Blood Sampling and Hormone Measurement for Determining the Stage in the Ovarian Cycle in Marmosets

Podsumowanie

This protocol describes blood and urine sampling for measuring progesterone/estradiol and chorionic gonadotropin levels to determine the ovarian cycle stage. The hormone levels are used to predict and determine the timing of ovulation and hormones are injected to regulate the ovarian cycle and oocyte growth.

Streszczenie

Common marmosets are small New World monkeys. Since many of their biological mechanisms are similar to those of humans, marmosets are potentially useful for medical and human biology research across a range of fields, such as neuroscience, regenerative medicine, and development. However, there is a lack of literature describing methods for many basic experiments and procedures. Here, detailed methods for determining the levels of sex hormones (progesterone, estradiol, and chorionic gonadotropin) in marmosets are described. The measurement of these hormones enables the prediction of the stage in the ovarian cycle, which is typically 26-30 days in marmosets; accurate determination is essential for the harvesting of oocytes/zygotes at the correct time point and for the preparation of host females for the generation of genetically modified marmosets.

Additionally, the measurement of sex hormone levels is useful for endocrinology, ethology, early development, and reproductive biology studies. This protocol provides a detailed description of the methods for blood sampling from the femoral vein, separation of plasma for hormone measurement, measuring chorionic gonadotropin levels using urine and plasma, resetting the ovarian cycle using injections of a prostaglandin F2α analog to shorten and synchronize the cycle, and promoting follicular growth and ovulation by injecting follicle-stimulating hormone and chorionic gonadotropin. Using these protocols, the stages in the ovarian cycle can be determined for the timely collection of oocytes/zygotes.

Wprowadzenie

The common marmoset (Callithrix jacchus) is a small New World monkey with many characteristics similar to those of humans, and the duration of its ovarian cycle is 26-30 days^1,2. Studies on the early development and the generation of genetically modified marmosets require the harvesting of oocytes and zygotes at specific stages in the ovarian cycle. Thus, accurate determination of the stage is crucial and can be estimated by measuring the blood levels of the hormones progesterone (P4) and estradiol (E2)^2,3. These hormones promote endometrial growth, which is necessary for implantation. P4 is produced from the corpus luteum, which forms in ovaries immediately following ovulation. E2 is secreted by the ovarian follicles in response to follicle-stimulating hormone (FSH) from the hypothalamus-pituitary complex in the brain. E2 levels increase as the follicle matures, peaking before ovulation³. High E2 levels cause the pulsed release of luteinizing hormone (LH) via the hypothalamus-pituitary complex in humans; this LH surge induces ovulation. However, in marmosets, the LH gene underwent degeneration during evolution, and ovulation is instead induced by the release of chorionic gonadotropin (CG), which has a similar structure to LH's, from the pituitary gland^4,5.

The ovarian cycle can be controlled by hormone injections. FSH injections, in humans, act on ovarian FSH receptors and are used to promote estrogen synthesis and follicle growth⁶. The injection of human CG (hCG) as a substitute for LH at the end of the follicular phase is used to stimulate ovulation in humans⁷. CG injections are also used to treat human infertility because CG stimulates the corpus luteum in early pregnancy, resulting in increased P4 production. Prostaglandin F2α (PGF2α) injections reset the ovarian cycle⁸. In domestic cattle, PGF2α injection is used to shorten the luteal phase and synchronize the estrus cycle for reproductive management.

Although marmosets and humans have similar biological mechanisms, making them ideal model animals, there is a lack of literature describing basic methods for many often-used techniques. Blood sampling is one of the most often used techniques^9,10,11,12. However, beginners sometimes have trouble finding the vein. Hence, this study conducted anatomical analyses of the femoral vein region. Based on anatomical observations, this protocol introduces the proximal region of the femoral triangle as an easy site for venipuncture.

Protokół

All methods involving marmosets utilized high ethical and welfare standards and were approved by the Institutional Animal Care and Use Committee at the National Center for Child Health and Development. Animals used here were single-housed or paired-housed (one female and one male) with 12 h of light per day.

