Oestrous sheep serum balances ROS levels to supply in vitro capacitation of ram spermatozoa

Reactive oxygen species (ROS) are fundamental for intracellular signalling. In spermatozoa, they are involved both to apoptosis and to capacitation, and changes in ROS levels can alter the balance between these two processes. Oestrous sheep serum (OSS) is considered an efficient agent for in vitro capacitation of ram spermatozoa. We have explored the effects of OSS on ram sperm physiology, especially on ROS production, during in vitro capacitation. Semen samples from 15 rams were cryopreserved. After thawing, samples were submitted to four treatments: control (CTL), 10% OSS supplementation for in vitro sperm capacitation, caspase inhibitor (INH, Z‐VAD‐FMK 100 μM) and OSS (10%) plus caspase inhibitor (I + E). Sperm samples were incubated for 30 min at 38.5°C and 5% CO₂ and evaluated motility and kinetic parameters by computer‐assisted semen analysis (CASA) and viability (propidium iodide), apoptotic‐like membrane changes (YO‐PRO‐1), acrosomal status (PNA‐FITC), intracellular calcium (FLUO‐3), membrane fluidity (M540) and ROS production (CM‐H₂DCFDA) by flow cytometry. OSS induced changes in kinetic parameters compatible with capacitation, with a decrease in the percentage of progressive motility and linearity, and an increase in the amplitude of the lateral displacement of the sperm head (p < .05). Moreover, OSS increased the proportion of M540+ viable spermatozoa, YO‐PRO‐1+ and acrosome‐reacted spermatozoa (p < .05). After incubation, OSS and I+E achieved lower ROS levels (p < .05). Ca²⁺ levels did not change with the incubation, but were slightly higher (p < .05) when both OSS and the inhibitor were present. We suggest that OSS may modulate ROS levels, allowing intracellular signalling for capacitation to occur while preventing higher levels that could trigger apoptosis.     

Ceramide enhances acrosomal exocytosis triggered by calcium and the calcium ionophore A23187 in boar spermatozoa

Mammalian spermatozoa must undergo acrosomal exocytosis prior to penetration of the oocyte at fertilization. The mechanisms underlying acrosomal exocytosis have not yet been fully elucidated. This study explored the possible involvement of ceramide in exocytosis of the boar sperm acrosome. Ejaculated boar spermatozoa, stored with the Beltsville TS extender at 17 degrees C for up to 3 days, were washed and preincubated for 10 min with C2-ceramide, an analogue of endogenous ceramide, C2-dihydroceramide (C2-DH-ceramide), a negative control to C2-ceramide, or with (1S,2R)-D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol (D-erythro-MAPP), an inhibitor of alkaline ceramidase, followed by incubation and stimulation with 3 mM Ca2+ and 0.3 microM A23187 (Ca2+/A23187) at 37 degrees C in air in a water bath. Spermatozoa fixed at specific intervals were examined, and the % of acrosomal exocytosis was monitored. Stimulation of spermatozoa with Ca2+/A23187 resulted in a time-dependent increase. There were no obvious changes at 5 min, but this was followed by a rapid increase at 10 min, reaching nearly a maximum level after 15 min or more of incubation. Preincubation with C2-ceramide or D-erythro-MAPP enhanced acrosomal exocytosis triggered by Ca2+/A23187 in a dose-dependent manner, whereas C2-DH-ceramide was without effect. These results suggest the possibility that ceramide may be involved in the mechanisms underlying acrosomal exocytosis.     

Changes in Actin Distribution of Ram Spermatozoa under Different Experimental Conditions

The purpose of this study was to determine the presence of actin in ejaculated ram spermatozoa and the changes of localization that actin undergoes as a consequence of certain in vitro -induced physiological states. Using indirect immunofluorescence (IIF), three different patterns of staining (defined immunotypes) were established in ejaculated sperm. The three sperm immunotypes showed actin labelling in flagellum, neck and post-acrosomal area, differing on the labelling in the acrosomal region that was complete in immunotype 1, partial (frequently concentrated in the apical area, punctuate form) in immunotype 2, and totally absent in immunotype 3. The main subpopulation in ejaculate was immunotype 1 that represented 68% of total sperm, while 21% corresponded to immunotype 2 and only 10% corresponded to immunotype 3. Selection of high-quality sperm using a dextran/swim-up procedure hardly influenced the proportion of each immunotype resulting in a slight increase in type 1 sperm. Cold-shock treatment and in vitro capacitation induced a partial loss of actin labelling in the acrosomal area, whereas the ionophore-induced acrosomal exocytosis provoked a total loss of the acrosomal actin labelling, a phenomenon partially inhibited by phalloidin.     

