• Clinical and Experimental Reproductive Medicine
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• v.49 no.4
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• pp.270-276
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• 2022

Objective: The present study assessed the biological characteristics of human spermatozoa at different time intervals (0, 1, 1.5, and 2 hours) after incubation at 37℃. Methods: Twenty-five normozoospermic semen samples were incubated at 37℃. Incubation was performed at four time intervals of 0 (after liquefaction), 1, 1.5, and 2 hours. The samples were evaluated for sperm parameters at each time interval. Results: The rate of sperm progressive motility decreased at 1.5 hours compared to 0 hours as well as 2 hours compared to 1 hour and 0 hours. The rate of non-motile spermatozoa also decreased after 2 hours compared to after 0 hours. No significant changes were observed in sperm viability (p=0.98) and non- progressive motility (p=0.48) at any time intervals. Abnormal sperm morphology increased at 1.5 hours of incubation time (p<0.001). No significant changes were observed in DNA fragmentation at 1 hour compared to 0 hours (median [interquartile range]: 19.5 [4] vs. 19 [4]), as well as at 1.5 hours compared to 1 hour (20 [5]). However, a significant increase in DNA fragmentation was observed at 1.5 hours compared to 0 hours. The mitochondrial membrane potential decreased remarkably after 1 hour of incubation time. No significant differences were observed in the acrosome reaction or malonaldehyde levels at any time point (p=0.34 and p=0.98, respectively). Conclusion: The incubation of normozoospermic samples before use in assisted reproductive technology should be less than 1.5 hours to minimize the destructive effects of prolonged incubation time on general and specific sperm parameters.

• Reproductive and Developmental Biology
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• v.34 no.3
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• pp.185-191
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• 2010

Plasminogen activators (PAs) are serine protease that cleave plasminogen to form the active protease plasmin and may participate in mammalian fertilization. Although correlations have been reported between reactive oxygen species (ROS) and sperm function, the relationship between PA activity and ROS is unknown. We determined the effects of ROS on sperm function and PA activities in boar spermatozoa preincubated under the X-XO system. When spermatozoa were treated with the X+XO group, a significant increase (p<0.05) was observed in the percentage of acrosome reacted spermatozoa compared with that of the control group. However, when antioxidants were added to the medium with X+XO, the rate of acrosome reaction tended to decrease. Also, a significantly lower percentage of acrosome reacted spermatozoa was observed in the X+XO+catalase group at 6 hr of incubation compared with that of X+XO group. The density of malondialdehyde (MDA) was higher in the X+XO group than in different treatment groups. In another experiment, incubation of spermatozoa in medium with X+XO was associated with a significant (p<0.05) increase in activity of tPA-PAI and tPA compared with the control group. Antioxidants decreased the increased activity of tPA-PAI and tPA by preincubation in the X-XO system. Also, a significantly lower (p<0.05) activities of tPA-PAI and tPA were observed in the X+XO+catalase group compared with the X+XO group. No significant differences, however, were observed in the activity of uPA. These results suggest that the increase of acrosome reaction by the X-XO system resulted in increase of PAs activity in the sperm incubation medium.

• Applied Microscopy
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• v.31 no.3
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• pp.223-233
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• 2001

The spermiogenesis of a Korean octopus, Octopus minor, inhabiting western of Korea Sea was observed by electron microscopy . The obtained results are as follows: The spermiogenesis of Octopus miner proceeds through four stages; early- , mid- , and late-spermatid, and mature sperm. An early spermatid is a spherical cell looking light due to the low electron density. The acrosome formed from Golgi complex of the upper nucleus looks dark due to the high electron density. The extra-nuclear rod (enr) stemming from proximal centriole is transformed from round shape into oval shape, elongating to the upper nucleus. In our observation, the axoneme was being formed from distal centriole, and the manchette composed of a number of microtubules is also found around nuclear membrane. In a mid-spermatid, chromatins in the nucleus contract shaping fine threads, and the manchette is also observed around nuclear membrane. Especially, the spherical acrosome is transformed into long oval one which is tinged with a number of horizontal stripes and has the middle electron density. In a late-spermatid, chromatins in the nucleus contract thick and short. Furthermore, the mitochondrial sleeve, in which the axoneme is surrounded with mitochondria, is observed at middle piece. The axoneme has a typical structure of 9+2 and around it, 9 coarse fibers are observed. Also in the acrosome cavity of mature sperm, horizontal striation is found. However, regularly spaced processes are peculiarly observed in there. A sperm is about 390 fm long, whose head is bent a little like a banana while the acrosome region is helical. In the middle piece of sperm, $11\sim12$ mitochondria are surrounding coarse fibers that reach the main piece of tail, while nothing but 9+2 structured axoneme is found in the end piece.

