M.M. and C.A.R. acknowledge the financial support of the Ministero della Salute, Ricerca Finalizzata/giovani ricercatori anno 2009 of the Fondazione IRCCS Ospedale San Matteo, Pavia (I). M.M and C.A.R. wish to thank Marianna Longo for helping with the images preparation.

이 프로토콜은 유럽의 원칙 (EU 2천10분의 63)과 과학 연구에 사용되는 동물을 보호하는 이탈리아어 (2천14분의 26) 법에 따라 수행된다. 경추 탈구 안락사는 난소 분리를 위해 사용되며, 이는 본 연구를 덮는 승인 프로토콜에 나열된 개인 전문가에 의해 수행되고 훈련된다. 1. 동물 <ol><li> 제어 온도와 습도 방에 6 주령의 암컷 마우스에 성인 3을 유지한다 (12L의 단계와 : 12D와 공급 임의로). </li><li> 난포 자극 호르몬 (FSH)의 효과를 모방하여 모낭의 성장을 자극하는 임신 한 암말의 혈청 성선 자극 호르몬의 2.5 IU (PMSG) 마우스에 복강 주사. 이후 전 정부 난자 수집 48 시간의 난소를 분리합니다. 하여 기관 가이드 라인에 따라, 이전에 PMSG 주입 암컷 마우스를 안락사. </li><li> 신속 체강에서 난소를 제거하고 배양 접시의 계속에 그들을 배치후속 난자 격리를위한 M2 매체 aining (섹션 3 참조). <ol><li> 해부 멸균 가위를 사용하여 복막에 절개 하였다 복벽을 덮는 피부에 절개를. 방광에서 시작하여 V 자 모양을 형성, 복부를 노출 절개를 열고 멸균 핀셋을 사용하여 한쪽에 용기를 이동하고 자궁 튜브를 지역화. </li><li> 신장 아래에있는 난소를 관찰한다. 멸균 트위터와 각 튜브를 잡고 그들을 해제 난소의 하단에 작은 절개를합니다. 미리 평형화 매체를 함유하는 페트리 접시의 하단 양쪽 난소를 전송. </li></ol></li></ol> 2. 호르몬 준비 <ol><li> 적절한 주입 부피 0.1 mL의 2.5 IU 호르몬의 작용 농도를 얻기 위해 멸균 등​​장 성 식염수로 (다른 스톡 농도에서 사용 가능) PMSG 희석. 참고 : 호르몬 솔루션은 0.5 ml의 튜브에 분주하고 저장해야합니다 -2사용 일까지 0 ° C. -20 ° C 3 개월 이상 희석 분취 량을 보관하지 마십시오. 해동 후에 직렬 주사를 수행하는 경우, +4 ℃에서 호르몬 용액의 분취 량을 저장 4 일 이내에이를 사용. </li></ol> 3. 전 정부 난 모세포 분리 적절한 난 모세포 분리의 경우 : <ol><li> 난자 분리 절차를 시작하기 전에 최소한 1 시간 동안 5 % CO 2, 37 ℃에서 13 M2 매체 (M2)를 평형. 두 개의 배양 접시 (35mm X 10mm), 난소 천공과에서 분리 한 후 난자 전송을 위해 미네랄 오일 (~ 1.5 ml)로 덮여 M2의 작은 방울과 두 번째 (20 μL)에 대한 (M2) 1 ml의 하나를 준비합니다 난소 (3.4-3.6 참조). 준비가 사용되는 때까지 5 % CO 2, 37 ° C에서 인큐베이터에서 모두 배양 접시를 유지합니다. </li><li> 난 모세포의 분리 과정에서 실체 현미경을 사용합니다. </li><li> 스트레치에 의한 난자를​​ 수집하는 얇은 입 유리 피펫을 준비힝 유리 파스퇴르 피펫은 분젠 버너에서 오는 수직 화염 (그림 1)을 통해, (두 손으로 피펫의 끝을 잡고) 수평으로 유지했다. <ol><li> 유리가 녹기 시작하면, 불꽃에서 피펫을 원하는 피펫을 얻기 위해 빠른 당기는 운동을 수행합니다. 