Cleaved PARP‐1, an Apoptotic Marker,can be Detected in Ram Spermatozoa

The presence of apoptotic features in spermatozoa has been related to lower quality and functional impairment. Members of the poly‐ADP‐ribose polymerases (PARP) familyare involved in both DNA repair and apoptosis, playing important roles in spermatogenesis. Poly‐ADP‐ribose polymerase can be cleaved by caspases, and the presence of its cleavage product (cPARP) in spermatozoa has been related to chromatin remodelling during spermatogenesis and to the activation of apoptotic pathways. There are no reports on immunodetection of cPARP in ram spermatozoa; thus, we have tested a commercially available antibody for this purpose. cPARP was microscopically detected in the acrosomal ridge of some spermatozoa (indirect immunofluorescence). A preliminary study was carried out by flow cytometry (direct immunofluorescence, FITC). Ram semen was extended in TALP and incubated for 4 h with apoptosis inducers staurosporine (10 μm ) or betulinic acid (200 μm ). Both inducers and incubation caused a significant increase in cPARP spermatozoa (0 h, control: 21.4±3.3%, inducers: 44.3±1.4%; 4 h, control: 44.3±2.4%, inducers: 53.3±1.4%). In a second experiment, we compared the sperm fractions after density gradient separation (pellet and interface). The pellet yielded a slightly lower proportion of cPARP spermatozoa (28.5±1.2% vs 36.2±2.0% in the interface; p < 0.001), and a 12‐h incubation increased cPARP similarly in both fractions (p < 0.001). cPARP seems to be an early marker of apoptosis in ram semen, although its presence in untreated samples was weakly related to worse quality (pellet/interface). We suggest to study the relationship of PARP and cPARP levels with between‐male differences on sperm fertility.     

The Acidic Probe LysoSensor™ is not Useful for Acrosome Evaluation of Cryopreserved Ram Spermatozoa

To try new acrosomal probes for assessing ram spermatozoa, we compared the LysoSensor^? probe, which labels acidic organelles, with the frequently used peanut agglutinin acrosomal probe (PNA‐PE; phycoerythrin as fluorescent moiety). The previous microscopic observations showed a lack of relationship of LysoSensor^? with acrosomal status. Semen was obtained from five rams and frozen in four pools. Each pool was analysed carrying out a triple staining propidium ioide/PNA‐PE/LysoSensor^? Green DND‐189 to test acrosome labelling, and a double staining SYBR‐14/PI, to assess sperm viability. Stained samples were analysed by flow cytometry. All measurements were replicated. Data were processed using agreement and repeatability tests. LysoSensor^? labelling did not agree with PNA (mean of differences: 30.8%; coefficient of agreement: 22.6%), confirming microscopic observations. Nevertheless, when LysoSensor^? was compared with SYBR‐14/PI, the agreement was high (mean of differences: ?0.05%; coefficient of agreement: 5.07%). Repeatability of both methods was high and similar. LysoSensor^? did not seem to specifically stain the acrosome, but it may accumulate in the cytoplasm and label viable spermatozoa. Therefore, LysoSensor^? might not be used as an acrosomal probe in ram spermatozoa, but it could be used in other kind of studies, taking advantage of its pH sensitivity.     

