Light and electron microscopic morphology of the fertilized egg and fertilized egg envelope of Poropanchax normani, Poeciliidae, Teleostei

김동희 한국현미경학회 2022 Applied microscopy Vol.52 No.1

We examined the morphology of the fertilized egg and the fine structure of fertilized egg envelopes of Poropanchax normani belonging to the family Poeciliidae, also known as Norman’s lampeye using light and electron microscopes. The fertilized eggs with narrow perivitelline space were found to be spherical and demersal, additionally containing small oil droplets in the vitelline membrane. Further, a bundle of adhesive filaments was observed to be present on one side of the fertilized egg. These filaments possessed remarkably high elasticity and were approximately 1-3 mm in length. The size of the fertilized egg was determined to be about 1.49 ± 0.07 mm ( n = 30). The outer surface appeared smooth, and adhesive filaments originating at different location of the surface of the envelope were found to be distributed around the egg envelope and were joined together to form a single long bundle in scanning electron microscopic observation. A peak-like structure formed of several straight wrinkles was observed around the micropyle. However, the complete structure of the micropyle could not be studied due to the depth at which it was located. Additionally, the total thickness of the egg envelope was ascertained to be approximately12.5–14.5 μm. The egg envelope consisted of two distinct layers, an outer electron dense layer and an inner lamellar layer, further consisting of 10 sublayers of varying thicknesses. Collectively, it was observed that the morphological characteristics of the fertilized egg, fine structures surrounding the micropyle, outer surface, adhesive structure consisting adhesive filaments, and sections of fertilized egg envelope displayed species specificity.

경골어류 카라신과 Hyphessobrycon serpae의 수정란 난막 미세구조

김동희,등영건,이규재,Kim, Dong-Heui,Deung, Young-Kun,Lee, Kyu-Jae 한국현미경학회 2005 Applied microscopy Vol.35 No.2

카라신과(Characidae)에 속하는 Hemigrammus ocellifer, Gymnocorymbus ternetzi 및 Hemigrammus caudovittatus 의 경우 동물극 쪽에 정자의 통로인 난문 (micropyle)이 있으며, 3종 모두 난문 주위에 난막의 융기선이 방사형으로 위치하여 공통적인 특징을 보이지만 난막의 단면구조는 종에 따라서 서로 다른 것으로 알려져 있다. 그러나 난문의 구조가 카라신과 어류의 공통적인 과의 특성인지는 아직 밝혀져 있지 않다. 따라서 본 연구는 카라신과 어류에서 난문의 구조가 과의 공통적인 특징인지 아니면 종만이 가지는 종특성인지를 확인하고 계통분류학적 기초 자료를 얻기 위하여 Hyphessobrycon serpae의 수정란과 난막의 외부 및 내부형태를 광학현미경, 주사전자현미경 및 투과전자현미경을 이용하여 관찰하고자 하였다. Hyphessobrycon serpae의 수정란은 구형의 무색투명한 부착성 및 침성란으로 유적 (oil droplet)과 부속사는 관찰되지 않았다. 동물극 쪽에 수정을 위한 정자의 통로인 한 개의 난문 (micropyle)이 관찰되었고, 난문 주위에는 난막의 융기선이 방사형으로 배열하고 있었으며 난막의 융기선은 $13{\sim}15$개로 수정란마다 약간의 차이를 보였다. 난막의 표면은 산란상에 부착하는 기능을 수행하는 것으로 생각되는 망상형의 섬유상 구조물들이 분포하고 있었으며 이 망상구조물 내부에 pore canal이 산재하고 있었다. 수정란 난막의 두께는 $0.9{\sim}1.0{\mu}m$였으며 3층으로 구성되어 있었다. 외층은 부착기능을 하는 전자밀도가 가장 높은 섬유상층이었고, 중층은 표면에서 관찰되었던 pore canal들이 중층이 끊어진 형태로 완전히 관통되어 있었으며 내층은 전자밀도가 서로 다른 $6{\sim}7$층의 다층구조를 하고 있었다. 이상과 같이 Hyphessobrycon serpae의 수정란 난막의 미세구조적 특징은 이 종만이 가지는 독특한 형태학적 형질로서 종을 분류하는데 사용될 수 있으며 난문 주위에 난막의 융기선이 방사형으로 배열하고 있는 것은 카라신과 어류의 공통적인 과 (Family)의 특성으로 생각된다. The ultrastructures of the fertilized egg envelope from Hyphessobrycon serpae belonging to Characidae was studied using scanning and transmission electron microscopes to get systematic fundamental data for classification of species and to confirm whether micropyle is a common trait of Characidae or not. The fertilized egg was of colorless, transparent, spherical, adhesive and demersal type. There were not oil droplets in vitelline membrane and attached structures in the outside of fertilize egg envelope. The egg envelope had a single micropyle resembling the pathway of sperm in the area of the animal pole. The micropyle was surrounded by 13 to 15 protruded lines of the egg envelope in a radiated form. The outer surface of fertilized egg envelope was covered by reticular adhesive fibrous structures and irregularly arranged by pore canals. The fertilized egg envelope consisted of three distinct layers an outer adhesive fibrous layer with high electron density, a middle layer with pore canals, and an inner layer consisting of 6 to 7 lamellae alternating layers with interlamellae of lower electron density. These ultrastructural characters of fertilized egg envelope form Hyphessobrycon serpae can be utilized in taxonomy of teleost, and as fundamental data for study on early development of fertilized egg. It seems that the morphology of micropyle is a common trait of Characidae

