This work was supported by the Major Project Plan of the Special Development Fund for the Shanghai Zhangjiang National Independent Innovation Demonstration Zone (ZJ2022-ZD-006), Shanghai Municipal Science and Technology Commission Targeted Funding Project (22DX1900400), Youth Program of Shanghai Municipal Health Commission (20204Y0276). the National Natural Science Foundation of China (32070849).

The protocol of ICSI-ET described below follows the guidelines and has been approved by the Animal Ethical Review of the Shanghai Institute of Planned Parenthood research. Safety Procedures: Always wear appropriate personal protective equipment (PPE) when handling chemicals or biological materials. Use of Hoods: Perform all procedures involving volatile chemicals or aerosol generation within a certified fume hood or biosafety cabinet. Use female mice (6-8 weeks old B6D2F1 strain) for the superovulation procedure.
1. ICSI in mice
<ol>
	<li>Superovulation of mice
	<ol>
		<li>Administer an intraperitoneal injection of pregnant mare serum gonadotropin (PMSG) at a dose of 7.5 IU per mouse at around 20:00 on the first day.</li>
		<li>Administer an intraperitoneal injection of human chorionic gonadotropin (HCG) at a dose of 7.5 IU per mouse at around 20:00 (48 hours after PMSG injection) on the third day.</li>
	</ol>
	</li>
	<li>Prepare HTF medium into 1 mL aliquots and leave them uncovered for overnight equilibration at 37 &#176;C in a 5% CO2 incubator on the same day of HCG injection in female mice.</li>
	<li>Preparation of ICSI fertilization droplets
	<ol>
		<li>On the same day of HCG injection in female mice, divide a 50 x 9 mm cell culture dish lid into upper and lower halves. Use the upper half to create 4-6 droplets of 5 &#181;L each, using 10% polyvinylpyrrolidone (PVP) for sperm placement. Use the lower half to create 8-10 droplets of 5 &#181;L each, using M2 medium for the ICSI procedure.</li>
		<li>Cover the PVP and M2 medium droplets with approximately 3 mL of mineral oil (Figure 1).</li>
	</ol>
	</li>
	<li>Preparation of culture dishes
	<ol>
		<li>On the same day of HCG injection in female mice, dispense 12 drops of KSOM medium (MR-020P-D) of 20 &#181;L evenly in a 35 x 10 mm cell culture dish. Cover the drops with approximately 3 mL of mineral oil.</li>
	</ol>
	</li>
	<li>Sperm harvesting:
	<ol>
		<li>Euthanize male mice by cervical dislocation at approximately 9:00 am on the fourth day (13 h after HCG injection).</li>
		<li>Isolate the epididymal cauda and thoroughly clean it to eliminate any adipose tissue and blood remnants by gently blotting with sterile gauze and rinsing in prewarmed HTF medium.</li>
		<li>Cut open the cauda epididymis to allow the sperm to freely swim out into the HTF medium. Gently squeeze the cauda using forceps to release the sperm, collect them in preequilibrated HTF tubes, and keep them uncovered in a 37 &#176;C, 5% CO2 incubator for sperm capacitation for further use.</li>
	</ol>
	</li>
	<li>Oocyte retrieval
	<ol>
		<li>Euthanize female mice stimulated for oocyte retrieval by cervical dislocation at around 9:30 am on the fourth day. Carefully make an incision along the midline of the abdominal skin and muscle layer to expose the abdominal cavity.</li>
		<li>Use fine forceps to gently isolate tissue from both sides of the oviducts, especially the swollen ampulla regions, and transfer it into M2 medium in a sterilized culture dish. Repeat this procedure until the oviducts from all the designated mice are collected.</li>
		<li>Under a dissecting microscope, use a 1 mL syringe needle to puncture the transparent section of the swollen ampulla of the oviduct gently, facilitating the release of cumulus-oocyte complexes (COCs) into the medium.</li>
		<li>Using the same syringe, carefully pick and transfer the COCs into a new dish containing 100 &#181;L of fresh M2 medium. Repeat this step until all COCs are successfully transferred from the original oviduct segments.</li>
		<li>Wipe away blood and tissue fluids, and under a dissecting microscope, tear open the ampulla of the oviduct using a 25 G needle, resulting in the release of numerous COCs.</li>
		<li>Remove the oviduct and tissue fragments, transfer the COCs into droplets of M2 medium containing hyaluronidase (HY), and prewarm them to 37 &#176;C.</li>
		<li>After incubation for approximately 5 min, carry out gentle pipetting to facilitate the separation of cumulus cells and oocytes. Transfer the metaphase II (MII) oocytes from the HY-containing solution to fresh M2 medium; then, perform the sequential transfer into multiple droplets of M2 medium.</li>
		<li>After 2-3 rounds of washes, transfer the oocytes to droplets of KSOM culture medium and keep them in a 37 &#176;C, 5% CO2 incubator for further use.</li>
	</ol>
	</li>
	<li>Around 10:00 am on the fourth day, sonicate 100 &#181;L of the capacitated sperm (see step 1.5.2.) for 5 s to separate the heads from the tails at a frequency of 46 kHz, with a power setting of 50 watts. 
	NOTE: When sonicating sperm to truncate the tail, be cautious to control the time, which should not exceed 5 s. A few seconds after sonication, sperm can be observed under an inverted microscope; stop sonicating when approximately half of the sperm show truncated tails.</li>
	<li>ICSI procedure
	<ol>
		<li>Use a commercially available micromanipulation needle with a flat tip and an inner diameter of 6 &#181;m, set at a 25&#176; angle, for the injection. Fill the needle with the operating fluid with the filled portion measuring a length of 0.5 cm inside the needle. Ensure that the level of the fluid reaches ~0.5 cm. 
		NOTE: Maintain room temperature at 19 &#176;C during injection to avoid high temperatures that may reduce the survival rate of fertilized oocytes. The precision in ensuring the fluid level reaches 0.5 cm is crucial for maintaining the integrity of the injection process and ensuring the viability of the oocytes.</li>
		<li>Install the injection needle on the right arm of the micromanipulator, securely fixed by tightening the holding cap. Install a commercially available flat-tipped micromanipulation holding pipette designed for mouse oocytes on the left arm of the micromanipulator, and position the injection needle and the holding pipette in the microscope&#39;s field of view.</li>
		<li>Add 1 &#181;L of sonicated sperm to a droplet of PVP solution using a pipette.</li>
