A study of the uterine protein variations during the estrous cycle in the cow: molecular weights determination

This study was carried out to investigate molecular weights (MW) of the uterine protein content as the first step in their identification, and their variations during the estrous cycle as a part of the uterine adjustments for provision of an appropriate environment for physiological events in the bovine. Of the 24 pro-estrous, 21 estrous, 24 met-estrous and 46 diestrous uterine fluid samples prepared for our previous work, five samples in each phase were selected randomly and subjected to SDS-PAGE analysis in parallel with standard marker proteins to separate their protein content into fractions. By comparing these fractions with those of the standards, MWs of <14.4, 20, 30, 38, 40, 46, 67, 75, 85, 90, 160, 200, 210, 270 and 330 kiloDaltons (kDa) were calculated, respectively. Proteins with MWs of less than 14.4kDa [5.3 mg/ml (7.5% of total protein)] were observed only in met-estrus. Proteins with the MW of 20 kDa [14.0 mg/ml (19.4%)] were found only in diestrus. Proteins with MW of 30 kDa in met-estrus [20.6 mg/ml (28.9%)] and diestrus [4.5 mg/ml (6.3%)], and 38 kDa proteins were observed in a considerable amount in pro-estrus [41.4 mg/ml (44.5%)] and estrus [40.0 mg/ml (44.2%)]. Forty kiloDaltons proteins were observed only in diestrus [5.5 mg/ml (7.6%)], and 46 kDa proteins were observed only in met-estrus [5.3mg/ml (7.6%)]. Sixty seven, 75 and 330 kDa proteins were observed in all the phases of the cycle with different densities, the higher values recorded in diestrus, pro- and met-estrus, respectively. Proteins with MW of 85 kDa were observed in all the phases but diestrus [9.8 mg/ml (10.5%), 8.6 mg/ml (9.5%) and 7.3 mg/ml (10.2%) for pro-estrus, estrus and met-estrus, respectively], and proteins with MW of 90 kDa were observed in estrus [20.0mg/ml (22.1%)] and diestrus [3.3mg/ml (4.6%)]. Proteins of 160 kDa fraction were observed in pro-estrus [9.8 mg/ml (10.5%)] and met-estrus [10.7 mg/ml (15%)], 200 kDa proteins were observed only in diestrus [6.3mg/ml (8.7%)]; finally, 210 and 270 kDa proteins were observed only in estrus [3.4 mg/ml (3.8%) and 2.0 mg/ml (2.2%), respectively]. It was concluded that protein profile of the bovine uterine lumen changes during the estrous cycle, both in quality and quantity, which may be a part of uterine adaptation for the physiological events. Further identification of these proteins would give us a better insight into their role in the bovine reproduction.     

Electrophoretic protein analysis in the conditioned captive wild coyote, Canis latrans

Total protein, albumin and serum protein values were determined on 19 male and 14 female captive, vaccinated, wild coyotes. Male coyotes had significantly higher total protein, alpha 1 and alpha 2 globulin levels than female coyotes. Captive, wild coyotes had lower values for total protein, albumin and beta globulins, and higher values for alpha 2 and gamma globulins than similar values for laboratory dogs. Albumin values determined by bromcresol green were slightly higher than values derived by electrophoresis. This difference was non-significant.     

Maintenance of plasma-derived proteins at much lower concentrations in the uterine lumen of the rabbit: a kinetic study of passage.

Human serum albumin (HSA) and human gamma globulin (HGG) in serum and uterine fluid of nonpregnant rabbits at various times after an i.v. injection (100 mg/kg) were measured by a radial immunodiffusion test using specific antisera. The HSA concentration in uterine fluid rose to a peak at 12 hr when it was 11% of the serum concentration and then declined, whereas HGG reached a peak at 18 hr (3.2% of serum level) and decreased thereafter. The HSA passed 2 1/2 times faster than HGG, but both proteins equilibrated with uterine fluid in about 12-18 hr. Steady state levels of HSA and HGG indicated that uterine fluid: serum ratios were 1:10 and 1:20, respectively. Similar ratios were found for total protein and rabbit serum albumin (1:10) and rabbit gamma globulin (1:20). Therefore, except when there is a local immune response, the uterine lumen contains only about 5% of the serum antibody concentration. Available data in the mouse, rat and dog also indicate disparity between serum and uterine fluid protein levels.     

