In vitro fertilization in mice
The in-vitro fertilization in mice is the in vitro fertilization in mice .
properties
While in the case of human in vitro fertilization the aim is to support the realization of a couple's desire to have children, in medical-biological research the purpose of mouse in vitro fertilization is to generate mouse offspring with a specific genotype and thereby to avoid the time-consuming crossing of the corresponding parental generations.
method
Induction of superovulation
To artificially increase the number of ovulating follicles in the sexually mature female mouse ( superovulation ), the serum gonadotropin from pregnant mares ( Pregnant Mare's Serum Gonadotropin , PMSG) is injected into the mouse. In addition, human chorionic gonadotropin (hCG) is injected. This peptide hormone induces ovulation , leads to the formation of the corpus luteum - and in the male mouse to the formation of the interstitial cells of the testicles . The Aschheim-Zondek reaction was one of the first pregnancy tests and used the release of chorionic gonadotropin to determine pregnancy.
The multinucleated syncytiotrophoblast synthesize chorionic gonadotropin which the in ovarian located luteum ( the corpus luteum ) to the release of progesterone stimulates. This sex hormone is responsible for the structure of the uterine mucosa and also prevents new ovulations by means of negative feedback to the pituitary gland of the ovaries.
Plate preparation
The in-vitro fertilization of mouse is usually on special plates. For this purpose, various drops of medium are pipetted onto them using a micropipette and then covered with liquid paraffin .
A drop of preincubation medium is used for the sperm plate. The same procedure is used to prepare the fertilization plate with a drop of the fertilization medium.
Four drops of Modified Human Tubal Fluid Medium - HEPES buffer (mHTF) are placed on a plate, enclosed in liquid paraffin and serve as a washing solution. mHTF increases the efficiency of the method and stabilizes the pH value for optimal, stress-free conditions during fertilization .
Collecting spermatozoa
To obtain the spermatozoa , the tail of the epididymis of a sexually mature male mouse is placed in a sperm plate and the vas deferens cut. The sperm are then released using a dissecting needle and then transferred to the preincubation medium on the sperm plate.
Collecting oocytes
The fallopian tube of a superovulating, sexually mature mouse is also transferred to the fertilization plate and the cumulus oocyte complexes (COCs), a complex of an oocyte surrounded by specialized granulosa cells , are isolated using a dissecting needle. In this case, too, the transfer into the fertilization medium follows.
insemination
For insemination, part of the sperm suspension is transferred into the fertilization medium containing COC. After a three-hour incubation phase, the oocytes are washed three times in the mHTF drops on the wash plate. The oocyte transfer to the fertilization plate must be carried out extremely carefully, without residues of the fertilization medium. In the last of the three washing steps, the pronuclei of the oocytes are examined in order to sort out parthenogenic oocytes that have only one pronucleus. In contrast, successfully fertilized oocytes have two pronuclei, one male and one female. On the following day, the two-cell stages are transferred to the last of the four drops of mHTF on the wash plate. In the following, their vitrification , their further cultivation or their transfer into a recipient female is possible.
The recipient female
The recipient female is a pseudo-pregnant female whose hormone production corresponds to that of a pregnant female. The hormone production is induced by the copulation of the female with a vasectomized and therefore sterile male.
Ethical background for the extraction of the rut synchronization hormone
The PMSG hormone is not only used for in-vitro fertilization in mice, but is also used in large quantities by the European meat industry in pig production. The animal welfare organization "Animal Welfare Foundation" published a video in 2015 that shows how pregnant mares are handled in South America when their blood is drawn. For the greatest possible profit, it is advantageous to fertilize the mares as often as possible in order to be able to take the blood serum that is valuable for the industry. The blood sample can be taken up to twice a week and includes the extraction of 10 liters of blood, which is almost a quarter of the total blood volume of a mare.
Individual evidence
- ↑ Chorionic Gonadotropin - Lexicon of Biochemistry . ( Spektrum.de [accessed on August 6, 2017]).
- ↑ Seiji Kito, Yuki Ohta: Medium effects on capacitation and sperm penetration through the zona pellucida in inbred BALB / c spermatozoa . In: Zygote (Cambridge, England) . tape 13 , no. 2 , May 2005, ISSN 0967-1994 , p. 145-153 , PMID 16128410 .
- ↑ Jason E. Swain, Marlane Angle, Nadir Ciray, Juergen Liebermann, Thomas Pool: A Commercially Available Dual - Buffered IVF Handling Medium Containing HEPES and MOPS Maintains Stable pH and Supports Human Sperm Survival, Normal Fertilization Following ICSI and Embryo Development. (PDF) Retrieved August 6, 2017 .
- ↑ How pharmaceutical companies do business with horse blood. In: sueddeutsche.de. September 29, 2015, accessed August 20, 2018 .