We extend our gratitude to Wenjie Zhao for her invaluable assistance in sorting round spermatids through flow cytometry and to Fang Wang for her expertise in mouse embryo transfer. This work received partial support from the Natural Science Foundation of Jilin Province (No. YDZJ202301ZYTS461). We thank Bullet Edits Limited for the linguistic editing and proofreading of the manuscript.

B6D2F1 (C57BL/6 x DBA/2), C57BL/6, and ICR mice used in this experiment were purchased from Beijing Vital River Laboratory Animal Technologies Co. Ltd. (Beijing, China). All animal treatments adhered to the experimental procedures and standards approved by the Experimental Animal Ethics Committee of the First Hospital of Jilin University (approval number: 20200435).
1. Preparation of relevant reagents
<ol>
	<li>Acquire some reagents commercially and self-prepare the remaining reagents.
	<ol>
		<li>Obtain M2, a buffer system for in vitro processing of round spermatids (RS), spermatozoa, and oocytes from the supplier.</li>
		<li>Prepare the Ca2+-free C.L. Chatot, C.A. Ziomek, and B.D. Bavister (CZB) medium for AOA following the previously published protocol20.</li>
		<li>Obtain the potassium simplex optimized medium supplemented with amino acids (KSOMaa) solution for embryo culture from the supplier.</li>
		<li>Prepare hyaluronidase, with a working concentration of 0.1% (1 g/L), by dissolving 0.1 g of hyaluronidase in 1 mL of embryo water. Take 10 &#181;L of concentrated storage solution and dissolve in 990 mL of M2, which could be stored at 4 &#176;C for 1 week.</li>
		<li>Prepare polyvinylpyrrolidone (PVP), with an average molecular weight of 360,000 and a mass concentration of 12%, by dissolving 1.2 g of PVP in 10 mL of M2. The resulting solution could be stored at 4 &#176;C for 1 week.</li>
		<li>Store the cytochalasin B (CB) stock solution (2 &#181;L), soluble in 18 &#181;L of DMSO at -20 &#176;C. Take 5 &#181;L of the concentrated storage solution and dissolve in 995 mL of M2. The resulting solution could be stored at 4 &#176;C for 1 week.</li>
	</ol>
	</li>
</ol>
2. Preparation of oocytes
<ol>
	<li>Acclimate female B6D2F1 mice, aged 6-8 weeks, to their new environment for at least 1 week. Inject intraperitoneally with 7.5 IU of pregnant mare serum gonadotropin (PMSG) at 5 pm, followed by 7.5 IU of human chorionic gonadotropin (HCG) 48 h later. Ensure no contact with male mice occurs after the injection. 
	NOTE: The specified time (5 pm) is not mandatory. It is just for work convenience. The 14 hours after 5 pm is 7 am, when the oocytes are ready, and the injection is performed, so the injection time is scheduled for the morning. The start time of the injection can be adjusted according to the laboratory&#39;s daily schedule.</li>
	<li>After 14 h of HCG injection, euthanize the mice using the cervical dislocation method. Open the abdominal cavity and locate the uterus. Identify the ovaries along the uterus and sever the fallopian tubes near the ovaries using scissors. Repeat the same procedure on the other side and place the fallopian tubes in M2, which is preheated at 37 &#176;C in advance.</li>
	<li>Under an upright microscope (10x), locate the enlarged part of the fallopian tube, showing a transparent and swollen appearance. Use a 1 mL syringe to secure the fallopian tube while cutting the enlarged part, collecting oocyte coronal cumulus complexes (OCCCs).</li>
	<li>Place OCCCs into the preheated M2 operating solution containing 0.1% hyaluronidase and remove granulosa cells by gently blowing through an oral pipette. 
	NOTE: Keep the OCCCs in 0.1% hyaluronidase for a short duration, approximately 2 min, if needed.</li>
	<li>After removing the granulosa cells, transfer the oocyte to KSOMaa, rinse 3x, and place in a carbon dioxide incubator for later use (37 &#176;C, 5% CO2).</li>
</ol>
3. Preparation of round spermatids and spermatozoa
<ol>
	<li>Collect round spermatids and spermatozoa from the testis and epididymis of male C57BL/6 mice (8-10 weeks), respectively.
	<ol>
		<li>For spermatozoa, euthanize the mice by cervical dislocation. Open the abdominal cavity, locate the epididymis near the testis, and gently excise with scissors.</li>
		<li>Place the epididymis in M2 medium and gently excise using a 1 mL syringe to allow spermatozoa to flow out under an upright microscope (10x).</li>
		<li>Siphon the flowing spermatozoa and M2 suspension into the bottom of a tube containing 0.5 mL of M2, allowing the spermatozoa to flow upstream naturally. Set them aside for later use.</li>
		<li>For round spermatids, remove the testicles from the abdominal cavity and place them in M2. Gently cut the white membrane through using a 1 mL syringe and squeeze the convoluted seminiferous tubules out under an upright microscope (10x).</li>
		<li>Excise the seminiferous tubules carefully using two 1 mL syringes. Extract the suspension and filter with a 400-mesh sieve (38 &#181;m).</li>
	</ol>
	</li>
	<li>Centrifuge the filtered cells, discard the supernatant, and add 200 &#181;L of Hoechst 33342 for staining, followed by incubation at 37 &#176;C for 10 min.</li>
	<li>Perform fluorescence-activated cell sorting (FACS) to distinguish round spermatids from other cell types. Place the stained cells into a flow cytometry tube and perform a round spermatid screening.
	<ol>
		<li>Different models of flow cytometry machines have different regulatory parameters and can be communicated with technical support during operation. As the first step, adjust the voltage and select the cell population according to forward scatter (FSS) and side scatter (SSC; see Supplementary Table 1).</li>
		<li>For the second step, remove cell adhesions according to FSC and trigger pulse width.</li>
		<li>As the third step, select the target haploid round spermatids. Hoechst 33342 binds to DNA, and cells with different ploidy exhibit different fluorescence intensities. Set the excitation wavelength to 355. Select two detection channels, 460/50 and 670/30, under 355 wavelength laser to sort round spermatids. Subsequently, store the sorted round spermatids in a 4 &#176;C refrigerator for later use. 