Equation for osmotic pressure of serum protein (fractions).

The colloid or protein osmotic pressure (Pi) is a function of protein molarity (linear) and of Donnan and other effects. Albumin is the major osmotic protein, but also globulins influence Pi. Equations based on concentrations of albumin and nonalbumin (globulin concentration + fibrinogen concentration) protein approximate Pi better than albumin alone. Globulins have a wide range of molecular weights, and a 1956 diagram indicated that Pi of globulin fractions decreased in the order alpha1-, alpha2-, beta-, and gamma-globulin. The molecular weight of the serum protein fractions had been extrapolated, so van't Hoff's law and nonlinear regression analysis of the curves permitted expression of the diagram as an equation: product Pi(s,Ott,2 degrees C,cmH2O)=x(alb)(0.338C(tot)+0.00339C(tot)(2))+x(alpha1)(0.518C(tot)+0.0107C(tot)(2))+x(alpha2)(0.203C(tot)+0.00155C(tot)(2))+x(beta)(0.187C(tot)+0.000577C(tot)(2))+x(gamma)(0.161C(tot)+0.000223C(tot)(2)), where Pi(s,Ott,2 degrees C,cmH2O) is Pi of serum at 2 degrees C (in cmH2O) computed from the 1956 diagram, C(tot) is the concentration (g/l) of total protein in serum, and x(alb), x(alpha1), x(alpha2), x(beta), and x(gamma) are the fractions of albumin, alpha1-, alpha2-, beta-, and gamma-globulin, respectively. At one and the same concentration of fractions, Pi("Ott") decreases in the order alpha1-globulin, albumin, alpha2-globulin, beta-globulin, and gamma-globulin.     

Electrophoretic and immunoelectrophoretic studies of sera from normal, tuberculous, and noninfected tuberculin-sensitive guinea pigs

Dardas, T. J. (Michigan State University, East Lansing), and V. H. Mallmann. Electrophoretic and immunoelectrophoretic studies of sera from normal, tuberculous, and noninfected tuberculin-sensitive guinea pigs. J. Bacteriol. 92:76-81. 1966.-Normal guinea pig serum was separated into seven fractions by electrophoresis on cellulose acetate membranes. Thirty antigens were found by immunoelectrophoresis: albumin, 6 alpha(1) globulins, 11 alpha(2) globulins, 6 beta(1) globulins, 5 beta(2) globulins, and gamma globulin. Hyper-alpha(2)-globulinemia was detected in sera from guinea pigs 14 days after inoculation with viable virulent Mycobacterium bovis. An additional alpha(2) globulin, not demonstrable prior to infection, was detected concomitantly with the hyper-alpha(2)-globulinemia by immunoelectrophoresis. The additional alpha(2) globulin was tentatively named alpha(2)-T. It persisted until the death of the guinea pigs. Neither hyper-alpha-globulinemia nor the alpha(2)-T antigen was detected by cellulose acetate electrophoresis and immunoelectrophoresis of sera from guinea pigs sensitized with heat-killed M. bovis. Both changes were due to the disease, not to delayed sensitivity alone.     

An electrophoretic characterization of serum proteins of the collared peccary (Tayassu tajacu)

Serum proteins of the collared peccary (Tayassu tajacu) were analyzed by agarose gel electrophoresis for wild adult males and females, nursing young, and reproductively-active females in captivity. Electrophoretic profiles of the adult peccary showed at least six distinct protein bands corresponding to the fractions: albumin, alpha-1, alpha-2, beta-1, beta-2, and gamma globulin. Globulin fractions of the peccary had different mobilities from the domestic swine. The only sexual dimorphisms were associated with the beta globulin:albumin ratio and the albumin:globulin ratio. Ingestion of colostrum in 1-day-old neonates was marked by a very large increase in gamma globulins. The only significant difference between pregnant and lactating females was in the alpha globulin:beta globulin ratio. Lactating females had higher concentrations of alpha-2 globulin than non-pregnant females.     