단단히 길이와 내부 모세관의 직경을 조정 손톱으로 피펫의 좁은 점에 가까운 초과 유리를 잘라. 얇은 모세관은 난자의 직경보다 약간 큰 ~ 100 ㎛의 내부 직경을 가지고 있는지 확인합니다 (~ 80 μm의). </li><li> 입 피펫, 적절한 팁을 이용하여 뽑아 피펫에 흡인기 관의 한쪽 끝을 연결하고 이빨을 확보 입 타단 장소. </li></ol></li><li> 무균 핀셋을 사용하여, 난소를 수집하고 세포 배양 용 배양 접시 (35mm X 10mm)의 바닥에이를 증착. M2 1ml의 이전에 37 ℃에서 평형화 그들을 덮5 % CO 2 (그림 2). </li><li> 부드럽게 M2 (그림 3)에서 전 정부 난자를 해제 멸균 인슐린 주사기 바늘로 난소의 난포에 구멍. </li><li> 부드럽게 입 여포 세포 및 다른 가능한 조직 파편 (도 4a)를 제거하고 이전 (배아 배양에 적합한 광유 덮여 M2의 작은 방울 (20 μL)의 하단에이를 정착 좁은 파스퇴르 피펫을 통해 난자를 피펫 준비도 4B). 주 : (M2)의 여러 다른 방울을 통해 지속적으로 피펫 팅에 의한 난자의 투명대에 연결된 모든 난구 세포를 제거하는 것이 중요하다. 매질의 pH의 변화를 피하기 위해 가능한 한 빨리이 단계를 수행한다. </li></ol> Hoechst33342 4. 전 정부 난 모세포 염색 참고 : 전 정부 난자는 SN (둘러싸인 핵소체), N​​SN (핵소체를 둘러싸인되지 않음) 또는 구별 할 수있다다른 중간 형태로, Hoechst33342의 supravital 농도 (또는 염기에 대한 구체적인 다른 마커 AT) (14) 염색 후 자신의 염색질 조직을 기반으로. NSN은 따라서,하지의 이름을하면서 사실, SN 난자는 핵소체 주위 헥스 양성 반지를 보여줍니다. 적절한 난자 염색의 경우 : <ol><li> 어둠 속에서 10 분 동안 50 NG / μL의 supravital 농도 Hoechst33342 (20 μL) (M2)에 희석 방울로 입 피펫을 사용하여 난자를 전송합니다. </li><li> Hoechst33342 함께 배양 후, 조직 염색질 (SN 또는 NSN)에 기초한 정렬, 광유 덮여 M2의 작은 방울 (5 μL)의 바닥에 입을 피펫으로 각 단일 난자 옮긴다. 난자의 염색질 구성을 시각화 Hoechst33342 필터 (발광 파장 486 ㎚)을 갖추고있는 형광 현미경을 사용합니다. 주 : 사용 된 형광 현미경의 종류에 따라, 난T는 이미지 수집을 위해 유리 바닥 배양 접시를 사용하는 것이 필수적 될 수 있습니다. 35mm 요리에 대한 시편 홀더를 장착 공 초점 현미경 올림푸스 Fluoview Fv10i는 0.17 mm의 표준 두께 허용 0.13 0.21 mm 정도의 범위와 이미지 수집에만 유리 바닥 요리가 필요합니다. </li><li> 아마도, 더 이상보다 5-10 초 동안 존재 핵소체 주위의 링 부재 (도 5)를 가시화하기에 충분한 시간을 난 모세포를 자극. </li><li> 체외 성숙을 유도 또는 분자 나 세포 분석을위한 적절한 버퍼에 보관 5 % CO 2, 37 ℃에서 (M2)에서, 문화 전 정부의 SN과 NSN 난자를 정렬 한 후. </li></ol>

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The authors have nothing to disclose