		NOTE: It is also possible to directly select round spermatids based on their morphology under a microscope, but training is required to ensure selection accuracy.</li>
	</ol>
	</li>
</ol>
4. Round spermatid injection (ROSI)
<ol>
	<li>Carry out the ROSI procedure using an inverted microscope with a micromanipulation system.</li>
	<li>In the initial preparation step, make holding and injection needles according to the previously described procedure21. Keep the inner diameter of the injection needle between 6-7 &#181;m, designed for the extraction of round spermatids, and for the holding needle, have an inner diameter of 20 &#181;m for securing the oocyte.</li>
	<li>In the subsequent preparation step, create an injection vessel by placing 10 &#181;L of M2 droplets containing CB to soften oocytes and 10 &#181;L of M2 droplets for storing round spermatids. Use PVP droplets to moisten and cleanse the injection needles. 
	NOTE: Adding CB here is not a necessary step, but adding CB can greatly improve the survival rate after injection.</li>
	<li>Extract round spermatids using an injection needle.</li>
	<li>Rotate the oocyte with a holding needle, positioning the oocyte&#39;s polar body at either the 12 o&#39;clock or 6 o&#39;clock position.</li>
	<li>Place the injection needle carefully at the 3 o&#39;clock position on the oocyte, tightly adhering to the zona pellucida. Use a PiezoXpert device to create a hole in the zona pellucida. Adjust the strength from small to large, which is approximately intensity = 5 and speed = 15 here.</li>
	<li>Gently advance the injection needle through the zona pellucida and enter the oocyte horizontally. When it passes the center of the oocyte, apply a piezo (intensity = 1 and speed = 1), resulting in the rupture of the oocyte membrane and the gradual injection of the round spermatid into the oocyte&#39;s cytoplasm.</li>
	<li>Withdraw the injection needle and aspirate a small amount of the oocyte&#39;s membrane near the opening to seal it. This step is crucial, as failure to seal would result in cytoplasmic leakage and oocyte death. 
	NOTE: It is worth noting that ROSI required AOA either before or after the injection of round spermatids, which we have discussed in subsequent sections.</li>
</ol>
5. Intracytoplasmic sperm injection (ICSI)
<ol>
	<li>Carry out the same steps for ICSI as for ROSI, with the only difference being the diameter of the ICSI injection needle, which measures 9-10 &#181;m.</li>
	<li>Place spermatozoa on a PVP runway to reduce their swimming speed and facilitate sperm extraction.</li>
	<li>Perform separation of the spermatozoa head and tail for ICSI. Aspirate the spermatozoa from the tail and position the spermatozoa&#39;s neck precisely at the mouth of the injection needle. Apply a piezo (multiple piezo could be used as needed, intensity = 5 and speed = 15) to separate the head and tail of the spermatozoa.</li>
</ol>
6. Assisted oocyte activation (AOA)
<ol>
	<li>According to the protocol, prepare a CZB medium without calcium and magnesium.</li>
	<li>Use strontium chloride hexahydrate (SrCl26H2O), with a molecular weight of 266.62, at a concentration of 10 mM. To prepare it, dissolve 0.26662 g of SrCl26H2O in 1 mL of embryo water. At the time of use, add 10 &#181;L of concentrated storage solution to 990 &#181;L of Ca2+-free CZB medium and use immediately.</li>
	<li>Place the oocytes in the Ca2+-free CZB medium containing SrCl26H2O for activation; each droplet has a volume of 20 &#181;L, and 20 oocytes are placed each time. Incubate for 20 min.</li>
	<li>Simultaneously, establish a group for parthenogenesis activation without injecting round spermatids or spermatozoa. It should be noted that the parthenogenesis activation group is shown without statistical analysis, and the control group is ICSI.</li>
</ol>
7. Embryo transfer
<ol>
	<li>House the ligated ICR male and female mice in a ratio of 1:2. The following morning, examine the female mice for the presence of a vaginal plug and select those with a vaginal plug for transplantation.</li>
	<li>Inject the transferred embryos consisting of 2-cell embryos the previous morning and transfer them the next afternoon.</li>
	<li>Make an incision on the back of the female mouse, expose the white fat and ovaries, and locate the enlarged part of the fallopian tube under an upright microscope (10x).</li>
	<li>Use a 1 mL syringe to create an opening in the enlarged part of the fallopian tube and aspirate the embryos through an oral pipette. Gently introduce the embryos in the direction of the uterus. Suture the wound, using 4 x 10 models of curved needles with thread, with a thickness of 5-0 (0.09-0.11 mm). The suture is made in two layers, with one layer for the peritoneum and one layer for the outer skin.</li>
	<li>For surrogate mice, perform cesarean sections based on the experimental schedule. Euthanize the mice by the cervical dislocation method, disinfect with alcohol, and open the abdominal cavity. Gently and quickly cut open the uterus and open the amniotic cavity to show the fetus and placenta. Remove the umbilical cord and cut from the side close to the fetus; the fetus was fed by the same period of breastfeeding mice. 
	NOTE: Due to the need to calculate birth rates, cesarean section is more accurate regarding production capacity. In natural childbirth, the offspring may be eaten by the mother, resulting in inaccurate statistics. The cesarean section is performed on surrogate mice transplanted in the early stage, and euthanasia cesarean section is performed before delivery.</li>
</ol>
8. Statistical analysis
<ol>
	<li>Use GraphPad Prism 5 to analyze. Data are expressed in mean &#177; SD; P &#60; 0.05 indicates a statistical difference, and P &#60; 0.01 indicates a significant statistical difference.</li>
</ol>

