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On August 11, 2022, Zhao Xiaoyang's research group of Southern Medical University, Changgang research group of Shenzhen University and Li Lin's research group of Southern Medical University made new progress in optimizing male infertility assisted reproductive technology using mice as a model, and the research results were published in
the journal Science Progress.
For the first time, the researchers systematically resolved the reprogramming defect
during the prokaryotic phase of mouse Round spermatid injection (ROSI) embryos.
This study further found that abnormal H3K9me2 modification mediated by histone methyltransferase G9A is an important reason for the inefficient development of ROSI embryos, and the intervention of A366, a small molecule inhibitor of G9A, can significantly improve the blastocyst development rate and live birth rate
of ROSI embryos.
This study has found a new way to improve the efficiency of ROSI embryonic development, and provides new ideas
for the optimization of ROSI technology.
Background Introduction
Azoozoa is an important factor in male infertility, accounting for about 1% of
the total male population.
Mature sperm or elongated sperm cells are often not available in the testicles and epididymis in patients with severe azoospermia, so these patients cannot obtain intracytoplasmic sperm injection (ICSI) through in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI).
Obtain its biological offspring
.
Nevertheless, round sperm cells can still be observed in the testicles of some azoospermia patients, and ROSI technology can bring good news
to these patients.
Renowned scientist Ryuzo Yanagimachi established the ROSI technique (1) in 1993 and used it to obtain healthy fertile mice (2).
However, ROSI technology has not yet been widely used in clinical practice, in large part because of the widespread development inefficiency of ROSI embryos (3).
Previous studies have reported abnormal epigenetic modifications, such as histone modifications, DNA methylation, or chromatin concentration in ROSI embryos (4,5), but the mechanisms of inefficient ROSI embryonic development and whether small molecule compounds can be used to improve their developmental efficiency are unclear
.
The researchers first used single-cell multi-omics sequencing technology to simultaneously detect the transcriptome, chromatin open status and DNA methylation of ROSI and ICSI preimplantation
embryos.
Through comparison, it is found that the prokaryotic period is a key stage of ROSI embryonic development and reprogramming: the expression of the minor ZGA gene in the prokaryotic ROSI embryo is abnormally down-regulated, and the abnormalities of DNA methylation and chromatin opening are mainly concentrated in the prokaryotic period, and some of the down-regulated minors are also regulated ZGA genes are strongly associated with abnormal DNA hypermethylation and chromatin shutdown, both of which are involved in biological events
such as chromatin and cytoskeletal assembly.
In addition, the researchers found that the H3K9me2 modification in the male pronucleus of ROSI embryos was significantly higher than that of control ICSI embryos and was smaller in diameter (Figure 1).
The above experiments have proved that ROSI embryos have obvious epigenetic modification reprogramming abnormalities
in the prokaryotic stage.
For epigenetic modification defects in the prokaryotic period of ROSI embryos, the researchers screened 178 related small molecule compounds and found that the small molecule inhibitor A366 of G9A (EHMT2) was able to increase the blastocyst development rate and live birth rate of mouse ROSI embryos by about two times
.
EHMT2, as a methyltransferase of histones, is directly involved in regulating the establishment of H3K9me2 modifications and is closely related to DNA methylation modifications (6).
The researchers further found that the expression of Ehmt2 in the PN3 period of ROSI embryos was significantly higher than that in ICSI embryos
in the control group.
Intracytoplasmic injection of siRNA targeting Ehmt2 can significantly improve the blastocyst development rate of mouse ROSI embryos.
Conversely, overexpression of Ehmt2 in mouse ROSI embryos would further reduce the blastocyst development rate
.
However, the addition of A366 to overexpression of Ehmt2 in ROSI embryos cannot improve the developmental efficiency
of embryos.
The above experiments confirm that EHMT2-mediated abnormal modification of H3K9me2 is an important reason for
the inefficient development of ROSI embryos.
Finally, the researchers found that the growth period weight and litter size of A366-treated ROSI live born mice were not statistically different from the control ICSI embryos, which somewhat demonstrated the biosafety of A366-treated mice (Figure 2).
