms. Azocaseinolytic activity progressively increased during the exponential growth phase and reached its highest values (approximately 65-70% of those determined in lesion amastigotes) at the stationary phase; the association of total proteinase activity with increased expression of cysteine proteinases was indicated by the strong inhibition of azocasein hydrolysis by E-64, the intensified banding of the 28-, 31-, and 35-kDa proteinases in gelatin gels, and the higher susceptibility of stationary-phase AL organisms to L-leucine methyl ester. Although overall axenic amastigotes were less infective to BALB/c mice than were lesion-derived parasites, stationary-phase AL organisms were more infective than were log-phase parasites. Medium pH increased during the exponential growth phase, but dropped in the stationary phase, when the observed morphological, biochemical, and biological changes became apparent.     

Nuclear pore formation and the cell cycle in Saccharomyces cerevisiae

The total number of nuclear pore complexes/nucleus was determined at intervals through the first cycle of synchronous growth in the yeast, Saccharomyces cerevisiae, using electron micrographs of freeze-fracture replicas of nuclei. Nuclear pore number increased in early G0 phase, attaining a plateau by late G0 which was maintained throughout the S phase. This was followed by a second increase at the time of nuclear division. The significance of these changes in relation to other events of the cell cycle is discussed.     

Effect of X-Irradiation at Different Stages in the Cell Cycle on Individual Cell–Based Kinetics in an Asynchronous Cell Population

Using an asynchronously growing cell population, we investigated how X-irradiation at different stages of the cell cycle influences individual cell–based kinetics. To visualize the cell-cycle phase, we employed the fluorescent ubiquitination-based cell cycle indicator (Fucci). After 5 Gy irradiation, HeLa cells no longer entered M phase in an order determined by their previous stage of the cell cycle, primarily because green phase (S and G2) was less prolonged in cells irradiated during the red phase (G1) than in those irradiated during the green phase. Furthermore, prolongation of the green phase in cells irradiated during the red phase gradually increased as the irradiation timing approached late G1 phase. The results revealed that endoreduplication rarely occurs in this cell line under the conditions we studied. We next established a method for classifying the green phase into early S, mid S, late S, and G2 phases at the time of irradiation, and then attempted to estimate the duration of G2 arrest based on certain assumptions. The value was the largest when cells were irradiated in mid or late S phase and the smallest when they were irradiated in G1 phase. In this study, by closely following individual cells irradiated at different cell-cycle phases, we revealed for the first time the unique cell-cycle kinetics in HeLa cells that follow irradiation.     

Tetraploid cycle in ageing solid tumours

When the mouse mammary adenocarcinoma 755 (Ca-755) reaches the plateau phase of growth, non-cycling cells with a G2-DNA content can be observed. They may belong to the diploid cell cycle but they could also be blocked in G0 or G1 of a tetraploid cycle. This hypothesis was tested in three ways: (1) non-cycling G2 nuclei were stained with a combination of Feulgen and naphthol yellow which revealed two populations, one with a low protein content and the other with a high protein content--the latter may represent nuclei ready to begin a new phase of DNA synthesis; (2) Feulgen staining and autoradiography were performed after tritiated thymidine had been administered to mice continuously: this showed that there were cells synthesizing DNA with a DNA index above 2; and (3) cells having 80 chromosomes, corresponding to the tetraploid cycle, were found almost exclusively in the plateau phase tumours. On the other hand, the use of texture and DNA parameters of the Feulgen stained nuclei showed that they were concentrated in a diploid cycle for tumours in the exponential phase of growth and were divided between a diploid and tetraploid cycle for 'plateau' cells. Neither the cause for, nor the role played by, polyploid cells is known.     

