STUDY ON THE PROLIFERATIVE STATE OF HAEMOPOIETIC STEM CELLS (CFU)

Hydroxyurea was used to study the proliferation rate of haemopoietic stem cells (CFUJ in normal mice, after irradiation or transplantation into irradiated recipients. It was demonstrated that the proliferation rate of endogenous CFU_S (endo-CFU,) and exogenous CFU_S (exo-CFU_s) are identical. After irradiation (650 R) the surviving endo-CFU_s begin to proliferate immediately. By contrast exo-CFU, transplanted into the irradiated recipient mouse (850 R), begin to proliferate only after about 30 hr. However, injection of isoproterenol (which stimulates adenyl cyclase) or dibutyryl cyclic adenosine 3′,5′-monophosphate shortly after marrow cell graft, triggers the transplanted CFU_S into cell cycle as shown by an almost immediately increased sensitivity to hydroxyurea. Isoproterenol is capable of inducing DNA synthesis also in stem cells of normal mice but it takes about 20 hr before CFU, become to be increasingly sensitive to hydroxyurea.     

Haemopoietic modulation in tumour-bearing animals: enhanced progenitor-cell production in femoral marrow

Altered haematopoiesis in the femoral marrow was observed in mice bearing the Lewis lung carcinoma (LLca). During tumour growth, a marked reduction was observed in the myeloperoxidase-positive cells (granulocytes) of the marrow 7 days after inoculation of the LLca tumour reaching a nadir (17% of control) by day 28. Accompanying this suppression of mature white cells was a gradual expansion of the CFU_c-GM compartment followed by an increase in the number of femoral CFU_s. Humoral-stimulating activity (HSA) increased through day 14 in the serum of these animals; then returned to control levels by day 28. During this same interval, the more primitive erythroid progenitor (BFU_e) compartment expanded to 168% of control, while the more differentiated (CFU_e) compartment was reduced (45% of control at day 28). Reductions in both ⁵⁹Fe-incorporation and erythroblasts/femur confirmed the suppression of erythroid differentiation in marrow during tumour growth. Similar results were observed following the daily injection (188 mg equivalent dose; q 24 hr × 10) of the supernatant prepared from LLca tissue. Marked differences were observed between the response of the spleen and the marrow to the supernatant. the data suggest that the growth of the LLca tumour results in a dissociation of the normal continuity of haematopoietic steady-state differentiation in the marrow of tumour-bearing animals.     

THE EFFECT OF E TYPE PROSTAGLANDINS ON THE PROLIFERATION OF HAEMOPOIETIC STEM CELLS IN VIVO

The ability of protaglandins E₁ and E₂ to stimulate the proliferation of haemopoietic stem cells (CFU_s) was studied in vivo. PGE₂, in a dose range of 10^-4 to 10^-1μg/g body weight and PGE₁ in a dose range of 10^-5 to 10^-1μg/g body weight, produced a rapid cycling wave of CFU_s. The increase in the number of CFU_s in S phase was not followed by a rise in the femoral CFU_s content, and except for a transient increase in femoral CFU_c level, no increase in differentiation was found either. Therefore, it is proposed that haemopoiesis after PG-induced CFU stimulation is ineffective. PGE₂ did not stimulate regeneration of CFU_s in a perturbed state (after sublethal irradiation). All these findings support the idea that PGEs might represent potent stimulators of the haemopoietic stem cells acting in physiological doses. However, if acting concurrently with physiological control systems PGs lead to ineffective haemopoiesis (under normal conditions) or do not exert any measurable effect (after sublethal irradiation).     

RECOVERY OF PROLIFERATING HAEMOPOIETIC PROGENITOR CELLS AFTER KILLING BY HYDROXYUREA

Two doses of 1 mg/g of hydroxyurea (HU), injected 7 hr apart into irradiated mice in which CFU-S were proliferating during marrow regeneration, killed about 90% of CFU-S. This same dose regime injected into normal female mice, with non-proliferating CFU-S killed 92 % of CFU-C, 99 % of ESC and only 30 % of CFU-S. One day after the treatment CFU-S had decreased to 50 % and remained at about this level for a further day then returned to normal values. In spleen the increase in CFU-S was delayed by a day and showed a marked overshoot. During the period that CFU-S were decreased in number they were actively proliferating. Marrow CFU-C recovered in an exponential manner with a doubling time of 16 hr. Spleen CFU-C recovered 1 day later than marrow and showed a pronounced overshoot. ESC recovered very rapidly with doubling time of 5 hr. The changes in ⁵⁹Fe incorporation into RBC, and the peripheral blood picture, were a delayed reflection of the changes in ESC and CFU-C.     