이 프로토콜은 분리 및 마우스 전 정부 GV 난자를 분류하기 위해 사용 된 방법을 설명한다. 몇 가지 예외를 제외하고, 밑줄 특별히 중요한 단계가 없습니다. ) 난자의 활력을 유지하고 b)에 사용되는 배지에서 pH 변화를 방지 : 첫째 :이 절차는 가능한 한 빨리 수행되어야한다. 둘째 : 난자는 IVM과 체외 수정은 다음 단계는 특히, 염색질 손상 및 난자의 후속 죽음을 피하기 위해 Hoechst33342에 (5 ~ 10 초) ?...

난소 전 정부 구획실로부터 분리 전 정부 완전히 자란 난자 (GV, 새싹 소포)는 일상적 같은 다른 목적을 위해 사용되는 예를 들면, 중기 II 단계까지 시험 관내 성숙 뒤에 분자 세포 메카니즘의 연구. 그들의 아름다운 외관에도 불구하고 대부분의 전 정부 난자, 전부는 아니지만, 감수 분열을 완료하고 포배기에 도달 할 수있다; 시비 3-5 발생할 경우 실제로 인간 1,2- 포함한 많은 포유류 종에서, 이들 중 약 30 %, 2 단계에서 셀들이 개발 체포. 현재까지, 몇몇 파라미터는 그렇게 할 수없는 것과 배아 발달을 유지할 수 난자를 구별하기 위해 사용된다. 예를 들어, 크레 실 블루 염색 (BCB)은 BCB + 또는 BCB- 염색 관련이 정렬 방법의 유용성이 있지만 글루코오스 -6- 포스페이트 탈수소 효소의 활성에 기초하여 난자를 정렬하는 방법으로서 유효한여전히 6-8 의존 실험실. 또 다른 잘 확립 된 방법은 염색질 조직에있는 (Hoechst33342 같은) 어떤 DNA의 인터 염료로 염색하는 경우, 전 정부 난자의 70 %가 핵소체 주위에 염료 긍정적 인 반지를 보여 핵소체 (SN)를 둘러싸인라고 30 %를 유지하면서 더 발견 긍정적 인 신호를 보여주고 (NSN) 3-5하지 둘러싸인 핵소체라고합니다. 최근에 우리는 보여 주었다 세포질 그릴 (9) (CPLS, 포유류의 난자와 배아 모두 모계 유래의 mRNA와 리보솜에 중요한 저장 사이트)의 부재 (10)의 존재와 함께 (CPLS 형성 (11)에 대한 필수) 이잖아요의 하향 조절, 그들의 세포질 9,12- 지질 방울, 2 세포 단계 이상으로 진행 NSN 난자의 무능력과 관련된 다른 원인이 될 수 있습니다. 불행하게도, 몇몇 기술은 전 정부 SN과 NSN의 난자와 정렬을 적용 할 수 있습니다,이러한 이유로, (핵 염색, 정착 절차 또는 입 피펫으로 조작없이) 덜 침습적 방법의 개발이 모두 인간과 동물의 배아 발달의 속도를 높이기 위해 매우 중요해질 수있다. 본 논문에서는 세부 간단하고 빠른 프로토콜은 광고 정렬 SN과 여성의 생쥐에서 NSN 전 정부 난자를 격리하는 데 사용됩니다 그것은 여성 배우자, 난자의 성숙과 배아 개발의 연구에 관심이있는 모든 과학자들에게 해결됩니다.

