This work was supported by the National Institutes of Health grants (R01 HD093778 to E.B-F and T32 HD007065 to R.B). We thank Zachary Boucher for his assistance with radiation experiment. We thank Mary Ann Handel for critical reading of the manuscript. We gratefully acknowledge the contribution of Sonia Erattupuzha and the Microscopy Core Service at The Jackson Laboratory for expert assistance with the microscopy work described in this publication and Jarek Trapszo from the Scientific Instrument Services at The Jackson Laboratory for designing the 3D-printed adaptor slide.

The authors have no conflicts of interest to disclose.

This article presents a detailed 3D immunostaining and imaging protocol for prenatal and postnatal ovaries for high-throughput and comparative studies for germ cell quantification and protein localization. We developed this protocol to analyze oocyte numbers in ovaries (N=6-12) at six developmental time points in 10-16 different strains, where 2-4 24-well plates are typically processed at one time. This method can be adapted for other organs or cellular markers. For example, this protocol can be used to label and visuali...

Most mammalian females are born with a finite number of meiotically arrested oocytes stored within primordial follicles, constituting the ovarian reserve (OR)1,2. The OR determines the overall female reproductive lifespan and health3. The OR normally declines in size with aging and can be prematurely depleted upon exposure to certain genotoxic agents (radiation/chemotherapy) and environmental stresses (malnutrition), leading to infertility4,5,6. Idiopathic female infertility can often be attributed to the genetic and physiological quality of eggs developing from the OR and remains poorly understood7,8. Because female follicle endowment is largely predetermined by birth, it is essential to understand the regulatory mechanisms involved in the OR establishment and maintenance.
In mice, OR formation starts with the specification of primordial germ cells (PGCs) around Embryonic day (E) 7.52. The PGCs migrate to the genital ridges, where they will reside by approximately E10.59. The following extensive proliferation occurs with incomplete cytokinesis resulting in the formation of cysts that will be broken down later in development10,11. At approximately E12.5, gonadal sex is determined, and PGC proliferation halts in ovaries. In females, PGCs, now oocytes, enter meiotic prophase I (MPI) at approximately E13.512,13.&#160;Oocytes progress through extended MPI and arrest at the dictyate stage around the time of birth. During the first week after birth, each arrested oocyte is surrounded by granulosa cells, thereby forming a primordial follicle.
The number of primordial follicles in the OR of a female depends on how many oocytes survived the waves of oocyte elimination that occur before and during MPI arrest through apoptosis, autophagy, or necrosis14,15. The first wave occurs during fetal development and is known as FOA. FOA is an evolutionarily conserved process in females (mammalian and non-mammalian), whereby an estimated 50-80% of the oocytes are eliminated depending on the female species16,17,18,19. In mice, FOA occurs during E15.5 to E18.5 and has been attributed to the reactivation and expression of retrotransposon LINE-1 sequences causing oocyte death20,21. The second wave of oocyte elimination occurs through a meiotic checkpoint that eliminates oocytes with meiotic defects such as unrepaired DNA double-strand breaks (DSBs)22,23. The next wave of oocyte elimination occurs during cyst breakdown, culminating during the formation of primordial follicles, each of which contains a single oocyte10,11,24,25.
