CN112652222A - Intra-ovarian fertilization process of female silkworm moths and model display method of ovaries - Google Patents
Intra-ovarian fertilization process of female silkworm moths and model display method of ovaries Download PDFInfo
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Abstract
The invention discloses a method for the fertilization process in the ovary of female silkworm moths and the model display of the ovary, which comprises the following steps: obtaining an ovary sample; ovarian sample transparency: placing the obtained ovary into a centrifugal tube, adding an acrylamide hydrogel solution, oscillating in a constant-temperature oscillator, replacing the acrylamide hydrogel solution once within a specified interval time, oscillating, replacing the acrylamide hydrogel solution with an SDS (sodium dodecyl sulfate) removing solution, oscillating again, and finally obtaining a transparent finished ovary sample; the ovary specimens after the completion of clearing were subjected to DAPI staining. The invention has simple use and convenient operation, and can clearly observe the internal condition of the ovary of the tissue organ to obtain direct observation evidence.
Description
Technical Field
The invention relates to a technology of an ovarian fertilization process of female silkworm moths and an ovarian model display method.
Background
Silkworm is an important lepidoptera model insect, breeds in an oviparous mode, and researches on ovaries are beneficial to understanding the whole reproductive physiology process in a female body after mating of male and female moths. At present, people mainly carry out detailed observation on reproductive organs of silkworm moths through anatomy, speculate the reproductive process, but lack direct observation evidence.
Disclosure of Invention
The invention aims to provide an ovarian internal fertilization process of female silkworm moths and an ovarian model display method, which are simple to use and convenient to operate, can clearly observe the internal conditions of the ovaries of tissue organs and obtain direct observation evidence.
The purpose of the invention is realized by the following technical scheme.
A method for the fertilization process in the ovary and the model display of the ovary of female silkworm moths is characterized by comprising the following steps:
obtaining an ovarian sample: selecting mated female moths to completely expose the ovarian tubes, clamping the ovaries, rinsing until the ovarian tubes are basically separated, cutting off the ovarian tubes, removing impurities, removing the adnexal glands and the ovarian tubes, and keeping the middle oviducts, the lateral oviducts, the fertilized sacs and the mating sacs;
ovarian sample transparency: placing the obtained ovary into a centrifugal tube, adding an acrylamide hydrogel solution, oscillating in a constant-temperature oscillator, replacing the acrylamide hydrogel solution once within a specified interval time, oscillating, replacing the acrylamide hydrogel solution with an SDS (sodium dodecyl sulfate) removing solution, oscillating again, and finally obtaining a transparent finished ovary sample;
DAPI staining was performed on ovarian specimens after clearing was complete: taking out the ovary samples after the completion of transparency, grouping the ovary samples, then respectively placing the ovary samples into a centrifuge tube, adding PBT (polybutylene terephthalate) for oscillation, replacing the PBT in the tube after oscillating for a set time period, oscillating again, dyeing by using a DAPI (Dapi) solution, oscillating in a dark place during dyeing, sucking out a dyeing solution after dyeing is finished, adding a PBT solution for cleaning, requiring continuous oscillation in the dark place, finally placing the ovary samples on a glass slide, dripping glycerol, and observing under a fluorescence microscope;
SDS scavenging solution, through SDS powder, boric acid powder is dissolved in distilled water, and the alkaline solution obtained by adjusting pH value with sodium hydroxide solution, the said SDS is sodium dodecyl sulfate;
PBT is a PBS solution of Triton X-100, 1mL of Triton X-100 and 99mL of PBS;
DAPI, referred to as 4',6-diamidino-2-phenylindole, is specified in Zink Daniele, Sadoni Nicolas, Stelzer error, visualization chromatography and chromosomes in living cells [ J ]. Methods,2003,29(1), and is a fluorescent dye for strongly binding DNA, i.e. deoxyribonucleic acid, deoxyribonic acid, without specific chemical equations.