Comparison of Membrane‐Permeant Fluorescent Probes for Sperm Viability Assessment in the Ram

This study was designed to compare the effectiveness and usability of four permeant fluorochromes (CFDA; SYBR‐14; Hoechst‐33342; and acridine orange), combined with propidium iodide to assess sperm membrane integrity. Three different experiments were conducted. The first trial was designed to study the optimal dye concentration and minimum incubation time required to achieve optimum fluorescence intensities and contrast for each fluorochrome combination using ram fresh semen samples. Both SYBR‐14 and acridine orange allowed a direct assessment of sperm membrane integrity, without the need of incubating samples, whereas a minimum of 4 and 6 min of incubation at 37°C was necessary to achieve optimum fluorescence intensities in the CFDA and Hoechst groups, respectively. In the second trial, fresh semen samples were mixed with different volumes of membrane‐affected sperm (semen treated with three cycles of freezing to ?20°C and thawing at room temperature) to produce semen samples with known proportions of damaged spermatozoa. The results were compared with the theoretical values predicted on the basis of the estimations made on fresh and frozen samples. The proportions of damaged sperm in each sample determined using the four fluorochrome combinations agreed with the predicted theoretical values, with the acridine orange/propidium iodide providing the best adjustment. The third experiment was performed to compare the results of sperm membrane integrity using the four fluorochrome combinations. The proportions of plasmalemma‐intact sperm determined by acridine orange and SYBR‐14 were greater (p < 0.0001) than the proportions of intact sperm determined by CFDA and Hoechst stains. It was concluded that the most efficient combinations to be used in ram sperm were AO/PI and SYBR/PI because it allowed a direct assessment of sperm viability without the need to incubate samples and obtaining reliable results.     

Effect of Albumin and Polyvinyl Alcohol on the Vitality, Motility and Acrosomal Integrity of Canine Spermatozoa Incubated in vitro

Sperm culture media used for in vitro fertilization (IVF) procedures are important factors concerning the viability, motility and acrosomal integrity of spermatozoa. The aim of this study was to investigate the effects of three different sperm diluting media, tissue culture medium (TCM‐199), sperm culture medium (Sp‐TALP) and human tubular fluid (HTF) supplemented with varying concentrations of bovine serum albumin (1, 4 and 6%) or polyvinyl alcohol (0.8%) on the acrosomal integrity, motility and viability of canine spermatozoa. Ejaculates collected from four dogs were diluted in all media and spermatozoa were separated from seminal plasma by the swim‐up technique. Sperm progressive motility was assessed using a phase contrast microscope. Viability and acrosomal integrity were evaluated using a dual stain technique (Giemsa–Trypan blue). The results demonstrated that the number of live canine spermatozoa was similar in culture media supplemented or not supplemented with macromolecules. A minimal concentration of albumin (1%) in the three media showed similar effects on vitality, motility and acrosomal integrity, as had higher concentrations (4 and 6%). The percentage of acrosome‐intact spermatozoa was markedly higher after HTF (94.1%) than after TCM‐199 (70.1%) or Sp‐TALP (71.0%) without supplementation. It is concluded that serum bovine albumin, irrespective of the concentration, preserved sperm viability and function, and HTF is the most suitable medium for preserving the acrosome in canine spermatozoa prepared for in vitro manipulation through short incubation.     

Ascorbic Acid,Catalase and Chlorpromazine Reduce Cryopreservation‐induced Damages to Crossbred Bull Spermatozoaa