		<li>Place 20 MII oocytes in a droplet of M2 medium.</li>
		<li>Use the injection needle to aspirate 10-20 sperm heads in PVP solution. Then, move the injection needle into the droplet containing the oocytes.</li>
		<li>Directly determine the position of the metaphase spindle within the oocyte under a polarization microscope to ensure that the spindle apparatus is not on the side of injection, with the polar body typically located at either the 12 o&#39;clock or 6 o&#39;clock position relative to the oocyte&#39;s orientation.</li>
		<li>Keep the oocyte in contact with the bottom of the culture dish. Adjust the focal plane so that the zona pellucida of the oocyte and the injection needle are in the same plane.</li>
		<li>Upon contact of the injection needle with the zona pellucida, apply a slight negative pressure to the injection needle, drawing in a portion of the zona pellucida.</li>
		<li>Simultaneously activate piezo pulses with an intensity setting of 5 and a frequency of 1, effectively creating a perforation in the zona pellucida and allowing the injection needle to pass through.</li>
		<li>Advance the injection needle into the perivitelline space, pushing the sperm head to the tip of the needle. Insert the injection needle into the oocyte until it reaches the opposite side of the zona pellucida.</li>
		<li>Use piezo pulses set to an intensity of 1 and a frequency of 1 to create a small pore in the plasma membrane, ensuring the sperm head is injected into the ooplasm with minimal entry of PVP and optimal preservation of the oocyte&#39;s integrity.</li>
		<li>Repeat step 1.8.11 until all 20 oocytes are injected, followed by a 15 min incubation at 19 &#176;C.</li>
		<li>Transfer the surviving oocytes to KSOM medium and place them in the incubator. Observe 2-cell, 4-cell, morula, and blastocyst formation rates. 
		NOTE: Timing for intracytoplasmic single sperm injection (ICSI) into the oocyte should be controlled, aiming to complete the procedure within 14-18 h after the injection of HCG.</li>
	</ol>
	</li>
</ol>
2. Uterine transplantation of mouse blastocysts
<ol>
	<li>Preparation of vasectomized male mice 
	NOTE: Use ICR male mice aged 6-8 weeks for the study.
	<ol>
		<li>Induce anesthesia by intraperitoneal injection of 1.25% 2,2,2-tribromoethanol at a dosage of 0.2 mL/10 g of body weight. The absence of any pain response indicates successful anesthetization. 
		NOTE: While the current protocol does not include the application of vet ointment to the eyes of anesthetized animals, it is recommended as a best practice to prevent eye dryness and ensure the health and comfort of the animals during procedures. Throughout the surgical and recovery process, all surgical instruments were sterilized before use, and the surgical area was prepared using antiseptic solutions to ensure a clean operating environment.</li>
		<li>To safeguard against potential stress or injury, house the mice that have undergone surgery individually during the initial recovery phase.</li>
		<li>Administer buprenorphine at a dosage of 0.05 mg/kg of body weight to ensure optimal pain relief. 
		NOTE: To ensure the well-being of the mice, closely monitor the mice during the recovery period and do not leave them unattended until they regain sufficient consciousness to maintain sternal recumbency.</li>
		<li>Prepare the surgical site at the lower abdominal region, superior to the testes. Remove the fur and disinfect with povidone-iodine. Using sterile scissors, make a 1 cm longitudinal incision along the linea alba. Gently remove any blood with sterile, dust-free paper and proceed to bluntly dissect the abdominal muscles along the linea alba and access the abdominal cavity.</li>
		<li>Using curved forceps, gently grasp and expose the left testis and its associated fat pad. Identify the vas deferens and carefully isolate it using pointed forceps, creating a 10 mm segment for manipulation.</li>
		<li>Sterilize the curved forceps by holding them in the outer flame of an alcohol lamp for several seconds. Gently grasp the vas deferens, causing it to become flattened and whitened due to cauterization. Create a second cauterization point 3-5 mm distal to the first. Using sterile surgical scissors, excise the segment of the vas deferens between the two cauterization points.</li>
		<li>Carefully inspect the cauterized ends of the vas deferens, ensuring that a short, flattened, whitened segment remains. Using curved forceps, gently return the testis and its associated fat pad to the abdominal cavity.</li>
		<li>Repeat the procedure described in step 2.1.6 on the contralateral (right) side.</li>
		<li>Close the surgical site using sutures, typically placing one stitch in the muscle layer and three stitches in the skin. Ensure proper alignment of the surgical incision and disinfect with povidone-iodine. Transfer the animal to a heating pad for observation, monitoring for 10-15 min until the animal regains consciousness. Once recovered, return the animal to its housing cage. 
		NOTE: Following vasectomy surgery, mice require a 14-day recovery period, followed by a 1-week trial mating experiment.</li>
		<li>During this week, continuously mate the vasectomized males with females and observe the pregnancy status of all females presenting with copulatory plugs. Successful mating is indicated by the presence of a yellowish-white, blocky vaginal plug at the vaginal orifice of the female mouse.
		<ol>
			<li>Pair the mice between 4:00 PM and 6:00 PM on the first day and check the vaginal plugs between 7:00 AM and 9:00 AM on the second day. Consider females with vaginal plugs to be in a pseudopregnant state at 0.5 days and use them for subsequent embryo transfer experiments. Return females without vaginal plugs to the recipient group. 
			NOTE: These females without vaginal plugs can continue to be used for mating to prepare pseudopregnant females. Ensure that all plugged females do not become pregnant; only then can the vasectomized males be used for formal experiments.</li>
			<li>While vasectomized males can mate daily, allow for 1 day of rest between mating sessions. 
			NOTE: Based on our experience, vasectomized males can maintain a high copulatory plug rate for over a year. However, vasectomized males that fail to produce copulatory plugs in mating females for 4-6 consecutive attempts should be removed from the study.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Preparation of pseudopregnant females