<table><tbody><tr><td>PMSG</td><td>Sigma</td><td>G4527</td><td></td></tr><tr><td>EmbrioMax M2</td><td>Millipore</td><td>MR-015-PD</td><td></td></tr><tr><td>Hoechst33342</td><td>Thermo Scientific</td><td>62249</td><td></td></tr><tr><td>Mineral Oil</td><td>Sigma</td><td>M8410</td><td></td></tr><tr><td>Cell Culture Dish 35mmx10mm</td><td>Corning</td><td>430165</td><td></td></tr><tr><td>&amp;micro;-Dish 35mm, high glass bottom</td><td>Ibidi</td><td>81158</td><td></td></tr><tr><td>Pipette Pasteur Borosilicate</td><td>Corning</td><td>7095D-9</td><td></td></tr><tr><td>Aspirator tube assemblies for calibrated micropillary pipettes</td><td>Sigma</td><td>A5177</td><td></td></tr></tbody></table>

isolation and characterization of mouse antral oocytes based on nucleolar chromatin organization

This protocol describes a simple and quick method to isolate and characterize mouse antral GV (Germinal Vesicle) oocytes as able (SN, Surrounded Nucleolus) or unable (NSN, Not Surrounded Nucleolus) to develop to the blastocyst stage after in vitro maturation (IVM) and in vitro fertilization (IVF). It makes use of Hoeschst33342 (or any other DNA intercalating dye) able to bind to the heterochromatin of the nucleolus showing a ring in the SN oocytes or not, like in the NSN oocytes. This represents the easiest and quickest way to sort both antral oocytes that can be eventually used for IVM or IVF procedures. 
Briefly, the protocol consists of the following steps: hormone injection to stimulate follicular growth; isolation of the oocytes at the GV stage from the antral compartment by puncturing the ovary with a sterile needle; preparation of thin glass pipettes for mouth pipetting of the oocytes; sorting of the oocytes with Hoechst33342 prepared at a supravital concentration; IVM, IVF or any other molecular/cellular analysis.
Unfortunately there are still few evidences to sort SN and NSN oocytes using less invasive techniques. If and once they will be identified, they could be potentially applied to human assisted reproductive technologies, although with several aspects that should be modified. To date, this technique has potential implications to dramatically increase IVM and IVF successful procedures in both endangered and species with economic interest.

다음 이미지 Hoechst33342 의해 난자 &#39;정렬에 피펫 준비부터 마우스 난소 전 정부로부터 난자를 분리하기 위해 사용 된 방법을 보여준다. 이 방법은 매우 간단하고, CO2 인큐베이터, 실체 현미경과 Hoechst33342의 관측을위한 올바른 필터를 갖춘 형광 현미경에 장착 된 임의의 실험실에서 수행 될 수있다 (1) 수치는 피펫 제조의 초기 단계를 보여준다. 유리 ?...

Watch this Scientific Journal Video about Isolation and Characterization of Mouse Antral Oocytes Based on Nucleolar Chromatin Organization at JoVE.com

Isolation and Characterization of Mouse Antral Oocytes Based on Nucleolar Chromatin Organization

여기에서 우리는 핵소체 조직을 기반으로 마우스의 전 정부 난자의 특성에 대한 프로토콜을 제시한다.

핵소체 염색질 조직을 바탕으로 마우스 전 정부 난 모세포의 분리 및 특성

This protocol describes a simple and quick method to isolate and characterize mouse antral GV (Germinal Vesicle) oocytes as able (SN, Surrounded ...

isolation-characterization-mouse-antral-oocytes-based-on-nucleolar

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

13.3K 조회수.  Fondazione IRCCS Policlinico San Matteo.  여기에서 우리는 핵소체 조직을 기반으로 마우스의 전 정부 난자의 특성에 대한 프로토콜을 제시한다. 

비디오: Isolation and Characterization of Mouse Antral Oocytes Based on Nucleolar Chromatin Organization

Chromatin Spread Preparations for the Analysis of Mouse Oocyte Progression from Prophase to Metaphase II

