The addition of calcium ion chelator,EGTA to thawing solution improves fertilizing ability in frozen–thawed boar sperm

In our previous study, seminal plasma effectively suppressed the induction of sperm to capacitation‐like status and acrosome loss during the thawing process. However, because boar seminal plasma is contaminated with various kinds of bacteria and/or viruses, it is necessary to develop a thawing solution without animal‐derived materials. In this study, we focused on the increase of intracellular Ca²⁺ ([Ca²⁺]_i) in sperm after thawing and the negative effects of sperm qualities. After thawing, the fluorescent intensity of [Ca²⁺]_i indicator, Fluo‐3/AM, and the level of phosphorylated tyrosine residue of protein were increased in the sperm. Next, we investigated whether the addition of Ca²⁺ chelators (ethylenediaminetetraacetic acid (EDTA) and ethyleneglycoltetraacetic acid (EGTA)) improved post‐thawed sperm motility. When the frozen–thawed sperm were treated with 6 mmol/L EDTA + 6 mmol/L EGTA, sperm motility was significantly increased as compared with control (6 mmol/L EDTA alone) at all incubation periods (P < 0.05). The combinational treatment significantly suppressed the elevation of [Ca²⁺]_i and the tyrosine phosphorylation, which improved the acrosomal status and fertilizing ability in vitro. Furthermore, when the thawing method was applied for artificial insemination, the fertilization rate was significantly higher than control (P < 0.05, 33% vs. 82%). Thus, we conclude that the addition of EDTA + EGTA to thawing solution is a beneficial tool for artificial insemination using frozen–thawed boar sperm.     

Growth hormone release induced by an amino acid mixture from primary cultured anterior pituitary cells of goats

The effects of amino acids on growth hormone (GH) release and cytosolic calcium concentration ([Ca²⁺]_i) were investigated in caprine anterior pituitary cells cultured for 3 d in Dulbecco modified Eagle medium. The addition of an amino acid mixture consisting of seven nonessential amino acids (NEAA: l-Asp, Gly, l-Ala, l-Ser, l-Pro, l-Asn, and l-Glu; concentration of each 12.5–200 μmol/l) in the medium significantly raised GH release from the cultured cells in a concentration-dependent manner with the maximum release at 200 μmol/l NEAA. Although an addition of l-Asp (0.1–100 μmol/l) caused a significant rise in GH release in a concentration-dependent manner, neither the individual amino acids contained in NEAA except l-Asp nor others (l-Leu, l-Phe, l-Gln, l-Met, and l-Arg) caused a rise in GH release when added alone to the medium. The rise in GH release induced by NEAA (200 μmol/l) and GH-releasing hormone (GHRH, 10 nmol/l) was significantly reduced by the addition of EGTA (l.8 mmol/l) and nifedipine (1 μmol/l) to the medium, respectively. The addition of NEAA (200 μmol/l) caused a rapid and transient [Ca²⁺]_i increase, followed thereafter by a steady increase. The prior addition of nifedipine (1 μmol/l), which itself significantly reduced the basal [Ca²⁺]_i, completely abolished the response induced by NEAA or GHRH. From these findings, we conclude that: 1) NEAA raises GH release and [Ca²⁺]_i in cultured caprine anterior pituitary cells, and 2) Ca²⁺ influx from the medium may be responsible for the cellular action of NEAA.     

pH-dependent modulation of intracellular free magnesium ions with ion-selective electrodes in papillary muscle of guinea pig