Murine Round Spermatid Injection Procedure to Study Embryonic Development

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The authors declare no conflicts of financial or other interests.

Assisted oocyte activation 
A critical prerequisite for ROSI is AOA since round spermatids alone cannot initiate oocyte activation. Currently, the most established method in mice involves the use of strontium chloride23,24, while the most advanced human application employs electrical activation13,14. The timing of oocyte activation is also of great significance. As reported in the literature, the most optimal activation approach in mice involves initially activating the oocyte and then injecting the round spermatid25. This contrasts with the conventional fertilization process, where sperm initially enters the oocytes, subsequently releasing phospholipase C&#160;&#950; to activate the oocyte26. Specialized research conducted by Satoshi Kishigami and colleagues emphasizes the differential ability of ROSI to form a male nucleus in pre- or post-activated oocytes. To achieve an efficient offspring production rate, both injection types must be carried out before the oocytes enter the G1 phase25.
The diameter of the injection needle 
In the context of ROSI, it is important to note that a larger diameter of the injection needle does not necessarily translate to better outcomes. The injection needle used has a 6-7 &#181;m diameter, slightly smaller than a round spermatid. Therefore, when a round spermatid is aspirated, the cell membrane is subjected to a squeezing effect, potentially leading to the direct exposure of the nucleus. This direct exposure may be conducive to depolymerization and the subsequent formation of the male pronucleus27.
Sealing treatment 
Mouse oocytes exhibit less cytoplasmic viscosity compared to other species. Even a small breach in the cell membrane can cause the cytoplasm to flow out easily, resulting in oocyte degeneration28. To mitigate the risk, after injecting the round spermatid into the oocyte cytoplasm and withdrawing the injection needle, it is imperative to aspirate a small portion of the cell membrane near the opening of the oocyte cell membrane to seal the space, akin to the procedure in ICSI21. This sealing process significantly reduces the risk of oocyte degeneration.
Application of ROSI as a model 
Beyond clinical applications, ROSI technology has various other applications as a model. It can expedite mouse generation times29, rescue female lethality resulting from a paternally inherited Xist deletion in mice17, generate mice after round spermatid injection into haploid parthenogenetic two-cell blastomeres15, generate transgenic mouse offspring16, and preserve fertility in adolescent children before cancer treatment30,31. Diligent research on mouse ROSI can substantially contribute to advancements in reproductive health research.
Limitations of the article 
This article serves as a methodological guide with several limitations, including the absence of a direct comparative selection of RS under a microscope for injection and a perceived lack of innovation.

The final stage of spermatogenesis involves the transformation of a round spermatid into a fully developed spermatozoon, characterized by distinct head, neck, and elongated tail structures1. This transformation encompasses significant changes in cell morphology, such as the condensation of chromatin in the nucleus, replacement of histones by protamine, acrosome formation, mitochondrial sheath development, centriole migration and loss, tail structure formation, and the removal of cellular residues2.
In 1992, the first human fetus was successfully born through intracytoplasmic sperm injection (ICSI) technology3. Since then, researchers have been exploring the potential of utilizing round spermatids, which share the same haploid genetic composition as mature spermatozoa, to fertilize oocytes and sustain viable pregnancies2,4. Subsequently, in 1996, the first human fetus conceived via round spermatid injection (ROSI) technology was delivered5,6. It is worth noting that studies involving ICSI and ROSI in mice lagged behind those in humans due to the susceptibility of the mouse oocyte membrane to damage during the injection process. This issue was successfully resolved with the introduction of the Piezo membrane-breaking device. Consequently, in 1995, the first mouse conceived through ROSI technology was born. Additionally, research on ROSI in various other animals is also underway7,8.
Currently, research on ROSI primarily centers around the following aspects: clinical application, mechanism elucidation, and strategies to enhance developmental efficiency, along with broader applications of ROSI technology. In the context of clinical applications, despite the birth of the first ROSI human fetus through ROSI in 1996, progress has been marked by a series of successes and failures9,10,11,12. To date, ROSI technology has not achieved widespread clinical implementation, largely due to its low efficiency and the need for further validation regarding the safety of fetuses conceived through ROSI technology. Incomplete statistics indicate that globally, fewer than 200 human ROSI-conceived fetuses have been delivered. A turning point in understanding the potential of ROSI technology occurred in 2015 when Tanaka and colleagues reported on the successful birth of 14 fetuses through ROSI technology, instilling renewed confidence in its clinical application and feasibility13,14. ROSI technology holds substantial promise for addressing reproductive biology challenges, particularly in non-obstructive azoospermia patients. In addition to its clinical applications, ROSI serves as a valuable tool for studying the intricate mechanisms of embryonic development15,16,17.
Numerous animal studies have been conducted to investigate the underlying factors contributing to the low efficiency of ROSI in achieving full embryonic development. These factors encompass the choice of assisted oocyte activation (AOA) methods and their timings, abnormalities in genomic stability, and, particularly, abnormalities in epigenetic modifications. It is important to recognize that round spermatids are immature germ cells, differing significantly from mature spermatozoa in various physiological aspects. Mizuki Sakamoto and colleagues indicated that H3K27me3, derived from round spermatids, is associated with chromatin that is less accessible and leads to impaired gene expression in ROSI embryos18. In a related study by Jing Wang and colleagues, reprogramming defects in ROSI embryos at the pronuclear stages were predominantly associated with the misexpression of a cohort of the genes responsible for minor zygotic genome activation19. They also found that introducing a selective euchromatic histone lysine methyltransferase 2 inhibitor, A366, could potentially enhance the overall developmental rate by approximately twofold.
The mouse stands as one of the most valuable model animals for studying embryonic development. This article elaborates on how to perform ROSI on mice. This comprehensive protocol encompasses the selection of suitable mice, detailed ovulation induction procedures, AOA techniques, injection techniques, and the preparation of surrogate mice. Furthermore, we present a comparative analysis of the effects of two injection regimens on birth efficiency: AOA followed by ROSI (A-ROSI; first regimen) and ROSI followed by AOA (ROSI-A; second regimen). We aim to encourage researchers to conduct mouse ROSI experiments with greater precision, offering more robust support for their clinical application and the fundamental research of embryonic development mechanisms.

