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An objective method for evaluating Angus and Hereford sperm motility.

Angus and Hereford sperm motility was evaluated by an objective spectrophotometric procedure and by a conventional subjective ranking system. Semen was collected by electroejaculation and divided so that one group contained 6 Angus samples and one group contained 12 Hereford samples. Objective procedures indicated that Angus sperm was twice motile as Hereford sperm in 2.9% sodium citrate. This objective procedure depends on the orientation of sperm in a flowing liquid and then spectrophotometrically measuring the sperm's ability to return to randomness when the flow is stopped. During preparation for freezing with liquid nitrogen, microscopic subjective ranking of sperm motility was carried out. Three conditions were studied subjectively: (I) arrival at the laboratory, (II) pre-freeze, and (III) thawed samples. No significant differences in subjective evaluation of sperm motility was found between conditions or breeds. Preliminary results indicate that this objective procedure can distinguish between the sperm motility in 2.9% sodium citrate from two breeds of cattle. Use of this objective procedure for studies relating to fertilization and artificial insemination is evident and there is no theoretical reason why the procedure can not be used with other species.

Animals

Adenine nucleotide changes at initiation of bull sperm motility.

Testicular and cauda epididymal sperm were obtained via catheters previously implanted in the rete testis and proximal vas deferens of bulls and were used to examine the relationships among sperm motility, cyclic adenosine 3':5'-monophosphate (cAMP) level, adenine nucleotide levels, and rates of glucose and oxygen consumption. Testicular, cauda epididymal, and ejaculated sperm contain cAMP-stimulated protein kinase, adenylate cyclase, and nucleotide phosphodiesterase. Treatment of the nonmotile testicular sperm with phosphodiesterase inhibitors resulted in a doubling of cellular cAMP concentration and a 25% increase in their glucose consumption. No change in motility, ATP level, or rate of oxygen consumption was observed. Sperm in neat cauda epididymal semen had flagellating tails but no progressive motility. Dilution of these sperm into glucose-containing buffer resulted in an increase in intracellular cAMP concentration and a decrease in ATP level with concomitant increases in ADP and AMP levels. These biochemical changes occurred within 30 s after dilution and apparently preceded the initiation of progressive motility by most cells. Since sperm in neat cauda epididymal semen became progressively motile when diluted with neat cauda epididymal plasma as well as accessory sex gland fluid or buffer, composition of the fluid surrounding the sperm is not responsible for the initiation of progressive motility upon dilution nor does cauda epididymal plasma contain an inhibitory factor. Perhaps release from contact immobilization provides the stimulation for the initial acquisition of progressive motility by cauda epididymal sperm. We conclude that during epididymal passage sperm develop from a cell physically unresponsive to changes in cAMP concentration to a form which initiates progressive motility upon changes in cAMP concentration.

3',5'-Cyclic-AMP Phosphodiesterases

Initiation of hamster sperm motility from quiescence: effect of conditions upon flagellation and respiration.

The point within the male reproductive tract where sperm motility originates varies with mammalian species. Premotile sperm from hamster, a species whose sperm are still quiescent in the epididymis, were used here to investigate further the parameters involved in the initiation of sperm motility. Two types of motility were produced: (1) partial, weak flagellation by simple dilution; (2) strong, complete motility by inducers (calcium, cyclic adenosine 3':5'-monophosphate) in the presence of amplifiers (caffeine, spermine). Expansion of initiation conditions to test tube volumes revealed that, at high sperm dilutions, bicarbonate could also induce motility. The respiratory consequences of sperm motility induction were measured. A Large, short-term burst of oxygen consumption occurred at a time paralleling the previously reported shifts in nucleotide levels associated with this event.

Animals

Stabilization of adenylate energy charge and its relation to human sperm motility.