In mice, the primordial follicle reserve is largely established by puberty, after which it decreases as primordial follicles are activated for growth during regular reproductive cycles. The OR size varies among individual women and among different genetic strains of mice; yet, the genetic regulation of OR size is not well understood26,27,28,29. Genetic studies of OR regulation are hampered by the lack of standardized protocols to study the waves of oocyte elimination during prenatal and postnatal development. Several oocyte quantification methodologies have been developed in mice, with the most common and widely used being histomorphometric evaluation of histological sections30,31. In this technique, oocytes are identified on serial sections with histological stains, such as hematoxylin and eosin (H&#38;E) and periodic acid-Schiff (PAS) or fluorescent markers. This technique is reliable if all conditions remain constant, including section thickness, efficient recovery of all sections throughout the ovary, and the counting schemes of individual laboratories. However, numbers reported by different laboratories often differ significantly and thus are not easily comparable.
Moreover, given genetic differences, the use of different mouse strains can also influence oocyte counts. Additional computational approaches have been developed for histomorphometric evaluation and include the automated detection of oocytes using the fractionator approach, automatic counting using computational algorithms, and 3D reconstruction of histological images to prevent multiple counts of the same oocyte31,32,33,34,35,36. Even with these improvements added to histomorphometric evaluation, the technique is relatively labor-intensive, particularly for large-scale and high-throughput studies. The data collected may not be reproducible and comparable between studies due to differences in counting schemes, computer algorithms, and software used.
Recently, accelerated by the development of new medium-resolution multiphoton and light sheet microscopy and optical tissue clearing methods, 3D modeling and analysis techniques for intact ovaries are becoming the method of choice to efficiently quantify oocyte numbers and study protein localization and dynamics37,38. These 3D methods are typically advantageous compared to histological methods as tissues and organs are better preserved and kept intact. Moreover, 3D analysis and modeling provide additional insights into function and interactions within and between cell niches or substructures within the organ that may be missed in 2D analysis.
3D analysis of whole organs requires optimization of fixation, immunostaining, and optical clearing protocols for individual organs, such as ovaries, without tissue distortion or damage. Additional optimization of sample mounting for imaging is required for high-resolution microscopy and may depend on the imaging platform available. Finally, imaging of the whole intact ovary generates a large amount of data for subsequent computational analyses. Therefore, there is a need to develop standardized 3D methods for counting oocytes for comparative studies and across developmental stages.
This protocol uses standard immunostaining and previously reported clearing protocols, focusing on a simple, user-friendly, and high-throughput approach38,39,40,41. The protocol is optimized to analyze large numbers of prenatal and postnatal ovaries up to postnatal day 28 (P28) and varying sizes of ovaries from different mouse genetic backgrounds. The immunostaining steps are similar for all stages; however, the clearing protocols differ for pubertal ovaries due to their larger size, ScaleS4(0) and CUBIC for small and large ovaries, respectively40,41. Further, whole-body perfusion is performed in P28 mice before fixation to prevent autofluorescence from blood cells. A multiphoton microscope was built on the Leica DIVE/4Tune platform as an alternative to light sheet microscopy to acquire images, and IMARIS 3D Visualization and Analysis software with various analytical tools was chosen for this protocol. This protocol is simple to follow and less hands-on, hence time-saving. Moreover, oocyte quantification is relatively quick, depending on the size of the ovary and arrangement of oocytes.