Chromatin spread techniques have been widely used to assess the dynamic localization of various proteins during gametogenesis, particularly for spermatogenesis. These techniques allow for visualization of protein and DNA localization patterns during meiotic events such as homologous chromosome pairing, synapsis and DNA repair. While a few protocols have been described in the literature, general chromatin spread techniques using mammalian prophase oocytes are limited and difficult due to the timing of meiosis initiation in fetal ovaries. In comparison, prophase spermatocytes can be collected from juvenile male mice with higher yields without the need for microdissection. However, it is difficult to obtain a pure synchronized population of cells at specific stages due to the heterogeneity of meiotic and post-meiotic germ cell populations in the juvenile and adult testis. For later stages of meiosis, it is advantageous to assess oocytes undergoing meiosis I (MI) or meiosis II (MII), because groups of mature oocytes can be collected from adult female mice and stimulated to resume meiosis in culture. Here, methods for meiotic chromatin spread preparations using oocytes dissected from fetal, neonatal and adult ovaries are described with accompanying video demonstrations. Chromosome missegregation events in mammalian oocytes are frequent, particularly during MI. These techniques can be used to assess and characterize the effects of different mutations or environmental exposures during various stages of oogenesis. As there are distinct differences between oogenesis and spermatogenesis, the techniques described within are invaluable to increase our understanding of mammalian oogenesis and the sexually dimorphic features of chromosome and protein dynamics during meiosis.

Oogenesis in mammals is known to be error-prone, particularly due to chromosome missegregation. This manuscript describes chromatin spread preparation methods for mouse prophase, metaphase I and II-staged oocytes. These fundamental techniques allow for the study of chromatin-bound proteins and chromosome morphology throughout mammalian oogenesis.

Chromatin 확산 마우스 Oocyte 진행 의향에서 분열 II의 분석에 대 한 준비

Chromatin spread techniques have been widely used to assess the dynamic localization of various proteins during gametogenesis, particularly for ...

연구

유전학

Using Mouse Oocytes to Assess Human Gene Function During Meiosis I

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively correlated with maternal age, age alone is not always predictive of the risk of generating an aneuploid embryo. Therefore, gene variants might account for incorrect chromosome segregation during oogenesis. Given that access to human oocytes is limited for research purposes, a series of assays were developed to study human gene function during meiosis I using mouse oocytes. First, messenger RNA (mRNA) of the gene and gene variant of interest are microinjected into prophase I-arrested mouse oocytes. After allowing time for expression, oocytes are synchronously released into meiotic maturation to complete meiosis I. By tagging the mRNA with a sequence of a fluorescent reporter, such as green fluorescent protein (Gfp), the localization of the human protein can be assessed in addition to the phenotypic alterations. For example, gain or loss of function can be investigated by establishing experimental conditions that challenge the gene product to fix meiotic errors. Although this system is advantageous in investigating human protein function during oogenesis, adequate interpretation of results should be undertaken given that protein expression is not at endogenous levels and, unless controlled for (i.e. knocked out or down), murine homologs are also present in the system.

As the genetic variants associated with human disease begin to become uncovered, it is becoming increasingly important to develop systems with which to rapidly evaluate the biological significance of those identified variants. This protocol describes methods for evaluating human gene function during female meiosis I using mouse oocytes.

감수 분열 동안 인간 유전자 기능을 평가 하기 위해 마우스 Oocytes를 사용 하 여 나

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively ...

Analysis of Chromosome Segregation, Histone Acetylation, and Spindle Morphology in Horse Oocytes

The field of assisted reproduction has been developed to treat infertility in women, companion animals, and endangered species. In the horse, assisted reproduction also allows for the production of embryos from high performers without interrupting their sports career and contributes to an increase in the number of foals from mares of high genetic value. The present manuscript describes the procedures used for collecting immature and mature oocytes from horse ovaries using ovum pick-up (OPU). These oocytes were then used to investigate the incidence of aneuploidy by adapting a protocol previously developed in mice. Specifically, the chromosomes and the centromeres of metaphase II (MII) oocytes were fluorescently labeled and counted on sequential focal plans after confocal laser microscope scanning. This analysis revealed a higher incidence in the aneuploidy rate when immature oocytes were collected from the follicles and matured in vitro compared to in vivo. Immunostaining for tubulin and the acetylated form of histone four at specific lysine residues also revealed differences in the morphology of the meiotic spindle and in the global pattern of histone acetylation. Finally, the expression of mRNAs coding for histone deacetylases (HDACs) and acetyl-transferases (HATs) was investigated by reverse transcription and quantitative-PCR (q-PCR). No differences in the relative expression of transcripts were observed between in vitro and in vivo matured oocytes. In agreement with a general silencing of the transcriptional activity during oocyte maturation, the analysis of the total transcript amount can only reveal mRNA stability or degradation. Therefore, these findings indicate that other translational and post-translational regulations might be affected.
Overall, the present study describes an experimental approach to morphologically and biochemically characterize the horse oocyte, a cell type that is extremely challenging to study due to low sample availability. However, it can expand our knowledge on the reproductive biology and infertility in monovulatory species.