	<ol>
		<li>Generate pseudopregnant female mice by mating ICR female mice (aged 7-10 weeks, weighing over 25 g) with vasectomized male mice (3-6 months old) at a 1:1 ratio. 
		NOTE: The vasectomized males were used to stimulate the uterine cervix of the female mice, resulting in the activation of the corpora lutea.</li>
	</ol>
	</li>
	<li>Preparation of the pipette for transferring blastocysts
	<ol>
		<li>Take the embryos to be transplanted out from the incubator and transfer them into a droplet of M2 medium at a temperature of 37 &#176;C.</li>
		<li>Use a transfer pipette to aspirate a small volume of liquid, draw a small amount of air, and aspirate 6-10 embryos. Finally, aspirate another small volume of air to seal the pipette. 
		NOTE: The length of the fluid in the transfer pipette should not exceed 0.5 cm.</li>
	</ol>
	</li>
	<li>Uterine transplantation (blastocysts)
	<ol>
		<li>Weigh the pseudopregnant female mice (2.5 days post mating) and anesthetize them by intraperitoneal injection of 1.25% 2,2,2-tribromoethanol at a dose of 0.2 mL/10 g of body weight. Gently press the ear with forceps.</li>
		<li>After administering anesthesia to the mice and ensuring asepsis through alcohol disinfection, make a midline longitudinal incision on the ventral abdomen. Taking advantage of the loose subcutaneous tissue, shift the incision to the left or right side as required for the transplant. Use small scissors to perform blunt dissection of the abdominal wall muscle layer at ~0.5 cm lateral to the lumbar spine to access the peritoneal cavity.</li>
		<li>Using atraumatic forceps, carefully exteriorize the ovaries, oviducts, and uterus.</li>
		<li>Using the tip of a syringe needle, make a small, upward-angled incision on the uterine wall below the utero-tubal junction, avoiding blood vessels. This incision provides access to the uterine cavity; gently insert the oviduct 2-3 mm through the small hole.</li>
		<li>Carefully expel the small air bubble at the end of the transfer pipette while cautiously watching the transfer pipette during the slow injection of the embryos. Once the central portion of the culture medium is confirmed to be expelled, halt the procedure and gently remove the transfer pipette.</li>
		<li>Reposition the ovaries, oviducts, and uterus back into the abdominal cavity. Suture the muscle layer and skin separately. Place the mice on a heating pad until they recover from anesthesia. To ensure the well-being of the mice, closely monitor the mice during the recovery period and do not leave them unattended until they regain sufficient consciousness to maintain sternal recumbency.</li>
	</ol>
	</li>
	<li>Observation of pregnancy
	<ol>
		<li>Following the surgery, observe the mice for 18-21 days to monitor the number of offspring born. In case of dystocia (difficult birth), perform a cesarean section. Allow the newborn mice to be fostered by lactating dams of the same age group.</li>
	</ol>
	</li>
</ol>
3. Random blood glucose, fasting blood glucose, and glucose tolerance test
<ol>
	<li>Tracking random blood glucose levels during mouse growth
	<ol>
		<li>Since the birth of the mice, monitor the random blood glucose level every 4 weeks at 9 am after a good night of feeding and fun.
		<ol>
			<li>Puncture the mouse tail tip to obtain tail vein blood.</li>
			<li>The first drop of blood has a higher sugar measurement value due to the presence of the tissue fluid mixed with it. Therefore, wipe the first drop of blood using clean absorbent paper gently.</li>
			<li>Measure the blood glucose from the second drop (~4 &#181;L) from the tail vein with a handheld glucometer.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Tracking changes in fasting blood glucose and glucose tolerance during mice development 
	NOTE: Considering that the first blood sampling time point is 15 min, all mice need to be injected within 15 min. Since each mouse injection took 20-30 s, to ensure that the injections are completed within this timeframe, the maximum number of mice that can be used in each test cannot exceed 30.
	<ol>
		<li>Perform the fasting blood glucose and glucose tolerance tests every 4 weeks after birth in overnight-fasted mice.
		<ol>
			<li>On the first day, prepare fresh irradiated shavings bedding, clean cages, lids, and water bottles. Transfer the mice into a fresh cage without food. Add do not feed tags to the cages.</li>
			<li>Prepare sufficient 10% glucose injection (sterile and pyrogen-free) and 1 mL syringes.</li>
			<li>Ensure that the mice in each group fast for 16 h, from 5:00 pm on day 1 to 9:00 am on day 2, but allow free access to water. Measure the fasting blood glucose levels as described in steps 3.1.1.1-3.1.1.3 using a glucose meter.</li>
		</ol>
		</li>
		<li>Weigh the mice and mark their tails to facilitate rapid identification. Prepare glucose syringes with 10 &#181;L/g of mouse weight.</li>
		<li>At 30 s intervals, inject 10 &#181;L/g glucose into the intraperitoneal cavity of the mouse. 
		NOTE: Special attention needs to be paid to injection techniques here to avoid inaccurate liquid injection volume. The key points of operation are as follows:
		<ol>
			<li>Immobilize the mouse and restrain the tail with one hand. Keep the head slightly lower than the buttocks so that the organs in the abdominal cavity will shift towards the head, leaving enough injection space.</li>
			<li>Position the needle between the midline and the hip bone, insert it through the skin at a 45-degree angle, then gently guide the needle deeper into the abdominal cavity while keeping it aligned parallel to the skin surface for ~0.5 cm. 
			NOTE: Pay attention to any resistance during injection to determine whether the internal organs are accidentally punctured.</li>
			<li>Eject the syringe into the abdominal cavity.</li>
			<li>Record the time when the injection started and use a timer to control the injection rate. Set the timer to 90 s for three mice to avoid losing time in handling the timer.</li>
		</ol>
		</li>
		<li>Take the blood glucose measurement from the tail vein at the desired intervals (0 prior to glucose administration, 15, 30, 60, 90, and 120 min after the glucose loading). 
		NOTE: Subsequent blood glucose testing should also take 20-30 s for each mouse.</li>
		<li>Return the mice to their home cages equipped with food and water after the experiment.</li>
	</ol>
	</li>
</ol>

