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Microinsemination and Nuclear Transfer in Mammals

1. Microinsemination

Inability to conceive after one year of adequate unprotected intercourse (six months if the woman is over age 35)

Male Factor Infertility

Anatomic defects Genetics Causes Trauma Infection Endocrine disorders Varicocele

Male Factor Infertility

Vol. > 2 ml, Conc. > 20x106, Mot. >50%, Morph. > 30%. Total motile sperm per milliliter > 10 x 106


Solution to male infertility factor :

Intrauterine Insemination In vitro fertilization (IVF) Intracytoplasmic Sperm Injection (ICSI)

Invitro Fertilization : Watch video

Tugas minggu depan : Jelaskan mengapa sperma tidak bisa membuahi sel telur yang telah dibuahi

Intracytoplasmic Sperm Injection

Laboratory setting for micromaipulation


Rate of success in mouse

Rate of success in rabit

The youngest and oldest records

Microinsemination with round spermatids from 17 day-old males Miki et al., J. Reprod. Dev., 2004

The youngest and oldest records

Microinsemination with round spermatids From azoospermic 33 month-old males Tanemura et al., Lab. Anim. Sci., 1997)

1. Nuclear transfer

The reproduction of a cell and, more generally, of a whole living organism without any modification of its genotype. Basically, non-sexual reproduction is cloning.
This phenomenon occurs widely in nature (bacteria and yeast reproduce according to thisProcess)

The same situation is encountered with the somatic cells of pluricellular Organisms
Plants can multiply independently of sexual reproduction Until 40 years ago, No phenomenon of this kind has ever been observed in vertebrates

Embryo development

In higher animals, it is recognized that the initial cell of the zygote formed just after fertilization (two or four cells of the young embryo) is totipotent. Totipotent : the cell can generate all the cells of the organism. Each of these cells can develop to generate a living organism when introduced into the zona pellucida of an oocyte

Beyond the blastocyst stage, the embryonic cells become progressively specialized. This process is known as embryonic differentiation

A blastocyst, is composed of two categories of cells : 1. The cells forming a unicellular layer along the zona pellucida, known as the trophectoderm, which is the precursor of the placenta.

2. The cells forming a compact block, known as inner cell mass are pluripotent.

The inner cell mass can be transplanted into blastocyst and generating a chimaera.

This kind of organism must not be confused with a hybrid.

A chimaera results from a mixture of cells that are superimposed, each one keeping its own genome. The gametes and offspring of chimaerae are derived from only one cell type of the original embryo.

Their genes are therefore not a combination of those from two embryos. In some cases, chimaeric animals may be obtained with cells originating from two different species. The animals are then formed by a mixture of cells from both species, but not a mixture of both. Interspecific chimaerae have been obtained with goats and sheep, quail and chickens.

The basic idea of nuclear transfer

Oocyte cytoplasm capable of favouring or inducing cell totipotency. Indeed, the sperm nucleus is transcriptionally inactive Oocyte cytoplasm thus has a strong capacity to reprogramme a genome for embryo development.

The dedifferentiation of a somatic cell to give a totipotent cell implies the reactivation of numerous silent genes. The phenomenon is referred to as genome reprogramming The mechanisms involved in this phenomenon are not known

First Nucleus transfer

Isolated nuclei from Xenopus embryonic cells were transferred by micromanipulation into the cytoplasm of enucleated oocytes.

Enucleation is imperative to eliminate the oocyte genome and to prevent the embryo from becoming triploid after the transfer of a diploid nucleus.

Enucleation was carried out by aspiration using a micropipette.

These pioneer experiments were not successful. It was sugested due to the utilization of an isolated nucleus, which may have lost essential components during the micromanipulation process.

Alternative method
Injecting a diploid embryonic cell between the zona pellucida and the plasma membrane of the enucleated oocyte.
This first step was followed by a fusion of the plasma membranes of the cell and the oocyte using electric current.

This treatment destabilizes the cell membranes, which then spontaneously fuse The nucleus of the cell is thus transferred to the oocyte cytoplasm without any micromanipulation.


Fertilization is not only the transfer of the sperm genome into the oocyte. Sperm also contains factors that induce calcium uptake by the oocyte. This is an essential signal to trigger embryo development

The electric field creates pores in the oocyte membrane, making it possible for the calcium in the medium to enter the cell.

This mimics the activation of the oocyte by sperm. The reconstituted embryo can thus start its development. Xenopus was finaly succesfully cloned in this manner 40 years ago

The success rate of these experiments was and is still low, even if it has been slightly improved after 15 years. Only about one per cent of the embryos reconstituted by nuclear transfer give rise to normal animals.

Possible cause
Cloning fails so frequently may result from the discordance between the stages of the division cycle of the donor cells and the recipient oocytes.

Cell synchronization can be carried out by depleting the culture medium of growth factors and serum.
The cells stop their division and enter the G0 phase. The addition of serum and growth factors to the medium reinitiates the division cell cycle in a synchronized manner. This methodological study initially revealed that sheep pluripotent cells cultured for several weeks were able to generate living lambs after nuclear transfer (Campbell et al., 1996).

Essentially, the same technique was used to generate lambs from foetal and adult cells. Cells used for this study were maintained at the G0 phase of the division cycle. This experiment led to the birth of several lambs from foetal cells and one, Dolly, from adult cells.

Before Dolly's birth, it is believed that cloning was possible only using pluripotent cells
The birth of the cloned animal using nuclei from somatic cells was successfully repeated and extended to two other ruminants, cows and goats, without any major adaptation

Factors affecting cloning success

1. Animal species Attempts to clone mice started several decades ago and failed A new protocol was developed. Nuclei were transferred to enucleated oocytes by direct microinjection of the isolated nuclei, rather than by cell fusion as is the case in other species.

Factors affecting cloning success

A specific method of zygote activation was also required. The activation was achieved by adding ions such as calcium for controlled periods of time to the culture medium after nuclear transfer. Attempts to use it on ruminants led to low success rates.

Factors affecting cloning success :

2. Type of cells
Some cell types give much better cloning results than others. Batches of cells from the same foetal sampling but cultured under slightly different conditions show quite different capacities for generating clones after nuclear transfer

Are cloned animal normal ?

Dolly's cells have abnormally short telomeres

Telomeres protect DNA from degradation by exonucleases.

Are cloned animal normal ?

Telomeres are constantly degraded and restored. The balance becomes negative as the cells age, leading to a degradation of chromosomes and to cell death after about 50 multiplications Gamete maturation might also include a lengthening of telomeres to ensure a long life for offspring. In all the cloned animals obtained after Dolly has normal or longer telomeres

Hypothesis :
The defect in cells may be of genetic origin. Inappropriate genome reprogramming is the essential cause of failure in animal cloning

Cloned animals may be genetically identical to each other but not strictly to the nuclear donor. Mitochondria found in clones are those of the recipient oocytes but not of the donor cells


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