In order to further elucidate the mechanism of A366 in improving the developmental efficiency of ROSI embryos, the researchers used single-cell multi-omics sequencing technology to find that A366 treatment could partially repair the abnormal expression of minor ZGA gene in mouse ROSI embryos, and partially repair the differential DNA methylation and chromatin open regions, and the related genes and sites were related to the assembly of the microtubule skeleton, and the morphology of the male prokaryotic of ROSI embryos could be recovered
after A366 treatment 。 The above experiments demonstrate that A366 can partially repair reprogramming defects in the prokaryotic stage by changing epigenetic and transcriptome states, thereby improving the developmental efficiency
of ROSI embryos.
Dr.
Wang Jing and Dr.
Zhou Wei of Southern Medical University, Gao Shuai researcher of China Agricultural University, master Song Xiuling, and Yang Xinyan, doctoral student are the joint first authors
of this paper.
Professor Zhao Xiaoyang of Southern Medical University, Changgang researcher of Shenzhen University and Professor Li Lin of Southern Medical University are the co-corresponding authors
of this paper.
This work is supported
by the National Key R&D Program of China, the National Natural Science Foundation of China, the Natural Science Foundation of Guangdong Province, the Key R&D Project of Guangzhou Laboratory of Regenerative Medicine and Health, the Key Project of Guangzhou Science and Technology Plan and the Key Project of Shenzhen Basic Research.
References:
About the corresponding author Zhao
Xiaoyang: Ph.
D.
, Deputy Dean of the School of Basic Medicine of Southern Medical University, Jiang Scholar Distinguished Professor of the Ministry of Education, National Outstanding Youth, Young Changjiang Scholar of the Ministry of Education, and Selected for
the Top Young Talents of the National Ten Thousand People Program.
His research interests include the use of a variety of omics techniques to study the human spermatogenesis process and the pathogenesis of infertility.
Use stem cell technology and tissue engineering technology to construct artificial testicular tissue to achieve sperm cell regeneration; This model is used for small molecule compound screening drugs
that promote spermatogenesis.
He has published nearly 60 SCI papers in journals such as Nature, Cell Stem Cell, Cell Research, Nature Communications, Science Advances, etc.
, and applied for 11 patents
.
Among the students trained, they were respectively awarded the National Excellent Youth and the Key R&D Youth Chief
of the Ministry of Science and Technology.
Chang Gang: Ph.
D.
, researcher of Shenzhen University Medical School, master tutor, high-level talent
in Shenzhen.
He graduated from Institute of Zoology/Beijing Institute of Biological Sciences, Chinese Academy of
Sciences in 2009.
He joined Shenzhen University Health Science Center
in 2013.
His research interests mainly include the development of germ cells, the pathogenesis of male infertility, etc.
, and he has published more than 20 research papers in journals including Cell Stem Cell, Cell Research, Nature Communications, Science Advances, Stem Cell Reports, Stem Cell Research & Therapy, etc.
as the corresponding author/first author
。
Li Lin: Ph.
D.
, Professor of the Department of Pathophysiology, School of Basic Medical Sciences, Southern Medical University, doctoral supervisor and postdoctoral co-supervisor
。 His research direction mainly includes single-cell omics technology development and mammalian germline development, presided over a total of 4 scientific research projects at or above the provincial level such as the National Key R&D Program and the Guangdong Outstanding Youth Project, and published 7 original papers in SCI journals such as Cell, Nature Cell Biology and Cell Research, among which the work of human primitive germ cells was selected as "Top Ten Advances in Chinese Science" and "Top Ten Progress in China's Life Science Field" in 2015
。
This article is submitted by the author team, and the views expressed in this article are only the views of the author team and do not represent the position
of Science/AAAS.
Welcome to pay attention to the official scientific public account
full quick look
On August 11, 2022, Zhao Xiaoyang's research group of Southern Medical University, Changgang research group of Shenzhen University and Li Lin's research group of Southern Medical University made new progress in optimizing male infertility assisted reproductive technology using mice as a model, and the research results were published in
the journal Science Progress.
For the first time, the researchers systematically resolved the reprogramming defect
during the prokaryotic phase of mouse Round spermatid injection (ROSI) embryos.
This study further found that abnormal H3K9me2 modification mediated by histone methyltransferase G9A is an important reason for the inefficient development of ROSI embryos, and the intervention of A366, a small molecule inhibitor of G9A, can significantly improve the blastocyst development rate and live birth rate
of ROSI embryos.