This manuscript describes an experimental approach to morphologically and biochemically characterize horse oocytes. Specifically, the present work illustrates how to collect immature and mature horse oocytes by ultrasound-guided ovum pick-up (OPU) and how to investigate chromosome segregation, spindle morphology, global histone acetylation, and mRNA expression.

말의 난 모세포에서의 염색체 분리, 히스톤 아세틸 화 및 스핀들 형태의 분석

The field of assisted reproduction has been developed to treat infertility in women, companion animals, and endangered species. In the horse, assisted ...

발생학

A Neural Network-Based Identification of Developmentally Competent or Incompetent Mouse Fully-Grown Oocytes

Infertility clinics would benefit from the ability to select developmentally competent vs. incompetent oocytes using non-invasive procedures, thus improving the overall pregnancy outcome. We recently developed a classification method based on microscopic live observations of mouse oocytes during their in vitro maturation from the germinal vesicle (GV) to the metaphase II stage, followed by the analysis of the cytoplasmic movements occurring during this time-lapse period. Here, we present detailed protocols of this procedure. Oocytes are isolated from fully-grown antral follicles and cultured for 15 h inside a microscope equipped for time-lapse analysis at 37 &#176;C and 5% CO2. Pictures are taken at 8 min intervals. The images are analyzed using the Particle Image Velocimetry (PIV) method that calculates, for each oocyte, the profile of Cytoplasmic Movement Velocities (CMVs) occurring throughout the culture period. Finally, the CMVs of each single oocyte are fed through a mathematical classification tool (Feed-forward Artificial Neural Network, FANN), which predicts the probability of a gamete to be developmentally competent or incompetent with an accuracy of 91.03%. This protocol, set up for the mouse, could now be tested on oocytes of other species, including humans.

Here, we present a protocol for non-invasive assessment of oocyte developmental competence performed during their in vitro maturation from the germinal vesicle to the metaphase II stage. This method combines time-lapse imaging with particle image velocimetry (PIV) and neural network analyses.

발달 능력 또는 무능 한 마우스 가득 Oocytes의 신경 네트워크 기반 식별

Infertility clinics would benefit from the ability to select developmentally competent vs. incompetent oocytes using non-invasive procedures, thus ...

Ovarian Tissue Culture to Visualize Phenomena in Mouse Ovary

Mammalian females periodically ovulate an almost constant number of oocytes during each estrus cycle. To sustain such regularity and periodicity, regulation occurs at the hypothalamic-pituitary-gonadal axis level and on developing follicles in the ovary. Despite active studies, follicle development mechanisms are not clear because of the several steps involved from the dormant primordial follicle activation to ovulation, and because of the regulation complexity that differs at each follicular stage. To investigate the mechanisms of follicle development, and the dynamics of follicles throughout the estrus cycle, we developed a mouse ovarian tissue culture model that can be used to observe follicle development using a microscope. Systematic follicle development, periodical ovulation, and follicle atresia can all be reproduced in the cultured ovary model, and the culture conditions can be experimentally modulated. Here, we demonstrate the usefulness of this method in the study of the regulatory mechanisms of follicle development and other ovarian phenomena.

Ovarian tissue cultures can be used as models of follicle development, ovulation, and follicle atresia and indicate regulatory mechanisms of dynamic ovarian processes.