The present study evaluated the effectiveness of ascorbic acid, catalase, chlorpromazine and their combinations in reducing the cryodamages to crossbred bull (Bos taurus × Bos indicus) spermatozoa. A total of 32 ejaculates (eight each from four bulls) were diluted in Tris–citric acid–fructose–egg yolk–glycerol extender. Each ejaculate was split into six parts (five treatment and one control). Treatment groups included 10 mm ascorbic acid, 0.1 mm chlorpromazine, 200 IU/ml catalase, 10 mm ascorbic acid + 0.1 mm chlorpromazine or 200 IU/ml catalase + 0.1 mm chlorpromazine in the extender. Fluorescent probes (Fluorescein isothiocyanate–Pisum sativum agglutinin + Propidium iodide) were used for the assessment of spermatozoa viability and acrosomal status. The proportion of acrosome intact live (AIL), acrosome intact dead, acrosome reacted live and acrosome reacted dead sperm was assessed in fresh, equilibrated and frozen‐thawed semen. The functional status of the sperm was assessed using hypo‐osmotic sperm swelling test (HOSST). Activities of acrosin and hyaluronidase enzyme were also determined. Lipid peroxidation level was assayed based on the melonaldehyde (MDA) production. In cryopreserved semen, the values of AIL spermatozoa, HOSST response, hyaluronidase and acrosin activity were reduced by 53%, 47%, 34% and 54%, respectively from their initial values in fresh semen. However, MDA level was threefold higher in the frozen‐thawed sperm compared with fresh sperm. Significant (p < 0.05) improvement in motility, viability, HOSST response, retention of hyaluonidase and acrosin and reduction in MDA was recorded in ascorbic acid, catalase, ascorbic acid + chlorpromazine and catalase + chlorpromazine incorporated groups. The percentage of AIL sperm was significantly (p < 0.05) higher in ascorbic acid, catalase and ascorbic acid + chlorpromazine incorporated groups compared with the control. Chlorpromazine alone did not improve the post‐thaw semen quality but when combined with either ascorbic acid or catalse, improvement in semen quality was noticed. It was inferred that incorporation of ascorbic acid, catalase and ascorbic acid + chlorpromazine in semen extender improved the post‐thaw semen quality in crossbred bulls.     

Post-thaw Survival and Longevity of Bull Spermatozoa Frozen with an Egg Yolk-based or Two Egg Yolk-free Extenders after an Equilibration Period of 18 h

The aim of the present study was to determine the suitability of using two egg yolk-free commercial extenders, Andromed and Biociphos Plus as compared with the Tris-egg yolk based diluent Biladyl, for the cryopreservation of bull spermatozoa when the freezing protocol involved holding the extended semen at 4 degrees C for 18 h before the freezing. Six ejaculates from each of 10 Holstein bulls were collected by using artificial vagina. The ejaculates were evaluated for volume, sperm concentration and motility, divided in to three equal volumes, and diluted, respectively, with the three extenders as specified above. Extended semen was equilibrated for 18 h at 4 degrees C and frozen in 0.25-ml straws. After thawing, 100-mul aliquots of semen were labelled with SYBR-14, PI and PE-PNA (Phycoerythrin-conjugated Peanut agglutinin) and analysed by flow cytometry at 0, 3, 6 and 9 h after incubation at 37 degrees C. A General Lineal Model procedure for repeated measures was used to determine the effects of extender, bull, replicate and the interaction between them, on sperm viability and acrosomal integrity. Semen samples frozen with Biladyl showed higher (p < 0.001) sperm survival after 0 h (47.9%) and 9 h (30.3%) of incubation than those frozen with Andromed (38.5% and 17.3%, after 0 and 9 h respectively) or Biociphos Plus (34.9% and 21.6%, after 0 and 9 h respectively). The bull and replicate had significant effects (p < 0.001) on both sperm viability and acrosomal integrity, but the interactions between bull and extender and between replicate and extender were not significant. It was concluded that, when holding the semen overnight before freezing, the use of Biladyl results in higher sperm survival and longevity than the use of Andromed or Biociphos Plus.     

Validation of the sperm quality analyzer and the hypo-osmotic swelling test for frozen-thawed ram and minke whale (Balaenoptera bonarensis) spermatozoa

The object of the present study was to investigate the validation of the sperm quality analyzer (SQA) and the hypo-osmotic swelling (HOS) test with standard sperm analysis methods in frozen-thawed ram and minke whale spermatozoa. In rams, highly significant correlations were observed in the percentage of motile spermatozoa (P<0.01) and sperm concentration (P<0.01) between the standard and SQA methods. But, the percentage of morphologically normal spermatozoa did not significantly correlate between the standard and SQA methods. The percentages of swollen spermatozoa at 15 minutes by the HOS test were significantly correlated with the motility by the standard (P<0.05) and by the SQA (P<0.05) methods. For minke whale spermatozoa, the SVI (sperm viability index) values by the standard method were significantly (P<0.001) correlated with the sperm motility index (SMI) values by SQA. The percentage of motile spermatozoa was also significantly correlated (P<0.01) with the motility measured by SQA. Using different hypo-osmotic solutions and incubation times, the HOS test with 25, 100 and 150 mOsM did not show significant variations. Motility observed by the standard method and the percentage of swollen spermatozoa were significantly correlated (P<0.05). These results indicate that the SQA and HOS test can be utilized to assess the post-thawing motility of ram and minke whale spermatozoa, and that the SQA and HOS test values are significantly correlated in ram spermatozoa. However, sperm concentration and morphologically normal spermatozoa are not assessed accurately by SQA in minke whales.     

