• Asian-Australasian Journal of Animal Sciences
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• v.8 no.2
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• pp.123-127
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• 1995

In vitro embryo production (IVP) is affected by various factors during in vitro maturation, fertilization, and development. In this experiment, the effect of ovary type, quality of follicular oocyte, medium used for fertilization, presence of hormone in medium, sperm concentration on in vitro maturation and fertilization were examined for effective IVP. In vitro maturation was carried out using TCM-199 supplemented with 15% FCS and hormones in 5% $CO_2$ incubator for 24h. In vitro fertilization was performed with frozen-thawed sperm in modified mTALP medium containing 0.3% BSA, $10{\mu}g/ml$ heparin, and 5mM/ml caffeine for 24h. The fertilized embryos were co-cultured on monolayer of cumulus cells in TCM-199. When oocytes were collected from functionally active and inactive ovaries, maturation rate was 76.9 and 7.7%, respectively. When oocytes were classified morphologically to good and poor grades, maturation rate was 75 and 58.8%, respectively. FSH + LH + $E_2$ (86.4%) showed higher maturation rate than control (53.0%) and FSH (73%). The fertilization rate was 28.2, 100 and 91.7% in $1.6{\times}10^5$, $5.0{\times}10^5$ and $10.0{\times}10^5$ sperm concentration per ml. When oocytes were fertilized in mTALP and BO media, fertilization and cleavage rates of oocytes in mTALP were higher (84.3 and 56.9%) than those (67.4 and 23.3%) in BO medium. In this experiment, in vitro maturation, fertilization and development of oocytes were affected by type of ovary, grade of oocyte, hormones, sperm concentration and fertilization medium.

• Reproductive and Developmental Biology
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• v.34 no.1
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• pp.21-25
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• 2010

The purpose of this study was to investigate the effects of the collection time, co-culture and sperm penetration of canine oocytes on in vitro maturation and fertilization. The oocytes were cultured in TCM-199 media containing hormonal supplements (10% FCS, 10 IU/ml HCG, 10 IU/ml PMSG) at 5% $CO_2$, 95% air, $38^{\circ}C$. The in vitro maturation rate to MII stage of in vitro oocytes recovered from ovaries that collected at follicular, luteal and inactive phases of the reproductive phase for 44~72 hrs were 19.2%, 12.2%, and 6.0%, respectively. Follicular phases oocytes had a significantly higher in vitro maturation rate than oocytes collected at luteal and anestrus stage (p<0.05). The in vitro maturation rates to the MII stage of canine oocytes after 48 hrs of culture with glutathione, pyruvate, or glutathione + pyruvate were 12.5%, 10.7%, and 17.5%, respectively. This was higher than that in both alone or the combination of the two compared to the control group (19.0%). The sperm penetration rates of in vitro matured oocytes by fresh and frozen semen were 29/80 (36.3%) and 18/80 (22.5%), respectively. Although there are limited reports about canine oocytes co-culture and in vitro fertilization, our results on in vitro maturation is comparable to the results from other researches.

• Asian-Australasian Journal of Animal Sciences
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• v.24 no.9
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• pp.1199-1203
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• 2011

The objective of this study was to examine the binding potential of sperm to the epithelium of the sperm storage tubules (SST) in vitro and in vivo to assess the functional maturation of fowl sperm. Sperm from the testis, epididymis, as well as the proximal, middle and distal vas deferens were incubated in vitro with either the uterovaginal junction (UVJ)- or infundibular tissue containing SST at $39^{\circ}C$ for 30 min. Aliquots of sperm were also artificially inseminated into the uteri of hens, and the UVJ and infundibulum were collected 24 h post artificial insemination (AI). After incubation and AI, tissues were washed to remove loosely adhered sperm and subjected to fluorescence staining with 4', 6-diamidino-2-phenylindole, dihydrochloride (DAPI) for counting the number of bound sperm per 0.25 mm2 of surface area. Sperm from the testis, epididymis, and the three segments of the vas deferens exhibited their differential (p<0.05) binding capacity, which increased gradually from the testicular to distal vas deferens sperm under both in vitro and in vivo conditions. Existing similar trend, sperm, regardless of their source had a lesser affinity to bind to the epithelium of the infundibular SST than to the UVJ-SST. These experimental results suggested that fowl sperm may undergo gradual changes in the process of functional maturation, whereby they gain the ability to bind to the epithelium of the SST during their passage through the male reproductive tract (MRT).

