Proportion Of Capacitated Spermatozoa Research Articles

Nirmatrelvir has detrimental effects on sperm function by altering the PI3K/PDK1/AKT signaling pathway

Nirmatrelvir (NMV) is a recently developed selective inhibitor of the main protease of Sars-Cov-2 that reduces the severity of infection. Despite its widespread use and various side effects, NMV's effect on male fertility is still unclear. This study was thus established to investigate how NMV affects male fertility. For experiments, Duroc spermatozoa were incubated with various concentrations of NMV (0, 0.1, 1, 10, 50, and 100 μM). Then, sperm motility, motion kinematics, capacitation status, intracellular ATP level, and cell viability were evaluated. In addition, the expression levels of phospho-PKA substrates, tyrosine-phosphorylated proteins, and PI3K/PDK1/AKT signaling pathway-related proteins were measured by western blotting. Our results showed that sperm motility, motion kinematics, proportion of capacitated spermatozoa, and intracellular ATP level were significantly decreased by NMV in a dose-dependent manner. Moreover, PKA activation was significantly suppressed by NMV, and expression levels of PI3K, phospho-PDK1, AKT, and phospho-AKT (Thr308 and Ser473) were significantly increased in a dose-dependent manner. Combining these findings, it is suggested that NMV has detrimental effects on sperm function by inducing abnormal changes in the PI3K/PDK1/AKT signaling pathway, resulting in PKA deactivation. Therefore, there is a need to pay particular attention to its male reproductive toxicity when NMV is administered.

Effects of supplemental antioxidants on in vitro fertility measures for cryopreserved boar spermatozoa

This work aims to evaluate how supplementing a commercial freezing media with butylated hydroxytoluene (BHT), or reduced glutathione (GSH), or their combination affected in-vitro measures of boar sperm after cryopreservation. One ejaculate was collected from 30 high-fertility boars in a weekly collection rotation. Samples were diluted 1:1 in an extender and cooled before overnight shipping at 17 °C to the freezing lab. On arrival, samples were split into the treatments with the following additions before cryopreservation; 1) semen without additional antioxidants (Control), 2) semen with 1 mM BHT, 3) semen with 2 mM GSH, and 4) semen with 1 mM BHT+2 mM GSH. Semen was evaluated for motility kinetics at 30, 120, and 240 min after thawing. Flow cytometry assessments were performed at 60 min after thawing. At all-time points evaluated, total and progressive motility were greater (P ≤ 0.05) in semen cryopreserved with GSH than in Control. No (P > 0.05) differences between Control and other treatment groups were observed in viability, or acrosomal and mitochondrial membrane integrity; however, the proportion of capacitated spermatozoa were reduced (by −21.17%) in semen treated with BHT + GSH compared to Control (P ≤ 0.05). In contrast, there was a higher (P ≤ 0.05, +21.18%) superoxide anion production in the Control than in the BHT + GSH. For IVF, semen cryopreserved with both antioxidants (BHT + GSH) had a negative (P < 0.05) impact on fertilization rate (−54.11%) compared to Control. However, for the blastocysts rate, there were more (+22.75%) blastocysts (P ≤ 0.05) for BHT compared to Control. These results indicate that commercial media supplemented with GSH increased motility but impaired in vitro fertilization rate. On the other hand, media supplemented with BHT improved the in vitro fertilizing ability of the frozen-thawed sperm cells. Therefore, we suggest the supplementation with 1 mM of BHT in the formula of commercial freezing media used in the present experiment.

Sperm functional attributes and oviduct explant binding capacity differs between bulls with different fertility ratings in the water buffalo (Bubalus bubalis).

We report here the differences in sperm functional attributes and sperm-oviduct binding index in bulls with different field fertility ratings. Cryopreserved spermatozoa from Murrah buffalo bulls (n=9) with different fertility ratings were evaluated for membrane integrity, capacitation status, acrosome intactness and protein tyrosine phosphorylation status. Frozen--thawed spermatozoa were incubated with oviduct explants for 1h under 5% CO2, 38.5°C with 95% relative humidity and the number of spermatozoa bound to the unit area of oviduct explants (binding index; BI) was assessed using 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1) fluorescent staining. The proportion of membrane-intact and acrosome-intact spermatozoa was significantly (P<0.05) higher and the proportion of capacitated spermatozoa was significantly (P<0.05) lower in high-fertile bulls compared with medium- and low-fertile bulls. The relationship between BI and bull fertility was significant and positive (r=0.69; P=0.04). BI was negatively and significantly (r=-0.83; P=0.01) related to membrane-compromised spermatozoa. It was concluded that the sperm-oviduct explant binding index was positively related to (1) the proportion of membrane-intact spermatozoa in a given semen sample and (2) invivo fertility of the buffalo bull, indicating the possibility of developing a fertility prediction tool using a sperm-oviduct explant binding model, once validated on a greater number of bulls.