泛素激活酶抑制剂对猪精子获能状态、膜重构及体外受精的影响

【目的】评估泛素激活酶(E1)对猪精子获能、膜重构和体外受精的影响。【方法】选取5头长白猪采集精液,以硫醇酯法评估泛素激活酶(ubiquitin activating enzyme, E1)抑制剂(TAK-243)对精子中泛素(ubiquitin, Ub)与E1结合的影响;将精子分为鲜精组(Fresh)、DMSO组(DMSO)、获能组(Capacitated)及2.4、4.8和7.2 nmol/L TAK-243组,鲜精组用2 mL PBS重悬精子沉淀;获能组用2 mL获能液重悬精子沉淀;DMSO组及2.4、4.8和7.2 nmol/L TAK-243组分别用2 mL含0.07%DMSO及2.4、4.8和7.2 nmol/L TAK-243的获能液重悬精子沉淀,使用微生物动(静)态图像检测系统检测精子的动力学参数;以Western blotting法检测精子的酪氨酸磷酸化水平;Zn²⁺荧光探针检测精子的Zn²⁺含量;花生凝集素染色检测精子的顶体膜重构;免疫荧光法检测顶体表面精子黏附蛋白(AQN-1、AWN)的表达;体外受精法检测TAK-243...     

Effect of adding different levels of caffeine in the extender on some biochemical constituents,enzymatic activities and physical characteristics of chilled and frozen ram semen

Two experiments were carried out to determine the efficiency of supplementation of ram semen extender with caffeine on chilled storage and frozen capacity of spermatozoa. In the first experiment, eighty ejaculates were collected by an artificial vagina from five adult Barki rams, aged 2–3 years and weighted 45.0 ± 2.0 kg throughout the experimental period (January to February 2017). The ejaculates were pooled and diluted (1:10) with tris‐citric egg yolk extender and were split into five groups. Group 1 served as control, whereas groups 2‐5 were supplemented with 0.1, 0.2, 0.3 and 0.4 mM caffeine. All diluted semen specimens were evaluated for physical characteristics immediately after dilution (T₀) and throughout preservation period of 48 hr at 4°C. Simultaneously, oxidative stress and indices such as total antioxidant capacity (TAC), malondialdehyde concentrations (MDA) and alkaline transaminase (AKP) concentrations and value of resazurin reduction test (RRT) were determined. In the second experiment, the raw pooled ejaculates were diluted (1:10) with glycerolated tris‐citric egg yolk extender, receiving the previously mentioned caffeine levels. The post‐thaw assessment of cryopreserved spermatozoa, in all groups, was conducted by a computer‐assisted sperm analysis (CASA) system. The results revealed that adding caffeine to ram semen extender at low (0.1 mM) or medium (0.2 mM) levels had positive impact on both physical characteristics of ram sperm and the enzymatic activities compared to the other semen groups. Caffeine supplementation also enhanced post‐thaw sperm dynamics, which implies its potential as an exogenous antioxidant supplement.     