A change in pH can alter the intracellular concentration of electrolytes such as intracellular Ca²⁺ and Na⁺ ([Na⁺]_i) that are important for the cardiac function. For the determination of the role of pH in the cardiac magnesium homeostasis, the intracellular Mg²⁺ concentration ([Mg²⁺]_i), membrane potential and contraction in the papillary muscle of guinea pigs using ion-selective electrodes changing extracellular pH ([pH]_o) or intracellular pH ([pH]_i) were measured in this study. A high CO₂-induced low [pH]_o causes a significant increase in the [Mg²⁺]_i and [Na⁺]_i, which was accompanied by a decrease in the membrane potential and twitch force. The high [pH]_o had the opposite effect. These effects were reversible in both the beating and quiescent muscles. The low [pH]_o-induced increase in [Mg²⁺]_i occurred in the absence of [Mg²⁺]_o. The [Mg²⁺]_i was increased by the low [pH]_i induced by propionate. The [Mg²⁺]_i was increased by the low [pH]_i induced by NH₄Cl-prepulse and decreased by the recovery of [pH]_i induced by the removal of NH₄Cl. These results suggest that the pH can modulate [Mg²⁺]_i with a reverse relationship in heart, probably by affecting the intracellular Mg²⁺ homeostasis, but not by Mg²⁺ transport across the sarcolemma.     

Molecular characteristics of horse phospholipase C zeta (PLCζ)

A sperm‐specific phospholipase C (PLC), PLCzeta (PLCζ), is thought to underlie the initiation of calcium ([Ca²⁺]_i) oscillations that induce egg activation in mammals. In large domestic species, only bovine, porcine and recently equine PLCζ have been cloned, and the physiological functions of these molecules have not been fully characterized. Here, we evaluated the physiological functions of equine PLCζ (ePLCζ) in mouse oocytes. ePLCζ was cloned from testis using RT‐PCR. The expression of ePLCζ messenger RNA was confirmed in testis but not in other tissues. Microinjection of ePLCζ complementary RNA (cRNA) into mouse oocytes induced long‐lasting [Ca²⁺]_i oscillations, and most of the injected oocytes formed pronuclei (PN). The injection of cRNAs encoding horse, mouse, human and cow PLCζ into mouse oocytes showed that ePLCζ had the highest [Ca²⁺]_i oscillation‐inducing activity among the species tested. Mutation of D202R, which renders the protein inactive, abrogated the activity of ePLCζ. The nuclear translocation ability of ePLCζ was defective when expressed in mouse oocytes. Taken together, our findings show for the first time that ePLCζ has highest activity of the mammalian species studied to date. Our findings will be useful for the improvement of reproductive technologies in the horse.     

Inositol-1,4,5-Trisphosphate Receptor-1 and -3 and Ryanodine Receptor-3 May Increase Ooplasmic Ca2+ During Quail Egg Activation

We previously reported that egg activation in Japanese quail is driven by two distinct types of intracellular Ca²⁺ ([Ca²⁺]i): transient elevations in [Ca²⁺]i induced by phospholipase Czeta 1 (PLCZ1) and long-lasting spiral-like Ca²⁺ oscillations by citrate synthase (CS) and aconitate hydratase 2 (ACO2). Although the blockade of inositol 1,4,5-trisphosphate receptors (ITPRs) before microinjections of PLCZ1, CS, and ACO2 cRNAs only prevented transient increases in [Ca²⁺]i, a microinjection of an agonist of ryanodine receptors (RYRs) induced spiral-like Ca²⁺ oscillations, indicating the involvement of both ITPRs and RYRs in these events. In this study, we investigated the isoforms of ITPRs and RYRs responsible for the expression of the two types of [Ca²⁺]i increases. RT-PCR and western blot analyses revealed that ITPR1, ITPR3, and RYR3 were expressed in ovulated eggs. These proteins were degraded 3 h after the microinjection of PLCZ1, CS, and ACO2 cRNAs, which is the time at which egg activation was complete. However, degradation of ITPR1 and ITPR3, but not RYR3, was initiated 30 min after a single injection of PLCZ1 cRNA, corresponding to the time of the initial Ca²⁺ wave termination. In contrast, RYR3 degradation was observed 3 h after the microinjection of CS and ACO2 cRNAs. These results indicate that ITPRs and RYR3 differentially mediate in creases in [Ca²⁺]i during egg activation in Japanese quail, and that downregulation of ITPRs and RYR3-mediated events terminate the initial Ca²⁺ wave and spiral-like Ca²⁺ oscillations, respectively.     