Comparing and observing the ovary sample after being transparent with the ovary which is not subjected to transparent treatment, wherein the whole ovary which is not subjected to transparent treatment is in original white, the state and the outline of the ovum in the ovary can not be seen clearly in the faint yellow state at the position with the ovum, the position of the ovum can only be seen through the color difference, after the treatment of the acrylamide hydrogel solution, the tissue is observed to be primarily transparent, but the whole ovary surface is in fuzzy, the outline of the ovum is observed under the illumination of a dissecting mirror, the outline of the ovum is compared with a fertilized sac tube according to the inclination direction of the ovum, whether the ovum in the ovary sample is in the state of being fertilized is judged, and the ovary sample is observed, screened and confirmed one by one. After the clearing procedure was completed, the ovaries were more clear and the interior eggs were observed through the ovarian epidermis.
When observed by a fluorescence microscope, a highlight blue strip is clearly observed between the ovum and the fertilized sac tube through the fluorescence microscope, and a small amount of blue bright spots are observed at the periphery of the ovum; when the sperms are discharged from the fertilized sac and fertilized, most sperms move to the egg pores, are dyed by DAPI and present blue highlight strips under the excitation of ultraviolet light, and part of sperms which cannot enter the eggs diffuse to the periphery of the eggs, and present highlight blue spots after being dyed and excited.
Drawing an ovary structure by using 3dsMax, namely drawing an ovary of a female silkworm moth, wherein the ovary consists of a fertilized sac, a mating sac, two accessory glands, a fertilized sac gland, a middle oviduct, two lateral oviducts, eight ovarian tubes, sperm guide tubes between the mating sac and the middle oviduct, the eight ovarian tubes are respectively jointed at one end of the two lateral oviducts, and the two lateral oviducts are then jointed with the middle oviduct; the fertilized sac is connected with the middle part of the middle oviduct, and the fertilized sac gland is arranged on the connecting pipe; the mating sac is directly connected with the oviposition hole, a tiny sperm duct is arranged between the mating sac and the middle oviduct, and the duct enables the secondary supplement of the sperm to be possible; and a fertilization animation video is produced. When male and female moths are mated, the male moths inject sperms into mating sacs for storage, and a part of sperms enter a fertilized sac through a sperm catheter. When a female moth ovulates, eight ova sequentially ovulate, the ova enter an atrium through an oviduct, the part of the ova is inclined towards the direction of the fertilized sac, a small amount of sperms are discharged from the fertilized sac, part of the sperms enter the ova, the other part of the sperms overflow to the periphery, and the sperms entering the ova further complete fertilization; finally completing the fertilization process in the ovary of female silkworm moths and the 3D model display of the ovary.
When preparing the hydrogel solution, dissolving paraformaldehyde in distilled water and adding PBS buffer, and observing that a large amount of paraformaldehyde is in a suspended or precipitated state. Adding a little sodium hydroxide particles to dissolve a large amount of paraformaldehyde; stirring by using a magnetic heating stirrer, clarifying the solution, and completely dissolving paraformaldehyde; adding acrylamide, methylene bisacrylamide and VA-044 initiator into the solution respectively, refrigerating the prepared solution for storage, and solidifying at normal temperature, wherein the PBS is phosphate buffer solution and the main component is Na2HPO4、KH2PO4NaCl and KCl, VA-044, 2-azabicyclo (2-imidazoline) dihydrochloride, formula C12H24Cl2N6。
8g of acrylamide, 0.08g of methylene bisacrylamide, 8g of paraformaldehyde, VA-0440.4 g and 50mL of 10 XPBS in the hydrogel solution are added, and finally the volume is determined to be 500 mL.
Compared with the prior art, the invention has the following advantages:
the invention utilizes acrylamide hydrogel solution transparent technology to make the ovary transparent, dyes the sperm with DAPI dye solution to show the trace of the sperm, and observes the process that the sperm enters the egg hole from the germ cell under a fluorescence microscope; and combining the images to obtain the moving track of the sperms, and drawing the three-dimensional structure of the whole ovary and the fertilization animation video by using 3 dsMax.
The invention utilizes three-dimensional animation software to simulate the reproductive physiology of silkworms, and uses the new expression form to display the ovary structure in a three-dimensional and vivid manner.
Drawings
FIG. 1 is a diagram showing the effect of the degree of transparency of three stages of the ovary of the method for the fertilization process in the ovary of female silkworm moth and the model display of the ovary of the present invention;
FIG. 2 is a diagram showing the effect of DAPI staining in the process of fertilization in the ovary of female silkworm moth and the method for displaying the ovary model;
FIG. 3 is a structural diagram of the fertilization process in the ovary of female silkworm moth and the model display method of the ovary of the present invention.
