I thank the authors contributing to the initial work which is the basis of this publication. Thanks to Aitor Aguirre, Genevieve E. Ryan and Erica L. Schoeller for help preparing the manuscript. Thanks to Jessica Sora Lee and Austin Chin for technical assistance with the manuscript. H.M.H. was supported by Eunice Kennedy Shriver National Institute of Child Health &#38; Human Development of the National Institutes of Health under Award Number R00HD084759.

All methods described here have been approved by the Institutional Animal Care and Use Committee of Michigan State University and conducted in accordance with the Guide for the Care and Use of Laboratory Animals.
1. Determine Pubertal Onset
<ol>
	<li>Follow institutional guidelines for clothing, at a minimum, it is necessary to wear a clean lab coat and clean gloves. Always handle mice wearing clean gloves.</li>
	<li>Prepare the work area by placing a pad on the table.
	<ol>
		<li>Place a cl...

<ol>
	<li>Schneider, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Energy+balance+and+reproduction.">Energy balance and reproduction.</a> Physiology and Behavior. 81 (2), 289-317 (2004).</li><li>Walton, J. C., Weil, Z. M., Nelson, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+photoperiod+on+hormones,+behavior,+and+immune+function.">Influence of photoperiod on hormones, behavior, and immune function.</a> Frontiers Neuroendocrinology. 32 (3), 303-319 (2012).</li><li>Hoffmann, H. M., Mellon, P. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leptin+accelerates+the+onset+of+puberty+in+normal+female+mice.">Leptin accelerates the onset of puberty in normal female mice.</a> Journal of Clinical Investigation. 99 (3), 391-395 (1997).</li><li>Bohlen, T. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+short-day+photoperiod+delays+the+timing+of+puberty+in+female+mice+via+changes+in+the+kisspeptin+system.">A short-day photoperiod delays the timing of puberty in female mice via changes in the kisspeptin system.</a> Frontiers in Endocrinology. 9 (FEB), 1-9 (2018).</li><li>Shahab, M., Mastronardi, C., Seminara, S. B., Crowley, W. F., Ojeda, S. R., Plant, T. 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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Neurokinin+B+in+the+Control+of+Female+Puberty+and+Its+Modulation+by+Metabolic+Status.">Role of Neurokinin B in the Control of Female Puberty and Its Modulation by Metabolic Status.</a> Journal of Neuroscience. 32 (7), 2388-2397 (2012).</li><li>Hoffmann, H. M., Tamrazian, A., Xie, H., P&#233;rez-Mill&#225;n, M. I., Kauffman, A. S., Mellon, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterozygous+deletion+of+ventral+anterior+homeobox+(Vax1)+causes+subfertility+in+mice.">Heterozygous deletion of ventral anterior homeobox (Vax1) causes subfertility in mice.</a> Endocrinology. 155 (10), 4043-4053 (2014).</li><li>Kauffman, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Kisspeptin+Receptor+GPR54+Is+Required+for+Sexual+Differentiation+of+the+Brain+and+Behavior.">The Kisspeptin Receptor GPR54 Is Required for Sexual Differentiation of the Brain and Behavior.</a> Journal of Neuroscience. 27 (33), 8826-8835 (2007).</li><li>Teles, M. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brief+report:+A+GPR54-activating+mutation+in+a+patient+with+central+precocious+puberty.">Brief report: A GPR54-activating mutation in a patient with central precocious puberty.</a> New England Journal of Medicine. , (2008).</li><li>Korenbrot, C. C., Huhtaniemi, I. T., Weiner, R. 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C., Valenzuela, N., Fai, S., Bennett, S. A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Performing+Vaginal+Lavage,+Crystal+Violet+Staining,+and+Vaginal+Cytological+Evaluation+for+Mouse+Estrous+Cycle+Staging+Identification.">Performing Vaginal Lavage, Crystal Violet Staining, and Vaginal Cytological Evaluation for Mouse Estrous Cycle Staging Identification.</a> Journal of Visualized Experiments. (67), 4-9 (2012).</li><li>Hedrich, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Laboratory Mouse. , (2012).</li><li>Hoffmann, H. M., Trang, C., Gong, P., Kimura, I., Pandolfi, E. C., Mellon, P. 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The overall wellbeing of the mice is critical for a successful fertility assay21. When performing a fertility assay, it is important to not physically check on the mice every day as this can cause stress. Further avoid frequent cage changes, as these are also stressful. Ideally cage changes will be done no more than 1-2 times per week. Light exposure during the dark phase negatively impacts breeding in nocturnal rodents. Do not turn on lights in the breeding room during the dark hours. If entry to...

