All-level follicle counting method for evaluating quality of experimental animal oocyte

Abstract

The invention discloses a method for counting follicles at all levels for evaluating the quality of oocytes of experimental animals, which comprises the following steps of: equally dividing the diameter of a long axis of an ovary tissue of an experimental animal into 4n parts along the long axis direction of the ovary tissue of the experimental animal, clockwise rotating the 4n parts of the ovary tissue by 90 degrees along the cutting direction, fixedly arranging the ovary tissue in an embedding box according to a sequence, and dehydrating the ovary tissue, then embedding and fixing the follicles in the same wax block, then slicing, dyeing and sealing, and finally counting the number of the follicles at each level through microscopic examination. According to the experimental animal follicle counting method provided by the invention, the slicing process is simplified, different areas of the oocyte are displayed on one slice, and the number of each level of follicles of the whole oocyte can be counted through one slice, so that the manpower for slicing and counting is saved, the loss of slides is reduced, and the working efficiency is improved. The counting efficiency and the scientificity of oocyte quality evaluation are improved, and the problems that the oocyte quality evaluation in the market is relatively high in limitation and cannot be popularized on a large scale are solved to a great extent.

Description

Technical Field

[0001] The present invention belongs to the field of female reproductive safety evaluation in toxicological evaluation of new drug development, and relates to ovarian function evaluation therein, and specifically relates to a method for counting follicles at various levels for evaluating the quality of oocytes in experimental animals. Background Art

[0002] When conducting nonclinical safety evaluations, especially in the field of female reproductive safety evaluation, the evaluation of the ovaries of experimental animals is crucial. The loss of oogonia, oocytes, or supporting somatic cells caused by the test substance may have adverse effects on reproduction. In reproductive toxicity studies, the detection of female reproductive toxicity relies on a combination of methods, including the assessment of estrous cycle duration, oocyte fertility assessment, corpus luteum counts, organ weights, and histopathology. In order to fully evaluate the ovaries, it is necessary to understand all morphological components of the ovaries and understand the changes that occur during the normal estrous cycle and aging. Usually, the first step in the evaluation of ovarian histopathology is a thorough qualitative histological examination by a toxic pathologist using sections stained with conventional stains (such as hematoxylin and eosin), including the discovery of morphological features associated with most functional changes, including changes in the relative number or density of ovarian components. However, histological examination can only evaluate the morphology of oocytes and cannot quantitatively and intuitively evaluate the quality of oocytes.

[0003] The main function of the ovary is to produce eggs and secrete estrogen and progesterone. Oogonia divide to form oocytes, which complete the maturation process before egg fertilization in the ovary. Oocytes are surrounded by epithelial cells to form primordial follicles. Primordial follicles gradually develop into primary follicles, secondary follicles and mature follicles. The quality of oocytes is directly related to the fertilization rate, the cleavage rate of fertilized eggs, the survival rate of embryos, the establishment of pregnancy, and the development and health of the fetus. The evaluation of oocyte quality is a key issue in the evaluation of ovarian function and directly reflects the reproductive function of the ovary. The evaluation of oocyte quality mainly analyzes the morphological structure and maturity of oocytes. The basis of the evaluation is the structural and biochemical changes of oocytes during growth and development. At present, the methods for evaluating oocyte quality mainly include invasive and non-invasive evaluation methods. In toxicological pathology evaluation, non-invasive methods are usually used to evaluate oocyte quality, including ovarian weight and follicle size evaluation methods, perivitelline space, and first polar body morphology evaluation methods. However, literature indicates that the relationship between oocyte morphology and embryonic developmental capacity remains to be discussed, and other morphological characterization methods have the defects of short existence time and difficulty in capturing in tissue sections, making them difficult to apply in a large number of toxicological assessments.