1. Blood sampling of the femoral vein

1. Prepare a 1 mL syringe (the short type is easy to use) with a 25 G needle attached blade-side up. To avoid blood clogging, heparinize the syringe by drawing 200 µL of undiluted heparin sodium solution into the syringe. Coat the inside of the syringe evenly by pulling it up and down several times; then, expel the heparin solution from the syringe.
   NOTE: Since changing to a new syringe is often necessary, prepare a few additional heparinized syringes.
2. Prepare absorbent cotton and alcohol swabs. Switch on an adjustable lamp to illuminate the area where the marmoset will be placed for blood sampling.
3. Prepare the restraint device (420 x 85 x 85 mm, Figure 1A), which is commercially available (see Table of Materials for details). To place a marmoset into the device, open the retention part with the sponge belt, which secures marmosets. Insert the marmoset into the restraint device facing up.
   NOTE: Marmosets are usually calm in restrain devices which may negate the need for restraint acclimation or training.
4. Capture the marmoset; insert the marmoset into the cylindrical part; and secure it by pressing down the sponge belt. Place the leg from which blood is to be collected on top of the other (Figure 1A). Hold the legs using the non-dominant hand; place the middle and ring fingers inside each leg to fasten them and the other fingers outside each leg to fix them.
   NOTE: Bite gloves are recommended when capturing marmosets. If a restraining device is not present, perform blood collection under anesthesia or with another person restraining the marmoset.
5. Check if the femoral vein is visible near the base of the thigh. If not, palpate to find the pulsating artery and use it as a landmark to check that the vein runs inside it (Figure 1B-D).
   NOTE: Desk illumination and hair shaving are recommended to improve visibility. Visibility can also be improved by rubbing with alcohol swabs. Lymph nodes in the triangle are often located close to the vein and show a dark blue color as a vein. How to distinguish them is described in the Representative Results section.
6. Disinfect the puncture site using an alcohol swab. Insert the needle blade-side up at an angle of 15°-20°. To prevent the needle from slipping out of the blood vessel during blood collection, stabilize the hand holding the syringe, for example, by resting it on the other hand.
7. Gently pull back the plunger to apply negative pressure (Figure 1E). Push the needle tip forward. Once blood enters the syringe, maintain the position of the needle tip until the required volume (500-700 µL) has been collected.
   1. When blood does not enter the syringe, change the site of puncture. If the blood comes out when pulling out the needle, stop bleeding by applying pressure to the puncture site for 3 min. After stopping the bleeding, restart the venipuncture.
   2. If the blood being drawn into the syringe stops flowing during the drawing process, slowly push the needle tip forward and then pull it back to find the blood vessel. This may restore the flow of blood into the syringe. If not, pull out the needle and perform venipuncture using a new syringe.
      NOTE: Use the thigh on the other leg when it is difficult to collect blood from the same side.
8. Carefully pull out the needle while pressing lightly on the puncture site using the little finger. Then, using an absorbent cotton swab, immediately apply pressure to the puncture site for 3 min to stop the bleeding. Invert the syringe to mix blood and heparin.
   NOTE: Apply pressure for a longer time (5 min) while cooling when arterial blood is drawn, and carefully confirm the ceasure of bleeding to prevent the formation of hematoma, which can sometimes result in a fatal outcome.
9. After confirming that bleeding has ceased, remove the sponge belt, hold the animal's waist using one hand, and rotate the animal so that one can hold the animal's underarm using another hand from the backside.
10. Return the marmoset to its cage. To reduce stress and facilitate repeated blood sampling, provide the marmoset with its favorite food (e.g., biscuits, marshmallows, or sponge cake). Check the occurrence of hematoma occasionally.
    NOTE: When hematoma is found at the early stage, apply a pressure bandage to prevent the progression of hematoma. When it is found after a long time, surgical removal of hematoma with ligation of the femoral artery and blood transfusion may be needed^9,13.
11. Detach the needle from the syringe to prevent hemolysis. Then, slowly expel the collected blood along the inside wall of a 1.5 mL microtube.
    NOTE: The collected blood can be stored at 4 ^oC for up to 24 h before plasma separation for measuring P4/E2 levels.