Detailed Description
The present invention is described in further detail below with reference to the attached drawing figures.
The invention relates to a method for the fertilization process in the ovary of female silkworm moths and the model display of the ovary, which comprises the following steps:
obtaining an ovarian sample: selecting mated female moths to completely expose the ovarian tubes, clamping the ovaries, rinsing until the ovarian tubes are basically separated, cutting off the ovarian tubes, removing impurities, removing the adnexal glands and the ovarian tubes, and keeping the middle oviducts, the lateral oviducts, the fertilized sacs and the mating sacs;
ovarian sample transparency: placing the obtained ovary into a centrifugal tube, adding an acrylamide hydrogel solution, oscillating in a constant-temperature oscillator, replacing the acrylamide hydrogel solution once within a specified interval time, oscillating, replacing the acrylamide hydrogel solution with an SDS (sodium dodecyl sulfate) removing solution, oscillating again, and finally obtaining a transparent finished ovary sample;
the ovary specimens after the completion of clearing were subjected to DAPI staining: the ovary sample after being transparent is taken out and grouped, then the ovary sample is placed in a centrifuge tube respectively, PBT is added to vibrate, PBT in the tube is replaced after the vibration is carried out for a set time period, the tube is vibrated again, DAPI solution is used for dyeing, vibration is carried out in a dark place during dyeing, after dyeing is finished, dyeing liquid is sucked out, the PBT solution is added to clean, vibration in a dark place is required to continue, finally the ovary sample is placed on a glass slide, glycerol is dripped, and observation is carried out under a fluorescence microscope.
Comparing and observing the ovary sample after being transparent with the ovary which is not subjected to transparent treatment, wherein the whole ovary which is not subjected to transparent treatment is in original white, the state and the outline of the ovum in the ovary can not be seen clearly in the faint yellow state at the position with the ovum, the position of the ovum can only be seen through the color difference, after the treatment of the acrylamide hydrogel solution, the tissue is observed to be primarily transparent, but the whole ovary surface is in fuzzy, the outline of the ovum is observed under the illumination of a dissecting mirror, the outline of the ovum is compared with a fertilized sac tube according to the inclination direction of the ovum, whether the ovum in the ovary sample is in the state of being fertilized is judged, and the ovary sample is observed, screened and confirmed one by one. After the clearing procedure was completed, the ovaries were more clear and the interior eggs were observed through the ovarian epidermis.
When observed by a fluorescence microscope, a highlight blue strip is clearly observed between the ovum and the fertilized sac tube through the fluorescence microscope, and a small amount of blue bright spots are observed at the periphery of the ovum; when the sperms are discharged from the fertilized sac and fertilized, most sperms move to the egg pores, are dyed by DAPI and present blue highlight strips under the excitation of ultraviolet light, and part of sperms which cannot enter the eggs diffuse to the periphery of the eggs, and present highlight blue spots after being dyed and excited.
Drawing an ovary structure by using 3dsMax, namely drawing an ovary of a female silkworm moth, wherein the ovary consists of a fertilized sac, a mating sac, two accessory glands, a fertilized sac gland, a middle oviduct, two lateral oviducts, eight ovarian tubes, sperm guide tubes between the mating sac and the middle oviduct, the eight ovarian tubes are respectively jointed at one end of the two lateral oviducts, and the two lateral oviducts are then jointed with the middle oviduct; the fertilized sac is connected with the middle part of the middle oviduct, and the fertilized sac gland is arranged on the connecting pipe; the mating sac is directly connected with the oviposition hole, a tiny sperm duct is arranged between the mating sac and the middle oviduct, and the duct enables the secondary supplement of the sperm to be possible; and a fertilization animation video is produced. When male and female moths are mated, the male moths inject sperms into mating sacs for storage, and a part of sperms enter a fertilized sac through a sperm catheter. When a female moth ovulates, eight ova sequentially ovulate, the ova enter an atrium through an oviduct, the part of the ova is inclined towards the direction of the fertilized sac, a small amount of sperms are discharged from the fertilized sac, part of the sperms enter the ova, the other part of the sperms overflow to the periphery, and the sperms entering the ova further complete fertilization; finally completing the fertilization process in the ovary of female silkworm moths and the 3D model display of the ovary.