Effects of Hydroxyurea On the Mitotic Activity and the G2 Phase In Hairless Mouse Epidermis

Hairless mice were given 5 mg hydroxyurea (HU) intraperitoneally (i.p.) followed by 0.15 mg Colcemid® at various times after HU. the animals were killed at 2 and 4 hr after Colcemid, the epidermal mitotic counts in dorsal skin were determined and the mitotic rates calculated. These were compared with the normal mitotic rates, and the ratios between the results from HU-treated and -untreated animals were calculated. Hydroxyurea caused a considerable reduction in the mitotic rate with a trough at 6 hr, followed by a wave of increased mitotic rate with a peak at 14 hr, followed by a secondary drop at 20 hr, and then a return to normal. Another group of mice were given HU only, and the fraction of epidermal cells in G₂ was measured by flow cytometry. From these animals, without previous injection of Colcemid, we also determined the mitotic counts and calculated the mitotic durations. Cells piled up in G₂ for the first 6 hr after HU injection, then the G₂ compartment was emptied. the results are discussed in relation to previous results from this department showing the effect of the same dose of HU on DNA synthesis in the same mouse strain. It is concluded that HU not only blocks or retards DNA synthesis in epidermal cells, but also affects the movement of cells through G₂ and M. the cell kinetic effects of HU thus seem to be very complex.     

Bone Marrow Response to Damage Induced By Hydroxyurea Or Colchicine

The extent of bone marrow damage caused by the administration of single or repeated doses of either hydroxyurea (1000 mg/kg b.w.) or colchicine (1 mg/kg b.w.) are comparable. This conclusion is based on serial studies of bone marrow cellularity and of the CFUc numbers in the bone marrow. the proliferation response of the pluripotential haemopoietic stem cells, determined by the cells forming colonies in the spleen of lethally irradiated mice (CFUs) markedly differs if the bone marrow damage is caused by hydroxyurea or colchicine. While hydroxyurea administration stimulates a large proportion of the resting G₀ cells into the cell cycle, the damage induced by colchicine is followed by only a mild increase in the CFUs proliferation rate. The seeding efficiency of the spleen colony technique has been determined after both hydroxyurea and colchicine administration. This parameter, important for the estimation of the number of the pluripotential haemopoietic stem cells in blood forming organs, is significantly affected by hydroxyurea administration, but also by repeated injections of colchicine. Following a single dose of hydroxyurea, the time-course of the CFUs numbers, which were corrected for the change in the seeding efficiency, shows an overshoot occurring after 18–20 hr. At the other time periods, the number of pluripotential haemopoietic stem cells is little affected by a single hydroxyurea injection. This poses a question about the nature of the stimulus, which after hydroxyurea administration triggers the CFUs from the resting G₀ state into the cell cycle. There is evidence that this stimulus is probably not represented by the damage caused to the various intensively proliferating cell populations of the bone marrow. This evidence is based on experiments which show that colchicine induced damage, of a degree similar to that after hydroxyurea, does not stimulate the CFUs proliferation rate to an extent comparable to hydroxyurea. The possibility that colchicine could block CFUs in the G₀ state or that it could interfere with the progress of CFUs through the G₁ and S phases of the cell cycle have been ruled out by experiments which demonstrated that colchicine (1 mg/kg b.w.), administered 10 min before hydroxyurea, does not reduce the number of CFUs triggered into the cell cycle as the consequence of hydroxyurea administration.     

HAEMOPOIETIC STEM CELL (CFU) KINETICS IN THE CULTURE

Haemopoiesis continued for over 2 months in organ culture of embryonal mouse liver, and haemopoietic stem cells (CFU_s) capable of DNA-synthesis were found in it all that time. Between the 10th and 40th day the number of stem cells in the culture was sustained in a steady state. Both in normal and in regenerating adult bone marrow haemopoiesis ceased within a short time in the culture. Induction of proliferation in haemopoietic stem cells combined with undamaged or improved micro-environment resulted in a little better maintenance of CFU_s in the adult bone marrow culture, The results are discussed in the light of current concepts of haemopoietic stem cell regulation.     

