Técnicas de substituição de DNA mitocondrial para reduzir a transmissão da Síndrome de Leigh / Mitochondrial DNA replacement techniques to reduce Leigh syndrome transmission

A Síndrome de Leigh (SL) é uma doença neuro-metabólica congênita, que faz parte do grupo das encefalopatias fatais, com progressão e morte dentro de 2 anos, em média. A SL é causada por mutações no DNA que causam alterações na geração de ATP celular pelas mitocôndrias. As mitocôndrias contêm seu próprio DNA (mtDNA) e, ao contrário do DNA nuclear, o mtDNA é herdado somente da mãe. Mulheres portadores de mutações causadoras da SL podem vivenciar experiências muito tristes ao tentarem realizar o sonho da maternidade. As técnicas de substituição de mtDNA mutado com mtDNA saudável de doadora, oferecem a essas mulheres a possibilidade de terem uma criança geneticamente relacionada sem a SL. O desenvolvimento e a aplicação clínica de terapias de substituição de mtDNA já são uma realidade, tendo o primeiro bebê gerado a partir da técnica nascido em 2016. Mas será que essas técnicas são seguras? Neste trabalho, revisamos a SL e algumas técnicas de substituição de mtDNA já aplicadas em humanos, que envolvem a transferência de pronúcleos de zigotos ou de fuso acromático de oócitos. Concluímos que, apesar dos resultados promissores, ainda é cedo para assegurar a aplicabilidade clínica de técnicas de substituição de mtDNA em seres humanos. (AU)

Leigh syndrome (SL) is a congenital neurometabolic disease included in the group of fatal encephalopathies, with progression and death within 2 years on average. SL is caused by mutations in the DNA that cause changes in the generation of cellular ATP by mitochondria. Mitochondria contain their own DNA (mtDNA) and, unlike nuclear DNA, mtDNA is inherited only from the mother. Women with SL mutations may experience mournful situations when attempting to fulfill the dream of motherhood. Techniques for replacing mutant mtDNA with healthy donor mtDNA provide these women with the possibility of having a genetically related child without SL. The development and clinical application of mtDNA replacement therapies is a reality, and the first baby generated using the technique was born in 2016. However, are these techniques safe? In this article, we review SL and some mtDNA replacement techniques that have been used in humans, which involve zygote pronuclear transfer or oocyte spindle transfer. We conclude that, despite the promising results, it is too early to ensure that mtDNA replacement techniques are clinically applicable to humans. (AU)

A Case of True Hermaphroditism, 46, XY DSD

A 26 year old student reared as a female, presented with inability to menstruate and increased facial hair growth. On examination , patient had hyperandrogenic features including hirsutism, low pitched voice, microphallus with hypospadias. Investigations revealed a 46 XY karyotype with increased testosterone and imaging revealed both ovaries and testes with a hypoplastic uterus. The patient was managed with bilateral testicular gonadectomy, feminising genitoplasty and hormonal therapy.

Mãe, obrigada pelos microRNAs / Mom, thanks for the microRNAs

Os microRNAs (miRNAs) são pequenas moléculas de RNA não codificante que têm grande importância nos mais diversos processos celulares, pois atuam na regulação da expressão gênica pós-transcricional. Estima-se que estes RNAs tenham controle de, em média, 30% da regulação de genes codificantes de proteínas em mamíferos. Da mesma forma, na fase zigótica do desenvolvimento embrionário, os miRNAs maternos desempenham funções notáveis e são fundamentais para a degradação dos próprios transcritos maternos. Este evento é determinante para a transição materno­zigótica, momento onde o zigoto passa a expressar completamente e de maneira independente seus próprios mRNAs, e; portanto, são vitais para o desenvolvimento inicial do embrião. O presente estudo, através de uma revisão narrativa de literatura, busca descrever os mecanismos de ação de miRNAs maternos presentes em zigotos de diversas espécies durante o desenvolvimento embrionário. Foram selecionados estudos disponíveis na base de dados PubMed através da busca utilizando palavras­chave descritas pelos Descritores em Ciências da Saúde (DeCS). (AU)

MicroRNAs (miRNAs) are small molecules of non-coding RNA that have great importance in the most diverse cellular processes, since they act in the post-transcriptional regulation of gene expression. It is estimated that these RNAs have a control of, on average, 30% of the regulation of protein-encoding genes in mammals. Likewise, in the zygotic phase of embryonic development, maternal miRNAs perform remarkable functions and are fundamental for the degradation of the maternal transcripts themselves. This event is determinant for the maternal-to-zygotic transition, at which moment the zygote begins to express completely and independently its own miRNAs, and is therefore vital for the initial development of the embryo. The present study, through a review of the literature, aims to describe the mechanisms of action of maternal miRNAs present in zygotes of different species during embryonic development. We selected only the studies listed in the PubMed database through the search using keywords described by the Health Sciences Descriptors (DeCS). (AU)