Dissolving paraformaldehyde in distilled water, adding a PBS buffer solution, observing that a large amount of paraformaldehyde is in a suspended or precipitated state, and adding a small amount of sodium hydroxide particles to dissolve the paraformaldehyde in a large amount; stirring by using a magnetic heating stirrer, clarifying the solution, and completely dissolving paraformaldehyde; and respectively adding acrylamide, methylene bisacrylamide and VA-044 initiator into the solution, refrigerating the prepared solution, and solidifying at normal temperature.
As described in more detail below:
the ovary of female silkworm moth is formed from mating sac, middle oviduct, fertilized sac gland, adnexal gland, lateral oviduct, ovarian tube and sperm duct between mating sac and middle oviduct, the middle oviduct is connected with two lateral oviducts, and 4 ovarian tubes are respectively connected on two lateral oviducts. When male and female moths are mated, the male moths genitals extend into the mating bursa and penetrate into sperms. Sperm then enters the fertilized sac through a sperm duct connecting the mouth of the mating sac with the main fallopian tube. When ovulation is carried out, 8 ovarian tubes sequentially ovulate, the top egg hole inclines towards the fertilized sac when the eggs enter the atrium, and the fertilized sac discharges part of sperms under the action of muscles to enter the eggs from the egg hole to finish fertilization.
At present, understanding about physiological activities of reproduction in ovaries after mating of silkworms mainly comes from anatomical observation of the structures of the ovaries of the silkworms, and the process is proved by means of a clear lipid-exchanged acrylamide hybridization fine imaging compatible tissue hydrogel technology (clear lipid-exchanged acrylamide-hybridized tissue imaging-compatible tissue-hydrogel, called simply "class hydrogel transparency technology") and a DAPI dyeing technology, and 3dsMax is used for visual three-dimensional display. In the study of lepidopteran insects, there is no precedent that the tissues are transparent.
The Clarity hydrogel transparency technique is a tissue transparency technique. The tissues and organs in the organism all take lipid, protein and water as main components, the lipid, the protein and the water have obvious refractive indexes, and most of transmitted light is blocked when the lipid, the protein and the water are combined together, so that the tissues and organs can be observed by people through reflected light. When it is necessary to observe the internal state of a tissue organ, in addition to dissection, the effect of transparency can be achieved by reducing the difference in refractive index between the main constituent substances. Tissue proteins are the main morphological material of tissue structure, while water is an important material to support tissue morphology, and therefore, the goal can only be achieved by treating lipids. The Clarity transparent technique is a technique in which a hydrogel mainly composed of acrylamide is used to replace lipids in a tissue to make the tissue and organ transparent, and the internal condition of the tissue and organ can be clearly observed by combining with other staining techniques.
DAPI：
Namely 4',6-diamidino-2-phenylindole (4',6-diamidino-2-phenylindole), is a fluorescent dye capable of strongly binding DNA, and can be used for dyeing living cells and fixed cells because the fluorescent dye can permeate through intact cell membranes. When DAPI binds to double-stranded DNA, the DAPI dye has a maximum absorption peak at 358nm (ultraviolet) and a maximum emission peak at 461nm (blue) under fluorescent microscopy, with the emission light in the wavelength range covering blue to cyan, so that DAPI is illuminated by ultraviolet light and detected by a blue or cyan filter in fluorescent microscopy.
The 3dsMax is three-dimensional animation software based on a PC platform developed by the internet Logic company, and is mainly widely applied to the fields of advertisement, movie and television, industrial design, architectural design, three-dimensional animation, multimedia production, games, engineering visualization and the like. In 1999, Discreet Logic was purchased from Autodesk and combined with the Kinetix division issued by Home responsibility 3ds as the Discreet division. With the continuous development of software, many users are owned at home and abroad by virtue of higher cost performance and simple operation.
The feasibility of the technical scheme is described through an experimental method.
Experimental Material
Sample preparation: female moths in post-mating ovulatory phase.
The instrument comprises the following steps: dissecting mirror (NIKON-SZ1 type dissecting mirror), constant temperature oscillator (Shaking Incubator-LCD), fluorescence microscope (AMG AMF4032), magnetic heating stirrer (IKA C-MAG HS7)
Medicine preparation: paraformaldehyde powder, acrylamide powder, methylene bisacrylamide powder, 10 XPBS buffer solution, VA-044 initiator powder, sodium hydroxide particles, SDS powder, boric acid powder, PBT solution and 5 mu g/mL DAPI solution.