Involvement of the Ca2+ signaling pathway in osteoprotegerin inhibition of osteoclast differentiation and maturation

The purpose of this study was to determine whether the Ca²⁺ signaling pathway is involved in the ability of osteoprotegerin (OPG) to inhibit osteoclast differentiation and maturation. RAW264.7 cells were incubated with macrophage colony-stimulating factor (M-CSF) + receptor activator of nuclear factor-κB ligand (RANKL) to stimulate osteoclastogenesis and then treated with different concentrations of OPG, an inhibitor of osteoclast differentiation. The intracellular Ca²⁺ concentration [Ca²⁺]_i and phosphorylation of Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) in the different treatment groups were measured by flow cytometry and Western blotting, respectively. The results confirmed that M-CSF + RANKL significantly increased [Ca²⁺]_i and CaMKII phosphorylation in osteoclasts (p < 0.01), and that these effects were subsequently decreased by OPG treatment. Exposure to specific inhibitors of the Ca²⁺ signaling pathway revealed that these changes varied between the different OPG treatment groups. Findings from the present study indicated that the Ca²⁺ signaling pathway is involved in both the regulation of osteoclastogenesis as well as inhibition of osteoclast differentiation and activation by OPG.     

Calcium homeostasis and its relationship to superoxide production in blood and milk neutrophils of lactating goats

Polymorphonuclear neutrophils (PMN), which comprise over 70% of the somatic cells in goat milk, are a major cellular component of innate immunity in the goat mammary gland. However, the function of milk PMNs is modified after diapedesis compared to PMNs in blood. As many aspects of PMN activity depend directly on intracellular Ca²⁺ concentration ((Ca²⁺)_i), the present study aimed to determine the changes in Ca²⁺ homeostasis of milk PMNs from lactating goats compared to autologous blood PMNs, and to examine the significance of these variations to the immuno-competency of milk PMNs. The intracellular Ca²⁺ store of freshly prepared milk cells was estimated from the elevation of (Ca²⁺)_i after ionomycin treatment, which was found to be significantly less than blood PMNs. Replenishment of the intracellular Ca²⁺ store in milk cells after intracellular Ca²⁺ depletion by Bapta-AM followed by spiking with 2.5 mM Ca²⁺ for 20 min was also compared to that of blood PMNs, showing that after depletion/spiking the intracellular Ca²⁺ store in milk cells was much less than blood PMNs. The production of superoxide anion (O₂^?) in vitro in response to (Ca²⁺)_i-dependent or (Ca²⁺)_i-independent modulators was used to evaluate the relevance of altered Ca²⁺ homeostasis on the immuno-competency of milk cells compared to blood PMNs. The results indicated that milk cells produced similarly low levels of O₂^? as blood PMNs when treated with ionomycin. However, the amount of O₂^? produced by milk cells in response to phorbol 12-myristate 13-acetate (PMA) stimulation, although greater than ionomycin treatment, was significantly less than that of blood PMNs. The capacity for O₂^? production by both cell types in response to PMA reverted to the resting state with use of the protein kinase C (PKC) inhibitor, staurosporine. In conclusion, the current study demonstrated an irreversible shortage of intracellular Ca²⁺ in the milk PMNs of lactating goats compared to blood PMNs. It also showed that preliminary O₂^–production, primed by ionomycin treatment, remained unchanged in milk PMNs, despite the shortage in intracellular Ca²⁺, but decreased O₂^? production capacity, mediated via the PKC pathway, in milk PMN. It is suggested that the defects in Ca²⁺ homeostasis in milk PMNs of lactating goats is partially attributable for the post-diapedesis functionality modifications.     

Characterization of Equine Phospholipase C Zeta: A Review and Preliminary Results on Expression Defects in Subfertile Stallions