Serum protein changes in lead exposed mice infected with Hymenolepis nana

Significant decrease in serum globulin took place in Hymenolepis nana mouse model treated with lead prior to infection. Decrease of alpha, beta and gamma globulins was maximum on day 4 in comparison to that of control, gradually tapering off by day 100 when suppression was minimum with the experimental values almost reaching the same level as those of the control. Thus, suppression of globulins was neither total nor permanent.     

Experimental infection with Ancylostoma caninum larvae in mice. IV. Serum protein pattern during primary infection.

Serum protein of Swiss albino mice infected with Ancylostoma caninum larvae, were analysed electrophoretically. Twenty mice were infected with a dose of 1000 larvae and the alterations in serum protein during an infection period of 30 days were recorded and compared with uninfected controls. The result showed a significant decrease in albumin and gamma globulin with an increase in beta globulin. There were no significant changes in the alpha-1 and alpha-2 globulins. The total globulins increased and A/G ratio decreased. These changes appeared to be remarkable on the 9th day after infection.     

Evaluation of serum protein electrophoresis in greater rhea (<Emphasis Type="Italic">Rhea americana</Emphasis> Linnaeus, 1758)

Serum proteins from 47 healthy greater rheas (Rhea americana; male and female) were separated by electrophoresis in order to characterize normal reference ranges. Determination of total protein concentration was performed through biuret reaction. The mean value of total serum protein was 4.4 g/dL. Absolute concentrations of serum proteins were determined by agarose gel electrophoretic fractioning. Five fractions were analyzed: albumin, alpha, beta1, beta2, and gamma globulins, and the average values in grams per deciliter were 2.39, 0.32, 0.45, 0.30, and 0.79, respectively.     

Distribution and Regulation of ENaC Subunit and CFTR mRNA Expression in Murine Female Reproductive Tract

The present study investigated the regional distribution and cyclic changes in the mRNA expression of epithelial Na+ channel (ENaC) subunit and cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated Cl- channel, in adult female mouse reproductive tract. In situ hybridization revealed that in contrast to the abundant expression of CFTR, ENaC (alpha, beta, gamma) mRNA signal was not detected throughout the estrus cycle in the ovary and oviduct. Messenger RNA for all ENaC subunits was abundantly detected in the cervical and vaginal epithelia throughout the estrus cycle but for CFTR, mRNA was found only at proestrus. In the uterine epithelium, alphaENaC mRNA was detected at diestrus but not found at any other stage, while CFTR mRNA was only detected at early estrus but not other stages. Semi-quantitative RT-PCR detected mRNA for all ENaC subunits in the uterus throughout the cycle with maximal expression at diestrus and CFTR mRNA was only found in the early stages of the cycle. The involvement of ENaC and CFTR in Na+ absorption and Cl- secretion was demonstrated in cultured endometrial epithelia using the short-circuit current technique and found to be influenced by ovarian hormones. Taken together, these data indicate a main secretory role of the ovary and oviduct and a predominantly absorptive role of the cervix and vagina. The present results also suggest an ability of the uterus to secrete and absorb at different stages of the estrus cycle. Variations in the fluid profiles may be dictated by the regional and cyclic variations in expression of ENaC and CFTR and are likely to contribute to various reproductive events in different regions of the female reproductive tract.     

Disruption of the tissue inhibitor of metalloproteinase-1 gene results in altered reproductive cyclicity and uterine morphology in reproductive-age female mice

Tissue inhibitor of metalloproteinase-1 (TIMP-1) is a multifunctional protein expressed in the uterus of essentially all species, yet the function of this protein is uncertain. To assess the role of TIMP-1 in the uterine events that occur during the murine estrous cycle, mature female TIMP-1 wild-type and null mice were monitored for reproductive cyclicity. Mice were sacrificed in each stage of the estrous cycle, and peripheral blood was collected and assayed for serum estradiol and progesterone content by RIA. Uterine morphology and TIMP-1, TIMP-2, TIMP-3, and TIMP-4 mRNA expression were also examined between genotypes in each stage of the estrous cycle. Disruption of the TIMP-1 gene product was associated with an altered reproductive cycle characterized by a significant decrease in the length of the estrus period in the null mice. Also during the period of estrus, null mice expressed significantly lower levels of uterine TIMP-3 mRNA expression, altered uterine morphology, significantly higher serum estradiol levels, and significantly lower serum progesterone levels compared to their wild-type counterparts. It is concluded from this study that TIMP-1 has a multifaceted role in regulating the murine reproductive cycle, and this control appears to be at the level of both the uterus and the ovary.     