[0004] Ovarian follicle counts are an important method for assessing ovarian toxicity in reproductive toxicity studies. The functional lifespan of the ovaries is largely dependent on the number of oocytes present, and the number of primordial follicles in female animals is usually constant, and the number of primordial follicles decreases continuously during follicular development. Drugs with reproductive toxicity have specific effects on primordial and primary follicles, leading to the loss of oocytes and ultimately adversely affecting fertility. Therefore, the U.S. Environmental Protection Agency (EPA), the U.S. Food and Drug Administration (FDA), and the Organization for Economic Cooperation and Development (OECD) guidelines for reproductive studies recommend quantitative assessment of primordial follicles in the ovaries. Currently, regulatory guidelines for general toxicity studies do not require follicle counts. However, properly performed follicle counts can complement qualitative ovarian assessments to characterize ovarian toxicants and understand their site of action.

[0005] Reproductive toxicity studies are an important part of the nonclinical safety evaluation of drugs and an important link for drugs to enter clinical research and market. In the preclinical general toxicology evaluation stage, the histopathological evaluation of the ovary usually examines tissue changes on a single slice. In the preclinical reproductive toxicity study stage, the cell changes of the ovary need to be examined in more detail. The most commonly used methods for follicle quantification are direct counting and stereology. The direct counting method uses the whole ovary embedded in paraffin and sliced ​​as a whole. The pathologist directly counts the follicles on some slices according to the set sampling method. This time-consuming method is mostly used for experimental animals with smaller ovarian diameters, such as rodents. For experimental animals with larger ovaries, this method of full cutting and full examination will be very time-consuming, material-consuming and inefficient. Another stereology method quantifies the number of tissue cells through designed tissue sampling and cell counting methods. This method does not require all tissue sections and examinations, but only examines some of the designed cells, and predicts the actual number of the entire tissue by calculation. The use of stereological methods requires some specialized equipment, including a microscope with a motorized stage driven by stereo software, and potentially more time-consuming tissue manipulation, such as resin embedding, sectioning using a microtome equipped with a glass knife, etc. Although this method improves efficiency, its increased cost, equipment requirements, and required expertise have led to less practical application of this technology in the evaluation of reproductive toxicity of drugs.

[0006] There is an urgent need for a practical method to evaluate the quality of oocytes in medium-sized and large experimental animals. Based on this problem, the present invention combines the randomness and scientific sampling principles of stereology with direct film reading to quantify various oocytes, and establishes an efficient method for quantifying rabbit oocytes. Compared with traditional methods, this method not only provides reliable data, but also significantly reduces evaluation time and cost. This study provides a new method for reducing costs and increasing efficiency for evaluating the effects of drugs on the quality of female oocytes in larger animals. Summary of the invention

[0007] Purpose of the invention: The technical problem to be solved by the present invention is to provide a method for counting follicles at various levels for evaluating the quality of oocytes in experimental animals. The ovaries are divided according to the principle of stereology and then the follicles are counted, which saves labor and glass slide loss and reduces the risk of excessive differences when counting a large number of counts.

[0008] Technical solution: In order to solve the above technical problems, the present invention provides a method for counting follicles at different levels for evaluating the quality of oocytes in experimental animals, comprising the following steps: dividing the long axis diameter of the ovarian tissue of the experimental animal into 4n parts along the long axis direction, and then rotating the 4n parts of the ovary by 90° clockwise along the cutting direction, arranging them in an embedding box in order for dehydration, and then embedding and fixing them in the same wax block, and then slicing, staining, and sealing, and finally counting the number of follicles at different levels in the 4n sections by microscopic examination and adding them up to obtain the number of follicles at different levels, where n=1 and a positive integer greater than 1.

[0009] As an optional embodiment, New Zealand rabbits are commonly used experimental animals in reproductive safety assessments, but primates and rodents are also commonly used in reproductive experiments.

[0010] Wherein, the thickness of the slice is 3-6 μm.

[0011] Wherein, the ovary includes bilateral ovaries. The method can be used to sample bilateral ovaries and make slices to count follicles at different levels.

[0012] The ovaries of the experimental animals are ovarian tissues fixed in neutral formalin, and the concentration of the neutral formalin is 10%.