Spermatogonial Multiplication In the Chinese Hamster. Iv. Search For Long Cycling Stem Cells

ABSTRACT In the Chinese hamster, 17 days, i. e. one cycle of the seminiferous epithelium, after two injections of [³H]TdR given 24 hr apart, labelled cells were found among all types of spermatogonia, including stem cells (A_s). These labelled A_s spermato-gonia derive from one or more self-renewing divisions of the stem cells that originally incorporated [³H]TdR. In the steady state, half of the divisions of the A_s will be self-renewing and the other half will give rise to A_pr spermatogonia that will ultimately become spermatozoa. Theoretically, the labelling index (LI) after 17 days will be similar to that after 1 hr, and in this study twice as high as for the 1-hr interval since only one injection was given. However, experimental values only half that of the theoretical LI were found after 17 days. the following causes for the loss of labelled stem cells are discussed: (1) dilution of label because of division; (2) influx of unlabelled components of false pairs (i. e. newborn stem cells that still have to migrate away. mostly during G₁, from their sister cells and are scored as A_pr spermatogonia) between 1 hr and 17 days; (3) the existence of long- and short-cycling stem cells, probably combined with preferential differentiation of the short-cycling elements; (4) selective segregation of DNA at stem cell mitosis; and (5) irradiation death of radiosensitive labelled stem cells. As it is not impossible that factors 1, 2, 4 and 5 together account for the total loss of labelled stem cells, LI results do not provide evidence for the existence of separate classes of short- and long-cycling stem cells. The distributions of the LIs of the A_s, A_pr and A_al spermatogonia over the stages of the epithelial cycle at 17 days are similar to those at 1 hr after injection. Hence the regulatory mechanisms that govern the stimulation and inhibition of proliferation of A_s that give rise to new A_s for the next epithelial cycle are similar to those of the A_s that will divide into A_pr spermatogonia during the same epithelial cycle. Grain counts revealed that more [³H]TdR is incorporated into A_s, A_pr and A_al spermatogonia that are in S phase during epithelial stages X-IV than in stages V-IX.     

Influence of the donors' clinical status on in vitro and in vivo tumor-cytotoxic activation of interleukin-2-exposed lymphocytes and their circulation in different organs

Summary In-vitro-generated lymphokine-activated killer (LAK) cells of BALB/c mice, bearing the syngeneic colon carcinoma C-26 for 7 days, were as efficient as those from normal mice in lysing C-26 cells whereas LAK cells from 14-day tumor-bearing and 5- and 14-day tumor-resected animals had a lower C-26 cytotoxicity. The level of C-26 lysis returned to normal values 30 days after surgery. To identify the best source of LAK cells in vivo, groups of normal mice were treated with 10⁴, 3×10⁴ or 10⁵ U/day of interleukin 2 (IL-2) for 7 days intraperitoneally (i. p.) or intravenously (i. v.) (3×10⁴ dose only). The highest lysis on C-26 was obtained from peritoneal exudate cells of mice given 3×10⁴ and 10⁵ U whereas spleen cells were lytic only when taken from mice treated with 10⁵ U IL-2. Peripheral blood lymphocytes lacked any cytotoxicity except for the group of mice which received IL-2 i. v. The kinetics of in vivo LAK activation in different organs showed a peak of anti-(C-26) lytic activity at day 5 in peritoneal exudate cells and spleen cells of mice given IL-2 for 5 days whereas administration of LAK cells alone had no effect; IL-2 plus LAK cells gave a lower peak of LAK activity as compared with IL-2 alone. A lower level of in vivo LAK activation was found in mice whose tumor was resected 5 days before; such impairment was evident even 14 days after surgery. Homing experiments were carried out with i. v. injected ⁵¹Cr-labelled LAK cells in normal or tumor-resected mice. In normal mice the highest radioactivity at 30 min was found in the lungs; liver and spleen also showed high radioactivity whereas blood had a negligible amount of radioactivity. Radioactivity declined rapidly in lungs (less than 10% after 24 h) while remaining at appreciable levels in the liver after 24 h and 48 h; spleen showed constant levels of 12%–15%. Homing of LAK cells was altered in mice receiving IL-2 i. p. for 5 days with slower and lower radioactivity peaks in the lung and higher levels in liver. In tumor-excised mice lower levels of radioactivity were found in lungs. These results show that: (a) alterations in LAK activity occur in early-tumor-resected and large-tumor-bearing animals; (b) the route of IL-2 administration is critical in LAK activation in vivo; (c) treatment with IL-2 modifies LAK homing.This study was in part supported by grant no. 87.01565.44 of the Finalized Project Oncology of CNR (Rome, Italy)     