<table><tbody><tr><td>Benchtop Incubator</td><td>Benchmark Scientific</td><td>H2200-H</td><td>37 &amp;deg;C incubator</td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>VWR</td><td>97061-416</td><td></td></tr><tr><td>C57BL/6J</td><td>The Jackson Laboratory</td><td>000664</td><td>mouse inbred strain</td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Sigma-Aldrich</td><td>D1435</td><td>Hazardous material</td></tr><tr><td>D-Sorbitol</td><td>Sigma-Aldrich</td><td>S6021</td><td></td></tr><tr><td>Dumont #5 Forceps</td><td>FST</td><td>91150-20</td><td></td></tr><tr><td>FastWells Reagent Barriers</td><td>GraceBio</td><td>664113</td><td>Sticky and flexible silicone gasket (adhesive well)</td></tr><tr><td>Fine Scissors</td><td>FST</td><td>91460-11</td><td></td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>G2025</td><td></td></tr><tr><td>Glycine</td><td>ThermoFisher Scientific</td><td>BP381-500</td><td></td></tr><tr><td>Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 647</td><td>Invitrogen</td><td>A-21246</td><td>Dilution 1:1000</td></tr><tr><td>Goat anti-Rat IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 555</td><td>Invitrogen</td><td>A-21434</td><td>Dilution 1:1000</td></tr><tr><td>Goat serum</td><td>Sigma-Aldrich</td><td>G9023</td><td></td></tr><tr><td>IMARIS Software</td><td>Oxford Instruments</td><td>Version 9.7.0</td><td>Image visualization and analysis software</td></tr><tr><td>Insight X3</td><td>Spectra-Physics</td><td>InSight&amp;nbsp;X3 Tunable Ultrafast Laser</td><td>Laser for Multiphoton Imaging</td></tr><tr><td>LASX software</td><td>Leica</td><td>Version 3.5.6</td><td>Image acquisition software</td></tr><tr><td>Leica DIVE/4TUNE/FALCON</td><td>Leica</td><td>Leica Dmi8, 2P-M-ready: # 158005406</td><td>Multiphoton Microscope</td></tr><tr><td>MaiTai HP</td><td>Spectra-Physics</td><td>Mai Tai DeepSee One Box Ultrafast Laser</td><td>Laser for Multiphoton Imaging</td></tr><tr><td>Masterflex Pump Controller</td><td>SPW Industrial</td><td>Model: 7553-50</td><td>Peristaltic pump for perfusion</td></tr><tr><td>Mayo Scissors</td><td>FST</td><td>14010-17</td><td>5&amp;rdquo; &amp;ndash;7&amp;rdquo; blunt/blunt scissors for decapitation</td></tr><tr><td>Micro Cover Glasses, Square, No. 1.5 25x25mm</td><td>VWR</td><td>48366-249</td><td></td></tr><tr><td>Mini BioMixer</td><td>Benchmark Scientific</td><td>B3D1020</td><td>shaker/nutator for 37 &amp;deg;C incubator</td></tr><tr><td>Nikon Ergonomic SMZ1270</td><td>Leica</td><td>&amp;nbsp;SMZ1270</td><td>stereomicroscope</td></tr><tr><td>Paraformaldehyde 16% (formaldehyde aqueous solution)</td><td>Electron Microscopy Sciences</td><td>15710</td><td>Hazardous material</td></tr><tr><td>PBS Tablets, Phosphate-buffered Saline</td><td>ThermoFisher Scientific</td><td>BP2944100</td><td>Dissolve in Milli-Q water</td></tr><tr><td>Penicillin-Streptomycin, 200x, Dual Antibiotic Solution</td><td>ThermoFisher Scientific</td><td>ICN1670249</td><td></td></tr><tr><td>Polyvinyl alcohol (PVA)</td><td>Sigma-Aldrich</td><td>P8136</td><td></td></tr><tr><td>Quadrol (N,N,N&amp;prime;,N&amp;prime;-Tetrakis(2-Hydroxypropylethylenediamine)</td><td>Sigma-Aldrich</td><td>122262</td><td></td></tr><tr><td>Rabbit anti-DDX4/MVH</td><td>Abcam</td><td>ab27591</td><td>Dilution 1:500</td></tr><tr><td>Rabbit anti-LINE-1 ORF1p</td><td>Abcam</td><td>ab216324</td><td>Dilution 1:500</td></tr><tr><td>Rat anti-TRA98/GCNA</td><td>Abcam</td><td>ab82527</td><td>Dilution 1:500</td></tr><tr><td>Sodium azide</td><td>Sigma-Aldrich</td><td>S2002</td><td>Hazardous material</td></tr><tr><td>Sodium borohydride</td><td>Sigma-Aldrich</td><td>452882</td><td>Hazardous material</td></tr><tr><td>Sucrose</td><td>ThermoFisher Scientific</td><td>S0389</td><td></td></tr><tr><td>Tekmar Orbital Shaker</td><td>Bimedis</td><td>VXR-S10</td><td>shaker for room temperature</td></tr><tr><td>Triethanolamine</td><td>Sigma-Aldrich</td><td>90279</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>X100</td><td></td></tr><tr><td>UNOLOK Infusion Set</td><td>MYCO Medical</td><td>7001-23</td><td>needles for perfusion</td></tr><tr><td>Urea</td><td>Amresco</td><td>97061-920</td><td></td></tr><tr><td>X-Cite 120LED</td><td>Excelitas</td><td>S/N XT640-W-0147</td><td>low-power LED fluorescence lamp</td></tr></tbody></table>