Identification of biomarker candidates for fertility in spermatozoa of crossbred bulls through comparative proteomics

Associations between expression of some proteins in spermatozoa and fertility have been sought in recent years to identify the male fertility markers. Since the incidence of sub-fertility is high in crossbred bulls, the present investigation was carried out on high- and low-fertile crossbred bulls to identify fertility markers in spermatozoa through proteomics approach. Sperm proteome of high-fertile bulls were compared with low-fertile bulls using 2D-DIGE and MALDI-TOF-MS techniques and the results were validated with immuno-blotting. The proteins MDH2, ENO1, RIBC1, CAPN7, ATP5D, LacA like protein-2 like, NCAPD3, DECR1, GCNT2, GDI2, TOP and USP12 were over expressed in high-fertile spermatozoa, whereas DST like isoform 1, TMEM43 and BSP1 were over expressed in low-fertile spermatozoa (P < 0.05). The differential expression ranged from 1.57 (GDI2) to 5.1 (BSP1) fold between the two groups. Based on the GO annotation, majority of them were involved in cellular and metabolic processes, with catalytic and binding activities, and localized in cell and organelles. Among these proteins, ENO1 and BSP1 were selected based on the degree of differential expression and reliability in identification, for further validation. Immuno-blotting studies indicated that ENO1expression was positively correlated (P < 0.05) while the expression of BSP1 was negatively (P < 0.01) correlated with bull fertility. The proportion of capacitated spermatozoa in frozen thawed spermatozoa of low-fertile bulls was higher (P < 0.05) as compared to high-fertile bulls. Collectively, the study identified some potential molecules in spermatozoa of bulls, which may act as a panel of biomarkers for fertility.

Caffeine induces ram sperm hyperactivation independent of cAMP‐dependent protein kinase

We have previously shown that a cocktail-containing phosphodiesterase inhibitors (theophylline and caffeine), a phosphatase inhibitor (okadaic acid) and dibutyryl-cAMP promoted specific protein tyrosine phosphorylation in ram spermatozoa during incubation in capacitating conditions. Here, we show, for the first time, that this cocktail induced a progressive time-dependent increase in the capacitated-sperm subpopulation. The addition of either the analogue of adenosine, 2-chloro-2'-deoxyadenosine (Cl-Ado) or caffeine provided a significant increase in the proportion of capacitated spermatozoa and total tyrosine phosphorylation. Computer-assisted semen analysis was used to identify hyperactivated spermatozoa by setting maximum threshold for linearity (< or =45%) and minimum for amplitude of lateral head displacement (> or =3.5 microm). Our results showed that ram spermatozoa can be capacitated in vitro without displaying hyperactivated movement. Among the above-mentioned compounds, only caffeine was able to induce hyperactivation that achieved the maximal response at 8 min of incubation, with a significant increase in hyperactivated spermatozoa of 44.4 +/- 5.6% related to control samples. Flow cytometry analyses showed that caffeine induced a significant increase in the content of calcium in viable spermatozoa during the time-course of incubation in capacitating conditions. BAPTA-AM, a cell-permeable calcium chelator, did not suppress the caffeine-dependent hyperactivation. Quantitative analysis revealed that the addition of caffeine or Cl-Ado accounted for an increase in intracellular cAMP level. However, this increase in cAMP does not seem to be responsible for the caffeine-induced hyperactivation because the cAMP-elevating agents (cocktail) did not promote hyperactivation either, although they greatly induced capacitation and protein tyrosine phosphorylation. The inhibition of PKA with H89 reduced both capacitation and protein tyrosine phosphorylation although hyperactivation increased. These results suggest that calcium from internal stores would be enough to initiate the hyperactivated movement, and that protein tyrosine phosphorylation implicated in ram sperm hyperactivation would be regulated by calcium rather than by PKA-dependent cAMP.

