The apparent dissociation constant (Kd) was determined to be 70.15 ± 5.25 nM, and 1.49 ± 0.06 X 106 binding sites/ cell was estimated from the maximum binding capacity (Bmax). The latter is comparable to that estimated from the isotherm saturation.
We examined the development of P-12 binding sites during sperm generation in the testis. In contrast to the cell morphology in the tissue (Fig. 9A), no P12 appeared in the seminiferous tubules or Leydig cells (Fig. 9C). P12 could be immunolocalized (Fig. 9B) in spermatids and spermatozoa but remained undetectable in Leydig cells, Sertoli cells, and precursor cells such as spermatogonia and spermatocytes in the tissue slices preincubated with P12 (Fig. 9B).
Suppression of Calcium Transport into Spermatozoa by P12
We measured 45Ca2+ uptake by epididymal mouse spermatozoa incubated in modified Tyrode’s solution (1.0 X 107 cells/ml) in the absence or presence of 1.5 ^M P12 or recombinant P12 at 37°C. On the basis of the study by Co-ronel and Lardy, cell surface binding was represented by the uptake of 45Ca2+ by fresh spermatozoa without cell capacitation. ampicillin antibiotic
Thus, the induced 45Ca2+ uptake, which reflected the calcium transport into cells, was estimated by subtraction of the cell surface binding from the total 45Ca2+ uptake. At any incubation time, the induced 45Ca2+ uptake by cells incubated in the presence of P12 was less than by those incubated in the absence of P12 (Table 2). About 50% of Ca2+ transport to the control cells during a 60-min incubation could be suppressed by P12. Recombinant P12 was as strong as the naturally occurring P12 in suppressing the incorporation of Ca2+ by spermatozoa. These data support the identification of P12 as a caltrin.
FIG. 9. Demonstration of the P12-binding cells in the seminiferous tubules of testis. The slices from testis of mice (8 wk) were histochemically stained. A) The slices were stained with hematoxylin and eosin to reveal the morphology of seminiferous tubules: a, spermatogonia; b, spermatocytes; c, spermatids; d, spermatozoa; e, Sertoli cells; f, Leydig cells. B) The slices were incubated in order with P12, P12 antiserum, and antirabbit IgG conjugated with alkaline phosphatase. The activity staining for alkaline phosphatase was performed. C) The slices were treated as described in B except not incubated with P12. Bar = 100 ^m.
TABLE 2. The inhibitory effect of P12 on Ca2+ uptake by the capacitated spermatozoa.3
|Inhibitor||45Ca2||+ uptake||Induced||uptakeb||Inhibition (%)c|
|Recombinant P12||7.9||9.4||3.4||4.9||3 ± 5 51 ± 9|
a I, 10 min incubation; II, 60 min incubation. The data in terms of nanomoles of 45CaCl2 per 108 cells were the average of results of triplicate experiments.
b Estimated by subtraction of cell-surface binding, which was determined to be 4.5 nmol 45CaCl2/108 cell from fresh cells without preincubation, from the value obtained at a specified condition.
c The inhibitory effect of P12 or recombinant P12 on 45Ca2+ uptake by spermatozoa was represented by percentage of 45Ca2+ uptake of the controlled cells; data were calculated from three experiments.