In all mammalian species studied thus far, fertilization results in a series of intracellular Ca²⁺ ([Ca²⁺]_i) increases, referred to as oscillations, responsible for driving oocyte activation and embryonic development. Current evidence supports the notion that sperm-borne phospholipase C zeta (PLCZ) is responsible for the initiation of these [Ca²⁺]_i oscillations. Although this appears to be a highly conserved mechanism for oocyte activation, differences in PLCZ sequence, activity, and expression do exist among different species. Herein, we summarize the information supporting PLCZ as the oocyte-activating factor in mammals and present our current knowledge regarding the characterization of this protein in the horse. The equine sequence yielded a protein of high relative [Ca²⁺]_i-releasing activity. Equine PLCZ was expressed over the head region overlying the acrosome, equatorial segment, connecting piece between the head and midpiece, and on the principal piece of the flagellum of stallion sperm. Equine PLCZ expressed both over the head and tail sperm regions was catalytically active, with the latter representing a characteristic unique to the horse. We also present preliminary data in subfertile stallions displaying PLCZ expression defects, although further research is required to establish a clear association between these defects and fertility problems in the horse. In summary, the information presented raises the questions of whether equine PLCZ could play diverse roles in sperm physiology and/or become a marker for the evaluation of stallion fertility, both of which are worthy of further investigation.     

Phosphorylation of the MAPK Pathway has an Essential Role in the Acrosome Reaction in Miniature Pig Sperm

In almost all animal species, sperm acrosome reaction (AR) is a crucial step for fertilization. The step is a Ca²⁺‐dependent secretory event that must be completed before fertilization. Many researchers have reported that several chemicals (such as ionomycin, thapsigargin and caffeine) artificially induce this step by increasing [Ca²⁺]_i. However, little information has been known on events that occur following Ca²⁺ induced initiation of the sperm AR. We show here for the first time that phosphorylation of the mitogen‐activated protein kinase (MAPK) pathway is required for the AR in miniature pig sperm. Following caffeine treatment artificially inducing the AR in miniature pig sperm, Raf was phosphorylated and then MAP kinase kinase (MEK) and extracellular‐signal regulated kinase1 (ERK1) were also phosphorylated in a time‐dependent manner. However, the total ERK1 level did not change during the culture. Pre‐treatment of sperm with U0126, a MEK inhibitor, significantly suppressed both the AR and phosphorylation of MEK/ERK1 in a dose‐dependent manner. Additionally, pre‐incubation of the sperm with seminal vesicle (SV) fluid, which is known to contain a decapacitation factor, suppressed both the AR and MEK/ERK1 phosphorylation. These results suggest that phosphorylation of MAPK pathway plays an important role in the AR in miniature pig sperm. Moreover, the SV fluid may have an inhibitory effect on the AR via the suppression of the MAPK pathway.     

The concentration of ionized magnesium in serum during the periparturient period of non-paretic dairy cows

Ion-selective electrodes have recently been designed for determining the ionized concentration of magnesium (Mg²⁺) in serum. This development may allow new insights into some metabolic diseases of cattle. For this report, the concentrations of Mg²⁺, total magnesium (Mg_tot), ionized calcium (Ca²⁺), total calcium (Ca_tot), and inorganic phosphate (P_i) were determined in sera from seventeen 3-to 16-year-old Brown Swiss and crossed Simmental/Red Holstein cows during the periparturient period. In each animal, a transient increase of Mg²⁺ and Mg_tot serum concentrations was observed in association with the transient decrease in serum concentrations of Ca²⁺, Ca_tot and P_i after parturition. On average, throughout the study, the serum Mg²⁺ concentrations were 68.5% of those of Mg_tot, whereas the serum Ca²⁺ concentrations were 52% of those of Ca_tot. The possible mechanisms involved in the transient increase of Mg²⁺ and Mg_tot serum concentrations are discussed.Abbreviations Ca²⁺ ionized calcium - Ca_tot total calcium - Mg²⁺ ionized magnesium - Mg_tot total magnesium - P_i inorganic phosphate - PTH parathyroid hormone - PTHrP parathyroid homrone related protein     

Cytoskeletal and mitochondrial properties of bovine oocytes obtained by Ovum Pick‐Up: the effects of follicle stimulation and in vitro maturation