钙调素对DNA合成的影响

利用钙调素ｃａｌｍｏｄｕｌｉｎ,ＣａＭ）拮抗剂─三氟拉嗪（ｔｒｉｆｌｕｏｐｅｒａｚｉｎｅ,ＴＦＰ）对Ｇ０期小鼠Ｃ３Ｈ１０Ｔ１／２成纤维细胞进入Ｓ期和ＤＮＡ合成进行了研究．Ｇ０期细胞进入Ｓ期时,大量钙调素进入细胞核,其水平为Ｇ０期的２倍。ＴＦＰ处理的细胞被阻抑在Ｇ１期,不仅使Ｓ期细胞群体下降,而且３Ｈ－ＴｄＲ掺入ＤＮＡ强度受到明显抑制．同时,ＴＦＰ处理的细胞胸腺嘧啶核苷激酶（ｔｈｙｍｉｄｉｎｅｋｉｎａｓｅ,ＴＫ）基因表达及ＴＫ活性亦明显下降,但不影响Ｓ期细胞核内的钙调素水平,结果表明钙调素功能之抑制不仅阻抑细胞从Ｇ１期至Ｓ期的进程,而且对细胞ＤＮＡ合成强度亦有抑制作用．     

DNA synthesis in the second and third cell cycles of mouse preimplantation development. A cytophotometric study

Female Swiss mice were sacrificed at 2 h intervals between 16–30 and 40–56 h after insemination. One-, 2- and 4-cell embryos were stained by the Feulgen method and cytophotometric measurement of their nuclear DNA content was carried out. The cells with 2C and 4C DNA content were assumed to be in G1 and G2 phase and those with intermediate DNA content in S phase of the cell cycle. The fractions of cells which had passed a given phase of the cell cycle were calculated for various times after insemination and utilized for measurements of the second and third cell cycle timing. Results of measurements for the second cell cycle: G1 phase 1.3 h, S phase 6.1 h, G2 phase 15.4 h, whereas for the third cell cycle: G1 phase 1.6 h, S phase 7.4 h, G2 phase 0.5 h. The first cleavage division was calculated as 1.6 h, the second as 1.3 h and the third as 1.2 h. Complete intra-embryonic synchronization of the DNA-synthesizing nuclei was preserved during the entire synthesis phase of 2-cell embryos, while in 4-cell embryos they were slightly asynchronized. Among mitotic cells of the first cleavage division and G1 cells of 2-cell embryos a slight interembryonic asynchronization was found which deepened during subsequent cell cycle phases.     

Stage of the estrous cycle at the time of pregnant mare's serum gonadotropin injection affects pre-implantation embryo development in vitro in the mouse

The present study aims to analyze in the mouse the effect of the stage of the estrous cycle at the time of pregnant mare's serum gonadotropin (PMSG) injection on fertilization of ovulated cumulus-enclosed oocytes and later embryo development in vitro to the blastocyst stage. Quality of blastocysts was evaluated by staining and counting of total number of nuclei, mitotic index, percentage of apoptotic nuclei, and cell allocation to the inner cell mass (ICM) and trophectoderm (TE) lineage. Superovulation of hybrid (C57Bl/6JIco female x CBA/JIco male) female mice of 4-6 weeks of age was induced by a priming injection of PMSG at different stages of the estrous cycle followed after a 48-hr interval by human chrorionic gonadotropin. Our data indicate that injection of PMSG at the estrus phase gives the best outcome whereas injection of PMSG at the diestrus-1 or diestrus-2 phase provides the worst results. In fact, (1) total number of oocytes ovulated, number of ovulated oocytes enclosed by cumulus cells, and number of TE cells in day-5 blastocysts were significantly lower in diestrus-1 females than in estrus, diestrus-2 and proestrus mice; (2) percentage of day-5 blastocysts and total number of cells in day-5 blastocysts were lower in diestrus-1 and diestrus-2 females than in estrus and proestrus mice; and (3) percentage of apoptotic nuclei in day-5 blastocysts was lower in estrus mice than in diestrus-1, diestrus-2, or proestrus females. These data endorse previous studies suggesting that administration of gonadotropins in mice should be synchronized with the innate estrous cycle of females.     