The role of Synaptotagmin1 in mouse oocyte fertilization / 天津医药

@#Objective To investigate the dynamics of cortical granules exocytosis (CGE), and the role of Synaptotagmin1 (Syt1) in mouse fertilization. Methods The dynamics of mouse CGE were filmed on Perkin Elmer precisely Ulta VIEW VOX confocal Imaging System. The Syt1 functions on mouse fertilization and expression in zygotes were analyzed by immunofluorescence, Western blot assy and qRT-PCR. Results The dynamic process of mouse CGE was observed after oocyte activation, and the exact pot seems to be close to the polar body. The Syt1 expression was gradually increased after fertilization. The mouse fertilization rate after Syt1 knockdown was significantly lower than that of the control group (P＜ 0.05). Conclusion The dynamic process of CGE is studied. It is found that Syt1 is involved in mouse fertilization process.

Preparation and detection of pipettes for microinjection / 西安交通大学学报(医学版)

Objective To introduce an optimized practical method of making and detecting pipettes for microinjection.Methods Transfer pipette was made from hard glass capillary. We softened the hard glass capillary by rotating it in a spirit-lamp flame,then moved out from the flame and quickly pulled it into two transfer pipettes.After broken by a grinding wheel,the tip of the pipette was fire-polished by quickly touching the flame to make a fine opening.A hard glass capillary (1.0 mm,ouside diametre)was pulled into two holding pipettes by pipette Puller.The pipette shoulder was broken at desired position with a grinding wheel,then the fine pipette tip opening was heated by a microforge and shrinked into a diameter -15 μm.Injection pipette could be made directly from a capillary with filament by Puller.The solution loaded injection pipette and holding pipette were assembled into the micromanipulator and could be checked before use.We transfered both pipettes into the zygotes media drop,touched the holding pipette with the tip of the injection pipette to make a "suitable"opening.Then we switched injection pipette to the mineral oil and applied injection pressure through the injector to check whether the solution could come out of the tip smoothly and at a proper speed.It could be further verified by pronucleus microinjection of zygotes.Results The results showed that the method introduced in this paper could produce suitable pipettes for zygote microinjection.In particular,the method of detecting the opening of the injection pipette was helpful for achieving high efficiency of zygote microinjection.Conclusion The method introduced here to make and detect pipettes for microinjection is very helpful for establishing a standard microinjection manipulation procedure and improving the efficiency of zygote microinjection.

Preparation and detection of pipettes for microinjection / 西安交通大学学报(医学版)

Objective To introduce an optimized practical method of making and detecting pipettes for microinjection.Methods Transfer pipette was made from hard glass capillary. We softened the hard glass capillary by rotating it in a spirit-lamp flame,then moved out from the flame and quickly pulled it into two transfer pipettes.After broken by a grinding wheel,the tip of the pipette was fire-polished by quickly touching the flame to make a fine opening.A hard glass capillary (1.0 mm,ouside diametre)was pulled into two holding pipettes by pipette Puller.The pipette shoulder was broken at desired position with a grinding wheel,then the fine pipette tip opening was heated by a microforge and shrinked into a diameter -15 μm.Injection pipette could be made directly from a capillary with filament by Puller.The solution loaded injection pipette and holding pipette were assembled into the micromanipulator and could be checked before use.We transfered both pipettes into the zygotes media drop,touched the holding pipette with the tip of the injection pipette to make a "suitable"opening.Then we switched injection pipette to the mineral oil and applied injection pressure through the injector to check whether the solution could come out of the tip smoothly and at a proper speed.It could be further verified by pronucleus microinjection of zygotes.Results The results showed that the method introduced in this paper could produce suitable pipettes for zygote microinjection.In particular,the method of detecting the opening of the injection pipette was helpful for achieving high efficiency of zygote microinjection.Conclusion The method introduced here to make and detect pipettes for microinjection is very helpful for establishing a standard microinjection manipulation procedure and improving the efficiency of zygote microinjection.