Polymyxin B enhances acrosomal exocytosis triggered by calcium and the calcium ionophore A23187 in ejaculated boar spermatozoa

Polymyxin B (PMB) is beneficial for boar semen storage since it neutralizes the endotoxin of bacteria. However, the direct effect of PMB on boar spermatozoa has been unknown. This study aimed to examine the effect of PMB on acrosomal exocytosis, an essential process for successful fertilization in boar spermatozoa. Ejaculated spermatozoa stored with BTS extender at 17°C were washed and incubated with 0–100 μM PMB for 20 min and then examined for % total motililty, vigor grade and viability. None of the parameters was significantly different between 0 and 50 μM PMB with a gradual decline at higher concentrations. Thus the effect on acrosomal exocytosis was investigated at 0–50 μM of PMB. Spermatozoa were preincubated with PMB for 10 min, incubated for stimulation of acrosomal exocytosis with Ca²⁺ and the calcium ionophore A23187 and then fixed with glutaraldehyde at 5, 10 and 15 min. Preincubation with PMB at 0.01–50 μM and 0.05–50 μM resulted in significant enhancement of acrosomal exocytosis at 10 min and 15 min of incubation, respectively. Preincubation with PMB followed by incubation without A23187 did not affect acrosomal exocytosis. These results suggest that PMB exerts effects on the acrosomal exocytosis triggered by Ca²⁺ and A23187 in boar spermatozoa.     

Effect of different egg yolk-based extenders on the quality of ovine cauda epididymal spermatozoa during storage at 4°C

Cauda epididymal spermatozoa were obtained from testicles collected from abattoir(s). The pooled sperm samples were divided into four aliquots. Each aliquot was washed separately with the buffer of respective extender and finally extended with the four extenders viz. egg yolk–citrate (EYC), egg yolk–citrate–fructose (EYCF), Tris–citric acid–egg yolk–fructose (TCEYF) and egg yolk–Mcillvaine glucose (EYMG) and preserved at 4°C. The per cent sperm motility for EYC, EYCF, TCEYF and EYMG at 0 h was 50.83%, 56.67%, 75.00% and 31.67%, respectively, and at 72 h was 24.17% (EYC), 30.83% (EYCF), 51.67% (TCEYF) and 7.50% (EYMG). The corresponding figures for live sperm count at 0 h was 83.17%, 86.33%, 90.42% and 81.75% and at 72 h was 64.75%, 73.92%, 76.00% and 57.67%. The corresponding figures for mean per cent intact acrosome at 0 h was 95.33%, 95.50%, 90.92% and 97.25% and at 72 h was 86.17%, 83.92%, 77.58% and 86.33%. The sperm motility was significantly (p < 0.05) higher for TCEYF at different h of preservation from 0 h through 72 h. The sperm motility, live sperm count and per cent intact acrosome declined significantly (p < 0.05) with the advancement of storage time in all the four extenders. Our study concluded that TCEYF was best out of the extenders studied for preservation of cauda epididymal spermatozoa after double centrifugation and extension at 4°C up to 72 h of preservation. However, EYCF also has better potential for the preservation of cauda epididymal spermatozoa as viability was in close proximity and acrosomal integrity was higher compared with TCEYF extender.     

Tunica dartos index as a parameter for measurement of adaptability of rams to subtropical conditions of Egypt

The aim of the present study was to investigate the effects of hot summer climatic conditions (compared to winter) on the libido, semen quality traits, level of serum testosterone hormone and some physiological and body measurements of Egyptian Suffolk rams. A parameter for the tolerance of the rams to adverse hot climatic conditions, designated as the tunica dartos index (TDI), was estimated as the multiplication of the percentage change in scrotal length and that of the percentage difference between rectal temperature and scrotal skin temperature. The relationships between the TDI and semen characteristics were also estimated. The results showed that in summer, the reaction time, pH, percentage dead and abnormal spermatozoa, acrosomal damage and temperatures of rectum, body skin and scrotal skin and scrotal length were significantly higher, while sperm motility, sperm concentration, scrotal circumference and testis length were significantly lower than in winter. The correlation coefficient (r) values estimated between the TDI and each of the semen pH, ejaculate volume, sperm motility and sperm cell concentration were positive, and those estimated between the TDI and each of the reaction time, dead and abnormal spermatozoa and acrosomal damage were negative. The best values of libido (low reaction time), semen pH, semen motility percentage, sperm cell concentration and testosterone level were recorded with a high TDI level (7.5 and more), while those of percentage of dead spermatozoa, sperm abnormalities and acrosomal damage were recorded with a low TDI level (less than 4.5). It can be concluded that the TDI correlates well with the reproductive ability of the ram and can be a reliable parameter to indicate the tolerance of rams to hot subtropical conditions in Egypt.     