The transition through puberty is required to attain sexual maturity and reproductive competence. The pubertal transition and the maintenance of fertility in adulthood is regulated by the reproductive axis, also termed the hypothalamic-pituitary-gonadal axis (Figure 1). The timing of pubertal onset and maintenance of fertility is tightly regulated by internal as well as environmental factors to increase the chances of survival of offspring and parents1,2. This protocol provides a non-invasive approach to determine pubertal onset in mice and rats to confirm sexual maturity prior to setting up a fertility study to assess reproductive competence.
A fertility study is performed in sexually mature animals and can be initiated after the animals have gone through puberty. Prior to pubertal onset, the reproductive axis is quiescent, and the key driver of sexual maturation, gonadotropin-releasing hormone (GnRH), is released onto the pituitary in insufficient amounts to initiate puberty (Figure 1). Pubertal onset is a complex process that results in increased GnRH release at the median eminence. GnRH promotes luteinizing hormone (LH) and follicle stimulating hormone (FSH) secretion from the pituitary, two hormones essential for gonadal maturation and reproductive function (Figure 1)3,4,5.
Insults to the reproductive axis result in reduced fertility and can also advance or delay pubertal onset. Conditions known to influence the timing of pubertal onset and reproductive competence include the exposure to endocrine disrupting chemicals6,7, increased/decreased body weight1,8, changes in day length2,9 and genetic mutations10,11,12,13,14,15.
The onset of sexual maturity is a critical step that needs to be completed prior to setting up a fertility assay. The advantages of determining pubertal onset through preputial separation, vaginal opening and first estrus, are the non-invasive characteristics of these procedures, as they do not require blood collection or sacrifice of the animal16,17.
After pubertal onset is determined, correctly setting up a fertility study will provide important information about the integrity of the reproductive axis, and usually has the second advantage of generating experimental animals for further studies (refinement)18. The fertility study setup described in this protocol can detect both minor and major deficits in reproductive competence in males and females. Key parameters evaluated include 1) time to the first litter, 2) number of litters generated in a given time frame and 3) litter size. Finally, recommendations for the type of follow up studies which can be conducted to identify the cause of fertility impairment are included.
The described protocol refers to mice and the representative data reflect work done in transgenic mice. However, all the included protocols are equally valid in rats.

<table border="0" cellpadding="0" cellspacing="0" width="1092">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Sterile Cotton Balls</td>
			<td style="width:167px;">Fisher</td>
			<td style="width:280px;">22456885</td>
			<td style="width:429px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Surface protector</td>
			<td>Fisher</td>
			<td>1420637</td>
			<td style="width:429px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Light meter</td>
			<td>VWR</td>
			<td>21800-014</td>
			<td style="width:429px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Methylene blue&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>M9140</td>
			<td style="width:429px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Microscope Slides</td>
			<td>Genesee Scientific</td>
			<td>29-101</td>
			<td style="width:429px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Optimouse rack with cages</td>
			<td>AnimalCare systems</td>
			<td>C89100</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Water Bottle Basket&#160;</td>
			<td>AnimalCare systems</td>
			<td>C61011</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Filtered Cage Tops</td>
			<td>AnimalCare systems</td>
			<td>C78210</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Optimice Standard Feeder</td>
			<td>AnimalCare systems</td>
			<td>C40100SG</td>
			<td style="width:429px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Cage Card Holder</td>
			<td>AnimalCare systems</td>
			<td>C43251</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Cage Cards</td>
			<td>AnimalCare systems</td>
			<td>M52010</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Bottle Assambley</td>
			<td>AnimalCare systems</td>
			<td>C79122P</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Bed R&#39;Nest Nesting</td>
			<td style="width:167px;">The Andersons</td>
			<td style="width:280px;">BRN4WSR</td>
			<td style="width:429px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">1/8&#34; Corn Cob bedding&#160;</td>
			<td style="width:167px;">The Andersons</td>
			<td style="width:280px;">8B</td>
			<td style="width:429px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Standard mouse chow</td>
			<td>Teklad</td>
			<td style="width:280px;">7904 (7004)</td>
			<td style="width:429px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Scale</td>
			<td>VWR</td>
			<td style="width:280px;">10205-004</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Polypropylene Beaker</td>
			<td>Fisher</td>
			<td>14-955-111F</td>
			<td></td>
		</tr>
	</tbody>
</table>