난소 조직 문화 현상을 마우스 난소에서 시각화 하

Mammalian females periodically ovulate an almost constant number of oocytes during each estrus cycle. To sustain such regularity and periodicity, ...

Multi-Photon Laser Ablation of Cytoplasmic Microtubule Organizing Centers in Mouse Oocytes

The fidelity of oocyte meiosis is critical for generating developmentally competent euploid eggs. In mammals, the oocyte undergoes a lengthy arrest at prophase I of the first meiotic division. After puberty and upon meiotic resumption, the nuclear membrane disassembles (nuclear envelope breakdown), and the spindle is assembled mainly at the oocyte center. Initial central spindle positioning is essential to protect against abnormal kinetochore-microtubule (MT) attachments and aneuploidy. The centrally positioned spindle migrates in a time-sensitive manner toward the cortex, and this is a necessary process to extrude a tiny polar body. In mitotic cells, spindle positioning relies on the interaction between centrosome-mediated astral MTs and the cell cortex. On the contrary, mouse oocytes lack classic centrosomes and, instead, contain numerous acentriolar MT organizing centers (MTOCs). At the metaphase I stage, mouse oocytes have two different sets of MTOCs: (1) MTOCs that are clustered and sorted to assemble spindle poles (polar MTOCs), and (2) metaphase cytoplasmic MTOCs (mcMTOCs) that remain in the cytoplasm and do not contribute directly to spindle formation but play a crucial role in regulating spindle positioning and timely spindle migration. Here, a multi-photon laser ablation method is described to selectively deplete endogenously labeled mcMTOCs in oocytes collected from Cep192-eGfp reporter mice. This method contributes to the understanding of the molecular mechanisms underlying spindle positioning and migration in mammalian oocytes.

An optimized protocol is presented that enables the depletion of cytoplasmic microtubule organizing centers in mouse oocytes during metaphase I using a near-infrared femtosecond laser.

마우스 난모세포에서 세포질 미세소관 조직 센터의 다광자 레이저 제거

The fidelity of oocyte meiosis is critical for generating developmentally competent euploid eggs. In mammals, the oocyte undergoes a lengthy arrest at ...

마우스의 Prophase-Arrested 난모세포에서 DNA 손상 확인

Detection of DNA Double-Stranded Breaks in Mouse Oocytes

Oocytes are amongst the biggest and most long-lived cells in the female body. They are formed in the ovaries during embryonic development and remain arrested at the prophase of meiosis I. The quiescent state may last for years until the oocytes receive a stimulus to grow and obtain the competency to resume meiosis. This protracted state of arrest makes them extremely susceptible to accumulating DNA-damaging insults, which affect the genetic integrity of the female gametes and, therefore, the genetic integrity of the future embryo. 
Consequently, the development of an accurate method to detect DNA damage, which is the first step for the establishment of DNA damage response mechanisms, is of vital importance. This paper describes a common protocol to test the presence and progress of DNA damage in prophase-arrested oocytes during a period of 20 h. Specifically, we dissect mouse ovaries, retrieve the cumulus-oocyte complexes (COCs), remove the cumulus cells from the COCs, and culture the oocytes in &#924;2 medium containing 3-isobutyl-1-methylxanthine to maintain the state of arrest. Thereafter, the oocytes are treated with the cytotoxic, antineoplasmic drug, etoposide, to engender double-strand breaks (DSBs). 
By using immunofluorescence and confocal microscopy, we detect and quantify the levels of the core protein &#947;H2AX, which is the phosphorylated form of the histone H2AX. H2AX becomes phosphorylated at the sites of DSBs after DNA damage. The inability to restore DNA integrity following DNA damage in oocytes can lead to infertility, birth defects, and increased rates of spontaneous abortions. Therefore, the understanding of DNA damage response mechanisms and, at the same time, the establishment of an intact method for studying these mechanisms are essential for reproductive biology research.