This study has found a new way to improve the efficiency of ROSI embryonic development, and provides new ideas
for the optimization of ROSI technology.
Background Introduction
Azoozoa is an important factor in male infertility, accounting for about 1% of
the total male population.
Mature sperm or elongated sperm cells are often not available in the testicles and epididymis in patients with severe azoospermia, so these patients cannot obtain intracytoplasmic sperm injection (ICSI) through in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI).
Obtain its biological offspring
.
Nevertheless, round sperm cells can still be observed in the testicles of some azoospermia patients, and ROSI technology can bring good news
to these patients.
Renowned scientist Ryuzo Yanagimachi established the ROSI technique (1) in 1993 and used it to obtain healthy fertile mice (2).
However, ROSI technology has not yet been widely used in clinical practice, in large part because of the widespread development inefficiency of ROSI embryos (3).
Previous studies have reported abnormal epigenetic modifications, such as histone modifications, DNA methylation, or chromatin concentration in ROSI embryos (4,5), but the mechanisms of inefficient ROSI embryonic development and whether small molecule compounds can be used to improve their developmental efficiency are unclear
.
The researchers first used single-cell multi-omics sequencing technology to simultaneously detect the transcriptome, chromatin open status and DNA methylation of ROSI and ICSI preimplantation
embryos.
Through comparison, it is found that the prokaryotic period is a key stage of ROSI embryonic development and reprogramming: the expression of the minor ZGA gene in the prokaryotic ROSI embryo is abnormally down-regulated, and the abnormalities of DNA methylation and chromatin opening are mainly concentrated in the prokaryotic period, and some of the down-regulated minors are also regulated ZGA genes are strongly associated with abnormal DNA hypermethylation and chromatin shutdown, both of which are involved in biological events
such as chromatin and cytoskeletal assembly.
In addition, the researchers found that the H3K9me2 modification in the male pronucleus of ROSI embryos was significantly higher than that of control ICSI embryos and was smaller in diameter (Figure 1).
The above experiments have proved that ROSI embryos have obvious epigenetic modification reprogramming abnormalities
in the prokaryotic stage.
Figure 1: Single-cell multiomics sequencing revealed that ROSI embryos had severe reprogramming defects during the prokaryotic period
For epigenetic modification defects in the prokaryotic period of ROSI embryos, the researchers screened 178 related small molecule compounds and found that the small molecule inhibitor A366 of G9A (EHMT2) was able to increase the blastocyst development rate and live birth rate of mouse ROSI embryos by about two times
.
EHMT2, as a methyltransferase of histones, is directly involved in regulating the establishment of H3K9me2 modifications and is closely related to DNA methylation modifications (6).
The researchers further found that the expression of Ehmt2 in the PN3 period of ROSI embryos was significantly higher than that in ICSI embryos
in the control group.
Intracytoplasmic injection of siRNA targeting Ehmt2 can significantly improve the blastocyst development rate of mouse ROSI embryos.
Conversely, overexpression of Ehmt2 in mouse ROSI embryos would further reduce the blastocyst development rate
.
However, the addition of A366 to overexpression of Ehmt2 in ROSI embryos cannot improve the developmental efficiency
of embryos.
The above experiments confirm that EHMT2-mediated abnormal modification of H3K9me2 is an important reason for
the inefficient development of ROSI embryos.
Finally, the researchers found that the growth period weight and litter size of A366-treated ROSI live born mice were not statistically different from the control ICSI embryos, which somewhat demonstrated the biosafety of A366-treated mice (Figure 2).
Figure 2: Small molecule compound A366 can significantly improve the developmental efficiency of mouse ROSI embryos
In order to further elucidate the mechanism of A366 in improving the developmental efficiency of ROSI embryos, the researchers used single-cell multi-omics sequencing technology to find that A366 treatment could partially repair the abnormal expression of minor ZGA gene in mouse ROSI embryos, and partially repair the differential DNA methylation and chromatin open regions, and the related genes and sites were related to the assembly of the microtubule skeleton, and the morphology of the male prokaryotic of ROSI embryos could be recovered
after A366 treatment 。 The above experiments demonstrate that A366 can partially repair reprogramming defects in the prokaryotic stage by changing epigenetic and transcriptome states, thereby improving the developmental efficiency
of ROSI embryos.
Figure 3: Small molecule A366 improves epigenetic and transcriptome status of ROSI prokaryotic embryos
Dr.
