The role of erythropoietin in the production of principal erythrocyte proteins other than hemoglobin during terminal erythroid differentiation

Erythropoietin (EP) controls the terminal phase of differentiation in which proerythroblasts and their precursors, the colony forming units-erythroid (CFU-e), develop into erythrocytes. Biochemical studies of this hormone-directed terminal differentiation have been hindered by the lack of a homogeneous population of erythroid cells at the developmental stages of CFU-e and proerythroblasts that will synchronously differentiate in response to EP. Such a population of cells can be prepared from the spleens of mice with the acute erythroblastosis resulting from infection with anemia-inducing Friend virus (FVA). Using these FVA-infected erythroid cells, which were induced to differentiate with EP, four proteins other than hemoglobin that have key functions in mature erythrocytes were monitored during the 48-hour period of terminal differentiation. Synthesis of spectrin and membrane band 3 proteins were determined by immunoprecipitation and SDS-polyacrylamide gel electrophoresis; accumulation of the cytoskeletal protein band 4.1 was monitored by immunoblotting; carbonic anhydrase activity was measured electrometrically. Band 3 synthesis and band 4.1 accumulation could be detected only after exposure of the cells to EP. Spectrin synthesis was ongoing prior to culture with EP, but it did increase after exposure to the hormone. Carbonic anhydrase-specific activity changed very little throughout the terminal differentiation process. These results reveal at least three patterns of production of principal erythrocyte proteins during EP-mediated terminal differentiation of FVA-infected erythroid cells. Depending on the specific protein examined, de novo synthesis can be induced by EP, an ongoing production can be enhanced by EP, or the production of a protein can be completed at a developmental stage prior to EP-mediated differentiation in these cells.     

Apoptosis in erythroid progenitors deprived of erythropoietin occurs during the G1 and S phases of the cell cycle without growth arrest or stabilization of wild-type p53.

Erythropoietin (Epo) inhibits apoptosis in murine proerythroblasts infected with the anemia-inducing strain of Friend virus (FVA cells). We have shown that the apoptotic process in FVA cell populations deprived of Epo is asynchronous as a result of a heterogeneity in Epo dependence among individual cells. Here we investigated whether apoptosis in FVA cells correlated with cell cycle phase or stabilization of p53 tumor suppressor protein. DNA analysis in nonapoptotic FVA cell subpopulations cultured without Epo demonstrated little change in the percentages of cells in G1,S, and G2/M phases over time. Analysis of the apoptotic subpopulation revealed high percentages of cells in G1 and S, with few cells in G2/M at any time. When cells were sorted from G1 and S phases prior to culture without Epo, apoptotic cells appeared at the same rate in both populations, indicating that no prior commitment step had occurred in either G1 or S phase. Steady-state wild-type p53 protein levels were very low in FVA cells compared with control cell lines and did not accumulate in Epo-deprived cultures; however, p53 protein did accumulate when FVA cells were treated with the DNA-damaging agent actinomycin D. These data indicate that erythroblast apoptosis caused by Epo deprivation (i) occurs throughout G1 and S phases and does not require cell cycle arrest, (ii) does not have a commitment event related to cell cycle phase, and (iii) is not associated with conformational changes or stabilization of wild-type p53 protein.     

Nonsurgical collection of blastocysts from dairy goats

In two trials, eight attempts were made to collect fertilized ova from dairy goats by nonsurgical methods. In both trials the cervix of each doe was dilated by inserting a Laminaria japonica tent device into the cervical canal prior to flushing. In Trial 1, an attempt was made to collect embryos from four nonsuperovulated does by flushing phosphate-buffered saline (PBS) through a rigid pipette. Little fluid and no embryos were recovered from the does. All four donors were in estrus two days after the procedure. In the second trial, FSH-superovulated does were collected on day 5 following estrus. The donors were anesthetized, and a modified Foley catheter was passed through the cervical canal. In three does, a 24 ga. two-way Foley was stiffened with a size 10 (French) polypropylene catheter which penetrated the Foley, extending 7 cm beyond the tip. Recovery of flushing medium with this device was minimal, and laparotomy of one doe revealed a punctured uterus. Replacement of this device with a different catheter, through which a polypropylene catheter (size 5 Fr.) penetrated only 1 to 2 cm, resulted in satisfactory return of infused PBS, and recovery of two blastocysts and one degenerated ovum from this doe. Use of the same device on a second doe without laparotomy resulted in collection of seven blastocysts and three degenerated ova. Of three observed donors that received Laminaria tents (including one which was not flushed) two were in estrus three days after the procedure, while unused synchronized recipients showed normal cycle lengths. Surgical transfer of two blastocysts from each donor to each of two synchronized recipients resulted in the birth of twin kids from one recipient doe. The study demonstrates the feasibility of embryo collection from dairy goats by nonsurgical means.     