[0013] The specific steps of dehydration are as follows: soaking the tissue embedding box in corresponding solutions for corresponding time in sequence, the immersion solution should completely cover the embedding box, soaking the tissue in 70% ethanol for 60 minutes, 80% ethanol for 240 minutes, 90% ethanol for 70 minutes, 95% ethanol for 60 minutes, and 95% ethanol for 50 minutes in sequence; then transferring to anhydrous ethanol for immersion for 30 minutes, taking out the tissue, transferring to new anhydrous ethanol for immersion for 40 minutes, taking out the tissue, transferring to anhydrous ethanol for immersion for 45 minutes, taking out the tissue, transferring to xylene for immersion for 30 minutes, then transferring to xylene for immersion for 30 minutes, and finally transferring to liquid paraffin for immersion for 180 minutes.

[0014] The specific steps of embedding are: rotating 4n parts of ovarian tissue by 90 degrees clockwise along the cutting direction to expose four different sections, injecting paraffin after confirming the position, and the height of the injected paraffin is half of the thickness of the ovarian tissue. This process is carried out on a heating table, transferring the embedding box to a position that is easy to cool, gently pressing and straightening the ovarian tissue with tweezers, covering the embedding mold with the corresponding embedding box, and injecting paraffin again to make the paraffin completely cover the ovarian tissue.

[0015] The specific steps of the microscopic examination are: conducting a comprehensive examination of the 4n partial sections, and performing graded counting and statistical summation of the primordial follicles, primary follicles, secondary follicles and mature follicles.

[0016] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: the experimental animal follicle counting method provided by the present invention simplifies the slicing process, and different areas of the oocyte are displayed on one slice. The number of follicles at all levels of the entire oocyte can be counted through one slice, which saves manpower for slicing and counting and saves the loss of glass slides, improves the counting efficiency and the scientific nature of oocyte quality assessment, and largely solves the problem that oocyte quality assessment in the market is highly limited and cannot be promoted on a large scale. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Schematic diagram of the method for evaluating the quality of New Zealand rabbit oocytes;

[0018] Figure 2 The figure is a comparison of the statistics of the number of primordial follicles between the traditional method and the method of the present invention. DETAILED DESCRIPTION

[0019] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0020] New Zealand rabbits were purchased from Qingdao Kangda Aibo Biotechnology Co., Ltd., with an age of about 5 to 6 months, i.e., the stage of sexual maturity, and a body weight of about 2.5 to 3.5 kg.

[0021] Example 1

[0022] 1. During autopsy, the ovaries of both sides of the New Zealand rabbit were collected and distinguished into left and right. After the tissues were collected, they were fixed in 10% neutral formalin. The ovaries of the New Zealand rabbit were long oval and about 1.2 cm to 2.0 cm in length.

[0023] 2. When sampling, keep the whole ovary, select the section perpendicular to the long axis of the ovary for segmentation, and divide the ovary into 4 parts according to the long axis diameter of the tissue. Rotate the 4 parts of the ovary 90° clockwise along the cutting direction to ensure that four different sections are exposed. Figure 1 , fix and arrange the four parts of the ovary in order in the embedding box.

[0024] 3. Dehydration: Dehydration in a broad sense is commonly used in pathological techniques, that is, the tissues that have been sampled are dehydrated, transparent, and waxed. During the dehydration process, you can use a dehydrator to set the following program for dehydration or manual dehydration: soak the tissue embedding box in the corresponding solution for the corresponding time in turn, and the soaking solution should completely cover the embedding box. After taking out the embedding box, put it into the next solution and process it according to the following steps:

[0025] 70% ethanol (60 min) → 80% ethanol (240 min) → 90% ethanol (70 min) → 95% ethanol (60 min) → 95% ethanol (50 min) → anhydrous ethanol (30 min) → anhydrous ethanol (40 min) → anhydrous ethanol (45 min) → xylene (30 min) → xylene (30 min) → paraffin (50 min) → paraffin (50 min) → paraffin (60 min) → paraffin (20 min).

[0026] 4. Embedding: In order to improve the efficiency of slice making, the embedding process is optimized. Take out the dehydrated embedding cassette and put it into a suitable embedding mold. Rotate the four ovarian tissues 90° clockwise along the cutting direction to expose four different sections. After confirming the position, inject paraffin (this process is recommended to be performed on a heating table). Move the embedding cassette to a position that is easy to cool. Use tweezers to gently press and straighten the tissue. Cover the embedding mold with the corresponding embedding cassette and inject paraffin. After the paraffin is fully solidified, peel off the embedding mold and clean off the excess paraffin attached to the edge of the embedding cassette in preparation for sectioning.