Follicle stimulation by follicular stimulating hormone (FSH) is known to improve developmental competence of bovine oocytes obtained by Ovum Pick‐Up (OPU); however, the exact factors in oocytes affected by this treatment have remained unclear. We compared in vitro matured (IVM) oocytes obtained at the immature stage from cows by OPU either without or with stimulation with FSH (non‐stimulated and stimulated OPU, respectively) to those obtained by superstimulation and in vivo maturation in terms of cytoskeleton morphology, mitochondrial distribution, intracellular adenosine triphosphate (ATP) content and H₂O₂ levels at the metaphase‐II stage and intracellular Ca²⁺ levels after in vitro fertilization (IVF). Confocal microscopy after immunostaining revealed reduced size of the meiotic spindle, associated with increased tendencies of microfilament degradation and insufficient mitochondrial re‐distribution in non‐stimulated OPU‐derived IVM oocytes compared with those collected by stimulated OPU, which in turn resembled in vivo matured oocytes. However, there was no difference in mitochondrial functions between oocytes obtained by stimulated or non‐stimulated OPU in terms of ATP content, cytoplasmic H₂O₂ levels, base Ca²⁺ levels and the frequencies and amplitudes of Ca²⁺ oscillations after IVF. Larger size of metaphase spindles in oocytes obtained by stimulated OPU may reflect and potentially contribute to their high developmental competence.     

Influence of prostaglandin F2α analogues on the secretory function of bovine luteal cells and ovarian arterial contractility in vitro

Although prostaglandin (PG) F_2α analogues are routinely used for oestrus synchronisation in cattle, their effects on the function of the bovine corpus luteum (CL), and on ovarian arterial contractility, may not reflect the physiological effects of endogenous PGF_2α. In the first of two related experiments, the effects of different analogues of PGF_2α (aPGF_2α) on the secretory function and apoptosis of cultured bovine cells of the CL were assessed. Enzymatically-isolated bovine luteal cells (from between days 8 and 12 of the oestrous cycle), were stimulated for 24 h with naturally-occurring PGF_2α or aPGF_2α (dinoprost, cloprostenol or luprostiol). Secretion of progesterone (P4) was determined and cellular [Ca²⁺]_i mobilisation, as well as cell viability and apoptosis were measured.Naturally-occurring PGF_2α and dinoprost stimulated P4 secretion (P < 0.05), whereas cloprostenol and luprostiol did not influence P4 synthesis. The greatest cytotoxic and pro-apoptotic effects were observed in the luprostiol-treated cells, at 37.3% and 202%, respectively (P < 0.001). The greatest effect on [Ca²⁺]_i mobilisation in luteal cells was observed post-luprostiol treatment (200%; P < 0.001).In a second experiment, the influence of naturally-occurring PGF_2α and aPGF_2α on ovarian arterial contraction in vitro, were examined. No differences in the effects of dinoprost or naturally-occurring PGF_2α were found across the studied parameters. The effects of cloprostenol and luprostiol on luteal cell death, in addition to their effects on ovarian arterial contractility, were much greater than those produced by treatment with naturally-occurring PGF_2α.     

Cellular mechanisms by which oxytocin mediates uterine prostaglandin F2α synthesis in bovine endometrium: role of calcium

The objective of these experiments was to determine the role of Ca²⁺ during oxytocin-stimulated prostaglandin (PG) F_2α release from bovine endometrial tissue in vitro. Uteri were collected from dairy cows on the day after spontaneous luteal regression. Caruncular endometrial explants were dissected and incubated in vitro to determine phospholipase C activity or PGF_2α release. A23187 (a calcium ionophore) and maitotoxin (an activator of voltage-gated L-type calcium channels) stimulated release of PGF_2α in a concentration-dependent manner (P < 0.05). Thapsigargin (induces accumulation of Ca²⁺ in the cytoplasm by inhibiting endoplasmic reticulum Ca²⁺/ATPase pumps) stimulated release of PGF_2α in a concentration-dependent manner as well (P < 0.13). Oxytocin (10⁻⁶ M), AlF₄⁻ (a nonspecific activator of G-proteins; 10⁻⁵ M), A23187 (10⁻⁵ M), and melittin (a stimulator of phospholipase A₂; 10⁻⁴ M) stimulated PGF_2α release when explants were incubated in Ca²⁺-free medium (P < 0.10); however, oxytocin, A23187, or melittin were unable to stimulate PGF_2α release when explants were incubated in Ca²⁺-free medium containing the calcium chelator EGTA (P < 0.10). This treatment did not prevent oxytocin or AlF₄⁻ from stimulating phospholipase C activity (P < 0.08). CoCl₂ (a nonspecific Ca²⁺ channel blocker) and methoxyverapamil (a specific voltage-gated L-type Ca²⁺ channel blocker) prevented oxytocin from stimulating PGF_2α release (P < 0.05). Our results suggest that both extracellular and intracellular Ca²⁺ may be required for oxytocin to stimulate PGF_2α secretion in bovine endometrial tissue.     