Preliminary studies on bovine embryo survival following short-term storage at 4 degrees C

A total of 113 non-surgically collected bovine embryos, 5-8 days of age, were stored for 48 hours at 4 degrees C in a modified phosphate-buffered saline solution (PBS). Following storage, embryos were cultured for 8-12 hours at 37 degrees C, and those which were morphologically normal were transferred to synchronized recipients by several methods designed to achieve twin pregnancies. Embryos which were collected and transferred on the same day served as controls. Of 113 embryos stored, 47 (42%) appeared to be transferable after the brief culture period. There was a marked breed effect on viability after refrigeration, with Hereford embryos surviving significantly better than Angus embryos (71% vs. 12%, respectively, p < .001). Post-transfer embryo survival of stored and control embryos, based on actual calvings, was 34 and 48 percent, respectively, a difference which was not significant (p=0.3). A marked difference in pregnancy rate following non-surgical transfer by 2 different technicians was noted (50% vs. 21.7%, respectively).     

Multiple ovulation resulting from low level administration of PMSG to cattle at different stages of the oestrous cycle

Two experiments were carried out to determine whether differences in sensitivity to exogenous gonadotrophin which exist during the oestrous cycle can be used effectively in the induction of multiple pregnancy in cattle. In Experiment I, Hereford heifers and cows were injected with 800 IU pregnant mare serum gonadotrophin (PMSG) on approximately day 10 of the oestrous cycle, followed 48 h later by 30 mg prostaglandin F_2α (PGF_2α). Controls were treated with PGF_2α alone. Mean ovulation rates based on rectal palpation were 1.33 ± 0.10 (range: 1–2) and 3.05 ± 0.68 (range: 1–13) for 21 control and 21 treated animals, respectively (P < 0.02). In Experiment II, Hereford cows were treated with 800 IU PMSG on either day 5 (14 cows) or day 10 (12 cows) of the oestrous cycle, followed 48 h later by PGF_2α. Mean numbers of ovulations for animals that became pregnant were 1.50 ± 0.26 (range 1–3) and 3.80 ± 0.74 (range 1–7), respectively (P < 0.02). A high incidence of embryonic wastage occurred by 120 days of gestation in animals treated on day 10. The results of this study indicate that taking advantage of an animal's reduced responsiveness to exogenous gonadotrophin during the early portion of the oestrous cycle may be useful in developing methods for inducing multiple births with exogenous gonadotrophins.     

Metal accumulation in two contiguous eutrophic peri-urban lakes,Chivero and Manyame,Zimbabwe

Concentrations of aluminium, cadmium, chromium, cobalt, copper, iron, lead, nickel and zinc were determined in surface water, benthic sediments, and the gills, liver and stomach muscle tissues of Oreochromis niloticus and Clarias gariepinus in peri-urban lakes Chivero and Manyame, Zimbabwe. Five sites were sampled in each lake once per month in November 2015, February, May, August and November 2016. Pollution load index detected no metal contamination, whereas the geo-accumulation index reflected heavy to extreme sediment pollution, with Fe, Cd, Zn, Cr, Ni and Cu present in both lakes. Significant spatial temporal variations were detected for Al, Cr, Cu and Pb across sites within and between the two lakes. High Fe, Al and Cr concentrations in water and sediments in lakes Chivero and Manyame derive from geogenic background sources in addition to anthropogenic loads and intensity. Elevated concentrations of Al, Pb, Cu, Cd, Fe and Zn detected in gills, liver and stomach tissue of catfish corroborate concentrations in water and sediments, and pose the highest ecological and health risk for hydrobionts in lakes Chivero and Manyame. Contiguity of peri-urban lakes exposes them to similar threats, necessitating creative water management strategies, which ensure ecological continuity.