R. Bathgate

Proteomic characterization and cross species comparison of mammalian seminal plasma

UNLABELLED Seminal plasma contains a large protein component which has been implicated in the function, transit and survival of spermatozoa within the female reproductive tract. However, the identity of the majority of these proteins remains unknown and a direct comparison between the major domestic mammalian species has yet to be made. As such, the present study characterized and compared the seminal plasma proteomes of cattle, horse, sheep, pig, goat, camel and alpaca. GeLC-MS/MS and shotgun proteomic analysis by 2D-LC-MS/MS identified a total of 302 proteins in the seminal plasma of the chosen mammalian species. Nucleobindin 1 and RSVP14, a member of the BSP (binder of sperm protein) family, were identified in all species. Beta nerve growth factor (bNGF), previously identified as an ovulation inducing factor in alpacas and llamas, was identified in this study in alpaca and camel (induced ovulators), cattle, sheep and horse (spontaneous ovulators) seminal plasma. These findings indicate that while the mammalian species studied have common ancestry as ungulates, their seminal plasma is divergent in protein composition, which may explain variation in reproductive capacity and function. The identification of major specific proteins within seminal plasma facilitates future investigation of the role of each protein in mammalian reproduction. BIOLOGICAL SIGNIFICANCE This proteomic study is the first study to compare the protein composition of seminal plasma from seven mammalian species including two camelid species. Beta nerve growth factor, previously described as the ovulation inducing factor in camelids is shown to be the major protein in alpaca and camel seminal plasma and also present in small amounts in bull, ram, and horse seminal plasma.

Characterization and Functional Significance of Calcium Transients in the 2-cell Mouse Embryo Induced by an Autocrine Growth Factor

Growth of preimplantation embryos is influenced by autocrine trophic factors that need to act by the 2-cell stage, but their mode of action is not yet described. This report shows that late zygote and 2-cell stage mouse embryos responded to embryo-derived platelet-activating factor (PAF) with transient increases in intracellular calcium concentration ([Ca2+] i ). [Ca2+] i transients were single global events and were specifically induced by embryo-derived PAF. They were blocked by inhibition of phospholipase C (U 73122) and an inositol trisphosphate (IP3) receptor antagonist (xestospongin C), indicating the release of calcium from IP3-sensitive intracellular stores. Transients were also inhibited by the absence of calcium from extracellular medium and partially inhibited by treatment with dihydropyridine (nifedipine, 10 μm), but not pimozide (an inhibitor of an embryonic T-type calcium channel). (±)BAY K8644 (an L-type channel agonist) induced [Ca2+] i transients, yet these were completely inhibited by nifedipine (10 μm). The complete inhibition of BAY K8644, but only partial inhibition of PAF by nifedipine shows that L-type channels were only partly responsible for the calcium influx. Depolarization of 2-cell embryos by 50 mm K+ did not inhibit PAF-induced calcium transients, showing that the influx channels were not voltage-dependent. Depletion of intracellular calcium stores by thapsigargin revealed the presence of store-operated channels. The interdependent requirement for IP3-sensitive internal calcium stores and extracellular calcium in the generation of PAF-induced transients may be explained by a requirement for capacitative calcium entry via store-operated channels. A functionally important role for the PAF-induced transients is supported by the observation that inhibition of [Ca2+] i transients by a PAF-antagonist (WEB 2086) or an intracellular calcium chelator (1,2-bis(2-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid tetrakis-acetoxymethyl ester; BAPTA-AM) caused marked inhibition of early embryo development. Growth inhibition by BAPTA-AM was relieved by addition of exogenous PAF.