ICSI-ET Mouse Model to Study Long-Term Impacts on Glucose Metabolism

<ol>
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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+health+outcomes+of+human+offspring+conceived+by+assisted+reproductive+technologies+(art).">The health outcomes of human offspring conceived by assisted reproductive technologies (art).</a> J Dev Orig Health Dis. 8 (4), 388-402 (2017).</li><li>Heber, M. F., Ptak, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+assisted+reproduction+technologies+on+metabolic+health+and+diseasedagger.">The effects of assisted reproduction technologies on metabolic health and diseasedagger.</a> Biol Reprod. 104 (4), 734-744 (2021).</li><li>Ma, R. C. W., Ng, N. Y. H., Cheung, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assisted+reproduction+technology+and+long-term+cardiometabolic+health+in+the+offspring.">Assisted reproduction technology and long-term cardiometabolic health in the offspring.</a> PLoS Med. 18 (9), e1003724 (2021).</li><li>De Waal, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cumulative+effect+of+assisted+reproduction+procedures+on+placental+development+and+epigenetic+perturbations+in+a+mouse+model.">The cumulative effect of assisted reproduction procedures on placental development and epigenetic perturbations in a mouse model.</a> Hum Mol Genet. 24 (24), 6975-6985 (2015).</li><li>Carlin, J., George, R., Reyes, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methyl+donor+supplementation+blocks+the+adverse+effects+of+maternal+high+fat+diet+on+offspring+physiology.">Methyl donor supplementation blocks the adverse effects of maternal high fat diet on offspring physiology.</a> PLoS One. 8 (5), e63549 (2013).</li><li>Hiraoka, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Piezo-icsi+for+human+oocytes.">Piezo-icsi for human oocytes.</a> J Vis Exp. (170), e60224 (2021).</li><li>Sullivan-Pyke, C. S., Senapati, S., Mainigi, M. A., Barnhart, K. 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The authors have no conflicts of interest to disclose.

This study integrated mouse intracytoplasmic sperm injection (ICSI) and embryo transfer (ET) techniques to comprehensively recapitulate human assisted reproductive technology (ART) and examine the impact of ICSI in conjunction with ET on offspring development. The application of the ICSI technique with normal mouse sperm yielded high fertilization (86.76%) and 2-cell rates (88.48%). Following ET, the birth rate of offspring mice was approximately 42.50%, indicating the robustness of the technical platform. The most notew...