Capacitation status of fresh and frozen-thawed buffalo spermatozoa in relation to cholesterol level, membrane fluidity and intracellular calcium

The present study was conducted to assess the capacitation status of fresh and frozen-thawed buffalo spermatozoa and its relationship with sperm cholesterol level, membrane fluidity and intracellular calcium. Semen from seven buffalo bulls (eight ejaculates each) was divided into two parts. Part I was used as fresh semen and part II was extended in Tris–egg yolk extender, equilibrated (4 °C for 4 h) and frozen at −196 °C in LN 2. The fresh and frozen-thawed spermatozoa were assessed for capacitation status using chlortetracycline (CTC) fluorescent assay, membrane fluidity using merocyanine 540/Yo-Pro-1 assay and intracellular calcium using Fluo-3 AM with flowcytometry. Results revealed a significant ( P < 0.01) increase in capacitated sperm population in frozen-thawed semen compared to fresh semen (42.21% vs 14.32%). Similarly, a significantly ( P < 0.01) higher proportion of frozen-thawed live spermatozoa showed high membrane fluidity (53.62% vs 25.67%) and high intracellular calcium (43.68% vs 11.72%) compared to fresh semen. The sperm cholesterol was significantly ( P < 0.01) reduced after freezing–thawing as compared to fresh semen. The proportion of capacitated spermatozoa (CTC pattern B) was positively correlated with the proportion of sperm with high intracellular calcium ( r = 0.81) and high membrane fluidity ( r = 0.65), and negatively correlated with cholesterol level ( r = −0.56) in frozen-thawed semen. The membrane fluidity was also strongly associated with the cholesterol level and intracellular calcium. The study concluded that changes in buffalo spermatozoa and established the relationship among capacitation status, sperm cholesterol level, membrane fluidity and intracellular calcium concentration in frozen-thawed spermatozoa.

Heparin and quercitin generate differential metabolic pathways that involve aminotransferases and LDH-X dehydrogenase in cryopreserved bovine spermatozoa

Heparin and quercetin induce capacitation in spermatozoa through membrane receptor binding and inhibition of Ca-ATPase of the plasma membrane, respectively. Although capacitation is energy intensive, ammonia from amino acid metabolism can inhibit respiration and Krebs cycle activity. The objective was to determine activities of key enzymes in bull spermatozoa that contribute to the redox state and supply energy for capacitation. Malate dehydrogenase (MDH-NAD +), alanine and aspartate aminotransferases (ALT, AST), and lactate dehydrogenase-X (LDH-X) were measured spectrophotometrically (340 nm); mean (±S.D.) activities in control spermatozoa were 7.65 ± 1.67, 0.45 ± 0.05 and 0.74 ± 0.14 × 10 −2 U/10 8 spermatozoa for MDH-NAD +, ALT and AST, respectively, and were 2.83 ± 0.66 U/10 8 spermatozoa for LDH-X. Heparin decreased ( P < 0.05) activities of MDH-NAD +, ALT, AST and LDH-X (78, 53, 66 and 66% of control levels, respectively); we inferred that amino acid catabolism was decreased. Quercetin decreased ( P < 0.05) activities of MDH-NAD + and ALT (60 and 49% of control levels), but activities of AST and LDH-X were not significantly different from controls; apparently maintenance of LDH-X activity supplied pyruvate for cellular metabolism. The proportion of capacitated spermatozoa in controls (8.5 ± 1.73%) was substantially increased ( P < 0.05) by treatment with either heparin (36.2 ± 4.5%) or quercetin (32.8 ± 4.7%), there was no significant difference among groups for acrosomal integrity and sperm viability. In conclusion, heparin- or quercetin-induced capacitation affected different metabolic pathways that modulated the redox state and oxidative metabolism in cryopreserved bovine spermatozoa.

Effect of sperm survival and CTC staining pattern on in vitro fertilization of porcine oocytes