Procedure of experiment
Preparing a reagent: preparing a hydrogel solution: 8g of paraformaldehyde is dissolved in 450mL of distilled water and 50mL of PBS buffer solution is added, a large amount of paraformaldehyde is observed to be in a suspended or precipitated state, a small amount of sodium hydroxide particles are added, and the paraformaldehyde is dissolved in a large amount because the paraformaldehyde is easily dissolved in weak acid and weak base. Stirring at 60 deg.C with a magnetic heating stirrer for 30min, clarifying the solution, and completely dissolving paraformaldehyde. Then 8g of acrylamide, 0.08g of methylene bisacrylamide and 0.4g of VA-044 initiator are added into the solution respectively. The prepared solution can be refrigerated and stored at 4 ℃ and can be solidified at normal temperature.
SDS scavenging solution: dissolving SDS powder 40g and boric acid powder 12.4g in distilled water 1L, and adjusting pH to 8.5 with 10mol/L sodium hydroxide solution, wherein SDS is sodium dodecyl sulfate and molecular formula C12H25O4NaS。
And (3) experimental operation:
obtaining the ovary: selecting female moths 6h after mating, cutting the female moths from the sixth abdominal segment, placing the female moths under a dissecting mirror, cutting the female moths from the back, gradually peeling off the exocuticle, and observing that the ovarian tubes are completely exposed. The ovaries were held with forceps and rinsed in water until the ovaries were substantially separated. The redundant ovarian duct is cut off, and the mating sac, the fertilized sac, the accessory gland and the middle oviduct can be clearly seen. And removing redundant impurities. The accessory glands and the ovarian ducts are further removed, and the middle oviduct, the lateral oviduct, the fertilized sac and the mating sac are reserved.
Transparency of ovary: dissected ovaries were placed into 50mL centrifuge tubes and 10 volumes of acrylamide hydrogel solution were added. The sample tube was shaken at 60r/min for 3 days in a constant temperature shaker at 10 ℃ and the hydrogel solution was changed every 24 hours. After 3 days, the hydrogel solution was changed to SDS-scavenger and shaken at 37 ℃ and 60r/min for 5 days.
DAPI staining of ovaries: and taking out the transparent samples, putting 3 samples in a group into a plurality of 1mL centrifuge tubes respectively, adding PBT, vibrating for 5min at 80r/min, replacing PBT in the tubes after 5min, and vibrating for 5min again. And dyeing with 5 mu g/mL DAPI solution for 15min, wrapping the centrifuge tube with tinfoil paper during dyeing, and shaking in the dark at 80 r/min. After dyeing is finished, sucking out the dyeing solution, adding 1mL of PBT solution, cleaning for three times, and continuously wrapping in the tin foil paper for oscillation. Finally, the sample was placed on a glass slide, a drop of glycerol was added, and observed under a fluorescence microscope.
The PBT is a PBS solution of Triton X-100, 1mL of Triton X-100 and 99mL of PBS, the DAPI is 4',6-diamidino-2-phenylindole, specifically, see Zink Daniele, Sadoni Nicolas, Stelzer Ernst]Methods,2003,29(1), without specific chemical equations, for fluorescent dyes that strongly bind DNA, deoxyribonucleic acid. The PBS (phosphate buffered saline) mainly contains Na2HPO4、KH2PO4NaCl and KCl, VA-044, 2-azabicyclo (2-imidazoline) dihydrochloride, formula C12H24Cl2N6。
Results of the experiment
And (4) clear observation of ovaries: before the ovary is subjected to the transparent treatment, the whole ovary presents an original white color, and the ovary is hidden and hidden in a faint yellow color at the position with the ovary. The state and contour of the egg in the ovary cannot be clearly seen, and the position of the egg can be identified only through color difference. After hydrogel treatment, it was observed that the tissue had been initially transparent, but was overall hazy, and the ovarian surface had a frosty texture. Under the light irradiation of the dissecting mirror, the outline of the ovum can be seen, the ovum is compared with the fertilized sac tube according to the inclined direction of the ovum, whether the ovum in the sample is in the state of being fertilized is judged, and the sample is observed, screened and confirmed one by one. After SDS treatment, the whole experiment was cleared and the ovary was observed in comparison with the previous ovary, which was seen to be more clear than the previous one. The ovaries, except for the eggs, are already substantially clear. The eggs inside can be clearly observed through the ovarian epidermis.