Effects of IL-6 on the development of zygote in mice after controlled ovarian hyperstimulation / 解放军医学杂志

ObjectiveTo study the effect of IL-6 on the development of zygotes of mice after controlled ovarian hyperstimulation. Methods The present experiment included three parts: a) Addition of IL-6: 80 female ICR mice were divided into 7 groups by random number table including 6 groups of superovulation (10 each) and a group of natural ovulation cycle (n=20). According to addition of IL-6 in different concentration to culture media, the superovulated ICR mice were divided into superovulation control group (0pg/ml IL-6 group), 1pg/ml IL-6 group, 5pg/ml IL-6 group, 10pg/ml IL-6 group, 25pg/ml IL-6 group, and 50pg/ml IL-6 group, with ICR mice in natural ovulation cycle served as control. b) Addition of IL-6 receptor antibody (RA): 90 female ICR mice were divided into 7 groups according to the random number table, including 5 groups of superovulation (10 each) on the basis of addition of different concentrations IL-6 and IL-6 RA to culture media (0pg/ml IL-6+RA groups, 1pg/ml IL-6+RA group, 5pg/ml IL-6+RA group, 10pg/ml IL-6+RA group, 25pg/ml IL-6+RA group), and 2 groups of normal natural cycle (20 each), including control group and the control group+IL-6 RA (100pg/ml) group. Mice in normal control group conceived naturally while those in superovulation group conceived after superovulation. The zygotes were collected and cultured in vitro for 1 day till the formation of 2-cell embryos, then the rate of 2-cell formation was observed under microscope. Experiments of each group were repeated three times. c) Immunofluorescence identification: 10 female ICR mice were divided into control group and superovulation group (5 each) by random number table method. The expressions of IL-6 in zygotes were determined with confocal immunofluorescence method. Results IL-6 addition experiment: the rate of 2-cell formation was significantly lower (P<0.05) in superovulated control group, 1pg/ml IL-6, 25pg/ml IL-6 and 50pg/ml IL-6 groups than in control group (P=0.023, P=0.026, P=0.000 and P=0.000, respectively). IL-6 receptor antibody (RA) addition experiment: compared with normal control group, the rate of 2-cell formation decreased in superovulation group (P=0.017), so did in other IL-6 RA groups (P=0.000). Immunofluorescence identification: the expression of IL-6 in zygote was obviously lower in superovulation group than in control group. Conclusion Controlled superovulation can reduce the expression of IL-6 in zygote, and it may be related to its effect on embryonic development of mice.

Establishment of transgenic mouse model with cardiac overexpression of PRKAG2-G100S / 第二军医大学学报

Objective To establish a novel transgenic mouse model of human PRKAG2 cardiac syndrome that overexpresses a PRKAG2G100S mutation, so as to lay a foundation for further studying the role of human PRKAG2 gene in the development, morphology, and function of mouse heart. Methods Human PRKAG2 with G100S mutation was sub-cloned into a multiple cloning site located in the downstream of α-myosin heavy chain (a-MHC) promoter of the plasmid. After the construction of the transgenic expressing vector, C57BL/6J mice were selected as the genetic background, and the transgenic mouse model of PRKAG2-G100S mutation was buitt by microinjection. Genotype was further confirmed using specific primer PCR. Real time PCR and Western blotting analysis were used to examin the expression of human PAKAG2(G100S) mRNA and protein, respectively. Results Two strains of transgenic mice were successfully developed using backcross breeding, which specifically overexpressed the human PRKAG2-G100S mutation in the cardiac tissues of F2 generations by the methods qPCR and Western blotting at both mRNA and protein levels. Moreover, the PRKAG2-G100S mutation was successfully passed steadily. Conclusion We have successfully established a human PRKAG2-G100S transgenic mouse model, which can help to further explore the role of PRKAG2-G100S mutation in the development and function of mouse cardiac tissue in the PRKAG2- G100S cardiac syndrome.

Aborto y embrión en pre-singamia / Abortion and embryo in pre-syngamic period

El artículo refiere brevemente el desarrollo del embrión antes de la primera división de segmentación. Penetrado el espermio en el oocito, de inmediato empiezan fenómenos de interacción entre componentes materno y paterno. Por lo tanto, el desarrollo del zigoto antes de la primera división de segmentación ya tiene características temporales y espaciales de un organismo de la especie humana. Por todo esto, el autor afirma que querer eliminar la palabra aborto de la interferencia con la vida en esta etapa del desarrollo no tiene ninguna base en los hechos científicos ni en su valoración ética, y se limita a dejar ver una preferencia de lenguaje.

This article deals with the development of the embryo before the first segmentation division. Once the sperm enters the oocyte, the interaction of paternal and maternal elements begins, following the patterns proper to the human species. The elimination of the word abortion when referring to interference with the life of the embryo at this stage can find no support in scientific evidence.