<table><tbody><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;2H&lt;sub&gt;2&lt;/sub&gt;O&amp;nbsp;</td><td>Sigma</td><td>C7902&amp;nbsp;</td><td>Preparation of CZB</td></tr><tr><td>Glucose&amp;nbsp;</td><td>Sigma</td><td>G6152&amp;nbsp;</td><td>Preparation of CZB</td></tr><tr><td>HEPES-Na (basic)&amp;nbsp;</td><td>Sigma</td><td>H3784&amp;nbsp;</td><td>Preparation of CZB</td></tr><tr><td>Hoechst 33342</td><td>Beyotime&amp;nbsp;</td><td>C1025</td><td>FACS</td></tr><tr><td>human chorionic gonadotropin (HCG)</td><td>Ningbo Second Hormone Company</td><td>HCG</td><td>Ovulation promoting drugs</td></tr><tr><td>Hyaluronidase</td><td>Sigma</td><td>H3506</td><td>Removing granulosa cells around the oocyte</td></tr><tr><td>KCl&amp;nbsp;</td><td>Sigma</td><td>P5405</td><td>Preparation of CZB</td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;&amp;nbsp;</td><td>Sigma</td><td>P5655&amp;nbsp;</td><td>Preparation of CZB</td></tr><tr><td>KSOMaa</td><td>Caisson Labs</td><td>IVL04-100ML</td><td>Potassium simplex optimized medium supplemented with amino acids</td></tr><tr><td>L-glutamine&amp;nbsp;</td><td>Sigma</td><td>G8540&amp;nbsp;</td><td>Preparation of CZB</td></tr><tr><td>M2</td><td>Sigma</td><td>M7167-50ML</td><td>Operating fluid</td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;7H&lt;sub&gt;2&lt;/sub&gt;O&amp;nbsp;</td><td>Sigma</td><td>M1880&amp;nbsp;</td><td>Preparation of CZB</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;-EDTA2H&lt;sub&gt;2&lt;/sub&gt;O&amp;nbsp;</td><td>Sigma</td><td>E5134&amp;nbsp;</td><td>Preparation of CZB</td></tr><tr><td>NaCl&amp;nbsp;</td><td>Sigma</td><td>S5886</td><td>Preparation of CZB</td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma</td><td>S5761&amp;nbsp;</td><td>Preparation of CZB</td></tr><tr><td>Na-lactate 60% syrup d = 1.32 g/L</td><td>Sigma</td><td>L7900&amp;nbsp;</td><td>Preparation of CZB</td></tr><tr><td>Na-pyruvate&amp;nbsp;</td><td>Sigma</td><td>P4562&amp;nbsp;</td><td>Preparation of CZB</td></tr><tr><td>Piezo drill tips (ICSI)&amp;nbsp;</td><td>Eppendorf&amp;nbsp;</td><td>piezoXpert</td><td>Piezoelectric membrane rupture</td></tr><tr><td>pregnant mare serum gonadotropin (PMSG)</td><td>Ningbo Second Hormone Company</td><td>PMSG</td><td>Ovulation promoting drugs</td></tr><tr><td>PVA&amp;nbsp;</td><td>Sigma</td><td>P8136</td><td>Preparation of CZB</td></tr></tbody></table>

mouse round spermatid injection

Round spermatids, characterized by their haploid genetic content, represent the precursor cells to mature spermatozoa. Through the innovative technique of round spermatid injection (ROSI), oocytes can be successfully fertilized and developed into viable fetuses. In a groundbreaking milestone achieved in 1995, the first mouse fetus was born through ROSI technology. ROSI has since emerged as a pivotal tool for unraveling the intricate mechanisms governing embryonic development and holds significant potential in various applications, including the acceleration of mouse generation and the production of genetically modified mice. In 1996, a milestone was reached when the first human fetus was born through ROSI technology. However, the clinical applications of this method have shown a fluctuating pattern of success and failure. To date, ROSI technology has not found widespread application in clinical practice, primarily due to its low birth efficiency and insufficient validation of fetal safety. This article provides a comprehensive account of the precise methods of performing ROSI in mice, aiming to shed new light on basic research and its potential clinical applications.