[0027] 5. Slicing: Fix the wax block on the slicer, adjust the wax block and the knife to the appropriate position, and move the wax block fixing device so that the wax block contacts the blade.

[0028] ① Rough trimming of sections: Turn the hand wheel, set the thickness to 10-20 μm, and perform rough trimming until the four pieces of ovarian tissue are completely exposed (the cortex and medulla of the ovarian tissue can be seen under the microscope).

[0029] ② Fine sectioning: Set the thickness to 6μm and slice. After cutting out an ideal slice, gently pick it up with a brush and move it to a water bath at about 42℃ for expansion.

[0030] ③After unfolding, stick the tissue on a glass slide, dry it slightly in the air, and then dry it in a dryer at about 65°C. After the slide is dry, it is ready for staining.

[0031] 6. Staining: Soak the tissue sections in the corresponding solutions for the corresponding time. The immersion solution should ensure that the tissue sections are completely covered. In the middle, the tissue sections can be taken out on filter paper or paper towels to properly shake off the solution of the previous step, and then put them into the solution of the next step. Stain the tissue sections according to the following steps:

[0032] Xylene (10min) → xylene (5min) → xylene (5min) → anhydrous ethanol (5min) → anhydrous ethanol (2min) → 95% ethanol (2min) → 80% ethanol (2min) → water wash (2min) → hematoxylin (5min) → water wash (2min) → 0.5% hydrochloric acid alcohol (3s) → water wash (10min) → water wash (10min) → eosin (5min) → 95% ethanol (10s) → anhydrous ethanol (30s) → anhydrous ethanol (1min) → anhydrous ethanol (2min) → xylene (2min) → xylene (2min) → xylene (2min).

[0033] After staining, a preliminary inspection of the slide shows that the nucleus and cytoplasm are clearly separated, the red and blue are moderate, the cell nucleus is blue, and the cytoplasm is red, which is a qualified stained section.

[0034] 7. Sealing: Take the slide out of xylene, wipe off the excess xylene on filter paper, then drop an appropriate amount of gum on the slide, slowly put a cover slip on the slide, and use filter paper to absorb the excess gum that overflows around the cover slip. If there are bubbles in the slide, use tweezers to squeeze the cover slip to make the bubbles overflow. After the gum is dry and fixed, it is ready for microscopic examination.

[0035] 8. Microscopic examination: The four parts of the slices were observed under microscopic photography to count the number of follicles of each level in both ovaries (Olympus optical microscope, BX53, Japan), including the graded counting of primordial follicles, primary follicles, secondary follicles, and mature follicles.

[0036] from Figure 1 The operation schematic diagram of the present invention can be seen. Figure 1 As shown in the figure, the ovary of the New Zealand rabbit is oblong. The ovary is divided into four equal parts according to the long axis of the ovary, and then embedded after being rotated 90° clockwise, presenting four sections on the same slice.

[0037] Comparative example: Direct follicle counting method

[0038] 1. During autopsy, the ovaries of both sides of the New Zealand rabbit were collected and distinguished into left and right. After the tissues were collected, they were fixed in 10% neutral formalin. The ovaries of the New Zealand rabbit were long oval and about 1.2 cm to 1.8 cm in length.

[0039] 2. The left and right ovaries of three New Zealand rabbits were sliced, dehydrated, embedded, serially sliced, HE stained, and sealed according to the method of the embodiment; wherein, the slices were continuously cut with a thickness of 6 μm, and according to the currently commonly used "direct counting method", the ovaries appeared in the slices as the starting point, and the first slice was retained in every ten slices, and 84, 129, and 266 equidistant slices were obtained respectively. The diameter of the rabbit's primordial follicle oocyte is 23.86 ± 2.23 μm, and the interval distance of the slice sampling is greater than the diameter of the primordial follicle. Under this condition, the repeated counting of the primordial follicle can be effectively avoided. Only the nucleated follicles were counted during the counting.