PP1γ2对树鼩精子成熟和运动性的调控作用研究

为了阐明树鼩精子中是否存在丝氨酸/苏氨酸蛋白磷酸酶1γ2（PP1γ2）及其在附睾精子中的存在形式,进而探究PP1γ2对精子成熟和运动性的调控作用,本试验以树鼩为研究对象,采用Western blotting分析了不同条件下树鼩附睾头和附睾尾精子中PP1γ2的存在形式和磷酸化程度,探讨了双丁酰环腺苷酸（db-cAMP）、3-异丁基-1-甲基黄嘌呤（IBMX）或Ca²⁺对树鼩精子中PP1γ2磷酸化表达水平的影响,并进一步研究了磷酸酶抑制剂冈田酸（okadaic acid,OA）和花萼海绵诱癌素A （calyculin A,CA）对树鼩精子中PP1γ2磷酸化程度的影响及其对树鼩附睾头和附睾尾精子运动度的影响。结果显示,在树鼩附睾头和附睾尾精子中均存在PP1γ2,且在等量的附睾头和附睾尾精子蛋白中,PP1γ2在附睾尾精子的磷酸化程度远高于附睾头精子;db-cAMP、IBMX或Ca²⁺不改变PP1γ2的磷酸化水平;磷酸酶抑制剂OA和CA能明显提高附睾头和附睾尾中PP1γ2磷酸化的程度,且能显著提高精子（尤其是附睾头精子）的运动度（P<0.05）,OA和CA的最佳作用浓度分别为1 μmol/L和10 nmol/L,最佳作用时间分别为15、20 min。本研究结果表明,蛋白磷酸酶PP1γ2对树鼩精子成熟及运动性具有重要的调控作用,其主要通过磷酸化和去磷酸化的变化发挥作用。     

A 32-kDa tyrosine-phosphorylated protein shows a protease-dependent increase in dead boar spermatozoa

Boar sperm TyrP32 is a 32-kDa tyrosine-phosphorylated protein that increases during the capacitation and acrosome reaction and during cryocapacitation. However, it is still unclear whether the increase in TyrP32 is an event that is limited to the process of sperm fertilization, including cryocapacitation. The aims of the present study were to demonstrate that TyrP32 is increased in dead spermatozoa after freeze-thawing without a cryoprotectant and to find the causal factors for this increase. Washed spermatozoa were resuspended in a salt solution and then frozen. The frozen samples were rapidly thawed in a warm water bath and then used for sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE)/Western blotting to detect TyrP32, SDS-PAGE/silver staining of sperm proteins and staining of acrosomal contents with fluorescein isothiocyanate (FITC)-conjugated peanut agglutinin (PNA). In the samples before freezing, TyrP32 was barely detectable, and the distribution of the acrosomal contents was normal in most spermatozoa. One cycle of freeze-thawing induced an increase in TyrP32, a decrease in major sperm proteins and disorder in the acrosomal contents. However, the addition of a protease inhibitor (APMSF, 1 mM) suppressed the increase in TyrP32 and the decrease in the major sperm proteins, although it did not have any influence on the disorder in the acrosomal contents. Additionally, the spermatozoa did not exhibit any flagellar movement after freeze-thawing, which showed that almost all of them were dead. These results indicate that TyrP32 can show a protease-dependent increase in dead spermatozoa after freeze-thawing without a cryoprotectant even though the dead spermatozoa do not undergo cryocapacitation.     