Sevoflurane inhibits equine myeloperoxidase release and activity in vitro

Objective To investigate the effects of the volatile anaesthetic sevoflurane on the release of total and active myeloperoxidase (MPO) by non‐stimulated and stimulated polymorphonuclear neutrophils (PMNs) in whole blood from healthy horses. Study design In vitro experimental study. Animals Adult healthy horses. Methods Samples of whole venous blood were collected and incubated in air or in air plus 2.3% or 4.6% sevoflurane for 1 hour. PMNs were stimulated with N‐formyl‐methionyl‐leucyl‐phenylalanine (fMLP), with a combination of cytochalasin B (CB) and fMLP or with phorbol myristate acetate (PMA). Total and active MPO contents released by PMNs in blood were measured by enzyme‐linked immunosorbent assay (ELISA) and specific immunological extraction followed by enzymatic detection (SIEFED) respectively. Additional experiments were performed to assess the effect of sevoflurane on the peroxidase and chlorination cycles of purified equine MPO using Amplex Red and 3’‐(p‐aminophenyl) fluorescein as fluorogenic substrates respectively. Results As compared with air alone, 1 hour exposure of whole blood to 4.6% sevoflurane in air significantly inhibited the release of total and active MPO by unstimulated and both fMLP‐ and CB + fMLP‐stimulated PMNs but not by PMA‐stimulated PMNs. Although 2.3% sevoflurane had no effect on total MPO release by unstimulated and stimulated PMNs, it significantly reduced the release of active MPO by unstimulated and fMLP‐stimulated PMNs. Additionally, sevoflurane reversibly inhibited the activity of MPO, especially the peroxidase cycle of the enzyme. Conclusions and clinical relevance Although our experimental study was not designed to assess the effects of sevoflurane in vivo, this inhibition of MPO release and activity may have relevance for anaesthetized horses and deserves further studies to examine the clinical importance of these findings.     

Extracellular signal-regulated kinase (ERK) activation in chicken heterophils stimulated with phorbol 12-myristate 13-acetate (PMA), Formyl-methionylleucyl-phenylalanine (fMLP) and lipopolysaccharide (LPS)

The signaling pathways leading to the activation of extracellular signal-regulated kinase (ERK) by phorbol 12-myristate 13-acetate (PMA), formyl-methionylleucyl-phenylalanine (fMLP) and lipopolysaccharide (LPS) in chicken heterophils were examined. To determine the mechanism of ERK's activation and its relation with the influx of calcium ions, heterophils were stimulated by PMA, fMLP and LPS. ERK was not activated by fMLP. LPS- and PMA-stimulated activation of ERK, based on Western blotting with antibodies against the phosphorylated form of ERK, was attenuated by the pretreatment of cells with the intracellular calcium chelator BAPTA/AM (1,2-bis (o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid) but not with the extracellular calcium chelator EGTA (glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid). Exposure of cells to the protein kinase C (PKC) inhibitor GF109203X inhibited the LPS- and PMA-stimulated phosphorylation of ERK in a concentration-dependent manner. The LPS-stimulated phosphorylation was inhibited by pretreatment with the phospholipase C (PLC) inhibitor U73122 but not the phosphatidylinositol 3-kinase (PI₃K) inhibitor LY294002. These results indicate that the LPS-induced phosphorylation of ERK in the chicken heterophils is mediated by PLC, PKC and intracellular calcium, and the PMA-stimulated phosphorylation is dependent on intracellular calcium ion and PKC.     