whole ovary immunofluorescence, clearing, and multiphoton microscopy for quantitative 3d analysis of the developing ovarian reserve in mouse

Female fertility and reproductive lifespan depend on the quality and quantity of the ovarian oocyte reserve. An estimated 80% of female germ cells entering meiotic prophase I are eliminated during Fetal Oocyte Attrition (FOA) and the first week of postnatal life. Three major mechanisms regulate the number of oocytes that survive during development and establish the ovarian reserve in females entering puberty. In the first wave of oocyte loss, 30-50% of the oocytes are eliminated during early FOA, a phenomenon that is attributed to high Long interspersed nuclear element-1 (LINE-1) expression. The second wave of oocyte loss is the elimination of oocytes with meiotic defects by a meiotic quality checkpoint. The third wave of oocyte loss occurs perinatally during primordial follicle formation when some oocytes fail to form follicles. It remains unclear what regulates each of these three waves of oocyte loss and how they shape the ovarian reserve in either mice or humans.
Immunofluorescence and 3D visualization have opened a new avenue to image and analyze oocyte development in the context of the whole ovary rather than in less informative 2D sections. This article provides a comprehensive protocol for whole ovary immunostaining and optical clearing, yielding preparations for imaging using multiphoton microscopy and 3D modeling using commercially available software. It shows how this method can be used to show the dynamics of oocyte attrition during ovarian development in C57BL/6J mice and quantify oocyte loss during the three waves of oocyte elimination. This protocol can be applied to prenatal and early postnatal ovaries for oocyte visualization and quantification, as well as other quantitative approaches. Importantly, the protocol was strategically developed to accommodate high-throughput, reliable, and repeatable processing that can meet the needs in toxicology, clinical diagnostics, and genomic assays of ovarian function.

Immunostaining and imaging of the whole ovary enables the visualization and quantification of oocytes or protein expression in ovaries at different developmental stages using the same technique and markers (Figure 3). This protocol was developed for a large-scale project in which analysis of ovaries at multiple stages and from multiple mouse strains was required. Here, we present data gathered for the C57BL6/J strain, a standard strain for genetic analysis. The technique presented here is st...

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Whole Ovary Immunofluorescence, Clearing, and Multiphoton Microscopy for Quantitative 3D Analysis of the Developing Ovarian Reserve in Mouse

Here, we present an optimized protocol for imaging entire ovaries for quantitative and qualitative analyses using whole-mount immunostaining, multiphoton microscopy, and 3D visualization and analysis. This protocol accommodates high-throughput, reliable, and repeatable processing that is applicable for toxicology, clinical diagnostics, and genomic assays of ovarian function.

Female fertility and reproductive lifespan depend on the quality and quantity of the ovarian oocyte reserve. An estimated 80% of female germ cells ...

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4.6K Views.  The Jackson Laboratory. Deze methode kan helpen bij het beantwoorden van een van de belangrijkste vragen op het gebied van voortplanting, namelijk hoe en wanneer genetisch gereguleerde processen de grootte van de ovariële reserve beïnvloeden waarvan we weten dat ze van invloed zijn op vrouwen, reproductieve levensduur Dit protocol kan worden aangenomen voor grootschalige studies met dezelfde technieken voor intacte eierstokken uit verschillende ontwikkelingsstadia. Het genereert produceerbare gegevens door een eff...

Video: Whole Ovary Immunofluorescence, Clearing, and Multiphoton Microscopy for Quantitative 3D Analysis of the Developing Ovarian Reserve in Mouse

Culture and Co-Culture of Mouse Ovaries and Ovarian Follicles

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together within a basement membrane. A finite pool of follicles is laid down during embryonic development, when oocytes in meiotic arrest form a close association with flattened granulosa cells, forming primordial follicles. By or shortly after birth, mammalian ovaries contain their lifetime&#8217;s supply of primordial follicles, from which point onwards there is a steady release of follicles into the growing follicular pool.
The ovary is particularly amenable to development in vitro, with follicles growing in a highly physiological manner in culture. This work describes the culture of whole neonatal ovaries containing primordial follicles, and the culture of individual ovarian follicles, a method which can support the development of follicles from an immature through to the preovulatory stage, after which their oocytes are able to undergo fertilization in vitro. The work outlined here uses culture systems to determine how the ovary is affected by exposure to external compounds. We also describe a co-culture system, which allows investigation of the interactions that occur between growing follicles and the non-growing pool of primordial follicles.