The Effects of Hoechst 33342 Staining and the Male Sample Donor on the Sorting Efficiency of Canine Spermatozoa

The aim of this study was to evaluate the influence of Hoechst 33342 (H‐42) concentration and of the male donor on the efficiency of sex‐sorting procedure in canine spermatozoa. Semen samples from six dogs (three ejaculates/dog) were diluted to 100 × 10⁶ sperm/ml, split into four aliquots, stained with increasing H‐42 concentrations (5, 7.5, 10 and 12.5 μl, respectively) and sorted by flow cytometry. The rates of non‐viable (FDA+), oriented (OS) and selected spermatozoa (SS), as well as the average sorting rates (SR, sorted spermatozoa/s), were used to determine the sorting efficiency. The effects of the sorting procedure on the quality of sorted spermatozoa were evaluated in terms of total motility (TM), percentage of viable spermatozoa (spermatozoa with membrane and acrosomal integrity) and percentage of spermatozoa with reacted/damaged acrosomes. X‐ and Y‐chromosome‐bearing sperm populations were identified in all of the samples stained with 7.5, 10 and 12.5 μl of H‐42, while these two populations were only identified in 77.5% of samples stained with 5 μl. The values of OS, SS and SR were influenced by the male donor (p < 0.01) but not by the H‐42 concentration used. The quality of sorted sperm samples immediately after sorting was similar to that of fresh samples, while centrifugation resulted in significant reduction (p < 0.05) in TM and in the percentage of viable spermatozoa and a significant increase (p < 0.01) in the percentage of spermatozoa with damage/reacted acrosomes. In conclusion, the sex‐sorting of canine spermatozoa by flow cytometry can be performed successfully using H‐42 concentrations between 7.5 and 12.5 μl. The efficiency of the sorting procedure varies based on the dog from which the sperm sample derives.     

Effects of acrosomal conditions of frozen-thawed spermatozoa on the results of artificial insemination in Japanese Black cattle

The purposes of this study were to examine the relationship between male artificial insemination (AI) fertility and sperm acrosomal conditions assessed by new and conventional staining techniques and to identify possible reproductive dysfunctions causing low conception rates in AI using frozen-thawed spermatozoa with poor acrosomal conditions in Japanese Black bulls. We investigated individual differences among bulls in the results concerning (1) acrosomal conditions of frozen-thawed spermatozoa as assessed by not merely peanut agglutinin-lectin staining (a conventional staining technique) but also immunostaining of acrosomal tyrosine-phosphorylated proteins (a new staining technique), (2) routine AI using frozen-thawed spermatozoa as assessed by pregnancy diagnosis, (3) in vivo fertilization of frozen-thawed spermatozoa and early development of fertilized eggs as assessed by superovulation/AI-embryo collection tests and (4) in vitro fertilization of frozen-thawed spermatozoa with oocytes. The percentages of frozen-thawed spermatozoa with normal acrosomal conditions assessed by the abovementioned staining techniques were significantly correlated with the conception rates of routine AI, rates of transferable embryos in superovulation/AI-embryo collection tests and in vitro fertilization rates. These results are consistent with new suggestions that the distribution of acrosomal tyrosine-phosphorylated proteins as well as the acrosomal morphology of frozen-thawed spermatozoa are AI fertility-associated markers that are valid for the prediction of AI results and that low conception rates in AI using frozen-thawed spermatozoa with poor acrosomal conditions result from reproductive dysfunctions in the processes between sperm insemination into females and early embryo development, probably failed fertilization of frozen-thawed spermatozoa with oocytes.     

白藜芦醇对奶牛性控冻精品质和体外受精能力的影响

本研究旨在探究不同浓度白藜芦醇处理对奶牛性控冻精品质和体外受精能力的影响。在解冻后的奶牛性控冻精中分别添加0、10~(-3)、10~(-4)、10~(-5) mol/L的白藜芦醇,各组精子在受精液中获能孵育1.5 h后,测定精子质量和体外受精能力。结果表明:添加白藜芦醇均可有效降低性控冻精中活性氧(ROS)含量、提高顶体完整活精子比例(P0.05),其中10~(-3)、10~(-4) mol/L的白藜芦醇处理对降低ROS含量最为显著;10-4 mol/L的白藜芦醇处理可显著降低丙二醛(MDA)含量并提高性控冻精的卵裂率和囊胚率(P0.05)。综上所述,白藜芦醇作为一种外源性天然抗氧化剂,通过降低性控精液中过量的ROS水平、抑制精子脂质过氧化反应、保护顶体完整性,从而提高性控精子质量和体外受精能力。