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

15.0K visualizações.  Michigan State University. Este método pode ajudar a responder a perguntas-chave no campo reprodutivo, como como determinar o aparecimento pubertal em camundongos e ratos através de separação pré-putial, abertura vaginal e estabelecer primeiros estrus antes de realizar um estudo de fertilidade. A principal vantagem dessa técnica é que, ao estabelecer o início pubertal utilizando a abordagem não invasiva demonstrada, é possível acompanhar e realizar um estudo de fertilidade em animais sexualmente maduros. As ...