[0040] 3. Three New Zealand rabbits of similar weight and age were selected for statistics, and the number of follicles at each level in the ovaries of the New Zealand rabbits was counted by the traditional direct counting method and the innovative counting method of Example 1. The direct counting method was counted using the following formula: the total number of follicles at each level on each slice (Q) was calculated, and this number was multiplied by the reciprocal of the sampling interval number (f) to obtain the total number of follicles at each level in the entire ovarian tissue.

[0041] N=Q / (1 / f)

[0042] N: total number of follicles in ovarian tissue

[0043] Q: The total number of follicles of each level in the extracted counting sections (each type of follicle is counted separately)

[0044] f: sampling interval number. In the present invention, one slice is selected for counting every 10 slices, so the sampling interval number is 10.

[0045] 4. Comparison of the total number of primordial follicles and the number of follicles per unit area obtained by the direct counting method and the counting method of the present invention. The results are as follows: Figure 2 , as shown in Table 1. Figure 2 It can be seen that the total number of primordial follicles by the method of the present invention is compared with the total number of primordial follicles by direct counting method. "ns" means no significant difference.

[0046] Table 1 Comparison of the number of follicles per unit area by the method of the present invention and the number of follicles per unit area by the direct counting method

[0047]

[0048] Drawing on the example of the direct counting method of rodents, a correction factor of 10 is used, that is, the interval is 60μm for counting. However, secondary follicles or mature follicles in the same position can be seen from the selected continuous sections. Combined with the diameter of rabbit secondary follicles and mature follicles (>60μm), the comparative practice of the direct counting method shows that it is very easy to repeat the counting of the same secondary / mature follicle on adjacent sections. At this time, the correction factor is introduced again, and the number of follicles counted repeatedly is magnified again, increasing the error. Therefore, although the direct counting method has avoided the repeated counting of primitive follicles, it cannot avoid the repeated counting of primary, secondary, and mature follicles with larger diameters. The result after multiplying by the correction factor is inaccurate. Multiplying by the correction factor may amplify the data, and the error of the relative estimated value will also be enlarged. The number of secondary follicles and mature follicles obtained is also obviously inconsistent with the actual situation. Therefore, although the direct counting method is currently a commonly used method for evaluating the number of follicles, the accuracy of the direct counting method is still questionable.

[0049] The present invention statistically analyzes the total number of primordial follicles obtained by introducing the correction factor, and the results are as follows: Figure 2 At this time, we introduce the "correction factor" of the innovative counting method based on the long axis diameter of the rabbit ovary to calculate the total number of follicles in each ovary; for example, if the ovary diameter is 1.5 cm, selecting four surfaces for statistics is equivalent to selecting one section for every 625 sections, and the correction factor is 625. Figure 2 The results showed that there was no significant difference (P>0.05) between the results of the ovarian follicles of 3 New Zealand rabbits counted using the direct counting method and the results of the 3 rabbits counted using the innovative counting method. And according to the distribution of the data, for ovaries with similar long axis diameters, the total number of primordial follicles ranged from 3×10 5 Span to 7.2×10 5 , the data fluctuates greatly, therefore, the animal's own primordial follicle storage and physiological condition will also affect the number of primordial follicles. Although the order of magnitude of follicles at other levels is smaller than that of primordial follicles, there are more repeated counting phenomena in the direct counting method. In the statistics of the innovative counting method, no more repeated counting phenomena were found for secondary follicles and mature follicles with larger diameters. In order to exclude the influence of individual animals on the comparison of the two methods, we selected sections in the slides of the direct counting method at a ratio of 25%, 50%, 75%, and 100%, that is, we used the two methods to compare the same individual at the same time and calculated the number of primordial follicles per unit area. The results show that there is no significant difference in the results of the two methods, and they have a high consistency. The density of follicles at all levels per unit area can also better reflect the distribution of follicles at all levels in the ovaries.

[0050] According to the principle of random sampling, the four sections extracted by the present invention represent 25%, 50%, 75%, and 100% of the ovarian sections, respectively, and can represent the sampling area and follicle distribution of the four ovarian sections after segmentation. From the above comparison, it can be seen that the follicle statistics obtained by our innovative counting method can characterize the number of follicles. The innovative counting method can greatly improve the efficiency of film making and statistics while ensuring the accuracy rate, and effectively solve the problem that the traditional direct counting method cannot effectively count and quantify secondary follicles / mature follicles, and realize accurate counting of follicles at all levels of New Zealand rabbits.