Fertility issues have emerged as a major focus of medical and sociological concern, especially in modern society where declining fertility rates and the increasing severity of infertility prevalence and severity have risen to prominence. Assisted Reproductive Technology (ART) provides a wide array of possibilities to tackle these challenges, with Intracytoplasmic Sperm Injection (ICSI) commonly utilized as a therapeutic intervention.
Since Palermo reported the first successful pregnancy achieved through Intracytoplasmic Sperm Injection (ICSI) in 1992, ICSI has become a pivotal technique in Assisted Reproductive Technologies (ART)1. However, considering that ICSI has been used in clinical settings for only 30 years, a relatively brief period compared to the human lifespan, the long-term effects of ICSI, especially on offspring development, have not been extensively investigated and elucidated. At present, mice characterized by their uniform genetic background and shorter lifespan, have emerged as a widely utilized alternative model in medical research. Moreover, the mouse model can recapitulate the entire human lifespan within a compressed time frame, where one year in mice roughly corresponds to 40 years in humans2.
Over the past decade, several small-scale studies have reported that individuals conceived through ICSI may be at an increased risk of developing metabolic syndromes, such as abnormal blood sugar levels, later in life3,4. Although the evidence is not definitive, this finding has nonetheless raised serious concerns within the scientific community regarding the long-term health implications of ICSI. This situation underscores the pressing need for more rigorous assessments of ART and its long-term health consequences. Particularly in light of the limitations and ethical considerations of human studies, developing animal models that can precisely recapitulate the development of human offspring following ICSI has become increasingly crucial. In this context, the mouse ICSI-ET (Intracytoplasmic Sperm Injection-Embryo Transfer) model, owing to its capacity to mimic the human ICSI and facilitate long-term monitoring of offspring health outcomes, has become an effective tool for assessing the potential health risks of ICSI technology to offspring5.
This study aims to investigate the impact of ICSI-ET technology on a prevalent metabolic phenotype, namely offspring glucose metabolic health, by employing random blood glucose monitoring, fasting blood glucose testing, and glucose tolerance tests to assess the glucose metabolic state of mice. Random blood glucose monitoring is utilized to capture the natural fluctuations in glucose metabolism during normal physiological activities, whereas fasting blood glucose and glucose tolerance tests are employed to assess potential prediabetic states.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.25% avertin (2,2,2-tribromoethanol)</td>
			<td>Nanjing Aibei</td>
			<td>M2960</td>
			<td>for anesthetization</td>
		</tr>
		<tr>
			<td>Bacteriological Petri Dishes 35 x 10 mm style w/tight lid, crystal-grade virgin polystyrene, sterile</td>
			<td>BD</td>
			<td>353001</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacteriological Petri Dishes 50 x 9 mm style w/tight lid, crystal-grade virgin polystyrene, sterile</td>
			<td>BD</td>
			<td>351006</td>
			<td></td>
		</tr>
		<tr>
			<td>Biosafety Cabinet</td>
			<td>ESCO</td>
			<td>class <img alt="Equation 1" src="/files/ftp_upload/66212/66212eq01.jpg" style="margin: auto;" /> BSC</td>
			<td>Aseptic operations, making culture dishes, aliquoting reagents, etc.</td>
		</tr>
		<tr>
			<td>CO2 Incubator</td>
			<td>Thermo</td>
			<td>8000DH</td>
			<td>Embryo culture</td>
		</tr>
		<tr>
			<td>Dissection Microscope</td>
			<td>Olympus</td>
			<td>SZX16</td>
			<td>Use in mouse embryo transfer</td>
		</tr>
		<tr>
			<td>Fluorinert Fc-770</td>
			<td>SIGMA</td>
			<td>F3556</td>
			<td>Fluorinert FC-770 is a thermally stable fully fluorinated liquid with high dielectric strength and resistivity, used as operating fluid</td>
		</tr>
		<tr>
			<td>handheld glucometer</td>
			<td>Roche</td>
			<td>AccuChek performa</td>
			<td>Blood glucose measurement</td>
		</tr>
		<tr>
			<td>HTF</td>
			<td>Merck</td>
			<td>MR-070-D</td>
			<td>The EmbryoMax Human Tubal Fluid (HTF) (1x), liquid designed for use with Mouse IVF is available in a 50 mL format and has been optimized and validated for Embryo Culture.</td>
		</tr>
		<tr>
			<td>Hydraulic Microinjector</td>
			<td>Eppendorf</td>
			<td>CellTram 4r Oil, 5196000030</td>
			<td>For sperm injection</td>
		</tr>
		<tr>
			<td>Inverted Microscope</td>
			<td>Nikon</td>
			<td>TI2-U</td>
			<td>Micromanipulation observation host</td>
		</tr>
		<tr>
			<td>KSOM</td>
			<td>Merck</td>
			<td>MR-020P-D</td>
			<td>(1x), Powder, w/o Phenol Red, 5 x 10 mL</td>
		</tr>
		<tr>
			<td>M2</td>
			<td>Merck</td>
			<td>MR-015-D</td>
			<td>EmbryoMax M2 Medium (1x), Liquid, with Phenol Red</td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>NARISHIGE</td>
			<td>NTX-N4</td>
			<td>Micromanipulation arm</td>
		</tr>
		<tr>
			<td>mineral oil&#160;</td>
			<td>SIGMA</td>
			<td>M8410</td>
			<td>Mineral oil is suitable for use as a cover layer to control evaporation and cross-contamination in various molecular biology applications.</td>
		</tr>
		<tr>
			<td>Needle Cutter</td>
			<td>Nanjing Aibei</td>
			<td>Sutter MF-800</td>
			<td>Use for fabricating micromanipulation needles</td>
		</tr>
		<tr>
			<td>Needle Puller</td>
			<td>Nanjing Aibei</td>
			<td>Sutter model p-100</td>
			<td>Use for making micromanipulation needles</td>
		</tr>
		<tr>
			<td>Piezo Drill Trip Mouce ICSI</td>
			<td>Eppendorf</td>
			<td>5195000.087</td>
			<td>Application of ICSI injection needle.</td>
		</tr>
		<tr>
			<td>Piezoelectric Micromanipulator (Membrane Breaker)</td>
			<td>Eppendorf</td>
			<td>Eppendorf PiezoXpert</td>
			<td>Use of micro-pulses to break the zona pellucida and oolemma of the oocyte</td>
		</tr>
		<tr>
			<td>Pneumatic Microinjector</td>
			<td>Eppendorf</td>
			<td>CellTram 4r Air, 5196000013</td>
			<td>For fixing the oocyte</td>
		</tr>
		<tr>
			<td>Ready-to-use Human Chorionic Gonadotropin (Hcg)</td>
			<td>Nanjing Aibei</td>
			<td>M2520</td>
			<td>Sterilization reagent, intraperitoneal injection. 50 IU/mL</td>
		</tr>
		<tr>
			<td>Ready-to-use Pregnant Mare&#39;s Serum Gonadotropin (PMSG)</td>
			<td>Nanjing Aibei</td>
			<td>M2620</td>
			<td>Sterilization reagent, intraperitoneal injection. 50 IU/mL</td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Olympus</td>
			<td>SZX7</td>
			<td>Oocyte retrieval and observation of embryo development</td>
		</tr>
	</tbody>
</table>

monitoring blood glucose in mouse offspring after intracytoplasmic sperm injection

Human lifespan is considerably long, while mouse models can simulate the entire human lifespan in a relatively short period, with one year of mouse life roughly equivalent to 40 human years. Intracytoplasmic sperm injection (ICSI) is a commonly used assisted reproductive technology in clinical practice. However, given its relatively recent emergence about 30 years ago, the long-term effects of this technique on human development remain unclear. In this study, we established the ICSI combined with embryo transfer (ET) method using a mouse model. The results demonstrated that normal mouse sperm, after undergoing in vitro culture and subsequent ICSI, exhibited a fertilization rate of 89.57% and a two-cell rate of 87.38%. Following ET, the birth rate of offspring was approximately 42.50%. Furthermore, as the mice aged, fluctuations in glucose metabolism levels were observed, which may be associated with the application of the ICSI technique. These findings signify that the mouse ICSI-ET technique provides a valuable platform for evaluating the impact of sperm abnormalities on embryo development and their long-term effects on offspring health, particularly concerning glucose metabolism. This study provides important insights for further research on the potential effects of the ICSI technique on human development, emphasizing the necessity for in-depth investigation into the long-term implications of this technology.