The effect of conglutinin on production of reactive oxygen species in bovine granulocytes

Conglutinin is a high molecular-weight lectin originally detected in bovine serum. It belongs to the family of collectins that bind sugar residues in a Ca²⁺-dependent manner and are effector molecules in innate immunity. Conglutinin appears to play an important role in immune defense mechanisms, showing antiviral and antibacterial activities when tested in vivo and in vitro. The present study evaluated the effect of conglutinin on the respiratory bursts in bovine peripheral phagocytes. Using nitroblue tetrazolium and hydrogen peroxide assays, we showed that sugar ligand-bound conglutinin stimulated the production of superoxide and H₂O₂ in granulocytes whereas the non-sugar-bound form of conglutinin inhibited these processes. These results indicate that both forms of conglutinin are able to interact with surface leukocyte receptors but have opposite effects on phagocytic activity. Our findings suggest that conglutinin bound to sugar residues on microbial surfaces can induce oxygen burst in phagocytes, and thereby mediates the elimination of pathogens and prevents the spread of infection.     

Activation of Mouse Oocytes Following Intracytoplasmic Injection of Chicken Sperm Extract

The cytosolic factor from the sperm of a wide variety of species has been reported to induce [Ca²⁺]i increase and/or activation in oocytes. Although many species have been studied, there is no data available on the chicken sperm factor responsible for activation of oocytes. This study was aimed to verify the activation of mouse oocyte by intracytoplasmic injection of chicken sperm extract (CSE). Survival rate of oocytes without rupture or lytic degeneration following injection was greater than 80% regardless of the presence or absence of CSE. Among the intact oocytes, activation rates following injection were 27.5% (11 of 40) in the sham operation group. Injection of CSE resulted in significantly higher activation rates in the 1/10 dilution group (57.8%, 26 of 45) compared with the sham operation group (p = 0.0045). Calculated amount of injected sperm extract of 1/10 dilution of CSE was equivalent of 12 chicken sperm. When the 1/10 diluted mouse sperm extract (MSE) was injected, survival rate of injected oocytes and activation rate of the survived oocytes was 82% (41 of 50) and 78% (32 of 41), respectively. Activation rate of MSE‐injected oocytes was a little but significantly higher than that of CSE‐injected oocytes (p < 0.037). In conclusion, it suggests that CSE can activate the mouse oocyte and that oocyte activating sperm factor(s) may be common between avian and mammal.     

Involvement of Ca2+-ATPase in suppressing the appearance of bovine helically motile spermatozoa with intense force prior to cryopreservation

In cattle, cryopreserved spermatozoa are generally used for artificial insemination (AI). Many of these specimens exhibit helical movement, although the molecular mechanisms underlying this phenomenon remain unclear. This study aimed to characterize helically motile spermatozoa, investigate the involvement of Ca²⁺-ATPase in suppressing the appearance of these spermatozoa prior to cryopreservation, and examine the potential of helical movement as an index of sperm quality. In the cryopreserved semen, approximately 50% of spermatozoa were helically motile, whereas approximately 25% were planarly motile. The helically motile samples swam significantly faster than those with planar movement, in both non-viscous medium and viscous medium containing polyvinylpyrrolidone. In contrast, in non-cryopreserved semen, planarly motile spermatozoa outnumbered those that were helically motile. Fluorescence microscopy with Fluo-3/AM and propidium iodide showed that flagellar [Ca²⁺]_i was significantly higher in cryopreserved live spermatozoa than in non-cryopreserved live ones. The percentage of non-cryopreserved helically motile spermatozoa was approximately 25% after washing, and this increased significantly to approximately 50% after treatment with an inhibitor of sarcoplasmic reticulum Ca²⁺-ATPases (SERCAs), “thapsigargin.” Immunostaining showed the presence of SERCAs in sperm necks. Additionally, the percentages of cryopreserved helically motile spermatozoa showed large inter-bull differences and a significantly positive correlation with post-AI conception rates, indicating that helical movement has the potential to serve as a predictor of the fertilizing ability of these spermatozoa. These results suggest that SERCAs in the neck suppress the cytoplasmic Ca²⁺-dependent appearance of helically motile spermatozoa with intense force in semen prior to cryopreservation.     