Production of Interferon in Mice: Effect of Altered Gaseous Environments

Effects of altered gaseous environments (parabarosis) on interferon production in mice were studied, with Newcastle disease virus (NDV) as the inducer. Increased levels of interferon in lung tissue were observed when mice were exposed to 11% O₂ in N₂ for 3 days before and after, or only after, injection of NDV. However, serum interferon levels remained unchanged. Exposure of mice to 77% O₂ for up to 7 days did not affect the response to interferon induction as assayed in lungs or sera. Interferon levels were significantly depressed in mice exposed to a simulated depth of 213 ft in seawater [with normal partial pressure of O₂ (pO₂) in N₂] for 2 or 4 weeks. Whereas definite depression of interferon was also observed in mice maintained at a simulated altitude of 37,000 ft (with normal pO₂) for 2 weeks, those maintained at the same condition for 4 weeks showed a normal level of interferon. The results obtained with hypoxia are compatible with other reports on the influence of O₂ tension on viral infection. The factors responsible for alterations observed in interferon level in mice kept in normal pO₂, but under altered pressure, have not yet been identified.     

Survival of human bone marrow progenitor cells after freezing: Improved detection in the colony-formation assay

If cryopreserved suspensions of human bone marrow were stimulated by human placental conditioned medium in the same way as fresh unseparated marrows, less than 40% of granulopoietic progenitor cells (CFU_c) was identified. By adding α-thioglycerol (0.6 mM) to the culture medium, the concentration of detectable CFU_c in cryopreserved bone marrow was increased by a factor of 3.4, and the recovery of CFU_c after cryopreservation rose to 90%. The low recovery of CFU_c after freezing in the absence of α-thioglycerol is due to the destruction of accompanying cells. Noncolony-forming cells normally present in the fresh human marrow promote colony formation in cultures stimulated by placental conditioned medium. Their effect can be replaced by α-thioglycerol. It is concluded that, in order to detect all CFU_c independent of the cellular composition of the marrow suspension, this supplement is essential for CFU_c cultures stimulated by conditioned medium.     

Nippostrongylus brasiliensis: Peripheral blood leucocyte response of rats,with special reference to basophils

Changes in peripheral blood leucocytes were followed in male August rats given one or two infections with the parasitic nematode, Nippostrongylus brasiliensis. During the initial infection, there was a biphasic increase in total numbers of leucocytes, lymphocytes, neutrophils, large mononuclear cells, and eosinophils. All except eosinophils fell rapidly to normal levels as the parasites were expelled, but eosinophils were elevated much longer. All these cell types increased in number to a single peak 5 days after reinfection. Basophils were detected at very low levels in uninfected rats (0.06% or $\text{1}\text{1600}$ leucocytes) and increased in number to a peak 13 days after initial infection, at which time they represented about 4.5% of total leucocytes, an 80-fold increase compared with the number in normal rats. In reinfected rats, the basophilia occurred more rapidly than in a primary infection, suggesting that the appearance of these cells in the circulation is probably an immunologically mediated event.     

Induced abortion in cattle

Several procedures were used to abort cattle during the second and third trimesters of gestation. The treatment to abortion interval was better (P<0.05) when dexamethasone trimethyiacetate (DTMA) injections repeated at either 6 or 4 day intervals than when a single injection of DTMA was followed 6 days later by the administration of stilboestrol. The treatment to abortion interval was not significantly shorter when DTMA was repeated after 4 days rather than 5 days (0.10<P>0.05). Prostaglandin F_2α produced abortion 1 to 4 days following direct administration into the foetai fluids.Peripheral plasma progesterone concentration had a tendency to rise immediately following the second injection of DTMA given at a 6 day interval. This was followed by a decline. Two injections of DTMA given at 4 day intervals resuited in a decline in progesterone concentration. Abortion occurred when plasma progesterone concentrations were about 1 ng/ml in cows treated with DTMA. In cows treated with prostagiandin F_2α the plasma progesterone concentration fell rapidly within one day of administration to approximately 2 ng/ml, at which concentration abortion took place.     