Wang Jing and Dr.
Zhou Wei of Southern Medical University, Gao Shuai researcher of China Agricultural University, master Song Xiuling, and Yang Xinyan, doctoral student are the joint first authors
of this paper.
Professor Zhao Xiaoyang of Southern Medical University, Changgang researcher of Shenzhen University and Professor Li Lin of Southern Medical University are the co-corresponding authors
of this paper.
This work is supported
by the National Key R&D Program of China, the National Natural Science Foundation of China, the Natural Science Foundation of Guangdong Province, the Key R&D Project of Guangzhou Laboratory of Regenerative Medicine and Health, the Key Project of Guangzhou Science and Technology Plan and the Key Project of Shenzhen Basic Research.
References:
1.
A.
Ogura, R.
Yanagimachi, Round spermatid nuclei injected into hamster oocytes from pronuclei and participate in syngamy.
Biologyof reproduction 48,219 (Feb, 1993).
2.
A.
Ogura, J.
Matsuda, R.
Yanagimachi, Birth of normal young after electrofusion of mouse oocytes with round spermatids.
Proceedings of the National Academy of Sciences of the United States of America 91,7460 (Aug 2, 1994).
3.
A.
Tanaka et al.
, Fourteen babies born after round spermatid injection into human oocytes.
Proceedings of the National Academy of Sciences of the United States of America 112,14629 (Nov 24, 2015).
4.
S.
Kishigami et al.
, Epigenetic abnormalities of the mouse paternal zygotic genome associated with microinsemination of round spermatids.
Dev Biol 289,195 (Jan 1, 2006).
5.
Y.
K.
Kurotaki et al.
, Impaired active DNA demethylation in zygotes generated by round spermatid injection.
Hum Reprod 30,1178 (May, 2015).
6.
G.
Auclair et al.
, EHMT2 directs DNA methylation for efficient gene silencing in mouse embryos.
Genome research 26,192 (Feb, 2016).
About the corresponding author Zhao
Xiaoyang: Ph.
D.
, Deputy Dean of the School of Basic Medicine of Southern Medical University, Jiang Scholar Distinguished Professor of the Ministry of Education, National Outstanding Youth, Young Changjiang Scholar of the Ministry of Education, and Selected for
the Top Young Talents of the National Ten Thousand People Program.
His research interests include the use of a variety of omics techniques to study the human spermatogenesis process and the pathogenesis of infertility.
Use stem cell technology and tissue engineering technology to construct artificial testicular tissue to achieve sperm cell regeneration; This model is used for small molecule compound screening drugs
that promote spermatogenesis.
He has published nearly 60 SCI papers in journals such as Nature, Cell Stem Cell, Cell Research, Nature Communications, Science Advances, etc.
, and applied for 11 patents
.
Among the students trained, they were respectively awarded the National Excellent Youth and the Key R&D Youth Chief
of the Ministry of Science and Technology.
Chang Gang: Ph.
D.
, researcher of Shenzhen University Medical School, master tutor, high-level talent
in Shenzhen.
He graduated from Institute of Zoology/Beijing Institute of Biological Sciences, Chinese Academy of
Sciences in 2009.
He joined Shenzhen University Health Science Center
in 2013.
His research interests mainly include the development of germ cells, the pathogenesis of male infertility, etc.
, and he has published more than 20 research papers in journals including Cell Stem Cell, Cell Research, Nature Communications, Science Advances, Stem Cell Reports, Stem Cell Research & Therapy, etc.
as the corresponding author/first author
。
Li Lin: Ph.
D.
, Professor of the Department of Pathophysiology, School of Basic Medical Sciences, Southern Medical University, doctoral supervisor and postdoctoral co-supervisor
。 His research direction mainly includes single-cell omics technology development and mammalian germline development, presided over a total of 4 scientific research projects at or above the provincial level such as the National Key R&D Program and the Guangdong Outstanding Youth Project, and published 7 original papers in SCI journals such as Cell, Nature Cell Biology and Cell Research, among which the work of human primitive germ cells was selected as "Top Ten Advances in Chinese Science" and "Top Ten Progress in China's Life Science Field" in 2015
。
This article is submitted by the author team, and the views expressed in this article are only the views of the author team and do not represent the position
of Science/AAAS.
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