Ultrastructure of the Fertilized Egg Envelope from Pseudobagrus fulvidraco, Bagridae, Teleostei

Sohn, Joon Hyung,Kwon, Ohyun,Kim, Dong Heui Korean Society of Microscopy 2016 Applied microscopy Vol.46 No.3

The ultrastructure of fertilized egg envelope from Pseudobagrus fulvidraco belongs to Bagridae was investigated using light and electron microscopes. The fertilized egg was compressed spherical, light-yellowish, demersal, and adhesive. The size of fertilized egg is about $1.85{\pm}0.13mm$, perivitelline space is not well developed, and there were no appendicular structures on the outer surface of egg envelope and oil droplets in vitelline membrane under light microscope. The micropyle was located in the animal pole of fertilized egg. Adhesive reticular fiber was covered fertilized egg envelope. The thickness of egg envelope was about $3.7{\sim}4.2{\mu}m$, and the egg envelope consisted of two layers: an outer, electron-dense adhesive fibers layer and an simple inner layer with pore. Therefore, the ultrastructure of cross section of the fertilized egg envelope showed species specificity, but studies on the other species belongs to Bagridae were need to get correct information about common traits in family.

수정에 의한 Mouse egg의 세포막전류 변화

홍성근,박춘옥,한재희,김익현,하대식,권종국,Hong, Seong-geun,Park, Choon-ok,Han, Jae-hee,Kim, Ik-hyun,Ha, Dae-sik,Kwun, Jong-kuk 대한수의학회 1992 大韓獸醫學會誌 Vol.32 No.2

Changes in the both inward current and conductance of membrane by the fertilization were observed using the one microelectrode voltage clamp(or switch clamp) technique. Unfertilized eggs and both 1- and 2-cell stage eggs after fertilization were donated from the superovulated mouse (ICR, more than 6 weeks old) treated with PMSG(pregnant mare serum gonadotropin, Sigma) and HCG(human chorionic gonadotropin, Sigma) and naturally mated ones, respectively in this experiment. Membrane potential was held at -90mV and the voltage step was applied from -80mV to 50mV with interval of 10mV or 20mV for 300ms. since both of amplitudes and time courses in the membrane currents were various according to the states of cells and clamping condition, results were presented by their $averages{\pm}SEM$(standard mean error)and ratios or percentages. Inward currents began to appear in response to the step depolarization from -60mV and reached its maximum at -50mV. However, since the potential was not clamped evenly during the voltage step, current-voltage(I-V) relationship might be positively shifted 10 or 20mV. From the steady-state currents plotted in the I-V curve, outward rectification was markedly observed. Peak inward currents$(i_{in})$ at -50mV were $-0.62{\pm}0.23nA$(n=4),$-0.52{\pm}0.25nA$(n=5) and $-0.37{\pm}0.25nA$(n=6), in the 1-cell stage, 2-cell stage fertilized eggs and in the unfertilized eggs, respectively. Pure inward current (difference between steady-state and peak, $i_{in. pure}$) were $-1.01{\pm}0.23nA$, $-0.69{\pm}0.43nA$ and $-0.68{\pm}0.29nA$, respectively in the 1-cell stage fertilized eggs, unfertilized eggs and 2-cell stage fertilized eggs. These results suggested that the outward current in fertilized eggs of 2-cell stage was more increased than those in the unfertilized eggs. Pure inward currents in the all stages of eggs showed a similar fashion in the I-V relationship from -50mV to 50mV and reversal potential at 50mV. Time constant of inactivation$({\tau})$ in the inward current was decreased as the membrane potential was depolarized in the unfertilized and 2-cell stage eggs but in the 1-cell stage eggs t was not likely to be affected significantly. Slope conductances were 14.2nS, 8.9n5 and 7.7nS in the 1-cell, 2-cell stage fertilized eggs and the unfertilized eggs, respectively. Membranes between two cells within a zona pellucida seem to be electrical-connected in the 2-cell stage eggs from the observation made in the analysis for the electronic spread and decay to the current stimuli. Both of inward current and membrane conductance were increased after fertilization in the mouse eggs. Inward current seems to be carried by the same ion or through the same channels up to the 2-cell stage and ion that carried inward current was thought to play important function after fertilization in the mouse eggs.