In our laboratory, we have achieved a fertilization rate of 89.57% and a 2-cell rate of 87.38% using ICSI with epididymal caudal sperm in mice. The birth rate of offspring following ET is approximately 42.50%. Remarkably, all the fertilization rates, 2-cell rates, and offspring birth rates are comparable to the levels achieved in human ART, enabling a comprehensive simulation of different stages of human ART techniques in mice. Further details are provided in Table 1 and Table 2. There w...

Read this Scientific Article Protocol about Monitoring Blood Glucose in Mouse Offspring after Intracytoplasmic sperm injection - Embryo Transfer at JoVE.com

Author Spotlight: Exploring the Long-Term Health Impacts of Intracytoplasmic Sperm Injection on Offspring

Here, we present a protocol to establish an intracytoplasmic sperm injection (ICSI)-embryo transfer (ET) mouse model, allowing us to observe age-related changes in glucose metabolism that may be attributed to ICSI, providing insights into its potential long-term impacts on human development.

Monitoring Blood Glucose in Mouse Offspring After Intracytoplasmic Sperm Injection

Human lifespan is considerably long, while mouse models can simulate the entire human lifespan in a relatively short period, with one year of mouse life ...

monitoring-blood-glucose-mouse-offspring-after-intracytoplasmic-sperm

Research

JoVE Journal

Developmental Biology

434 Views.  Shanghai-MOST Key Laboratory of Health and Disease Genomics. Here, we present a protocol to establish an intracytoplasmic sperm injection (ICSI)-embryo transfer (ET) mouse model, allowing us to observe age-related changes in glucose metabolism that may be attributed to ICSI, providing insights into its potential long-term impacts on human development.

Video: Author Spotlight: Exploring the Long-Term Health Impacts of Intracytoplasmic Sperm Injection on Offspring

In Utero Intra-cardiac Tomato-lectin Injections on Mouse Embryos to Gauge Renal Blood Flow

The formation and perfusion of developing renal blood vessels (apart from glomeruli) are greatly understudied. As vasculature develops via angiogenesis (which is the branching off of major vessels) and vasculogenesis (de novo vessel formation), perfusion mapping techniques such as resin casts, in vivo ultrasound imaging, and micro-dissection have been limited in demonstrating the intimate relationships between these two processes and developing renal structures within the embryo. Here, we describe the procedure of in utero intra-cardiac ultrasound-guided FITC-labeled tomato lectin microinjections on mouse embryos to gauge the ontogeny of renal perfusion. Tomato lectin (TL) was perfused throughout the embryo and kidneys harvested. Tissues were co-stained for various kidney structures including: nephron progenitors, nephron structures, ureteric epithelium, and vasculature. Starting at E13.5 large caliber vessels were perfused, however peripheral vessels remained unperfused. By E15.5 and E17.5, small peripheral vessels as well as glomeruli started to become perfused. This experimental technique is critical for studying the role of vasculature and blood flow during embryonic development.

In Utero Intra-cardiac Tomato-lectin Injections on Mouse Embryos to Gauge Renal Blood Flow

This manuscript describes a technique for visualization of the developing vasculature. Here we utilized in utero intra-cardiac FITC-labeled tomato lectin microinjections on mouse embryos. Using this technique, we delineate the perfused and unperfused vessels throughout the embryonic kidney.

The formation and perfusion of developing renal blood vessels (apart from glomeruli) are greatly understudied. As vasculature develops via angiogenesis ...

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during oogenesis. In order to study roles of such maternal factors in early embryonic development, it is desirable to manipulate their functions from the very beginning of embryonic development. Conventional ways of gene interference are achieved by injection of antisense oligonucleotides (oligos) or mRNA into fertilized eggs, enabling under- or over-expression of specific proteins, respectively. However, these methods normally require more than several hours until protein expression is affected, and, hence, the interference of gene functions is not effective during early embryonic stages. Here, we introduce an experimental system in which expression levels of maternal proteins can be altered before fertilization. Xenopus laevis oocytes obtained from ovaries are defolliculated by incubating with enzymes. Antisense oligos or mRNAs are injected into defolliculated oocytes at the germinal vesicle (GV) stage. These oocytes are in vitro matured to eggs at the metaphase II (MII) stage, followed by intracytoplasmic sperm injection (ICSI). By this way, up to 10% of ICSI embryos can reach the swimming tadpole stage, thus allowing functional tests of specific gene knockdown or overexpression. This approach can be a useful way to study roles of maternally stored factors in early embryonic development.

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

We describe methods of manipulating Xenopus laevis immature oocytes, in vitro maturation of oocytes to eggs, and intracytoplasmic sperm injection. This protocol allows degradation of some maternal proteins and overexpression of genes of interest at fertilization, and hence is valuable to study roles of specific factors in early embryonic development.

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during ...