저자 스포트라이트: 포유류 난모세포와 착상 전 배아의 DNA 손상 반응 이해 

Maintaining oocyte genome integrity is necessary to ensure the genetic fidelity in the resulting embryo. Here, we present an accurate protocol for detecting DNA double-strand breaks in mammalian female germ cells.

마우스 난모세포에서 DNA 이중 가닥 절단 검출

Oocytes are amongst the biggest and most long-lived cells in the female body. They are formed in the ovaries during embryonic development and remain ...

Mouse Oocyte Microinjection, Maturation and Ploidy Assessment

Mistakes in chromosome segregation lead to aneuploid cells. In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads to infertility, miscarriages or developmental disorders like Down syndrome. Haploid gametes form through species-specific developmental programs that are coupled to meiosis. The first meiotic division (MI) is unique to meiosis because sister chromatids remain attached while homologous chromosomes are segregated. For reasons not fully understood, this reductional division is prone to errors and is more commonly the source of aneuploidy than errors in meiosis II (MII) or than errors in male meiosis 1,2. 
In mammals, oocytes arrest at prophase of MI with a large, intact germinal vesicle (GV; nucleus) and only resume meiosis when they receive ovulatory cues. Once meiosis resumes, oocytes complete MI and undergo an asymmetric cell division, arresting again at metaphase of MII. Eggs will not complete MII until they are fertilized by sperm. Oocytes also can undergo meiotic maturation using established in vitro culture conditions 3. Because generation of transgenic and gene-targeted mouse mutants is costly and can take long periods of time, manipulation of female gametes in vitro is a more economical and time-saving strategy. 
Here, we describe methods to isolate prophase-arrested oocytes from mice and for microinjection. Any material of choice may be introduced into the oocyte, but because meiotically-competent oocytes are transcriptionally silent 4,5 cRNA, and not DNA, must be injected for ectopic expression studies. To assess ploidy, we describe our conditions for in vitro maturation of oocytes to MII eggs. Historically, chromosome-spreading techniques are used for counting chromosome number 6. This method is technically challenging and is limited to only identifying hyperploidies. Here, we describe a method to determine hypo-and hyperploidies using intact eggs 7-8. This method uses monastrol, a kinesin-5 inhibitor, that collapses the bipolar spindle into a monopolar spindle 9 thus separating chromosomes such that individual kinetochores can readily be detected and counted by using an anti-CREST autoimmune serum. Because this method is performed in intact eggs, chromosomes are not lost due to operator error.

Oocytes are prone to aneuploidy due to errors in chromosome segregation during meiotic maturation. Aneuploid eggs can cause infertility, miscarriages or developmental disorders like Down syndrome. Here, we describe methods to introduce materials of choice into oocytes and methods to study meiotic maturation and assess ploidy.

마우스 Oocyte의 Microinjection, 성숙과 Ploidy 평가

Mistakes in chromosome segregation lead to aneuploid cells.  In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads to ...

핵소체 염색질 조직을 바탕으로 마우스 전 정부 난 모세포의 분리 및 특성

요약

더 많은 비디오 탐색

이 비디오의 챕터

Chromatin 확산 마우스 Oocyte 진행 의향에서 분열 II의 분석에 대 한 준비

감수 분열 동안 인간 유전자 기능을 평가 하기 위해 마우스 Oocytes를 사용 하 여 나

말의 난 모세포에서의 염색체 분리, 히스톤 아세틸 화 및 스핀들 형태의 분석

발달 능력 또는 무능 한 마우스 가득 Oocytes의 신경 네트워크 기반 식별

난소 조직 문화 현상을 마우스 난소에서 시각화 하

마우스 난모세포에서 세포질 미세소관 조직 센터의 다광자 레이저 제거

마우스 난모세포에서 DNA 이중 가닥 절단 검출

마우스 난모세포에서 DNA 이중 가닥 절단 검출

마우스 난모세포에서 DNA 이중 가닥 절단 검출

마우스 Oocyte의 Microinjection, 성숙과 Ploidy 평가