Effects of hydroxyurea on the mitotic activity and the G2 phase in hairless mouse epidermis

Hairless mice were given 5 mg hydroxyurea (HU) intraperitoneally (i.p.) followed by 0.15 mg Colcemid at various times after HU. The animals were killed at 2 and 4 hr after Colcemid, the epidermal mitotic counts in dorsal skin were determined and the mitotic rates calculated. These were compared with the normal mitotic rates, and the ratios between the results from HU-treated and -untreated animals were calculated. Hydroxyurea caused a considerable reduction in the mitotic rate with a trough at 6 hr, followed by a wave of increased mitotic rate with a peak at 14 hr, followed by a secondary drop at 20 hr, and then a return to normal. Another group of mice were given HU only, and the fraction of epidermal cells in G2 was measured by flow cytometry. From these animals, without previous injection of Colcemid, we also determined the mitotic counts and calculated the mitotic durations. Cells piled up in G2 for the first 6 hr after HU injection, then the G2 compartment was emptied. The results are discussed in relation to previous results from this department showing the effect of the same dose of HU on DNA synthesis in the same mouse strain. It is concluded that HU not only blocks or retards DNA synthesis in epidermal cells, but also affects the movement of cells through G2 and M. The cell kinetic effects of HU thus seem to be very complex.     

Difference in the development of tolerance to morphine and d-ala2-methionine-enkephalin in C57 BL/6J and DBA/2J mice

The analgesic effect elicited by intracerebroventricular (icv) administration of either morphine or d-ala²-methionine-enkephalin (d-ala²-met-enk) was studied during the onset and offset of morphine tolerance in DBA/2_J (DBA) and C₅₇ BL/6_J (C₅₇) strains of mice. DBA mice become tolerant to the analgesic effect of morphine icv injected after receiving 8 subcutaneous (sc) injections (2 injections daily × 4 days) of the ED₅₀ of morphine for analgesia. In c₅₇ mice tolerance to morphine icv-administered is evident after only a single sc injection of morphine ED₅₀. On the contrary the development of cross-tolerance to the analgesic effect of d-ala²-met-enk is similar in both strains of mice. With respect to the offset period, the recovery of the analgesic effect of morphine and d-ala²-met-enk is slower in C₅₇ than in DBA mice; in C₅₇ mice tolerance to both morphine and d-ala²-met-enk is still present 10 days after morphine withdrawal. These results suggest the existence of a strain dependent rate in the onset of tolerance to the analgesic effect of morphine. C₅₇ mice represent an interesting tool to investigate tolerance to opiates and opioid peptides.     

STUDY OF THE EFFECT OF HYDROXYUREA ON THE ERYTHROPOIETIN-SENSITIVE CELLS

The response of polycythaemic mice to a standard dose of erythropoietin has been measured at various, time intervals after single or repeated injections of hydroxyurea. The results exclude S phase of the cell cycle as the period responsive to erythropoietin. They suggest the existence of feedback mechanisms within the cell cycle, operating at the G₁-S boundary and within the G₁ phase. Hydroxyurea given to polycythaemic mice at various time intervals after erythropoietin induced characteristic changes in the response. These changes can be explained if both gradual transit of differentiated cells into the DNA synthesis (S phase) and changes in amount of the erythropoietin sensitive cells caused by the feedback mechanisms operating in the cell cycle are considered.     

Pyruvate Kinase Activity of Wheat Plants Grown under Potassium Deficient Conditions

The activity of pyruvate kinase was determined in the first leaves of wheat plants grown under K⁺-deficient conditions. An enhancement of the enzyme activity compared with the normal plants was found to start from eighth day of growth, and about 4-fold increase in the enzyme activity was observed in 14-day wheat leaves. The addition of K₂SO₄ to the nutrient solution given to the K⁺-deficient plants at tenth day resulted in the restoration of the enzyme activity to the normal level after 3 days. The levels of K⁺ as well as carbohydrates and chlorophyll were found to return normal over the same period. These findings are discussed in relation to the metabolic pattern of plants at the early stages of K⁺-deficiency.     