The degree of transparency of the three stages is shown in particular in FIG. 1 below, which is labeled as pre-ovarian clearing 1; indicated is ovarian hydrogel post treatment 2; indicated is 3 after ovarian SDS treatment.
And (3) observation by a fluorescence microscope: after the transparent sample is washed by PBT, dyed by DAPI and washed by PBT, the sample is excited by ultraviolet rays under a fluorescence microscope to emit blue light. As in fig. 2, by fluorescence microscopy, marked with eggs 4; the overflowing sperm 5; sperm band 6. A highlight blue band can be clearly observed between the egg and the fertilized sac tube, and a small amount of blue bright spots can be observed at the periphery of the egg. When the sperms are discharged from the fertilized sac and fertilized, most sperms move to the egg pores, are dyed by DAPI and present blue highlight strips under the excitation of ultraviolet light, and part of sperms which cannot enter the eggs diffuse to the periphery of the eggs, and present highlight blue spots after being dyed and excited.
Drawing a three-dimensional structure of a silkworm moth ovary and fertilizing an animation video:
the ovary of the female silkworm moth consists of a fertilized sac, a mating sac, 2 accessory glands, a fertilized sac gland, a middle oviduct, 2 lateral oviducts, 8 ovarian tubes and a sperm guide tube between the mating sac and the middle oviduct, wherein the 8 ovarian tubes are respectively jointed at one end of the two lateral oviducts, and the two lateral oviducts are further jointed with the middle oviduct; the fertilized sac is connected with the middle part of the middle oviduct, and the fertilized sac gland is arranged on the connecting pipe; the mating sac is directly connected with the oviposition hole, and a tiny sperm duct is arranged between the mating sac and the middle oviduct, so that secondary supplement of the sperm is possible. When male and female moths are mated, the male moths inject sperms into mating sacs for storage, and a part of sperms enter a fertilized sac through a sperm catheter. When the female moth ovulates, 8 ova are sequentially ovulated, enter the atrium through the oviduct, the egg hole part inclines to the direction of the fertilized sac, and the sperm from the fertilized sac is received to finish fertilization.
The ovarian structure was mapped via 3dsMax, as in fig. 3, labeled ovarian ducts 7; the lateral fallopian tube 8; the middle fallopian tube 9; a fertilized sac 10; the accessory glands 11; mating capsule 12; the fertilized sac gland 13.
And (4) analyzing results: from the above observations, we can conclude that: during ovulation, the egg hole is inclined toward the fertilized sac when the egg passes through the atrium. At this time, the fertilized sac discharges a small amount of sperm, a part of the sperm enters the egg hole, and the other part overflows to the periphery, and the sperm entering the egg hole further completes fertilization. The method is consistent with the guess of the reproductive physiology of the silkworm moth ovary, the research result directly shows the fertilization process of the silkworm eggs in the ovary, and provides a practical basis for the hypothesis and animation production of the reproductive physiology process. The fertilization process of the egg in the ovary can be clearly seen in the animation. But how do the sperm enter the fertilized capsule? The mating sac and the fertilized sac are respectively connected with the middle oviduct through the sperm duct and the fertilized sac tube, but how do the sperm move from the outlet of the sperm duct to the inlet of the fertilized sac tube in the middle oviduct? This is a problem to be studied in the future.
The following further explains the related art:
the transparent effect and the protein loss degree of different hydrogel formulas are different, and the main substance in the structure is protein due to the purpose of observing the ovary structure after being transparent, so that the hydrogel formula with the lowest protein loss amount is selected, and the protein loss amount of the A4B4P4 group is the lowest by comparing the application effects of 4 hydrogels with different concentrations in the transparentization of mouse brain, liver and kidney tissues. Thus, A4B3.2P4 hydrogel was selected: 8g of acrylamide, 0.08g of methylene bisacrylamide, 8g of paraformaldehyde, VA-0440.4 g and 50mL of 10 XPBS, and finally the volume is determined to be 500 mL.