We initiated our investigation by examining AOA&#39;s effect on embryos&#39; developmental capability. A schematic illustration of the experimental design is shown in Figure 1A. Before the spermatozoon injection, the oocytes underwent either AOA (A-ICSI) or remained untreated (ICSI). Detailed data on embryonic development is presented in Table 1. The results revealed no significant differences in cleavage, blastocyst, or birth rates between the A-ICSI and ICSI groups (P &#62; 0.05; Figure 1B). These findings indicate that AOA using 10 mM SrCl2 for 20 min did not affect the developmental potential of the embryos.
It was previously reported that there were no discernible differences in the developmental efficiency of ROSI embryos selected either by FACS or by direct visual examination under a microscope22. Our experiments employed FACS technology to identify RS (Figures 2A,B). Under the microscope, mouse round spermatids were approximately 10 &#181;m in diameter and displayed a protrusion-like nucleolus structure in the middle (Figure 2C). We posit that selection through FACS is more accurate than direct visual examination under a microscope. Additionally, the literature supports the direct exploration of RS through morphological differences. ROSI embryos were generated using two distinct methods: the A-ROSI group, where oocytes were subjected to AOA before the round spermatid injection, and the ROSI-A group, where oocytes were subjected to AOA after the round spermatid injection. The schematic diagram of the experimental design is shown in Figure 1C. Notably, no significant differences in the cleavage and blastocyst rates were found among the A-ROSI, ROSI-A, and ICSI groups (P &#62; 0.05; Figure 1D). The blastocyst rates of the A-ROSI, ROSI-A, and ICSI groups were significantly higher than those of the activation group (P &#60; 0.05; Figure 1D). However, the birth rate of the ROSI group was lower than that of the ICSI group, irrespective of whether the oocytes were activated before or after injection (P &#60; 0.05; Figure 1D). Importantly, the birth rate of the A-ROSI group was slightly higher than that of the ROSI-A group (Figure 1D). Further details on embryo development data are presented in Table 1.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65898/65958fig01.jpg" /> 
Figure 1: ROSI embryos displayed reduced developmental efficiency compared to ICSI embryos. (A) Schematic illustration of the experimental protocol assessing the effect of activation on ICSI embryonic development. Blue = Oocytes were not activated; Yellow = Oocytes were activated. (B) Developmental efficiency of embryos derived from A-ICSI and ICSI. (C) Schematic illustration of the experimental protocol for generating different types of embryos. (D) Developmental efficiency of embryos derived from A-ROSI, ROSI-A, A, and ICSI. **, P &#60; 0.01. Abbreviations: A= Activation E= Embryonic day; ROSI= Round spermatid injection; ICSI= Intracytoplasmic sperm injection. The error bars show standard deviation. The comparison of rates is conducted using the chi-square test. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65898/65958fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65898/65958fig02.jpg" /> 
Figure 2: Round spermatids were selected via flow cytometric sorting. (A) A flow cytometric sorting diagram was utilized for the selection of round spermatids. (B) Representative images showcasing the selection of round spermatids. Scale bar: 10 &#181;m. Abbreviations: FSC = Forward scatter; SSC = side scatter; 355 is the excitation wavelength; 460/50 and 670/30 are two detection channels under 355 wavelength laser. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65898/65958fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td rowspan="2">Groups</td>
			<td colspan="3">Pre-implantation development</td>
			<td colspan="2">Post-implantation development</td>
		</tr>
		<tr>
			<td>Replicates</td>
			<td>Cleavage rate (%)</td>
			<td>Blastocyst rate (%)</td>
			<td>2-cell embryos transferred/ No. of recipients&#160;</td>
			<td>Birth rate (%)</td>
		</tr>
		<tr>
			<td>A-ICSI</td>
			<td>5</td>
			<td>99.33 (149/150)</td>
			<td>75.17 (112/149)</td>
			<td>49/5</td>
			<td>48.98 (24/49)</td>
		</tr>
		<tr>
			<td>A-ROSI</td>
			<td>5</td>
			<td>99.33 (149/150)</td>
			<td>73.83 (110/149)</td>
			<td>44/5</td>
			<td>18.18 (8/44) **</td>
		</tr>
		<tr>
			<td>ROSI-A</td>
			<td>5</td>
			<td>98.00 (147/150)</td>
			<td>64.63 (95/147)</td>
			<td>45/5</td>
			<td>13.33 (6/45) **</td>
		</tr>
		<tr>
			<td>Activation</td>
			<td>5</td>
			<td>100.00 (150/150)</td>
			<td>12.00 (18/150) **</td>
			<td>51/5</td>
			<td>0.00 (0/51) **</td>
		</tr>
		<tr>
			<td>ICSI</td>
			<td>5</td>
			<td>98.00 (147/150)</td>
			<td>73.47 (108/147)</td>
			<td>46/5</td>
			<td>52.17 (24/46)</td>
		</tr>
	</tbody>
</table>
Table 1. The developmental efficiency of embryos derived from different groups.
Supplementary Table 1. The proportion of different cell populations sorted by flow cytometry.P3 part is round spermatids. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65898/Supplementary Table 1_RE.zip" target="_blank">Please click here to download this File.</a>

Read this Scientific Article Protocol about Mouse Round Spermatid Injection at JoVE.com

Mouse Round Spermatid Injection

Here, we introduce a method for performing round spermatid injection (ROSI) in mice, a technique with promising clinical applications and utility for investigating the mechanisms underlying embryonic development.