Effect of melatonin on 24h changes in plasma protein levels during the preclinical phase of Freund's adjuvant arthritis in rats

The 24h rhythms in plasma protein concentration were examined in rats on the third day after injection of Freund's complete adjuvant or adjuvant's vehicle, performed 3h after light on. In rats treated with adjuvant's vehicle, peak values of albumin and gamma globulin occurred during the nocturnal activity span (P < .02 and P < .0001, respectively), while those of alpha-1, alpha-2, and beta globulins were found late during the rest span (P < .002, P < .0001, and P < .0004, respectively). Freund's adjuvant administration abolished temporal changes in plasma albumin and beta globulin levels. It also decreased the amplitude of daily changes in alpha-1 and alpha-2 globulin (P < .05) and diminished mean values of alpha-2 globulin (P < .01). Pretreatment of rats with melatonin (30 microg daily) for 11 days, 11h after light on, counteracted mycobacterial adjuvant-induced suppression of the 24h rhythms in albumin and alpha-1, alpha-2, and beta globulins. The results further support the existence of preventive properties of a pharmacological dose of melatonin in situations in which a lost of circadian rhythmicity is expected.     

Association between blood plasma urea nitrogen levels and reproductive fluid urea nitrogen and ammonia concentrations in early lactation dairy cows

Two experiments were conducted to study the relationship of blood plasma urea nitrogen (PUN) concentrations with NH3, urea nitrogen, K, Mg, P, Ca, and Na concentrations in fluid of preovulatory follicles (experiment 1) and the relationships of PUN concentration and stage of estrus cycle with ammonia and urea nitrogen concentrations in uterine fluids (experiment 2) in early lactation dairy cows. Mean PUN levels were used to distribute cows into two groups: cows with PUN>or=20 mg/dl (HPUN), and cows with PUN<20 mg/dl (LPUN). In experiment 1, blood and follicular fluids from preovulatory follicles of 38 early lactation dairy cows were collected on the day of estrus (day 0) 4h after feed was offered. Follicular fluid NH3 was higher (P<0.01) in HPUN cows (339.0 micromol/L+/-72.2) compared to LPUN cows (93.9 micromol/L+/-13.1). Follicular fluid urea N was higher (P<0.001) in HPUN cows (22.4 mg/dl+/-0.4) compared to LPUN cows (17.0 mg/dl+/-0.3). PUN and follicular fluid urea N were correlated (r2=0.86) within cows. In experiment 2, blood and uterine fluids were collected from 30 cows on day 0 and on day 7. Uterine fluid NH3 was higher (P=0.05) in HPUN cows (1562 micromol/L+/-202) than in LPUN cows (1082 micromol/L+/-202) on day 7, but not on day 0. Uterine fluid urea N was higher (P<0.001) in HPUN cows than in LPUN cows on day 0 (26.9 mg/dl+/-1.3 and 20.4 mg/dl+/-0.7) and day 7 (26.5 mg/dl+/-1.1 and 21.4 mg/dl+/-1.1). There was a correlation (r2=0.17) between PUN and uterine fluid urea N within cows. The results of this study indicate that high PUN concentrations were associated with elevated NH3 and urea N concentrations in the preovulatory follicular fluids on the day of estrus and in the uterine fluid during the luteal phase of the estrous cycle in early lactation dairy cows. Elevated NH3 or urea N concentrations in the reproductive fluids may contribute to reproductive inefficiency in dairy cows with elevated plasma urea nitrogen due to embryo toxicity.     