Structure-function Studies in Mouse Embryonic Stem Cells Using Recombinase-mediated Cassette Exchange

Gene engineering in mouse embryos or embryonic stem cells (mESCs) allows for the study of the function of a given protein. Proteins are the workhorses of the cell and often consist of multiple functional domains, which can be influenced by posttranslational modifications. The depletion of the entire protein in conditional or constitutive knock-out (KO) mice does not take into account this functional diversity and regulation. An mESC line and a derived mouse model, in which a docking site for FLPe recombination-mediated cassette exchange (RMCE) was inserted within the ROSA26 (R26) locus, was previously reported. Here, we report on a structure-function approach that allows for molecular dissection of the different functionalities of a multidomain protein. To this end, RMCE-compatible mice must be crossed with KO mice and then RMCE-compatible KO mESCs must be isolated. Next, a panel of putative rescue constructs can be introduced into the R26 locus via RMCE targeting. The candidate rescue cDNAs can be easily inserted between RMCE sites of the targeting vector using recombination cloning. Next, KO mESCs are transfected with the targeting vector in combination with an FLPe recombinase expression plasmid. RMCE reactivates the promoter-less neomycin-resistance gene in the ROSA26 docking sites and allows for the selection of the correct targeting event. In this way, high targeting efficiencies close to 100% are obtained, allowing for insertion of multiple putative rescue constructs in a semi-high throughput manner. Finally, a multitude of R26-driven rescue constructs can be tested for their ability to rescue the phenotype that was observed in parental KO mESCs. We present a proof-of-principle structure-function study in p120 catenin (p120ctn) KO mESCs using endoderm differentiation in embryoid bodies (EBs) as the phenotypic readout. This approach enables the identification of important domains, putative downstream pathways, and disease-relevant point mutations that underlie KO phenotypes for a given protein.

Proteins often contain multiple domains that can exert different cellular functions. Gene knock-outs (KO) do not consider this functional diversity. Here, we report a recombination-mediated cassette exchange (RMCE)-based structure-function approach in KO embryonic stem cells that allows for the molecular dissection of various functional domains or variants of a protein.

Gene engineering in mouse embryos or embryonic stem cells (mESCs) allows for the study of the function of a given protein. Proteins are the workhorses of ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...

An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity

Spermatogenesis is the development of spermatogonia into mature spermatozoa in the seminiferous tubules of the testis. This process is supported by Sertoli cell junctions at the blood-testis barrier (BTB), which is the tightest tissue barrier in the mammalian body and segregates the seminiferous epithelium into two compartments, a basal and an adluminal. The BTB creates a unique microenvironment for germ cells in meiosis I/II and for the development of postmeiotic spermatids into spermatozoa via spermiogenesis. Here, we describe a reliable assay to monitor BTB integrity of mouse testis in vivo. An intact BTB blocks the diffusion of FITC-conjugated inulin from the basal to the apical compartment of the seminiferous tubules. This technique is suitable for studying gene candidates, viruses, or environmental toxicants that may affect BTB function or integrity, with an easy procedure and a minimal requirement of surgical skills compared to alternative methods.

An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity

Here, we present a protocol to assess the blood-testis barrier integrity by injecting inulin-FITC into testes. This is an efficient in vivo method to study blood-testis barrier integrity that can be compromised by genetic and environmental elements.

Spermatogenesis is the development of spermatogonia into mature spermatozoa in the seminiferous tubules of the testis. This process is supported by ...

Fluorimetric Techniques for the Assessment of Sperm Membranes

Standard spermiograms describing sperm quality are mostly based on the physiological and visual parameters, such as ejaculate volume and concentration, motility and progressive motility, and sperm morphology and viability. However, none of these assessments is good enough to predict the semen quality. Given that maintenance of sperm viability and fertilization potential depends on membrane integrity and intracellular functionality, evaluation of these parameters might enable a better prediction of sperm fertilization competence. Here, we describe three feasible methods to evaluate sperm quality using specific fluorescent probes combined with fluorescence microscopy or flow cytometry analyses. Analyses assessed plasma membrane integrity using 4&#39;,6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI), acrosomal membrane integrity using fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA) and mitochondrial membrane integrity using 5,5&#39;,6,6&#39;-tetra-chloro-1,1&#39;,3,3&#39;-tetraethylbenzimidazolyl carbocyanine iodide (JC-1). Combinations of these methods are also presented. For instance, use of annexin V combined with PI fluorochromes enables assessing apoptosis and calculating the proportion of apoptotic sperm (apoptotic index). We believe that these methodologies, which are based on examining spermatozoon membranes, are very useful for the evaluation of sperm quality.

Here, we present methodologies to evaluate spermatozoan membrane integrity, a cellular feature associated with sperm fertilization competence. We describe three techniques for the fluorimetric assessment of sperm membranes: simultaneous staining with specific fluorescent probes, fluorescence microscopy, and advanced sperm-dedicated flow cytometry. Examples of combining the methodologies are also presented.

Standard spermiograms describing sperm quality are mostly based on the physiological and visual parameters, such as ejaculate volume and concentration, ...

Assessment of Ultrastructural Neuroplasticity Parameters After In Utero Transduction of the Developing Mouse Brain and Spinal Cord

The present study combines in utero transduction with transmission electron microscopy (TEM) aiming at a precise morphometrical analysis of ultrastructural parameters in unambiguously identified topographical structures, affected by a protein of interest that is introduced into the organism via viral transfer. This combined approach allows for a smooth transition from macrostructural to ultrastructural identification by following topographical navigation maps in a tissue atlas. High-resolution electron microscopy of the in-utero-transduced tissue reveals the fine ultrastructure of the neuropil and its plasticity parameters, such as cross-sectioned synaptic bouton areas, the number of synaptic vesicles and mitochondria within a bouton profile, the length of synaptic contacts, cross-sectioned axonal areas, the thickness of myelin sheaths, the number of myelin lamellae, and cross-sectioned areas of mitochondria profiles. The analysis of these parameters reveals essential insights into changes of ultrastructural plasticity in the areas of the nervous system that are affected by the viral transfer of the genetic construct. This combined method can not only be used for studying the direct effect of genetically engineered biomolecules and/or drugs on neuronal plasticity but also opens the possibility to study the in utero rescue of neuronal plasticity (e.g., in the context of neurodegenerative diseases).