This protocol describes the primary culture/co-culture of mouse ovarian tissue, using ovaries from neonatal mice and individual ovarian follicles from prepubertal mice. The culture techniques support development in a highly physiological manner, allowing investigation of the effect of extrinsic agents on the ovary, and of interactions between ovarian follicles.

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together ...

Analysis of Cardiomyocyte Development using Immunofluorescence in Embryonic Mouse Heart

During heart development, the generation of myocardial-specific structural and functional units including sarcomeres, contractile myofibrils, intercalated discs, and costameres requires the coordinated assembly of multiple components in time and space. Disruption in assembly of these components leads to developmental heart defects. Immunofluorescent staining techniques are used commonly in cultured cardiomyocytes to probe myofibril maturation, but this ex vivo approach is limited by the extent to which myocytes will fully differentiate in culture, lack of normal in vivo mechanical inputs, and absence of endocardial cues. Application of immunofluorescence techniques to the study of developing mouse heart is desirable but more technically challenging, and methods often lack sufficient sensitivity and resolution to visualize sarcomeres in the early stages of heart development. Here, we describe a robust and reproducible method to co-immunostain multiple proteins or to co-visualize a fluorescent protein with immunofluorescent staining in the embryonic mouse heart and use this method to analyze developing myofibrils, intercalated discs, and costameres. This method can be further applied to assess cardiomyocyte structural changes caused by mutations that lead to developmental heart defects.

Mutations that lead to congenital heart defects benefit from in vivo investigation of cardiac structure during development, but high-resolution structural studies in the mouse embryonic heart are technically challenging. Here we present a robust immunofluorescence and image analysis method to assess cardiomyocyte-specific structures in the developing mouse heart.

During heart development, the generation of myocardial-specific structural and functional units including sarcomeres, contractile myofibrils, intercalated ...

Organ Culture and Whole Mount Immunofluorescence Staining of Mouse Wolffian Ducts

Tubal morphogenesis is a fundamental requirement for the development of most mammalian organs, including the male reproductive system. The epididymis, an integral part of the male reproductive tract, is responsible for sperm storage, maturation, and transport. The adult epididymis is a highly coiled tube that develops from a simple and straight embryonic precursor known as Wolffian duct (WD). Proper coiling of the epididymis is essential for male fertility, as sperm in the testis are unable to fertilize an oocyte. However, the mechanism responsible for epididymal development and coiling remains unclear, partially due to the lack of whole organ culture and imaging methods. In this study, we describe an in vitro culture system and whole mount immunofluorescence protocol to better visualize the process of WD coiling and development, which may also be applied to study other tubular organs.

We present a method for isolation and culture of the mouse Wolffian duct (WD). We also demonstrate a detailed procedure for whole mount immunostaining of cultured/freshly isolated WDs with fluorescently tagged antibodies. Together, these techniques enable the study of WD development, coiling, and differentiation.

Tubal morphogenesis is a fundamental requirement for the development of most mammalian organs, including the male reproductive system. The epididymis, an ...

Correlative Super-resolution and Electron Microscopy to Resolve Protein Localization in Zebrafish Retina

We present a method to investigate the subcellular protein localization in the larval zebrafish retina by combining super-resolution light microscopy and scanning electron microscopy. The sub-diffraction limit resolution capabilities of super-resolution light microscopes allow improving the accuracy of the correlated data. Briefly, 110 nanometer thick cryo-sections are transferred to a silicon wafer and, after immunofluorescence staining, are imaged by super-resolution light microscopy. Subsequently, the sections are preserved in methylcellulose and platinum shadowed prior to imaging in a scanning electron microscope (SEM). The images from these two microscopy modalities are easily merged using tissue landmarks with open source software. Here we describe the adapted method for the larval zebrafish retina. However, this method is also applicable to other types of tissues and organisms. We demonstrate that the complementary information obtained by this correlation is able to resolve the expression of mitochondrial proteins in relation with the membranes and cristae of mitochondria as well as to other compartments of the cell.

