Intracellular calcium responses in bovine oocytes induced by spermatozoa and by reagents

The objectives of the present study were to clarify and compare the characteristics of the transient rises in intracellular calcium concentrations ([Ca2+]i) induced either by spermatozoa or by stimulation with artificial activators in bovine oocytes. These transient rises in [Ca2+]i in oocytes matured in vitro were recorded with Ca2+ imaging using the Ca2+ indicator fura-2. During fertilization, a series of transient rises in [Ca2+]i was observed. The first Ca2+ response peaked at a concentration of 521 +/- 39 nM (n = 20) and lasted for 4 min, while the subsequent Ca2+ responses were significantly smaller and shorter, with a peak of 368 +/- 13 nM (n = 23) and a duration of 2 min. Injection of inositol 1,4,5- triphosphate (InsP3) into unfertilized oocytes caused a transient rise in [Ca2+]i in a dose-dependent manner. The maximum response was induced by 20 nA x 1 sec injection of InsP3. Thimerosal, a sulfhydryl reagent, induced the repetitive transient rises in [Ca2+]i. The peak and the duration of the rises in [Ca2+]i induced by InsP3 or thimerosal were smaller and shorter, respectively, than those of the first rise induced by spermatozoa. Ethanol and Ca2+ ionophore IA23187, which are general parthenogenetic activators of unfertilized oocytes, each induced a single transient rise in [Ca2+]i. The duration of the rise in [Ca2+]i by ethanol or Ca2+ ionophore was significantly longer than that by spermatozoa at fertilization, although the peaks were smaller. These results clarified the characteristics of the rises in [Ca2+]i induced by spermatozoa and by several artificial reagents, and showed that the first rise in [Ca2+]i induced by spermatozoa had a higher peak [Ca2+]i and a longer duration compared with each the subsequent rises in [Ca2+]i and the rises in [Ca2+]i induced by artificial reagents. These indicate that a mode like as the first rise in [Ca2+]i induced by spermatozoa is an effective trigger for artificial activation of oocytes.     

Effects of some steroid hormones on head-to-head association in bovine spermatozoa

In the presence of 2 × 10^?6 M Ca²⁺ in Tris-buffered medium 0.5 × 10^?6 M, oestradiol-17β or corticosterone significantly increased the head-to-head association of washed bull spermatozoa; in the same concentration, testosterone and 5α-dihydrotestosterone had no significant effects, whereas progesterone significantly dissociated the associated spermatozoa. At 8 × 10^?6 M Ca²⁺ in the same medium, all five hormones increased the association to about the same level. In Tyrode solution with a Ca²⁺ concentration of 1.4 × 10^?3 M, oestradiol-17β and corticosterone acted as above, whereas progesterone and the two testosterones effected dissociation. In Tyrode solution each of the dissociating hormones was combined with oestradiol-17β. In each case a sum of the effects of the two hormones was obtained without any stimulation or inhibition. All five hormones still produced significant effects at 5 × 10^?7 M in Tyrode solution. A corresponding value for ATP was found at 1 × 10^?5 M.     

Capacitation of bovine spermatozoa by oviduct fluid

Oviduct fluid collected from chronically cannulated oviducts of heifers was evaluated for its effect on capacitation of bovine sperm in vitro. Capacitation was determined by the ability of sperm to fertilize bovine oocytes in vitro and to undergo an acrosome reaction (AR) upon exposure to lysophosphatidylcholine (LC). After incubation of sperm with 0-25% (v/v) estrual oviduct fluid (collected +/- 1 day from estrus) for 4 h, addition of LC (100 micrograms/ml) for an additional 0.25 h resulted in an increasing percentage of acrosome-reacted sperm as the concentration of oviduct fluid increased. Sperm incubated 4 h with 25% estrual oviduct fluid fertilized more oocytes than sperm incubated in medium alone (p less than 0.05) but was not different from sperm incubated with 10 micrograms/ml heparin (p greater than 0.05). Glucose inhibited the ability of LC to induce ARs in sperm incubated 4 h with heparin or estrual oviduct fluid. Incubation of sperm with 25% oviduct fluid collected at various days over the estrous cycle demonstrated that peak capacitating activity was found at estrus but was also present +/- 1 day from estrus. The active capacitating factor in oviduct fluid was found to be heat stable. In addition, when extraction procedures were applied in sequential order, oviduct fluid capacitating activity was resistant to protease digestion, precipitable by ethanol, size-excluded by Sephadex G-25, and destroyed by nitrous acid. These results suggest that a heparin-like glycosaminoglycan from the oviduct is a potential in vivo capacitating agent in the bovine.     

