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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 parents^1,2. 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 chemicals^6,7, increased/decreased body weight^1,8, changes in day length^2,9 and genetic mutations^{10,11,12,13,14,15}.

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 animal^16,17.

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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Protokół

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 clean mouse cage top with the grid facing upwards on the pad.
   2. Set up a scale in the work area. Place a clean 500 – 1000 mL beaker on the scale and tare.
   3. Place a sheet next to the work area to record body weight and preputial separation, vaginal opening or first estrus.
3. Identify the mice for the study. Perform daily inspections for pubertal onset until puberty is reached. Perform the inspection at the same time of day throughout the study. Pubertal onset can occur as early as postnatal day 11-12¹⁹, however, in most experimental setups, pubertal onset monitoring starts at ~22 days.
4. Place the mouse cage in the work area (Step 1.2). Open the cage and place the lid, water bottle and food holder on the table.
5. Determining preputial separation in male mice.
   1. While holding the mouse by the tail, place it on the clean mouse cage top from Step 1.2.1. While gently holding onto the tail, let the mouse explore the grid of the cage top for ~5 s.
   2. Gently pull the tail backward; this usually results in the animal moving forward while holding on to the grid with its forelegs. Approach the other hand to the skin close by the neck and ears.
   3. Grasp the loose skin between the shoulders and neck with the thumb and forefinger. It is important to get a good hold of the skin, as it will prevent the mouse from turning its head around to bite.
   4. Grab the skin on the back with the remaining fingers and hold it by pressing against the hand. Hold firmly without obstructing breathing, blood flow, or damaging bones, muscles or skin. Turn the hand to expose the belly of the mouse, while supporting the back of the mouse within the palm of the hand.
   5. With the free hand, gently push back the skin around the penis without forcing. At preputial separation, the preputial skin slides backward exposing the glans penis (Figure 2A, arrow showing preputial separation). Record the presence or absence of preputial separation.
   6. Weigh the mouse by gently placing it in the 500-1000 mL beaker from Step 1.2.2 and record the weight.
   7. Reintroduce the mouse in the housing cage by holding the beaker 0-2 cm over the cage floor and slowly tip over the beaker allowing the mouse to exit on its own. Clean the beaker with water and mild detergent and dry it off before returning it to the scale and taring the scale.
6. Determining vaginal opening in female mice.
   1. Place a cotton ball, beaker and a bottle of sterile water in the work area (Step 1.2). Pour the water into the beaker. Humidify the cotton ball with the sterile water and place it next to the cage top from Step 1.2.1.
   2. Place the mouse cage in the work area (Step 1.2) and transfer the mouse from its home cage to the cage top (Step 1.2.1) by holding it at the base of the tail. Gently pull back the tail to encourage a forward movement of the mouse.
   3. Lift the tail while supporting the hips with the free fingers. Allow the hindlimbs to maintain contact with the top of the cage. If the mouse moves too much, hold it in the hand as described in Steps 1.5.1-1.5.4.
   4. Gently clean the vulva with the water humidified cotton ball from Step 1.6.1. Use a clean humidified cotton ball for each mouse. To determine vaginal opening, examine the vulva and determine whether the vagina is completely open (Figure 2A, female vaginal opening).
   5. Record if vaginal opening has occurred. Weigh the mouse and return it to the home cage as described in Steps 1.5.6-1.5.7.
7. First estrus an indicator of first ovulation.
   1. Place a beaker with sterile water, a labeled glass slide and a 200 µL pipette with a clean tip in the work area (Step 1.2).
   2. Hold the female as described in Step 1.6.2-1.6.3. Place the tip of the pipette containing ~50 µL of sterile water at the vaginal opening.
   3. Gently flush the cells from the vaginal wall by introducing and reabsorbing ~50 µL of water 2-4 times. Smear the content from the pipette tip onto a labeled glass microscope slide.
   4. Carefully observe the cellular morphology on a brightfield microscope using a 10X or 20X objective or let the smear airdry for ~1 h and then counterstain with a 0.1% methylene blue solution (dissolved in ddH₂O) (Figure 2B). To counterstain, dip the dried slide into the 0.1% methylene blue solution for ~ 30 s. Let the slide air dry for 1 h.
   5. Observe the slides from Step 1.7.4 on a bright field microscope at 10X or 20X. Establish the stage of the estrous cycle (Figure 2B)²⁰ by observing the cell morphology which allows to distinguish the four distinct stages of the estrous cycle (Figure 2B).
      Note: Metestrus is characterized by a mixture of cornified squamous epithelial cells and leukocytes, diestrus by leukocytes, proestrus by a mixture of leukocytes and nucleated epithelial cells, and estrus by cornified epithelial cells (Figure 2B)²⁰.
   6. Record the body weight and return the mouse to its home cage as described in Steps 1.5.6-1.5.7. Vaginal smears are terminated on the day when the mouse has its first estrus (Figure 2B).