2. Separation of plasma and determination of hormonal levels

1. Centrifuge the blood in a 1.5 mL tube at 1,100 × g for 5 min at 4 ^oC.
2. Transfer the separated plasma (supernatant) from the 1.5 mL tube to a new tube/cup, carefully avoiding the inclusion of blood cells (sediment).
3. Measure the P4 and E2 levels using an ELISA kit or an automatic analyzer. If the amount of plasma is not enough for the automatic analyzer, use a sample dilution solution.
   NOTE: For the measurement using an automatic analyzer, >175 µL of plasma is required for determining only a P4 level, and >250 µL of plasma is required for determining both P4 and E2.

3. Urine CG measurement to detect ovulation and pregnancy

NOTE: CG levels in marmosets can be measured using an immunochromatographic kit test for both ovulation and pregnancy. In the case of ovulation, a positive result can be obtained 0-2 days before ovulation. In the case of pregnancy, a positive result is detected from days 15-20 to approximately day 100 of pregnancy. The test requires a small amount (90 µL) of urine (one drop of urine is ~30 µL).

1. Tray method: If more than two animals are in the same cage, move the target marmoset (or the other animals) to another cage the day before. Place a clean tray at the bottom of the cage the night before or before lighting. Urine is not drained from the bladder during the night. Therefore, urine is usually released from the bladder shortly after lighting.
   NOTE: Entering the room in the morning before lighting may disrupt the cohort's sleep cycle. Urine can usually be collected within approximately 30 min after lighting.
2. Squeezing method: Prepare the washed trays for urine collection. After locating the marmoset's bladder, carefully squeeze it from the front and both sides using the entire length of the fingers (Figure 1F). Collect urine just before lighting up the room in the morning.
   NOTE: If urine collection is unsuccessful due to the absence of urine in the bladder, wait a while and try again. Be careful not to use excessive force because this will injure the animal.
3. Immediately after collection, place the urine sample in the well of the immunochromatographic test kit. Read the result after 10 min according to the manufacturer's instructions.

4. Control and determination of the ovarian cycle stage for the collection of oocytes, zygotes, and embryos

1. Germinal vesicle (GV) oocyte collection from the ovaries
   1. Administer an intramuscular injection of 3 µL of 0.263 mg/mL cloprostenol (a synthetic PGF2α analog) diluted in 150 µL of saline¹⁴ (Figure 2) to reset the ovarian cycle at the end of the luteal phase (i.e., ≥10 days after the initiation of the luteal phase).
      NOTE: Making the diluted solution in a larger volume may be convenient as the diluted cloprostenol stays stable for, at least, several weeks at +4 °C.
   2. On the next day (day 1), confirm the initiation of the follicular phase by checking that the P4 level has dropped.
      NOTE: Cloprostenol injection has been reported to dramatically decrease P4 levels (usually <10 ng/mL) within 24 h³.
   3. From day 1, inject FSH (25 IU, intramuscular) once every 2 days for a total of 5x (days 1, 3, 5, 7, and 9). On day 10, inject hCG (75 IU, intramuscular) in the afternoon.
   4. Collect GVs from the ovaries on day 11 by follicular aspiration under anesthesia according to the literature^15,16.
      NOTE: Sometimes, ovulation occurs earlier than expected. Thus, it is recommended to check CG levels from day 8. If the CG test is positive, perform GV collection on this day.
2. Collection of metaphase II (MII) oocytes, zygotes, and early embryos
   1. Reset the ovarian cycle using cloprostenol as described in step 4.1.1.
   2. On the next day (day 1), confirm the initiation of the follicular phase by checking that the P4 level has dropped.
   3. For the collection of zygotes and embryos, house female marmosets together with male marmosets for mating from day 6.
   4. From day 7, check the blood P4/E2 levels and urine CG levels of the females. Detection of CG is an indication of ovulation within a few days (usually the next day). On the day that P4 levels increase and E2 levels decrease compared with the previous day, collect MII oocytes or embryonic day 0 (E0) zygotes from the oviducts^17,18.
   5. For embryo collection, as described in the literature, perform flushing either from the oviducts (E1-E3, 1-8 cells)^17,18 or the uterus (E5-E10, 8 cells-blastocyst)^19,20,21,22 at the appropriate time point, depending on the targeted stage.