Antibacterial activity of mare uterine fluid

Luminal fluid from the mare uterus was used to investigate its relation to antibacterial defenses. Uterine flushings were collected at Day 3 of estrus, Day 8 postovulation and Day 15 postovulation. Uterine proteins were concentrated by ultrafiltration, dialyzed and examined for chemotactic activity to neutrophils and for antibacterial properties. Serum taken at the time of flushing was dialyzed and studied in a similar manner. Neutrophil migration in response to serum from Day 3 estrus and Day 8 postovulation was increased (P less than 0.05) above controls. Uterine protein from Day 8 postovulation and from Day 3 of estrus also stimulated neutrophil migration (P less than 0.05) above values of controls. Antibacterial activity was measured by incubation of S. zooepidemicus with concentrated uterine flushing or serum. Serum from all three estrous cycle intervals diluted 1:10 or used at a protein concentration equal to the protein concentration of uterine fluid did not inhibit growth. After 4 h of incubation, bacterial growth in estrous serum was significantly greater (P less than 0.01) than serum taken at Day 8 and Day 15 postovulation. Uterine flushings from Day 8 postovulation significantly decreased bacterial colony-forming units (P less than 0.01). Heating flushings at 56 degrees C for 30 min did not abolish the antimicrobial activity, while heating flushings for 30 min at 80 degrees C removed this activity. The antibacterial activity does not appear to be due to agglutinating antibody.     

Effects of anabolic steroids on acute phase responses in intra-abdominal sepsis

The acute phase response is an important adaptive response to sepsis and injury. As anabolic steroids increase protein synthesis we postulated that these agents might also increase hepatic acute phase protein synthesis. Male Wistar rats were pretreated with testosterone or danazol for 48 h prior to caecal ligation and puncture (CLP). Thirty-six h following surgery the animals were killed and blood taken for full blood count, total protein, albumin, alpha, beta and gamma globulin fractions on serum electrophoresis, complement C(3) and transferrin levels. Danazol increased the alpha1, alpha2 and beta1 globulin serum protein fractions in comparison with no surgery and CLP alone groups. These results indicate that danazol increases plasma acute phase proteins, as measured by electrophoresis, in this model of intra-abdominal sepsis.     

Cyclic variation in rats's uterine fluid phosphatase level after administration of lyndral (17 alpha-ethinyl estradiol).

An oral administration of antifertility drug Lyndral (17 alpha-ethinyl estradiol) at doses 5 micrograms/rat/a week for 3 consecutive weeks altered the internal biochemical milieu of uterus, showing a cyclic variation in both acid and alkaline phosphatase of the uterine fluid with high levels being present at proestrus and estrus in control rats. In Lyndral treated rats acid phosphatase showed a tendency to increase in estrus and metestrus rats, whereas alkaline phosphatase increased significantly during proestrus and estrus stages of the cycle. All these altered phosphatase levels in uterine fluid, after Lyndral treatment, gives some insight into the hormonal sensitiveness of these enzymes.     

Development of resistance in laboratory animals to adults of the tick Rhipicephalus evertsi evertsi

Acquired resistance to adults of the two-host tick Rhipicephalus evertsi evertsi Neumann has been demonstrated in guinea-pigs and rabbits. Four infestations of both hosts with R.e.evertsi adults resulted in a significant reduction in the mean weight of the engorged females. A decline in the mean weight of the female ticks was also observed in ticks which fed on a rabbit previously injected with serum taken from a rabbit resistant to R.e.evertsi. This suggests that humoral immunity may have an important role in the acquired resistance to ticks. In both hosts an increase in the level of serum globulins was recorded. In rabbits the level of gamma globulin was affected, while in guinea-pigs an increase was observed in the concentration of alpha 1 and beta globulins. The differences in host responses to the R.e.evertsi adults are discussed.     