determination of reproductive competence by confirming pubertal onset and performing a fertility assay in mice and rats

Assessment of reproductive competence is critical for understanding the impact of a treatment or genetic manipulation on the reproductive axis, also termed the hypothalamic-pituitary-gonadal axis. The reproductive axis is a key integrator of environmental and internal input adapting fertility to favorable conditions for reproduction. Prior to embarking upon a fertility study in mice and rats, sexual maturity is evaluated to exclude the possibility that the observed reproductive phenotypes are caused by delayed or absent pubertal onset. This protocol describes a non-invasive approach to assess pubertal onset in males through the determination of preputial separation, and in females through vaginal opening and first estrus. After the confirmation of the completion of puberty and the achievement of sexual maturity, a fertility study can be initiated. The procedure describes the optimal breeding conditions for mice and rats, how to set up a fertility study, and what parameters to evaluate and determine if the treatment or gene deletion has an impact on fertility.

The presented results are from two different transgenic mouse models where the transcription factor Ventral anterior homeobox 1 (Vax1) has been deleted in the whole body on one allele, here referred to as heterozygote mice (HET)13, or Vax1 has been conditionally deleted within GnRH neurons22, here termed conditional KO (cKO). Prior to setting up the fertility study, it is important to confirm pubertal onset in all the mice....

Watch this Scientific Journal Video about Determination of Reproductive Competence by Confirming Pubertal Onset and Performing a Fertility Assay in Mice and Rats at JoVE.com

Determination of Reproductive Competence by Confirming Pubertal Onset and Performing a Fertility Assay in Mice and Rats

Many treatments and genetic mutations impact the timing of sexual maturity and fertility. This protocol describes a non-invasive method to evaluate pubertal onset in mice and rats prior to setting up a fertility study in sexually mature animals.

Assessment of reproductive competence is critical for understanding the impact of a treatment or genetic manipulation on the reproductive axis, also ...