• Journal of Embryo Transfer
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• v.16 no.1
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• pp.35-40
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• 2001

Artificial insemination (AI) with frozen or cooled semen is widely used in commercial fields of cattle and pig. Little is known about characteristics of canine sperm after freezing or cooling. For both practical and commercial goal, the canine semen treated with cooling and freezing should be carried out to exam the fundamentals, including sperm motility, survivability and fertilizing capacity. The aim of this study, thus, was to identify the effects of extended exposure to 4$0^{\circ}C$ on canine semen by motility, survivability, acrosomal changes following different duration. Fifteen ejaculates collected by digital manipulation twice per week from 3 dogs (Shih-Tzu) were divided to 16 aliquots after adding Tris-egg yolk (TE) buffer formulated by our laboratory, and cooled from 37 to 4$^{\circ}C$, by ramp rate of 0.6$^{\circ}C$/min. Each sample was evaluated by their motility, survivability and the acrosomal status at 0h (control), 2h, 12h and 1 d~10 d, respectively. The motility of spermatozoa was graded to 6 levels using the modified method of Seager. The survivability of sperm was assessed using an epifluorescence microscope after Fert/Light (Mole-cular Probes Inc.) staining. To estimate the proportion of the spermatozoa of intact acrosome, 200 spermatozoa were assessed in randomly selected fields, using epifluorescence microscope after FITC/PSA (Sigma) staining. At 2 h after cooling, the motility of most spermatozoa were assessed to be grade 0 and 1. At 12 h, high number of sperm were in grade 0 to 1, however, it was significantly (P<0.05) lower than that of 2 h. From 1 d to 4 d, ~50% of sperm was assessed to grade 0 to 1. On day 7, a little sperm were in grade 0 to 1. No sperm showed motility on day 10. Sperm motility was rapidly reduced by the percent of 10% of grade 0 to 1. From 2 h to 6 h, the number of live sperm was 90% and the sperm chilled for 10 days lived>50%. Acrosomal intact of spermatozoa exposed to 4$^{\circ}C$ for 2 h was 51%, supposed the sperm of control was 100%. Our results suggest that 1) this is easy to transfer and preservation for short periods 2) AI can be used by semen chilled for 6-Day.

• Reproductive and Developmental Biology
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• v.39 no.4
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• pp.117-125
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• 2015

Iron is required for cell viability but is toxic in excess. While the iron-mediated malfunction of testicular cells is well appreciated, the underlying mechanism(s) of this effect and its relationship with fertility are poorly understood. Ferritin is a ubiquitous intracellular protein that controls iron storage, ferroxidase activity, immune response, and stress response in cells. Ferritin light chain protein (FTL) is the light subunit of the Ferritin. Previously, we had identified the FTL in bovine spermatozoa following capacitation. In present study, to investigate the role of Ferritin in sperm function, mice spermatozoa were incubated with multiple doses (1, 10 and $100{\mu}M$) of sodium nitroprusside (SNP), an iron donor. SNP was increased Ferritin levels in a dose-dependent manner. The Ferritin was detected on the acrosome in spermatozoa by immunocytochemistry. Short-term exposure of spermatozoa to SNP increased tyrosine phosphorylation and the acrosome reaction (AR). Finally, SNP affected a significant decrease in the rate of fertilization as well as blastocyst formation during early embryonic development. On the basis of these results, we propose that the effects of Ferritin on the AR may reduce overall sperm function leads to poor fertility in males and compromised embryonic development.

• Applied Microscopy
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• v.34 no.3
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• pp.179-184
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• 2004

Normal and abnormal morphology of the epididymal spermatozoa in the big white-toothed shrew, Crocidura lasiura were studied with the light and electron microscopy. Normal spematozoa were observed with a few abnormal spermatozoa. This indicates that abnormal morphology is no absolute indicator of infertility. However, the existence of the abnormal spermatozoa is related to infertility. Especially abnormal morphology of the sperm head is definitely associated with infertility. The following types of abnormal head morphology of the epididymal spermatozoa in the wild healthy adults of the C. lasiura were described: 1) Nucleus with lack of condensation of the nucleoplasm 2) Destructed acrosome 3) Folded acrosome and plasma membrane 4) Separation of the acrosome from the nucleus 5) Acrosome with irregular condensation 6) Wrongly located granules of the apical body.

• Applied Microscopy
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• v.31 no.2
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• pp.129-141
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• 2001

To investigate the spermiogenesis of the Saghalien Pygmy shrew (Sorex minutus gracillimus), the testis obtained from mature male shrew was studied by electron microscopy, and the following results obtained based on the morphological characteristics of cell differentiation of the seminiferous epithelium in the testis. According to the fine structural differentiation, spermiogenesis of S. minutus gracillimks was divided into Golgi, cap, acrosome, maturation and spermiation phases. Beside, the Golgi and cap phases were subdivided into three steps of early, middle and late phase respectively, and acrosome phase into two steps of early and late phase , and maturation and spermiation phases has only one step respectively. Thus, the spermiogenesis of S. minutus gracillimus was divided into a total of ten steps. The chromatin granules begin to be condensed in the acrosome phase, and a perfect nucleus of sperm was formed at the spermiation phase. Mancette were appeared from the late acrosome phase to the maturation phase. The formation of sperm tail began to develop in the late Golgi phase, and completed at the spermiation phase. Multivesicular bodies were appeared from the Golgi phase to the maturation phase, recognized with pale, pale and moderate, and dense at Golgi, cap and acrosomal and matulation phases respectively.