Cryopreservation of epididymal alpaca (Vicugna pacos) sperm: a comparison of citrate-, Tris- and lactose-based diluents and pellets and straws

Epididymal spermatozoa were harvested from male alpacas and frozen after extension and cooling to 4 degrees C in citrate-, Tris- and lactose-based diluents (Experiment 1) and as pellets in 0.25- and 0.5-mL straws on either dry ice or over liquid nitrogen vapour (Experiment 2) to determine the effects diluents and packaging on their motility and acrosome integrity. In Experiment 1, sperm motility was higher after cooling to 4 degrees C and after freeze-thawing (0 but not 3 h post-thaw) for spermatozoa extended in the lactose- than the citrate- or Tris-based diluent (P < 0.05). Post-thaw acrosome integrity after cooling to 4 degrees C and post-thaw (0 h) was reduced for spermatozoa frozen in citrate- compared with lactose- or Tris-based diluents, but was similar for all groups 3 h after thawing. In Experiment 2, sperm motility immediately after thawing was higher for pellet freezing than for 0.25- or 0.5-mL straws on dry ice or liquid nitrogen vapour (P < 0.05), although by 3 h post-thaw motility was similar for pellets and straws (P > 0.05). Acrosome integrity was similar for all groups immediately after thawing and 3 h post-thaw. Cryopreservation of epididymal alpaca spermatozoa is feasible, with retained motility and acrosome integrity post-thaw. Freezing as pellets in a lactose-based diluent is recommended.

Antioxidant mechanisms and their benefit on post-thaw boar sperm quality.

While being an important component of normal cellular function, excess levels of reactive oxygen species (ROS) cause cell damage and death. The ability to protect sperm against oxidative damage is of particular importance in the artificial reproduction industry because of the increased production of ROS by the sperm cell during processing. This review discusses the formation of ROS and the use of antioxidants in protecting boar sperm against oxidative damage.

Ligand-Activated Signal Transduction in the 2-Cell Embryo

Abstract Platelet-activating factor (PAF) is an autocrine trophic/survival factor for the preimplantation embryo. PAF induced an increase in intracellular calcium concentration ([Ca2+]i) in the 2-cell embryo that had an absolute requirement for external calcium. L-type calcium channel blockers (diltiazem, verapamil, and nimodipine) significantly inhibited PAF-induced Ca2+ transients, but inhibitors of P/Q type (ω-agatoxin; ω-conotoxin MVIIC), N-type (ω-conotoxin GVIA), T-type (pimozide), and store-operated channels (SKF 96365 and econazole) did not block the transient. mRNA and protein for the α1-C subunit of L-type channels was expressed in the 2-cell embryo. The L-type calcium channel agonist (±) BAY K 8644 induced [Ca2+]i transients and, PAF and BAY K 8644 each caused mutual heterologous desensitization of each others responses. Depolarization of the embryo (75 mM KCl) induced a [Ca2+]i transient that was inhibited by diltiazem and verapamil. Whole-cell patch-clamp measurements detected a voltage-gated channel (blocked by diltiazem, verapamil, and nifedipine) that was desensitized by prior responses of embryos to exogenous or embryo-derived PAF. Replacement of media Ca2+ with Mn2+ allowed Mn2+ influx to be observed directly; activation of a diltiazem-sensitive influx channel was an early response to PAF. The activation of a voltage-gated L-type calcium channel in the 2-cell embryo is required for normal signal transduction to an embryonic trophic factor.

In vitro characteristics of frozen-thawed, sex-sorted bull sperm after refreezing or incubation at 15 or 37°C.

The objective was to determine the in vitro characteristics of frozen-thawed dairy bull sperm after sex-sorting and refreezing and thawing (0, 2, and 4h post-thaw; 37 degrees C) or post-sort incubation at 15 or 37 degrees C for 30 and 24h, respectively. These sperm were compared with nonsorted frozen-thawed sperm (control) and with nonsorted sperm undergoing two cryopreservation procedures (FF; 0, 2, and 4h). Frozen-thawed sex-sorted (FS) sperm maintained at 15 or 37 degrees C had higher (P<0.001) progressive motility (PM), velocity, mitochondrial function, viability, and acrosome integrity than that of control sperm but similar total motility at 0 and 2h of incubation. Frozen-thawed sex-sorted sperm incubated at 15 degrees C maintained high levels of motility (66.5+/-1.6%) and viability/acrosome integrity (64.9+/-1.2%) at 24h incubation and, after rewarming and further 6h incubation at 37 degrees C, acceptable levels of motility (35.8+/-1.6%) and viability/acrosome integrity (51.2+/-1.2%) were maintained. Frozen-thawed sex-sorted sperm maintained at 37 degrees C had lower levels of motility, integrity, mitochondrial respiration, and velocity from 4h of incubation onward than that of those incubated at 15 degrees C. However, when frozen-thawed sex-sorted sperm were refrozen (FSF), motility and velocity were depressed at all hours compared with levels exhibited by control sperm, but membrane viability/acrosome integrity and mitochondrial respiration were similar at 0 and 2h post-thaw. Acrosome integrity of sperm in all groups undergoing sorting was exceptionally high at 0h (> or =90%), even after re-cryopreservation and 4h of incubation (77.5+/-1.3%). Double frozen-thawed nonsorted sperm (FF) had similar motility to FSF sperm at 0 and 2h post-thaw but at all time points had the lowest (P<0.001) levels of acrosome intact/viable sperm and mitochondrial respiration and the lowest velocity at 0 h. In conclusion, whereas sex-sorting improved the functionality of frozen-thawed sperm, refreezing depressed motility, viability, and velocity but not acrosome integrity after extended incubation compared with that of control sperm. Furthermore, frozen-thawed, sex-sorted sperm may be stored for transport at 15 degrees C for up 24h without detrimental effects on in vitro sperm characteristics.