경골어류 등목어과 Three-spot gourami, Pearl gourami 및 Marble gourami의 수정란 난막 미세구조 비교

김동희,등영건,김완종,류동석,캉송젠,Kim, Dong-Heui,Deung, Young-Kun,Kim, Wan-Jong,Reu, Dong-Suck,Kang, Song Jian 한국현미경학회 1999 Applied microscopy Vol.29 No.3

등목어과에 속하는 어류의 경우 수정란 난막이 형태학적으로 같은 과이기 때문에 종간에 유사성이 있는지 또는 같은 과에 속한다고 하더라도 종에 따라 서로 다른 구조를 하고 있는지를 알아보고, 과 또는 종의 기준이 되는 형태학적 특징들을 확인하기 위해서 three-spot gourami (Trichogaster trichopterus), pearl gourami (Trichogaster leeri) 및 marble gourami (Trichogaster trichopterus trichopterus)를 실험재료로 하여 수정란과 난막구조를 광학현미경, 투과전자현미경 및 주사전자현미경을 이용하여 관찰 비교하였다. 3종의 수정란은 모두 구형의 무색투명한 부착성란으로 부성란이었고 난막 표면에 부속사는 없었으며 난황장 중앙에 하나의 유적이 있었다. 3종 모두 정자의 통로로 생각되는 난문이 관찰되었는데 경사가 완만한 수많은 흠을 보유하고 있는 깔대기 형태였으나 pearl gourami의 경우 꽃 모양의 구조물이 난문 안쪽에 위치하고 있었고 three-spot gourami와 아종인 marble gourami의 경우 형태는 서로 유사했다. 난막의 표면은 홈들이 난막 전체에 분포하고 있었고, 3종 모두 난막은 2층으로 부착층인 외층 및 전자밀도가 높은 한 층인 내층으로 구성되어 있었다. 이상과 같이 수정란의 형태, 난문의 구조, 난막표면 및 난막의 단면구조는 3종 모두 매우 유사한 형태로 등목어과 어류의 수정란 및 난막의 공통적인 특징으로 생각된다. The structures of the fertilized egg envelope from three species, three-spot gourami (Trichogaster trichopterus), pearl gourami (Trichogaster leeri) and marble gourami (Trichogaster trichopterus trichopterus) belong to Belontiidae were observed, utilizing light, scanning and transmission electron microscopes. In all three species, the fertilized eggs were the colorless, transparent, spherical, adhesive and pelagic type. A large oil droplet was located in vitelline membrane of the fertilized egg. The egg envelopes have a single micropyle, which is thought to the pathway of sperm in the area of the animal pole. Specially, the micropyle of three-spot gourami was similar to that of marble gourami which is subspecies of three-spot gourami. An outer surface of the fertilized egg envelope was arranged by grooves in all three species. The fertilized egg envelopes consists of two distinct layers; an adhesive outer layer and an inner layer with high electron density. In conclusion, the morphological similarity of the fertilized egg, micropyle, outer surface and transverse section of the fertilized e9g envelope seems to be an indication of the Belontiidae.

Voltage Dependent N Type Calcium Channel in Mouse Egg Fertilization

Eum, Jin Hee,Park, Miseon,Yoon, Jung Ah,Yoon, Sook Young The Korean Society of Developmental Biology 2020 발생과 생식 Vol.24 No.4