This method can help answer key questions in the reproductive field, such as how to determine pubertal onset in mice and rats through preputial separation, vaginal opening, and establishing first estrus prior to performing a fertility study. The main advantage of this technique is that by establishing pubertal onset using the demonstrated non-invasive approach, it is possible to follow and conduct a fertility study in animals that are sexually mature. The implications of this technique extend towards diagnosis of deficits in reproduction, as caused by genetic mutations or treatments susceptible to impact reproductive competence, such as endocrine disrupting chemicals.Though this method can provide insight into reproductive competence in mice, it can also be applied to other model systems, such as rats and hamsters. Visual demonstration of this method is critical, as it requires good and delicate animal handling skills. It is particularly important to hold the mice correctly for the following visual identification of preputial separation and vaginal opening.To prepare the work area, place a pad on the table. Then place a clean mouse cage top with the grid facing up, on the pad. Next, set up a scale in the work area.Place a clean 500 to 1000 milliliter beaker on the scale and tare it. Then place a sheet next to the work area to record the data. After this, place the mouse cage in the work area.Open the cage and remove the lid, water bottle, and food holder to the table. While holding the mouse by the tail, place it on the clean mouse cage top. While maintaining hold of the tail, allow the mouse to explore the grid of the cage top.Gently pull the tail backward and approach the other hand to the skin close to the neck and ears. Grasp the loose skin between the shoulders and neck with the thumb and forefinger. Then grab the skin on the back with the remaining fingers and hold the mouse by gently pressing it against the hand.With the free hand, gently push the skin around the penis back. At preputial separation, the preputial skin slides backwards, exposing the glans penis. After examination, record the presence or absence of preputial separation.After this, weigh the mouse and record its weight. Then reintroduce the mouse to the housing cage by holding the beaker over the cage floor and allowing the mouse to exit on its own. Place a cotton ball, a large beaker, and a bottle of sterile water in the work area.Pour sterile water into the beaker. Then, humidify the cotton ball with the sterile water in the beaker, and place it next to the clean cage top. Place the mouse cage in the work area.Then transfer the mouse to the cage top by holding it by the base of its tail. Gently pull on the mouse's tail to encourage a forward movement by the mouse. After this, lift the tail while supporting the hips with three fingers.Gently clean the vulva with the humidified cotton ball, then examine the vulva to determine if the vagina is completely open. Record if vaginal opening has occurred. Then weigh the mouse and return it to the home cage.Prepare a clean mouse cage with lid, water, food, bedding, and nesting material for each breeding pair of mice. Then fill out the cage cards with the requisite information. Place the prepared bedding cage on a clean table, then slowly introduce a gloved hand into the mouse cage and leave it in the cage for a short period of time.Slowly approach the mice with the gloved hand. When the mice run over the hand, grab the tail of one mouse between the thumb and index finger, then lift the mouse by the tail. The mouse can be held by the tail without support of the body for up to two to three seconds.Place the mouse in the prepared breeding cage, then close the cage after ensuring that the male and female mice have food, water, and nesting material. Using the presented method, two different transgenic mouse models were studied. Vaginal opening and preputial separation occurred at the same age and weight in both the heterozygous mice and the control mice.Conversely, vaginal opening and preputial separation were significantly delayed in conditional knockout females and males, and associated with increased body weight. At 4.5 weeks of age, female controls and the conditional knockout mice had comparable body weight, while the conditional knockout males were slightly heavier than the controls. This indicates that the delay in vaginal opening and preputial separation of the conditional knockout mice was not associated with a delay in weight gain.After completion of pubertal onset, the fertility study was started at age 10 to 16 weeks. Both male and female heterozygote mice were sub-fertile. Heterozygote females generated fewer litters in the three month study, and both male and female heterozygote mice generated smaller litters.To illustrate the importance of confirming pubertal onset prior to setting up a fertility assay, reproductive competence was evaluated in conditional knockout mice. During the 70 day fertility study, neither conditional knockout males nor females generated any litters. While attempting this procedure, it is important to remember to handle the mice with care and remain calm while approaching and handling the animals.Following this procedure, other methods like measuring circulating sex hormone levels can be performed to answer additional questions, like whether infertility or other subfertility was caused by abnormal testosterone, estrogen, follicle stimulating hormone, and luteinizing hormone levels. Don't forget that working with mice can be hazardous due to the risk of biting and development of allergies. To reduce exposure, precautions such as wearing gloves and a lab coat, and careful handling of the mice, should always be taken while performing this procedure.

determination-reproductive-competence-confirming-pubertal-onset

Research

JoVE Journal

Biology

14.9K ビュー.  Michigan State University. この方法は、前接分離、膣開き、および不妊治療研究を行う前に最初の発情を確立することによって、マウスおよびラットの思春期発症を決定する方法など、生殖分野の重要な質問に答えるのに役立ちます。この技術の主な利点は、実証された非侵襲的アプローチを用いて思春期発症を確立することによって、性的に成熟した動物に従って不妊治療研究を行うことは可?...

ビデオ: Determination of Reproductive Competence by Confirming Pubertal Onset and Performing a Fertility Assay in Mice and Rats

Manual Restraint and Common Compound Administration Routes in Mice and Rats

Being able to safely and effectively restrain mice and rats is an important part of conducting research. Working confidently and humanely with mice and rats requires a basic competency in handling and restraint methods. This article will present the basic principles required to safely handle animals. One-handed, two-handed, and restraint with specially designed restraint objects will be illustrated. Often, another part of the research or testing use of animals is the effective administration of compounds to mice and rats. Although there are a large number of possible administration routes (limited only by the size and organs of the animal), most are not used regularly in research. This video will illustrate several of the more common routes, including intravenous, intramuscular, subcutaneous, and oral gavage. The goal of this article is to expose a viewer unfamiliar with these techniques to basic restraint and substance administration routes. This video does not replace required hands-on training at your facility, but is meant to augment and supplement that training.