Wyniki

Details related to the animals used in this study are listed in Table 1.

Anatomical analyses of the femoral vein
Anatomical analyses of the femoral vein were performed using a 2-year-old male common marmoset (I 7713M) undergoing euthanasia. The femoral veins and arteries are located in the femoral triangle. The femoral triangle is formed at the boundaries between the abdominal wall and thigh muscles (Figure 1B-

Dyskusje

Locating the vein is the most critical step in blood collection. Based on anatomical observations, this protocol introduces the proximal area in the femoral triangle as an easy site for blood collection in marmosets. Using this area, blood sampling from a large vein can be easily performed. However, even using this protocol, injury to an artery sometimes occurs. When injuring an artery, complete stopping of bleeding by applying pressure for >5 min is suggested to prevent hematoma. In addition, while applying pressure...

Materiały

Name	Company	Catalog Number	Comments
AIA-360	Tosoh Corporation	0019945	Hormone measurement (P4/E2)
AIA-PACK DILUENT CONCENTRATE	Tosoh Corporation	0020956	Hormone measurement (P4/E2)
AIA-PACK SUBSTRATE SET II	Tosoh Corporation	0020968	Hormone measurement (P4/E2)
AIA-PACK WASH CONCENTRATE	Tosoh Corporation	0020955	Hormone measurement (P4/E2)
CMS-1	CLEA Japan		Marmoset food
Estrumate	MSD Animal Health		PGF2alpha analog (cloprostenol)
Gonal-f Subcutaneous Injection 150	Merck Biopharma Co., Ltd.		FSH
Gonatropin for intramuscular injection 1000	ASKA Pharmaceutical Co., Ltd.	872413	hCG
Heparin sodium injection solution 5,000 units/5 mL	Mochida Pharmaceutical Co., Ltd.	224122458	Blood collection
Immunochromatographic Test Kit for Detection of Common Marmoset Chorionic Gonadotropin (Dual Checker)	CLEA Japan, Inc.		Determining CG level
Low-profile double-arm microscope illumination LPF-SD	SHIOKAZE GIKEN		Desk lamp for blood collection
Marmoset blood collection restraint device	JIC Japan	JM-1006	Blood collection http://www.jic-japan.jp/prd/marmoset/prd016.html email: vi@jic-japan.jp
Metacam 0.05%	Boehringer Ingelheim Animal Health Japan Co., Ltd.		Hematoma treatment
Sample Cup, 3 mL, PS, for Tosoh 360 and AIA-600 II, 1000/Bag	Globe Scientific	110913	Hormone measurement (P4/E2)
ST AIA-PACK iE2	Tosoh Corporation	0025224	Hormone measurement (P4/E2)
ST AIA-PACK iE2 CALIBRATOR SET	Tosoh Corporation	0025324	Hormone measurement (P4/E2)
ST AIA-PACK iE2 SAMPLE DILUTING SOLUTION	Tosoh Corporation	0025524	Hormone measurement (P4/E2)
ST AIA-PACK PROGIII	Tosoh Corporation	0025240	Hormone measurement (P4/E2)
ST AIA-PACK PROGIII CALIBRATOR SET	Tosoh Corporation	0025340	Hormone measurement (P4/E2)
ST AIA-PACK PROGIII SAMPLE DILUTING SOLUTION	Tosoh Corporation	0025540	Hormone measurement (P4/E2)
Syringe with 25 G (0.50 x 25 mm) needle	TERUMO	SS-01T2525	Blood collection
Tensolvet 5.000 I.E. gel.	Dechra Pharmaceuticals	14033492	Hematoma treatment
TOSOH MULTI-CONTROL SET	Tosoh Corporation	0015965	Hormone measurement (P4/E2)