• Clinical and Experimental Reproductive Medicine
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• v.18 no.1
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• pp.23-34
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• 1991

In order to study the sperm-egg interaction during fertilization process in mouse, the effects of sperm concentration, the duration of capacitation and insemination, the stages of maturation and development of eggs, and sperm extracts and BSA on sperm binding to egg were examined. Sperm-egg binding was increased depending on sperm concentration within the range of $10^3-10^6$ sperm/ml. It showed the most numbers of sperm-egg binding at 60min from the beginning of preincubation(capacitation) and insemination, respectively. During sperm capacitation, sperm-egg binding inhibitor was released from sperm into the incubation medium. Sperm extracts containing trypsin-like enzyme which is secreted through the acrosome reaction increased the binding. BSA in the culture medium showed a positive effect on the binding. It is suggested that physicochemical alterations of zona pellucida in the process of maturation and fertilization of eggs leaded to inhibition of sperm-egg binding.

• Asian-Australasian Journal of Animal Sciences
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• v.23 no.11
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• pp.1421-1426
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• 2010

We investigated the effects of various concentrations of ${\beta}$-hydroxybutyrate (BHB, 0, 0.1, 1 and 10 mM), a ketone body, added to chemically-defined maturation medium with or without energy substrates (glucose, pyruvate and lactate) on nuclear maturation rates up to the metaphase stage of the second meiotic division (M-II stage). In addition, we also assessed the influence of BHB on glutathione content, sperm penetration rate and embryonic development up to the blastocyst stage of oocytes matured under the presence of these energy substrates. Nuclear maturation rates up to the M-II stage of oocytes matured with BHB in each concentration group did not show a significant increase compared with the control (0 mM) groups in both the presence and absence of energy substrates. Although glutathione contents were not significantly different in each BHB concentration group, the sperm penetration rate in the 1 mM BHB group was significantly higher (p<0.05) and the embryonic development rate of oocytes up to the blastocyst stage was significantly lower (p<0.05) than the respective values of the control groups. These results suggest that BHB added to a chemically-defined maturation medium may stimulate sperm penetration while inhibiting embryonic development of porcine oocytes.

• Korean Journal of Animal Reproduction
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• v.20 no.1
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• pp.63-69
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• 1996

Bovine immature oocytes cultured for various times in TC-199 medium were inseminated with frozne-thawed spermatozoa in TC-199 medium supplemented with caffeine(5mM) and heparin(10$\mu\textrm{g}$/ml). Sperm penetraton was possible in oocytes at any stage of maturation, but penetration rates were lower in oocytes inseminated 0~16h (60~76%) than 20h (98%) after culture. Formation of male and female pronuclei were first observed in oocytes inseminated 8h after cultrue. Formation of male and female pronuclei were first observed in oocytes inseminated 8h after culture. The proportions of polyspermy were high(50~76%) in oocytes inseminated at any stage of maturation. Sperm penetration into oocytes at the GV stage started at 8h after insemination and the penetration rates gradually increased as time after insemination proceeds. The proportion(35%) of oocytes matured beyond metaphase-II 20h after sperm-oocytes incubation was low. When oocytes were incubated without spermatozoa in TC-199 medium, maturation rates were significantly higher (P<0.001) in those without(45 and 84% for 16 and 20 h) than with (0 and 36% for 16 and 20 h) caffeine and heparin. These results indicate that TC-199 medium with caffeine and heparin is not suitable for maturation and fertilization of immature oocytes and may inhibit male pronuclear formation in the cytoplasm.

• Asian-Australasian Journal of Animal Sciences
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• v.12 no.1
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• pp.22-27
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• 1999

Bovine oocytes with compact and complete cumulus cells were cultured in 6 groups for up to 24h in TCM199 buffered with 25 mmol/1 HEPES and supplemented with 10% FCS, 1 mg/ml $17{\beta}$-estradiol, 20 IU/ml hCG. Half of the oocytes at each group cultured in the presence of $25{\mu}g/ml$ cycloheximide at different times during maturation (0, 6, 12, 18, 20, 22 h) were fixed at 24 h of maturation to examine the nuclear progression. The rests of them were inseminated with frozen-thawed spermatozoa in medium BO with 10 mg/ml BSA and 10 mg/ml heparin and fixed after additional 18-20 h culture to evaluate the sperm head transformation. When a protein synthesis inhibitor was added at the onset of the maturation, the oocytes were prevented to proceed GVBD. A few of the oocytes (16%) were able to be penetrated and sperm head decondensation was inhibited either. Addition of cycloheximide after 6-12 h of culture resulted in an increasing percentage of GVBCD (more than 80%), but the oocytes became arrested in M-I (69.2%). More than half of the oocytes was penetrated with a decondensing sperm head. Formation of male pronucleus was first obtained at 12 h of culture in the presence of cycloheximide. When cycloheximide was added from 18 h of culture onwards, nuclear progression to M-II was increasingly restored (80.4-85.5%). The proportion of male and female pronuclear formation increased from 17.9% to 46.2%. It is concluded that protein synthesis is necessary not only for GVBD and development from M-I to M-II, but also for sperm head decendensation and male pronuclear formation in bovine oocytes.