Working safely and humanely with research rodents requires a core competency in handling and restraint methods. This article will present the basic principles required to safely handle and effectively administer compounds to mice and rats.

Being able to safely and effectively restrain mice and rats is an important part of conducting research.  Working confidently and humanely with mice and ...

Diagnostic Necropsy and Selected Tissue and Sample Collection in Rats and Mice

There are multiple sample types that may be collected from a euthanized animal in order to help diagnose or discover infectious agents in an animal colony. Proper collection of tissues for further histological processing can impact the quality of testing results. This article describes the conduct of a basic gross examination including identification of heart, liver, lungs, kidneys, and spleen, as well as how to collect those organs. Additionally four of the more difficult tissue/sample collection techniques are demonstrated. Lung collection and perfusion can be particularly challenging as the tissue needs to be properly inflated with a fixative in order for inside of the tissue to fix properly and to enable thorough histologic evaluation. This article demonstrates the step by step technique to remove the lung and inflate it with fixative in order to achieve optimal fixation of the tissue within 24 hours. Brain collection can be similarly challenging as the tissue is soft and easily damaged. This article demonstrates the step by step technique to expose and remove the brain from the skull with minimal damage to the tissue. The mesenteric lymph node is a good sample type in which to detect many common infectious agents as enteric viruses persist longer in the lymph node than they are shed in feces. This article demonstrates the step by step procedure for locating and aseptically removing the mesenteric lymph node. Finally, identification of infectious agents of the respiratory tract may be performed by bacterial culture or PCR testing of nasal and/or bronchial fluid aspirates taken at necropsy. This procedure describes obtaining and preparing the respiratory aspirate sample for bacterial culture and PCR testing.

This article describes the procedures for conducting a basic postmortem examination of a mouse or rat, and the collection of basic organs, as well as more challenging sample types from for histological, microbiological, and PCR evaluation.

There are multiple sample types that may be collected from a euthanized animal in order to help diagnose or discover infectious agents in an animal ...

Ovariectomy and 17&beta;-estradiol Replacement in Rats and Mice: A Visual Demonstration

Estrogens are a family of female sexual hormones with an exceptionally wide spectrum of effects. When rats and mice are used in estrogen research they are commonly ovariectomized in order to ablate the rapidly cycling hormone production, replacing the 17&beta;-estradiol exogenously. There is, however, lack of consensus regarding how the hormone should be administered to obtain physiological serum concentrations. This is crucial since the 17&beta;-estradiol level/administration method profoundly influences the experimental results1-3. We have in a series of studies characterized the different modes of 17&beta;-estradiol administration, finding that subcutaneous silastic capsules and per-oral nut-cream Nutella are superior to commercially available slow-release pellets (produced by the company Innovative Research of America) and daily injections in terms of producing physiological serum concentrations of 17&beta;-estradiol4-6. Amongst the advantages of the nut-cream method, that previously has been used for buprenorphine administration7, is that when used for estrogen administration it resembles peroral hormone replacement therapy and is non-invasive. The subcutaneous silastic capsules are convenient and produce the most stable serum concentrations. This video article contains step-by-step demonstrations of ovariectomy and 17&beta;-estradiol hormone replacement by silastic capsules and peroral Nutella in rats and mice, followed by a discussion of important aspects of the administration procedures.

Ovariectomy and 17&amp;#946;-estradiol Replacement in Rats and Mice: A Visual Demonstration

Ovariectomy and subsequent replacement of 17&beta;-estradiol by means of silastic capsules and peroral Nutella are demonstrated in rats and mice.

Estrogens are a family of female sexual hormones with an exceptionally wide spectrum of effects. When rats and mice are used in estrogen research they are ...