Metabolism of fatty acids by bovine spermatozoa

The incorporation of (14)C-labelled myristic, palmitic, stearic, oleic and linoleic acids in vitro into the lipids of bovine spermatozoa was measured at intervals from 2min to 2h. All acids were rapidly incorporated into diglycerides, myristic acid being metabolized to the greatest extent. Whereas the low incorporation of acids into total phospholipids reflected the relative stability of the major phospholipid fractions in sperm, the minor phospholipids, particularly phosphatidylinositol, showed comparatively high metabolic activity. Although, in general, saturated acids were incorporated more actively than unsaturated substrates, stearic acid was poorly incorporated into all lipids except phosphatidylinositol. In regard to fatty acid composition of sperm lipids it was notable that diglycerides contained myristic acid as the major component, and this acid was also a prominent moiety of phosphatidylinositol. Docosahexaenoic acid was the principal fatty acid of the major phospholipid classes. These findings have been discussed in relation to the role of lipids in the metabolism of spermatozoa.     

Protamine-DNA association in mammalian spermatozoa

We have previously identified two subsets of basic nuclear proteins of mouse sperm: the protamines and a group of less basic proteins and, with the aid of a polyvalent antiserum, we have demonstrated their differential extractibility by NaCl in reducing solution (Rodman et al., J cell sci 53 (1982) 227) [9]. By affinity purification with isolated mouse sperm protamines we have obtained a protamine-specific fraction of that antiserum and a fraction that contains antibodies to the subset of less basic proteins. With those immunochemical probes we have shown the following The antigenic sites recognized by the protamine-specific antibodies are accessible, intranuclearly, only after the DNA has been removed by DNase I. The antibodies and DNA compete for binding sites on the protamines. DNA removal and consequent availability of the antigenic sites of the protamine molecules to the antibodies are possible only after displacement of the less basic proteins and chromatin decondensation have been induced. Immunoreactivity by the less basic proteins takes place without intervention of DNase. Those data indicate that the protamines are DNA-bound but that the less basic proteins are not or, alternatively, their putative DNA-binding sites do not coincide with their immuno-reactive sites. Those data also suggest that a function of the subset of less basic proteins may be to provide a shield for the protamine-DNA complex. The mouse protamine-affinity-bound antibodies are highly cross-reactive with protamines of other mammalian sperm suggesting that, despite considerable molecular diversity among mammalian protamines, the DNA-binding sites are conserved.     

Uptake and metabolism of androgens by bovine spermatozoa

Spermatozoa from bovine ejaculates and cauda epiditymidis were incubated with either tritiated 17 beta-hydroxy-5 alpha-androstane-3-one (DHT) or 5 alpha-androstane-3 alpha, 17 beta-diol (3 alpha-diol). Examination of the medium incubations demonstrated metabolic conversion of both DHT and 3 alpha-diol when these steriods were incubated with ejaculated sperm. In addition to this interconversion, the following metabolities were identified: 5 alpha-androstane-3 beta, 17 beta-diol, (3 beta-diol), androsterone and 5 alpha-androstane-3, 17-dione (5 alpha-A-dione). Incubations with cauda spermatozoa showed similar metabolic patterns. Androgen binding was exhibited by both sperm types. Examination of the washed cauda sperm pellet, following incubations with 3 alpha-diol showed that the incubated steroid was the most abundantly bound. DHT and 5 alpha-androst-16-en-3 alpha-ol (delta 16-3 alpha-ol1 were also detected. The major part of the radioactivity bound in the sperm pellet was identified as DHT when this steroid was used as the substrate; the remaining radioactivity consisted of 3 alpha-diol and delta 16-3 alpha-ol. Investigations of ejaculated sperm pellets gave similar results apart from the additional identification of 5 alpha-androst-16-en-3 one (delta 16-3-one) and 5 alpha-androst-16-en-3 beta-ol (delta 16-3 beta-ol (delta 16-3 beta-ol).     

Capacitation of bovine spermatozoa by lysophospholipids and trypsin

Bovine spermatozoa were incubated in vitro with lysophosphatidylserine (LPS), lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), lysophosphatidylinositol (LPI), or trypsin. Capacitation of sperm was evaluated by penetration of the zonae pellucidae of dead bovine oocytes. Capacitation times could be shortened to 3 h or less by treatment of spermatozoa with each of these lysophospholipids (LPLs) (P < .05). The maximum oocyte penetration percentages for individual LPLs were 40% for 10 μM LPS, 24% for 160 μM LPC, 31% for 320 μM LPE, and 19% for 320 μM LPI. Capacitation also was facilitated (P < .01) by trypsin treatment of spermatozoa. Spermatozoa treated with 250 or 2,500 units/ml of trypsin penetrated more oocytes (17 and 18%) than spermatozoa treated with 0 or 25 units/ml of trypsin (0 and 3%). Spermatozoa treated with increasing concentrations of LPL showed a decrease in both the percentage of intact acrosomes and of progressively motile spermatozoa. Increasing levels of trypsin in the incubation medium also led to a decrease (P < .05) in the percentages of intact acrosomes and a decrease (P < .01) in the percentages of progressively motile spermatozoa. Percentages of live, ovulated oocytes fertilized by spermatozoa incubated for 1 h in LPS (86%, 6/7) were not different from those incubated for 24 h in control medium (71%, 5/7). Percentages of oocytes fertilized with both of these capacitation treatments were higher (P < .05) than for oocytes exposed or killed or uncapacitated sperm. Rapid induction of capacitation and the acrosome reaction can be accomplished by exogenous treatment of bovine sperm with lysophospholipids or trypsin.     

