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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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

  1. 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.
  2. Prepare the work area by placing a pad on the table.
    1. Place a cl.......

Representative Results

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........

Discussion

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.......

Acknowledgements

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 & Human Development of the National Institutes of Health under Award Number R00HD084759.

....

Materials

NameCompanyCatalog NumberComments
Sterile Cotton BallsFisher22456885
Surface protectorFisher1420637
Light meterVWR21800-014
Methylene blue Sigma-AldrichM9140
Microscope SlidesGenesee Scientific29-101
Optimouse rack with cagesAnimalCare systemsC89100
Water Bottle Basket AnimalCare systemsC61011
Filtered Cage TopsAnimalCare systemsC78210
Optimice Standard FeederAnimalCare systemsC40100SG
Cage Card HolderAnimalCare systemsC43251
Cage CardsAnimalCare systemsM52010
Bottle AssambleyAnimalCare systemsC79122P
Bed R'Nest NestingThe AndersonsBRN4WSR
1/8" Corn Cob bedding The Andersons8B
Standard mouse chowTeklad7904 (7004)
ScaleVWR10205-004
Polypropylene BeakerFisher14-955-111F

References

  1. Schneider, J. E. Energy balance and reproduction. Physiology and Behavior. 81 (2), 289-317 (2004).
  2. Walton, J. C., Weil, Z. M., Nelson, R. J. Influence of photoperiod on hormones, behavior, and immune function. Frontier....

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