Polyspermy in pig oocytes fertilized in vitro remains unacceptably high. In this study, we evaluated the effects of gamete coincubation time, and determined if the proportion of capacitated spermatozoa would be predictive of the fertilizing ability of frozen-thawed semen in vitro. Cumulus–oocyte complexes were collected from slaughterhouse prepubertal gilt ovaries and matured in vitro for 44 h in TCM199, with EGF, FSH, cysteamine and follicular fluid. Fertilization was induced with 2×10 5 frozen-thawed spermatozoa/ml in TBM. Penetration of oocytes as well as polyspermic fertilization occurred 2 h after insemination. A strong correlation between penetration and polyspermic fertilization rates has been demonstrated, but there was no correlation between the proportion of capacitated spermatozoa, as assessed by chlortetracycline staining, at the time of insemination and fertilization rates. We also compared the results of IVF in three IVF media: TBM, m199 and TALP. Penetration and polyspermy were very different in these three media: 71±19% and 25±13% in TBM, 37±11% and 6±2% in m199, 10±2% and 0% in TALP, respectively. Nevertheless, survival of spermatozoa or modifications of the capacitation status were not different in these media after 6 h incubation. We concluded that survival and capacitation characteristics of the semen used for IVF could not be predictive of the IVF results. It seems necessary to act at the oocyte level to control both variability between replicates and the incidence of polyspermy. Improving the spermatozoa penetration blocking system of the oocytes and reducing the number of sperm-binding sites on the zona pellucida (ZP) are our further objectives.

Relationship between the proportion of capacitated spermatozoa present in frozen-thawed bull semen and fertility with artificial insemination.

The objective of the present study was to confirm the presence of prematurely capacitated spermatozoa in frozen-thawed bull semen and to investigate the relationship of premature capacitation to the fertility of the respective semen. Twenty batches of frozen semen from young AI bulls of the Swedish Red and White breed with known fertility (expressed as 56-day non-return rates; 56 d-NRR) were tested using a Chlortetracycline (CTC) assay to assess capacitation status in frozen-thawed spermatozoa. The status of capacitation, as evidenced in this experiment, was further tested based on the hypothesis that capacitated spermatozoa present in frozen-thawed semen should undergo the acrosome reaction (AR) on co-incubation with homologous zona pellucida (ZP) glycoproteins. The percentage (mean +/- SEM) of uncapacitated, capacitated and acrosome-reacted spermatozoa in the frozen-thawed semen (n = 20) were 49.3 +/- 11.9, 36.3 +/- 8.3 and 14.2 +/- 11.9, respectively. On co-incubation with ZP, there was a significant increase (p = 0.001) in the proportion of spermatozoa undergoing the AR compared to the control with a concurrent decrease in the proportion of capacitated spermatozoa, suggesting that a proportion of capacitated spermatozoa were undergoing the AR. The proportion of viable, uncapacitated spermatozoa present in the frozen-thawed semen was correlated to the 56 d-NRR (n = 20, r = 0.5, p = 0.03). In conclusion, a proportion of spermatozoa in frozen-thawed semen was capacitated and the proportion of viable, uncapacitated spermatozoa present in semen was positively correlated to fertility.

Sperm-oviduct interaction: induction of capacitation and preferential binding of uncapacitated spermatozoa to oviductal epithelial cells in porcine species.

After mating, inseminated spermatozoa are transported to the oviduct. They attach to and interact with oviductal epithelial cells (OEC). To investigate sperm-OEC interactions, we used chlortetracycline to study the capacitation status of boar spermatozoa in coculture with homologous OEC and cells of nonreproductive origin (LLC-PK1, porcine kidney epithelial cell line). Boar spermatozoa were cocultured with OEC and LLC-PK1 cells for 15, 60, 120, or 240 min. The proportion of capacitated spermatozoa in coculture with the isthmic and ampullar cells increased significantly (p < 0.05) during incubation. However, most spermatozoa in coculture with LLC-PK1 cells or blank (medium only) remained uncapacitated. In addition, preferential binding of uncapacitated, capacitated, or acrosome-reacted boar spermatozoa to OEC and the other cell type was investigated. Our approach was to vary the proportions of uncapacitated, capacitated, or acrosome-reacted boar spermatozoa in suspension using long preincubation and lysophosphatidylcholine treatment of semen prior to a very short incubation with OEC or LLC-PK1 cells. The results showed that the majority of spermatozoa that were bound to OEC or LLC-PK1 cells were uncapacitated and that a significant relationship existed between the relative proportion of uncapacitated spermatozoa in the control samples and those bound to LLC-PK1 cells (r2 = 0.43, p < 0.005). However, there was no correlation between the proportion of uncapacitated spermatozoa in the control samples and the proportion of those bound to isthmic or ampullar cells. In conclusion, the results clearly demonstrated the specific nature of the sperm-OEC interaction in the porcine species. This interaction is initiated by uncapacitated spermatozoa binding to OEC and is continued by the induction of capacitation in cocultured spermatozoa.

