Characterisation of the progesterone receptor on canine spermatozoa.

The present study was conducted to characterise and localise the progesterone receptor (PR) on canine spermatozoa. Using a progesterone-bovine serum albumin-fluorescein isothiocyanate conjugate (PBF) and different monoclonal antibodies (C262 and NCL-PGR against the steroid binding domain and N-terminus of intracellular PR, respectively, and h151 against the hinge domain of the intracellular oestrogen receptor), the PR was identified on the plasma membrane over the acrosomal region. Two proteins (54 kDa and 65 kDa) were detected by recognition of the three monoclonal antibodies using Western blotting. PBF labelling was observed in the majority of cauda epididymal spermatozoa (63 +/- 4%), but this labelling was markedly reduced (33 +/- 17%) after the addition of canine seminal plasma. Over a 7-h capacitation, the proportion of ejaculated spermatozoa exhibiting PBF labelling (indicating the presence of the PR) increased from 18 +/- 10% (onset) to 59 +/- 7% by 5 h, where it plateaued. Progesterone (P 4 ) induced the acrosome reaction (AR) in a dose-dependent manner (0, 0.1, 1 and 10 ug/mL P 4 corresponding to 10 +/- 5%, 16 +/- 9%, 23 +/- 7% and 30 +/- 7%). Pre-treatment of capacitated spermatozoa with canine seminal plasma reduced the incidence of the P 4 -induced AR (12 +/- 5%). In addition, treatment with the monoclonal antibodies significantly reduced the incidence of the P 4 -induced AR (10 microg/mL) in capacitated ejaculated spermatozoa from 19 +/- 6% to 11 +/- 4% (h151, 1 : 10) and 12 +/- 6% (C262, 1 : 10), respectively. A typical Scatchard plot revealed one binding with high affinity and low capacity, and another binding with low affinity and high capacity, suggesting at least two different characteristic PR. Taken together, these results demonstrate that P 4 induced the AR in a dose-dependent manner via functional transmembranal receptors in the acrosomal region of the canine sperm plasma membrane. The characteristics of this membrane receptor seem similar to those of other mammalian spermatozoa, and it shows structural homology to the intracellular PR.

Effects of cryo-injury on progesterone receptor(s) of canine spermatozoa and its response to progesterone.

The integrity of sperm progesterone (P4) receptor(s) and its response to steroid stimulation might be crucial for the maintenance of sperm fertilizing ability after cryopreservation. The aim of the current investigation was to study the effect of cryo-procedures on canine sperm P4 receptor(s). In addition, alteration of P4 receptor(s) at the molecular level and their functional integrity following cryo-procedures was evaluated. Fresh and frozen-thawed semen samples (n=6 same dogs) after capacitation were treated with 10 microg/mL P4 to induce the acrosome reaction (AR, FITC-PNA staining). Parallel samples were treated with 50% canine seminal plasma (SP) prior to AR induction with P4. The percentages of AR in capacitated fresh and frozen-thawed semen samples after treatment with P4 were 31.0+/-6.7 and 21.6+/-4.1% (P<0.05), respectively. The percentage of AR in fresh and frozen-thawed semen samples pretreated with SP and incubated with P4 was; 11.5+/-4.8 and 16.5+/-2.0% (P<0.05), respectively. The incidence of the spontaneous AR (P>0.05) in fresh and frozen-thawed semen samples at the onset (5.5+/-2.2 and 6.1+/-1.8%; respectively) and after a 2h (9.6+/-5.1 and 10.4+/-2.7%; respectively) capacitation, avoiding P4 stimulation, were not different. The percentage of progesterone-BSA-FITC staining over the acrosomal region was 18.3+/-10.3% in fresh semen, 36.0+/-11.9% in capacitated (P<0.05) and less than 5% in SP treated spermatozoa. This staining was barely visible in frozen-thawed spermatozoa regardless of capacitation status. In western blot analysis, mAb C262 recognized two bands (54 and 65 kDa). Digitonin treated fresh and frozen-thawed spermatozoa, labeled with [3H]-progesterone, revealed that the P4 binding capacity decreased from 6.0+/-4.4 in fresh to 3.0+/-2.1 nM in frozen-thawed spermatozoa. In nearly all samples tested (except one) 65 kDa protein band decreased significantly after freeze-thaw procedures while the 54kDa protein was increased. These results indicate that the reduced incidence of AR in response to P4 in frozen spermatozoa is possibly due to the conformational changes of P4 receptor(s) and/or reduced P4 receptor density derived from freezing injury.

Serum haptoglobin concentration in cattle.

To obtain a basal concentration of serum Haptoglobin (Hp) in cattle in Taiwan, Hp concentrations were measured from serum samples collected from 10 healthy heifers, every week for one year. The values were also compared with those collected from 15 cows diagnosed with postpartum metritis. The heifers were successfully impregnated by artificial insemination six months after the tests. Hp concentrations were also measured in the serum collected from 11 other cows within 3 weeks after parturition. The Hp assay developed in this study gave a good correlation (r=0.893)with Western blotting. The Hp concentration of 454 serum samples from the 10 heifers had a mean value of 83.6 +/- 34.1 mg/l, and there was no significant difference among individual heifers. The basal value of Hp in heifers was calculated as less than 73.6 mg/l. No significant difference in Hp concentration was observed among the 10 heifers during cold and warm seasons (19.8 +/- 2.2 degrees C vs 27.3 +/- 1.4 degrees C), or before and after pregnancy. The mean serum Hp concentration from cows suffering from postpartum reproductive disorders was 1133.5 +/- 627.1 mg/l, which was significantly greater than the serum of healthy heifers and postpartum cows (104.6 +/- 61.0 mg/l) (P<0.05). These results demonstrate that Hp concentration may be a useful indicator for cows with postpartum reproductive disorders.

Apoptosis and oxidative stress of infiltrated neutrophils obtained from mammary glands of goats during various stages of lactation.

OBJECTIVE: To examine apoptosis in infiltrated neutrophils during involution of mammary glands and compare them with those obtained during late and peak lactation, and to measure oxidative stress and activities of antioxidant enzymes and determine involvement of free radicals in apoptosis of infiltrated neutrophils. SAMPLE POPULATION: Neutrophils from mammary gland secretions of 8 goats at 4 stages (late and peak lactation and 1 and 2 weeks after end of lactation). PROCEDURE: DNA fragmentation was evaluated to characterize apoptosis. Concentration of thiobarbituric acid reactive substances (TBARS) was used to evaluate oxidative stress. Activities of superoxide dismutase and glutathione peroxidase were determined. RESULTS: Neutrophils from secretions obtained after end of lactation of all goats and from late-lactation milk of some goats underwent prominent apoptosis, whereas neutrophils from peak lactation secretions did not. Higher lipid peroxidation and lower antioxidant enzyme activities in neutrophils during involution were observed, compared with those during late and peak lactation. A significant negative correlation existed between TBARS concentrations and antioxidant enzyme activities during the nonlactating period. CONCLUSIONS AND CLINICAL RELEVANCE: Apoptosis is a feature of infiltrated neutrophils during involution of mammary glands in goats. This feature may allow prompt resorption and clearance of infiltrated neutrophils without damaging surrounding tissues. Increased oxidative stress in infiltrated neutrophils from secretions obtained after end of lactation is probably related to a deficiency in antioxidant enzyme activities. Understanding the relationship between apoptosis and oxidative stress will lead to new strategies for manipulating involution and reducing tissue damage.