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

Published: July 05, 2024

doi:

¹National Center for Child Health and Development

Summary

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.

Abstract

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.

Introduction

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 days¹^,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 gland⁴^,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 techniques⁹^,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.

Protocol

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 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 syr…

Representative Results

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-</stro…

Discussion

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…

Divulgations

The authors have nothing to disclose.

Acknowledgements

We would like to thank Chunshen Shen, Hiroko Akutsu, Fumiyo Sugiki, Yuuna Hashimoto, Hina Naritomi, Yuuki Sakamoto, and Mikiko Horigome for their support in establishing this protocol and in the day-to-day care of marmosets; Takayuki Mineshige for comments on the manuscript; Yukiko Abe and the members of Aiba lab for sharing zygote collection techniques; CIEA for sharing the information on marmosets housing and experiments that they have cultivated over 40 years. This research was supported by AMED, JST, and KAKENHI under the grant Nos. JP19gm6310010, JP20gm6310010, JP21gm6310010, and JP22gm6310010 (AMED), JPMJPR228B (JST), 20H05764, 20H03177, and 22K18356 (KAKENHI).

Materials

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)

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