Wherein the components of acrylamide, methylene bisacrylamide, paraformaldehyde, VA-044 and PBS have the functions:
acrylamide is a polymer chain monomer;
methylene bisacrylamide is a cross-linking agent, so that a plurality of acrylamide polymer chains are cross-linked to form a three-dimensional structure;
formaldehyde: crosslinking the tissue, covalently linking the hydrogel monomers to biomolecules including proteins, nucleic acids, and small molecules;
VA-044: thermal initiator to polymerize hydrogel monomer to make the tissue and hydrogel become one hybrid structure to support the tissue structure physically;
PBS: a buffer solution;
when the methylene diacrylamide is low, the crosslinking degree of the hydrogel is low, the formed hydrogel has large pores, and the formed hydrogel is favorable for removing liquid to enter tissues and remove lipid, so that a better transparent effect is achieved, but if the methylene diacrylamide is too low and the crosslinking effect is too weak, the tissue structure cannot be supported well, and the protein loss is increased. Therefore, the invention adopts A4B3.2P4 (acrylamide 4%, methylene bisacrylamide 0.04%, paraformaldehyde 4%) to achieve the ideal effect.
Whether the transparency is a fuzzy concept can not be determined, the transparency is called transparency, and secondly, the tissue transparency experiment can only be divided into different concentration points.
The invention combines the CLARITY hydrogel transparent technology, the three-dimensional model and the tissue and organ research of lepidoptera insects, is an unprecedented innovation, shows vivid images and animations for the structural research of silkworm moth ovaries and provides reference for the structural research of other lepidoptera insects. The Clarity hydrogel transparent technology is a technology for replacing lipid in tissues by using solidified acrylamide to realize tissue transparency, is based on the fact that the form of a silkworm moth ovary is small, and has a structure which is more complex compared with brain, liver, kidney and intestinal tracts. The smaller size results in the ovaries not being sliced as large pieces of tissue; the complex structure makes it difficult to separate the ovaries from the hydrogel after coagulation. According to the above two points, the treatment of the tissue with the hydrogel solution at 10 ℃ which does not coagulate was attempted, and finally, the transparency was still accomplished. The existing teaching resources are mainly resources such as pictures, texts, videos and the like. Traditional teaching modes such as pictures and characters are abstract and not easy to understand, the three-dimensional animation model can vividly and comprehensively represent knowledge points, and the problems that the knowledge points are not easy to observe through picture recording and the like can be found through multi-angle observation of the three-dimensional model, so that the learning interest of students can be stimulated, and scientific problems can be explored.
The above product example is one of the more preferable embodiments of the present invention, and those skilled in the art can make general changes and substitutions within the technical scope of the present invention and shall be included in the protection scope of the present invention.
Claims (6)
1. A method for the fertilization process in the ovary and the model display of the ovary of female silkworm moths is characterized by comprising the following steps:
obtaining an ovarian sample: selecting mated female moths to completely expose the ovarian tubes, clamping the ovaries, rinsing until the ovarian tubes are basically separated, cutting off the ovarian tubes, removing impurities, removing the adnexal glands and the ovarian tubes, and keeping the middle oviducts, the lateral oviducts, the fertilized sacs and the mating sacs;
ovarian sample transparency: placing the obtained ovary into a centrifugal tube, adding an acrylamide hydrogel solution, oscillating in a constant-temperature oscillator, replacing the acrylamide hydrogel solution once within a specified interval time, oscillating, replacing the acrylamide hydrogel solution with an SDS (sodium dodecyl sulfate) removing solution, oscillating again, and finally obtaining a transparent finished ovary sample;
the ovary specimens after the completion of clearing were subjected to DAPI staining: taking out the ovary samples after the completion of transparency, grouping the ovary samples, then respectively placing the ovary samples into a centrifuge tube, adding PBT (polybutylene terephthalate) for oscillation, replacing the PBT in the tube after oscillating for a set time period, oscillating again, dyeing by using a DAPI (Dapi) solution, oscillating in a dark place during dyeing, sucking out a dyeing solution after dyeing is finished, adding a PBT solution for cleaning, requiring continuous oscillation in the dark place, finally placing the ovary samples on a glass slide, dripping glycerol, and observing under a fluorescence microscope;
the SDS scavenging solution is an alkaline solution obtained by dissolving SDS powder and boric acid powder in distilled water and adjusting the pH value by using a sodium hydroxide solution, wherein the SDS is sodium dodecyl sulfate;
the PBT is a PBS solution of Triton X-100, and the PBS is a phosphate buffer solution;
the DAPI refers to 4',6-diamidino-2-phenylindole, a fluorescent dye for strongly binding DNA.