Birth of offspring after artificial insemination of heifers with frozen-thawed, sex-sorted, re-frozen-thawed bull sperm.

Two field trials were conducted to determine the fertilising capacity of (i) frozen-thawed, sex-sorted re-frozen-thawed (FSF) dairy bull sperm inseminated close to the time of ovulation, (ii) FSF sperm following large dose insemination, and frozen-thawed, sex-sorted (FS) sperm inseminated within 12h after sorting. In Trial 1, 24 heifers in synchronised oestrus were observed for standing heat over a 30-min period once every 3h. Upon observation of standing heat, the size of the pre-ovulatory follicle was tracked by ultrasound every 6h until ovulation was judged to be imminent. Heifers were inseminated with 4 x 10(6) X-bearing FSF or Control sperm within 6h of ovulation. Ovaries were scanned 6h after AI to ensure ovulation had occurred. All 24 heifers displayed standing oestrus and 20 of these subsequently ovulated. The mean length of standing oestrus was 16.8+/-0.4h and ovulation occurred 27.6+/-1.1h after the onset of standing heat from a pre-ovulatory follicle with a diameter of 16.1+/-0.3mm. All 12 heifers that received FSF sperm returned to oestrus<26d after AI. Of 8 heifers that received Control sperm, 6 (75%) were confirmed pregnant by ultrasound 7 wk after AI, confirming that the method of AI and herd fertility were sound. In Trial 2 the number of sperm inseminated and the effect of eliminating the post-sort cryopreservation step were investigated. Heifers (n=21) were synchronised for oestrus, and inseminated 24h after the onset of standing oestrus with 10 x 10(6) X-bearing FSF, 4 x 10(6) X-bearing FS, or 10 x 10(6) non-sorted frozen-thawed (Control) sperm. Heifers were observed for return to oestrus from 21d, and diagnosed for pregnancy 7 wk after AI. Of the 7 heifers that received FSF sperm, one was confirmed pregnant (14.3%) and delivered a female calf. Four heifers inseminated with control sperm became pregnant and calved, but no pregnancies were obtained using FS sperm. The birth of a calf following AI with FSF sperm demonstrates the potential of sorting from frozen-thawed semen, and with further work, may be a promising technique that will give producers access to sexed sperm from a greater range of bulls.

Embryo production after in vitro fertilization with frozen-thawed, sex-sorted, re-frozen-thawed bull sperm

The objective of this study was to determine the in vitro fertilizing capacity of bull sperm derived from fresh or frozen samples and subjected to sex sorting and re-cryopreservation. Four sperm types were assessed for their ability to fertilize and sustain early embryo development in vitro. Semen from three Bos taurus bulls of different breeds (Jersey, Holstein and Simmental) was collected and either sorted immediately and then frozen (SF) or frozen for later sorting. Frozen sperm destined for sorting were thawed, sex-sorted, and re-frozen (FSF) or thawed, sex-sorted (FS), and used immediately for in vitro fertilization (IVF). Frozen-thawed nonsorted semen from the same ejaculate was used as a control. Oocytes from donor cows were aspirated via ovum pick-up and matured in vitro prior to IVF and culture. On average, 19.0+/-1.7 (mean+/-SEM) oocytes were aspirated per donor cow, of which 74.4+/-2.2% were selected for maturation. The proportion of cleaved embryos (Day 3) did not differ between sperm groups (P=0.91). Likewise, IVF with FSF sperm resulted in similar Day 7 blastocyst rates (as a percentage of total oocytes) as those of control, SF, and FS sperm (FSF, 34.5+/-4.7; control, 32.2+/-4.6; SF, 35.9+/-4.8; and FS, 26.9+/-4.1%; P=0.23). These encouraging results show that frozen-thawed sex-sorted sperm may be re-frozen and used for in vitro embryo production with similar blastocyst production as that of nonsorted frozen-thawed and sex-sorted frozen-thawed sperm.