The combination of transmission electron microscopy and in utero transduction is a powerful approach for studying morphological changes in the fine ultrastructure of the nervous system during development. This combined method allows deep insights into changes in structural details underlying neuroplasticity with respect to their topographical representation.

The present study combines in utero transduction with transmission electron microscopy (TEM) aiming at a precise morphometrical analysis of ...

Measuring Sperm Guidance and Motility within the Caenorhabditis elegans Hermaphrodite Reproductive Tract

Successful fertilization is fundamental to sexual reproduction, yet little is known about the mechanisms that guide sperm to oocytes within the female reproductive tract. While in vitro studies suggest that sperm of internally fertilizing animals can respond to various cues from their surroundings, the inability to visualize their behavior inside the female reproductive tract creates a challenge for understanding sperm migration and mobility in its native environment. Here, we describe a method using C. elegans that overcomes this limitation and takes advantage of their transparent epidermis. C. elegans males stained with a mitochondrial&#160; dye are mated with adult hermaphrodites, which act as modified females, and deposit fluorescently labeled sperm into the hermaphrodite uterus. The migration and motility of the labeled sperm can then be directly tracked using an epi-fluorescence microscope in a live hermaphrodite. In wild-type animals, approximately 90% of the labeled sperm crawl through the uterus and reach the fertilization site, or spermatheca. Images of the uterus can be taken 1 h after mating to assess the distribution of the sperm within the uterus and the percentage of sperm that have reached the spermatheca. Alternatively, time-lapse images can be taken immediately after mating to assess sperm speed, directional velocity and reversal frequency. This method can be combined with other genetic and molecular tools available for the C.elegans to identify novel genetic and molecular mechanisms that are important in regulating sperm guidance and motility within the female reproductive tract.

Measuring Sperm Guidance and Motility within the Caenorhabditis elegans Hermaphrodite Reproductive Tract

Sperm must successfully navigate through the oviduct to fertilize an oocyte. Here, we describe an assay for measuring sperm migration within the C. elegans hermaphrodite uterus. This assay can provide quantitative data on sperm distribution within the uterus after mating, as well as on speed, directional velocity, and reversal frequency.

Successful fertilization is fundamental to sexual reproduction, yet little is known about the mechanisms that guide sperm to oocytes within the female ...

Evaluation of Fertilization State by Tracing Sperm Nuclear Morphology in Arabidopsis Double Fertilization

Flowering plants have a unique sexual reproduction system called &#8216;double fertilization&#39;,&#160;in which each of the sperm cells precisely fuses with an egg cell or a central cell. Thus, two independent fertilization events take place almost simultaneously. The fertilized egg cell and central cell develop into zygote and endosperm, respectively. Therefore, precise control of double fertilization is essential for the ensuing seed development. Double fertilization occurs in the female gametophyte (embryo sac), which is deeply hidden and covered with thick ovule and ovary tissues. This pistil tissue construction makes observation and analysis of double fertilization quite difficult and has created the present situation in which many questions regarding the mechanism of double fertilization remain unanswered. For the functional evaluation of a potential candidate for fertilization regulator, phenotypic analysis of fertilization is important. To judge the completion of fertilization in Arabidopsis thaliana, the shapes of fluorescence signals labeling sperm nuclei are used as indicators. A sperm cell that fails to fertilize is indicated by a condensed fluorescence signal outside of the female gametes, whereas a sperm cell that successfully fertilizes is indicated by a decondensed signal due to karyogamy with the female gametes&#8217; nucleus. The method described here provides a tool to determine successful or failed fertilization under in vivo conditions.

Evaluation of Fertilization State by Tracing Sperm Nuclear Morphology in Arabidopsis Double Fertilization

We demonstrate a method to determine successful or failed fertilization on the basis of sperm nuclear morphology in Arabidopsis double fertilization using an epifluorescence microscope.

Flowering plants have a unique sexual reproduction system called &#8216;double fertilization&#39;,&#160;in which each of the sperm cells precisely fuses ...

Vascular Casting of Adult and Early Postnatal Mouse Lungs for Micro-CT Imaging

Blood vessels form intricate networks in 3-dimensional space. Consequently, it is difficult to visually appreciate how vascular networks interact and behave by observing the surface of a tissue. This method provides a means to visualize the complex 3-dimensional vascular architecture of the lung.
To accomplish this, a catheter is inserted into the pulmonary artery and the vasculature is simultaneously flushed of blood and chemically dilated to limit resistance. Lungs are then inflated through the trachea at a standard pressure and the polymer compound is infused into the vascular bed at a standard flow rate. Once the entire arterial network is filled and allowed to cure, the lung vasculature may be visualized directly or imaged on a micro-CT (&#181;CT) scanner.
When performed successfully, one can appreciate the pulmonary arterial network in mice ranging from early postnatal ages to adults. Additionally, while demonstrated in the pulmonary arterial bed, this method can be applied to any vascular bed with optimized catheter placement and endpoints.

The aim of this technique is ex vivo visualization of pulmonary arterial networks of early postnatal and adult mice through lung inflation and injection of a radio-opaque polymer-based compound via the pulmonary artery. Potential applications for casted tissues are also discussed.

Blood vessels form intricate networks in 3-dimensional space. Consequently, it is difficult to visually appreciate how vascular networks interact and ...