• The Korean Journal of Zoology
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• v.24 no.2
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• pp.65-75
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• 1981

The formation of the acrosome during spermatogenesis in Gerris paludum was studied. The Golgi bodies are dispersed randomly in the cytoplasm at the early stage of the spermatocyte and get together to form several group of many bodies, and then they are equally divided into the spermatids by the meiotic divisions. The acroblast first appears in the form of a vesicle and soon an acrosomal granule is differentiated within it. The acroblast is separated from the acrosomal granule at the posterior of the nucleus and is finally sloughed off along the tail filament. The acrosome, after moving to the side of the nucleus opposite the mitochondrial derivatives, differentiates into two zones. The two basal bodies and the differentiated tip originate from the sheath. The basal bodies appear at the proximal part of the sheath simply in contact with the core on one side. During elongation and and narrowing of the acrosomes of the spermatids, they surround the one side at the base of the acrosome and finally all the other are immediately adjacent to the nucleus. The differentiated tip continues to the sheath at the anterior of the cores and is elongated prior to the two basal bodies. They appear to be contiguous twin-tubes, not a single granule in the later stage of the spermatids, and a group of the basal bodies in the sperm bundle.

• Korean Journal of Animal Reproduction
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• v.4 no.1
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• pp.20-27
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• 1980

This experiment was to study semen properties of Charolais for the a, pp.ication ot artificial insemination. The result obtained were summarized as follows: 1. In the preservation of liquid semen for 6 days, the survival rates of Charolais semen averaged 57.14% in skim milk solution and 58.17% in tris buffer solution. There were not differences. 2. Recovery of semen after thawing was vigorous in the semen that was diluted and frozen in 48 hrs. 3. The real rates of survival sperm for Charolais averaged 83% after living sperm was diluted and stained for 6 days. 4. Methylene blue reduction test diluted semen was fresh when it was diluted within 48 hrs. 5. If the diluted semen was preserved below 5$^{\circ}C$ in Charolais, the pH decreased by 0.2 in a day. 6. Diluted semen was more resistant to the cold shock than fresh semen. 7. In resistance against hot shock, sperm was almost dead in 20 minutes in 46.5$^{\circ}C$ in diluted semen, while it was dead in 30 minutes in 42.5$^{\circ}C$ in diluted semen. 8. In examination of morphological changes of sperm acrosome for 6 days, normal sperm in skim milk solution and tris buffer solution was 80% and 76.97% respectively, swelling sperm 12.8% and 15.27%, deficient sperm 0.6% and 0.97% abnormal staining 3.07% and 5.25%, immature sperm 0.28%, and 0.23%, whereas other abnormal sperm was 1.28% and 1.42%.

• Clinical and Experimental Reproductive Medicine
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• v.29 no.2
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• pp.105-115
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• 2002

Objective : To evaluate the effects of the reactive oxygen species (ROS) generated with a xanthine (X) and xanthine oxidase (XO) system on sperm function, the change of sperm characteristics, lipid peroxidation, and DNA fragmentation in bovine spermatozoa. Materials and Methods: ROS were produced using a combination of 1000 uM X and 50 mU/ml XO. The ROS scavengers: superoxide dismu tase (SOD) (200 U/ml) and catalase (500 U/ml) were also tested. Spermatozoa were incubated for 2 hours in BWW medium with a combination of X-XO supplemented with or without ROS scavengers at $37^{circ}C$ under 5% $CO_2$ incubator. Sperm movement characteristics by CASA (computer-aided sperm analysis), HOST (hypoosmotic swelling test), Caionophore induced acrosome reaction, malondialdehyde formation for the analysis of lipid peroxidation, the percentage of DNA fragmentation using the method of TdT-mediated nick end labelling (TUNEL) by flow cytometry were determined after 2 hours incubation. Results: The action of ROS on bovine spermatozoa resulted in a decreased in capacity for sperm motility, Ca-ionophore induced acrosome reaction and membrane integrity, an increased in malondialdehyde formation and the percentage of sperm with DNA fragmentation. In the effects of antioxidant, catalase completely alleviated the toxic effects induced by the ROS in terms of sperm function and characteristics, however SOD exhibited no capacity to reduce the toxic effects. Conclusion: The ROS can induce significant damages to sperm functions and characteristics. The useful ROS scavengers can minimized the defects of sperm function and various damages of spermatozoa.