RNA polymerase activity in bovine spermatozoa

Washed mature spermatozoa from bulls incorporate ribonucleoside triphosphates into RNA using an endogenous template. Maximum incorporation was observed at 31 degrees C in the presence of MgCl2, all four ribonucleoside triphosphates, beta-mercaptoethanol, and glycine sodium hydroxide buffer at pH 9.0. The amount of synthesis was linearly dependent upon the concentration of spermatozoa and continued for at least 4 h. Digestion studies revealed the RNA to be present in a protected (intracellular?) location in the spermatozoa. The RNA synthesis was inhibited by ethidium bromide, rifampicin, acriflavine, actinomycin D, and caffeine, but not by alpha-amanitine or rifamycin SV. Fractionation of the spermatozoa by sonication and separation of the heads and tails by centrifugation through a discontinuous gradient revealed that more than half of the total RNA polymerase activity was associated with the tail fraction.     

Motility of bovine spermatozoa studied by laser light scattering

Laser light scattering has been employed to determine the swimming speed distribution and the fraction of motile cells in samples of bovine spermatozoa. As predicted from theory, average trajectory velocities determined by laser light scattering were approximately four times the average translational speed estimated using light microscopy. The proportion of motile spermatozoa decreased with time at the same rate when samples were prepared in either HEPES or phosphate buffers. However, whereas the mean swimming velocity declined slowly in HEPES buffer, it dropped rapidly when phosphate buffer was used. Dilution (in the range 40–0.4×10⁶ spermatozoa·ml^-1) in either of these two buffers reduced the fraction of motile spermatozoa in the sample, but the mean swimming velocity of the remaining active spermatozoa was unchanged. Lowering the temperature from 37° C to 15° C reduced the mean swimming speed by a factor of 2–3 and the fraction of motile cells by a factor of 4–5.     

Cryopreservation of flow-sorted bovine spermatozoa

Experiments were designed to maximize sperm viability after sorting by flow cytometry and cryopreservation. Experiments concerned staining sperm with Hoechst 33342 dye, subsequent dilution, interrogation with laser light, and postsort concentration of sperm. Concentrating sorted sperm by centrifugation to 10 to 20 x 10(6) sperm/ml reduced adverse effects of dilution. Exposing sperm to 150 mW of laser light resulted in lower percentages of progressively motile sperm after thawing than did 100 mW. Sorted sperm extended in a TRIS-based medium had higher postthaw sperm motility after incubation for 1 or 2 h than sperm extended in egg-yolk citrate (EYC) or TEST media, and equilibrating sperm at 5 degrees C for 3 or 6 h prior to freezing was superior to an equilibration time of 18 h. For sorting sperm 4 to 7 h postcollection, it was best to hold semen at 22 degrees C neat instead of at 400 x 10(6)/ml in a TALP buffer with Hoechst 33342. Current procedures for sexing sperm using flow cytometry result in slightly lower postthaw motility and acrosomal integrity compared to control sperm. However, this damage is minor compared to that caused by routine cryopreservation. Fertilizing capacity of flow-sorted sperm is quite acceptable as predicted by simple laboratory assays, and sexed bovine sperm for commercial AI may be available within 2 years.     

Penetration in vitro of bovine oocytes during maturation by frozen-thawed spermatozoa

Bovine immature oocytes cultured for various times in TC-199 medium were inseminated with frozen-thawed spermatozoa in Medium BO with caffeine (5 mM) and heparin (10 micrograms/ml). Very high penetration rates (95-100%) were obtained in all oocytes which had been cultured for 0-20 h. When oocytes cultured for 0 and 4 h were inseminated, 100% of them were penetrated and had a decondensing sperm head and most of the oocytes remained at the stage of condensed germinal vesicle (GV) to telophase-I 20-22 h after insemination. The formation of male and female pronuclei was first observed in oocytes inseminated 8 h after culture. The proportions of polyspermy and average number of spermatozoa in penetrated oocytes gradually decreased as oocyte maturation proceeded. Penetration of at least one spermatozoon with a decondensing head into oocytes at the GV stage (without culture) was almost completed up to 8 h after insemination and at that time most of the penetrated oocytes were still at the stage of GV or condensed GV. These results indicate that maturation of bovine oocytes is not required for sperm penetration into the vitellus or for sperm nuclear decondensation under the in-vitro conditions used.