Tyrosine phosphorylation of a 95 kDa protein and induction of the acrosome reaction in human spermatozoa by recombinant human zona pellucida glycoprotein 3.

Protein tyrosine phosphorylation and induction of the acrosome reaction (AR) in non-capacitated and capacitated human spermatozoa was investigated in response to recombinant human zona pellucida glycoprotein (rhZP3) produced by Chinese hamster ovary cells transfected with a plasmid containing human ZP3 cDNA. rhZP3-containing medium promoted the AR in a high proportion of capacitated spermatozoa (48.6 +/- 3.2%; P < 0.01) compared with control (no rhZP3) samples (14.8 +/- 2.1%). However, rhZP3-containing medium did not cause increased acrosomal exocytosis in non-capacitated spermatozoa (16.8 +/- 3.0%). Induction of the AR was associated with increased tyrosine phosphorylation of a 95 +/- 5 kDa epitope only in capacitated spermatozoa. A dose-dependent increase in the protein phosphorylation of a 95 kDa epitope in response to rhZP3 was detected by [gamma-32P]-ATP labelling of detergent-solubilized sperm proteins. When spermatozoa were co-incubated with monoclonal antibody 97.25 (mAb 97.25) recognizing a 95 kDa tyrosine kinase epitope, there was no rhZP3 induction of tyrosine phosphorylation of the 95 kDa protein. Such co-incubation also markedly inhibited the AR (23.9 +/- 3.1%). These results support the model that initial interaction of the fertilizing spermatozoon with ZP3 involves the tyrosine phosphorylation of a 95 kDa tyrosine kinase protein and that this requires capacitation.

The relationship between membrane status and fertility of boar spermatozoa after flow cytometric sorting in the presence or absence of seminal plasma.

The motility, viability (percent live), capacitation status and in vitro fertility of boar spermatozoa were examined, after staining with Hoechst 33,342 and flow cytometric sorting in the absence or presence of seminal plasma. Viability was higher in unstained controls and when seminal plasma was present in the medium used to collect spermatozoa from the cell sorter than when seminal plasma was absent or in the staining extender only, but motility was highest when seminal plasma was included in the extender only, compared with the controls and other treatments. The proportions of capacitated spermatozoa were increased by sorting, but were lower when seminal plasma was present, rather than absent, from the staining extender and the collection medium. Compared with unstained controls, extension and staining without sorting only increased the proportion of capacitated spermatozoa after washing in preparation for in vitro fertilization. The percentages of polyspermic, penetrated and cleaved oocytes were lower when inseminated with unsorted (stained) than control (unstained) spermatozoa, regardless of the presence or absence of seminal plasma. These parameters were higher for sorted than for control spermatozoa in the absence of seminal plasma, but in its presence penetration and cleavage were substantially lower. The proportions of capacitated spermatozoa were lower when seminal plasma was present in the collection medium only than in the staining extender or when it was absent altogether, but the former treatment substantially reduced the proportions of polyspermic, penetrated and cleaved oocytes, and the proportion of blastocysts. These findings indicate that sperm capacitation associated with flow cytometric sorting can be reduced by the inclusion of seminal plasma in the collection medium, but this treatment reduces the ability of spermatozoa to fertilize oocytes in vitro under these conditions.

Effect of Taurine on In Vitro Fertilization and Embryo Development of BALB/c Mouse Strain.

Inbred BALB/c strain mouse was characterized by a low in vitro fertilization (IVF) rate. However, this strain has been used broadly for various researches, especially for researches in immunology. For improving the fertilization rate in this strain, we used taurine, a sulfur-containing β-amino acid that had been reported to be effective in improving fertilization in several species. Taurine (1 or 10 mM) did not change the proportion of capacitated spermatozoa in vitro. However, 1 mM taurine significantly (P<0.05) improved the fertilizaton rate compared to that of the control. The highest fertilizaton rate (78%) was obtained by addition of 1 mM taurine to TYH medium. The taurine effect on the early embryonic development was also examined in a simple culture medium (KSOM) for 4 days culture. There was no effect of taurine on embryo development. More than 50% of fertilized embryos developed to the blastocyst stage without taurine. The viability of fertilized embryos was proved by embryo transfer to ICR recipient mice. A total of 13 offsprings were born from two recipient mice. The present results indicate that taurine has a supporting effect on the improvement of IVF in inbred BALB/c mouse strain. Fertilized embryos developed to the blastocyst stage in in vitro culture and normal offspring after embryo transfer.