Repetitive changes in the intracellular calcium concentration ([Ca²⁺]i) triggers egg activation, including cortical granule exocytosis, resumption of second meiosis, block to polyspermy, and initiating embryonic development. [Ca²⁺]i oscillations that continue for several hours, are required for the early events of egg activation and possibly connected to further development to the blastocyst stage. The sources of Ca²⁺ ion elevation during [Ca²⁺]i oscillations are Ca²⁺ release from endoplasmic reticulum through inositol 1,4,5 tri-phosphate receptor and Ca²⁺ ion influx through Ca²⁺ channel on the plasma membrane. Ca²⁺ channels have been characterized into voltage-dependent Ca²⁺ channels (VDCCs), ligand-gated Ca²⁺ channel, and leak-channel. VDCCs expressed on muscle cell or neuron is specified into L, T, N, P, Q, and R type VDCs by their activation threshold or their sensitivity to peptide toxins isolated from cone snails and spiders. The present study was aimed to investigate the localization pattern of N and P/Q type voltage-dependent calcium channels in mouse eggs and the role in fertilization. [Ca²⁺]i oscillation was observed in a Ca²⁺ contained medium with sperm factor or adenophostin A injection but disappeared in Ca²⁺ free medium. Ca²⁺ influx was decreased by Lat A. N-VDCC specific inhibitor, ω-Conotoxin CVIIA induced abnormal [Ca²⁺]i oscillation profiles in SrCl₂ treatment. N or P/Q type VDC were distributed on the plasma membrane in cortical cluster form, not in the cytoplasm. Ca²⁺ influx is essential for [Ca²⁺]i oscillation during mammalian fertilization. This Ca²⁺ influx might be controlled through the N or P/Q type VDCCs. Abnormal VDCCs expression of eggs could be tested in fertilization failure or low fertilization eggs in subfertility women.

Expression of Yolk Processing Enzyme Genes in Fertilized Eggs from Artificially Matured Female Eel, Anguilla japonica

Oh, Hyeon Ji,Kim, Jung-Hyun,Mun, Seong Hee,Kim, Jin Hui,Kim, Dae-Jung,Kwon, Joon Yeong The Korean Society of Developmental Biology 2018 발생과 생식 Vol.22 No.3

Large quantity of eggs fail to be fertilized and many of fertilized eggs are unable to hatch in the eel, Anguilla japonica. Larvae of eel absorb egg yolk up to 8 days after hatching but the majority of hatched larvae die before they reach the stage of first feeding in this species. Genes of key enzymes for yolk processing (cathepsin B, D, L and lipoprotein lipase - abbreviated as ctsb, ctsd, ctsl and lpl, respectively) could be associated with egg quality. In this study, we investigated differences in the expression of these genes between floating eggs and sinking eggs, and also the relationship between the gene expressions of the enzymes and fertilization rates in the fertilized eggs obtained from artificially matured female eels. Expressions of yolk processing enzyme genes did not show significant difference between floating and sinking egg groups. Expression of ctsb decreased when fertilization rate was high. Expression of ctsd, ctsl and lpl, however, did not show any significant differences. These results suggest that ctsb expression could be an indicator of egg quality, and that some proteins prone to be digested by ctsb could be very important in the process of fertilization and normal cleavage in this species. Further study should identify these critical proteins to improve our understanding on the quality of fish eggs.

Expression of Yolk Processing Enzyme Genes in Fertilized Eggs from Artificially Matured Female Eel, Anguilla japonica

Hyeon Ji Oh,Jung-Hyun Kim,Seong Hee Mun,Jin Hui Kim,Dae-Jung Kim,Joon Yeong Kwon 한국발생생물학회 2018 발생과 생식 Vol.22 No.3

Large quantity of eggs fail to be fertilized and many of fertilized eggs are unable to hatch in the eel, Anguilla japonica. Larvae of eel absorb egg yolk up to 8 days after hatching but the majority of hatched larvae die before they reach the stage of first feeding in this species. Genes of key enzymes for yolk processing (cathepsin B, D, L and lipoprotein lipase - abbreviated as ctsb, ctsd, ctsl and lpl, respectively) could be associated with egg quality. In this study, we investigated differences in the expression of these genes between floating eggs and sinking eggs, and also the relationship between the gene expressions of the enzymes and fertilization rates in the fertilized eggs obtained from artificially matured female eels. Expressions of yolk processing enzyme genes did not show significant difference between floating and sinking egg groups. Expression of ctsb decreased when fertilization rate was high. Expression of ctsd, ctsl and lpl, however, did not show any significant differences. These results suggest that ctsb expression could be an indicator of egg quality, and that some proteins prone to be digested by ctsb could be very important in the process of fertilization and normal cleavage in this species. Further study should identify these critical proteins to improve our understanding on the quality of fish eggs.