Effect of Semen Quality on the Embryo Development / 华中科技大学学报（医学）（英德文版）

To investigate the influences of sperm quality on the zygotes and embryos development,as the role of the paternal factor in early human embryogenesis is gaining more attention because of the application of techniques such as intracytoplasmic sperm injection (ICSI) for the treatment of men infertility, 136 infertility couples with men factors (Group Ⅰ ) were included from May 2002 to January 2004. One hundred and seventy two infertility couples with tube factors (Group Ⅱ ) served as controls. The sperm parameters, gemmates and embryos quality, implantation rate and pregnant rate in both groups were analyzed. It was found that there was no significant differences in the number of oocytes retrieved, the fertilization rate and number of embryos transferred between two groups. Sperm concentration, percentage of motile sperm and percentage of sperm with normal morphology were significantly lower in group Ⅰ than in group Ⅱ (P＜0.01). The proportion of good quality zygotes and good quality embryos were significantly lower in the male infertility group than in the tubal disease group (P＜0.05). Implantation rate and pregnancy rate were similar in two groups. It was concluded that spermatozoa is involved in the embryo quality, even in the early stages of development, which limited the treatment potency of IVF procedure.

Higher pregnancy rate with intrafallopian tubal gamete or zygote transfer (GIFT or ZIFT) than intrauterine embryo transfer (IVF-ET).

From November 1996 to September 1998, we had treated infertile couples who required assisted reproductive conception by allocating to either one of the three treatment methods: IVF-ET for those with bilateral tubal obstruction; ZIFT for those with at least one tubal patency but poor sperm ; and GIFT for those with at least one tubal patency and normal sperm (unexplained infertility). The ovarian stimulation protocol was all the same by using GnRH analogue (Suprefactฎ) for pituitary suppression and daily hMG (Metrodinฎ) injection for ovarian stimulation. The oocyte pick up was due when the leading follicle reach 18 mm. For IVF-ET and ZIFT, the fertilization was obtained by conventional in vitro fertilization or by ICSI depending on the sperm quality. Laparoscopic intrafallopian tubal gamete or zygote transfer was preformed on day 0 ( ovum pick up day) for the GIFT or on day 1 for the ZIFT group. Intrauterine embryo transfer was performed on day 2- 3 for the IVF-ET group. Every treatment cycle was conducted by the investigator group to minimize the variation of technical bias. Of all the 213 treatment cycles, 82 were IVF-ET, 92 were ZIFT, and 39 were GIFT. The average female patient age in each groups was not different. The pregnancy rate achieved in the GIFT and ZIFT groups were significantly higher than the IVF-ET group ( 46.2%, 40.2% and 23.2% respectively , p < 0.05). For the pregnancy outcome, the abortion rate seemed to be highest in the IVF-ET group ( 36.8%) whereas the multiple pregnancy rate seemed to be higher in the fallopian tubal transfer group ( 27% for ZIFT and 38.9% for GIFT), although there were no statistical significance. The benefit of the higher pregnancy rate for the intrafallopian tubal transfer treatment group could be due to the more suitable environment for the early stage embryo and the the more synchronize of the endometrial receptivity and the embryo arrival timing provided by the fallopian tube. In conclusion, until the optimal in vitro embryo culture system can be developed, gamete and zygote intrafallopian tubal transfer should yield higher pregnancy rate than intrauterine embryo transfer.

FINE STRUCTURE OF FERTILIZATION AND CYTOPLASMIC COMPONENTS OF THE PRONUCLEAR EMBRYOS IN THE GOAT / 解剖学报

Light and electron microscopic observations revealed that most of the embryos collected 15-16 hours after fertilization were at the pronuclear stage. Many supernumerary spermatozoa were found on the surface and in the outer zone of the zona pellucida, but none of them got into the inner zone of the zona or the perivitelline space. Some spermatozoa on the zona surface were observed in acrosome reaction stage, and those penetrated into the zona always left some acrosome reaction vesicles behind on the surface of the zona. The fertilized ovum eliminated almost all of its cortical granules and where there were some granules left, in the area that the plasma membrane had fewer microvilli. The cortical cytoplasm of the pronuclear zygote was populated with clustered hooded mitochondria, SER vesicles, yolk vacuoles and lipid droplets. Directly surrounding the pronucleus were a variety of organelles including welldeveloped Golgi complex, SER, mitochondria and annulate lamellae. The significance of these morphological changes of the fertilized ovum was discussed.