Round spermatids, characterized by their haploid genetic content, represent the precursor cells to mature spermatozoa. Through the innovative technique of ...

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

603 Views.  First Hospital of Jilin University. Here, we introduce a method for performing round spermatid injection (ROSI) in mice, a technique with promising clinical applications and utility for investigating the mechanisms underlying embryonic development.

Video: Mouse Round Spermatid Injection

Harvesting Sperm and Artificial Insemination of Mice

Rodents of the genus Peromyscus (deer mice) are the most prevalent native North American mammals. Peromyscus species are used in a wide range of research including toxicology, epidemiology, ecology, behavioral, and genetic studies. Here they provide a useful model for demonstrations of artificial insemination. 

Methods similar to those displayed here have previously been used in several deer mouse studies, yet no detailed protocol has been published. Here we demonstrate the basic method of artificial insemination. This method entails extracting the testes from the rodent, then isolating the sperm from the epididymis and vas deferens. The mature sperm, now in a milk mixture, are placed in the female&#x2019;s reproductive tract at the time of ovulation. Fertilization is counted as day 0 for timing of embryo development. Embryos can then be retrieved at the desired time-point and manipulated.

Artificial insemination can be used in a variety of rodent species where exact embryo timing is crucial or hard to obtain. This technique is vital for species or strains (including most Peromyscus) which may not mate immediately and/or where mating is hard to assess. In addition, artificial insemination provides exact timing for embryo development either in mapping developmental progress and/or transgenic work. Reduced numbers of animals can be used since fertilization is guaranteed. This method has been vital to furthering the Peromyscus system, and will hopefully benefit others as well.

This protocol demonstrates methods for extracting sperm from the testes of males and then inseminating female mice. This procedure is useful when precise time is needed in developmental studies as well as transgenic work.

Rodents of the genus Peromyscus (deer mice) are the most prevalent native North American mammals.  Peromyscus species are used in a wide range of research ...

Biology

A Seminiferous Tubule Squash Technique for the Cytological Analysis of Spermatogenesis Using the Mouse Model

Meiotic progression in males is a process that requires the concerted action of a number of highly regulated cellular events. Errors occurring during meiosis can lead to infertility, pregnancy loss or genetic defects. Commencing at the onset of puberty and continuing throughout adulthood, continuous semi-synchronous waves of spermatocytes undergo spermatogenesis and ultimately form haploid sperm. The first wave of mouse spermatocytes undergoing meiotic initiation appear at day 10 post-partum (10 dpp) and are released into the lumen of seminiferous tubules as mature sperm at 35 dpp. Therefore, it is advantageous to utilize mice within this developmental time-window in order to obtain highly enriched populations of interest. Analysis of rare cell stages is more difficult in older mice due to the contribution of successive spermatogenic waves, which increase the diversity of the cellular populations within the tubules. The method described here is an easily implemented technique for the cytological evaluation of the cells found within the seminiferous tubules of mice, including spermatogonia, spermatocytes, and spermatids. The tubule squash technique maintains the integrity of isolated male germ cells and allows examination of cellular structures that are not easily visualized with other techniques. To demonstrate the possible applications of this tubule squash technique, spindle assembly was monitored in spermatocytes progressing through the prophase to metaphase I transition (G2/MI transition). In addition, centrosome duplication, meiotic sex chromosome inactivation (MSCI), and chromosome bouquet formation were assessed as examples of the cytological structures that can be observed using this tubule squash method. This technique can be used to pinpoint specific defects during spermatogenesis that are caused by mutation or exogenous perturbation, and thus, contributes to our molecular understanding of spermatogenesis.

The goal of this tubule squash technique is to rapidly assess cytological features of developing mouse spermatocytes while preserving cellular integrity. This method allows for the study of all stages of spermatogenesis, and can be easily implemented alongside other biochemical and molecular biological approaches for the study of mouse meiosis.

Meiotic progression in males is a process that requires the concerted action of a number of highly regulated cellular events. Errors occurring during ...

Enrichment of Pachytene Spermatocytes and Spermatids from Mouse Testes Using Standard Laboratory Equipment

To characterize each step of spermatogenesis, researchers must separate different subpopulations of germ cells from testes. However, isolating discrete populations is challenging, because the adult testis contains a complex mix of germ cells from all steps of spermatogenesis along with certain populations of somatic cells. Over the past few decades, different techniques such as centrifugal elutriation, fluorescence-activated cell sorting (FACS), and STA-PUT have been successfully applied to the isolation of germ cells. A drawback is that they all require dedicated devices and specialized training. Following principles underlying the STA-PUT method, a simple protocol has been developed for the isolation of pachytene spermatocytes, round spermatids, and elongating spermatids from mouse testes. After preparing a single cell suspension of testicular cells, specific cell populations are enriched by gravity sedimentation through a discontinuous bovine serum albumin (BSA) density gradient. The cell fractions are then manually collected and microscopically analysed. This modified density gradient for round spermatids (MDR) sedimentation protocol can be widely applied, because it requires only standard laboratory equipment. Furthermore, the protocol requires minimal starting materials, reducing its cost and use of laboratory animals.

Presented here is a protocol for enriching pachytene spermatocytes, round spermatids, and elongating spermatids from adult mouse testes using a discontinuous bovine serum albumin density gradient with standard laboratory equipment.

To characterize each step of spermatogenesis, researchers must separate different subpopulations of germ cells from testes. However, isolating discrete ...