This protocol describes the necessary steps to obtain subcellular protein localization results on zebrafish retina by correlating super-resolution light microscopy and scanning electron microscopy images.

We present a method to investigate the subcellular protein localization in the larval zebrafish retina by combining super-resolution light microscopy and ...

Whole-mount Clearing and Staining of Arabidopsis Flower Organs and Siliques

Due to its formidable tools for molecular genetic studies, Arabidopsis thaliana is one of the most prominent model species in plant biology and, especially, in plant reproductive biology. However, plant morphological, anatomical, and ultrastructural analyses traditionally involve time-consuming embedding and sectioning procedures for bright field, scanning, and electron microscopy. Recent progress in confocal fluorescence microscopy, state-of-the-art 3-D computer-aided microscopic analyses, and the continuous refinement of molecular techniques to be used on minimally processed whole-mount specimens, has led to an increased demand for developing efficient and minimal sample processing techniques. In this protocol, we describe techniques for properly dissecting Arabidopsis flowers and siliques, basic clearing techniques, and some staining procedures for whole-mount observations of reproductive structures.

Whole-mount Clearing and Staining of Arabidopsis Flower Organs and Siliques

In this protocol, we describe techniques for the proper dissection of Arabidopsis flowers and siliques, some basic clearing techniques, and selected staining procedures for whole-mount observations of reproductive structures.

Due to its formidable tools for molecular genetic studies, Arabidopsis thaliana is one of the most prominent model species in plant biology and, ...

Quantitative Whole-mount Immunofluorescence Analysis of Cardiac Progenitor Populations in Mouse Embryos

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development and organogenesis are two areas of research that have benefitted greatly from these advances, due to the high quality of data that can be obtained directly from imaging pre-implantation embryos or ex vivo organs. While pre-implantation embryos have yielded data with especially high spatial resolution, later stages have been less amenable to three-dimensional reconstruction. Obtaining high-quality 3D or volumetric data for known embryonic structures in combination with fate mapping or genetic lineage tracing will allow for a more comprehensive analysis of the morphogenetic events taking place during embryogenesis.
This protocol describes a whole-mount immunofluorescence approach that allows for the labeling, visualization, and quantification of progenitor cell populations within the developing cardiac crescent, a key structure formed during heart development. The approach is designed in such a way that both cell- and tissue-level information can be obtained. Using confocal microscopy and image processing, this protocol allows for three-dimensional spatial reconstruction of the cardiac crescent, thereby providing the ability to analyze the localization and organization of specific progenitor populations during this critical phase of heart development. Importantly, the use of reference antibodies allows for successive masking of the cardiac crescent and subsequent quantitative measurements of areas within the crescent. This protocol will not only enable a detailed examination of early heart development, but with adaptations should be applicable to most organ systems in the gastrula to early somite stage mouse embryo.

Here, we describe a protocol for whole mount immunofluorescence and image-based quantitative volumetric analysis of early stage mouse embryos. We present this technique as a powerful approach to qualitatively and quantitatively assess cardiac structures during development, and propose that it may be widely adaptable to other organ systems.

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development ...