Schistosoma japonicum: The design and experimental evaluation of a multivalent DNA vaccine

The aim of this study was to construct and evaluate the immunity efficacy of the DNA multivalent vaccine pVIVO₂SjFABP-23. The vaccine was constructed and produced as follows. Forty BALB/c mice were divided into four groups designated pVIVO₂, pVIVO₂Sj23, pVIVO₂SjFABP and pVIVO₂SjFABP-23. Each mouse was immunized with 100 μg of the corresponding plasmid DNA by intramuscular injection. 28 days post-vaccination, the mice were challenged with S. japonicum cercariae, and the worm and egg burdens were determined 42 days post-challenge. Serum samples were collected from all the mice before and after vaccination and at the end of the experiment, and used for antibody detection. The IFN-γ and IL-4 levels were quantified in the supernatants of specifically stimulated spleen cells. The number of worms was reduced by 52%, 40% and 42% in mice respectively immunized with pVIVO₂SjFABP-23, pVIVO₂Sj23 or pVIVO₂SjFABP. A respective 61%, 38% and 39% egg reduction was determined relative to those mice that only received the empty pVIVO2 plasmid. pVIVO₂SjFABP-23 immunization increased IgG levels against SWAP and SEA. Increased IFN-γ levels were detected in the supernatant of specific stimulated spleen cells from mice immunized with the 3 different constructs. The multivalent DNA vaccine developed induced higher levels of protection than the two monovalent tested vaccines.     

DMPS and N-acetylcysteine induced renal toxicity in mice exposed to mercury

Acute effects of mercuric chloride (HgCl₂) were evaluated on mice. Mice received a single dose of HgCl₂ (4.6 mg/kg, subcutaneously) for three consecutive days. Thirty minutes after the last injection with HgCl₂, mice received one single injection of 2,3-dimercapto-1-propanesulfonic acid (DMPS) or N-acetylcysteine (NAC) or diphenyl diselenide (PhSe)₂. DMPS, NAC and (PhSe)₂ were utilized as therapy against mercury exposure. At 24 h after the last HgCl₂ injection, blood, liver and kidney samples were collected. δ-Aminolevulinate dehydratase (δ-ALA-D) and Na⁺, K_-⁺ ATPase activities, thiobarbituric acid-reactive substances (TBARS), non-protein thiols (NPSH) and ascorbic acid concentrations were evaluated. Plasma aspartate (AST) and alanine (ALT) aminotransferase activities, as well as urea and creatinine levels were determined. The group of mice exposed to Hg + (PhSe)₂ presented 100% of lethality. Exposure with HgCl₂ caused a decrease on the body weight gain and treatments did not modify this parameter. δ-ALA-D, AST and ALT activities, TBARS, ascorbic acid levels and NPSH (hepatic and erythrocytic) levels were not changed after HgCl₂ exposure. HgCl₂ caused an increase in renal NPSH content and therapies did not modify these levels. Mice treated with (PhSe)₂, Hg + NAC and Hg + DMPS presented a reduction in plasma NPSH levels. Creatinine and urea levels were increased in mice exposed to Hg + NAC, while Hg + DMPS group presented an increase only in urea level. Na⁺, K_-⁺ ATPase activity was inhibited in mice exposed to Hg + DMPS and Hg + NAC. In conclusion, therapies with (PhSe)₂, DMPS and NAC following mercury exposure must be better studied because the formation of more toxic complexes with mercury, which can mainly damage renal tissue.     

Additional effects of postpuberal estrogen injections on the vaginal epithelium in neonatally estrogenized mice

In the ovariectomized mice given 10 injections of 100 micrograms 17 beta-estradiol at intervals of 2 weeks from 60 days of age, the vaginal epithelium was atrophic when killed more than 2 months after the last injection. If mice given 3 daily injections of 20 micrograms 17 beta-estradiol from the day of birth were similarly treated with estradiol after postpuberal ovariectomy, the vaginal epithelium was stratified and hyperplastic at autopsy performed more than 2 months later. These changes in the epithelium persisted for at least 30 days after transplantation of the vaginae to normal ovariectomized hosts. Neonatal treatments only did not produce such persistent vaginal changes. In view of these results, additional effects of neonatal and postpuberal injections of estrogen on the vaginal epithelium are evident. However, effects of such neonatal and postpuberal injections of estrogen might be transient on the uterine epithelium, since abnormal proliferation was not observed in it.