Leukotrienes Affect Secretory Function of Ovarian Cells In Vitro*

The aim of this study was to determine which cells are the source of production and target for leukotriene (LTs) action within the bovine ovary. Luteal (CL, days 14–16 of the oestrous cycle), steroidogenic cells (LSC) and endothelial cells (LEC) of the bovine corpus luteum (CL), and granulosa cells (GC) were isolated enzymatically, cultured in a monolayer and incubated with LTC₄, LTB₄, Azelastine (an antagonist of LTC₄) or Dapsone (an antagonist of LTB₄). Then cells were collected for determination of mRNA expression for LT receptors (LTRs) and 5‐lipoxygenase (5‐LO) by real time RT‐PCR, and media were collected for determination of prostaglandin (PG)E₂, F_2α, progesterone (P4; LSC only), endothelin‐1 (ET‐1; LEC only) and 17‐β oestradiol (E2; GC only). The greatest mRNA expression for LTR‐II and 5‐LO were found in LEC, whereas LTR‐I mRNA expression did not differ among cell types. The level of PGE₂ increased after LTs treatment in each type of ovarian cell, excluding LTC₄ treatment in LEC. The secretion of PGF_2α was also increased by LTs, but decreased after LTB₄ treatment of LSC. In GC cultures, both LTs stimulated E2 secretion; in LEC cultures, LTB₄ stimulated whereas LTC₄ inhibited P4 secretion; in LEC cultures, LTC₄ stimulated but LTB₄ inhibited ET‐1 secretion. The results show that LTs are produced locally and are involved in PGs production/secretion in all examined cells (LSC, LEC and GC) of bovine ovary. Leukotriene treatment modulate secretion of E2, by GC, P4 by LSC and ET‐1 by LEC, which indicates that LTs are involved in regulation of ovarian secretory functions.     

Leukotrienes are Auto‐/Paracrine Factors in the Bovine Corpus Luteum: An In Vitro Study

The aim of this study was to determine leukotrienes (LTs) functions in the bovine corpus luteum (BCL) during the oestrous cycle. In steroidogenic CL cells we examined the effect of luteotropic [LH, prostaglandin E₂ (PGE₂)] and luteolytic (PGF_2α, cytokines) factors on: the levels of LTB₄ and C₄, the expression of 5‐lipoxygenase (LO), LT receptors type I (LTR‐I) and LTR‐II, and the effects of LTB₄ and C₄ stimulations on the levels of progesterone (P4), PGE₂, F_2α and nitric oxide (NO) metabolites. Both luteolytic and luteotropic factors stimulated 5‐LO expression on days 2–4 and 17–19 of the cycle. Leukotriene receptors type I expression increased after PGE₂ and tumour necrosis factor α with interferon γ (TNF/IFN) stimulation on days 2–4 of the cycle. Leukotriene receptor type II expression increased after PGE_2α and TNF/IFN stimulation on days 2–4 and 17–19 of the cycle, and LTR‐II expression on days 8–10 of the cycle was unchanged after cell stimulation with any factor. Leukotriene B₄ level increased after BSC incubation with luteotropic factors during all examined days of the cycle and after cytokine stimulation at early‐ and mid‐luteal stages, whereas luteolytic factors stimulated LTC₄ secretion over the entire cycle. Leukotriene B₄ stimulated P4 secretion at the mid‐luteal stage and stimulated NO secretion during all examined phases. Leukotriene B₄ stimulated PGE₂ secretion at the early‐ and mid‐luteal stage. Leukotriene C₄ inhibited P4 secretion at the mid‐ and regressing‐luteal stage, stimulated NO (entire cycle) and PGF_2α at mid‐ and regressing‐luteal phases. Leukotrienes modulate steroidogenic cells functions, depending on the stage of the cycle. Leukotriene B₄ plays a luteotropic role stimulating P4 and PGE₂ secretions; LTC₄ stimulates the secretion of luteolytic factors and enhances the luteolytic cascade within BCL.