2. Desirable Breeding Room Conditions

1. Ensure that the breeding room has a temperature of ~20-24 °C, with 55 ± 10% humidity, and homogenous room light of an intensity of ~300-400 lux approximately 1 m above the floor²¹. This light intensity allows the desirable mid-cage light intensity of 25-80 lux. To test light intensity, use a light meter.
2. Use an automated system to control the lighting in the breeding room to ensure appropriate timing of lighting throughout the experiment. Optimal day-length conditions to assess fertility in rodents range from a 12 h day (12 h light and 12 h darkness, LD12:12), to “summer-like” conditions with 14 h of light and 10 h darkness (LD14:10)²¹.
3. Prepare breeding cages.
   1. Prepare a clean mouse cage with lid, water, food, bedding and nesting per breeding pair.
   2. Fill out the cage cards with all the required information, including sex, date of birth, mouse strain, and the date when the mating is set up.

3. Fertility Study

1. Identify the animals to be used for the fertility study. Ensure that the animals are sexually mature (Step 1). A typically used age range to set up a fertility study in mice is 10-16 weeks of age, an age where sexual maturation has been completed in most experimental conditions.
2. On a clean table, place the breeding cage prepared in Step 2.3.
3. Catch and hold the mouse.
   1. Slowly introduce a gloved hand into the mouse cage. Leave the hand in the cage for a short period of time (~5-30 s), allowing the mice to adapt to the smell of the glove and hand. Avoid hectic and jerky movements.
   2. Lower the cupped hand close to the bottom of the cage and slowly approach it to the mice. They will run away. When they run over the hand, trap the tail of one mouse between the thumb and index finger.
   3. Lift the mouse by the tail for 2-3 s. If the mouse needs to be held longer, hold onto the tail and place the mouse in the cupped hand maintaining the hold on the tail.
4. Transfer 1 male and thereafter 1 female into the breeding cage by holding them by the tail (Step 3.3). Ensure that the females are in the same estrous stage when setting up the fertility assay. Determine the estrous stage of the females by performing a vaginal smear as described in Step 1.7.
5. Close the cage, and ensure that the mice have food, water and nesting material. Attach the cage card holder with the cage card to the cage.
6. Place the breeding cage on the housing rack. Leave the breeding cage as undisturbed as possible for the full duration of the fertility study. Conduct the fertility study for 30 days. For each breeding pair, keep detailed records and note 1) the date the mating is set up, 2) the date litters are born and 3) the number of pups born, including dead and live pups. Perform litter checks daily throughout the study.
7. Prolong the fertility study for an additional 30-60 days if no or a weak impact on fertility is established in Step 3.6.

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Wyniki

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....

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Dyskusje

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...

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Materiały

Name	Company	Catalog Number	Comments
Sterile Cotton Balls	Fisher	22456885
Surface protector	Fisher	1420637
Light meter	VWR	21800-014
Methylene blue	Sigma-Aldrich	M9140
Microscope Slides	Genesee Scientific	29-101
Optimouse rack with cages	AnimalCare systems	C89100
Water Bottle Basket	AnimalCare systems	C61011
Filtered Cage Tops	AnimalCare systems	C78210
Optimice Standard Feeder	AnimalCare systems	C40100SG
Cage Card Holder	AnimalCare systems	C43251
Cage Cards	AnimalCare systems	M52010
Bottle Assambley	AnimalCare systems	C79122P
Bed R'Nest Nesting	The Andersons	BRN4WSR
1/8" Corn Cob bedding	The Andersons	8B
Standard mouse chow	Teklad	7904 (7004)
Scale	VWR	10205-004
Polypropylene Beaker	Fisher	14-955-111F