• Journal of Animal Reproduction and Biotechnology
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• v.35 no.1
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• pp.73-85
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• 2020

A high-cholesterol diet can reduce male fertility. However, it is not known whether a high-cholesterol diet can regulate the expression of genes involved in sperm maturation and sperm fertilizing ability. Quercetin, a natural product, is known to have cytoprotective effects by regulating lipid metabolism in various cell types. This study aimed to confirm the expression of genes involved in sperm maturation in the testes of mice fed a high-cholesterol diet and to determine whether quercetin can reverse the genetic regulation of cholesterol. Mice were divided into groups fed a normal chow diet and a high-cholesterol diet. Mice fed the high-cholesterol diet were dose-dependently supplemented with quercetin for 6 weeks. Investigations using quantitative PCR and in situ hybridization revealed that the high-cholesterol diet alters the expression of genes associated with sperm maturation in the testes of mice, and this was reversed with the supplementation of quercetin. In addition, the high-cholesterol diet regulated the expression of genes related to lipid metabolism in the liver of mice. Under a high-cholesterol diet, quercetin can improve male fertility by regulating the expression of genes involved in sperm maturation.

• Applied Microscopy
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• v.22 no.2
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• pp.97-117
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• 1992

In order to study process of spermiogenesis of the Korean greater horseshoe bat, Rhinolophus ferrumequinum korai, the cycle of seminiferous epithelium was examined by the light and electron microscope and the following results were obtained based on the epithelial cell differentiation. 1. Spermiogenesis occurred from early July to mid-Octber, and spermatogenic activity was vigorous from mid-August to late September. Spermatocytes including spermatogonia were found to be degenerated in only July. It is deduced that the degeneration serves as the mechanism to regulate effective use of energy to prepare for mating and hibernating periods, and regulation of breeding cycle. 2. Spermiogenesis of the Korean greater horseshoe bat was divided according to differentiation of the cell structure, into Golgi, cap, acrosome, maturation and spermiation phases; Golgi, cap and spermiation phases were further divided into two steps of early and late phase respectively, and acrosome phase into three steps of early, mid and late phases, and maturation phase has only one step. Hence, the spermiogenesis consists of ten phases. The first research was done in this article on the changes of chromatin with nucleus, the time of appearance and disappearance of chromatin granules, in case of Korean greater horseshoe bat (Rhinolophus ferrumequinum korai). Chromatin granule began to be condensed in late Golgi and the condensation proceeded to form an irregular mass of a electron-dense chromatin in a form of circular cylinder in the center of nucleus at the phase of maturation. Finally, the chromatin condensation proceeded and perfect nucleus of sperm with homogeneous density was formed when the sperm was separated from Sertoli cell. Therefore, appearance and disappearance of chromatin granules occurred in the period of time between late Golgi and maturation phase, The tail of sperm began to develop in early cap phase, Numerous lipid droplets were obseved in the cytoplasm of spermatids during the maturation phase, which seemed to be used as energy source necessary to make mature sperm during spermiogenesis.

• Clinical and Experimental Reproductive Medicine
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• v.42 no.3
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• pp.77-85
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• 2015

One of the key factors of early development is the specification of competence between the oocyte and the sperm, which occurs during gametogenesis. However, the starting point, growth, and maturation for acquiring competence during spermatogenesis and oogenesis in mammals are very different. Spermatogenesis includes spermiogenesis, but such a metamorphosis is not observed during oogenesis. Glycosylation, a ubiquitous modification, is a preliminary requisite for distribution of the structural and functional components of spermatids for metamorphosis. In addition, glycosylation using epididymal or female genital secretory glycans is an important process for the sperm maturation, the acquisition of the potential for fertilization, and the acceleration of early embryo development. However, nonemzymatic unexpected covalent bonding of a carbohydrate and malglycosylation can result in falling fertility rates as shown in the diabetic male. So far, glycosylation during spermatogenesis and the dynamics of the plasma membrane in the process of capacitation and fertilization have been evaluated, and a powerful role of glycosylation in spermatogenesis and early development is also suggested by structural bioinformatics, functional genomics, and functional proteomics. Further understanding of glycosylation is needed to provide a better understanding of fertilization and embryo development and for the development of new diagnostic and therapeutic tools for infertility.