실험실에서 사육된 고랑가리비 Chlamys swiftii 수정란 발생과 유생 성장

이주,김이청,김기승,남명모 한국패류학회 2013 The Korean Journal of Malacology Vol.29 No.4

고랑가리비의 산란을 유도하고 수정난의 발생과 유생의 성장과정을 조사하였다. 산란자극 방법으로 광 자극, 간출 자극,온도 자극, 그리고 간출 후 온도자극을 수온 16 ± 0.5℃에서각각 실시하였다. 광 자극은 암컷 1마리당 700-900천개, 온도자극은 700-800천개로 적은 산란량을 보인 반면 간출 자극은700-1,200천개로 높았으며 간출 자극 후 온도자극이1,000-1,500천개로 가장 높은 산란량을 보였다. 수정율에 있어 광 자극은 암컷 1마리당 71.7%, 온도자극은 73.4%, 간출자극이 73.6%이었으며 간출 자극 후 온도자극은 76.3%로 가장 높았으나 자극방법에 따른 수정율의 유의적인 차이는 없었다. 수정란부터 D상 유생으로의 수온별 발생과정을 조사하기위하여 비이커에 ml당 1,000개의 수정란을 수용하고 각각8℃, 12℃, 16℃, 20℃ 및 24℃에서 D상 유생으로 발생하는생존율을 광학현미경을 사용하여 30분 간격으로 관찰한 결과,8℃와 24℃의 실험군이 4.1%와 3.2%로 생존율이 낮은 편이었으며 16℃에서 32.7%로 가장 높은 생존율을 보여주었다. 수정란의 크기는 72 ± 2.1 μm, 담륜자 유생은 103 ± 3.8 μm,D상 유생은 129 ± 10.4 μm, 각정기 유생은 145 ± 16.8 μm, 후기 유생은 197 ± 13.6 μm이었으며 528시간 후에 각장245 ± 15.8 μm의 초기 부착종묘로 성장하였다. The development of swift's scallop Chlamys swiftii, reared in the laboratory, has been examined through the investigation of morphological characteristics in fertilized egg, larvae and juvenile. Eggs were fertilized with a dilute sperm solution to improve the survival of fertilized eggs. Developing larvae were maintained at a temperature of 16 ± 0.5℃ and salinity concentration of 33 ppt. We have investigated the fertilization rates and egg number spawned at several stimulating conditions such as sunlight exposure, air dry, seawater temperature rise (5℃) and seawater temperature rise (5℃) after exposure of air dry for spawning induction of swift's scallop Chamys swiftii. Stimulation treated with sunlight exposure and seawater temperature rise (5℃) have shown the spawning number of 700,000-900,000 and 700,000-800,000 per individual, respectively while stimulation treated with seawater temperature rise (5℃) after exposure of air dry have shown the high spawning number of 1,000,000-1,500,000 per individual. Survival rate of D-shaped larvae of swift's scallop put into the different seawater temperatures of 8℃, 12℃, 16℃, 20℃ and 24℃ has been 4.1%, 11.6%, 32.7%, 18.6% and 3.2%, respectively. Fertilized eggs with the diameter of 72 μm developed into trochophore larvae of 103 ± 3.8 μm shortly after 35 hours and to D larvae of 129 ± 10.4 μm shell length within 72 hours. It took 336 hours to become initial Umbo-stage larva of 145 ± 16.8 μm shell length. Post larvae, which have been 197 ± 13.6 μm shell length, spontaneously have settled in the attachment substances. It have required 528 hours from fertilized eggs to early attached juvenile to become initial juvenile size of 245 ± 15.8 μm shell length.

The Study on the Internal Change of Eggs During Incubation Using Magnetic Resonance Imaging Technique

( Seung Hoon Baek ),( Hak Sung Kim ),( Seong Min Kim ) 한국농업기계학회 2017 한국농업기계학회 학술발표논문집 Vol.22 No.2

There are some probability of non-fertilization in fertile eggs. It needs non-destructive or non-invasive technique to observe internal of egg changes and monitor the incubation process. It is an important advantage to sort the eggs in early detection. In this study, we used Magnetic Resonance Imaging (MRI) technology, one of the non-destructive measurement methods to investigate the changes of eggs during hatching. We used ‘Hy-line brown’ species eggs (Fertile: Yeoju Eden Farm, Non-fertile: Pyeongchang Seoul National University Farm) as a testing materials in experiment. A sample tray was used to fix the egg position constant. The magnetic resonance (MR) image data was obtained by magnetic resonance imaging system which installed at the Institution for Agricultural Machinery & ICT Convergence at Chonbuk National University. In MRI ‘Gradient Echo’ pulse was used and every time we peformed, we acquired 64 images in one series with each having 70×70 mm field of view (FOV) in axial image direction. The experiment was peformed in 3 days and tested almost identical intervals. From the images, we focused on yellow yolk. When with visual observation, it was confirmed that fertile egg’s upper part of the center of yolk became inflated at 22 hours after hatching, then began to shrink and disappeared a lot with the shape collapsed. On the other hand, non-fertile egg was little change. These results suggest that MRI technique has great advantage on analyzing the internal material changes in nondestructive way.