[0051] The counting method of the present invention greatly improves the efficiency of slide making and pathologist counting process on the basis of ensuring accuracy. When pathologists count the number of follicles, for a New Zealand rabbit, the direct counting method will produce at least 100 slices, including the statistics of 200 sections; while the innovative counting method only needs to count the number of follicles of 4 sections of the same slice, and the number of animals in toxicological experiments often ranges from 30 to 100. According to the method of the present invention, when the innovative counting method is used in the counting of follicles in conventional reproductive toxicity tests, 90% of the slides can be reduced, the amount of slide making and loss can be reduced, and the efficiency and accuracy of manual statistics can be improved.

Claims

1. A method for counting follicles at various levels for evaluating the quality of oocytes in experimental animals, characterized in that: The following steps are involved: The long axis diameter of the ovarian tissue of the experimental animal is divided into 4n parts along its long axis direction, and then the 4n parts of ovarian tissue are rotated 90° clockwise along the cutting direction, fixed and arranged in order in an embedding box for dehydration, and then embedded and fixed in the same wax block, and then sliced, stained, and sealed. Finally, the number of follicles of each level in the 4n sections is counted by microscopy and the sum is added to obtain the number of follicles of each level, where n is a positive integer greater than 1.

2. The method for counting follicles at various levels for evaluating the quality of oocytes in experimental animals according to claim 1, characterized in that: The experimental animals include rabbits, primates or rodents.

3. The method for counting follicles at various levels for evaluating the quality of oocytes in experimental animals according to claim 1, characterized in that: The experimental animals are New Zealand rabbits.

4. The method for counting follicles at various levels for evaluating the quality of oocytes in experimental animals according to claim 1, characterized in that: The thickness of the slice is 3-6 μm.

5. The method for counting follicles at various levels for evaluating the quality of oocytes in experimental animals according to claim 1, characterized in that: The ovarian tissue of the experimental animal is the ovarian tissue fixed in neutral formalin.

6. The method for counting follicles at various levels for evaluating the quality of oocytes in experimental animals according to claim 1, characterized in that: The specific steps of dehydration are: soaking the ovarian tissue embedding box in the corresponding solution for the corresponding time in sequence, the immersion solution should completely cover the embedding box, taking out the embedding box and then putting it into the next solution, and processing according to the following steps: soaking the tissue in 70% ethanol for 60 minutes, 80% ethanol for 240 minutes, 90% ethanol for 70 minutes, 95% ethanol for 60 minutes, and 95% ethanol for 50 minutes in sequence; Then transfer to anhydrous ethanol and soak for 30 min, take out the tissue, transfer to new anhydrous ethanol and soak for 40 min, take out the tissue, transfer to anhydrous ethanol and soak for 45 min, take out the tissue, transfer to xylene and soak for 30 min, then transfer to xylene and soak for 30 min, and finally transfer to liquid paraffin and soak for 180 min.

7. The method for counting follicles at various levels for evaluating the quality of oocytes in experimental animals according to claim 1, characterized in that: The specific steps of embedding are: rotating the 4n parts of ovarian tissue 90° clockwise along the cutting direction to expose four different sections, injecting paraffin after confirming the position, and the height of the paraffin injection is half of the thickness of the ovarian tissue. This process is carried out on a heating table, transferring the embedding box to a position that is easy to cool, gently pressing the ovarian tissue with tweezers, covering the embedding mold with the corresponding embedding box, and injecting paraffin again to completely cover the ovarian tissue. After the paraffin is fully solidified, the embedding mold is peeled off, and the excess paraffin attached to the edge of the embedding box is cleaned off.

8. The method for counting follicles at various levels for evaluating the quality of oocytes in experimental animals according to claim 1, characterized in that: The specific steps of the microscopic examination are: comprehensively examining the 4n partial sections, and performing graded counting and statistical addition of the primordial follicles, primary follicles, secondary follicles and mature follicles.

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