Determination of the Optimal Ammonium Sulfate Concentration for the Fractionation of Rabbit, Sheep, Horse, and Goat Antisera

Various ammonium sulfate concentrations and reaction conditions were employed in the fractionation of sera from rabbits, sheep, horses, and goats. Precipitates and supernatant fluids were analyzed by electrophoresis to study the effects of the controlled variables. At room temperature, the third precipitate in 35% saturated (NH(4))(2)SO(4) was the best fraction from both rabbit and sheep sera; 80 to 90% of the gamma globulins were recovered. The second and third precipitates of horse sera proteins in 30% saturated (NH(4))(2)SO(4) were both satisfactory, but only 44% of the gamma globulin was recovered after three precipitations. Goat sera yielded a very satisfactory fraction; 80% of the gamma globulin was recovered after two precipitations-the first in 30% and the second in 45% saturated (NH(4))(2)SO(4). The composition of these fractions was not influenced by the pH of the sulfate solutions (pH 5.8 and 7.2), by a range of normal room temperatures (20 to 30 C), or by diluting the sera before fractionation. Crude globulins and fluorescein isothiocyanate-labeled globulins were successfully refractionated by one precipitation in the optimal sulfate concentration for the appropriate animal species. The refractionated products contained considerably less beta and alpha globulins than did the original crude fractions and little or no albumin.     

The Metabolism of Serum Proteins in Neonatal Rabbits

1. Incorporation of S³⁵-labeled amino acids into serum proteins has been studied in neonatal and developing rabbits. It was found that, per unit weight, neonatal rabbits synthesized only about 1/36 of the gamma globulin, 1/7 of the beta globulin, ½ of the alpha globulin, and ⅛ of the albumin that an adult synthesized. The growing rabbit developed the ability to synthesize various serum proteins at different times. 2. Plasma volumes and serum protein concentrations were determined at different times during the growth period of the rabbit. Plasma volumes were found to be 1 and ½ times larger in newborn animals than in adults, with a gradual decline to the adult level. The total serum protein concentration at birth was about 60 to 65 per cent of the adult value and gradually increased with growth as the plasma volume decreased. 3. Half-lives of homologous albumin and gamma globulin were studied. The half-life of albumin in neonates was nearly twice as long as the half-life in adults, the latter value being reached at 1 month of age. The half-life of gamma globulin in neonates was more than twice as long as the half-life in adults and reached adult values at 2 to 3 months. 4. Attempts were made to alter serum protein metabolism. Gamma globulin synthesis early in life was augmented with antigen injections.     

Regulation of expression of fibroblast growth factor 7 in the pig uterus by progesterone and estradiol

Fibroblast growth factor 7 (FGF7) stimulates cell proliferation, differentiation, migration and angiogenesis. The consensus is that FGF7, expressed by mesenchymal cells, binds FGF receptor 2IIIb (FGFR2) on epithelia, thereby mediating epithelial-mesenchymal interactions. The pig uterus is unique in that FGF7 is expressed by the luminal epithelium (LE) and FGFR2 is expressed by the LE, glandular epithelium (GE), and trophectoderm to effect proliferation and differentiated cell functions during conceptus development and implantation. FGF7 expression by the uterine LE of pigs increases between Days 9 and 12 of the estrus cycle and pregnancy, as circulating concentrations of progesterone increase, progesterone receptors (PGR) in the uterine epithelia decrease, and the conceptuses secrete estradiol-17beta (E(2)), for pregnancy recognition. Furthermore, E(2) increases the expression of FGF7 in pig uterine explants. The present study investigates the relationships between progesterone, E(2), and their receptors and the expression of FGF7 in the pig uterus in vivo. Pigs were ovariectomized on Day 4 of the estrus cycle and injected i.m. daily from Day 4 to Day 12 with either corn oil (CO), progesterone (P4), P4 and ZK317,316 (PZK), E(2), P4 and E(2) (PE), or P4 and ZK and E(2) (PZKE). All gilts (n = 5/treatment) were hysterectomized on Day 12. The results suggest that: 1) P4 is permissive to FGF7 expression by down-regulating PGR in LE; 2) P4 stimulates PGR-positive uterine stromal cells to release an unidentified progestamedin that induces FGF7 expression by LE; 3) E(2) and P4 can induce FGF7 when PGR are rendered nonfunctional by ZK; and 4) E(2) from conceptuses interacts via estrogen receptor alpha, but not estrogen receptor beta in LE to induce maximal expression of FGF7 in LE on Day 12 of pregnancy in pigs.