Studies on the resistance/reactivation of Giardia muris cysts and Cryptosporidium parvum oocysts exposed to medium-pressure ultraviolet radiation

The ex vivo and in vivo reactivation of Giardia muris cysts and Cryptosporidium parvum oocysts after exposure to different doses of ultraviolet (UV) radiation was determined using animal infectivity. The infectivity of UV-treated parasites stored for 1-4 days (G. muris) or 1-17 days (C. parvum) at room temperature in the dark was similar to that of organisms administered immediately after UV treatment, indicating that the parasites did not reactivate ex vivo. In contrast, we observed in vivo reactivation of G. muris in three of seven independent animal infectivity experiments, when parasites were treated with relatively low doses of medium-pressure UV (<25 mJ/cm(2)). Our observations indicate that G. muris cysts and C. parvum oocysts exposed to medium-pressure UV doses of 60 mJ/cm(2) or higher did not exhibit resistance to and/or reactivation following treatment. This suggests that when appropriate doses of UV are used, significant and permanent inactivation of these parasites may be achieved.     

Apoptosis and cell cycle progression in an acidic environment after irradiation

Apoptosis and cell cycle progression in HL60 cells irradiated in an acidic environment were investigated. Apoptosis was determined by TUNEL staining, PARP cleavage, DNA fragmentation, and flow cytometry. The majority of the apoptosis that occurred in HL60 cells after 4 Gy irradiation took place after G(2)/M-phase arrest. When irradiated with 12 Gy, a fraction of the cells underwent apoptosis in G(1) and S phases while the rest of the cells underwent apoptosis in G(2)/M phase. The apoptosis caused by 4 and 12 Gy irradiation was transiently suppressed in medium at pH 7.1 or lower. An acidic environment was found to perturb progression of irradiated cells through the cell cycle, including progression through G(2)/ M phase. Thus it was concluded that the suppression of apoptosis in the cells after 4-12 Gy irradiation in acidic medium was due at least in part to a delay in cell cycle progression, particularly the prolongation of G(2)/M-phase arrest. Irradiation with 20 Gy indiscriminately caused apoptosis in all cell cycle phases, i.e. G(1), S and G(2)/M phases, rapidly in neutral pH medium and relatively slowly in acidic pH medium. The delay in apoptosis in acidic medium after 20 Gy irradiation appeared to result from mechanisms other than prolonged G(2)/ M-phase arrest.     

Cell cycle distributions, growth characteristics, and variation in prolactin and growth hormone production in cultured rat pituitary cells.

Clonal strains of rat pituitary tumour cells (GH3 cells) spontaneously produce and secrete prolactin and growth hormone. Chromosome analysis and DNA ploidy measurements revealed that the GH3 cells in the present study were triploid and had a decreased chromosome number compared to the parent strain. Monolayer cultures of these cells grow exponentially for 6-7 days with a mean doubling time of 54 h. Cell cycle distributions and phase durations were determined by micro-flow fluorometric measurements of cellular DNA content combined with computer calculations. During exponential growth the cell cycle distribution did not change (65.4% cells with a G1 phase DNA content, 24.9% with an S phase DNA content, and 9.7% with a (G2 + M) phase DNA content). Counting of mitoses gave 1.4% cells in M phase. The 3H-Tdr labeling indices were determined by autoradiography, and the results were in good agreement with the number of cells in S phase as calculated by micro-flow fluorometry. The phase durations were: Ts=15.9 h, TG2=6.2 h, TM=1.1 h, and TG1=30.9 h. TS and TM calculated from 3H-Tdr labeled and Colcemid treated cultures gave corresponding results. In plateau phase cultures the number of cells with a G1 DNA content increased to 80% and the number of cells with an S phase DNA content decreased to between 5% and 10%. The specific production of prolactin and growth hormone determined by radioimmunoassay showed two and four-fold increases respectively, during exponential growth. The hormone values decreased to initial or subinitial values (day 2 values) when approaching plateau phase. We conclude: that changes in the cell cycle distribution of the cell population cannot be responsible for the spontaneous alterations in hormone production during growth and that most of the hormone-producing cells must be in the G1 phase.