The adenylate energy charge of human ejaculated spermatozoa was studied when the sperm motility was perturbed by varying pH, prolonged incubation, and caffeine. Between pH 8 and 9, which was optimal for the sperm motility, the energy charge was in the physiological range of 0.8 to 0.9. Above pH 9, the mobility, ATP content, and adenine nucleotide pool declined rapidly but the energy charge was maintained slightly below 0.8. Below pH 8, the motility also dropped drastically, but the ATP, nucleotide pool, and energy charge fell only slightly. Prolonged incubations of the spermatozoa decreased the motility, ATP, and nucleotide pool. However, the energy charge would remain above 0.6. Caffeine stimulation of the motility caused a rapid fall of ATP and the reduction of the physiological energy charge by 0.2 unit, unless glucose was added. Imidazole which reduced the caffeine-stimulated motility did not alter the physiological energy charge of the spermatozoa. The study showed that the spermatozoa could maintain the energy charge above 0.6 under stress.

Adenine Nucleotides

Protamine gene expression is associated with sperm motility in rams: An integrative experimental and gene network analysis.

Protamine 1 (PRM1) and protamine 2 (PRM2) are essential regulators of sperm chromatin condensation and genome integrity, and their dysregulation has been associated with impaired male fertility. However, their role in rams remains insufficiently characterized. This study investigated the relationship between protamine gene expression and semen quality in rams and explored their potential upstream regulatory mechanisms using gene regulatory network (GRN) analysis. Fifteen ejaculates from five rams were analyzed. Based on total sperm motility using computer-assisted sperm analysis (CASA), ejaculates were classified into a high-motility group (n&#x202f;=&#x202f;8) and a low-motility group (n&#x202f;=&#x202f;7). PRM1 and PRM2 expression levels were quantified by RT-qPCR. Following normality confirmation (p&#x202f;>&#x202f;0.05), parametric tests were applied using the ejaculate as the biological experimental unit. Samples with reduced motility showed significantly lower expression of both protamines (p&#x202f;<&#x202f;0.01). Moreover, progressive sperm motility was strongly correlated with both PRM1 (r&#x202f;=&#x202f;0.71, p&#x202f;=&#x202f;0.019) and PRM2 (r&#x202f;=&#x202f;0.69, p&#x202f;=&#x202f;0.03) transcript levels. Cross-species GRN inference using scGeneRAI and a reference human spermatogenesis dataset identified several hypothesis-generating candidate transcription factors, including HMGB4, HMGB1, H2AFZ, NKX6-1, and SMC3, consistently supported across multiple bootstrap resampling runs. These findings demonstrate a strong association between protamine expression and sperm motility in rams. While the identified candidate regulators provide a valuable framework for future species-specific validation, they also highlight promising candidate molecular biomarkers of male fertility in livestock.

Gene regulatory networks

Improvement of sperm motility in patients with asthenozoospermia by kallikrein treatment.

Parenteral and oral application of kallikrein (EC 3.4.21.8)--a kinin-releasing proteinase from porcine pancreatic tissue--significantly stimulates quantitative and qualitative sperm motility in subfertile males with semen criteria of asthenozoospermia. Improvement of sperm motility was found to exist for at least 3 months following termination of the 7 week duration kallikrein treatment. Additionally, a significant increase in the number of spermatozoa was observed 3 and 5 months after starting parenteral application of kallikrein.

Administration, Oral

Effect of kallikrein on bull sperm motility in vitro.

The application of kallikrein--a kinin-releasing porcine pancreatic kininogenase--exerted considerable stimulation of bull sperm motility in vitro. The kallikrein was used in two concentrations: 1 and 100 KU/ml semen. Both concentrations gave similar effect: at the 4th hour of incubation at room temperature the motility rate of spermatozoa exceeded five and four times, respectively, that of the control samples. Higher penetrating ability of bull spermatozoa in cow's estral secretion was also recorded in the experimental samples treated with kallikrein in both concentrations at the 2nd and 4th hour of incubation in comparison with the controls.

Animals

COXFA4L3 enhances mitochondrial complex IV function to boost ATP synthesis and drive sperm motility.

COXFA4L3 is a testis-specific cytochrome c oxidase subunit that enhances mitochondrial complex IV activity during spermatogenesis. From the analysis of Coxfa4l3 knockout mice, the isoform switch from COXFA4 to COXFA4L3 may increase the potential COX activity, although this activity does not appear in the testis. This latent enhancement becomes evident in sperm, where COXFA4L3 promotes higher respiratory capacity, increasing sperm motility and ATP production. These findings indicate that COXFA4L3 is a key regulator of mitochondrial energy metabolism and may provide insights into the mechanisms underlying male infertility.