Transillumination-Assisted Dissection of Specific Stages of the Mouse Seminiferous Epithelial Cycle for Downstream Immunostaining Analyses

Spermatogenesis is a unique differentiation process that ultimately gives rise to one of the most distinct cell types of the body, the sperm. Differentiation of germ cells takes place in the cytoplasmic pockets of somatic Sertoli cells that host 4 to 5 generations of germ cells simultaneously and coordinate and synchronize their development. Therefore, the composition of germ cell types within a cross-section is constant, and these cell associations are also known as stages (I&#8211;XII) of the seminiferous epithelial cycle. Importantly, stages can also be identified from intact seminiferous tubules based on their differential light absorption/scatter characteristics revealed by transillumination, and the fact that the stages follow each other along the tubule in a numerical order. This article describes a transillumination-assisted microdissection method for the isolation of seminiferous tubule segments representing specific stages of mouse seminiferous epithelial cycle. The light absorption pattern of seminiferous tubules is first inspected under a dissection microscope, and then tubule segments representing specific stages are cut and used for downstream applications. Here we describe immunostaining protocols for stage-specific squash preparations and for intact tubule segments. This method allows a researcher to focus on biological events taking place at specific phases of spermatogenesis, thus providing a unique tool for developmental, toxicological, and cytological studies of spermatogenesis and underlying molecular mechanisms.

This protocol describes transillumination-assisted microdissection of segments of adult mouse seminiferous tubules representing specific stages of seminiferous epithelial cycle, and cell types therein, and subsequent immunostaining of squash preparations and intact tubule segments.

Spermatogenesis is a unique differentiation process that ultimately gives rise to one of the most distinct cell types of the body, the sperm. ...

Combining Imaging with a Stimulant Delivery System to Study Live Mouse Sperm

Real-Time Imaging of Acrosomal Calcium Dynamics and Exocytosis in Live Mouse Sperm

Acrosome exocytosis (AE), in which the sperm&#39;s single exocytotic vesicle fuses with the plasma membrane, is a complex, calcium-dependent process essential for fertilization. However, our understanding of how calcium signaling regulates AE is still incomplete. In particular, the interplay between intra-acrosomal calcium dynamics and the intermediate steps leading to AE is not well-defined. Here, we describe a method that provides spatial and temporal insights into acrosomal calcium dynamics and their relationship to membrane fusion and subsequent exocytosis of the acrosome vesicle. The method utilizes a novel transgenic mouse expressing an Acrosome-targeted Sensor for Exocytosis (AcroSensE). The sensor combines a genetically encoded calcium indicator (GCaMP) fused with mCherry. This fusion protein was specifically designed to enable the concurrent observation of acrosomal calcium dynamics and membrane fusion events. Real-time monitoring of acrosomal calcium dynamics and AE in live AcroSensE sperm is achieved using a combination of high frame-rate imaging and a stimulant delivery system that can target single sperm. This protocol also provides several examples of basic methods to quantify and analyze the raw data. Because the AcroSensE model is genetically encoded, its scientific significance can be augmented by using readily available genetic tools, such as crossbreeding with other mouse genetic models or gene-editing (CRISPR) based methods. With this strategy, the roles of additional signaling pathways in sperm capacitation and fertilization can be resolved. In summary, the method described here provides a convenient and effective tool to study calcium dynamics in a specific subcellular compartment-the sperm acrosome-and how those dynamics regulate the intermediate steps leading to membrane fusion and acrosome exocytosis.

Author Spotlight: A Live Cell Imaging Technique to Study Calcium Signaling and Acrosome Exocytosis in Mouse Sperm 

The AcroSensE mouse model and live cell imaging methods described here provide a new approach to studying calcium dynamics in the subcellular compartment of the sperm acrosome and how they regulate intermediate steps leading to membrane fusion and acrosome exocytosis.

Acrosome exocytosis (AE), in which the sperm&#39;s single exocytotic vesicle fuses with the plasma membrane, is a complex, calcium-dependent process ...

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

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.

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.

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 ...

Transgenic Rodent Assay for Quantifying Male Germ Cell Mutant Frequency

De novo mutations arise mostly in the male germline and may contribute to adverse health outcomes in subsequent generations. Traditional methods for assessing the induction of germ cell mutations require the use of large numbers of animals, making them impractical. As such, germ cell mutagenicity is rarely assessed during chemical testing and risk assessment. Herein, we describe an in vivo male germ cell mutation assay using a transgenic rodent model that is based on a recently approved Organisation for Economic Co-operation and Development (OECD) test guideline. This method uses an in vitro positive selection assay to measure in vivo mutations induced in a transgenic &#955;gt10 vector bearing a reporter gene directly in the germ cells of exposed males. We further describe how the detection of mutations in the transgene recovered from germ cells can be used to characterize the stage-specific sensitivity of the various spermatogenic cell types to mutagen exposure by controlling three experimental parameters: the duration of exposure (administration time), the time between exposure and sample collection (sampling time), and the cell population collected for analysis. Because a large number of germ cells can be assayed from a single male, this method has superior sensitivity compared with traditional methods, requires fewer animals and therefore much less time and resources.

De novo mutations in the male germline may contribute to adverse health outcomes in subsequent generations. Here we describe a protocol for the use of a transgenic rodent model for quantifying mutations in male germ cells induced by environmental agents.

De novo mutations arise mostly in the male germline and may contribute to adverse health outcomes in subsequent generations. Traditional methods for ...