A Rapid and Specific Microplate Assay for the Determination of Intra- and Extracellular Ascorbate in Cultured Cells

Vitamin C (ascorbate) plays numerous important roles in cellular metabolism, many of which have only come to light in recent years. For instance, within the brain, ascorbate acts in a neuroprotective and neuromodulatory manner that involves ascorbate cycling between neurons and vicinal astrocytes - a relationship that appears to be crucial for brain ascorbate homeostasis. Additionally, emerging evidence strongly suggests that ascorbate has a greatly expanded role in regulating cellular and systemic iron metabolism than is classically recognized. The increasing recognition of the integral role of ascorbate in normal and deregulated cellular and organismal physiology demands a range of medium-throughput and high-sensitivity analytic techniques that can be executed without the need for highly expensive specialist equipment. Here we provide explicit instructions for a medium-throughput, specific and relatively inexpensive microplate assay for the determination of both intra- and extracellular ascorbate in cell culture.

Ascorbate plays numerous important roles in cellular metabolism, many of which have only come to light in recent years. Here we describe a medium-throughput, specific and inexpensive microplate assay for the determination of both intra- and extracellular ascorbate in cell culture.

Vitamin C (ascorbate) plays numerous important roles in cellular metabolism, many of which have only come to light in recent years. For instance, within ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular physiology. Dysfunctional eNOS such as uncoupling of eNOS leads to decrease in NO bioavailability and increase in superoxide anion (O2.&#8722;) production, and in turn promotes cardiovascular diseases. Therefore, appropriate measurement of NO and O2.&#8722; levels in the endothelial cells are pivotal for research on cardiovascular diseases and complications. Because of the extremely labile nature of NO and O2.&#8722;, it is difficult to measure NO and O2.&#8722; directly in a blood vessel. Numerous methods have been developed to measure NO and O2.&#8722; production. It is, however, either insensitive, or non-specific, or technically demanding and requires special equipment. Here we describe an adaption of the fluorescence dye method for en face simultaneous detection and visualization of intracellular NO and O2.&#8722; using the cell permeable diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE), respectively, in intact aortas of an obesity mouse model induced by high-fat-diet feeding. We could demonstrate decreased intracellular NO and enhanced O2.&#8722; levels in the freshly isolated intact aortas of obesity mouse as compared to the control lean mouse. We demonstrate that this method is an easy technique for direct detection and visualization of NO and O2.&#8722; in the intact blood vessels and can be widely applied for investigation of endothelial (dys)function under (physio)pathological conditions.

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

In this article we describe an adapted relatively easy method using the fluorescence dye diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE) for en face simultaneous detection and visualization of intracellular nitric oxide (NO) and superoxide anion (O2.&#8722;) respectively, in freshly isolated intact aortas of an obesity mouse model.

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular ...

The Mouse Isolated Perfused Kidney Technique

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a prerequisite for studying the physiology of the isolated organ and for many innovative applications that may be possible in the future, including perfusion decellularization for kidney bioengineering or the administration of anti-rejection or genome-editing drugs in high doses to prime the kidney for transplantation. During the time of the perfusion, the kidney can be manipulated, renal function can be assessed, and various pharmaceuticals administered. After the procedure, the kidney can be transplanted or processed for molecular biology, biochemical analysis, or microscopy.
This paper describes the perfusate and the surgical technique needed for the ex vivo perfusion of mouse kidneys. Details of the perfusion apparatus are given and data are presented showing the viability of the kidney&#39;s preparation: renal blood flow, vascular resistance, and urine data as functional, transmission electron micrographs of different nephron segments as morphological readouts, and western blots of transport proteins of different nephron segments as molecular readout.

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. The buffers and surgical technique are described in detail.

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a ...

Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.

CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) is responsible for the oxidation of cytidine monophosphate-N-acetylneuraminic acids in mammals. However, humans cannot oxidize cytidine monophosphate-N-acetylneuraminic acid to cytidine monophosphate-N-glycolylneuraminic acid due to a primary exon deletion of the CMAH gene. To understand the effects and implications of the lack of CMAH activity in more detail, a Cmah knock-out model in mice is of keen interest in basic and applied research. The analysis method to determine the phenotype of this mouse model is herein described in detail, and is based on the detection of both N-acetylneuraminic acid and N-glycolylenuraminic acid in the liver and milk of wild-type and Cmah knock-out mice. Endogenous sialic acids are released and derivatized with o-phenylenediamine to generate fluorogenic derivatives, which can be subsequently analyzed by HPLC. The presented protocol can be also applied for the analysis of milk and tissue samples from various other origins, and may be of use to investigate the nutritional and health effects of N-glycolylneuraminic acid.

Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.

We describe a HPLC-based method for the determination of N-acetylneuraminic acid and N-glycolylenuraminic acid in mouse liver and milk.

CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) is responsible for the oxidation of cytidine monophosphate-N-acetylneuraminic acids in ...

Forced Salivation As a Method to Analyze Vector Competence of Mosquitoes

Vector competence is defined as the potential of a mosquito species to transmit a mosquito-borne virus (mobovirus) to a vertebrate host. Viable virus particles are transmitted during a blood meal via the saliva of an infected mosquito. Forced salivation assays allow determining the vector potential on the basis of single mosquitoes, avoiding the use of animal experiments. The method is suitable to analyze a large number of mosquitoes in one experiment within a short period of time. Forced salivation assays were used to analyze 856 individual mosquitoes trapped in Germany, including two different Culex pipiens pipiens biotypes, Culex torrentium as well as Aedes albopictus, which were experimentally infected with Zika virus (ZIKV) and incubated at 18 &#176;C or 27 &#176;C for two and three weeks. The results indicated the lack of vector competence of the different Culex taxa for ZIKV. In contrast, Aedes albopictus was susceptible to ZIKV, but only at 27 &#176;C, with transmission rates similar to an Aedes aegypti laboratory colony tested in parallel.

For efficient control of mosquito borne virus transmission, the knowledge of the vector potential of respective mosquitoes is of particular interest. We describe forced salivation as a method to analyze vector competence of Aedes albopictus and three different Culex taxa for the transmission of Zika virus.

Vector competence is defined as the potential of a mosquito species to transmit a mosquito-borne virus (mobovirus) to a vertebrate host. Viable virus ...

Reproductive Techniques for Ovarian Monitoring and Control in Amphibians

Ovarian control and monitoring in amphibians require a multi-faceted approach. There are several applications that can successfully induce reproductive behaviors and the acquisition of gametes and embryos for physiological or molecular research. Amphibians contribute to one-quarter to one-third of vertebrate research, and of interest in this context is their contribution to the scientific community&#39;s knowledge of reproductive processes and embryological development. However, most of this knowledge is derived from a small number of species. In recent times, the decimation of amphibians across the globe has required increasing intervention by conservationists. The captive recovery and assurance colonies that continue to emerge in response to the extinction risk make existing research and clinical applications invaluable to the survival and reproduction of amphibians held under human care. The success of any captive population is founded on its health and reproduction and the ability to develop viable offspring that carry forward the most diverse genetic representation of their species. For researchers and veterinarians, the ability to monitor and control ovarian development and health is, therefore, imperative. The focus of this article is to highlight the different assisted reproductive techniques that can be used to monitor and, where appropriate or necessary, control ovarian function in amphibians. Ideally, any reproductive and health issues should be reduced through proper captive husbandry, but, as with any animal, issues of health and reproductive pathologies are inevitable. Non-invasive techniques include behavioral assessments, visual inspection and palpation and morphometric measurements for the calculation of body condition indices and ultrasound. Invasive techniques include hormonal injections, blood sampling, and surgery. Ovarian control can be exercised in a number of ways depending on the application required and species of interest.

The study of amphibian biology provides valuable information on the reproductive, physiological, embryological and developmental processes that drive organisms of many taxonomic groups. Here, we present a comprehensive guide on different methodologies that can be used to study ovarian control and monitoring in amphibians.

Ovarian control and monitoring in amphibians require a multi-faceted approach. There are several applications that can successfully induce reproductive ...