Electron Transport Complex IV

A comparison of subjective measurements of human sperm motility and viability with two live-dead staining techniques.

Two commonly used live-dead stains (eosin-nigrosin (EN) and eosin-opal blue (EOB)) were compared with the estimated active spermatozoa in semen samples from patients attending an infertility service. Twenty-eight semen samples were analyzed throughout the day of their collection by estimating the number of active spermatozoa and by staining a portion of the incubated sample (37 degrees C) with each stain. The samples were analyzed 30, 60, 120, 240, and 360 minutes after the initial collection. At 30 minutes there were no significant differences between the estimated values and those of either stain. The slope of the EN stain closely paralleled that of the estimated measurements throughout the remainder of the time periods, while the EOB slope was somewhat steeper. Repeatability of values for semen samples obtained on different days was generally good for each stain. A comparison of semen samples from 85 patients, 30 minutes after collection, showed no significant differences between the numbers of estimated active sperm and the percentage live using the EN stain. Studies of 132 semen samples using the EN stain showed a positive correlation with the over-all quality of sperm motility. The results indicate that there is a role for live-dead staining in assessing semen quality.

Cell Biology

Mineral-driven molecular signatures of energy metabolism underpin sperm motility in buffalo.

The success of spermatogenesis depends on the interplay of various biomolecules that ultimately determine sperm quality. In this study, RNA-seq analysis of frozen-thawed buffalo sperm (n&#x202f;=&#x202f;8) revealed the presence of 263 mineral-associated genes (>1 FPKM) in high (n&#x202f;=&#x202f;4) and 181 in low motile (n&#x202f;=&#x202f;4) sperm groups. Among these, 177 mineral-associated genes were commonly expressed between them, and the majority were upregulated (>1 fold), LOC102391588 (ncRNA; 37-fold), ZNF699 (26.5-fold), MYZAP (13-fold), etc., in the high motile group. The expression of selected mineral-associated genes was validated. The top enriched functions in commonly expressed genes were regulation of transcription by RNA polymerase II (FDR: 4.7&#x202f;&#xd7; 10&#x207b;2; ZNF331, ZNF692, ZNF180, etc.), followed by spermatogenesis (FDR: 2.9&#x202f;&#xd7; 10&#x207b;2; CALR3, ADAM18, ADAM29, etc.), proton transmembrane transport (FDR: 4.0&#x202f;&#xd7; 10&#x207b;2; ATP6V0E1, ATP1A4, ATP6V1B2, etc.) and flagellated sperm motility (FDR: 2.3&#x202f;&#xd7; 10&#x207b;1; CATSPERD, EFCAB6, CABS1, etc.). Additionally, the chromatin remodeling pathway (FDR: 3&#x202f;&#xd7; 10&#x207b;2; PTP4A1, PPM1A, DUSP1, etc.) emerged as the most significant and may suggest that these minerals influence genome packaging and sperm functionality. Mineral-associated genes were predominantly associated with zinc (49%), followed by calcium (20%), phosphorus (10%), iron (5%), sodium (2%), potassium (2%), copper (1%) and other trace elements (11%). Although the current study uses frozen-thawed sperm, the findings indicate that mineral-associated genes are crucial for promoting membrane stability, energy production, motility and chromatin integrity, which may contribute to the superior fertilizing ability of sperm.

Animals

The effect of temperature on sperm motility. II. Is bacterial growth a factor?

The previous demonstration that sperm kept at body temperature (37 degrees C) had a marked deterioration in motility accompanied by an overgrowth of bacteria in the semen and a concomitant decrease in pH led to this study to test the hypothesis that the decrease in motility was caused by the bacteria or by bacterial alteration of seminal pH. Semen specimens from fertile prevasectomy patients with and without added antibiotics were maintained at 20 degrees C and 37 degrees C and evaluated at 3, 12, and 18 hours after collection. There was still a significant deterioration in spermatozoal motility in the samples kept at 37 degrees C even when bacterial growth and change in pH were prevented by buffered antibiotics. Although the decrease in spermatozoal motility at body temperature may in part be attributed to bacterial growth or the products of bacterial metabolism, clearly another factor is present related to time and temperature and independent of the presence of bacteria.

Humans