Whole Mount Immunofluorescence and Follicle Quantification of Cultured Mouse Ovaries

Research in the field of mammalian reproductive biology often involves evaluating the overall health of ovaries and testes. Specifically, in females, ovarian fitness is often assessed by visualizing and quantifying follicles and oocytes. Because the ovary is an opaque three-dimensional tissue, traditional approaches require laboriously slicing the tissue into numerous serial sections in order to visualize cells throughout the entire organ. Furthermore, because quantification by this method typically entails scoring only a subset of the sections separated by the approximate diameter of an oocyte, it is prone to inaccuracy. Here, a protocol is described that instead utilizes whole organ tissue clearing and immunofluorescence staining of mouse ovaries to visualize follicles and oocytes. Compared to more traditional approaches, this protocol is advantageous for visualizing cells within the ovary for numerous reasons: 1) the ovary remains intact throughout sample preparation and processing; 2) small ovaries, which are difficult to section, can be examined with ease; 3) cellular quantification is more readily and accurately achieved; and 4) the whole organ imaged.

Here, we present a protocol to quantify follicles in cultured ovaries of young mice without serial sectioning. Using whole organ immunofluorescence and tissue clearing, physical sectioning is replaced with optical sectioning. This method of sample preparation and visualization maintains organ integrity and facilitates automated quantification of specific cells.

Research in the field of mammalian reproductive biology often involves evaluating the overall health of ovaries and testes. Specifically, in females, ...

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.

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

Accurate Follicle Enumeration in Adult Mouse Ovaries

Sexually reproducing female mammals are born with their entire lifetime supply of oocytes. Immature, quiescent oocytes are found within primordial follicles, the storage unit of the female germline. They are non-renewable, thus their number at birth and subsequent rate of loss largely dictates the female fertile lifespan. Accurate quantification of primordial follicle numbers in women and animals is essential for determining the impact of medicines and toxicants on the ovarian reserve. It is also necessary for evaluating the need for, and success of, existing and emerging fertility preservation techniques. Currently, no methods exist to accurately measure the number of primordial follicles comprising the ovarian reserve in women. Furthermore, obtaining ovarian tissue from large animals or endangered species for experimentation is often not feasible. Thus, mice have become an essential model for such studies, and the ability to evaluate primordial follicle numbers in whole mouse ovaries is a critical tool. However, reports of absolute follicle numbers in mouse ovaries in the literature are highly variable, making it difficult to compare and/or replicate data. This is due to a number of factors including strain, age, treatment groups, as well as technical differences in the methods of counting employed. In this article, we provide a step-by-step instructional guide for preparing histological sections and counting primordial follicles in mouse ovaries using two different methods: [1] stereology, which relies on the fractionator/optical dissector technique; and [2] the direct count technique. Some of the key advantages and drawbacks of each method will be highlighted so that reproducibility can be improved in the field and to enable researchers to select the most appropriate method for their studies.

Here, we describe, compare, and contrast two different techniques for accurate follicle counting of fixed mouse ovarian tissues.

Sexually reproducing female mammals are born with their entire lifetime supply of oocytes. Immature, quiescent oocytes are found within primordial ...

Three-dimensional Reconstruction of the Vascular Architecture of the Passive CLARITY-cleared Mouse Ovary

The ovary is the main organ of the female reproductive system and is essential for the production of female gametes and for controlling the endocrine system, but the complex structural relationships and three-dimensional (3D) vasculature architectures of the ovary are not well described. In order to visualize the 3D connections and architecture of blood vessels in the intact ovary, the first important step is to make the ovary optically clear. In order to avoid tissue shrinkage, we used the hydrogel fixation-based passive CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/ Immunostaining/In situ-hybridization-compatible Tissue Hydrogel) protocol method to clear an intact ovary. Immunostaining, advanced multiphoton confocal microscopy, and 3D image-reconstructions were then used for the visualization of ovarian vessels and follicular capillaries. Using this approach, we showed a significant positive correlation (P &#60;0.01) between the length of the follicular capillaries and volume of the follicular wall.

Here we present an adaptation of the passive CLARITY and 3D reconstruction method for visualization of the ovarian vasculature and follicular capillaries in intact mouse ovaries.

The ovary is the main organ of the female reproductive system and is essential for the production of female gametes and for controlling the endocrine ...

Whole Ovary Immunofluorescence, Clearing, and Multiphoton Microscopy for Quantitative 3D Analysis of the Developing Ovarian Reserve in Mouse

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