Name: determination of reproductive competence by confirming pubertal onset and performing a fertility assay in mice and rats
Uploaded: 2018-10-13T15:00:00
Description: 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

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
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	<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.
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		<li>Place a cl...

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	<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. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+hypothalamic+GPR54+signaling:+A+potential+mechanism+for+initiation+of+puberty+in+primates.">Increased hypothalamic GPR54 signaling: A potential mechanism for initiation of puberty in primates.</a> Proceedings of the National Academy of Sciences. , (2005).</li><li>Hoffmann, H. M., 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=A+small+population+of+hypothalamic+neurons+govern+fertility:+the+critical+role+of+VAX1+in+GnRH+neuron+development+and+fertility+maintenance.">A small population of hypothalamic neurons govern fertility: the critical role of VAX1 in GnRH neuron development and fertility maintenance.</a> Neuroscience communications. 2, 5-9 (2016).</li><li>Navarro, V. 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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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pre-+and+postnatal+nutritional+histories+influence+reproductive+maturation+and+ovarian+function+in+the+rat.">Pre- and postnatal nutritional histories influence reproductive maturation and ovarian function in the rat.</a> PLoS ONE. , (2009).</li><li>Manual, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breeding+Strategies+for+Maintaining+Colonies+of+Laboratory+Mice.">Breeding Strategies for Maintaining Colonies of Laboratory Mice.</a> Management. , (2007).</li><li>Kennedy, G. 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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.

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			<td height="40" style="height:40px;width:216px;">Sterile Cotton Balls</td>
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			<td height="40" style="height:40px;">Methylene blue&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>M9140</td>
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			<td height="40" style="height:40px;">Microscope Slides</td>
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			<td>AnimalCare systems</td>
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			<td height="40" style="height:40px;">Optimice Standard Feeder</td>
			<td>AnimalCare systems</td>
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			<td>AnimalCare systems</td>
			<td>C43251</td>
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			<td height="40" style="height:40px;">Cage Cards</td>
			<td>AnimalCare systems</td>
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			<td height="40" style="height:40px;">Bottle Assambley</td>
			<td>AnimalCare systems</td>
			<td>C79122P</td>
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			<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>
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		<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>
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			<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>
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			<td height="40" style="height:40px;">Scale</td>
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			<td height="40" style="height:40px;">Polypropylene Beaker</td>
			<td>Fisher</td>
			<td>14-955-111F</td>
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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 ...

Research

JoVE Journal

Biology

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 ...

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 ...

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

Ovariectomy and 17ÃƒÅ½Ã‚Â²-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 ...

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 ...

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

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) ...

In Vivo Microinjection and Electroporation of Mouse Testis

In Vivo Microinjection and Electroporation of Mouse Testis

This video and article contribution gives a comprehensive description of microinjection and electroporation of mouse testis in vivo. This particular ...

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

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 ...

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 ...

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

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 ...

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 ...