In Vivo Microinjection and Electroporation of Mouse Testis

This video and article contribution gives a comprehensive description of microinjection and electroporation of mouse testis in vivo. This particular transfection technique for testicular mouse cells allows the study of unique processes in spermatogenesis.
The following protocol focuses on transfection of testicular mouse cells with plasmid constructs. Specifically, we used the reporter vector pEGFP-C1, which expresses enhanced green fluorescent protein (eGFP) and also the pDsRed2-N1 vector expressing red fluorescent protein (DsRed2). Both encoded reporter genes were under the control of the human cytomegalovirus immediate-early promoter (CMV).
For performing gene transfer into mouse testes, the reporter plasmid constructs are injected into testes of living mice. To that end, the testis of an anaesthetized animal is exposed and the site of microinjection is prepared. Our preferred place of injection is the efferent duct, with the ultimately connected rete testis as the anatomical transport route of the spermatozoa between the testis and the epididymis. In this way, the filling of the seminiferous tubules after microinjection is excellently managed and controlled due to the use of stained DNA solutions. After observing a sufficient filling of the testis by its colored tubule structure, the organ is electroporated. This enables the transfer of the DNA solution into the testicular&#160;cells. Following 3 days of incubation, the testis is removed and investigated under the microscope for green or red&#160;fluorescence, illustrating transfection success.
Generally, this protocol can be employed for delivering DNA- or RNA- constructs into living mouse testis in order to (over)express or knock down genes, facilitating in vivo gene function analysis. Furthermore, it is suitable for studying reporter constructs or putative gene regulatory elements. Thus, the main advantages of the electroporation technique are fast performance in combination with low effort as well as the moderate technical equipment and skills required compared to alternative techniques.

In Vivo Microinjection and Electroporation of Mouse Testis

This article describes microinjection and electroporation of mouse testis in&#160;vivo as a&#160;transfection technique for testicular mouse cells to study unique processes of spermatogenesis. The presented protocol involves steps of glass capillary preparation, microinjection via the efferent duct, and transfection by electroporation.

This video and article contribution gives a comprehensive description of microinjection and electroporation of mouse testis in vivo. This particular ...

Preparation of Meiotic Chromosome Spreads from Mouse Spermatocytes

Mammalian meiosis is a dynamic developmental process that occurs in germ cells and can be studied and characterized. Using a method to spread nuclei on the surface of slides (rather than dropping them from a height), we demonstrate an optimized technique on mouse spermatocytes that was first described in 1997. This method is widely used in laboratories to study mammalian meiosis because it yields a plethora of high quality nuclei undergoing substages of prophase I. Seminiferous tubules are first placed in a hypotonic solution to swell spermatocytes. Then spermatocytes are released into a sucrose solution to create a cell suspension, and nuclei are spread onto fixative-soaked glass slides. Following immunostaining, a diversity of proteins germane to meiotic processes can be examined. For example, proteins of the synaptonemal complex, a tripartite structure that connects the chromosome axes/cores of homologs together can be easily visualized. Meiotic recombination proteins, which are involved in repair of DNA double-strand breaks by homologous recombination, can also be immunostained to evaluate progression of prophase I. Here we describe and demonstrate in detail a technique widely used to study mammalian meiosis in spermatocytes from juvenile or adult male mice.

Meiosis is the developmental process by which gametes are formed through a single round of DNA replication and two successive rounds of chromosome segregation. Mammalian meiosis can be examined by utilizing a technique to prepare meiotic chromosome spreads. Here, we demonstrate a method of preparing surface-spread nuclei from mouse spermatocytes.

Mammalian meiosis is a dynamic developmental process that occurs in germ cells and can be studied and characterized. Using a method to spread nuclei on ...

Functional Assessment of Kinesin-7 CENP-E in Spermatocytes Using In Vivo Inhibition, Immunofluorescence and Flow Cytometry

In eukaryotes, meiosis is essential for genome stability and genetic diversity in sexual reproduction. Experimental analyses of spermatocytes in testes are critical for the investigations of spindle assembly and chromosome segregation in male meiotic division. The mouse spermatocyte is an ideal model for mechanistic studies of meiosis, however, the effective methods for the analyses of spermatocytes are lacking. In this article, a practical and efficient method for the in vivo inhibition of kinesin-7 CENP-E in mouse spermatocytes is reported. A detailed procedure for testicular injection of a specific inhibitor GSK923295 through abdominal surgery in 3-week-old mice is presented. Furthermore, described here is a series of protocols for tissue collection and fixation, hematoxylin-eosin staining, immunofluorescence, flow cytometry and transmission electron microscopy. Here we present an in vivo inhibition model via abdominal surgery and testicular injection, that could be a powerful technique to study male meiosis. We also demonstrate that CENP-E inhibition results in chromosome misalignment and metaphase arrest in primary spermatocytes during meiosis I. Our in vivo inhibition method will facilitate mechanistic studies of meiosis, serve as a useful method for genetic modifications of male germ lines, and shed a light on future clinical applications.

Functional Assessment of Kinesin-7 CENP-E in Spermatocytes Using In Vivo Inhibition, Immunofluorescence and Flow Cytometry

This article reports an in vivo inhibition of CENP-E through abdominal surgery and testicular injection of GSK923295, a valuable model for male meiotic division. Using the immunofluorescence, flow cytometry and transmission electron microscopy assays, we show that CENP-E inhibition results in chromosome misalignment and genome instability in mouse spermatocytes.

In eukaryotes, meiosis is essential for genome stability and genetic diversity in sexual reproduction. Experimental analyses of spermatocytes in testes ...

Mouse Round Spermatid Injection

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Functional Assessment of Kinesin-7 CENP-E in Spermatocytes Using In Vivo Inhibition, Immunofluorescence and Flow Cytometry