2. The method for in-ovary fertilization and model display of an ovary according to claim 1, the method is characterized in that: comparing and observing the ovary sample after the completion of the transparence with the ovary without the transparence treatment, wherein the whole ovary without the transparence treatment presents the original white color, the state and the contour of the ovum in the ovary can not be seen clearly when the ovum is hidden and the position is hidden in the faint yellow, the position of the ovum can be identified only by the color difference, after the acrylamide hydrogel solution treatment, the tissues are observed to be initially transparent, but the whole body is still fuzzy, the ovary surface presents the texture of ground glass, observing the outline of the ova under the light irradiation of a dissecting mirror, comparing the outline with a fertilized sac tube according to the inclined direction of the ova, judging whether the ova in the ovary sample are in a state of being fertilized, and observing, screening and confirming the ovary sample one by one. After the clearing procedure was completed, the ovaries were more clear and the interior eggs were observed through the ovarian epidermis.
3. The method for exhibiting the fertilization process in the ovary and the model of the ovary of the female silkworm moth according to claim 1, wherein a blue band is clearly observed between the egg and the fertilized sac tube by a fluorescence microscope, and a small number of blue bright spots are observed around the egg; when the sperms are discharged from the fertilized sac and fertilized, most sperms move to the egg hole, are stained by DAP I and present a blue highlight strip under the excitation of ultraviolet light, and part of sperms which cannot enter the egg diffuse to the periphery of the egg, and present a highlight blue spot after being stained and excited.
4. The method for performing fertilization in the ovary and displaying a model of the ovary of female silkworm moth according to claim 1, wherein 3dsMax is used for drawing the structure of the ovary, namely drawing the ovary of the female silkworm moth, wherein the drawing is composed of a fertilized sac, a mating sac, two epiphyses, a fertilized sac gland, a middle oviduct, two lateral oviducts, eight ovarian tubes and a sperm duct between the mating sac and the middle oviduct, the eight ovarian tubes are respectively jointed at one end of the two lateral oviducts, and the two lateral oviducts are further jointed with the middle oviduct; the fertilized sac is connected with the middle part of the middle oviduct, and the fertilized sac gland is arranged on the connecting pipe; the mating sac is directly connected with the oviposition hole, a tiny sperm duct is arranged between the mating sac and the middle oviduct, and the duct enables the secondary supplement of the sperm to be possible; and a fertilization animation video is produced. When male and female moths are mated, the male moths inject sperms into mating sacs for storage, and a part of sperms enter a fertilized sac through a sperm catheter. When a female moth ovulates, eight ova sequentially ovulate, the ova enter an atrium through an oviduct, the part of the ova is inclined towards the direction of the fertilized sac, a small amount of sperms are discharged from the fertilized sac, part of the sperms enter the ova, the other part of the sperms overflow to the periphery, and the sperms entering the ova further complete fertilization; finally completing the fertilization process in the ovary of female silkworm moths and the 3D model display of the ovary.
5. The method for the in-ovary fertilization process and the model display of the ovary of the female silkworm moth according to claim 1, wherein: dissolving paraformaldehyde in distilled water, adding a PBS buffer solution, observing that a large amount of paraformaldehyde is in a suspended or precipitated state, and adding a small amount of sodium hydroxide particles to dissolve the paraformaldehyde in a large amount; stirring by using a magnetic heating stirrer, clarifying the solution, and completely dissolving paraformaldehyde; and respectively adding acrylamide, methylene bisacrylamide and a VA-044 initiator into the solution, refrigerating the prepared solution for storage, and solidifying at normal temperature, wherein the VA-044 is 2, 2-aza-bis (2-imidazoline) dihydrochloride.
6. The method for the in-ovary fertilization process and the model display of the ovary of the female silkworm moth according to claim 1, wherein: 8g of acrylamide, 0.08g of methylene bisacrylamide, 8g of paraformaldehyde, VA-0440.4 g and 50mL of 10 XPBS in the hydrogel solution are added, and finally the volume is determined to be 500 mL.
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