Seminal plasma aids the survival and cervical transit of epididymal ram spermatozoa

Seminal plasma purportedly plays a critical role in reproduction, but epididymal spermatozoa are capable of fertilisation following deposition in the uterus, calling into question the biological requirement of this substance. Through a combination of direct observation of spermatozoa in utero using probe-based Confocal Laser Endomicroscopy, in vivo assessment of sperm fertility and in vitro analysis of various sperm functional parameters, this study investigated the role of seminal plasma in spermatozoa transit through the cervix of the ewe. Following deposition in the cervical os, epididymal spermatozoa previously exposed to seminal plasma displayed an enhanced ability to traverse the cervix as evidenced by both significantly higher pregnancy rates and numbers of spermatozoa observed at the utero-tubal junction when compared with epididymal spermatozoa not previously exposed to seminal plasma. The beneficial effect of seminal plasma on sperm transport was clearly localised to transit through the cervix as pregnancy rates of spermatozoa deposited directly into the uterus were unaffected by exposure to seminal plasma. This phenomenon was not explained by changes to sperm motion characteristics, as seminal plasma had no effect on the motility, kinematic parameters or mitochondrial membrane potential of spermatozoa. Rather, in vitro testing revealed that seminal plasma improved the ability of epididymal spermatozoa to penetrate cervical mucus recovered from ewes in oestrus. These results demonstrate that the survival and transport of ram spermatozoa through the cervix of the ewe is not linked to their motility or velocity but rather the presence of some cervical penetration trait conferred by exposure to seminal plasma.

Flow-sorted ram spermatozoa are highly susceptible to hydrogen peroxide damage but are protected by seminal plasma and catalase

To determine whether flow sorting increased the susceptibility of spermatozoa to reactive oxygen species (ROS), ram semen was either diluted with Tris medium (100 x 10(6) spermatozoa mL(-1); D) or highly diluted (10(6) spermatozoa mL(-1)) before being centrifuged (DC) at 750g for 7.5 min at 21 degrees C or flow-sorted (S) before cryopreservation. Thawed spermatozoa were resuspended in graded concentrations of hydrogen peroxide to induce oxidative stress. In Experiment 1, following exposure to 30 or 45 muM hydrogen peroxide (H(2)O(2)), the total motility (%) of DC (41.0 +/- 7.3 or 25.7 +/- 6.7, respectively) and S spermatozoa (33.8 +/- 6.3 or 20.1 +/- 6.3, respectively) was lower (P < 0.001) than that of D spermatozoa (58.7 +/- 5.6 or 44.5 +/- 6.7, respectively). In Experiment 2, supplementation of samples containing H(2)O(2) with catalase (150 IU mL(-1)) or seminal plasma proteins (4 mg protein per 10(8) spermatozoa) negated oxidative stress, resulting in comparable values to samples receiving no H(2)O(2)in terms of the proportion of spermatozoa with stable plasmalemma (as determined using merocyanine-540 and Yo-Pro-1) in the D and S groups, the proportion of viable, acrosome-intact spermatozoa (as determined by fluorescein isothiocyanate and propidium iodide staining) in the D group and the motility of control (undiluted) and S spermatozoa. Neither H(2)O(2) nor sperm type (i.e. D, DC or S) had any effect on intracellular concentrations of ROS. These results show that flow sorting increases the susceptibility of spermatozoa to ROS, but the inclusion of anti-oxidants or seminal plasma as part of the sorting protocol improves resistance to oxidative stress.